Background: Fibroblasts are mesenchymal cells that can be readily cultured in the laboratory and play a significant role in epithelialmesenchymal interactions, secreting various growth factors and cytokines that have a direct effect on epidermal proliferation, differentiation and formation of extracellular matrix. They have been incorporated into various tissue-engineered and used for a variety of clinical applications, including the treatment of burns, chronic venous ulcers and several other clinical applications in dermatology and plastic surgery. Method: Isolated fibroblasts by the enzymatic process from foreskin were cultivated successively in a culture medium to establish cell banking. Foreskin and the last subcultured cells were checked for HBV, HCV, HIV, HSV I, HSV II, HTLV I, HTLV II, EBV, CMV, Treponema Pallidum, Mycoplasma sp. and Clamydia. The 1st, 5th and 10th subcultured cells were processed for immunocytochemistry studies using a panel of monoclonal antibodies including antibodies to MHC class I & II antigens for ensuring the elimination of superficial cell antigens during cultivation. Subcultured cells were karyotyped to find any chromosomal abnormalities. The best passages were chosen for culturing on silicone sheets provided by the Iran Polymer and Petrochemical Institute. Results: Evaluation for bacteria and viruses by molecular methods was negative. Karyotyping of cultured fibroblasts after the 10th passage showed some abnormalities. HLA expression was imperceptible in the cells obtained from the 10th sub-culture. The best passages were from 5th to 10th for banking and culturing on silicone sheets. Conclusion: Expression of HLA on fibroblast surfaces was diminished during subculturing. To prevent chromosomal abnormalities in fibroblast passaging, we should select the best colony that is expected to be chromosomally stable with the least antigenicity. In our study, the 5th to 10th sub-cultures were the best cells for the purpose of grafting and acceleration of the wound healing.