Innovative 3D culture model of human hepatocytes with large potential applications

Transcription

1 Innovative 3D culture model of human hepatocytes with large potential applications Sophie Langouët

2 Human liver Vital organ, involved in many functions: : Synthesis Storage Detoxification Sinusoïde Hépatocytes Veine centrale Lobules hépatiques Canal biliaire Veine porte Artère hépatique Travée hépatocytaire Non-parenchymental cells (5%) Stellate cells, Kuppfer cells, Parenchymental cells (75%) Hepatocytes Endothelial cells

3 Hepatocellular Carcinoma (HCC) Hepatitis virus B,C Xenobiotics (Alcohol, Mycotoxines,...) Obesity Normal Fibrosis Cirrhosis Hepatocellular Carcinoma F1 F F3 F4 Liver primary Cancer : 3 th cause of cancer death worldwide (HCC : 85-9%) 9% of HCC occur on cirrhotic liver, the ultime stage of fibrosis Increase of HCC in industrial countries Njei et al., 15 ; White et al., 17 Müller et al., 6 Identification of new mutations not associated with chemical carcinogens known to date Guichard et al., 1

4 Metabolism of xenobiotics in the liver Xenobiotic R Hydrophobic Molecules Phase I Functionnalization CYP Hydroxylated Metabolites ROH Phase II Conjugation NAT, SULT, UGT, GST Conjugated Metabolites RX Mutations Repair Phase III Elimination MRP, MDR RX Hydrosolubles Molecules

5 Interspecies specificities reinforce the need of the development of human hepatocyte cell models

6 Human Hepatic cell models Primary Human Hepatocytes PHH Hepatocyte differentiation 1 jdays Hepatocarcinoma cell lines Hepatocytes derived from stem cells Huh7, HepG HepaRG Proliferation Proliferation Proliferation Hepatocyte differentiation + DMSO Hepatocyte differentiation Hepatocytes geneticaly modified SV-4 / htert Upcyte E6/E7 du HPV Proliferation +OSM Hepatocyte differentiation -OSM Hepatocyte functions are rapidely decreased/ limited Proliferation and differentiation are always exclusives

7 D Culture.5 D Culture 3D Culture 3D Cell culture Without matrix with matrix low-attachment Plates Magnetic levitation Hanging-drop Bioreactors Natural Hydrogels and/or synthetic Tostoes et al., 1; Gunness et al., 13 ; Bell et al. 16

8 3D Cell culture in matrix 3D culture in a matrix allow to partly recreate the complex microenvironment of cells in vivo and to modified the tension forces Schrader et al., 11; Edmonson et al., 14; Antoni et al., 15

9 3D Cell culture model in collagen matrix Three distinct human hepatocytes cell types: HuH7 HepaRG PHH Transformed Normal

10 Collagen matrix with varying rigidity.5mg/ml.5mg/ml.75mg/ml 1 mg/ml 1.5 mg/ml mg/ml

11 Hepatic cells in 3D cultures d8 1µm TPEF Morphologic caracterisation d8 TPEF + SHG Human hepatocytes cultivated in collagen gels formed spheroids

12 Influence of 3D stiffness on growth and differenciation of human hepatocytes (HuH7) Proliferation Differenciation Soft Gel 1.5 Pa (.75) Rigid Gel 3.5 Pa (1.5) Rigidity influenced the cell phenotype and induced hepatic spheroid cluster development Proliferation and Biotransformation activities are increase in 3D gels Bomo et al, J Cell Biochem, 15

13 Tranformed Human Hepatocytes HepaRG Proliferation Hepatic differentiation 8 j 14 jours 14 jours 8 jours + DMSO % + DMSO % - DMSO Co-culture d hépatocytes et de cholangiocytes-like High expression of xenobiotic metabolism enzymes Regulation (AhR, PXR, CAR ) Functional capacities are stable during 4 weeks with DMSO Gripon et al., Aninat et al., 6 Differentiation after 8 days of culture DMSO is a CYP3A4 inducer Aninat et al., 6; Kanebratt and Anderson, 8 Differentiation decrease immediately after DMSO removal Aninat et al., 6

14 Transformed Human Hepatocytes HepaRG Morphologic caracterization d4 d15 d8 HES E cadherin mm N cadherin d8 mm MRP d8 1mm d8 1mm TPEF Collagen gel culture provides formation of proliferating and polarized spheroids

15 mrna expression mrna expression Tranformed Human Hepatocytes HepaRG Hepatic function caracterization D (D) D 3D DMSO % Temps de culture (jours) 8j - DMSO 8j 15j 8j, Biliary Marker 1,5 1,,5 CK19 D (D) d8 d15 d8 Epithelial Marker E-cadherin D (D) d8 d15 d8 Hepatic Marker Hepatic Transporter HNF1 3 1 MRP D (D) d8 d15 d8 D (D) d8 d15 d8 Long-term maintenance of hepatocyte differentiation

16 pmol/mn/od pmol/mn/od mrna expression Influence of matrix rigidity on hepatocyte differentiation Phase I Phase II 1, 1, CYP1A (3-MC) UGT1A1 UGT1A9 6 4,75mg/ml 1,5mg/ml,8,6,4, 1 d5 d15 d5 d15, D,75 1,5 D,75 1,5 mg/ml de collagène UGT1A1 b-estradiol 3-G UGTB b-estradiol 17-G d5 d15 Matrix collagen rigidity modulate CYP1A activity Differentated HepaRG cells rapidly and long term ( 4 semaines), without DMSO d8,6,5,4,3,,1, Optimum Matrix Rigidity Increasing the Expression and Activity of Phase II Enzymes d8

17 Median % Tail Intensity % ghax positive cells Adduits/1 7 bases DNA damages analysis Control + HAAs Cisplatine MMS AFB 1 4-ABP PhIP MeIQx AaC BREAKS ADDUCTS Comet Assay ghax LC-MS mM 1mM mm 1 8 1mM mm 4 3 1mM days of treatment 4h of treatment Relevant model for DNA damage detection (breaks, adducts) also after chronic treatment

18 HepaRG Model Optimization Obtention of proliferative and polarized spheroids Rapid and stable cell differentiation without DMSO Expression at short and long term of phase I, II et III xenobiotic metabolism enzymes Detection of DNA damages at dose-dependant manner High level of DNA adducts after 15 days of culture allowing chronic treatmets

19 Human Hepatic cell models (PHH) Gold standard: Primary Human Hepatocytes (PHHs) Differenciated hepatic functions +++ Interindividuals variations Genetic polymorphisms detectable Rapid lost of differentiation (1 days) Limited survival (15 days) Low availability Non proliferative in vitro

20 mrna expression Hepato-specific Factors (PHH) a-fetoprotein Albumin Aldolase B HNF4a D 3D D 3D 3 1 D 3D D 3D D 3D D 3D D 3D D 3D d4 d15 d4 d15 d4 d15 d4 d15 E-cadherin N-cadherin d15 d8 Albumin / Nucleus 3D culture in collagen gels promote the expression of liver markers

21 mrna expression pmol/mn/od Metabolism Hepatic Functions (PHH) Phase I,3 CYP1 CYP1 (3-MC) 3 Phase III MRP CDFDA MRP3,,1 1, D 3D D 3D d4 d15 D d8 3D D 3D D 3D d4 d15 D d8 3D d4 5mm Phase II 8 GSTa1/ UGT1A1 NAT D 3D D 3D D 3D D 3D D 3D D 3D d8 Albumin / Nucleus d4 d15 d4 d15 d4 d15 Our 3D culture allows the formation of spheroids which are functionals and polarized at long term

22 e culture de on D 3D LAP nulle Faible Characterization of the proliferation 3D en gels de collagène (PHH) Élevée Ki67 Cycline D1 BrdU D1 ne x 5mm 5mm 5mm 5mm d5 Cycline D1 / Albumin / Nucleus Ki67 / Albumin / Nucleus 1 % CyclinD1+/Alb+ cells 1 % Ki67+/Alb+ cells 5mm d Time of culture (days) d4 BrdU / Albumin / Nucleus % BrdU+/Alb+ cells 5mm Times of culture (days) Time of culture (days) PHH cultivated in 3D are highly proliferative

23 Two ways of proliferation (PHH) Proliferation Studies on 1 independant cell cultures demonstrated the ability of PHH to proliferate

24 % Proliferating cells Influence of matrix rigidity (PHH) D LAP,75mg/ml 1,5mg/ml 3mg/ml 4mg/ml Collagen gels Controlled 3D stiffness is essential for optimum proliferation

25 An innovative 3D model of proliferative and differenciated PHH Formation of polarized and functional spheroids Long term cultures ( 4 weeks) with highly differenciated hepatocyte functions Proliferation of Primary human hepatocytes Caracterisation of the proliferation : ways of proliferation during the first weeks of culture Identification of a optimal rigidity windows This model raises a major technology lock for development many pharmacological and toxicological applications and biotechnology European Patent No : Method of culturing proliferative hepatocytes, May 18 th 18

26 Équipe Dymec Dynamique du microenvironnement et cancer: structure, signal & contaminants Sophie LANGOUET Georges BAFFET Sophie ROSE Frédéric EZAN Marie CUVELLIER Nathalie THERET Maël CONAN Vincent LEGAGNEUX François TIHAO Dominique BONNIER Fida AZAR Bassil DEKKY Maxime FOLSCHETTE Cancer center Robert TURESKY Medjda BELLAMRI CRB Santé Alain FAUTREL Pascale BELLAUD Gevorg GHUKASYAN Marine SEFFAIS Roselyne VIEL Rémy LE GUEVEL Stéphanie DUTERTRE Xavier PINSON PNREST n

27 Georges