The method of 2D cell culture is not a true reflection of the physiological cell environment where cells connect to other cells and, also to the extracellular matrix (ECM). Therefore some of the processes studied in 2D culture such as gene expression, apoptosis and importantly drug uptake and toxicity may not be directly transferable to in vivo experiments. HCS Pharma has developed a physiological and natural hydroscaffold, based on Hyaluronic Acid (HA) biofunctionalized with ECM components as collagens I, IV or VI, fibronectin and other molecules of interest. We will present the use of this scaffold on an automated platform, with the development of a 3D cell model for steatosis and phospholipidosis assessment as an example. Automated HepG2 cells seeding, medium renewal, spheroids treatment, fixation, staining and imaging will be discussed. BIOMIMESYS® represents a new generation of mimetic hydroscaffold for 3D cell culture. Available in a ready-to-use format it enables the culture of cells under physiological conditions that are representative of the microenvironment found in whole tissues. The highly porous nature of the hydroscaffold allows the rapid uptake of nutrients, oxygen, etc. into the cells to create a reproducible study model for all downstream analytical techniques used with 3D cell culture. Biomimesys® scaffolds technology is an innovative tool, closely mimeting the ECM matrix for relevant 3D cell culture allowing advanced drug discovery, ADME-Tox profiling and disease modelling. It is physiological, easy to use and compatible with all standard analysis methods (fluorescent plate reader, microscopy, Immunofluorescence, direct cell lysis for nucleic acids and protein extraction, histology).

1. BIOMIMESYS® Hydroscaffold for 3D cell culture:
process automation from cell seeding to imaging
Lesaffre M., Vandenhaute E., Ferron P.-J., Souguir Z.* & Maubon N.
HCS Pharma, FRANCE
*zied.souguir@hcs-pharma.com
Abstract
Conclusions
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The growing interest in phenotypic screening,
together with evidence that the cellular
response to drugs in three-dimensional (3D)
systems more closely resembles in vivo
activity, has made high-throughput 3D
fluorescence imaging an attractive screening
option in the drug discovery process.
However, creating 3D structures compatible
with high-content screening (HCS) can be
time-consuming and expensive. HCS Pharma
provides BIOMIMESYS® Hydroscaffold, an
easy-to-use cell culture system allowing to
scale up and increase the power of 3D models.
Here we present the use of BIOMIMESYS® for
the culture, treatment and observation of
HepG2 cells in a 3D environment, using
automation throughout the process.
 BIOMIMESYS® is a range of
new patented Hyaluronic Acid-
based hydroscaffolds for 3D
cell culture.
 BIOMIMESYS® is made of
RGDS- and galactosamine-
grafted Hyaluronic Acid, Adipic
Acid Dihydrazide crosslinker
agent and extracellular matrix
(ECM) proteins (collagens type
I and type IV).
 BIOMIMESYS® is adapted to
automation thanks to its
format (96-well plates, 384-
well plates under
development) and the
thinness of the hydroscaffold.
200 µm 10µm 20µm
The controlled and reproductible size
of BIOMIMESYS® Hydroscaffolds
allows a fast and homogenous
automated cell seeding.
Using the VIAFLO 96/384 to seed
HepG2 cells into a 96-well plate
BIOMIMESYS® Liver plate.
0 µM
Hoechst CDFDA Phase contrast Merged
1 µM
10 µM
25 µM
50 µM
100 µM
1. Vizualization of bile canaliculi activity in CPZ-treated
HepG2 spheroids using CDFDA efflux assay (green). Cell
nuclei were stained with Hoechst (blue). Direct fluorescence
imaging and bright field microscopy showed that CDFDA was
effluxed by cells: this event can still be observed in the
presence of low concentrations of CPZ but not with higher
doses.
2. Texture index calculated
with MetaXpress ‘Granularity’
application module
After 10 days of culture,
HepG2 were treated with
increasing concentrations of
chlorpromazine (CPZ).
After 72 hours of treatment,
the activity of bile canaliculi
was observed using CDFDA
efflux assay (fluorescence and
bright field microscopy, 1 and
2) and and the viability
quantified using WST-1
reagent (3).
 BIOMIMESYS® Hydroscaffolds are adapted to automation, allowing easy cell seeding,
medium changes and cell treatments.
 Automated analyses are simple:
- no need to transfer the samples to other plates
- no need to remove the hydroscaffold
 The hydrogels are translucent, allowing imaging and plate reader measurements
Methods
Cells were treated, fixed
and stained on our robotic
platform integrated by
Molecular Devices.
Image acquisition was done
using ImageXpress Micro
Confocal High-Content
Imaging System and image
analysis was performed
with MetaXpress software
(Molecular Devices).
Automated HepG2 handling on BIOMIMESYS® Automation of drug treatment, staining and image acquisition
Results
Assessment of viability & cholestasis on HepG2 3D cell cultures following treatment with chlorpromazine (CPZ)
Scanning electron microscopy observation
of BIOMIMESYS® Hydroscaffold. The
porosity ranges from 100 to 200 μm.
3. Viability of HepG2 cells
assessed using WST-1 reagent
To know more about BIOMIMESYS® Hydroscaffold, visit our website: www.biomimesys.com
 +33(0) 769 999 137 hello@biomimesys.com