Cell sheets prepared via gel-sol transition of calcium RGD-alginate.

UNLABELLED

The formation of layered tissues through the use of cell sheet harvesting has recently emerged as a potentially viable approach for clinical tissue engineering applications. Since the demonstration of effective cell sheet formation using temperature responsive substrates, a number of different stimuli have been utilized to facilitate cell sheet detachment. Each approach has differing advantages and disadvantages. Herein we demonstrate the ability of calcium alginate hydrogels to function as an effective substrate for cell sheet formation. By conjugating the integrin binding peptide sequence RGD to the alginate and crosslinking it with calcium ions, the hydrogel formed supported the attachment and growth of both 3T3 fibroblasts and human corneal epithelial cells (HCECs). Once the cells had grown to confluence, exposing the calcium alginate to the chelating agent citrate caused the release of a consolidated cell sheet. When HCEC sheets were stacked, the cell layers adhered to each other and the cells began to integrate.

STATEMENT OF SIGNIFICANCE

Herein we describe a simple and inexpensive process for creating cell sheets using the ability of calcium alginate hydrogels to be dissolved under mild conditions. The alginate was first modified to possess cell attachment sites and in this demonstration of feasibility we employed an RGD peptide, but other peptides could be readily attached. The modified alginate was then formed into stable hydrogels through a drying and re-hydration step, and used as a substrate to grow confluent cell sheets of human corneal epithelial cells and 3T3 fibroblasts as examples. The cell sheets were released through chelating the calcium using citrate. The cell-cell connections were retained following this release and the cell sheets interconnect and grew following being stacked.