Establishment of standard human fibroblast bank

ABSTRACT

Fibroblasts are mesenchymal cells that can be readily cultured in the laboratory and play a significant role in epithelial-mesenchymal 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. Establishment of standard human fibroblast bank is the basis of some important researches. Foreskin was obtained aseptically from patient younger than 1 year old during surgical excision. Fibroblasts isolated by enzymatic treatment were cultivated successively in a culture medium to establish cell banking. The cells were checked to be negative for HBV, HCV, HIV, HSV-I, HSV-II, EBV, CMV, Treponema pallidum, Mycoplasma sp. and Chlamydia. First, 5[th] and 10[th] subcultured cells were processed for immunocytochemistry studies using a panel of monoclonal antibodies including antibodies to MHC class I and II antigens for checking of elimination of superficial cell antigens during cultivation. First, 5[th], 10[th] and 22[nd] subcultured cells were karyotyped to find any chromosomal abnormalities. The fibroblasts used in production of cell bank were checked for some bacteria and viruses by molecular methods and confirmed to be negative. The results of karyotyping of cultured fibroblasts after 22[nd] passages show abnormalities. Multiple centromeric fissions were present in the various metaphase spreads. There were multiple clones with variable rearrangements and chromosome fissions. Expression of HLA on the fibroblast surfaces was diminished during subculturing. Fifth to 10[th] subcultured fibroblasts were the best cells to establish a human fibroblast bank. To prevent chromosomal abnormalities in fibroblast passaging, the best colony that is chromosomally stable and has lower enzymatic separation time should be selected