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 maintaed 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.
Acrylic Resins/chemistry* ; Animal ; Coculture ; Cytological Techniques* ; Endothelium/cytology* ; Human ; Surface Properties ; Temperature*

Human marrow mesenchymal stem cells were cultured in a medium containing glycerophosphate, ascorbic acid, and dexamethasone (Dex) on alumina ceramic discs and on tissue culture polystyrene (TCPS) dishes. Cell proliferation followed by osteogenic differentiation was observed to be equal on both culture substrata. The differentiation resulted in the appearance of bone-forming osteoblasts, which fabricated mineralized matrices on these substrata. Stem cells kept at 4degrees Cfor 24 h outside a CO2 incubator maintained a viability level of more than 90%. The regenerative cultured bone outside the incubator also maintained high alkaline phosphatase activity for several hours. These results verified that cultured bone fabricated at a cell processing center can be transported to distant hospitals for use in hard tissue repair. To date, the tissue engineered cultured bone formed on alumina ceramics in this environment have already been used in clinical situations, such as total ceramic ankle replacements.
Adult ; Aged ; Aluminum Oxide ; Bone Marrow Cells/*cytology ; Cell Differentiation ; Cell Division ; Ceramics ; Humans ; Mesenchymal Stem Cells/*cytology ; Middle Aged ; *Osteogenesis ; Research Support, Non-U.S. Gov't ; *Tissue Engineering