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

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.

3D co-culture; Hepatocyte; Liver function; Liver organoid; Stem cells

The liver is an important organ that performs many functions, such as protein synthesis, drug biotransformation, and detoxification (Li et al., 2017; Uygun et al., 2017). While it has a strong regenerative ability, massive liver damage can lead to liver failure (Miyaoka and Miyajima, 2013; Mazza et al., 2017). Liver transplantation, the primary available therapy for liver failure, has many shortcomings, such as difficulty in finding suitable donors, a high cost, and the need for the long-term use of immunosuppression drugs, leading to large numbers of patients awaiting liver transplantation (Li et al., 2017).

Many alternative therapies have been developed to overcome these issues, including cell transplantation using hepatocytes and stem cells, the use of artificial livers, and, most recently, the use of bioartificial livers (BALs). Hepatocyte transplantation requires long-term immuno-suppression and is further limited by small numbers of donors, isolation difficulties, and low hepatocyte engraftment rates (Vacanti and Kulig, 2014). Mesenchymal stem cell (MSC) transplantation, despite being relatively safer, also has the potential to trigger thrombosis in some cases (Zheng et al., 2013). Artificial livers can be developed with a synthetic device using an extracorporeal perfusion system, but they are functionally limited to the removal of toxins. (Zhang et al., 2018). BALs combine an artificial liver with hepatocytes, using primary hepatocytes from pig livers or hepatoblastoma cell lines; BALs have a short lifespan because of limited primary hepatocyte proliferation and differences between the functions of hepatoma cell lines and hepatocytes (Vacanti and Kulig, 2014). These limitations require the development of new technologies overcome the remaining challenges facing existing BAL prototype materials, such as imparting equivalent liver functions, to meet the high demand for liver transplantation.

A liver organoid is defined as an in vitro liver reconstruction that mimics the microstructure and function of the liver in vivo. Reconstructing liver organoids requires cell components that replicate the liver’s in vivo microenvironment as well as culture techniques that can support long-term liver function. Techniques for the co-culture of hepatocytes with non-parenchymal cells are continuously being developed to obtain a 3D microenvironment and structure that mimic the liver’s in vivo microenvironment. Co-culture is carried out to obtain a 3D microenvironment, while culture methods are used to obtain a 3D structure. The culture medium must also be refined to ensure an optimal hepatocyte microenvironment (Monckton and Khetani, 2018).

The liver is a complex unit consisting mainly of parenchymal cells, which are hepatocytes, and non-parenchymal cells, including Kupffer cells, hepatic stellate cells (HSCs), and endothelial cells (Vacanti and Kulig, 2014). Hepatocytes are co-cultured with an HSC line (e.g., LX2 cells), umbilical cord-mesenchymal stem cells (UC-MSCs), and umbilical cord blood (UCB)-CD34 hematopoietic stem/progenitor cells with the aim of producing a microenvironment matching the liver’s in vivo microenvironment. A previous study reported the formation of a vessel-like structure in a co-culture of hepatocytes with endothelial cells and HSCs, with increased urea and albumin secretion as well as Cyp3A4 expression (Wang et al., 2018). Meanwhile, in a co-culture of MSCs and an endothelial cell line (HUVEC EA. hy 926), increases in EGF-A expression and cell viability and the formation of a tubular structure in the presence of CD31 expression were reported (Arutyunyan et al., 2016). MSCs have strong proliferation and differentiation abilities, allowing them to produce a paracrine effect that supports the endogenesis required by endothelial cell progenitors (CD34⁺) for neovascularization and biliary duct formation.

In this study, we combined four cellular components that have not been used before in combination for liver organoid reconstruction. We also used four culture media with basic supplementations to ensure that they could be applied in areas with limited resources. An optimal density ratio of the four cellular components and an optimal culture medium are required for liver organoid reconstruction to ensure that viability and liver function are maintained for an extended period of time. The results of this study are expected to provide proof of concept that with an optimal density ratio of hepatocytes, LX2 cells, MSCs, and CD34⁺ cells and an optimal medium, it is possible to reconstruct liver organoids. 

Our results indicate the successful reconstruction of liver organoids using an optimum ratio of hepatocytes:LX2cells:UC-MSCs:UCB-CD34⁺ cells. We also identified Williams E medium supplemented with platelet lysate, ITS, and dexamethasone as the optimum culture medium for reconstructing liver organoids, which is a simple and economical medium. The combination of these cellular components and culture medium provides a suitable microenvironment that mimics the in vivo liver microenvironment. This optimum ratio with a simple and economical medium can be used to develop and maintain liver organoid function for 14 days. As a future direction, these organoids can be used in BALs, although the organoids should be exchanged with new organoids every 14 days.

    This study was supported by a grant from the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia, PUSN 2018, contract no. 554/UN2.R3.1/HKP05.00/2018, and the subsequent grant Penelitian Pengembangan 2019, contract number NKB-1804/UN.R3.1/HKP.05.00/2019.  Publishing of this article is supported by Ministry of Finance, LPDP, to defray the publication cost.

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