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Characterisation of 3D cultured primary human hepatocytes and HepaRG for DMSO-free preclinical toxicity testing

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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.

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Characterisation of 3D cultured primary human hepatocytes and HepaRG for DMSO-free preclinical toxicity testing

  1. 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

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