Growing interest in phenotypic screening, together with evidence that the drug response of cells grown in three-dimensional (3D) structures more closely resembles in vivo activity, has made high throughput, 3D fluorescence imaging an attractive screening option for drug discovery. However, creating 3D spheroids compatible with high content screening can be a difficult and expensive process. BIOMIMESYS® is a range of patented hyaluronic acid scaffolds for 3D cell culture. They are made of RGDS- and galactosaminegrafted hyaluronic acid, using an adipic acid dihydrazide crosslinker and extracellular matrix proteins (eg. type I, IV or VI collagen) to accurately mimic the in vivo extracellular environment. BIOMIMESYS® is suitable for automated testing thanks to the uniform thickness of the scaffold – around 650 μm – with an average porosity of 100 to 200 μm This application note describes a straightforward workflow for 3D cell seeding and spheroid formation using HCS Pharma’s BIOMIMESYS® plates in combination with INTEGRA’s VIAFLO 96/384 pipetting system. This process ensures rapid, controllable and reproducible 3D cell cultures, providing researchers with a highly efficient method to produce physiologically-relevant cellular models in a high throughput format

1. HCS Pharma
Biocentre Fleming Bat A
250 rue Salvador Allende
59120 LOOS
BIOMIMESYS® is a registered trademark
of HCS Pharma
+33 769 999 137
www.biomimesys.com
hello@biomimesys.com
A groundbreaking 3D
cell culture technology
Application Note
3D CELL CULTURE GENERATION FOR HIGH CONTENT SCREENING
WITH BIOMIMESYS® AND THE VIAFLO 96/384
INTRODUCTION
Growing interest in phenotypic screening,
together with evidence that the drug response of
cells grown in three-dimensional (3D) structures
more closely resembles in vivo activity, has
made high throughput, 3D fluorescence imaging
an attractive screening option for drug
discovery. However, creating 3D spheroids
compatible with high content screening can be a
difficult and expensive process.
BIOMIMESYS® is a range of patented hyaluronic
acid hydroscaffolds™ for 3D cell culture. They
are made of RGDS- and galactosamine- grafted
hyaluronic acid, using an adipic acid dihydrazide
crosslinker and extracellular matrix proteins (eg.
type I, IV or VI collagen) to accurately mimic the
in vivo extracellular environment.
BIOMIMESYS® is suitable for automated testing
thanks to the uniform thickness of the
hydroscaffold™ – around 300 μm with an
average porosity of 100 to 200 μm (Figure 1)
This application note describes a straightforward
workflow for 3D cell seeding and spheroid
formation using HCS Pharma’s BIOMIMESYS®
plates in combination with INTEGRA’s VIAFLO
96/384 pipetting system. This process ensures
rapid, controllable and reproducible 3D cell
cultures, providing researchers with a highly
efficient method to produce physiologically-
relevant cellular models in a high throughput
format.
Figure 1 : Schematic of BIOMIMESYS® synthesis
(upper panel) and SEM image of the resulting
hydroscaffold™ (lower panel).
MATERIALS AND METHODS
Cell seeding onto BIOMIMESYS® plate with
VIAFLO 96/384
The VIAFLO 96/384 was used to both seed cells
and renew the growth medium. This system was
equipped with a 96-channel pipetting head –
using sterile 300 μl tips – and placed in a
microbiological safety cabinet to ensure sterile
cell handling. The initial cell suspension was
homogenized by pipetting, then ~5,000 HepG2
cells/well were seeded into the ready-to-use
BIOMIMESYS® Liver plate in 50 μl of culture
medium (DMEM supplemented with 10 % FBS, 2
mM L-glutamine, non-essential amino acids
(1X), 50 U/ml of penicillin and 50 μg/ml
streptomycin).

2. HCS Pharma
Biocentre Fleming Bat A
250 rue Salvador Allende
59120 LOOS
BIOMIMESYS® is a registered trademark
of HCS Pharma
+33 769 999 137
www.biomimesys.com
hello@biomimesys.com
A groundbreaking 3D
cell culture technology
Accurate dispensing is crucial to ensure
homogeneous seeding across the plate. Using
the VIAFLO 96/384 allowed the cells to be
seeded onto the center of each hydrogel, with a
consistent pipetting height (z-height limit: 2.9
mm) and speed (set on 2) (Figure 2).
Figure 2 :Using the VIAFLO 96/384 to seed HepG2
into a 96-well plate containing BIOMIMESYS Liver
matrix.
Following seeding, 150 μl/well of medium was
added (z-height limit: 3.2 mm), speed (set on 2)
and the plate was incubated at 37 °C under 5 %
CO2. Seeding and media addition took under two
minutes for a complete 96-well plate, indicating
the potential of this set-up for easy scale-up.
HepG2 viability assay
The cell proliferation reagent WST-1 was used to
determine the metabolic activity and viability of
HepG2 cells. 100 μl/well of medium was
removed, and replaced with 10 μl/well of WST-1.
After a further 1.5 h incubation, OD440
measurements were performed using an
Infinite® M200 PRO plate reader (Tecan).
Chlorpromazine treatment of HepG2 cells
The At Day 6, when the HepG2 cells have
formed multicellular spheroids, the cultures were
treated with the well-known hepatotoxic drug
chlorpromazine (CPZ). 100 μl/well of medium
was removed, and replaced with 100 μl/well of
medium containing various amounts of CPZ to
give final drug concentrations of 0,1, 10, 50,
100 and 250 μM (n=6/concentration; H=30 mm;
S=1). Cells were then incubated at 37 °C under
5 % CO2 for 24 hours.
Bile canaliculi activity of HepG2 cells
Bile canaliculi activity of HepG2 cells treated
with different concentrations of CPZ was
observed using 5(6)-carboxy-2',7'-
dichlorofluorescein diacetate (CDFDA), a
synthetic substrate for bile canaliculi. After
washing the hydrogels with PBS, cells were
incubated with 200 μl/well of 3 μM CDFDA
solution for 30 min. Cells were then washed in
PBS and imaged using an ImageXpress® Micro
Confocal High-Content Imaging System
(Molecular Devices).
RESULTS AND DISCUSSION
HepG2 viability
Thanks to the accuracy and ease of use of the
VIAFLO 96/384 system, 3D cell cultures were
successfully grown for six days after seeding
without disturbing the hydrogels or forming
multicellular spheroids. By enabling 96channel
transfers in a single step, the VIAFLO 96/384
also significantly improves the reproducibility of
the pipetting for both seeding and media
exchange. This is demonstrated by the
consistent cell viability between wells, with low
CVs of just 4 % on Day 3 and 7 % on Day 6
(Figure 3).
Figure 3 : Viability of HepG2 grown in BIOMIMESYS®
liver plate (n=16), measured with WST-1 reagent.

3. HCS Pharma
Biocentre Fleming Bat A
250 rue Salvador Allende
59120 LOOS
BIOMIMESYS® is a registered trademark
of HCS Pharma
+33 769 999 137
www.biomimesys.com
hello@biomimesys.com
A groundbreaking 3D
cell culture technology
The translucence of BIOMIMESYS®
hydroscaffold™ also means that HepG2 spheroid
growth can be observed directly using bright
field microscopy (Figure 4).
Figure 4 : Bright field image of multicellular HepG2
spheroid six days after seeding.
Chlorpromazine toxicity
Thanks to the accuracy and ease of use of the
The viability of HepG2 cells was measured after
24 hours treatment with the hepatotoxicity-
inducing drug CPZ (Figure 5). The results
indicate an IC50 of 34 μM, with excellent
reproducibility at concentrations above 1 μM.
Figure 5 : Viability analysis of HepG2 cultures treated
with chlorpromazine (n=6).
Bile canaliculi activity
Direct fluorescence imaging and bright field
microscopy showed that CDFDA efflux is efflux
by cells could still be observed following
treatment with low concentrations of CPZ.
However, at higher CPZ concentrations (≥10
μM), it accumulates inside HepG2 cells,
indicating that bile canalicular network formation
and maintenance is disrupted (Figure 6).
Figure 6 : Vizualization of bile canaliculi activity in
CPZ- treated HepG2 spheroids, using CDFDA (green)
and Hoechst staining (blue, for cell nuclei). Scale
bar=100 μm.

4. HCS Pharma
Biocentre Fleming Bat A
250 rue Salvador Allende
59120 LOOS
BIOMIMESYS® is a registered trademark
of HCS Pharma
+33 769 999 137
www.biomimesys.com
hello@biomimesys.com
A groundbreaking 3D
cell culture technology
CONCLUSIONS
The application described here demonstrates
that HCS Pharma’s BIOMIMESYS® plate is fully
compatible with INTEGRA’s VIAFLO 96/384
pipetting system. This combination provides a
straightforward, compact and cost-effective
solution for the generation of 3D multicellular
spheroids suitable for a wide range of high
content screening applications.
The VIAFLO 96/384 ensures accurate and
consistent cell seeding and medium exchanges,
leading to very low variability in cellular activity
between wells, which can be easily confirmed
using bright field microscopy.
This set-up simplifies maintenance and
treatment of 3D cell cultures, offering
researchers a straightforward workflow for
performing reproducible viability and functional
assays in a 96-well format. Throughput could be
further improved by switching to a 384-well
format, offering further productivity gains while
minimizing the time spent pipetting.
REFERENCE
(1) Ryan SL, et al. Drug discovery approaches
utilizing three-dimensional cell culture. Assay
Drug Dev Technol, 2016, 14(1), 19-28.