Investigation of Mitomycin-C-Treated Fibroblasts in 3-D Collagen Gel and Conditioned Medium for Keratinocyte Proliferation

Fibroblasts produce a spectrum of necessary growth factors essential for growth and proliferation of a variety of cell types. In this study, the paracrine effect of mitomycin-C- treated fibroblasts with various densities in collagen gel for keratinocyte proliferation was investigated from which an optimum cell density and optimum conditioned medium would be determined to expand keratinocyte without further differentiation for skin equivalent tissue engineering. The optimum cell density in collagen feeder gel for optimum collected medium preparation will be determined by checking the level of keratinocyte growth factor and granulocyte macrophage colony-stimulating factor in conventional medium. The results showed that the cell density of 1 x 10(5) cells /gel in the feeder gel is better to produce optimum collected medium. The conditioned medium is prepared by mixing together the optimum collected medium and molecular cellular and developmental biology (MCDB) 153 medium in different ratios for keratinocyte growth. The keratinocyte viability will be measured by 3- (4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay to determine the optimum conditioned medium. From the study, 67% conditioned medium was supposed as the better medium for keratinocyte proliferation. In this experiment, the optimum cell density in feeder gel to coculture with keratinocytes is also determined as 1 x 10(5) cells/gel. Keratin 10 (K10) and Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling stain will be used to check the cell differentiation and apoptosis, respectively. The results suggest that keratinocytes should not be cultured in postconfluent conditions due to undesired apoptosis and differentiation. The result of cell viability from passages to passages shows that the optimum feeder gel plays a more important role to the keratinocyte proliferation than that of optimum conditioned medium. Keratinocytes cultured with optimum feeder gel in 67% conditioned medium could effectively promote proliferation, inhibit apoptosis, and prevent differentiation. The combination of conditioned media and feeder gel to culture keratinocytes without external supplements can provide an inexpensive way for keratinocyte proliferation and construct an environment for real-time communication between the two cells. The results conclude that keratinocyte cultivation in feeder gel with modified medium should be feasible in the production of high quality keratinocytes for skin equivalents preparation