Confluent human aortic endothelial cells (HAECs) cultured on thermo-responsive culture dish grafted with poly (N-isopropylacrylamide) were recovered as a contiguous cell sheet. The double-layered co-culture was achieved by placing the recovered HAEC sheet onto the rat hepatocyte layer directly. The double-layered structure of HAEC and hepatocytes remained in tight contact during culture. Hepatocytes in the layered co-culture system with the HAEC sheet maintained the differentiated cell shape and the albumin expression for over 41 days of culture, whereas the functions disappeared within 10 days of culture in control hepatocytes without the HAEC sheet. The layered co-culture of hepatocytes and the HAEC sheets, which allows for the expression of differentiated functions of hepatocyte continuously, such as liver lobule, offers a major advancement in liver tissue engineering.

We have developed two novel cell co-culture system, without any on cell type combination limitation, utilizing a polymer surface which is temperature-sensitive with respect to its cell adhesion characteristics. One system involves a patterned co-culture of primary hepatocytes with endothelial cells utilizing patterned masked of the electron-beam cured, temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) by masked electron beam irradiation. Hepatocytes were cultured to confluency at 37 degrees C on these surfaces. When the culture temperature was reduced below 32 degrees C, cells detached from the PIPAAm-grafted areas without any need for trypsin. Endothelial cells were then seeded onto the same surfaces at 37 degrees C. These subsequently seeded endothelial cells adhered only to the now-exposed PIPAAm-grafted domains and could be co-cultured with the hepatocytes initially seeded at 37 degrees C in well-ordered patterns. The other system involves a double layered co-culture obtained by overlaying endothelial cell sheets of the designed shape onto hepatocyte monolayers. The endothelial cells adhered and proliferated on the PIPAAm-grafted surface, as on polystyrene tissue culture dishes at 37 degrees C. By reducing the temperature, confluent monolayers of cells detached from the PIPAAm surfaces without trypsin. Because the recovered cells maintained intact cell-cell junctions together with deposited extracellular matrix, the harvested endothelial cell sheets, with designed shapes, were transferable and readily adhered to hepatocyte monolayers. Stable double layered cell sheets could be co-cultivated. These two co-culture methods enabled long-term co-culture of primary hepatocytes with endothelial cells. Hepatocytes so co-cultured with endothelial cells maintained their differentiated functions, such as albumin synthesis for unexpectedly long periods. These novel two co-culture systems offer promising techniques for basic biologic researches upon intercellular communications, and for the clinical applications of tissue engineered constructs.

We describe a new culture system utilizing the temperature‐responsive polymer grafted surface for designing of cell position and layered tissue reconstruction. Organizing of the hepatic tissue structure by controlling the culture system, that is patterned co‐culture and layered cell sheet co‐culture achieved by moving the cultured cells from the culture surface, resulted in regulation of the hepatocyte function. The technique for cell sheet manipulation would promote the liver tissue engineering in quality.