1) A digital microfluidic (DμF) platform was developed to automate the liquid handling steps for differentiating embryonic stem cells into cardiomyocytes and assaying the resulting tissue. 2) The DμF platform improved upon existing manual methods by automating embryoid body growth, differentiation medium exchanges, and non-invasive assays of beating cardiomyocyte tissue using electric fields. 3) Beating cardiomyocyte embryoid bodies were cultured on the DμF platform for up to 8 weeks and responses to known chronotropic agents were measured non-invasively through impedance changes, demonstrating the potential of the system for screening cardiotoxicity.

1. Lab-on-a-Chip Platform for Culturing and Assaying Cardiomyocyte
Tissue Derived from Embryonic Stem Cells
Contact Pads
Driving
Electrodes
Brian F. Bender† and Robin L. Garrell*
Department of Bioengineering, University of California, Los Angeles
Los Angeles, CA USA 90095
† bfbender@ucla.edu
Digital Microfluidics
Tissue Growth Long-Term Culture Conditions
Acknowledgments
1G. Vunjak-Novakovic, et al., Tissue Eng, 2010, 16,
169-187.
2J.A. DiMasi, et al., J Health Econ, 2003, 22, 151-185.
3A. Aijian, et al., JALA, 2014, 20, 283-295.
4R. Fobel, et al., Applied Physical Letters, 2013, 102,
193513.
5J.L. Perez-Diaz, et al., J Colloid Interf Sci, 2012, 1, 180-
182.
Glass
PDMS
ITO Parylene-C
Cytop™ Aqueous Buffer Cell Media
Spacer
Side view schematic of DµF device for EB culture.3 Through-holes, or ‘wells,’ have been fabricated into
the middle plate of the DµF device. Droplets of cell-suspension are dispensed and delivered to the
wells (100-120 V at 18.5 kHz) and are pulled into the well via capillary forces. Cells aggregate and
compact into EBs after ~24 h in culture. A modified fabrication process allows multiple stacked layers.
Digital (droplet) microfluidics (DμF) is a liquid handling platform that enables the
manipulation (dispensing, translating, splitting, and mixing) of discrete pico-microliter
sized droplets of liquid on a planar array of photolithographically patterned electrodes
through the controlled application of electric fields.
OffOn
Electrowetting:
Conductive liquids respond
directly to the electric field
(applied voltage)
OffOn
Dielectrophoresis:
Non-conductive (dielectric,
insulating) liquids respond to
electric field gradient
This work was supported by UCLA research funds and a Dissertation Year
Fellowship to B.F.B. Confocal laser scanning microscopy was performed at
the UCLA CNSI Advanced Light Microscopy/Spectroscopy Shared Resource
Facility, which is supported by an NIH-NCRR shared resources grant (CJX1-
443835-WS-29646) and an NSF Major Research Instrumentation grant
(CHE-0722519). Cleanroom fabrication was performed at the UCLA CNSI
Integrated Systems & Nanofabrication Cleanroom (ISNC). Human
embryonic stem cells were graciously donated from the Nakano lab at
UCLA. Parts, training, and assistance with the DropBot System and µDrop
software were provided by the Wheeler lab at the University of Toronto.
References
Medium exchange from hanging drops.3 Medium
exchange was performed after 48 h periods with
differentiation medium.
Live/Dead Staining. EBs of
human fibroblasts (a) and
mouse mesenchymal stem cells
(b) maintained over 95% viability
after 48 h of incubation. A hESC-
EB (c) in an on-chip well. White
scale bar = 1 mm.
Embryoid
Body
Embryoid
Body
Differentiation
medium 2
1
3
Discarded
4
Liquid exchange. >50% exchange can be
achieved after two cycles, which is
sufficient for EB culture.
50
75
100
36.5
36.75
37
1 10 100 1000
RelativeHumidity(%)
Temperature(⁰C)
Time (log[min])
0
0.005
0.01
0 100 200 300 400 500
InstantaneousVelocity
(mm/s)
Time (ms)
60% RH
70% RH
80% RH
90% RH
0
0.005
0.01
0 100 200 300 400
InstantaneousVelocty
(mm/s)
Time (ms)
60% RH
90% RH
Velocity of DI water actuated at 80 V and 20
kHz from within an incubator, highlighting
that as the humidity rises the droplet
velocity decreases. The error bars correspond to
the standard error with n=6.
0
1
2
BeatingFrequency(Hz)
5 mM Epinephrine
5 µM Epinephrine
5 mM Caffeine
5 µM Caffeine
Media
A vertically stacked DµF design enabled EB
retrieval for downstream processing. Scale bar =
1 mm.
Capacitance and impedance measurements of an 8-day old beating
cardiomyocyte EB showing a more homogenous yet weaker beating profile
compared to the more mature but heterogeneous 8-week old beating
cardiomyocyte EBs.
A digital microfluidic (DµF) method for automating the liquid handling steps for
differentiating and assaying embryonic stem cell (ESC)-derived cardiomyocytes
was developed. This method improves upon existing methods by automating
the manual, multi-step, liquid handling protocols currently used for embryoid
body (EB) growth and differentiation. The electric fields used to manipulate
discrete droplets in DµF platforms were then used to non-invasively assay
phenotypic behavior in beating, 3D cardiomyocyte tissue.
1
Two Primary Motivations
Non-Invasive Assays
The stability over temperature,
humidity, and CO2 provided by
operation within an incubator helps
prevent evaporation, better maintain
solution concentrations, and mitigate
temperature shock.
Setup
Evaporation
Materials
Droplet Velocity
The material selection needed to be re-evaluated to avoid dielectric
breakdown and delamination.
Parylene-C is a CVD-deposited polymer commonly used as the dielectric
layer. After 24 hr of incubation, electrode actuation caused delamination
and device breakdown, revealing the need to readdress material selection.
SiO2 delaminated from ITO electrodes occurs without adherence to
strict cleanroom protocols. However, proper protocols produced
devices that could withstand incubated conditions for >1 month.
DµF operation from within an incubator has many advantages over the current
practice of moving a chip back and forth between the incubator and the benchtop.
Automating droplet sequencing requires a
precise understanding of droplet position and
translation speed for timing.
𝐹 𝑉 = 𝛾 𝐿𝐹 cos 𝜃 𝑎 𝑉
The surface tension of the water-air
interface, 𝛾 𝐿𝐹, decreases as the humidity and
the temperature rise.5 The actuation force,
𝐹 𝑉 , therefore decreases in the incubator.
Furthermore, the static contact angle, cos 𝜃 𝑜,
will decrease an the elevated humidity.
∆𝑃 =
𝛾 𝐿𝐹
𝑑
cos 𝜃 𝑎 − cos 𝜃 𝑜
𝐹(𝑉) = )𝑃𝑟 − 𝑃𝑎(𝑉 ∙ ℎ
The actuation force is therefore decreased
when modeling the difference in Laplace
pressure, ∆𝑃.
In a common, water-jacked incubator
the equilibrium conditions can take
hours to reach.
The concentration of solution constituents can change dramatically
even in samples placed into an incubator that must re-equilibrate.
This can alter cell behavior and confound results.
Differentiation Medium Day 3-5 Day 5-7 Day 7-10
Constituent Stock Final Final
No Chemical
KY-1 50 mM 3 µM 3 µM
XAV 10 mM 1 µM 1 µM
A419259 5 mM 0.3 µM 0.3 µM
AG1478 100 mM 8 µM 4 µM
Day 3 – 10: 0.4 % Albumin Media
Day 10+: 0.04 % Albumin Media
(C)
Known chronotropic and ionotropic agents were
delivered on-chip to beating cardiomyocyte EBs.
Video recordings revealed expected results.
Our understanding of cardiomyocyte maturation is not fully developed.1
Many methods have been developed to probe the differentiation and maturation
process of cardiomyocytes, such as molecular or genomic tagging, videographic
algorithms, micro-post arrays, and micro-electrode arrays. However, few methods have
been developed to holistically monitor 3D tissue samples, despite our awareness that
these systems can illicit more in-vivo-like behavior.
2 A need exists for streamlined tools for differentiating and assaying cardiomyocytes.1
A recent study has estimated the cost of developing a single new drug at over $2.6 billion
and taking over 25 years.2 In order to bring down the costs and timelines for screening
and developing new therapeutics, advanced cell-based assays that better mimic in-vivo
conditions are needed. DμF is capable of automating the liquid handling and non-
invasive assaying steps needed for a streamlined stem cell culture microenvironment.
Abstract
Conclusions
1
A new approach was taken to electrically monitoring EB beating behavior. Changes to
beating heterogeneity were observed in whole 3D samples by applying an electric field
around the entire sample and monitoring impedance changes.2
A DµF platform was used to create a streamlined lab-on-a-chip device capable of
forming EBs of human embryonic stem cells, long-term culturing in an incubator,
differentiation into functional cardiomyocytes, and performing non-invasive assays.
Epinephrine produced a ~4-5x
increase in beating amplitude
evidenced via similarly shaped
capacitance measurements.
8-day old beating EB 8-week old beating EB Controls
19-point weighted triangular smoothing
applied
Epinephrine Assay
Electrical
Visual EB Retrieval
~1-3 mm
~7-10 μL
4 μm
400 nm
110 nm1.7 mm
1 mm
2 mm
To CPU
300 μm
Beating cardiomyocyte EBs were positioned between electrodes, and an 80 V,
15 kHz AC signal was applied while the impedance/capacitance was recorded.
Embryoid Body
(EB)
Differentiation
DropBot4 equipment
and amplifier Cell culture
incubator
3D-printed plastic
holder for both the
DµF chips and the
printed circuit
board cabling
connections