Three-dimensional co-culture of human hepatocytes and mesenchymal stem cells: improved functionality in long-term bioreactor cultures

Abstract

The development of human cell models that can efficiently restore hepatic functionality and cope with the reproducibility and scalability required for preclinical development poses a significant effort in tissue engineering and biotechnology. Primary cultures of human hepatocytes (HHs), the preferred model for in vitro toxicity testing, dedifferentiate and have short-term viability in two-dimensional (2D) cultures. In this study, hepatocytes isolated from human liver tissue were co-cultured with human bone marrow mesenchymal stem cells (BM-MSCs) as spheroids in automated, computer-controlled, stirred-tank bioreactors with perfusion operation mode. A dual-step inoculation strategy was used, resulting in an inner core of parenchymal liver tissue with an outer layer of stromal cells. Hepatocyte polarization and morphology as well as the mesenchymal phenotype of BM-MSCs were maintained throughout the culture period and the crosstalk between the two cell types was depicted. The viability, compact morphology and phenotypic stability of hepatocytes were enhanced in co-cultures in comparison to monocultures. Gene expression of phase I and II enzymes was higher and CYP3A4 and CYP1A2 activity was inducible until week 2 of culture, being applicable for repeated-dose toxicity testing. Moreover, the excretory activity was maintained in co-cultures and the biosynthetic hepatocellular functions (albumin and urea secretion) were not affected by the presence of BM-MSCs. This strategy might be extended to other hepatic cell sources and the characterization performed brings knowledge on the interplay between the two cell types, which may be relevant for therapeutic applications. Copyright © 2015 John Wiley & Sons, Ltd.

Three‐dimensional liver‐derived extracellular matrix hydrogel promotes liver organoids function

“…Previous studies demonstrated that the co‐culture of hepatocytes with other cell types such as sinusoidal cells, MSCs, and fibroblasts improved maintenance of liver cell function. 3D matrices composed of natural collagen or synthetic biomaterials have been used to culture hepatocytes .…”

3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases, Drug Metabolism, and Toxicity: Emerging Culture Paradigms and Applications

“…In addition, cells in this study retained their RNA expression levels of various phase I (CYP1A2, CYP2C9, and CYP3A4) and phase II enzymes (GSTA1 and UGT2B7). Coculturing PHH in stirred bioreactors with an outer layer of bone marrow mesenchymal stem cells have been shown to further increase the expression of these cytochrome P450s (CYPs), whereas functional parameters (urea and albumin) were not affected . While these results are promising regarding the molecular phenotypes and activities of the cultured cells, bioreactors require large numbers of cells and form spheroids of heterogeneous sizes, which complicates analyses and is incompatible with high‐throughput testing of different conditions.…”

Concise Review: Liver Regenerative Medicine: From Hepatocyte Transplantation to Bioartificial Livers and Bioengineered Grafts

“…Coculture with mesenchymal stem cells (MSCs) provides primary human hepatocytes with direct structural and paracrine trophic support, resulting in improved viability and function . Another strategy that may improve hepatocyte functionality is 3D culture . Aggregation into organoid‐like structures has been tested in primary hepatocytes and stem cell‐derived HLCs with and without MSC coculture, yielding promising results both in terms of functionality and engraftment .…”