Standardization for mass production of allogeneic cultured dermal substitute by measuring the amount of VEGF, bFGF, HGF, TGF-β, and IL-8

Fibroblasts were isolated from a piece of donated skin measuring about 1 x 1 cm and were proliferated over nine successive cultivations. Fibroblasts obtained from the fourth cultivation were cryopreserved in 10 cryotubes as master cells. Fibroblasts obtained from the fifth to ninth cultivations were cryopreserved in 49 cryotubes in total as working cells. At the end of each procedure in the fourth to eighth cultivations, about three-quarters of the proliferated fibroblasts were cryopreserved, and the remaining one-quarter were proliferated in the next cultivation. All fibroblasts obtained from the ninth (final) procedure were cryopreserved as the last working cells. Two cryotubes of the last cryopreserved working cells (ninth cultivation) were thawed and proliferated. Cultured dermal substitute (CDS) was prepared by performing ten successive cultivations. After each cultivation, about three-quarters of the resulting fibroblasts were used to prepare CDS, and the remaining one-quarter were proliferated in the next cultivation for 1 week. Finally, all fibroblasts produced by the tenth (final) procedure were used to prepare CDS. In each of the first to ninth cultivations for CDS preparation, the density of fibroblasts remained relatively constant, and the levels of cytokines, i.e., vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, transforming growth factor-beta 1, and interleukin-8 released from CDS remained relatively constant. In total, 47 sheets of CDS measuring 10 x 10 cm were prepared. The present findings indicate that about 1000 sheets of CDS measuring 10 x 10 cm can be prepared using all the working cells prepared from a piece of donated skin measuring 1 x 1 cm.