Human iPSC-derived hepatocytes in 2D and 3D suspension culture for cryopreservation and in vitro toxicity studies

Autor: Saskia Altmaier, Ina Meiser, Emilie Lemesre, Benjamin Chanrion, Rachel Steeg, Lidia Elena Leonte, Bjørn Holst, Boye Schnack Nielsen, Christian Clausen, Katharina Schmidt, Anne Marie Vinggaard, Heiko Zimmermann, Julia Christiane Neubauer, Mikkel Aabech Rasmussen
Přispěvatelé: Publica
Rok vydání: 2022
Předmět:
Zdroj: Altmaier, S, Meiser, I, Lemesre, E, Chanrion, B, Steeg, R, Leonte, L E, Holst, B, Nielsen, B S, Clausen, C, Schmidt, K, Vinggaard, A M, Zimmermann, H, Neubauer, J C & Rasmussen, M A 2022, ' Human iPSC-derived hepatocytes in 2D and 3D suspension culture for cryopreservation and in vitro toxicity studies ', Reproductive Toxicology, vol. 111, pp. 68-80 . https://doi.org/10.1016/j.reprotox.2022.05.005
Reproductive Toxicology
ISSN: 0890-6238
DOI: 10.1016/j.reprotox.2022.05.005
Popis: Hepatocytes are of special interest in biomedical research for disease modelling, drug screening and in vitro toxicology. Human induced pluripotent stem cell (hiPSC)-derived hepatocytes could complement primary human hepatocytes due to their capability for large-scale expansion. In this study, we present an optimized protocol for the generation of hepatocyte-like cells (HLCs) from hiPSC in monolayer (2D) and suspension culture (3D) for production of organoids. A protocol was initially optimized in 2D using a gene edited CYP3A4 Nanoluciferase reporter hiPSC line for monitoring the maturity of HLCs and cryopreservation of definitive endoderm (DE) cells. The protocol was optimized for microwell cultures for high-throughput screening to allow for a sensitive and fast readout of drug toxicity. To meet the increasing demand of hepatic cells in biomedical research, the differentiation process was furthermore translated to scalable suspension-based bioreactors for establishment of hepatic organoids. In pilot studies, the technical settings have been optimized by adjusting the initial seeding density, rotation speed, inoculation time, and medium viscosity to produce homogeneous hepatic organoids and to maximize the biomass yield (230 organoids/ml). To speed up the production process, cryopreservation approaches for the controlled freezing of organoids were analysed with respect to cell recovery and marker expression. The results showed that cryopreserved organoids maintained their phenotype only when derived from hepatocyte progenitors (HPs) at day 8 but not from more mature stages. The establishment of robust protocols for the production of large batches of hepatocytes and hepatic organoids could substantially boost their use in biomedical and toxicology studies.
Databáze: OpenAIRE
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