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3D Co-Culture with Vascular Cells Supports Long-Term Hepatocyte Phenotype and Function In Vitro

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Abstract

Progress in liver tissue engineering depends on the ability to reliably culture hepatocytes in vitro. In this study, we designed an environment that can help support liver-specific functions of primary hepatocytes for long enough periods of time to use them in therapeutic devices and in vitro modeling. This was accomplished by encapsulating hepatocytes with endothelial cells (ECs) and pericytes in a cell-adhesive and enzymatically degradable poly(ethylene glycol) (PEG) hydrogel scaffold. We used these hydrogels to investigate the long-term effects of 3D co-culture of hepatocytes with non-parenchymal ECs and pericytes in the context of vascular networks. We found that 3D co-culture of hepatocytes with tubule-forming ECs and pericytes leads to the development of robust microvascular tubules with close approximation to hepatocytes, similar to structures found in vivo. Furthermore, hepatocytes help support vasculogenesis and long-term tubule stability. We show that 3D co-culture of hepatocytes with cells in these networks enhances and retains hepatocyte function and phenotype. Hepatocytes in our constructs were able to synthesize 6.3 ± 1.1 pg albumin/hepatocyte/day at day 7 and increased over 3-fold to 22.6 ± 1.5 pg albumin/hepatocyte/day by day 28. Other essential liver functions, like urea secretion and cytochrome P450 activity, were also retained in our constructs for at least 4 weeks. Finally, we found that some of the hepatocytes in these organoids expressed the proliferation marker, Ki-67. These results are promising for the development of new liver disease treatments, organ engineering, and drug-testing models.

Lay Summary

Hepatocytes are the parenchymal cells of the liver, and they are responsible for performing many essential liver functions. When they are isolated from the body and cultured without the correct biochemical cues, they tend to lose their liver-specific functions and dedifferentiate into fibroblast-like cells. Advancement in tissue engineering liver disease therapies and in vitro drug-testing models depends on the development of hepatocyte culture methods that can maintain hepatocyte phenotype and function. Here, we tailored a polymeric hydrogel in which we can 3D co-culture hepatocytes with stabilizing non-parenchymal cells and achieve organ-level albumin synthesis as well maintenance of urea secretion and cytochrome P450 enzymatic activity for at least a month.

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Acknowledgements

AZU was supported by the National Science Foundation Graduate Research Fellowship.

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    Asli Z. Unal, Sydney E. Jeffs & Jennifer L. West

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Unal, A.Z., Jeffs, S.E. & West, J.L. 3D Co-Culture with Vascular Cells Supports Long-Term Hepatocyte Phenotype and Function In Vitro. Regen. Eng. Transl. Med. 4, 21–34 (2018). https://doi.org/10.1007/s40883-018-0046-2

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