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Emilia Biffi (ed.), Microfluidic and Compartmentalized Platforms for Neurobiological Research, Neuromethods,
vol. 103, DOI 10.1007/978-1-4939-2510-0_1, © Springer Science+Business Media New York 2015
Chapter 1
The Fabrication of Microfluidic Platforms
with Pneumatically/Hydraulically Controlled PDMS Valves
and Their Use in Neurobiological Research
Bryson M. Brewer, Donna J. Webb, and Deyu Li
Abstract
Microfluidic technology has made a significant impact in neurobiological research. The new capabilities
offered by microfluidic devices allow researchers to investigate neurobiological phenomena in ways previ-
ously unachievable using traditional cell biology techniques. Here we detail the fabrication of a microflu-
idic cell coculture platform that uses pneumatically or hydraulically controlled valves to reversibly separate
cell populations. Using this platform, communication between cell populations in different culture cham-
bers can be enabled or restricted as desired. This allows for both growth of different cell types in each
respective optimal culture media and separate treatment of individual cell populations with transfecting
agents, growth factors, and drugs, etc. At a desired time-point, cell-cell interactions can be studied by
deactivating the valve. The device has been used previously to transfect neurons with different fluorescent
presynaptic and postsynaptic markers and then observe in real-time the subsequent process of synapse
formation. Additionally, the platform has been an effective tool for investigating the role of glia–neuron
communication on synaptic formation and stability. In this chapter, the design, fabrication, and operation
of the valve-enabled microfluidic cell coculture platform are clearly described so as to enable the reader to
replicate the device for use in future neurobiological research.
Key words Microfluidic cell coculture, Synapse formation, Neuron–glia coculture, Pneumatic/
hydraulic valve actuation, Soft lithography
1 Introduction
The ongoing development of microfluidic technology has produced
tools for neurobiological researchers that provide several advan-
tages over traditional methods and techniques [1]. The spatiotem-
poral control afforded by microfluidic devices makes the technology
well suited for manipulating the cellular microenvironment [2].
This capability is especially useful for in vitro studies at the single-
cell level [3], and as a result, an increasing number of microfluidic
platforms have been successfully employed for neurobiological
investigations [4]. A variety of applications have been demonstrated,](https://image.slidesharecdn.com/33718-250314204213-1755516e/75/Microfluidic-and-Compartmentalized-Platforms-for-Neurobiological-Research-1st-Edition-Emilia-Biffi-Eds-22-2048.jpg)
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including (1) a platform that incorporates a multi-electrode array
with a microfluidic chip to monitor the electrophysiological activ-
ity of cultured neuronal networks [5], (2) a gradient-generating
design that optimizes the proliferation of neural stem cells in cul-
ture [6], and (3) a device that utilizes fluidic isolation to separate
neuronal cell bodies while allowing axonal growth through con-
necting microgrooves in order to independently probe axonal
injury, regeneration, and transport [7]. These are just a few exam-
ples of many that demonstrate the potential of microfluidic tech-
nology in the field of neurobiology.
By far, the most popular material used to construct microfluidic
devices is polydimethylsiloxane (PDMS) [8]. This flexible, optically
transparent material is cost-effective, biocompatible, and capable of
rapid prototyping via soft lithography [9–12]. Standardized proce-
dures exist for attaching fabricated PDMS chips to different substrates
(with the most popular substrate being glass) [13], as well as bonding
and assembling multiple PDMS layers together, providing the capa-
bility of three-dimensional microfluidic designs [14]. In addition, the
flexibility of PDMS allows pneumatically or hydraulically controlled
valves to be easily integrated onto a microfluidic chip, allowing for
greater control over fluid flow within the microchannels [15]. These
properties of PDMS make it a suitable material for the fabrication of
the pneumatically/hydraulically valve-enabled microfluidic platform
described in this chapter.
The valve-enabled microfluidic device that is the focus of the
following sections was developed by the authors and can be used
for a variety of cell coculture applications. For example, the device
has been used in the monitoring of cancer and endothelial cell
cross-migration to investigate the effects of specific angiocrine fac-
tors on tumor cell motility [16]. The platform is particularly well
suited for a number of neurobiology applications, being used to
observe the dynamic formation of synaptic contacts between neu-
ronal populations [17] and to investigate the effects of glia cocul-
ture on both neuronal transfection efficiency [18] and synapse
formation and stability [19].
Figure 1 illustrates the key elements of the microfluidic plat-
form. The device consists of either two or four microfluidic cell
culture chambers that are connected by short, narrow barrier
regions. These barrier regions either take the form of an array of
microgrooves or a single continuous slit, depending on the appli-
cation. A pressure control chamber attached above the barrier
regions can be pressurized, forcing the flexible PDMS barriers to
collapse, effectively separating the cell culture chambers. This capa-
bility allows cell populations in each chamber to be treated inde-
pendently (with different culture media, transfection agents, etc…)
until the pressure is removed and the barriers are released from the
substrate, re-enabling communication among cells in different
culture chambers. Additionally, the microfluidic device uses a passive
Bryson M. Brewer et al.](https://image.slidesharecdn.com/33718-250314204213-1755516e/75/Microfluidic-and-Compartmentalized-Platforms-for-Neurobiological-Research-1st-Edition-Emilia-Biffi-Eds-23-2048.jpg)
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pumping method [20, 21] to continuously provide fresh media to
cell cultures, eliminating the need for external flow control equip-
ment such as syringe pumps. As a result, the entire platform can be
contained inside a petri dish, reducing the risk of contamination
during transport between culture hoods, incubators, and micro-
scopes. Finally, the device has been successfully used with a variety
of microscopy techniques, including high magnification confocal
microscopy employing objectives with small working distances.
The following sections describe the fabrication and operation
of the pneumatically/hydraulically valve-enabled microfluidic neu-
robiology platform. Three primary fabrication steps are detailed:
1. Photolithography—used to create an SU-8 replica mold on a
silicon wafer that will serve as the “negative” pattern for the
different PDMS layers. The mold can be used to make dozens
of PDMS devices before needing to be replaced.
2. Soft Lithography—used to fabricate the PDMS layers contain-
ing the cell culture chambers/connecting barriers, the pressure
control chamber, and tubing supports. This step involves pour-
ing liquid PDMS over the SU-8 mold, curing, and removing
to form the flexible, solid PDMS components of the device.
3. Assembly—used to combine all of the device components into
an operational microfluidic platform. Plasma bonding is
employed to irreversibly seal the different PDMS layers to a
Fig. 1 Overview of the two-chamber valve-enabled microfluidic platform. (a) The three-dimensional schematic
demonstrates the multiple layers of the device, as well as the location of the inlet/outlet reservoirs used for
loading cells and media and the inlet/outlet tubing for the pressure control chamber. (b) An overhead view of
the device clearly showcases the position of the cell culture chambers in relation to the pressure control cham-
ber. (c) A cross-sectional slice identifies the pressure control chamber and barrier region connecting the two
culture chambers. Note that the images here are not to scale
The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…](https://image.slidesharecdn.com/33718-250314204213-1755516e/75/Microfluidic-and-Compartmentalized-Platforms-for-Neurobiological-Research-1st-Edition-Emilia-Biffi-Eds-24-2048.jpg)
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This completes the fabrication of the pneumatic/hydraulic
valve-enabled microfluidic neurobiology platform. The devices can
typically be stored in Parafilm sealed petri dishes for weeks before
use, as long as the channels and chambers remain filled with water.
After assembly, the microfluidic device is ready to be used for
suitable neurobiological assays. The following steps demonstrate
how to prepare the device for use and activate/deactivate the pres-
sure valve (Fig. 6a, b). For more detailed examples of previously
documented biological protocols using this device (coating of
channels, cell loading, etc.), please see references [17–19].
1. First, each device should be sterilized using UV (ultra-violet)
irradiation found in most cell-culture hoods. UV exposure
should last for at least 1 h.
2. When ready to activate the pressure valve, a 5 mL plastic
syringe with a 23-gauge needle should be filled with either air
or water. Insert the syringe into the inlet tubing of the pressure
control chamber.
(a) For pneumatic pressurization, clamp the outlet tubing of
the pressure control chamber closed using a metal pinch
clamp. Then inject 0.2–0.3 mL of air into the pressure
control chamber and clamp the inlet tubing (see Note 25).
(b) For hydraulic pressurization, fill the entire pressure control
chamber and tubing with water. Then, clamp the outlet
tubing. Finally, inject an additional 0.2–0.3 mL of water
into the chamber and clamp the inlet tubing (see Note 26).
3. To deactivate the valve, unclamp the inlet and outlet tubing of
the pressure control chamber (see Note 27).
In order to check that your device is working properly, fluores-
cent dye or food coloring can be added to one of the channels after
pressurization. Any leakage through the barrier/microgrooves should
be apparent. Additionally, the dye should perfuse freely through the
barrier/microgroove region after releasing the pressure (see Fig. 7).
3.3 Device Operation
Fig. 6 Three-dimensional cross-section of a two-chamber, continuous barrier device before and after applying
pressure to the control chamber. (a) An unpressurized device is shown with the continuous barrier elevated
above the substrate. Here the two cell culture chambers are connected. (b) A device that has been pressurized
using either pneumatic or hydraulic pressure in which the barrier is pushed down to the substrate, effectively
separating the two cell culture chambers
Bryson M. Brewer et al.](https://image.slidesharecdn.com/33718-250314204213-1755516e/75/Microfluidic-and-Compartmentalized-Platforms-for-Neurobiological-Research-1st-Edition-Emilia-Biffi-Eds-35-2048.jpg)
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4 Notes
1. The fabrication of the molds or replica-masters to be employed
later in the soft-lithography step is achieved using standard
photolithography protocols. For best results, this step should
be conducted in a Class 1000 clean room (or better).
2. Although there are many available options for printing photo-
masks, the authors have had past success using CAD/Art
Services, Inc. for designs similar to those presented here.
3. The dimensions of the device features can be modified to fit a
particular application. For example, the cell culture chambers
could be made 1–10 mm long and/or 200–2,000 μm wide,
and the width of the barrier region varied from 50 to 500 μm
without sacrificing device performance. However, note that in
order to better control the location where cells will be seeded
within the channel, the cell culture chamber should be at least
twice as wide as the inlet/outlet microchannels. The flow velo-
city decreases in the wider cell culture region, slowing down
the cells and allowing them to settle onto the substrate. In
addition, the aspect ratio of the final mold must be considered
when designing a mask, as SU-8 features with a high aspect
ratio may be prone to breaking during later soft-lithography
steps. Concurrently, PDMS microchannels with a very low
aspect ratio may collapse in the absence of support pillars.
Finally, be mindful of the dimensions of the glass substrate you
will use when fabricating the final device. All device features
should be designed to be at least 3 mm from the edges of the
glass substrate after final device assembly.
Fig. 7 Demonstration of proper valve behavior using fluorescent dye. (a) Fluorescein isothiocyanate (FITC)
loaded into the bottom cell culture chamber is contained as long as the valve barrier is properly activated using
pneumatic/hydraulic pressure. (b) After deactivating the barrier, FITC is free to flow across the barrier region
into the top cell culture region. Figure reprinted with permission from ref. [18]
The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…](https://image.slidesharecdn.com/33718-250314204213-1755516e/75/Microfluidic-and-Compartmentalized-Platforms-for-Neurobiological-Research-1st-Edition-Emilia-Biffi-Eds-36-2048.jpg)
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20. Properly aligning the PDMS cell culture chamber layer with
the glass can be tricky initially. It is best to hold the PDMS
layer by its edges (being careful not to touch the plasma treated
face) and line up the top of the PDMS with the top of the
coverslip. Then, the PDMS layer can be gently dropped onto
the glass. On a dark background, the formation of the bond
between the two layers can be observed. Note that this bond is
irreversible, so care must be taken during alignment.
21. After plasma treatment, the surface of the PDMS changes from
its natural hydrophobic state to a hydrophilic state. However,
this change is temporary, and the PDMS will become hydro-
phobic again if left exposed to air [22]. Hydrophobic channel
walls make loading microchannels with water difficult, as pres-
sure must be applied to force the water through, and air bubbles
will often get trapped in the corners of channels and chambers.
On the other hand, hydrophilic channels can be loaded with
media using capillary action, and air bubbles are generally not a
problem.
22. Sometimes during device assembly the roof of the microgroove
or barrier region will collapse to the glass substrate. In order to
check if this has occurred, one should load only one channel
with DI water. Then, observe whether or not the water
flows through the microgroove/barrier region to the other
channel(s). If the water does not flow through, slight vacuum
or pressure can be applied to neighboring channels to force the
roof of the microgroove/barrier to release from the substrate,
allowing the water to flow freely. Generally, this problem
occurs more often in continuous barrier rather than micro-
groove devices but can usually be avoided by careful device
assembly.
23. The liquid PDMS mixture should be “painted” on top of the
tubing supporter around the tubing. This can be achieved
using the tip of a scalpel to apply a small drop around the
perimeter of the tubing. Be careful not to add too much, as
extra liquid PDMS could run off of the tubing supporters onto
the pressure control chamber and form a rounded layer over
the viewing window, potentially causing imaging problems in
bioassays.
24. Again, a scalpel can be used to apply the thin coating of liquid
PDMS to the cylinder. Make sure the surface of the PDMS is
dry around the inlet/outlet punched holes before attaching the
cylinder. Any water present on the surface at the attachment
point will result in a poor seal and likely lead to leakage.
25. Due to the compressibility of air, more air may need to be
injected to ensure that the valve is activated. By closely watch-
ing the walls of the pressure control chamber, a slight bulging
The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…](https://image.slidesharecdn.com/33718-250314204213-1755516e/75/Microfluidic-and-Compartmentalized-Platforms-for-Neurobiological-Research-1st-Edition-Emilia-Biffi-Eds-40-2048.jpg)
![26
to perform highly parallelized and reproducible analyses in a
high-throughput fashion. PDMS itself offers several advantageous
volume and surface properties such as optical transparency, bio-
compatibility, gas permeability, and ease of fabrication, making
microfluidic devices suitable for cell culture applications [1]. Addi-
tionally, microfluidic platforms offer the potentiality to precisely
monitor parameters in time- and space-controlled manners within
the cellular microenvironments, providing greater control over the
cell response to different stimuli [2]. Among other research fields,
microfluidic devices have been widely used in neuroscience [3–5]
thanks to their size-scale, compatible with neurons own size.
Particularly, the direct integration of PDMS microfluidic devices
with multielectrode arrays (MEAs) allows coupling the recording
of neuronal electrical activity together with the inherent high con-
trol of the cell microenvironment offered by the microfluidic
approach [6–8].
Currently, most of the microfluidic devices are fabricated by
bonding irreversibly a PDMS stamp containing the chambers and
microchannels to a second substrate layer (glass or polymer), by
activating both surfaces through air or oxygen plasma [9]. This
bonding technique allows for a reliable sealing able to withstand
relatively high pressures (200 kPa and above) without the addition
of any gluing material [10]. However, the irreversibility of the
bonding introduces several limitations in terms of possible device
exploitations, including cell and material patterning, surface func-
tionalization as well as capability of samples injection and retrieval
to perform secondary analyses and direct measurements not achiev-
able within the device. Moreover, the affordability of coupling
PDMS microfluidic devices with valuable substrates, like MEAs, is
subordinated to the ability of disassembling the devices and reus-
ing the substrate upon sterilization. For these reasons, many efforts
have been recently directed to the development of reversible-
bonding methods, which have to attain an adhesion between layers
sufficient to withstand the pressure required for the microfluidic
applications, while enabling a simple access to the “experimental
field” and a non-disposable use of the devices. Reversible sealing
between PDMS microfluidic layer and MEAs substrates have been
attempted through different techniques including self-adhesion
[6] and reversible plasma bonding [11]. However, these tech-
niques do not assure a high hydraulic tightness when relatively
high pressure are applied (<35 kPa), limiting their application to
static or quasi-static conditions [12].
In this chapter, we describe an inexpensive and reliable technique
for reversely bonding PDMS microfluidic device to different flat sub-
strates for long-term culture of cells [13]. The presented bonding
technique is based on magnetic forces which allows to obtain devices
Paola Occhetta et al.](https://image.slidesharecdn.com/33718-250314204213-1755516e/75/Microfluidic-and-Compartmentalized-Platforms-for-Neurobiological-Research-1st-Edition-Emilia-Biffi-Eds-45-2048.jpg)
![27
able to withstand a high range of working pressures (up to about
150 kPa), thus suitable for cell culture applications requiring a
continuous perfusion. In detail, a PDMS/iron micropowder layer is
aligned onto a microfluidic layer and coupled with a flat substrate
exploiting the either temporary or continuous use of a permanent
magnet (Fig. 1). The introduced reversibly sealing method is charac-
terized by compatibility with: (1) complex fluidic layer configurations,
(2) micrometer size channel expanding the range of application of
previously reported magnetic bonding technique, and (3) optical
transparency for flow visualization and inspection over channel
regions.
Specifically, neuronal networks were grown within the descri-
bed device reversibly coupled to a commercial flat MEA for long-
term primary neuronal cell cultures [14, 15]. However, the method
offers high versatility and the design can be easily adapted to differ-
ent biological applications. As an example, the same technique can
be applied to precisely functionalize flat surfaces through protein
perfusion or pattern either 2D cell sheets or 3D cellular structures
with a microscale precision.
Fig. 1 Exploded view of the reversible bonding microfluidic device. The device is mainly made of PDMS and it
is constituted by two layers: the bottom layer (fluidic layer) contains the fluidic features; the top layer (magnetic
layer) contains a magnetic filler solution and it is optically transparent in correspondence to the fluidic regions.
Once the device is assembled, channels are closed onto a cell culture substrate (e.g., histology glass slide) and
maintained tight through a uniform magnetic field generated by a permanent magnet
A Reliable Reversible Bonding Method for Perfused Microfluidic Devices](https://image.slidesharecdn.com/33718-250314204213-1755516e/75/Microfluidic-and-Compartmentalized-Platforms-for-Neurobiological-Research-1st-Edition-Emilia-Biffi-Eds-46-2048.jpg)
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- 6. NE U RO M E T H O D S Series Editor Wolfgang Walz University of Saskatchewan Saskatoon, SK, Canada For further volumes: http://www.springer.com/series/7657
- 8. Microfluidic and Compartmentalized Platforms forNeurobiological Research Edited by Emilia Biffi Bioengineering Laboratory, Scientific Institute IRCCS E.Medea, Bosisio Parini (LC), Italy DepartmentofElectronics,InformationandBioengineering,PolitecnicodiMilano, Milan, Italy
- 9. ISSN 0893-2336 ISSN1940-6045 (electronic) Neuromethods ISBN 978-1-4939-2509-4 ISBN 978-1-4939-2510-0 (eBook) DOI 10.1007/978-1-4939-2510-0 Library of Congress Control Number: 2015932873 Springer New York Heidelberg Dordrecht London © Springer Science+Business Media New York 2015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper Humana Press is a brand of Springer Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com) Editor Emilia Biffi Bioengineering Laboratory Scientific Institute IRCCS E. Medea Bosisio Parini (LC), Italy Department of Electronics Information and Bioengineering Politecnico di Milano, Milan, Italy
- 10. v Experimental life scienceshave two basic foundations: concepts and tools. The Neuromethods series focuses on the tools and techniques unique to the investigation of the nervous system and excitable cells. It will not, however, shortchange the concept side of things as care has been taken to integrate these tools within the context of the concepts and questions under investigation. In this way, the series is unique in that it not only collects protocols but also includes theoretical background information and critiques which led to the methods and their development. Thus it gives the reader a better understanding of the origin of the techniques and their potential future development. The Neuromethods publishing program strikes a balance between recent and exciting developments like those concerning new ani- mal models of disease, imaging, in vivo methods, and more established techniques, includ- ing, for example, immunocytochemistry and electrophysiological technologies. New trainees in neurosciences still need a sound footing in these older methods in order to apply a critical approach to their results. Under the guidance of its founders, Alan Boulton and Glen Baker, the Neuromethods series has been a success since its first volume published through Humana Press in 1985. The series continues to flourish through many changes over the years. It is now published under the umbrella of Springer Protocols. While methods involving brain research have changed a lot since the series started, the publishing environment and technology have changed even more radically. Neuromethods has the distinct layout and style of the Springer Protocols program, designed specifically for readability and ease of reference in a laboratory setting. The careful application of methods is potentially the most important step in the process of scientific inquiry. In the past, new methodologies led the way in developing new disci- plines in the biological and medical sciences. For example, Physiology emerged out of Anatomy in the nineteenth century by harnessing new methods based on the newly discov- ered phenomenon of electricity. Nowadays, the relationships between disciplines and meth- ods are more complex. Methods are now widely shared between disciplines and research areas. New developments in electronic publishing make it possible for scientists that encounter new methods to quickly find sources of information electronically. The design of individual volumes and chapters in this series takes this new access technology into account. Springer Protocols makes it possible to download single protocols separately. In addition, Springer makes its print-on-demand technology available globally. A print copy can there- fore be acquired quickly and for a competitive price anywhere in the world. Wolfgang Walz Series Preface
- 12. vii Microfluidics is atechnology which features the manipulation of small amounts of fluids in channels with dimensions of tens to hundreds of micrometers. Microfluidics takes advantage of both soft lithography and poly(dimethylsiloxane) (PDMS), a silicon-based elastomeric material which is cheap, easy to mold and with good optical properties, as well as nontoxic to cells and gas permeable, offering a suitable solution for cell and tissue culture experiments. The design of microfluidic devices as platforms with different compartments had been inspired by the so-called Campenot chambers, developed to compartmentalize axons and cell bodies. Microfabricated versions of these devices have already been used in a wide range of biological applications thanks to their low consumption of samples and reagents, the ability to precisely control parameters within the cellular microenvironment, and the capa- bility to perform highly concurrent and reproducible analyses. Thanks to their scale, com- patible with neuron size, compartmentalized microfluidic devices are nowadays a crucial tool in the field of neuroscience. They indeed provide a powerful platform for the manipu- lation of subpopulation of neuronal cells and the study of complex system dynamics associ- ated to the connectivity between two or more adjacent regions of the brain. Thanks to the feature of fluidically separated compartments, these devices are also suitable for local cell stimulation, for the creation of dynamic concentration gradients, or for high-content phar- macological screening. In this volume, the main cutting-edge techniques to design and fabricate compartmen- talized microfluidic devices are described in Part I. In Chap. 1, a microfluidic cell coculture platform that uses pneumatically or hydraulically controlled valves to reversibly separate cell populations is described. Chapter 2 illustrates the procedures to fabricate microfluidic devices reversibly sealed to different flat substrates through magnetic forces, which are a suitable approach for long-term cultures of neurons due to their reliable hydraulic tight- ness. In Chap. 3, microfluidic circuits for arraying neurons with single cell precision, enabling high-throughput experimentation, are presented as well as approaches to couple these minimalistic cocultures to open access reservoirs for electrophysiology recordings. Part II is dedicated to the topic of axon guidance and manipulation. Chapter 4 pro- vides details of a microfluidic chip with a modular design for highly defined isolation of axons, asymmetric genetic manipulation, and whole-cell patch-clamp recording. In Chap. 5 a unique laser cell-micropatterning system for the creation of a compartmentalized, axon- isolating, polarized neuron-growth platform at the single-cell level is described. Chapter 6 explains how Campenot cultures and microfluidics chambers can be coupled and used together for biochemical analysis and for high resolution imaging of Dorsal Root Ganglia (DRG) neuronal cell bodies and their extensive axons. In Part III, compartmentalized devices for synapse manipulation are described. Chapter 7 illustrates an easy and inexpensive technique based on microfluidics that provides a high degree of control in positioning and guiding cells, thereby enabling the laying down of desired cellular networks and facilitating the study of synaptic connections. In Chap. 8, the use of three-compartment microfluidic devices to model in vitro activity-dependent synap- tic plasticity with dual inputs is detailed and the synaptic competition model is presented. Preface
- 13. viii Part IV isdedicated to the study of how different cell populations interact in either physiological or pathological condition. Chapter 9 describes a six-compartment neuron- glia coculture microsystem platform, where interactions between the axon and glia can be studied in isolation. In Chap. 10, compartmentalized microfluidic devices to study periph- eral neuro-osteogenic interactions are discussed, and qualitative and quantitative analyses for two- or three-dimensions cocultures are also presented. Chapter 11 provides details of a compartmented in vitro model of the lower motor neuron-neuromuscular junction cir- cuit, incorporating primary spinal motor neurons, supporting glia and skeletal muscle, and spatially mimicking the unique anatomical and cellular interactions of this circuit. In Part V, compartmentalized devices for pharmacological research and drug discovery are described. Chapter 12 details a powerful tool based on microfluidics used to mimic the key pathological hallmarks of Alzheimer’s disease and observe the long-term disease spread- ing at the microscale. In Chap. 13 a device for long-term growth of twin neuronal networks and for their controlled biochemical stimulation and electrophysiological recording is described. Finally, in Chap. 14 the protocol and methodological considerations for devel- oping synapse microarrays enabling ultrasensitive, high-throughput and quantitative screening of small molecules involved in synaptogenesis are illustrated. The authors and I believe you will find this volume invaluable in gaining an under- standing of the practical skills needed to fabricate and use microfluidics and compartmen- talized platforms with cell cultures as well as the strengths of these exciting devices and their precious contribution in the field of neuroscience. Milan, Italy Emilia Biffi Preface
- 14. ix Series Preface .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi PART I DESIGNS AND METHODS 1 The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically Controlled PDMS Valves and Their Use in Neurobiological Research . . . . . . . 3 Bryson M. Brewer, Donna J. Webb, and Deyu Li 2 A Reliable Reversible Bonding Method for Perfused Microfluidic Devices . . . . 25 Paola Occhetta, Emilia Biffi, and Marco Rasponi 3 Bridging Two Cultures: Minimalistic Networks Prepared by Microfluidic Arraying, and Open Access Compartments for Electrophysiology . . . . . . . . . . 39 Jonathan West, Ngoc-Duy Dinh, Heike Hardelauf, Ya-Yu Chiang, Tracey A. Newman, Mariana Vargas-Caballero, Ayodeji A. Asuni, Katrin Deinhardt, and Martin Arundell PART II AXONAL GUIDANCE AND MANIPULATION 4 Asymmetric Genetic Manipulation and Patch Clamp Recording of Neurons in a Microfluidic Chip. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Prasanna Sakha, Cecilia Brunello, Joonas Heikkinen, Ville Jokinen, and Henri J. Huttunen 5 Development of a Compartmentalized Biochip for Axonal Isolation and Neuronal-Circuit Formation at the Single-Cell Level . . . . . . . . . . . . . . . . 83 Ting Huang, Russell K. Pirlo, Wan Qin, Yongliang Lin, Lina Wei, Lucas Schmidt, Nick Erdman, Tingfei Xi, Mauris N. DeSilva, and Bruce Z. Gao 6 Campenot Cultures and Microfluidics Provide Complementary Platforms for Spatial Study of Dorsal Root Ganglia Neurons . . . . . . . . . . . . . . 105 Sara J. Fenstermacher, Maria F. Pazyra-Murphy, and Rosalind A. Segal PART III MANIPULATION OF SYNAPSES 7 Development of Microfluidic Devices for the Manipulation of Neuronal Synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Anika Jain and Martha U. Gillette 8 Use of a 3-Compartment Microfluidic Device to Study Activity Dependent Synaptic Competition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Ainsley Coquinco and Max Cynader Contents
- 15. x PART IV INTERACTIONOF DIFFERENT CELL POPULATIONS 9 Multi-compartment Neuron–Glia Coculture Microsystem. . . . . . . . . . . . . . . . 149 Jaewon Park, Sunja Kim, Jianrong Li, and Arum Han 10 Compartmentalized Microfluidic Platforms as Tool of Choice to Study the Interaction Between Neurons and Osteoblasts. . . . . . . . . . . . . . . 161 Estrela Neto, Diogo Paramos-de-Carvalho, Ana Henriques Lourenço, Paulo Aguiar, and Meriem Lamghari 11 A Novel In Vitro Primary Culture Model of the Lower Motor Neuron–Neuromuscular Junction Circuit. . . . . . . . . . . . . . . . . . . . . . . 181 Katherine A. Southam, Anna E. King, Catherine A. Blizzard, Graeme H. McCormack, and Tracey C. Dickson PART V PHARMACOLOGICAL MANIPULATION AND SCREENING 12 Compartmentalized Microfluidics for In Vitro Alzheimer’s Disease Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Yufei Ren, Anja Kunze, and Philippe Renaud 13 Selective Biochemical Manipulation of Twin Neuronal Networks on Microelectrode Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 Giulia Regalia, Emilia Biffi, Marco Rasponi, and Alessandra Pedrocchi 14 Compartmentalized Synapse Microarray for High-Throughput Screening . . . . 231 Amol D. Jadhav, Wei Li, Zhen Xu, and Peng Shi Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 Contents
- 16. xi PAULO AGUIAR •INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal MARTIN ARUNDELL • Institute for Life Sciences, University of Southampton, Southampton, UK AYODEJI A. ASUNI • Institute for Life Sciences, University of Southampton, Southampton, UK; Centre for Biological Sciences, University of Southampton, Southampton, UK EMILIA BIFFI • Bioengineering Laboratory, Scientific Institute IRCCS E. Medea, Bosisio Parini (LC), Italy; Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy CATHERINE A. BLIZZARD • Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia BRYSON M. BREWER • Department of Mechanical Engineering, Vanderbilt University, Nashville, TN, USA CECILIA BRUNELLO • Neuroscience Center, University of Helsinki, Helsinki, Finland YA-YU CHIANG • Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Dortmund, Germany AINSLEY COQUINCO • Brain Research Centre, University of British Columbia, Vancouver, BC, Canada MAX CYNADER • Brain Research Centre, University of British Columbia, Vancouver, BC, Canada KATRIN DEINHARDT • Institute for Life Sciences, University of Southampton, Southampton, UK; Centre for Biological Sciences, University of Southampton, Southampton, UK MAURIS N. DESILVA • Naval Medical Research Unit San Antonio, Fort Sam Houston, TX, USA TRACEY C. DICKSON • Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia NGOC-DUY DINH • Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Dortmund, Germany NICK ERDMAN • Department of Bioengineering, Clemson University, Clemson, SC, USA SARA J. FENSTERMACHER • Department of Neurobiology, Harvard Medical School, Boston, MA, USA; Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA BRUCE Z. GAO • Department of Bioengineering, Clemson University, Clemson, SC, USA MARTHA U. GILLETTE • Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, IL, USA; Neuroscience Program, University of Illinois, Urbana-Champaign, IL, USA ARUM HAN • Department of Electrical and Computer Engineering, Texas A&M University, College Station, TX, USA; Department of Biomedical Engineering, Texas A&M University, College Station, TX, USA HEIKE HARDELAUF • Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Dortmund, Germany JOONAS HEIKKINEN • Department of Materials Science & Engineering, Aalto University, Espoo, Finland Contributors
- 17. xii TING HUANG •Department of Bioengineering, Clemson University, Clemson, SC, USA; Academy for Advanced Interdisciplinary Studies, Center for Biomedical Materials and Tissue Engineering, Peking University, Beijing, China HENRI J. HUTTUNEN • Neuroscience Center, University of Helsinki, Helsinki, Finland AMOL D. JADHAV • Department of Mechanical and Biomedical Engineering, City University of Hong Kong, Hong Kong SAR, China ANIKA JAIN • Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign, IL, USA VILLE JOKINEN • Department of Materials Science & Engineering, Aalto University, Espoo, Finland SUNJA KIM • Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA ANNA E. KING • Wicking Dementia Research and Education Centre, University of Tasmania, Hobart, TAS, Australia ANJA KUNZE • Di Carlo Laboratory, Department of Bioengineering, University of California, Los Angeles (UCLA), Los Angeles, CA, USA MERIEM LAMGHARI • INEB – Instituto de Engenharia Biomédica, Porto, Portugal; ICBAS - Institute for Biomedical Sciences Abel Salazar University of Porto, Porto, Portugal JIANRONG LI • Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA DEYU LI • Department of Mechanical Engineering, Vanderbilt University, Nashville, TN, USA WEI LI • Department of Mechanical and Biomedical Engineering, City University of Hong Kong, Hong Kong SAR, China YONGLIANG LIN • National Engineering Laboratory for Regenerative Implantable Medical Devices, Guangzhou, Guangdong, China ANA HENRIQUES LOURENÇO • Instituto de Engenharia Biomedica (INEB), University of Porto, Porto, Portugal; FEUP - Faculty of Engineering, University of Porto, Porto, Portugal GRAEME H. MCCORMACK • Wicking Dementia Research and Education Centre, University of Tasmania, Hobart, TAS, Australia ESTRELA NETO • INEB – Instituto de Engenharia Biomédica, Porto, Portugal; FMUP – Faculdade de Medicina da Universidade do Porto, Porto, Portugal TRACEY A. NEWMAN • Institute for Life Sciences, University of Southampton, Southampton, UK; Faculty of Medicine, University of Southampton, Southampton, UK; Clinical and Experimental Sciences, University of Southampton, Southampton, UK PAOLA OCCHETTA • Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy DIOGO PARAMOS-DE-CARVALHO • INEB - Instituto de Engenharia Biomédica, Universidade do Porto (ICBAS-UP), Porto, Portugal JAEWON PARK • Department of Electrical and Computer Engineering, Texas A&M University, College Station, TX, USA; Department of Electrical and Electronic Engineering, South University of Science and Technology of China, Shenzhen, China MARIA F. PAZYRA-MURPHY • Department of Neurobiology, Harvard Medical School, Boston, MA, USA; Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA ALESSANDRA PEDROCCHI • Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy Contributors
- 18. xiii RUSSELL K. PIRLO• Bioenergy and Biofabrication Section, Naval Research Laboratory, Washington, DC, USA WAN QIN • Department of Bioengineering, Clemson University, Clemson, SC, USA MARCO RASPONI • Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy GIULIA REGALIA • Department of Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy YUFEI REN • Microsystems Laboratory (LMIS4), Institute of Microengineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland PHILIPPE RENAUD • Microsystems Laboratory (LMIS4), Institute of Microengineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland PRASANNA SAKHA • Neuroscience Center, University of Helsinki, Helsinki, Finland LUCAS SCHMIDT • Department of Bioengineering, Clemson University, Clemson, SC, USA ROSALIND A. SEGAL • Department of Neurobiology, Harvard Medical School, Boston, MA, USA; Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA, USA PENG SHI • Department of Mechanical and Biomedical Engineering, City University of Hong Kong, Hong Kong SAR, China KATHERINE A. SOUTHAM • Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia MARIANA VARGAS-CABALLERO • Institute for Life Sciences, University of Southampton, Southampton, UK; Centre for Biological Sciences, University of Southampton, Southampton, UK DONNA J. WEBB • Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA; Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, TN, USA; Department of Cancer Biology, Vanderbilt University, Nashville, TN, USA LINA WEI • Department of Bioengineering, Clemson University, Clemson, SC, USA; Academy for Advanced Interdisciplinary Studies, Center for Biomedical Materials and Tissue Engineering, Peking University, Beijing, China JONATHAN WEST • Institute for Life Sciences, University of Southampton, Southampton, UK; Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Dortmund, Germany; Faculty of Medicine, University of Southampton, Southampton, UK TINGFEI XI • Academy for Advanced Interdisciplinary Studies, Center for Biomedical Materials and Tissue Engineering, Peking University, Beijing, China ZHEN XU • Department of Mechanical and Biomedical Engineering, City University of Hong Kong, Hong Kong SAR, China Contributors
- 20. Part I Designs andMethods
- 22. 3 Emilia Biffi (ed.),Microfluidic and Compartmentalized Platforms for Neurobiological Research, Neuromethods, vol. 103, DOI 10.1007/978-1-4939-2510-0_1, © Springer Science+Business Media New York 2015 Chapter 1 The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically Controlled PDMS Valves and Their Use in Neurobiological Research Bryson M. Brewer, Donna J. Webb, and Deyu Li Abstract Microfluidic technology has made a significant impact in neurobiological research. The new capabilities offered by microfluidic devices allow researchers to investigate neurobiological phenomena in ways previ- ously unachievable using traditional cell biology techniques. Here we detail the fabrication of a microflu- idic cell coculture platform that uses pneumatically or hydraulically controlled valves to reversibly separate cell populations. Using this platform, communication between cell populations in different culture cham- bers can be enabled or restricted as desired. This allows for both growth of different cell types in each respective optimal culture media and separate treatment of individual cell populations with transfecting agents, growth factors, and drugs, etc. At a desired time-point, cell-cell interactions can be studied by deactivating the valve. The device has been used previously to transfect neurons with different fluorescent presynaptic and postsynaptic markers and then observe in real-time the subsequent process of synapse formation. Additionally, the platform has been an effective tool for investigating the role of glia–neuron communication on synaptic formation and stability. In this chapter, the design, fabrication, and operation of the valve-enabled microfluidic cell coculture platform are clearly described so as to enable the reader to replicate the device for use in future neurobiological research. Key words Microfluidic cell coculture, Synapse formation, Neuron–glia coculture, Pneumatic/ hydraulic valve actuation, Soft lithography 1 Introduction The ongoing development of microfluidic technology has produced tools for neurobiological researchers that provide several advan- tages over traditional methods and techniques [1]. The spatiotem- poral control afforded by microfluidic devices makes the technology well suited for manipulating the cellular microenvironment [2]. This capability is especially useful for in vitro studies at the single- cell level [3], and as a result, an increasing number of microfluidic platforms have been successfully employed for neurobiological investigations [4]. A variety of applications have been demonstrated,
- 23. 4 including (1) aplatform that incorporates a multi-electrode array with a microfluidic chip to monitor the electrophysiological activ- ity of cultured neuronal networks [5], (2) a gradient-generating design that optimizes the proliferation of neural stem cells in cul- ture [6], and (3) a device that utilizes fluidic isolation to separate neuronal cell bodies while allowing axonal growth through con- necting microgrooves in order to independently probe axonal injury, regeneration, and transport [7]. These are just a few exam- ples of many that demonstrate the potential of microfluidic tech- nology in the field of neurobiology. By far, the most popular material used to construct microfluidic devices is polydimethylsiloxane (PDMS) [8]. This flexible, optically transparent material is cost-effective, biocompatible, and capable of rapid prototyping via soft lithography [9–12]. Standardized proce- dures exist for attaching fabricated PDMS chips to different substrates (with the most popular substrate being glass) [13], as well as bonding and assembling multiple PDMS layers together, providing the capa- bility of three-dimensional microfluidic designs [14]. In addition, the flexibility of PDMS allows pneumatically or hydraulically controlled valves to be easily integrated onto a microfluidic chip, allowing for greater control over fluid flow within the microchannels [15]. These properties of PDMS make it a suitable material for the fabrication of the pneumatically/hydraulically valve-enabled microfluidic platform described in this chapter. The valve-enabled microfluidic device that is the focus of the following sections was developed by the authors and can be used for a variety of cell coculture applications. For example, the device has been used in the monitoring of cancer and endothelial cell cross-migration to investigate the effects of specific angiocrine fac- tors on tumor cell motility [16]. The platform is particularly well suited for a number of neurobiology applications, being used to observe the dynamic formation of synaptic contacts between neu- ronal populations [17] and to investigate the effects of glia cocul- ture on both neuronal transfection efficiency [18] and synapse formation and stability [19]. Figure 1 illustrates the key elements of the microfluidic plat- form. The device consists of either two or four microfluidic cell culture chambers that are connected by short, narrow barrier regions. These barrier regions either take the form of an array of microgrooves or a single continuous slit, depending on the appli- cation. A pressure control chamber attached above the barrier regions can be pressurized, forcing the flexible PDMS barriers to collapse, effectively separating the cell culture chambers. This capa- bility allows cell populations in each chamber to be treated inde- pendently (with different culture media, transfection agents, etc…) until the pressure is removed and the barriers are released from the substrate, re-enabling communication among cells in different culture chambers. Additionally, the microfluidic device uses a passive Bryson M. Brewer et al.
- 24. 5 pumping method [20,21] to continuously provide fresh media to cell cultures, eliminating the need for external flow control equip- ment such as syringe pumps. As a result, the entire platform can be contained inside a petri dish, reducing the risk of contamination during transport between culture hoods, incubators, and micro- scopes. Finally, the device has been successfully used with a variety of microscopy techniques, including high magnification confocal microscopy employing objectives with small working distances. The following sections describe the fabrication and operation of the pneumatically/hydraulically valve-enabled microfluidic neu- robiology platform. Three primary fabrication steps are detailed: 1. Photolithography—used to create an SU-8 replica mold on a silicon wafer that will serve as the “negative” pattern for the different PDMS layers. The mold can be used to make dozens of PDMS devices before needing to be replaced. 2. Soft Lithography—used to fabricate the PDMS layers contain- ing the cell culture chambers/connecting barriers, the pressure control chamber, and tubing supports. This step involves pour- ing liquid PDMS over the SU-8 mold, curing, and removing to form the flexible, solid PDMS components of the device. 3. Assembly—used to combine all of the device components into an operational microfluidic platform. Plasma bonding is employed to irreversibly seal the different PDMS layers to a Fig. 1 Overview of the two-chamber valve-enabled microfluidic platform. (a) The three-dimensional schematic demonstrates the multiple layers of the device, as well as the location of the inlet/outlet reservoirs used for loading cells and media and the inlet/outlet tubing for the pressure control chamber. (b) An overhead view of the device clearly showcases the position of the cell culture chambers in relation to the pressure control cham- ber. (c) A cross-sectional slice identifies the pressure control chamber and barrier region connecting the two culture chambers. Note that the images here are not to scale The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…
- 25. 6 glass substrate, andliquid PDMS is used to attach tubing and cylindrical reservoirs to the microchannel inlets/outlets. Finally, operation of the device via activation/deactivation of the pneumatic/hydraulic valve barrier is discussed. 2 Materials and Instruments ● SU-8 negative photoresist (SU-8 2005, SU-8 2050, and SU-8 2150). ● Acetone. ● Isopropyl alcohol. ● Microchem SU-8 developer. ● 3″ polished silicon wafer. ● Spin coater. ● Two hot plates. ● Photolithography mask. ● Ultraviolet exposure system. Please see Note 1 for additional information. ● Dow Corning Sylgard 184 Silicone Encapsulant 0.5 kg Clear Kit (PDMS). ● 2 and 5 mm biopsy punches. ● 14-gauge blunt needle. ● 0.20″ I.D/0.60″ O.D. Tygon Microbore Tubing. ● 10 mm×10 mm pyrex cloning cylinders. ● Air/oxygen plasma cleaner. ● Vacuum pump. ● Vacuum chamber or desiccator. ● Oven. ● Deionized (DI) water. ● 5 mL plastic syringe. ● 23-gauge. ● Stainless steel metal pinch clamps. 3 Methods The first step in the fabrication of a pressure controlled valve- enabled microfluidic neurobiological coculture platform is the cre- ation of a master mold using photolithography with the negative photoresist SU-8. Using this SU-8 master, the PDMS-based device 2.1 Photolithography Using SU-8 Negative Photoresists 2.2 PDMS Based Soft-Lithography and Device Assembly 2.3 Device Operation 3.1 Photolithography Using SU-8 Negative Photoresists Bryson M. Brewer et al.
- 26. 7 layers can becreated. In addition, the master molds can be used numerous times (>100) before needing to be replaced. This allows for the fabrication of many microfluidic devices without spending much effort on the relatively time-consuming photolithography process. Note that all steps presented here (except the design of the photolithography mask) regarding the production of the mas- ter molds should be carried out in a clean room, preferably with a Class 1000 rating or better. This will decrease the likelihood of contamination (via dust or other airborne particles) during fabrica- tion that could damage the small features of the mold. In order to fabricate the SU-8 mold, a photolithography mask containing the channel features must be created. SU-8 is a negative photoresist; therefore, the channel features that are desired for the mold should be transparent on the mask, and all other areas should be opaque. First, CAD software (such as AutoCAD) is used to design the desired channel features. Then, a high-resolution pho- tomask (greater than 20,000 DPI) of the design is printed on a plastic transparency sheet (typically obtained using a CAD printing service, see Note 2). The fabrication of a valve-enabled neurobiology microfluidic platform requires at least three photomasks, two for the cell culture chamber PDMS layer and one for the pressure chamber PDMS layer. The two masks for the cell culture chamber layer include one for the taller channel/chamber features and one for the shorter barrier features. Figure 2a, b demonstrates a CAD drawing con- taining the designs for both the channel and barrier layers of standard two-chamber and four-chamber SU-8 molds. To begin with, alignment marks are found on the perimeter of both mask designs to aid in the alignment process during the actual photoli- thography step. For the channel-chamber layer mask, the inlet and outlet microchannels are 200 μm wide (w) and 10–20 mm long (l), and the inlet and outlet ports shown are 5 mm in diameter, though the actual size of the port will depend on a later fabrication step. The cell culture chambers are 1 mm (w)×5 mm (l) and contain several semi-lunar shaped support pillars that serve to prevent the roof of the chamber in the assembled device from collapsing upon the application of pressure. The separation distance between neigh- boring cell culture chambers (or width of the barrier region) shown in Fig. 2 is 100 μm. In the barrier layer mask, the connecting microgrooves are 50 μm long. The width of both the connecting microgrooves and the continuous barrier is dependent upon the width of the barrier region. Generally, the width of the grooves or continuous barrier on the mask is selected to be at least three times as wide as the barrier region itself; this additional tolerance makes the alignment of the two layers during the actual mold fabrication easy to achieve. Note that the dimensions mentioned here can be slightly modified if needed, as long as several design rules are followed (see Note 3). In addition to the photolithography masks 3.1.1 Design of the Photolithography Mask The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…
- 27. 8 for the channellayer, a mask must also be designed for the pressure control chamber layer. The designs for both the two- and four- channel pressure control chambers are shown in Fig. 2c. For the two-channel design, the chamber is 12 mm long×4 mm wide, while the four-channel design is 8×12 mm. The short inlet and outlet regions are 1×2 mm. After obtaining a high-resolution photomask with the desired features, the SU-8 master mold can be fabricated using photoli- thography. The steps listed here should be carried out in a clean room and will produce a master containing barrier features that are 3.1.2 Fabrication of the Cell Culture Chamber Layer Masters Fig. 2 Lithography photomasks for each device layer. (a) A CAD produced photomask containing the two- and four-chamber designs. Note the location of the alignment marks. (b) The mask used for making the connecting barrier features contains designs for both a continuous barrier and microgroove array. Identical marks to those in the channel design mask are used to facilitate proper alignment during fabrication. (c) Photomasks for the pressure control chambers corresponding to both two- and four cell culture chamber designs, which do not require alignment marks, as this mold requires only a single layer of SU-8.These designs are to scale (relevant dimensions listed in the text) Bryson M. Brewer et al.
- 28. 9 ~5 μm talland channel/chamber features ~100 μm tall. A summary of the protocol is presented in Fig. 3a, b. 1. Clean a 3″ silicon wafer by placing the wafer on a spin coater, washing the surface with acetone, and spinning dry at 2,000 RPM for 2 min. After drying, visually inspect the wafer for any impurities or remaining residue and re-clean if neces- sary before proceeding. 2. Deposit a ~1.5″ diameter drop of SU-8 2005 on the center of the wafer. 3. Program the spin coater for a two-step process. The first step is a spin at 500 RPM for 10 s at an acceleration of 100 RPM/s. This is followed by a 35 s spin at 2,000 RPM at an acceleration of 300 RPM/s. After running the program, a uniform SU-8 layer of ~5 μm tall should cover the surface of the silicon wafer. For other layer thicknesses, see Note 4. 4. Using a dry non-abrasive wipe, remove any excess SU-8 around the perimeter of the wafer by carefully placing the wipe against the bottom edge of the wafer with the vacuum on and slowly rotating the wafer one full revolution. 5. Turn off the vacuum on the spin coater and place the wafer on a hot plate set to 95 °C for 2 min. 6. Remove the wafer and allow it to cool to room temperature (usually 1–2 min). 7. Position the photomask containing the barrier features on top of the wafer with the inked side making contact with the wafer. Place a large cover glass (≥4″ diameter) on top of the mask to ensure clean contact with the wafer. Then expose the wafer using a UV source with a dose of 390 mJ/cm2 (see Note 5). 8. Remove the large cover glass and photomask. Then place the wafer on the 95 °C hot plate for 3 min. 9. Remove the wafer, let it cool for 1–2 min, and place it back on the spin coater and turn on the vacuum. 10. Spray SU-8 developer on top of the wafer until the entire sur- face is covered. Wait 30–45 s, then spin the wafer at 2,000 RPM until all excess SU-8 developer has spun off of the wafer surface (see Note 6). 11. Stop the spinner and visually inspect the wafer to determine if any SU-8 remains that is not crosslinked. If any excess SU-8 remains, spray another puddle of SU-8 developer on the sur- face and wait 20 s before spinning at 2,000 RPM. If no excess SU-8 remains, start spinning at 2,000 RPM immediately after applying another puddle of SU-8 developer. 12. After the SU-8 developer has spun off, spray the wafer with acetone, followed by a wash with isopropyl alcohol (with the wafer still spinning at 2,000 RPM through all washing steps). The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…
- 29. 10 Fig. 3 Summaryof fabrication of SU-8 replica molds using photolithography. This figure demonstrates the photolithography processes necessary to fabricate the SU-8 molds for the two- and four-chamber cell culture chamber layers, as well as the pressure control chamber. The cell culture chamber layer molds require mul- tiple mask layers, with the photolithography process essentially being performed twice with proper alignment between the barrier region and channel layers. Note that microgrooves can also be used with a two-chamber design, and vice-versa Bryson M. Brewer et al.
- 30. 11 13. Remove thewafer from the spin coater and blow dry with nitro- gen gas. Inspect the SU-8 features under a microscope to ensure that no defects are present, then place the wafer on a 95–150 °C hotplate for 5 min to anneal any minor surface cracks that are present. This concludes the fabrication of the 5 μm thick layer. 14. The process must now be repeated to fabricate the 100 μm channel/chamber layer on top of the 5 μm barrier layer. Again, clean the wafer as described in step 1. 15. Deposit a ~1.5″ diameter drop of the more viscous SU-8 2050 on the center of the wafer. 16. For this layer, the two-step spinning program is a spin at 500 RPM for 10 s at an acceleration of 100 RPM/s, followed by a 35 s spin at 1,650 RPM at an acceleration of 300 RPM/s. After running the program, a uniform SU-8 layer ~100 μm tall should cover the surface of the silicon wafer. 17. After removing excess SU-8 with a dry non-abrasive wipe, place the wafer on a 65 °C hot plate for 5 min, followed by 20 min on a 95 °C hot plate. 18. Remove the wafer, allow it to cool for 1–2 min, and place the wafer under the UV exposure system. 19. Place the photomask for the channel/chamber layer (either the two- or four-chamber design) on top of the wafer while care- fully overlapping the alignment marks from the 5 μm SU-8 layer with the marks on the photomask (see Note 7 for tips on achiev- ing proper alignment). Expose with a dose of 390 mJ/cm2 . 20. Place the exposed wafer on a 65 °C hot plate for 1 min and a 95 °C hot plate for 10 min. Then remove the wafer and allow it to cool for 1–2 min. 21. Repeat steps 10–12 to develop the non-cross-linked SU-8. However, for the thicker 100 μm SU-8 layer, the SU-8 devel- oper puddle should be left on the wafer for ~10 min to remove all of the excess photoresist (see Note 8). 22. Repeat step 13 to complete the fabrication of the SU-8 master mold for the two- or four-chamber cell culture chamber layer. The pressure control chamber master mold is fabricated in the same manner as the cell culture chamber layer master. However, the pres- sure control chamber mold should be ~400 μm in height. Thus, the following modifications to the above procedure can be used to fab- ricate a 400 μm tall SU-8 pressure control chamber master mold. Note that an alternative fabrication method can be used here that does not require the use of clean room (see Note 9). The steps are summarized in Fig. 3c. 1. Clean a 3″ silicon wafer using the previously described method. 2. Deposit a ~1.5″ diameter drop of SU-8 2150 on the center of the wafer. 3.1.3 Fabrication of the Pressure Control Chamber Layer Master The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…
- 31. 12 3. Program thespin coater for a two-step process. The first step is a spin at 500 RPM for 10 s at an acceleration of 100 RPM/s. This is followed by a 35 s spin at 1,625 RPM at an acceleration of 300 RPM/s. After running the program, a uniform SU-8 layer ~400 μm tall should cover the surface of the silicon wafer. 4. After removing excess SU-8 from the edge and bottom of the wafer, place it on a 65 °C hot plate for 8 min, followed by 105 min on a 95 °C hot plate. 5. Once the wafer has reached room temperature, position the photomask for the pressure control chamber on top of the SU-8 as previously described and expose using a 390 mJ/cm2 dose two separate times, waiting 1 min between doses. 6. Place the wafer on the 65 °C hotplate for 5 min, followed by 30 min on the 95 °C hotplate. Remove the wafer and allow it to cool to room temperature. 7. Development should be performed as described previously; however, for the ~400 μm layer, complete removal of the non- cross-linked photoresist will take ~20 min. 8. After placing the developed SU-8 mold on a 95–150 °C hotplate for several minutes to anneal any surface cracks, the fabrication of the pressure control chamber master mold is complete. After successfully fabricating the desired SU-8 masters, the newly constructed molds can be used to make each PDMS device layer. The following protocol can be used with any of the molds fabricated in Sect. 3.2 to produce the desired PDMS device layer. Figure 4 highlights the main components of this protocol. 1. Mix together the two components of the Sylgard 184 silicone encapsulant kit in a 10:1 w/w ratio (base polymer–curing agent). See Note 10. 2. Place the desired master mold in a petri dish and pour the PDMS mixture over it (see Note 11). 3. Place the mold in a vacuum chamber or desiccator at −80 kPa for 45–60 min (see Note 12). 4. With all air bubbles removed, place the mold in an oven at 70 °C for ~2 h to allow the PDMS to cure (see Note 13). 5. Remove the mold from the oven and allow it to cool to room temperature. 6. Using a scalpel or razor, cut the PDMS around the perimeter of the device layer while leaving plenty of clearance around the edges of the channel features. 7. Carefully and slowly peel off the cut PDMS layer from the SU-8 mold. 3.2 PDMS Based Soft-Lithography and Device Assembly 3.2.1 Fabrication of PDMS Device Layers Bryson M. Brewer et al.
- 32. 13 8. Place thePDMS on the center of a clean glass slide and trim the excess PDMS from the edges so that the entire PDMS layer fits cleanly on the slide (see Note 14). 9. Use a 5 mm biopsy punch to cut out the inlet and outlet res- ervoirs for all channels in the two- and four-chamber channel layers. Use a 2 mm biopsy punch to cut the holes in the inlet and outlet regions of the pressure control chamber layer. 10. Cover both the top and bottom surfaces of the PDMS layer with acrylic office tape (“Scotch Tape”). See Note 15. In addition to the molded PDMS device layers, tubing supports must also be cut out from a plain, ~2 mm thick layer of PDMS. Each tubing support should be 3 mm (l)×3 mm (w), and a 14 gauge needle should be used to punch out a hole in the center of each piece. Place each support on a piece of tape to maintain a clean surface (see Note 16). Fig. 4 Summary of soft lithography processes used to fabricate each device layer. Here the workflow required for fabrication of the PDMS cell culture chamber layer, pressure control chamber layer, and tubing supports are detailed. The schematics are not to scale, as in reality two cell culture chamber device layers and multiple pressure control chambers can be produced per 3″ silicon wafer The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…
- 33. 14 The final stepin fabricating the valve-enabled neurobiological platform is the assembly of all the fabricated pieces into a com- pleted device. This involves a combination of using a plasma cleaner to activate the surfaces of both PDMS and glass to facilitate irre- versible bonding, as well as using small amounts of uncured PDMS to “glue” and seal certain components (tubing and reservoirs) to the device. The following procedure outlines the details of device assembly and is summarized in Fig. 5. 1. Clean a glass coverslip or slide (see Note 17) using compressed nitrogen and place it inside the chamber of the plasma cleaner. 2. Remove the tape from the open channel side of the two- or four-chamber PDMS cell culture chamber layer and place it in the plasma cleaner with the exposed area face up (see Note 18). 3. Close the plasma cleaner chamber and expose the glass and PDMS to air plasma for 30 s by applying ~18 W to the RF coil (see Note 19). 3.2.2 Device Assembly Fig. 5 Summary of the assembly of components into a final valve-enabled microfluidic device. (a) The first assembly step requires attaching the PDMS cell culture chamber layer to a glass coverslip via air plasma bonding. (b) Next, plasma bonding is used to attach the pressure control chamber to the cell culture chamber layer. (c) Then, tubing supports are aligned and plasma bonded to the pressure control chamber. (d) Finally, liquid PDMS is used as a glue to attach inlet/outlet tubing and reservoirs to the device Bryson M. Brewer et al.
- 34. 15 4. Remove theglass coverslip and PDMS channel device layer from the plasma cleaner chamber. Carefully bond the open chamber side of the PDMS cell culture chamber layer to the plasma treated side of the coverslip by gently dropping the PDMS layer onto the glass (see Note 20). 5. Remove the tape from the open chamber side of the PDMS pressure control chamber layer and place it in the plasma cleaner chamber alongside the newly bonded coverslip/PDMS cell culture chamber layer. Perform the air plasma treatment as described in step 3. 6. After removing both pieces from the plasma cleaner, bond the PDMS pressure control chamber to the top of the PDMS cell culture chamber layer. Be careful to align the control chamber over the cell culture chambers. 7. Place the coverslip/cell culture chamber layer/pressure control chamber assembly back into the plasma cleaner, along with two PDMS tubing supporters. The side of the tubing sup- porter that was protected by tape during storage should be face up in the plasma cleaner. 8. Perform the standard plasma treatment as described in step 3. After removing from the cleaner, use forceps to place the tubing supporters on top of the pressure control chamber, aligning the holes in each component. Apply slight pressure to the top of the tubing supporter to promote a good seal. 9. Immediately fill the cell culture chamber with DI water using a pipette. Each chamber should require ~25–50 μL of water (see Notes 21 and 22). The pressure control chamber does not need to be filled with water. 10. Next, insert 0.02″ ID/0.06″ OD Tygon microbore tubing into the inlet and outlet tubing supporters of the pressure con- trol chamber using forceps. Each piece of tubing should be ~3″ long. Mix up 1–2 g of PDMS and curing agent at a 10:1 ratio, and use the liquid mixture to seal the insertion point of the tubing into the tubing supporters (see Note 23). 11. Attach 10×10 mm (diameter×height) Pyrex cloning cylinders to the inlet and outlet of each cell culture chamber by applying a thin layer of liquid PDMS mixture onto the bottom of a cylinder and gently pressing onto the punched hole of each chamber inlet/outlet (see Note 24). 12. Add 200–300 μL of DI water to each cylinder and place the entire device in a 70 °C oven for 45–60 min to allow the liquid PDMS seals to cure. Be careful not to allow the reservoirs or channels to dry out during this curing process. 13. Remove the device from the oven and allow it to cool down to room temperature. Store in a petri dish and cover in Parafilm to prevent evaporation before use. The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…
- 35. 16 This completes thefabrication of the pneumatic/hydraulic valve-enabled microfluidic neurobiology platform. The devices can typically be stored in Parafilm sealed petri dishes for weeks before use, as long as the channels and chambers remain filled with water. After assembly, the microfluidic device is ready to be used for suitable neurobiological assays. The following steps demonstrate how to prepare the device for use and activate/deactivate the pres- sure valve (Fig. 6a, b). For more detailed examples of previously documented biological protocols using this device (coating of channels, cell loading, etc.), please see references [17–19]. 1. First, each device should be sterilized using UV (ultra-violet) irradiation found in most cell-culture hoods. UV exposure should last for at least 1 h. 2. When ready to activate the pressure valve, a 5 mL plastic syringe with a 23-gauge needle should be filled with either air or water. Insert the syringe into the inlet tubing of the pressure control chamber. (a) For pneumatic pressurization, clamp the outlet tubing of the pressure control chamber closed using a metal pinch clamp. Then inject 0.2–0.3 mL of air into the pressure control chamber and clamp the inlet tubing (see Note 25). (b) For hydraulic pressurization, fill the entire pressure control chamber and tubing with water. Then, clamp the outlet tubing. Finally, inject an additional 0.2–0.3 mL of water into the chamber and clamp the inlet tubing (see Note 26). 3. To deactivate the valve, unclamp the inlet and outlet tubing of the pressure control chamber (see Note 27). In order to check that your device is working properly, fluores- cent dye or food coloring can be added to one of the channels after pressurization. Any leakage through the barrier/microgrooves should be apparent. Additionally, the dye should perfuse freely through the barrier/microgroove region after releasing the pressure (see Fig. 7). 3.3 Device Operation Fig. 6 Three-dimensional cross-section of a two-chamber, continuous barrier device before and after applying pressure to the control chamber. (a) An unpressurized device is shown with the continuous barrier elevated above the substrate. Here the two cell culture chambers are connected. (b) A device that has been pressurized using either pneumatic or hydraulic pressure in which the barrier is pushed down to the substrate, effectively separating the two cell culture chambers Bryson M. Brewer et al.
- 36. 17 4 Notes 1. Thefabrication of the molds or replica-masters to be employed later in the soft-lithography step is achieved using standard photolithography protocols. For best results, this step should be conducted in a Class 1000 clean room (or better). 2. Although there are many available options for printing photo- masks, the authors have had past success using CAD/Art Services, Inc. for designs similar to those presented here. 3. The dimensions of the device features can be modified to fit a particular application. For example, the cell culture chambers could be made 1–10 mm long and/or 200–2,000 μm wide, and the width of the barrier region varied from 50 to 500 μm without sacrificing device performance. However, note that in order to better control the location where cells will be seeded within the channel, the cell culture chamber should be at least twice as wide as the inlet/outlet microchannels. The flow velo- city decreases in the wider cell culture region, slowing down the cells and allowing them to settle onto the substrate. In addition, the aspect ratio of the final mold must be considered when designing a mask, as SU-8 features with a high aspect ratio may be prone to breaking during later soft-lithography steps. Concurrently, PDMS microchannels with a very low aspect ratio may collapse in the absence of support pillars. Finally, be mindful of the dimensions of the glass substrate you will use when fabricating the final device. All device features should be designed to be at least 3 mm from the edges of the glass substrate after final device assembly. Fig. 7 Demonstration of proper valve behavior using fluorescent dye. (a) Fluorescein isothiocyanate (FITC) loaded into the bottom cell culture chamber is contained as long as the valve barrier is properly activated using pneumatic/hydraulic pressure. (b) After deactivating the barrier, FITC is free to flow across the barrier region into the top cell culture region. Figure reprinted with permission from ref. [18] The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…
- 37. 18 4. Data sheetscontaining curves relating the spin-speed to layer thickness for each SU-8 model can be found on the MicroChem website: http://microchem.com/Prod-SU8_KMPR.htm. It is generally helpful to verify the thickness of the SU-8 layer being produced after fabrication by measuring the feature height using a profilometer, as some variation from the spin-speed/ thickness curve may be observed. 5. The actual required exposure dosage will vary depending on the model of SU-8 used, the thickness of the layer, wafer diam- eter, and the type of exposure system used. Again, the data sheets found on the MicroChem website, http://microchem. com/Prod-SU8_KMPR.htm, provide general guidelines on exposure dosage; however, it may be necessary to test a range of exposure doses to find the optimum value for a particular equipment setup. The authors typically use a Novacure 2100 Ultraviolet/Visible Spot Cure System to fabricate SU-8 master molds. 6. In order to determine when SU-8 developer, acetone, or IPA has been completely spun off of the wafer, look for a ring pattern on the wafer surface. When the ring pattern becomes visible, almost all of the excess liquid has been removed from the surface. After the rings disappear, the next washing step can be applied. 7. Often, it will be difficult to directly see the alignment marks on the 5 μm thick SU-8 layer with the photomask on top of the wafer. In this case, it is helpful to scratch the outline of the alignment marks directly into the surface of the 100 μm SU-8 layer before alignment. This is achieved by using a razor blade or scalpel to carefully score lines indicating the alignment marks visible from the 5 μm thick SU-8 layer. Then, alignment of the photomask with the new scored marks should be readily achieved. 8. For longer development times, it is useful to agitate the wafer to speed up the development process. This can be achieved by either manually rotating the spin coater back and forth through ~5 cm oscillations or by spinning the wafer at a slow spin speed (~50 RPM) during development. The slow spin speed will cause the SU-8 developer to oscillate on top of the wafer with- out being spun off of the surface. In addition, the 10 min development time for the 100 μm layer can be split into two 5 min developments where the SU-8 developer is spun off after the first 5 min and reapplied after stopping the spinner. 9. Unlike the cell culture chamber layer masters, the mold for the pressure control chamber layer does not contain any intricate features. Thus, a clean room is not necessary for the fabrication of the pressure control chamber mold. The following steps Bryson M. Brewer et al.
- 38. 19 should be followedto produce a glass master mold that will produce pressure control chambers of ~1 mm thickness: (a) On a standard 1 mm thick glass slide, use a diamond-tip pen to score and cut several glass rectangles measuring either 4 mm×12 mm (two-chamber design) or 8 mm× 12 mm (four-chamber design). (b) Using cyanoacrylate (“Super Glue”) or an equivalent low- viscosity epoxy, attach 2–3 of the cut glass rectangles to the center of another uncut glass slide (25×75 mm). Note that a conservative amount of glue should be used. As the cut glass rectangles are pressed against the glass slide, little to no glue should leak out around the edges. Any extra glue will mold unwanted features into the final PDMS layer, potentially causing bonding problems. (c) Place the glass mold in a petri dish for use in the standard soft-lithography protocol. 10. In the standard 0.5 kg kit, the base polymer comes in the larger container, while the curing agent is in the smaller bottle. The two components should be mixed thoroughly for 3–5 min to ensure uniformity. 11. For the cell culture chamber layer, a layer of PDMS 1–2 mm thick should cover the top of the SU-8 mold after pouring. For the pressure control chamber layer, a 3–5 mm thick layer of PDMS should cover the top of the mold features. 12. The mixing of the two components introduces air bubbles into the PDMS. Placing the mold in a vacuum removes those air bubbles. Note that after removing the mold from the vacuum, a few bubbles may still remain on the PDMS surface. These can be removed by applying a light blast of nitrogen gas to the PDMS surface. In addition, be careful to remove any air bub- bles trapped underneath the silicon wafer, as these bubbles can cause an uneven cell culture chamber layer. These trapped bubbles can usually be removed by lightly pressing on the center of the silicon wafer with forceps or a scalpel. 13. The curing process will transform the PDMS from the liquid state poured over the mold to a flexible, solid rubbery state. While the PDMS should be cured for at least 2 h, longer curing times (even overnight) should not affect device performance. 14. For the cell culture chamber layers, make sure that the edges of the PDMS are flat. Any raised portion should be trimmed so that the top of the PDMS layer is flat. Raised edges can become problematic when later attaching Pyrex reservoir cylinders. 15. The acrylic tape serves to both clean the surface of the PDMS (aiding in bonding together different layers), as well as protect The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…
- 39. 20 the surface fromfurther contamination during storage and transport before device assembly is performed. 16. The tubing supports serve to aid in the device assembly phase of fabrication. Additionally, they provide extra support to the connection between the tubing and pressure control chamber, helping prevent leakage and device failure when pressurizing the pressure control chamber. Note that the holes punched in the tubing supports are slightly smaller than the tubing itself to ensure a snug fit when inserting the tubing. 17. The typical coverslip used by the authors is 25×60×0.13 mm (width×length×thickness). Although the use of a thin coverslip can make fabrication slightly more difficult due to its fragility, a thin substrate is necessary when using high magnification microscope objectives with a short working distance (i.e., when using confocal microscopy to look at individual synapses). On the other hand, when a thin substrate is not needed, a thick (~1 mm) glass slide can be used instead in order to reduce the chance of cracking the substrate during fabrication. 18. An easy way to make sure that the tape is being removed from the correct side of the device is to inspect the tape after removal. The outline of the channels should be visible when the tape was removed from the open channel/chamber side of the device. 19. A variety of plasma cleaners exist and have been used in the fabrication of microfluidic devices. The plasma cleaner typically used by the authors is from Harrick Plasma (PDC-32G). It uses a 3″ diameter×6.5″ length Pyrex chamber and contains three power settings. For all bonding procedures, the maximum power setting is used (~18 W applied to the RF coil). In order for the plasma to be generated, a vacuum must be achieved in the plasma cleaner chamber. Thus, a typical bonding step requires closing the chamber door, turning on a vacuum pump to evacuate the chamber, sequentially stepping the power set- ting from low to high (approximately 10 s each at the two lower settings), and finally removing the vacuum after com- pleting the exposure to retrieve the samples. Often, the airflow into the plasma cleaner chamber is adjusted during operation by slightly opening or closing a needle valve connected to the chamber; this is done to maintain the intensity of the plasma in the chamber throughout the exposure. A visible purple color is emitted in the chamber during operation, and the brightness of the color can be used to gauge the plasma intensity. Finally, tuning of the exposure time and power may be required for different plasma cleaners; however, many protocols can be found in literature for guidance on the proper parameters. Bryson M. Brewer et al.
- 40. 21 20. Properly aligningthe PDMS cell culture chamber layer with the glass can be tricky initially. It is best to hold the PDMS layer by its edges (being careful not to touch the plasma treated face) and line up the top of the PDMS with the top of the coverslip. Then, the PDMS layer can be gently dropped onto the glass. On a dark background, the formation of the bond between the two layers can be observed. Note that this bond is irreversible, so care must be taken during alignment. 21. After plasma treatment, the surface of the PDMS changes from its natural hydrophobic state to a hydrophilic state. However, this change is temporary, and the PDMS will become hydro- phobic again if left exposed to air [22]. Hydrophobic channel walls make loading microchannels with water difficult, as pres- sure must be applied to force the water through, and air bubbles will often get trapped in the corners of channels and chambers. On the other hand, hydrophilic channels can be loaded with media using capillary action, and air bubbles are generally not a problem. 22. Sometimes during device assembly the roof of the microgroove or barrier region will collapse to the glass substrate. In order to check if this has occurred, one should load only one channel with DI water. Then, observe whether or not the water flows through the microgroove/barrier region to the other channel(s). If the water does not flow through, slight vacuum or pressure can be applied to neighboring channels to force the roof of the microgroove/barrier to release from the substrate, allowing the water to flow freely. Generally, this problem occurs more often in continuous barrier rather than micro- groove devices but can usually be avoided by careful device assembly. 23. The liquid PDMS mixture should be “painted” on top of the tubing supporter around the tubing. This can be achieved using the tip of a scalpel to apply a small drop around the perimeter of the tubing. Be careful not to add too much, as extra liquid PDMS could run off of the tubing supporters onto the pressure control chamber and form a rounded layer over the viewing window, potentially causing imaging problems in bioassays. 24. Again, a scalpel can be used to apply the thin coating of liquid PDMS to the cylinder. Make sure the surface of the PDMS is dry around the inlet/outlet punched holes before attaching the cylinder. Any water present on the surface at the attachment point will result in a poor seal and likely lead to leakage. 25. Due to the compressibility of air, more air may need to be injected to ensure that the valve is activated. By closely watch- ing the walls of the pressure control chamber, a slight bulging The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…
- 41. 22 can often beobserved when sufficient pressure has been applied to activate the valve. Note that PDMS is permeable to air, so using pneumatic pressure to activate the valve will only work for short periods of time (4–6 h). Hydraulic pressure should be used when long activation time-periods are required. 26. Again, slight bulging of the pressure control chamber walls generally indicates sufficient pressure has been applied to acti- vate the valve. Be careful not to over-pressurize using water, as the PDMS-PDMS seal is not as robust as the PDMS-glass seal. Thus, high pressures can cause delamination of the different PDMS layers. 27. Sometimes it may be necessary to only unclamp the inlet tubing first, then insert an empty syringe into the inlet tubing and pull a slight vacuum. This will promote proper barrier/ microgroove release from the glass substrate and fully deacti- vate the valve. References 1. Taylor AM, Jeon NL (2010) Micro-scale and microfluidic devices for neurobiology. Curr Opin Neurobiol 20:640–647 2. Meyvantsson I, Beebe DJ (2008) Cell culture models in microfluidic systems. Annu Rev Anal Chem (Palo Alto Calif) 1:423–449 3. Gross PG, Kartalov EP, Scherer A, Weiner LP (2007) Applications of microfluidics for neuro- nal studies. J Neurol Sci 252(2):135–143 4. Park JW, Kim HJ, Kang MW, Jeon NL (2013) Advances in microfluidics-based experimental methods for neuroscience research. Lab Chip 13(4):509–521 5. Biffi E, Piraino F, Pedrocchi A, Fiore GB, Ferrigno G, Redaelli A, Menegon A, Rasponi M (2012) A microfluidic platform for controlled biochemical stimulation of twin neuronal net- works. Biomicrofluidics 6(2):24106–2410610 6. Chung BG, Flanagan LA, Rhee SW, Schwartz PH, Lee AP, Monuki ES, Jeon NL (2005) Human neural stem cell growth and differen- tiation in a gradient-generating microfluidic device. Lab Chip 5(4):401–406 7. Taylor A, Blurton-Jones M, Rhee S, Cribbs DH, Cotman CW, Jeon NL (2005) A micro- fluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods 2(8): 599–605 8. Zhou J, Yan H, Ren K, Dai W, Wu H (2009) Convenient method for modifying poly (dimethylsiloxane) with poly(ethylene glycol) in microfluidics. Anal Chem 81(16):6627–6632 9. Duffy DC, McDonald JC, Schueller OJ, Whitesides GM (1998) Rapid prototyping of microfluidic systems in poly(dimethylsiloxane). Anal Chem 70(23):4974–4984 10. Mukhopadhyay R (2007) When PDMS isn’t the best. What are its weaknesses, and which other polymers can researchers add to their toolboxes? Anal Chem 79(9): 3248–3253 11. Toepke MW, Beebe DJ (2006) PDMS absorp- tion of small molecules and consequences in microfluidic applications. Lab Chip 6(12): 1484–1486 12. El-Ali J, Sorger PK, Jensen KF (2006) Cells on chips. Nature 442(7101):403–411 13. McDonald JC, Whitesides GM (2002) Poly(dimethylsiloxane) as a material for fabri- cating microfluidic devices. Acc Chem Res 35(7):491–499 14. Zhang M, Wu J, Wang L, Xiao K, Wen W (2010) A simple method for fabricating multi- layer PDMS structures for 3D microfluidic chips. Lab Chip 10(9):1199–1203 15. Unger MA, Chou H-P, Thorsen T, Scherer A, Quake SR (2000) Monolithic microfabricated valves and pumps by multilayer soft lithogra- phy. Science 288(5463):113–116 16. Brantley-Sieders DM, Dunaway CM, Rao M, Short S, Hwang Y, Gao Y, Li D, Jiang A, Shyr Y, Wu JY, Chen J (2011) Angiocrine factors modulate tumor proliferation and motility through EphA2 repression of Slit2 tumor Bryson M. Brewer et al.
- 42. 23 suppressor function inendothelium. Cancer Res 71(3):976–987 17. Gao Y, Majumdar D, Jovanovic B, Shaifer C, Lin PC, Zijlstra A, Webb DJ, Li D (2011) A versatile valve-enabled microfluidic cell co- culture platform and demonstration of its applications to neurobiology and cancer biol- ogy. Biomed Microdevices 13(3):539–548 18. Majumdar D, Gao Y, Li D, Webb DJ (2011) Co-culture of neurons and glia in a novel microfluidic platform. J Neurosci Methods 196(1):38–44 19. Shi M, Majumdar D, Gao Y, Brewer B, Goodwin C, McLean JA, Li D, Webb DJ (2013) Glia co- culture with neurons in microfluidic platforms promotes the formation and stabilization of syn- aptic contacts. Lab Chip 13(15):3008–3021 20. Lynn NS, Dandy DS (2009) Passive microfluidic pumping using coupled capillary/evaporation effects. Lab Chip 9(23):3422–3429 21. Walker GM, Beebe DJ (2002) An evaporation- based microfluidic sample concentration method. Lab Chip 2(2):57–61 22. Bodas D, Khan-Malek C (2007) Hydrophili- zation and hydrophobic recovery of PDMS by oxygen plasma and chemical treatment—an SEM investigation. Sensors Actuator B Chem 123(1):368–373 The Fabrication of Microfluidic Platforms with Pneumatically/Hydraulically…
- 44. 25 Emilia Biffi (ed.),Microfluidic and Compartmentalized Platforms for Neurobiological Research, Neuromethods, vol. 103, DOI 10.1007/978-1-4939-2510-0_2, © Springer Science+Business Media New York 2015 Chapter 2 A Reliable Reversible Bonding Method for Perfused Microfluidic Devices Paola Occhetta, Emilia Biffi, and Marco Rasponi Abstract Microfluidic devices made of poly(dimethylsiloxane) (PDMS) are suitable for cell culture applications, mainly due to both the advantageous volume and surface properties of the material itself. Bulk properties include optical transparency, gas permeability, and ease of fabrication, to name a few. On the other hand, silanol groups (SiOH) present on the surface can be easily activated through air/oxygen plasma treat- ments, and used to permanently bond to other materials, like silicon, glass or PDMS. The importance of a standard sealing method with no need of additional gluing materials is crucial for microfluidic applica- tions, where micrometer sized channels and chambers are involved. Despite the reliability of the plasma treatment to permanently seal microfluidic devices, reversible-bonding methods are sometimes desirable— e.g., high magnification microscopy, sample retrieval, and multiple usages of valuable substrates. For this purpose, common techniques rely either on weakening the plasma treatment (partial treatment, only involving one of the surfaces of interest) or on increasing the self-sealing properties of PDMS (by adjusting the ratio of pre-polymer and curing agent). However, the adhesion strength of these methods is low, thus making them suitable only for static or quasi-static conditions. Whenever there is the requirement for continuous perfusion, other techniques are needed. Here, we describe a PDMS microfluidic device for long-term culture of cells, which can be reversibly sealed to different flat substrates. The hydraulic tightness is guaranteed through magnetic forces, being the substrate interposed between a permanent magnet and the microfluidic device, locally enriched with fer- romagnetic material. In particular, neuronal networks were grown within the device, reversibly coupled to a flat Microelectrode Array (MEA). Thus, the proposed approach allows to combine the advantageous features of microfluidics and the multiple use of commercial MEA substrates. Indeed, it allows for electro- physiological investigations in highly controlled microenvironments. Key words Microfluidics, Reversible bonding, Microelectrode arrays, Neuronal culture 1 Introduction In the last decade, the interest in poly(dimethylsiloxane) (PDMS) microfluidic devices for chemistry and biotechnology has been exponentially increasing due to their peculiar advantages compared to traditional analytical instruments such as lower reagent con- sumption, reduced analysis time, lower fabrication costs and ability
- 45. 26 to perform highlyparallelized and reproducible analyses in a high-throughput fashion. PDMS itself offers several advantageous volume and surface properties such as optical transparency, bio- compatibility, gas permeability, and ease of fabrication, making microfluidic devices suitable for cell culture applications [1]. Addi- tionally, microfluidic platforms offer the potentiality to precisely monitor parameters in time- and space-controlled manners within the cellular microenvironments, providing greater control over the cell response to different stimuli [2]. Among other research fields, microfluidic devices have been widely used in neuroscience [3–5] thanks to their size-scale, compatible with neurons own size. Particularly, the direct integration of PDMS microfluidic devices with multielectrode arrays (MEAs) allows coupling the recording of neuronal electrical activity together with the inherent high con- trol of the cell microenvironment offered by the microfluidic approach [6–8]. Currently, most of the microfluidic devices are fabricated by bonding irreversibly a PDMS stamp containing the chambers and microchannels to a second substrate layer (glass or polymer), by activating both surfaces through air or oxygen plasma [9]. This bonding technique allows for a reliable sealing able to withstand relatively high pressures (200 kPa and above) without the addition of any gluing material [10]. However, the irreversibility of the bonding introduces several limitations in terms of possible device exploitations, including cell and material patterning, surface func- tionalization as well as capability of samples injection and retrieval to perform secondary analyses and direct measurements not achiev- able within the device. Moreover, the affordability of coupling PDMS microfluidic devices with valuable substrates, like MEAs, is subordinated to the ability of disassembling the devices and reus- ing the substrate upon sterilization. For these reasons, many efforts have been recently directed to the development of reversible- bonding methods, which have to attain an adhesion between layers sufficient to withstand the pressure required for the microfluidic applications, while enabling a simple access to the “experimental field” and a non-disposable use of the devices. Reversible sealing between PDMS microfluidic layer and MEAs substrates have been attempted through different techniques including self-adhesion [6] and reversible plasma bonding [11]. However, these tech- niques do not assure a high hydraulic tightness when relatively high pressure are applied (<35 kPa), limiting their application to static or quasi-static conditions [12]. In this chapter, we describe an inexpensive and reliable technique for reversely bonding PDMS microfluidic device to different flat sub- strates for long-term culture of cells [13]. The presented bonding technique is based on magnetic forces which allows to obtain devices Paola Occhetta et al.
- 46. 27 able to withstanda high range of working pressures (up to about 150 kPa), thus suitable for cell culture applications requiring a continuous perfusion. In detail, a PDMS/iron micropowder layer is aligned onto a microfluidic layer and coupled with a flat substrate exploiting the either temporary or continuous use of a permanent magnet (Fig. 1). The introduced reversibly sealing method is charac- terized by compatibility with: (1) complex fluidic layer configurations, (2) micrometer size channel expanding the range of application of previously reported magnetic bonding technique, and (3) optical transparency for flow visualization and inspection over channel regions. Specifically, neuronal networks were grown within the descri- bed device reversibly coupled to a commercial flat MEA for long- term primary neuronal cell cultures [14, 15]. However, the method offers high versatility and the design can be easily adapted to differ- ent biological applications. As an example, the same technique can be applied to precisely functionalize flat surfaces through protein perfusion or pattern either 2D cell sheets or 3D cellular structures with a microscale precision. Fig. 1 Exploded view of the reversible bonding microfluidic device. The device is mainly made of PDMS and it is constituted by two layers: the bottom layer (fluidic layer) contains the fluidic features; the top layer (magnetic layer) contains a magnetic filler solution and it is optically transparent in correspondence to the fluidic regions. Once the device is assembled, channels are closed onto a cell culture substrate (e.g., histology glass slide) and maintained tight through a uniform magnetic field generated by a permanent magnet A Reliable Reversible Bonding Method for Perfused Microfluidic Devices
- 47. 28 2 Equipment andMaterials ● Device fabrication: – Desktop CNC (Computer Numerical Control) milling machine (MDX40, Roland DG) with an 800 μm in diam- eter carbide end mill. – Clean room facility: mask aligner (MA56, Karl Suss) – Clean room facility: two flat hot plates – Clean room facility: fume-hood – Clean room facility: spin coater – Clean room facility: optical microscope – Clean room facility: nitrogen gas – Stereomicroscope – Spin coater – Plasma cleaner (Harrick Plasma Inc.) (see Note 1) – Oven – Vacuum chamber ● Neuronal culture and electrophysiology: – Humidified 5 % CO2 incubator – Recording setup: pre-amplifier stage (MEA-1060-Inv-BC- Standard, gain: 55, band width: 0.02 Hz–8.5 kHz, MCS GmbH); amplification and filtering stage (FA64, gain 20, bandwidth: 10 Hz–3 kHz, MCS GmbHz); data acquisi- tion system (USB-ME64, MCS GmbH) (see Note 2). – Programmable syringe pump Aladdin AL2000 (see Note 3). ● 4-in. polished silicon wafers ● Negative photoresist (SU-8 50, Microchem) ● SU-8 developer (Microchem) ● Poly(dimethylsiloxane) (PDMS) (Sylgard 184, Dow Corning) ● Ethanol, isopropyl alcohol (IPA), acetone, chloroform. ● Sharpened biopsy puncher (Diameter 500 μm) ● Scalpel ● Tape (Magic Tape, 3M) ● Disposable petri dish (100 and 120 mm, VWR International) ● 5 and 8 mm tick poly(methyl methacrylate) (PMMA) sheets ● Iron micropowder with grain size smaller than 212 μm (Sigma- Aldrich Corp) ● Neodymium magnet (40×20×10 mm, magnetization N42) 2.1 Equipment 2.2 Materials 2.2.1 Device Fabrication Paola Occhetta et al.
- 48. 29 ● Flat MicroelectrodeArray (60MEA200/30iR-Ti-w/o, Multi Channels System GmbH) (see Note 4) ● Tweezers for silicone wafer handling ● Stainless steel couplers (23 Gauge, 12 mm or more long) ● Blunt needles (23 Gauge) ● Tygon tubing (ID 0.02″, OD 0.06″) ● Syringes ● Plating medium (before cell seeding and 4 h after): Neurobasal medium (Invitrogen), 10 % Fetal Bovine Serum (FBS; Lonza), 1 % Penicillin and Streptomycin, (Gibco) ● Culturing medium (from 4 h after the cell seeding to the end of the culture): Neurobasal medium (Invitrogen), B-27 1× (Invitrogen), GlutaMAX 1 mM (Invitrogen), 1 % Penicillin and Streptomycin (Gibco). ● Bidistilled and autoclaved water (dH2O) 3 Methods 1. Design chip layout through CAD software (AutoCAD, Autodesk) (see Note 5) 2. Print out layout mask at high-res (greater than 20,000 dpi) on a transparency sheet (deep black for unexposed regions) (see Note 6) 3. Clean room operations: (a) Clean new wafer by rinsing with ethanol, IPA, and ace- tone, followed by drying with nitrogen gas. (b) Spin-coat SU-8 50 to the desired thickness (a thickness of 100 μm use a spin time of 30 s and a spin rate of 1,100 rpm). (c) Soft-bake the wafer through a two-step (two-hot plate) procedure. Move the coated wafer with tweezers on a 65 °C preheated hot plate. After 10 min, move rapidly the wafer on a 95 °C preheated hot plate. Bake it for at least 30 min and let it cool to room temperature. (d) Cut the mask around the designed wafer with a square shape, slightly greater than wafer diameter, and tape it upside-down to a quartz glass compatible with the mask holder of the mask aligner. (e) Place the wafer in the mask aligner, move it against the mask in soft-contact mode, and expose to a dose of 500 mJ/cm2 in the i-line region (see Note 7). 2.2.2 Neuronal Culture 3.1 Device Preparation 3.1.1 Bottom Layer Mold Fabrication (Fig. 2c) A Reliable Reversible Bonding Method for Perfused Microfluidic Devices
- 49. 30 (f) Post-bake thewafer in a similar fashion of the pre-bake: bake for 1 min at 65 °C and 10 min at 95 °C. (g) Develop the wafer in a SU-8 developer bath for about 10 min (h) Rinse briefly the wafer with IPA then dry it with a gentle stream of nitrogen gas 4. Place the wafer (final mold) in a petri dish and tie it with tape for further use. 1. Decide the dimensions of the visual inspection window (see Note 8), and accordingly design the borders of the mag- netic cavities. 2. Use a 3D CAD modeler to extrude the inspection window and the wall to a height of 5 mm, and export it to a CAM compat- ible file (e.g., iges format). 3.1.2 Top Layer Mold Fabrication (Fig. 2a) Fig. 2 Fabrication of the magnetic device. To obtain the magnetic layer a PMMA mold is realized with a CNC milling machine at a final depth of 5 mm (a). PDMS is cast and a ferromagnetic suspension (iron powder/PDMS mixture at ratio 4:1 as w/w) is manually plastered into specific cavities (b).The fluidic layer is obtained by spin- coating PDMS (thickness of 250 μm) on a silicon mold (d), previously realized with standard soft-lithography techniques (c). Finally the two layers are manually aligned under a microscope taking advantage of to the optical transparency of the top layer in the channel regions (e).The top view of the final assembled devices is sketched to show the culture channel, aligned to a MEA substrate, through the top layer’s cavity outline (f) Paola Occhetta et al.
- 50. Exploring the Varietyof Random Documents with Different Content
- 51. marvellous simplicity. Andthen the rest will have to follow, you know, one day.” “Oh yes,” said Maradick absently. His eyes were fixed on the opposite wall, but, out of the corners of them, he was watching for the moment when Mrs. Lester should look up. Now he could regard yesterday afternoon with perfect equanimity; it was only an inevitable move in the situation. He wondered at himself now for having been so agitated about it; all that mattered was how she took it. The dogged, almost stupid mood had returned. His eyes were heavy, his great shoulders drooped a little as he bent to listen to Lester. There was no kindness nor charity in his face as he looked across the floor. He was waiting; in a moment she would look up. Then he would know; afterwards he would see Morelli. “And so, you see,” said Lester, “Plato still has the last word in the matter.” “Yes,” said Maradick. Mrs. Lawrence was being entirely tiresome at the tea-table. The strain of the situation was telling upon her. She had said several things to Sir Richard and he had made no answer at all. He continued to look with unflinching gaze upon Tony. She saw from Lady Gale’s and Mrs. Lester’s curious artificiality of manner that they were extremely uneasy, and she was piqued at their keeping her, so resolutely, outside intimacy. When she was ill at ease she had an irritating habit of eagerly repeating other people’s remarks with the words a little changed. She did this now, and Lady Gale felt that very shortly she’d be forced to scream. “It will be such a nuisance,” said Mrs. Lester, still continuing the “flying” conversation, “about clothes. One will never know what to put on, because the temperature will always be so very different when one gets up.” “Yes,” said Mrs. Lawrence eagerly, “nobody will have the slightest idea what clothes to wear because it may be hot or cold. It all depends——” “Some one,” said Lady Gale, laughing, “will have to shout down and tell us.”
- 52. “Yes,” said Mrs.Lawrence, “there’ll have to be a man who can call out and let us know.” Tony felt his father’s eyes upon him. He had wondered why he had said nothing to him about his last night’s absence, but it had not really made him uneasy. After all, that was very unimportant, what his father or any of the rest of them did or thought, compared with what Morelli was doing. He was curiously tired, tired in body and tired in mind, and he couldn’t think very clearly about anything. But he saw Morelli continually before him. Morelli coming round the table towards him, smiling—Morelli . . . What was he doing to Janet? He wanted to speak to Maradick, but it was so hard to get to him when there were all these other people in the room. The gaiety had gone out of his eyes, the laughter from his lips. Maradick was everything now; it all depended on Maradick. “You’re looking tired,” Alice said. She had been watching him, and she knew at once that he was in trouble. Of course anyone could see that he wasn’t himself, but she, who had known him all his life, could see that there was more in it than that. Indeed, she could never remember to have seen him like that before. Oh! if he would only let her help him! She had not been having a particularly good time herself just lately, but she meant there to be nothing selfish about her unhappiness. There are certain people who are proud of unrequited affection and pass those whom they love with heads raised and a kind of “See what I’m suffering for you!” air. They are incomparable nuisances! Alice had been rather inclined at first to treat Tony in the same sort of way, but now the one thought that she had was to help him if only he would let her! Perhaps, after all, it was nothing. Probably he’d had a row with the girl last night, or he was worried, perhaps, by Sir Richard. “Tony,” she said, putting her hand for a moment on his arm, “we are pals, aren’t we?” “Why, of course,” pulling himself suddenly away from Janet and her possible danger and trying to realise the girl at his side.
- 53. “Because,” she wenton, looking out of the window, “I’ve been a bit of a nuisance lately—not much of a companion, I’m afraid—out of sorts and grumpy. But now I want you to let me help if there’s anything I can do. There might be something, perhaps. You know”— she stopped a moment—“that I saw her down on the beach the other day. If there was anything——” She stopped awkwardly. “Look here,” he began eagerly; “if you’re trying to find out——” Then he stopped. “No, I know, of course you’re not. I trust you all right, old girl. But if you only knew what a devil of a lot of things are happening——” He looked at her doubtfully. Then he smiled. “You’re a good sort, Alice,” he said, “I know you are. I’m damned grateful. Yes, I’m not quite the thing. There are a whole lot of worries.” He hesitated again, then he went on: “I tell you what you can do—keep the family quiet, you know. Keep them off it, especially the governor. They trust you, all of them, and you can just let them know it’s all right. Will you do that?” He looked at her eagerly. She smiled back at him. “Yes, old boy, of course. I think I can manage Sir Richard, for a little time at any rate. And in any case, it isn’t for very long, because we’re all going away in about a week; twenty-seventh or twenty-eighth, I think Lady Gale said.” Tony started. “Did she?” he said. “Are you sure of that, Alice? Because it’s important.” “Yes. I heard Lady Gale discussing it with Sir Richard last night.” “By Jove. I’m glad to know that. Well, anyhow, Alice, I’ll never forget it if you help us. We want it, by Jove.” She noticed the “we.” “Oh, that’s all right,” she said, smiling back at him. “Count on me, Tony.” At that moment a general move was made. The meal, to everyone’s infinite relief, was over. Mrs. Lester got up slowly from her chair, she turned round towards Maradick. For an instant her eyes met his; the corners of her mouth were raised ever so slightly— she smiled at him, then she turned back to his wife. “Mrs. Maradick,” she said, “do come over and sit by the window. There’ll be a little air there. The sun’s turned the corner now.”
- 54. But Mrs. Maradickhad seen the smile. Suddenly, in a moment, all her suspicions were confirmed. She knew; there could be no doubt. Mrs. Lester, Mrs. Lester and her husband—her husband, James. Dear, how funny! She could have laughed. It was quite a joke. At the same time, she couldn’t be well, because the room was turning round, things were swimming; that absurd carpet was rising and flapping at her. She put her hand on the tea-table and steadied herself; then she smiled back at Mrs. Lester. “Yes, I’ll bring my work over,” she said. The rest of the company seemed suddenly to have disappeared; Maradick and Tony had gone out together, Lady Gale and Alice, followed by Sir Richard and Lester, had vanished through another door; only Mrs. Lawrence remained, working rather dismally at a small square piece of silk that was on some distant occasion to be christened a table-centre. Mrs. Maradick sometimes walked on her heels to increase her height; she did so now, but her knees were trembling and she had a curious feeling that the smile on her face was fixed there and that it would never come off, she would smile like that always. As she came towards the table where Mrs. Lester was another strange sensation came to her. It was that she would like to strangle Mrs. Lester. As she smiled at her across the table her hands were, in imagination, stretching with long twisting fingers and encircling Mrs. Lester’s neck. She saw the exact spot; she could see the little blue marks that her fingers would leave. She could see Mrs. Lester’s head twisted to one side and hanging in a stupid, silly way over her shoulder. She would draw her fingers very slowly away, because they would be reluctant to let go. Of course it was a very stupid, primitive feeling, because ladies that lived in Epsom didn’t strangle other ladies, and there were the girls to be thought of, and it wouldn’t really do at all. And so Mrs. Maradick sat down. “It is quite cool,” she said as she brought out her work, “and after such a hot day, too.”
- 55. Mrs. Lester enjoyedthe situation very much. She knew quite well that Maradick had been watching her anxiously all the afternoon. She knew that he was waiting to see what she was going to do about yesterday. She had not been quite sure herself at first. In fact, directly after he had left her she had been furiously angry; and then she had been frightened and had gone to find Fred, and then had cried in her bedroom for half an hour. And then she had dried her eyes and had put on her prettiest dress and had come down to dinner intending to be very stiff and stately towards him. But he had not been there; no one had known where he was. Mrs. Maradick had more or less conveyed that Mrs. Lester could say if she wanted to, but of course she wouldn’t. However, she really didn’t know. The evening was stupid, tiresome, and very long. As the hours passed memories grew stronger. No one had ever held her like that before. She had never known such strength. She was crushed, gasping. There was a man! And after all, it didn’t matter; there was nothing wrong in that. Of course he oughtn’t to have done it. It was very presumptuous and violent; but then that was just like the man. It was the kind of thing that he did, the kind of thing, after all, that he was meant to do! In the Middle Ages, of course, would have been his time. She pictured him with some beautiful maiden swung across the crupper, and the husband, fist in air but impotent—that was the kind of man. And so she had smiled at him, to show him that, after all, she wasn’t very angry. Of course, she couldn’t be always having it; she didn’t even mean that she’d altogether forgiven him, but the whole situation was given an extra piquancy by the presence of Mrs. Maradick. She didn’t mean any harm to the poor little spectacle of a woman, but to carry him off from under her very nose! Well! it was only human nature to enjoy it! “You must come and see us, dear Mrs. Maradick, both of you, when you’re back in town. We shall so like to see more of you. Fred has taken enormously to your husband, and it’s so seldom that he really makes a friend of anyone.”
- 56. “Thank you somuch,” said Mrs. Maradick, smiling, “we’ll be sure to look you up. And you must come out to Epsom one day. People call it a suburb, but really, you know, it’s quite country. As I often say, it has all the advantages of the town and country with none of their disadvantages. A motor-van comes down from Harrods’ every day.” “That must be delightful,” said Mrs. Lester. “And Lord Roseberry living so near makes it so pleasant. He’s often to be seen driving; he takes great interest in the school, you know—Epsom College for doctors’ sons—and often watches their football!” “Oh yes,” said Mrs. Lester. Mrs. Maradick paused and looked out of the window. What was she going to do? What was she going to do? The great black elms outside the window swept the blue sky like an arch. A corner of the lawn shone in the sun a brilliant green, and directly opposite a great bed of sweet-peas fluttered like a swarm of coloured butterflies with the little breeze. What was she to do? She was feeling now, suddenly, for the first time in her selfish, self-centred life utterly at a loss. She had never been so alone before. There had always been somebody. At Epsom there had been heaps of people; and, after all, if the worst came to the worst, there had always been James. She had never, in all these years, very actively realised that he was there, because she had never happened to want him; there had always been so many other people. Now suddenly all these people had gone. Epsom was very, very far away, and, behold, James wasn’t there either! She realised, too, that if it had been some one down in the town, a common woman as she had at first imagined it, it would not have hurt so horribly. But that some one like Mrs. Lester should care for James, should really think him worth while, seemed at one blow to disturb, indeed to destroy all the theories of life in general and of James in particular that had governed her last twenty years. What could she see? What could any one of them see in him? she asked herself again and again.
- 57. Meanwhile, of course,it must all be stopped somehow. They must go away at once. Or perhaps it would be better to be quiet for a day or two and see. They would all be gone in a week or so. And then Epsom again, and everything as it had been and none of this— she called it “intrigue.” “I’m so glad,” said Mrs. Lester, smiling, “that Tony Gale has taken so strong a liking to your husband. It’s so good for a boy of that age to have some one older. . . . He’s a charming boy, of course, but they always need some one at that age just to prevent them from doing anything foolish.” This was fishing, Mrs. Maradick at once felt. She couldn’t see exactly what Mrs. Lester wanted, but she did want something, and she wasn’t going to get it. She had a sudden desire to prove to Mrs. Lester that she was a great deal more to her husband than appeared on the surface. A great deal more, of course, than any of the others were. For the first time in their married life she spoke of him with enthusiasm. “Ah! James,” she said, “is splendid with young men. Only I could really tell the world what he has been to some of them. They take to him like anything. There’s something so strong and manly about him —and yet he’s sympathetic. Oh! I could tell you——” She nodded her head sagaciously. “Yes,” said Mrs. Lester; “I can’t tell you how I admire him, how we all do, in fact. He must be very popular in Epsom.” “Well, as a matter of fact he rather keeps himself to himself there. They all like him enormously, of course; but he doesn’t want anything really except just the family—myself and the girls, you know. He’s a very domestic man, he always has been.” “Yes, one can see that,” said Mrs. Lester, smiling. “It’s delightful when one sees that nowadays. It so seldom happens, I am afraid. You must be very proud of him.” “I am,” said Mrs. Maradick. The impulse to lean over and take Mrs. Lester’s head and slowly bend it back until the bones cracked was almost too strong to be resisted.
- 58. Mrs. Maradick prickedher finger and stopped the blood with her handkerchief. Both ladies were silent. The last rays of the sun as it left the corner of the lawn fell in a golden shower upon the sweet- peas. Mrs. Lawrence could be heard counting her stitches. Meanwhile Mrs. Lester’s smile had had its effect upon Maradick. He had waited, tortured, for the smile to come, but now it was all right. They were still friends. He could not see it any farther than that. After all, why should he trouble to look at it any more deeply? They were friends. He would be able to talk to her again; he would see her smile again. If she did not want him to behave like that, if she did not want him to hold her hand, he was ready to obey in anything. But they were still friends. She was not angry with him. His depression took wings and fled. He put his arm on Tony’s shoulder as they went down the stairs. “Well, old chap,” he said, “I’m off to see Morelli now. You can bet that it will be all right. Things looked a bit funny last night. They always do when one’s tired and it’s dark. Last night, you see, you imagined things.” But Tony looked up at him quietly with grave eyes. “No,” he said, “there was nothing to imagine. It was just as I told you. Nothing happened. But I know now that there’s something in what the chaps in the town said. I believe in devils now. But my God, Maradick”—he clutched the other’s arm—“Janet’s down there. It isn’t for myself I care. He can do what he likes to me. But it’s her, we must get her away or there’s no knowing. . . . I didn’t sleep a wink last night, thinking what he might be doing to her. He may carry her away somewhere, where one can’t get at her; or he may do—God knows. But that’s what he said last night, just that! that she wasn’t for me or for anyone, that she was never for anyone—that he would keep her.” Tony broke off. “I’m silly with it all, I think,” he said, “it’s swung me off my balance a bit. One can’t think; but it would be the most enormous help if you’d go and see. It’s the uncertainty that’s so awful. If I could just know that it’s all right . . . and meanwhile I’m thinking out plans. It’s all got to happen jolly soon now. I’ll talk when you come back. It’s most awfully decent of you. . . .”
- 59. Maradick left himpacing the paths with his head down and his hands clenched behind his back. He found Morelli sitting quietly with Janet and Miss Minns in the garden. They had had tea out there, and the tea-things glittered and sparkled in the sun. It would have been difficult to imagine anything more peaceful. The high dark red brick of the garden walls gave soft velvet shadows to the lawn; the huge tree in the corner flung a vast shade over the beds and paths; rooks swung slowly above their heads through the blue spacious silence of the summer evening; the air was heavy with the scent of the flowers. Morelli came forward and greeted Maradick almost eagerly. “What! Have you had tea? Sure! We can easily have some more made, you know. Come and sit down. Have a cigar—a pipe? Right. I wondered when you were going to honour us again. But we had young Gale in yesterday evening for quite a long time.” Janet, with a smile of apology, went indoors. Miss Minns was knitting at a distance. This was obviously the right moment to begin, but the words would not come. It all seemed so absurd in this delicious garden with the silence and the peace, and, for want of a better word, the sanity of it all; all the things that Maradick had been thinking, Tony’s story and the fantastic scene in the market-place last night, that and the ideas that had sprung from it, were all so out of line now. People weren’t melodramatic like that, only one had at times a kind of mood that induced one to think things, absurd things. But Morelli seemed to be waiting for Maradick to speak. He sat gravely back in his chair watching him. It was almost, Maradick thought, as though he knew what he had come there for. It was natural enough that Morelli should expect him, but he had not imagined precisely that kind of quiet waiting for him. He had to clear all the other ideas that he had had, all the kind of picture that he had come with, out of his head. It was a different kind of thing, this sheltered, softly coloured garden with its deep shadows and high reds and browns against the blue of the sky. It was not, most emphatically it was not, melodrama.
- 60. The uncomfortable thoughtthat the quiet eyes and grave mouth had guessed all this precipitated Maradick suddenly into speech. The peace and silence of the garden seemed to mark his words with a kind of indecency. He hurried and stumbled over his sentences. “Yes, you know,” he said. “I thought I’d just come in and see you —well, about young Gale. He told me—I met him—he gave me to understand—that he was here last night.” Maradick felt almost ashamed. “Yes,” said Morelli, smiling a little, “we had some considerable talk.” “Well, he told me, that he had said something to you about your daughter. You must forgive me if you think that I’m intrusive at all.” Morelli waved a deprecating hand. “But of course I’m a friend of the boy’s, very fond of him. He tells me that he spoke about your daughter. He loves Miss Morelli.” Maradick stopped abruptly. “Yes,” said Morelli gently, “he did speak to me about Janet. But of course you must look on it as I do; two such children. Mind you, I like the boy, I liked him from the first. He’s the sort of young Englishman that we can’t have too much of, you know. My girl wouldn’t be likely to find a better, and I think she likes him. But of course they’re too young, both of them. You must feel as I do.” Could this be the mysterious terror who had frightened Tony out of his wits? This gentle, smiling, brown-faced little man lying back there so placidly in his chair with his eyes half closed? It was impossible on the face of it. Absurd! And perhaps, after all, who knew whether it wouldn’t be better to wait? If Morelli really felt like that about it and was prepared eventually to encourage the idea; and then after all Janet might be introduced gradually to the family. They would see, even Sir Richard must see at last, what a really fine girl she was, fine in every way. He saw her as she had stood up to meet him as he stepped across the lawn, slim, straight, her throat rising like a white stem of some splendid flower, her clear dark eyes pools of light. Oh! they must see if you gave them time. And, after all, this was rather carrying the matter with a high hand, this eloping and the
- 61. rest! The garden hada soothing, restful effect upon him, so that he began to be sleepy. The high red walls rose about him on every side, the great tree flung its shadow like a cloud across, and the pleasant little man smiled at him with gentle eyes. “Oh yes, of course, they are very young.” “And then there’s another thing,” went on Morelli. “I don’t know, of course, but I should say that young Gale’s parents have something else in view for him in the way of marriage. They’re not likely to take some one of whom they really know nothing at all. . . . They’ll want, naturally enough, I admit, something more.” He paused for a moment, then he smiled. “But perhaps you could tell me,” he said. Maradick had again the sensation that the man knew perfectly well about the whole affair, about the Gales and Alice and Tony, and even perhaps about himself. He also felt that whatever he could say would be of no use at all; that Morelli was merely playing with him, as a cat plays with a mouse. Meanwhile he had nothing to say. “Well, you see,” he began awkwardly, “as a matter of fact, they haven’t had the opportunity—the chance, so to speak, of knowing— of meeting Miss Morelli yet. When they do——” “They’ve been here,” broke in Morelli quietly, “some weeks now. Lady Gale could have called, I suppose, if she had been interested. But I gather that Gale hasn’t told her; hasn’t, indeed, told any of them. You see,” he added almost apologetically, “she is my only child; she has no mother; and I must, in a way, see to these things.” Maradick agreed. There was really nothing to be said. It was perfectly true that the Gales didn’t want Janet, wouldn’t, in fact, hear of her. The whole affair seemed to lose a great deal of its immediate urgency in this quiet and restful place, and the fact that Morelli was himself so quiet and restful was another motive for waiting. The girl was in no danger; and, strangely enough, Maradick seemed to have lost for the first time since he had known Morelli the sense of uneasy distrust that he had had for the man; he was even rather ashamed of himself for having had it at all.
- 62. “Well,” he saidslowly, “you don’t object to things being as they are for a time. I’m sure Tony will see it sensibly, and perhaps Miss Morelli might meet Lady Gale. It would be a pity, don’t you think, to put a stop altogether to the acquaintance?” “Ah yes,” said Morelli, “certainly. We’ll say no more about it for the present. It was very pleasant as it was. As I told you, I like young Gale; and who knows?—perhaps one day——” Maradick sat back in his chair and looked up lazily at the sky. It was all very pleasant and comfortable here in this delicious old garden; let the matter rest. And then Morelli proved himself a most delightful companion. He seemed to have been everywhere and to have seen everything. And it was not only knowledge. He put things so charmingly; he had a thousand ways of looking at things, a thousand ways of showing them off, so that you saw them from new points of view, and the world was an amusing, entertaining treasure-house of wonders. The minutes slipped by; the sun went down the sky, the shadow of the tree spread farther and farther across the lawn, the pinks and roses lay in bunches of red and pink and yellow against the dark background of the wall. Maradick got up to go and Morelli walked with him, his hat set back, his hands in his pockets. As they entered the house he said, “Ah, by the way, there was that Spanish sword that I promised to show you. It’s a fine thing and of some value; I’ll bring it down.” He disappeared up the stairs. Suddenly Janet was at Maradick’s elbow. He had not seen her coming, but she looked round with quick, startled eyes. Her white dress shone against the dark corners of the hall. He saw, too, that her face was very white and there were dark lines under her eyes; to his surprise she put her hand on his arm, she spoke in a whisper. “Mr. Maradick, please,” she said, “I must speak to you. There is only a minute. Please listen, it’s dreadfully important. Tony says you want to help us. There isn’t anyone else;” she spoke in little gasps and her hand was at her throat as though she found it difficult to breathe. “I must get away somehow, at once, I don’t know what will happen if I don’t. You don’t know father, and I can’t explain now, but
- 63. I’m terribly frightened;and he will suddenly—I can see it coming.” She was nearly hysterical; he could feel her whole body trembling. “Tony said something yesterday that made father dreadfully angry. Tony ought not to have come; anything might happen when father’s like that. If you can’t help me I will run away; but you must help.” She grew calmer but still spoke very rapidly, still throwing frightened glances at the stairs. “Listen; on the twenty-seventh— that’s Thursday—father’s going away. He’s going to Pendragon for the whole day; it was arranged long ago. He was to have taken me, but he has decided not to; I heard him tell Miss Minns—I——” But suddenly she was gone again, as quietly as she had come. He saw now that there had been a door behind her leading to some room. He looked up and saw that Morelli was coming downstairs carrying the sword. Five minutes afterwards he had left the house. It had all happened so suddenly, so fantastically, that it was some minutes before he could straighten it out. First he had the impression of her, very young, very frightened, very beautiful. But there was no question of the reality of her terror. All the feelings of danger that he had had with Tony last night came crowding back now. It was true then? It hadn’t only been Tony’s imagination. After all, Janet must know. She hadn’t lived with her father all those years without knowing more about it than he, Maradick, possibly could. She wouldn’t have been likely to have taken the risk of seeing him like that if there was nothing in it, if there was only the mere ordinary domestic quarrel in it. But above all, there was the terror in her eyes; that he had seen. He could not, he must not, leave her then. There was danger threatening her somewhere. The whole business had entirely changed from his original conception of it. It had been, at first, merely the love affair of a boy and girl, and he, from a pleasant sense of romance and a comfortable conviction that it was all good for his middle-aged solidity, had had his share in it. But now it had become suddenly a serious and most urgent affair, perhaps even a matter of life and of death. He turned, as he had turned before, to Punch. There was no time to lose, and he was the man to see about it; he must find him at
- 64. once. The lights werecoming out in the town as he passed through the streets; there were not many people about, and the twilight was lingering in the air so that all the colours of the sky and the houses and the white stretches of pavement had a faint pure light. The sky was the very tenderest blue, and the last gleam from the setting sun still lingered about the dark peaks and pinnacles of the houses. He was soon on the outskirts of the town, and at last he trod the white high road. At the farther turn were Punch’s lodgings. There was a full round globe of a moon, and below him he could hear the distant beating of the sea. Some one was walking rapidly behind him; he turned round, and to his astonishment saw, as the man came up to him, that it was the very person for whom he was looking. “Ah! that’s splendid, Garrick,” he said, “I was just coming for you. I’m a bit worried and I want your advice.” “I’m a bit worried too, sir, as a matter of fact,” said Punch, “but if there’s anything I can do——” Maradick saw now that the man was very different from his usual cheerful self. He was looking anxious, and his eyes were staring down the road as though he were expecting to see something. “What’s the matter?” said Maradick. “Well, it’s the dog,” said Punch, “Toby, you know. He’s missing, been gone all the afternoon. Not that there’s very much in that in the ordinary way. He often goes off by ’imself. ’E knows the neighbourhood as well as I do; besides, the people round ’ere know him and know his mind. But I’m uneasy this time. It’s foolish, perhaps, but when a man’s got only one thing in the world——” He stopped. “But why should you be uneasy?” said Maradick. The loss of a dog seemed a very small thing compared with his own affairs. “Well, as a matter of fact, it’s Morelli.” The lines of Punch’s mouth grew hard. “’E’s owed me a grudge ever since I spoke to ’im plain about them animals. And ’e knows that I know a good bit, too. He passed me in the market-place two days back, and stopped for an instant and looked at the dog. To them that don’t know Morelli that’s
- 65. nothing; but forthem that do—’e’d think nothing of having his bit of revenge. And it’s late now, and the dog’s not home.” The little man looked at Maradick almost piteously, as though he wanted to be reassured. “Oh, I expect it’s all right,” said Maradick. “Anyhow, I’ll come along with you and we can talk as we go.” In a few words he explained what had happened that afternoon. Punch stopped for a moment in the road and stared into Maradick’s face. “Get ’er away, sir,” he said, “whatever you do, get ’er away. I know the girl; she wouldn’t have spoken to you like that unless there was something very much the matter. And I know the man; there’s nothing ’e’d stop at when ’e’s roused.” “But why,” said Maradick, “if he feels like this about it did he let them go about together? He helped them in every way. He seemed to love to have Tony there. I can’t understand it.” “Ah, sir, if you take Morelli as an ordinary man you won’t understand ’im. But ’e’s a kind of survival. ’E loves to be cruel, as they did in the beginning of things when they didn’t know any better. It’s true. I’ve seen it once or twice in my life. It’s a lust like any other lust, so that your body quivers with the pleasure of it. But there’s more in it than that. You see ’e wants to have young things about ’im. ’E’s always been like that; will play with kittens and birds and puppies, and then p’r’aps, on a sudden, kill them. That’s why he took to young Gale, because of ’is youth. And ’e liked to watch them together; but now, when young Gale comes and talks of marriage, why, that means that they both leave ’im and ’e can’t play with them any more, so ’e’ll kill them instead. Take ’er away, sir, take ’er away.” They were out now upon the moor that ran between the woods and the sea; the world was perfectly still save for the distant bleating of some sheep and the monotonous tramp of the waves on the shore far below them. There was no sign of any other human being; the moon flung a white unnatural light about the place. Punch walked with his eyes darting from side to side; every now and again he whistled, but there was no answering bark.
- 66. “It may seema bit absurd to you, sir,” Punch said almost apologetically, “to be fussing this way about a dog, but ’e’s more to me than I could ever explain. If I hadn’t got ’im to talk to and have about at nights and kind o’ smile at when you’re wanting company the world would be another kind of place.” Maradick tried to fix his mind on Punch’s words, but the ghostliness of the place and the hour seemed to hang round him so that he could not think of anything, but only wanted to get back to lights and company. Every now and again he turned round because he fancied that he heard steps. Their feet sank into the soft soil and then stumbled over tufts of grass. Faint mists swept up from the sea and shadowed the moon. Behind them the lights of the town twinkled like the watching eye of some mysterious enemy. A bird rose in front of them with shrill protesting cries, and whirled, screaming, into the skies. Punch seemed to be talking to himself. “Toby, boy, where are you? Toby, old dog. You know your master and you wouldn’t hide from your master. It’s time to be getting home, Toby. Time for bed, old boy. Damn the dog, why don’t ’e come? Toby, old boy!” Every few minutes he started as though he saw it, and he would run forward a few paces and then stop. And indeed, in the gathering and shifting mist that went and came and took form and shape, there might have been a thousand white dogs wandering, an army of dogs, passing silently, mysteriously across the moor. “Toby, old boy, it’s time to be getting back. ’E was that used to the place you couldn’t imagine ’is being lost anywhere round about. ’E was that cunning . . .” But the army of dogs passed silently by, curving with their silent feet in and out of the mists. One new dog had joined their ranks. He fell in at the rear and went by with the others; but his master did not see them. Suddenly the mists broke and the moon shone out across the moor like a flame. The moon leapt into the light. A little to the right on a raised piece of ground lay something white. The army of dogs had vanished. The woods, the moor, the sea, were bathed in white colour.
- 67. Punch ran forwardwith a cry; he was down on his knees and his arms were round the dog’s body. He bent down, and for a moment there was perfect silence, only, in a far distant field, some sheep was crying. Then he looked up. The tears were rolling down his face; he lifted his hand and brushed them back. “It’s Toby. My dog! ’E’s been killed. Something’s torn ’im. . . .” He bent down and picked it up and held it in his arms. “Toby, old dog, it’s time to go back. It’s all right; ’e hasn’t hurt you, old boy. It’s all right.” He broke off. “Curse him,” he said, “curse him! ’E did it—I know his marks—I’ll kill ’im for it.” His hands fell down to his side. “Toby, old dog! Toby. . . .” The moon crept back again behind the mist. In the shadow the man sat nursing the dog in his arms. Far below him sounded the sea.
- 68.
- 69. CHAPTER XVI MRS. LESTER,TOO, WOULD LIKE IT TO BE THE TWENTY-SEVENTH BUT MARADICK IS AFRAID OF THE DEVIL On Monday the 24th the weather broke. Cold winds swept up from the sea, mists twisted and turned about the hotel, the rain beat in torrents against the panes. In all the rooms there were fires, and it seemed impossible that, only the day before, there should have been a burning, dazzling sun. It was after lunch, and Lady Gale and Tony were sitting over the fire in the drawing-room. Tony had been obviously not himself during these last few days, and his mother felt that her silence could last a very little time longer. However, matters were at length approaching a crisis. Things must decide themselves one way or the other in a day or two, for Sir Richard had, at lunch, announced his intention of departing on Saturday the 29th; that is, they had the inside of a week, and then Treliss, thank Heaven, would be left behind. Surely nothing very much could happen in a week. Her earlier feeling, that above all she did not want Tony to miss this girl if she were the right one for him, had yielded now to a kind of panic. All that she could think of now was to get him away. There was a look in his eyes that she had never seen in his face before. It was a look that aged him, that robbed him altogether of that delightful youth and vitality that had been his surprising, his charming gift! But there was more than a look of weariness and distress, there was positive fright there! She watched him when he was in the room with her, and she had seen him suddenly start and tremble, fling back his head as though he expected to find some one behind him. He, her boy Tony, who had never been afraid of anyone or anything. And then, too, she had seen a new look of determination in his mouth and eyes during these last days. His mind was made up to something, but to what she was too afraid to think!
- 70. She must gethim away, and she had heard her husband’s decision about Saturday with tremendous relief. She had watched Tony’s face at the announcement. But it had not changed at all; only, for a moment he had looked quickly across at Maradick; it had apparently not startled him. His indifference frightened her. If he was taking it so calmly then he must have decided on something that this date could not affect, on something probably before the date? But what could he do before Saturday? She seemed to miss altogether the obvious thing that he could do. But it had been seldom enough that she had had him to herself during these last weeks, and now she snatched eagerly at her opportunity. She sat on one side of the fire, one hand up to shield her face, her rings glittering in the firelight; her brown dress stood out against the white tiles of the fireplace and her beautiful snow- white hair crowned her head gloriously. Tony sat at her feet, one hand in hers. He stared straight before him into the fire. She had noticed during these last three days a delightful tenderness towards her. His attitude to her had always been charming, courteous, affectionate and yet companionable; but now he seemed to want to do everything that he could to show her that he loved her. And yet though she valued and treasured this it also frightened her. It was a little as though he were preparing for some departure, at any rate some change, that might hurt her. Well, they were going at the end of the week, only a few more days. He took her fingers and stroked them. His hand stopped at the wedding ring and he passed his thumb across it. “I say, mother,” he looked up in her face with a little laugh, “I suppose you’d say that you’d rather lose anything in the world than that.” “Yes, dear, it’s very precious;” but she sighed. “I suppose it is. It must be ripping having something that is just yours and nobody else’s, that you simply don’t share with anyone. It must be ripping having somebody that belongs to you and that you belong to; just you two.”
- 71. “Yes, But thatring means more to me than that. It means you and Rupert as well as your father. It means all those hours when you screamed and kicked, and the day when you began to talk, and the first adventurous hours when you tried to cross the nursery floor. And yes, a thousand things besides.” “Dear old mater,” he said softly. “It’s been just ripping having you. You’ve always understood so splendidly. Some chaps’ mothers I’ve seen, and they don’t know their sons in the very least. They do all the things that are most likely to drive them wild, and they never seem to be able to give them a bit what they want.” “Yes, but it works both ways,” she answered. “A son’s got to try and understand his mother too. It’s no use their leaving it all to her, you know.” “No, of course not.” Then he turned his body round and looked her in the face. “But you do understand so splendidly. You always have understood. You see, you trust a fellow.” Then he added quickly, “You’re trusting me now, aren’t you?” “Yes,” she answered, looking at him steadily, “perfectly. Only, just these last few days, perhaps I’ve been a little tiny bit worried. You haven’t been looking happy, and then I’m always worried; it’s so seldom that you’re not all right.” “But you’d rather not know—what’s going on, I mean. It’s all right, perfectly right, and if it wasn’t—if it wasn’t right for you, I mean, as well as for me—I wouldn’t go on with it for a moment. Only it’s dreadfully important.” “Yes, dear, I know. And if Mr. Maradick knows about it——” “He’s a brick, isn’t he?” Tony interrupted eagerly. “You know, so few middle-aged men can understand the point of view of a chap who’s only about twenty-five. They are either fatherly and patronising or schoolmasterly and bossing, or kind of wise and beneficent; but Maradick’s most awfully young really, and yet he’s wise too. He’s a ripper.” He stopped. They neither of them spoke for some minutes. “It will be quite all right, mother,” he said, “very soon. Just now things are a little difficult, but we’ll pull through.”
- 72. He got upand stood looking down at her. “You are a brick to trust me and not to ask,” he said. “It would make things so awfully difficult if you asked.” He bent down and kissed her. “It’s a bit of luck having you,” he said. But as soon as he had left the room his face was serious again. He passed Mrs. Lester on the stairs and smiled and hurried on. It was all very well; she was there, of course, real enough and all that sort of thing, but she simply didn’t count for him at that moment, she didn’t exist, really, any more than the hotel or the garden did. Nothing existed except that house in the town with Janet somewhere in it waiting for him to set her free. That was the one point on which his eyes were now fixed. In his earlier days it had, perhaps, been one of his failings—that he had run rather too eagerly after too many interests, finding in everything so immediate an excitement that he forgot the purpose of yesterday in the purpose of to-day. It had always been the matter with him that he had too many irons in the fire. Life was so full and such fun! —that had been the excuse. Now it was deadly earnest. But it was the first time that the world had so resolved into one single point for him. He was already years older; these last days had made him that, the uncertainties, the indecisions, the fluctuating enthusiasms, the passing from wonder to wonder. All these had solidified into one thing, and one thing only—Janet, how to get her out, how to marry her, how to have her for always; the rest of the world was in shadow. To-day was Monday; Tuesday, Wednesday, and then Thursday, Thursday the 27th. That was the day on which everything must be done. He was thinking it all out, they had got that one chance. If they missed it Morelli would be back, and for ever. They must not miss it. But he was perfectly calm about it. His agitation seemed curiously to have left him. He was cold and stern and absolutely collected. He and Maradick were going to pull it through. He could not find Maradick. He searched for him in the dining- room, the passages, the billiard-room.
- 73. No. The servantshadn’t seen him. Mrs. Maradick was with Mrs. Lawrence in one of the drawing-rooms; no, they hadn’t see him, he had disappeared after lunch. Mrs. Maradick smiled. “Find Mrs. Lester” was the advice that she would have given him. She went back to her novel with tightly closed mouth and refused to talk to Mrs. Lawrence. And then Tony suddenly remembered. Of course, he would be up in that old room where he so often went, the room with the gallery. Tony found him there. The rain was beating furiously against the panes, and there was a very dismal light that struggled across the floor and lost itself hopelessly in the dark corners under the gallery. Maradick was sitting close up against the window, reading in the rather feeble light. He looked up when he saw Tony and put his book down. “Ah, Tony, I was coming down to find you; Sir Richard’s decision at lunch pretty well settles things, doesn’t it? We must move at once.” He looked up at the boy and saw the age in his face. “Don’t worry,” he said. “We’re going to pull it through all right.” “Oh, I’m not worrying,” Tony answered shortly. “It’s too damned serious, and besides, there’s no time.” He paused as though he were collecting his thoughts, and then he went on. “Look here, I’ve thought it all out. I’ve been able to write to Janet and have had several letters from her. She’s plucky, my word, you can’t think! Anyhow, that beast’s all right for the moment, it seems, only he keeps looking at her as though he was meaning to do something, and she’s terribly frightened, poor little girl. But he’s going on Thursday all right, and that’s when we’ve got to do the trick.” “Yes,” said Maradick. “I’m absolutely at your service.” Their positions had changed. Tony was taking the lead. “Yes,” said Tony, very solemnly and speaking rather quickly. “It’s all got to be Thursday. I want you to go off this afternoon, if you don’t very much mind, to that parson I was telling you of—the parson at Tremnan. He knows me and he’s a real sportsman. He must do the trick. You can tell him, 1.30 Thursday. Then there’s the licence to be got. I’ll see to that. I’ve been here three weeks now, so
- 74. that’s all right.Then it only remains to think about that, I’m going to get ’em—the family, I mean—to go for an expedition on Thursday. Mother will understand if I ask her, and that will get them out of the way. Then we just take a cab, you and I and Janet and Miss Minns.” “Miss Minns?” broke in Maradick. “Yes,” said Tony, still very seriously. “The poor woman’s frightened out of her life, and Janet’s taken her into her confidence. We’re going to take her away with us. She’s going to live with us. That’ll be all right. She’s got more sense than you think. Well, we four drive out to the church and there the thing’s done. Then we get back and catch the three o’clock up to town. Then off to Paris that same night; and there you are!” He stopped and looked at Maradick for a moment. “The only thing,” he said, “is about you.” “About me?” Maradick looked up, smiling. “Yes. What are we going to do about you? Of course you can come off with us, too, if you like, but then there’s your wife and the girls. You couldn’t do that very well, I suppose?” “No,” said Maradick, “I couldn’t.” “Well, but, you know, if you’re left, why then, everybody’s got you, so to speak—Morelli, my people, everybody. There’s only you to turn on; you’ll have a pretty rotten time. It isn’t fair. And even now, you know, if you’d rather get out of it I expect I’ll manage.” Maradick said nothing. “I hadn’t really seen how damned selfish it all was until just now. I asked you to come and didn’t see it really a bit, what it would all lead to, I mean, and especially for you.” Maradick looked up, laughing. “My dear boy, do you suppose I, at any rate, haven’t seen? Why, from the beginning, from that first night of all when we talked about it, I was responsible; responsible to your mother at any rate, and she’s the only person who really matters. As to Morelli, he can do nothing. When I see a girl look as Janet looked the other night, why, then it was time some steps were taken by somebody to get her away.”
- 75. He put hishand on Tony’s arm. “And besides, whatever happened to me, do you suppose that I could ever cease to be grateful for all that you’ve done for me, your being with me, your showing me a new kind of life altogether? I’d be a bit of a cur if I wasn’t ready to help you after that. Nothing that I can do can quite repay you.” “That’s all right, then,” said Tony. He was a little impatient, just then, of Maradick’s approach to sentiment. It was off the mark; it hadn’t anything at all to do with Janet, and besides, it was all rot, anyway, to talk about all that he’d done. He’d done nothing. But he didn’t, in the least, want to be ungracious. “But that’s most awfully good of you, really, and I don’t suppose, as a matter of fact, they’ll do very much. They can’t, anyhow. I’m over age, and I shan’t have to go to the governor for money. Besides, it will be all right in a week or two. The governor’s like that; I know him, and once the thing’s over he’ll get over it, because he loathes things being uncomfortable; besides, mother will manage him. Anyhow, are you sure you don’t mind going off to the parson? I’d come, too, but I think it would be safer for me on the whole to hang round here this afternoon.” No, Maradick didn’t mind. Maradick would like to go; Maradick would do anything. And, as a matter of fact, he wanted to get out and away—away from the house and the people in it, where he could think undisturbed. He left Tony and started down to the town. His brain was still on fire with his meeting with Mrs. Lester on the evening before. During these last three days they had had very few opportunities of meeting, but the affair had nevertheless advanced with extraordinary rapidity. Then, last night, he had been alone with her, after dinner, in the garden. It had been terribly hot and oppressive, a prelude to the storm that came a few hours later. There was not a breath of wind; the world might have been of carved stone, so motionless was it. He had had her in his arms; her hands had crept round his neck and had pulled his head down until it rested on her breast. He had been on fire—the world had been on fire—and he had poured into her ear, in fierce hurried words, passion
- 76. such as itseemed to him no man had ever known before. He had told her the old, old arguments; things that seemed to him absolutely new and fresh. Their marriages had been, both of them, absurd. They had been joined, each of them, to persons who did not understand them, people who did not even care to understand them. After all, what were marriage vows? A few words spoken hurriedly when they could not possibly tell whether there was even a chance of their being able to keep them. They were not meant to keep them. They had made their mistake, and now they must pay for it; but it was better to break with those bonds now, to have done with them once and for all, than to go on for ever in hypercritical mockery, pretending what they could not feel, acting a lie before God and man. But now, if they could escape now, away from this stupid country with its stupid conventions, away to some place where they would be happy together for ever until death . . . and so on, and so on; and the leaves and the paths and the dark sky were held together, motionless, by the iron hand of God. And then some one had in a moment interrupted them; some fool from the hotel. Maradick’s fingers itched to be about his throat. “What a close night! Yes, a storm must be coming up. They’d heard very distant thunder; how solemn the sea sounded . . .” and so they had gone into the hotel. The rain had ceased. The streets stretched in dreary wet lines before him, the skies were leaden grey; from some room the discordant jingle of a piano came down to him, a cart bumped past him through the mud and dirt. And then suddenly the tower in the market-place sprung upon him. It was literally that, a definite springing out from all the depths of greyness and squalor behind it to meet him. On shining days, when the sky was very blue and the new smart hotel opposite glittered in all its splendour, the tower put on its most sombre cloak of grey and hid itself. That was no time or place for it when other things could look so brilliant, but now, in the absolutely deserted market-place, when the
- 77. cobbles glittered inthe wet and the windows, like so many stupid eyes, gave back the dead colour of the sky, it took its rightful place. It seemed to be the one thing that mattered, with its square and sturdy strength, its solidity that bid defiance to all the winds and rains of the world. Puddles lay about its feet and grey windy clouds tugged at its head, but it stood confidently resolute, while the red hotel opposite shrunk back, with its tawdry glitter damped and torn and dishevelled. So Maradick stood alone in the market-place and looked at it, and suddenly realised it as a symbol. He might have his room from which he looked out and saw the world, and he held it to be good; Tony had shown him that. He might have his freedom, so that he might step out and take the wonderful things that he had seen; Punch had shown him that. But he must also have—oh! he saw it so clearly—his strength, the character to deal with it all, the resolution to carve his own actions rather than to let his actions carve him; and the tower had shown him that! As he looked at it, he almost bowed his head before it. Foolish to make so much of an old thing like that! Sentimental and emotional with no atom of common-sense in it, but it had come out to meet him just when he wanted it most. It needed all his resolution to persuade himself that it had not a life of its own, that it did not know, like some old, sober, experienced friend, what danger he was running. He passed out into the country. Although the rain had ceased and the grass was scenting the air with the new fragrance that the storm had given it, the sky was dark and overcast, grey clouds like Valkyries rushed furiously before the wind, and the sea, through the mist, broke into armies of white horses. As far as the eye could reach they kept charging into the grey dun-coloured air and fell back to give way to other furious riders. The mist crept forward like live things, twisting and turning, forming into pillars and clouds, and then rent by the screaming wind into a thousand tatters. The road was at a high level, and he could see the coast for some miles bending round until it reached the headland; a line of
- 78. white foam stretched,with hard and clear outline, from point to point. This was a new Cornwall to him, this grey mysterious thing, hinting at so much, with a force and power almost terrible in its ominous disregard of human individuality. He had thought that the right light for Cornwall was on a day of gold and blue, now he knew that he had not seen half the wonder and fascination; it was here, with this crawling foam, the sharp rocks, the screaming wind and the turning, twisting mist, that she was rightly to be seen. The wind tore at his coat and beat him about the face. It was incredible that only yesterday there should have been heat and silence and dazzling colour. He pressed forward. His thought now was that he was glad Mrs. Maradick did not know. Until this morning he had not considered her at all. After all, she had given him a bad twenty years of it, and she had no right to complain. Other men did it with far less excuse. But there had been something when she had met him at breakfast this morning that he had not understood. She had been almost submissive. She had spoken to him at breakfast as she had never spoken to him in their married life before. She had been gentle, had told Annie not to jingle her teaspoon because it worried father, and had inquired almost timidly what were his plans for the day. He had felt yesterday that he rather wanted her to see that Mrs. Lester was fond of him; she had driven it into him so often that he was only accepted by people as her husband, that he had no value in himself at all except as a payer of bills. She had even chaffed him about certain ladies of whom she had ironically suggested that he was enamoured. And so it had seemed in its way something of a triumph to show her that he wasn’t merely a figurehead, a person of no importance; that there was somebody who found him attractive, several people, indeed. But now he was ashamed. He had scarcely known how to answer her when she had spoken to him so gently. Was she too under influence of the place? In fact, he did not know what he was going to do. He was tired, worn out; he would not think of it at all. He would see how things turned out.
- 79. The character ofthe day had changed. The mists were still on the sea, but behind them now was the shining of the sun, only as a faint light vaguely discerned, but the water seemed to heave gently as though some giant had felt the coming of the sun and was hurrying to meet it. The light was held back by a wall of mist, but in places it seemed to be about to break through, and the floor of the sea shone with all the colours of mother of pearl. The little church stood back from the cliff; it stood as though it had faced a thousand years of storm and rain, as an animal stands with its feet planted wide and its ears well back ready for attack. Its little tower was square and its stone was of weather-beaten grey, only the little windows with deep blue glass caught the haze from the sea and shone like eyes through the stone and across the grass. The little rectory stood on the other side of the road. It also was minute and absolutely exposed to the elements; here lived the Rev. Mark Anstey, aged eighty-two, quite alone except for the company of five dogs, six cats, three pigeons, a parrot, two tame rabbits, a hedgehog and a great many frogs, these last in a pond near by. When Maradick came up the road he saw the old man standing in his garden watching the sea. The mist had been drawn back, as a veil is drawn back by a mysterious hand, until it lay only on the horizon. The sea was still grey, but it hinted, as it were, at wonderful colours. You fancied that you could see blue and gold and purple, and yet when you looked again it was still grey. It was as though a sheet of grey gauze had been stretched over a wonderful glittering floor and the colours shone through. The old man was a magnificent figure of enormous height. He had a great white beard that fell almost to his waist and his snow- white hair had no covering. Three of his dogs were at his side and the five cats sat in a row on his doorstep. He was standing with his hands behind his back and his head up as though to catch the wind. Maradick introduced himself and stated his errand. The old man shook him warmly by the hand. “Ah, yes; come in, won’t you? Very pleased to meet you, Mr. Maradick. Come into my study and I’ll just take down details.”
- 80. His voice wasas clear as a bell, and his eyes, blue as the sea, looked him through and through. “Here, this is my room. Bit of a mess, isn’t it? But a bachelor can’t help that, you know; besides, I like a mess, always did.” Whatever it was, it was the right kind of mess. The fireplace was of bright blue tiles; there were books, mostly, it seemed, theological, fishing tackle cumbered one corner, guns another, a writing-desk took up a good deal of the room. The old man filled the place. He really was enormous, and he had a habit of snapping his fingers with a sharp, clicking noise like the report of a pistol. Two deerhounds were lying by the fireplace, and these came to meet him, putting their noses into his hands. “Ah, ha! Hum—where are we? Oh! yes! Sit down, Mr. Maradick, won’t you? Oh, clear those things off the chair—yes—let me see! Anthony Gale—Janet Morelli—what? Morelli? How do you spell it? What? M-o-r-e—oh! yes, thanks! Thursday—1.30. Yes, I know the boy; going to be married, is he? Well, that’s a good thing—can’t start breeding too young—improves the race—fill the country with children. Married yourself, Mr. Maradick? Ah! that’s good.” Maradick wondered whether the name, Morelli, would seem familiar to him, but he had obviously never heard it before. “We don’t have many weddings up in this church here, nowadays. They don’t come this way much. Just the people down at the cove, you know. . . . Have some tea—oh, yes! you must have some tea.” He rang the bell and a small boy with a very old face came and received orders. “Remarkable thing, you know,” said Mr. Anstey when the boy had gone out again. “That boy’s twenty-three. You wouldn’t think it, now, would you? But it’s true. Stopped growing, but he’s a good boy; rings the bell in the church, and digs in the garden and all the rest of it. We’ll have tea outside. It’s warm enough and it’s going to be fine, I think. Besides, I always must have my eyes on the sea if it’s possible.” They had tea in the little porch over the door; the honeysuckle was still in flower and there were still roses in the beds, a mass of red hollyhocks at the farther end of the garden stood out against the sky. The old man talked of Tony.
- 81. “Yes, I’ve methim several times; a splendid boy, a friend of Garrick’s who’s brought him up here. Ah, you know Garrick?” Yes, Maradick knew Garrick. “Well, there’s a man! God made that man all right, even though he isn’t often inside a church. He worships in his own kind way, you know, as most of us do, if you only look into it. God’s more tolerant than most of us parsons, I can tell you, and understands people a lot better, too. Not that we parsons aren’t a pretty good lot on the whole, but we’re a bit apt to have our eyes fixed on our little differences and our creeds and our little quarrels when we ought to be having our eyes on the sky. Ah, if I could get a few of those gentlemen who are quarrelling there up in London and just set them here in this garden in rows with that to look at!” He waved his hand at the sea. The hill bent at the end of the garden and disappeared, and beyond the bend there was nothing but the sea. The blue was beginning to steal into it in little lakes and rivers of colour. “That’s God’s work, you know; take your atheist and show him that.” He talked about Tony. “A nice boy, if ever there was one. But what’s this about marriage? Well, I suppose I mustn’t ask questions. You’re a friend of his and you’re looking after him. But that’s a boy who’ll never go wrong; I’d trust any woman to him.” Soon Maradick got up to go. This man had impressed him strangely; he had got that thing that Tony and Punch had got, but he had used it in the right way. There was not only the sentiment, the emotion of the view, there was the strength of the tower as well. Maradick left him standing gazing at the sea. His figure seemed to fill the sky. On his way back the sky grew clearer, and although the sun was never actually to be seen its light was felt in the air and over the sea. There was a freshness about everything around him. The sheaves on the hills, the grass waving on the moor, the sheep clustered in their pens, the hard white clean lines of the road surrounded him with new life. He felt suddenly as though he had
- 82. been standing duringthese last days in a dark, close room with the walls pressing about him and no air. And yet he knew, as he neared the town, that the fascination, the temptation was beginning to steal about him again. As the door of the hotel closed round him, the tower, the clear colours of the land and sky, the man standing gazing at the sea—these things were already fading away from him. He had nearly finished dressing when his wife came into his room. She talked a little, but had obviously nothing very much to say. He was suddenly conscious that he avoided looking at her. He busied himself over his tie, his shirt; it was not, he told himself angrily, that he was ashamed of facing her. After all, why should he be? All that he had done was to kiss another woman, and most men had done that in their time. He was no saint and, for that matter, neither was she. Nobody was a saint; but he was uncomfortable, most certainly uncomfortable. Looking into the glass as he brushed his hair, he caught sight of her staring at him in a strange way, as though she were trying to make up her mind about something. Puzzled—puzzled—puzzled about what? Perhaps it was just possible that she too was just discovering that she had missed something in all these years. Perhaps she too was suddenly wondering whether she had got everything from life that she wanted; perhaps her mind was groping back to days when there did seem to be other things, when there were, most obviously, other people who had found something that she had never even searched for. The thought touched him strangely. After all, what if there was a chance of starting again? Lord! what a fool he was to talk like that! Didn’t he know that in another two hours’ time he would be with the other woman, his pulses beating to a riotous tune that she, his wife, could never teach him; you couldn’t cure the faults, the mistakes, the omissions of twenty years in three weeks. Dinner that night was of the pleasantest. Tony was at his very best. He seemed to have recovered all his lost spirits. That white, tense look had left his face, the strain had gone out of his eyes; even the waiters could not keep back their smiles at his laughter.
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