Development of a novel vitrification method for chondrocyte sheets

Abstract

BackgroundThere is considerable interest in using cell sheets for the treatment of various lesions as part of regenerative medicine therapy. Cell sheets can be prepared in temperature-responsive culture dishes and applied to injured tissue. For example, cartilage-derived cell sheets are currently under preclinical testing for use in treatment of knee cartilage injuries. The additional use of cryopreservation technology could increase the range and practicality of cell sheet therapies. To date, however, cryopreservation of cell sheets has proved impractical.ResultsHere we have developed a novel and effective method for cryopreserving fragile chondrocyte sheets. We modified the vitrification method previously developed for cryopreservation of mammalian embryos to vitrify a cell sheet through use of a minimum volume of vitrification solution containing 20% dimethyl sulfoxide, 20% ethylene glycol, 0.5 M sucrose, and 10% carboxylated poly-L-lysine. The principal feature of our method is the coating of the cell sheet with a viscous vitrification solution containing permeable and non-permeable cryoprotectants prior to vitrification in liquid nitrogen vapor. This method prevented fracturing of the fragile cell sheet even after vitrification and rewarming. Both the macro- and microstructures of the vitrified cell sheets were maintained without damage or loss of major components. Cell survival in the vitrified sheets was comparable to that in non-vitrified samples.ConclusionsWe have shown here that it is feasible to vitrify chondrocyte cell sheets and that these sheets retain their normal characteristics upon thawing. The availability of a practical cryopreservation method should make a significant contribution to the effectiveness and range of applications of cell sheet therapy.

Efficient Production of Live Offspring from Mouse Oocytes Vitrified with a Novel Cryoprotective Agent, Carboxylated ε-poly-L-lysine

“…It seems that COOH-PLL has low permeability, because most of the FITC-tagged COOH-PLL was not observed inside the oocyte cytoplasm. Results from vitrification of chondrocyte sheets suggest COOH-PLL is non-permeable CPA [22]. It is unclear how COOH-PLL is involved in the improvement of oocyte vitrified with EG.…”

Cryopreservation method for spheroids and fabrication of scaffold-free tubular constructs

“…However, the cryopreservation solution remaining inside the spheroids may affect cell viability and spheroid fusion. Maehara et al reported a vitrification method for chondrocyte cell sheets [22]. The cell sheet was washed several times in equilibration solution and washing solution to help diffuse the cryopreservation solution inside the cell sheets after thawing to remove the cryopreservation solution and maintain osmotic pressure.…”

Vitrification and storage of oral mucosa epithelial cell sheets

“…Cell sheet technology has been developing for the past decade to target the delivery of cell sheets onto damaged organs. Success of both cell sheet engineering and transplantation have been reported in numerous articles; however, only three recent publications have reported the vitrification of cell sheets (Maehara et al, ; Ohkawara et al, ; Tani et al, ). Little is known about cell sheet vitrification; more studies are needed to increase our knowledge and to improve the vitrification methodology of cell sheets.…”