Human SLC10A1 Stable Cell Line - Huh7.5.1

It is estimated that approximately 2 billion people worldwide are infected with the hepatitis B virus (HBV). Although HBV is highly infectious within the body, research into the virus has long been hindered by the lack of an in vitro culture system that is readily susceptible to infection. The SLC10A1 gene (Solute Carrier Family 10 Member 1) encodes the sodium/taurocholate co-transporting polypeptide (NTCP), a key protein located on the basolateral membrane of hepatocytes. SLC10A1 is responsible for the uptake of bile acids from the bloodstream into hepatocytes and is critical for the enterohepatic circulation of bile acids as well as lipid metabolism. Overexpression of the NTCP bile acid transporter in hepatocarcinoma cells has been shown to enhance the efficiency of HBV infection. Here, researchers report a hepatocarcinoma cell culture system capable of supporting HBV infection without the use of dimethyl sulfoxide (DMSO). They first overexpressed NTCP in Huh7.5 cells and subsequently cultured these cells in a medium supplemented with human serum (HS)-rather than fetal bovine serum (FBS)-to induce differentiation. The results demonstrated that the human serum-supplemented culture environment significantly enhanced the susceptibility of Huh7.5-NTCP cells to HBV infection. Furthermore, culture in human serum elevated the expression levels of hepatocyte differentiation markers-such as albumin secretion-in Huh7.5-NTCP cells to levels comparable to those observed in primary human hepatocytes. The N-glycosylation modification of the NTCP protein, induced by the human serum culture, may play a facilitative role in the process of viral entry into the cells.

Here, researchers determined the optimal duration required for Huh7.5-NTCP cells to complete differentiation in a culture medium supplemented with HS. Prior to HBV infection, levels of HBV pgRNA increased as the differentiation period lengthened, reaching a peak after 14 to 21 days of differentiation in the HS-supplemented medium (Figure 1A). Subsequently, the researchers utilized a NanoLuc-recombinant virus and a NanoLuc luminescence assay as surrogate markers for the early stages of HBV infection. The results demonstrated that luminescence intensity reached its maximum when cells were infected with HBV after having undergone 21 days of differentiation in the HS-supplemented medium (Figure 1B). These findings indicate that culturing cells in an HS-supplemented medium for 14 to 21 days prior to HBV infection constitutes the optimal condition for achieving enhanced HBV infection. The researchers also employed an ELISA method to quantify albumin secretion-a commonly used marker for the differentiation status and viability of primary human hepatocytes (PHHs). Upon culturing Huh7.5-NTCP cells in the HS-supplemented medium, albumin secretion increased significantly, approaching the secretion levels observed in plated PHHs as well as PXB cells (human hepatocytes isolated from humanized chimeric liver mice and cultured in vitro) (Figure 1C). During the initial phase of culture with HS supplementation (the first 7 days), albumin secretion exhibited an upward trend. Subsequently, this high level of albumin secretion was sustained throughout the entire 28-day culture period (Figure 1C). These results suggest that the HS-supplemented medium is capable of inducing the differentiation of the Huh7.5-NTCP hepatocarcinoma cell line toward a hepatocyte-like phenotype.

Figure 1. Enhancement of HBV replication and expression of hepatocyte markers in Huh7.5-NTCP cells cultured in human serum. (Le C, et al., 2021)