3D Co-Culture of Hepatocyte, a Hepatic Stellate Cell Line, and Stem Cells for Developing a Bioartificial Liver Prototype

Abstract

A liver organoid is an in vitro reconstruction of the liver that mimics the in vivo liver microstructure and performs liver functions. Liver organoids can be used for drug testing, as a model of liver disease pathogenesis, and as a bioartificial liver prototype material to develop promising alternative therapies for liver failure. In this study, we reconstructed liver organoids using primary rat hepatocytes, a hepatic stellate cell line (LX2), human umbilical cord-mesenchymal stem cells (UC-MSCs), and human umbilical cord blood (UCB)-CD34+ hematopoietic stem/progenitor cells. Suspensions of primary rat hepatocytes, LX2 cells, UC-MSCs, and UCB-CD34+ cells were co-cultured using 11 ratio sets, and spheroid formation was evaluated for 2 days. Ratio sets with a positive liver organoid appearance were cultured in four different culture media, and after they were harvested, their viability was compared with that of a hepatocyte monoculture. Liver organoids were further analyzed for 14 days to assess albumin and urea production as well as relative gene expression. We found that a 5:1:2:2 cellular density ratio of hepatocytes:LX2 cells:UC-MSCs:UCB-CD34+ cells, respectively, and Williams E medium supplemented with platelet lysate, ITS, and dexamethasone were the most suitable conditions for liver organoid reconstruction. Expression of the albumin and GPT1 genes and CD31 in the liver organoid increased until day 14, while urea secretion increased until day 5. Liver organoids reconstructed through the 3D co-culture of primary rat hepatocytes, LX2 cells, UC-MSCs, and UCB-CD34+ cells at a specific cellular ratio using an economical medium with a simple composition maintained their functions until day 14. As a future direction, these organoids can be used to develop a bioartificial liver.

Polyethylene Terephthalate Fiber Modified with Type I Collagen as a 3D Scaffold Material for Bioartificial Liver

“…Ensuring a high-density, large-scale hepatocyte culture within the bioreactor while preserving its viability and function is pivotal for amplifying the therapeutic effectiveness of bioartificial livers [16][17][18]. Tissue-engineered three-dimensional cultures represent the evolving trend in artificial liver bioreactor technology [19,20]. Given that hepatocytes are polarized, anchorage-dependent cells that naturally proliferate within a three-dimensional network scaffold composed of diverse extracellular matrices in vivo, mimicking this native environment through the preparation of a three-dimensional porous scaffold could potentially facilitate tissue-engineered three-dimensional culture in vitro.…”

The Effects of Static and Dynamic Culture Systems on Cell Proliferation and Conditioned Media of Umbilical Cord-derived Mesenchymal Stem Cells

“…Static 2D culture is feasible for small-scale MSC expansion; however, this method is costly and quite challenging to adopt for large-scale cell production (Mizukami et al, 2018). The 3D culture system is a promising method for scaling up cell production using various types of bioreactors (Tsai et al, 2019;Sibuea et al, 2020;Nadhif et al, 2020). Dissolved oxygen in conventional culture can be maintained by limiting media depth.…”