Culturing Cells Of Tissue Engineering Skin And Evaluation Of Its Immunogenicity

Objective: In our study, we explored a suitable inoculation density, which could significantly shorten cultivation time and choosed a suitable medium to culture keratinocytes in the tissue engineering field. Langerhans cells and melanocytes in the different generations of keratinocytes were detected . To reveal how immunogenicity of cultured keratinocytes and fibroblast changed, those were mixed with allogenic lymphocytes. Methods:(1)The samples of circumcision were separated by 0.25% dispase, 0.125%Trypsin and 0.01%EDTA sequentially. According to different inoculation density, keratinocytes were cultured by K-SFM,FAD and basic medium . Observed initial generation keratinocytes confluence time and growth condition after passage. (2)The quantity of langerhans cells and melanocyte of different generations were detected by ATP-staining technique and immunohistochemical method (.3)The different generations of keratinocytes and fibroblasts were individulally mixed with allogenic lymphocytes in vitro to observe how the allogenic lymphocytes proliferated. Results:(1)Dermis and epidermis could be separated by dispase and trypsin easily.Living cells count was over 95 percent of total cells.When inoculation density for the initial passage keratinocyte was between 1×105/cm2 and 5×105/cm2, cells anchored well, proliferated fast and cell culture cycle was shortened significantly. Compared with FAD and basic medium ,cells cultured by K-SFM growed with single layer and growed fast. After several generations, morphous of cells was similar with that of initial generation. (2)ATP staining technique could only stain langerhans cells in stretched preparation of epidermis.Through immunohistochemical method ,there were 5.8 percent langerhans cells and 8.1 percent melanocytes in the initial generation keratinocytes and 2.1 percent langerhans cells and 2.8 percent melanocytes in the first generation keratinocytes. (3)With the passages of keratinocytes and fibroblasts gradually increased, the cpm gradually decreased. The cpm of initial passage and first passage descented sharply. The cpm of the second passage ,the third passage, the fourth passage and the fifth passage descented weakly,especially fibroblast. Conclusion:(1)In our study, we have explored a method of culturing keratinocytes, which was suitable for industrialization of tissue engineering skin, and established the purified human keratinocytes bank.(2)Mixed lymphocyte reacion demonstrated after cultured in vitro, the ability of keratinocytes and fibroblasts stimulating allogeneic lymphocytes proliferating decreased gradually. It indirectly reflected that the immunogenicity of cultured keratinocytes and fibroblasts decreased gradually. (3)K-SFM was suitable for culturing keratinocytes. Cells of bank should be beyond the fifth generation which growed well and low immunogenicity.