Hepatocyte and Non-Parenchymal Cell In Vitro Co-Cultivation Mimics the Native Hepatic Microenvironment

The native hepatic microenvironment is quite complex and very difficult to simulate through in vitro modelling. This mix of cells includes hepatocytes, non-parenchymal cells, immune cells, the extracellular matrix (ECM), and many chemical factors. Non-parenchymal cells (NPC) are a category of liver cells that include Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC), hepatic stellate cells (HSC), biliary cells, and lymphocyte (Seo & Jeong, 2016).

Each of these cells provides a specific and necessary function for the liver to property filter toxins and regulate most chemicals in the body.

Several studies have been able to monoculture hepatocytes in 2D and 3D configurations to study long-term liver function, hepatotoxicity, drug effects, etc. (Baze et al., 2018), showing evidence that primary human hepatocytes (PHH) cultured in 3D-spheroids lead to some success as a model system for liver toxicity screenings and liver function testing although they degrade over a short period of time, given that hepatocytes are unable to maintain their dedifferentiated phenotype in monoculture and increase ECM stiffness (Godoy et al., 2013). However, 3D spheroid co-culture of PHH and NPC have shown to be a stronger in vitro model for liver function testing. This is due to function based on the cell-to-cell interactions that occur in the liver (Bhatia et al., 1999). This intercellular communication is not completely different between in vivo and in vitro environments, considering that all cell types need one another to communicate and maintain a healthy looking and functioning organ, tissue or culture.

That is why cell-to-cell interactions are especially important when creating an in vitro microenvironment. This is apparent when co-culturing PHH and hepatic NPC that need one another to differentiate and create a sustainable model. Some results have shown that co-culture allows many models to last for weeks, compared to 14-days average monoculture, enhanced liver-specific functions, hepatocyte differentiated phenotype and increased functionality (Bhatia et al., 1999). This is due to the complex cell-to-cell interactions between the cell types which organize signaling pathways and coordinate growth and proliferation of different cell types, organization, and function. Recreating these connections through co-culture in a 3D model could replicate the signaling network and, therefore, mimicry and improve the functionality of hepatocytes, such as increased albumin secretion, among other functions (Baze et al., 2018).

In this regard, one major obstacle is determining the correct proportion of hepatocytes to non-parenchymal cells in culture. Although, hepatocytes constitute 60% of liver cells, as they are the basic structural component of the organ, and non-parenchymal cells constitute 40% of the remaining liver cells (Vekemans & Braet, 2005); in vitro models do not necessarily have to follow the in vivo cell ratio in order to mimic the liver functionality necessary for their use in studies. However, it is crucial to establish an adequate cell ratio and keep it to a minimum to allow for native cell patterning without overcrowding. To this end, different studies have been developed demonstrating the potential of in vitro patterning with different ratios, and depending on the final objective, this ratio may vary.

In this regard, one major obstacle is determining the correct proportion of hepatocytes to non-parenchymal cells in culture. Although, hepatocytes constitute 60% of liver cells, as they are the basic structural component of the organ, and non-parenchymal cells constitute 40% of the remaining liver cells (Vekemans & Braet, 2005); in vitro models do not necessarily have to follow the in vivo cell ratio in order to mimic the liver functionality necessary for their use in studies. However, it is crucial to establish an adequate cell ratio and keep it to a minimum to allow for native cell patterning without overcrowding. To this end, different studies have been developed demonstrating the potential of in vitro patterning with different ratios, and depending on the final objective, this ratio may vary.

Overall, the co-culture of primary human hepatocytes and liver non-parenchymal cells shows a remarkable potential in creating a functional liver model for a variety of testing.

Hepatocytes alone are not always efficient enough in monoculture to sustain some long-term tests of liver functions; since both cell types together are important not only for each other, but also for understanding communication pathways and liver functionality yet to be elucidated. Thereby, co-culture models have the potential to enable further study of diseased liver models, tissue constructs for medical use, cell communication, and organ development.

References used:

Baze, A., Parmentier, C., Hendriks, D. F. G., Hurrell, T., Heyd, B., Bachellier, P., Schuster, C., Ingelman-Sundberg, M., & Richert, L. (2018). Three-Dimensional Spheroid Primary Human Hepatocytes in Monoculture and Coculture with Nonparenchymal Cells. Tissue Engineering - Part C: Methods, 24(9), 534–545. https://doi.org/10.1089/ten.tec.2018.0134

Bhatia, S. N., Balis, U. J., Yarmush, M. L., & Toner, M. (1999). Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. In FASEB J (Vol. 13). Godoy, P., Hewitt, N. J., Albrecht, U., Andersen, M. E., Ansari, N., Bhattacharya, S., Bode, J. G., Bolleyn, J., Borner, C., Böttger, J., Braeuning, A., Budinsky, R. A., Burkhardt, B., Cameron, N. R., Camussi, G., Cho, C.-S., Choi, Y.-J., Craig Rowlands, J., Dahmen, U., … Hengstler, J. G. (2013). Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Archives of Toxicology, 87(8), 1315–1530. https://doi.org/10.1007/s00204-013-1078-5 Seo, W., & Jeong, W. il. (2016).

Hepatic non-parenchymal cells: Master regulators of alcoholic liver disease? In World journal of gastroenterology (Vol. 22, Issue 4, pp. 1348–1356). Baishideng Publishing Group Inc. https://doi.org/10.3748/wjg.v22.i4.1348 Vekemans, K., & Braet, F. (2005). Structural and functional aspects of the liver and liver sinusoidal cells in relation to colon carcinoma metastasis. In World Journal of Gastroenterology (Vol. 11, Issue 33, pp. 5095–5102). Baishideng Publishing Group Co. https://doi.org/10.3748/wjg.v11.i33.5095

• By CYTES BIOTECHNOLOGIES, SL.
• 15/07/2021
• microenvironment, Primary Human Hepatocytes, hepatic, cellinvitro