Hepatocytes in primary cultures are still the gold standard in the pharmaceutical industry ; however they are expensive and have a very short lifespan with a progressive loss of specific functions over time. The dipolar aprotic solvent, dimethyl sulfoxide (DMSO) is wildly used to stabilize the liver specific function of human hepatocytes in vitro. It is also used to potentialize the dfferentiation of the human HepaRG-hepatocytes-like cells that share functional characteristics with human hepatocytes. Besides its free radicals scavenging properties that contribute to hepatoprotection, DMSO has been shown to interfere with apoptosis signalling pathways. To avoid this disadvantage, we have analysed the functional properties of freshly isolated human hepatocytes (FIH) and HepaRG-hepatocyte-like cells in 3D cultures performed using Ultra Low Attachment Technology (ULA) plates.

1. Abstract
Hepatocytes in primary cultures are still the gold standard in the pharmaceutical industry ; however they are expensive and have a very short
lifespan with a progressive loss of specific functions over time. The dipolar aprotic solvent, dimethyl sulfoxide (DMSO) is wildly used to stabilize the
liver specific function of human hepatocytes in vitro. It is also used to potentialize the differentiation of the human HepaRG-hepatocytes-like cells
that share functional characteristics with human hepatocytes. Besides its free radicals scavenging properties that contribute to hepatoprotection,
DMSO has been shown to interfere with apoptosis signalling pathways. To avoid this disadvantage, we have analysed the functional properties of
freshly isolated human hepatocytes (FIH) and HepaRG-hepatocyte-like cells in 3D cultures performed using Ultra Low Attachment Technology
(ULA) plates.
Materials and Methods
Conclusions
Results
HCS Pharma – French startup focused on in vitro preclinial research development with a specialisation in cell imaging – http://www.hcs-pharma.com
CYP expression in 3D culture compared with classic 2D culture Nile red staining for steatosis evaluation
Characterisation of 3D cultured primary human hepatocytes and
HepaRG for DMSO-free preclinical toxicity testing
Savary C1, Jarnouen K1, Molez S, Ferron PJ2, Maubon N2, Corlu A1
1Institut NuMeCan - U1241 Inserm – U1341 Inra - Université de Rennes 12, rue Henri Le Guilloux – 35033 Rennes Cedex
2HCS Pharma, 6 rue Pierre Joseph Colin, 35000 Rennes, FRANCE
One month for
differentiation
HepaRG cells
3 7 21 days14
72 h with treatment
- CYP3A4 inducer : rifampicine (RIF)
- Genotoxic : benzo[a]pyren (B[a]P)
• CytochromeP450 mRNA expression and activity
• HistologyFreshly Human Hepatocytes
(FIH)
Characterisation Image based screening
With or
without
DMSO
0
5
10
15
20
HepaRG cells with DMSO
0
0,5
1
1,5
2
2,5
3
HepaRG cells without DMSO
0
1
2
3
4
5
Human Hepatocytes
FIH 03 7 14 21 FIH3 7 14 21 0 FIH3 7 14 21
DaysDaysDays
CYP3A4mRNA
Relativeexpression
0
20
40
60
80
100
120
140
0
10
20
30
40
50
60
70
80
90
100
2D
3D
FIH
0 FIH3 7 14 21
Days
0 FIH3 7 14 21
Days
0
1
2
3
4
FIH 3 7 14 21
Days
CYP1A2mRNA
Relativeexpression
HepaRG
+ DMSO
gamma
H2AX
CTR
B[a]P
CTR
B[a]P
100
µm
100
µm
HepaRG
without
DMSO
Gamma
H2Ax
HoechstHoechst
HepaRG
with
DMSO
Gamma
H2Ax
Genotoxicity response
RIFCTR
RIFCTR
Human Hepatocytes
7 jours
HepaRG without DMSO
14 days
100 µm
100 µm
CYP3A4 inductibility
Immunohistochemestryajoute
peutetrelatechnique
Seeding
days 0
The content of this poster is a part of InnovCell3D project. This project was funded, by « Région Bretagne », FRANCE.
Nuclei, neutral lipids
200X
Nuclei, neutral lipids
100X
Cell culture
Automated fluorescence
microscopy and image analysis :
• miniaturized in 96 well plates
• 2D and 3D imaging
• Nuclei and neutral lipid detection
• Single cells analysis
Z Stack
Using image based lipid droplets
detection, our assay was
validated on HepaRG cells with
DMSO in 2D and 3D. After 48
hours of exposure, we
specifically detect steatosis our
in house hepatotoxic compounds
library.
FIH and HepaRG are classically cultivated in 2D, with addition of DMSO to enhanced and maintained cell differentiation. Significant progress has been
made in 3D cell culture, in order to develop reproducible tissue-like models. Compared to FIH, HepaRG cultivated in 3D whithout DMSO showed a
better expression of CYP3A4. CYP expression of CYP3A4 and CYP1A2 were maintained thought time and tend to increase until 21 days of 3D cell
culture. 3D ULA culture allow to cultivate HepaRG in DMSO-Free conditions without decreasing CYP3A4 expression compared to FIH. CYP3A4
inducibility was maintained in DMSO-Free conditions, and shows a CYP3A4 repartition close to FIH spheroids. Drug metabolism toxicity was confirmed
for both DMSO and DMSO Free conditions, showing DNA double strand breaks induced by B[a]P, mostly in peripheral cells of spheroids. Steatosis has
been evaluate with high content screening approach, highlighting a better survival of HepaRG cells cultivate in 3D. HepaRG spheroids in 96 wells ULA
plates shown the expression of most metabolic markers requested to study xenobiotic metabolism. By maintaining those markers upon 21 days, this
model enable investigations of repeated exposure scenarios in a DMSO-Free conditions.
Single cell analysis