3D-spheroid primary human hepatocytes in mono- and co-culture with non-parenchymal cells.
Fiche publication
Date publication
août 2018
Journal
Tissue engineering. Part C, Methods
Auteurs
Membres identifiés du Cancéropôle Est :
Pr BACHELLIER Philippe, Pr HEYD Bruno
Tous les auteurs :
Baze A, Parmentier C, Hendriks DFG, Hurrell T, Heyd B, Bachellier P, Schuster C, Ingelman-Sundberg M, Richert L
Lien Pubmed
Résumé
Recent advances in the development of various culture platforms are promising for achieving more physiologically relevant in vitro hepatic models using primary human hepatocytes (PHH). Previous studies have shown the value of PHH 3D-spheroid models, cultured in low cell number (1330-2000 cells/3D-spheroid), to study long-term liver function as well as pharmacological drug effects and toxicity. Here we report that only plateable PHH aggregate and form compact 3D-spheroids with a success rate of 79%, with 96% reproducibility. Out of 3D-spheroid forming PHH, 65% were considered stable (less than 50% ATP decrease) over the subsequent 14 days of culture, with reproducibility of a given PHH lot being 82%. We also report successful co-culturing of PHH with human liver non-parenchymal cells (NPC). Crude P1c-NPC fractions were obtained by low centrifugation of the PHH supernatant fraction followed by a few-days in culture before harvesting and cryopreservation. At 3D-spheroid aggregation of PHH/P1c-NPC (2:1 ratio 3D-spheroids), liver sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells were successfully integrated and remained present throughout the subsequent 14 culture-period as revealed by the mRNA expression markers and immunostaining. Increased mRNA expression of ALB, APOB, CYP3A4 and of albumin secretion compared to PHH 3D-spheroid monocultures highlighted that in a 3D-spheroid co-culture configuration with, PHH functionality is increased. We thus achieved the development of a more integrated co-culture model system requiring low cell numbers, of particular interest due to the scarcity of human liver NPC.
Mots clés
3D spheroids, coculture, nonparenchymal cells, primary human hepatocytes
Référence
Tissue Eng Part C Methods. 2018 Aug 13;: