Development of Dry Preserved Fibroblast Cell Sheets for Regenerative Medicine to Promote Wound Healing

Abstract

To popularize cell sheet transplantation therapy, it is necessary to develop an easy‑to‑use cell sheet preservation method. Because cell viability determines therapeutic efficacy, cryopreservation, with a high viability rate, has been considered for preservation. We developed dry, preserved, multilayered fibroblast cell sheets（dry sheets） as a novel cell sheet preservation method. Dry sheets consist of dead cells with damaged cell membranes. However, dry sheets using allogeneic cells promote angiogenesis and wound healing in a full‑thickness skin defect model of diabetic mice through a novel mechanism of releasing intracellular growth factors by rehydration. The growth factors released from dry sheets had physiological activity, mainly through the action of fibroblast growth factor‑2（FGF‑2），as demonstrated by inhibition experiments using FGF‑2 neutralizing antibodies. Growth factors in dry sheets were stable for at least one month when stored in a refrigerator. In our investigation of the optimal cell source for human dry sheets, fibroblasts were considered the ideal cell source for because of their high FGF‑2 content, rapid cell proliferation, easy handling and availability, and low culture cost. Compared to conventional cell sheets, dry sheets are easier to handle and preserve, which is promising for treating cutaneous ulcers and performing tissue reinforcement during surgery.