In vitro production of human dermal equivalent

Abstract

Human dermal tissue is composed of loose and dense connective tissue. Main cell populations are fibroblasts and the dominant fibers are built from collagen type I. The aim of our study was to determine the precise method and time frame for the in vitro production of human dermal equivalent and to investigate the effects of ratio of structural elements and vitamin C on characteristics of the engineered tissue. Primary isolation of the foreskin fibroblasts was performed by explant method and enzymatic dissociation. Various collagen gels were obtained by mixing cells (from 25 x10(3) 200 x 10(3)/ml(3)) and neutralized collagen type I (from 2 to 4 mg/mI), with or without vitamin C. The routine histological and morphometrical examination was performed. Enzymatic dissociation of the foreskin proved to be a faster method for production of desired number of fibroblasts (7.5 x 10(5) for 4 days). The contraction of collagen-gels started from day one through day seven and was dependent on cell and collagen concentration with higher density gels being contracted to a greater extent, except for the lowest/highest values. The best result was achieved with 100 x 10(3) cells and 2 mg/ml collagen. Vitamin C at 50 microg/ml had no effect on speed of tissue formation. A precise approach that mimic the in vivo conditions is needed for the in vitro production of the dermal equivalent suitable for the possible treatment of tissue defects. Nearly ten days are necessary from the donor tissue dissociation to the final product.