Reproducible Preparation of Primary Rat Hepatocyte Sheets Using a Thermoresponsive Culture Dish

Hepatocyte transplantation has been utilized as a therapy for congenital metabolic liver diseases such as hemophilia and for liver function support in acute liver failure. Hepatocyte sheet technology using a thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-grafted dish is expected to provide an efficient cell transplantation method because the resulting hepatocyte sheet possesses extracellular matrix (ECM) on the basal surface, which enhances attachment to the target sites. However, the cultured hepatocytes consume large amounts of oxygen, leading to the loss of a few hepatocytes within the confluent culture sheet owing to a lack of oxygen. To circumvent this problem, this work demonstrates the shortening of diffusion distance, that is, the medium depth, to accelerate oxygen supply from the gas phase/medium interface to the cultured hepatocytes, allowing them to form a monolayer hepatocyte sheet. Incubation of hepatocytes with medium at a depth of 1.3 mm facilitates confluent culture of hepatocytes for 72 h, whereas viable hepatocytes decreased at 2.6 mm depth. Hepatocyte sheets are formed on a 0.5 μg/cm² fibronectin-physisorbed PIPAAm-grafted dish during 72 h incubation at 37°C. Detachment of the cultured hepatocyte sheet from the PIPAAm-grafted dish where the surface becomes hydrophilic at 20°C is accomplished by scraping the periphery of the sheet using a cell scraper. Furthermore, the apical side of the hepatocyte sheet can be physically grabbed using a gelatin-coated membrane, and the sheet with ECM on the basal surface can be readily transferred to the target site after melting the coated gelatin at 37°C. Both methods are beneficial for creating tissue models by layering with another type of cell sheets, and for quick transplantation, such as into the subcutaneous space and orthotopic transplantation on the surface of the liver.