Tissue-engineered Cell Sheets Research Articles

Creation of designed shape cell sheets that are noninvasively harvested and moved onto another surface.

We developed a novel method to obtain designed shape cell sheets for tissue engineering. Shaping of cell sheets were achieved by the use of poly(N-isopropylacrylamide) (PIPAAm) and poly(N,N'-dimethylacrylamide) (PDMAAm) for temperature-responsive cell adhesive and cell nonadhesive domains, respectively. These polymers were covalently grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation with mask patterns. At 37 degrees C, human aortic endothelial cells (HAECs) attached, spread, and proliferated to make a monolayer only on PIPAAm-grafted domains. HAECs did not adhere on PDMAAm-grafted domains for more than 1 month even under the serum-supplemented condition. By reducing the culture temperature below 32 degrees C, PIPAAm changed to hydrophilic and HAEC sheets were detached from PIPAAm-grafted surfaces without any need of an enzyme such as trypsin. Cell-cell junctions were retained in the recovered cell sheets and easily moved to virgin TCPS dishes with the aid of hydrophilically modified polyvinylidenefluoride membranes as a supporter during the transfer. Moved cell sheets rapidly adhered onto the dish surfaces, and the supporter was easily peeled off from the cell layers. HAEC sheets transferred to new dishes revealed the identical shape and size to those before transfer. This novel technique is the only way to create, harvest, and transfer designed shape cell sheets and would have promising applications in tissue engineering.

Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces.

Bovine aortic endothelial cells were cultured on surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), in the presence of serum. Cells adhered, spread, proliferated, and reached confluency as observed on ungrafted tissue culture polystyrene dishes. A decrease in culture temperature released cells only from the grafted surfaces without enzymatic or ethylenediaminetetraacetic acid treatment. Upon lowering temperature, the culture surfaces changed from hydrophobic to hydrophilic owing to the hydration of grafted PIPAAm and thus weakened the cell attachment to the dishes. Released cells maintained cell-cell junctions composing monolayer cell sheets. Immunoblotting and immunofluorescence microscopy revealed that fibronectin (FN) was deposited and accumulated on the grafted surfaces during the culture. Furthermore, the deposited FN matrix adhering to cell sheets was also recovered from temperature-responsive surfaces by low-temperature treatment, while trypsin treatment destroyed the matrix. The recovery of FN by low-temperature treatment was as high as by physical scraping with a rubber blade. Temperature-responsive surfaces can provide a novel method to use cultured confluent cell sheets for tissue engineering, and also to elucidate structure and function of deposited extracellular matrix during cell culture.