Abstract
Here, we aimed to enhance rat hepatocyte functions and durations in culture through improving both oxygen supply and its actual concentrations at the cell level. Rat primary hepatocytes were cultured in sandwich culture method with Matrigel on PDMS (Polydimethylsiloxane) membrane under various O2 concentrations (20%-O2 (+), 10%-O2 (+), 5%-O2 (+)) to investigate their effects on the performances of culture. In parallel, TCPS (Tissue culture treated-polystyrene) cultures were carried out as control groups (20%-O2 (-), 10%-O2 (-), 5%-O2 (-)).
Cellular level O2 tensions (pO2) on PDMS membranes were almost at the equilibrium concentrations in the incubator: 18-20%, 9-10% and 4-5% under the cultures of 20%-O2 (+), 10%-O2 (+) and 5%-O2 (+), which were remarkably lower on TCPS surfaces. HIF staining confirmed that hepatocytes were normoxic under 20%-O2 (+) and 10%-O2 (+) but hypoxia under other cultures. Albumin secretion increased greatly under 10%-O2 (+) from Day 2 and maintained at the highest level throughout the culture. CYP1A1/2 activity also increased greatly from Day 8 under 10%-O2 (+) and 5%-O2 (+) and maintained at a higher level on Day 14 under 10%-O2 (+). PCR array analysis illustrated that expression levels of various drug-metabolism genes under 20%-O2 (+) and 10%-O2 (+) were closest to those seen in the isolated rat liver before inoculation. These results demonstrate that it is important to satisfy cellular oxygen demand at in vivo-like physiological oxygen concentrations in in vitro hepatocyte culture and oxygen permeable membrane provide a simple methodology to realize this condition for in vitro toxicological research.
