Oxygen-permeable membrane-based direct oxygenation remarkably enhances functions and gene expressions of rat hepatocytes in both 3D and sandwich cultures

• Direct oxygenation meeting OCR remarkably enhanced hepatocytes maintenance and their functions.
• PDMS membranes could meet the intensive cellular requirement for oxygen.
• Microporous ePTFE membranes greatly improved the maintenance of hepatocytes in 3D culture.
• Sandwich culture with Matrigel is a promising culture model for hepatocytes.

Although there have been remarkable progresses in hepatocytes cultures in terms of mimicking microenvironments of in vivo liver, oxygen supply is still a critical issue. In this study, we investigated the effect of direct oxygenation through oxygen-permeable membranes on functionalities of hepatocytes in two widely accepted advanced culture models, sandwich culture and 3D culture. Rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes for 14 days in monolayer culture, sandwich culture with Matrigel and 3D culture with microporous expanded polytetrafluoroethylene (ePTFE) membranes in the presence and absence of direct oxygenation from the other side of the membranes. The present results showed remarkable enhancement of hepatocytes duration and their functions by oxygen transfer through PDMS membranes in all these three cultures. The hepatocytes cultured in sandwich with oxygen exhibited extended survival and highest maintenance of metabolic activities, such as albumin productivity and Cyp1a1/2 activity. Additionally, the expression levels of various drug-metabolism genes, as examined by PCR arrays, were also closest to those of freshly isolated hepatocytes. As the cellular maintenance has been greatly improved by microporous ePTFE membranes, the hepatocytes in 3D culture performed increased functions that comparable to those in sandwich culture. This study clearly illustrates that oxygenation is a critical factor to be considered in optimization of the microenvironments of hepatocytes cultures.