Enhanced Functionality of Human Hepatocytes using the Oxygen Permeable Culture Plate for Optimized Oxygen Supply in Co-culture Systems with Culture Inserts

Abstract

Co-culture systems with culture inserts are commonly used to study cell-cell interactions mediated by soluble factors. However, the inclusion of culture inserts reduces the air-medium interface for cells cultured in the bottom wells, potentially leading to insufficient oxygen supply. This study investigated the impact of culture conditions on human hepatocytes when cultured with culture inserts. The results showed that using culture inserts increased cell death and decreased mitochondrial activity in hepatocytes grown at the bottom wells of standard multi-well plates. Additionally, bile canaliculi formation was impaired in hepatocytes derived from a chimeric mouse with a humanized liver. To address these limitations, we replaced the standard culture plates with InnoCell^TM T-plates, which facilitate oxygen supply from the bottom of the wells, and assessed cellular functions. Using the InnoCell^TM T-plate significantly reduced cell death, increased the presence of mitochondria with high membrane potential, and restored bile canaliculi formation. Furthermore, CYP3A4 activity, one of the key drug metabolizing enzymes of hepatocytes, was notably enhanced. These findings demonstrate that the reduced functionality of hepatocytes in co-culture systems with culture inserts can be mitigated by improving oxygen delivery through the use of InnoCell^TM T-plates. This approach supports improved cell survival and enables more accurate observation of cell-cell interactions. Co-culture systems with culture inserts are increasingly recognized as part of Microphysiological Systems (MPS). The above approach will lead to the specification of the alternatives to animal experiments and the consideration points to be made in MPS.