The Possibility of Long‐term Cryopreservation of Cultured Dermal Substitute

Abstract

Allogeneic cultured dermal substitute (CDS) was prepared by cultivating fibroblasts on a two-layered spongy matrix of hyaluronic acid (HA) and atelo-collagen (Col). CDS can be cryopreserved and transported to other hospitals in a frozen state. To evaluate cell viability, cell growth, and release of VEGF after long-term cryopreservation, the CDS was cryopreserved at -85 degrees C or -152 degrees C for a given period. We measured cell viability immediately after thawing and cell growth in CDS that was recultured for 1 week after thawing. In addition, the amount of vascular endothelial growth factor (VEGF) released from CDS that was recultured for 1 week after thawing was measured. The cell viability and cell growth of control CDS that was thawed within 3 weeks after freezing was 56.2% and 132.7%, respectively. The cell viability and cell growth of the CDS that was cryopreserved at -85 degrees C for 6 months was 43.4% and 119.7%, respectively. When cryopreserved at -152 degrees C for 1 year, the cell viability and cell growth was 52.0% and 110.8%, respectively. These values were comparable to those of the control. The amount of VEGF released from CDS cryopreserved at -85 degrees C for 6 months (491.0 pg/mL) or at -152 degrees C for 1 year (586.8 pg/mL) was comparable to that of the control CDS (587.3 pg/mL). In contrast, the amounts of VEGF released from CDS cryopreserved at -85 degrees C for 1 year (322.5 pg/mL) or at -152 degrees C for 2 years (210.8 pg/mL) were low, with a marked decrease in cell viability and cell growth. These findings suggest that CDS cryopreserved at -85 degrees C for 6 months or at -152 degrees C for 1 year maintains sufficient cell viability and the ability to proliferate and release a significant amount of VEGF. The release of VEGF from CDS after long-term cryopreservation is a useful therapeutic effect, and is important for clinical use.