P values of 0.05 or smaller were considered significant. We noticed that cells grown in media supplemented with HS, compared to FBS, had reduced Selleckchem LY294002 growth rates, and show contact inhibition
after approximately 7 days. From this point on, cells could be kept in confluent monolayers without further subculturing. HS cells did not pile up or detach from the culture plate. Cells in HS media could be maintained in monolayers for at least 2 months with regular media changes. Determination of cell numbers over a 3-week period confirmed that cell numbers did not increase after approximately 7 days in HS media (Fig. 1A). Morphology of cells cultured in HS-containing media changed dramatically during the first 3 weeks (Fig. 1B-D). After approximately 21 days, these cells had many morphological features of cultured primary hepatocytes. They formed tightly packed monolayers that were strongly attached to the tissue culture substrate, with a pavement-like organization. Buparlisib datasheet Similar to primary hepatocytes in culture (Fig. 1D), HS cells were mono- or binucleated and had a granular appearance (Fig. 1C). The size of the cells also increased. These changes were most obvious after more than 21 days of culturing in HS media. To further investigate whether cells that were cultured in HS-supplemented media underwent differentiation to a hepatocyte-like cell,
we first examined transcript levels of hepatocyte differentiation markers alpha-1-antitrypsin (α1AT), ALB, and low-density lipoprotein receptor (LDL-R). No significant
changes were observed after culturing in HS for 7 days. However, after 21 days, messenger RNA (mRNA) levels of both α1AT (Fig. 2A) and ALB (Fig. 2C) were significantly higher than in FBS-cultured cells and were comparable to those in cultured human primary hepatocytes. We did not find an increase in LDL-R mRNA as a result of culturing in HS (Fig. 2B). We used quantitative ALB ELISA to confirm that the increase in ALB mRNA resulted in increased ALB secretion (Fig. MCE 2D). In line with mRNA levels, after 7 days in HS, no significant changes in ALB secretion were observed; however, after 21 days in HS, ALB secretion had increased approximately 6-fold. The presence of tight and adherens junctions are well-recognized features of hepatocytes in vivo and linked to increased liver-specific functionality in vitro; loss of cell-junction components is commonly associated with metastatic cell types. Cells that were grown in HS-supplemented media for 14 days or more became very strongly attached to the plate and to each other. They were difficult to release by trypsinization. Cells that were eventually released remained organized in large clumps, indicating strong cell–cell contacts. We determined the mRNA levels of the two main tight junction components, claudin-1 and occludin, and the chief component of cell adherens junctions, e-cadherin. In HS-supplemented media, Huh7.