Physiological oxygen tension reduces hepatocyte dedifferentiation in in vitro culture

Abstract

Primary hepatocytes cultured in vitro are a powerful tool to study the functions of hepatocytes and to evaluate the metabolism and toxicity of new drugs. However, in vitro culture of hepatocytes has proven to be very difficult. Ordinary culture conditions lead to dedifferentiation of hepatocytes, resulting in rapid change in cell morphology and significant reduction in specific cell functions. In the current study, we show that hepatocyte dedifferentiation is a rapid process under 21% O2 conditions. Hepatocytes cultured in 21% O2 undergo epithelial-to-mesenchymal transition (EMT), obtain fibroblast-like morphology, and show decreased hepatic functions. In contrast, 5% O2 is very effective in maintaining the epithelial morphology and many functions of the primary hepatocytes cultured in vitro for up to five days. These functions include albumin production, glycogen storage, LDL-uptake and CYP450-mediated drug metabolism. Furthermore, we find that 5% O2 can relieve the production of reactive oxygen species (ROS) and decrease the level of DNA damage in primary cultured hepatocytes. In addition, we also show that blocking the ERK and GSK-3β pathways can inhibit the dedifferentiation of hepatocytes to a certain extent. Lowering the oxygen tension in cell culture is easily achievable, we believe it could be combined with other methods, such as the use of small molecule cocktails and 3D culture, to maintain proliferation and functions of primary hepatocytes in vitro.

Domain‐specific distribution of gap junctions defines cellular coupling to establish a vascular relay in the retina

“…Less than 2% cross reactivity to bovine, human and pig albumin was detected. The specificity of the antibodies was determined in cultural primary mouse hepatocytes under control conditions and conditions when albumin synthesis was blocked (Guo, Xu, Lu, & Xie, ). In the retina, the staining for albumin was contained inside retinal blood vessels, which correspond to the location of the blood plasma albumin (our data; Iwase et al, ).…”

Defining Physiological Normoxia for Improved Translation of Cell Physiology to Animal Models and Humans

“…These studies in hepatocytes emphasize the need to understand monolayer oxygenation more closely. Ambient PO 2 during in vitro culture must be accurately tailored to the cell type to avoid exposing high O 2 -demanding cells to cellular hypoxia (204).…”

Cell Aggregation Culture Induces Functional Differentiation of Induced Hepatocyte-like Cells through Activation of Hippo Signaling

“…Thus, in vitro analyses of hepatocyte-specific properties are important for biomedical studies and drug development. However, because it is difficult to expand and maintain differentiated hepatocytes in culture (Desai et al, 2017;Guo et al, 2017;Khetani and Bhatia, 2008), alternative cell sources for hepatocytes are desirable for analyses of hepatic functions in vitro. As an alternative to hepatocytes, hepatocyte-like cells can be induced from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (Sancho-Bru et al, 2011;Si-Tayeb et al, 2010b).…”