一人皮肤成纤维细胞培养.docVIP

PRIMARY FIBROBLAST CULTURE FROM HUMAN SKIN BIOPSY This protocol describes the steps for obtaining a primary fibroblast cell line from human skin biopsies. Fibroblasts are derived directly from excised skin as explants; enzyme digestion by collagenase may help obtain cells in a shorter time. This protocol describes the different steps for obtaining a primary cell line from a skin biopsy. Equipment and materials Laminar flow hood CO2 incubator Inverted microscope Sterile surgical Instruments for microdissection BME fibroblast medium PBS 10x without Ca+2 or Mg+2 Collagenase type II 4mg/ml Petri dishes 100 mm Pasteur pipettes Culture flask, 25 cm2 15 ml sterile plastic tube BME Fibroblast medium BME 80 ml Foetal bovine serum 20 ml Penicillin-streptomycin solution 100x 1 ml Filter and store at +4°C, up to 1 month. The skin biopsy sample should be shaped as a diamond and about 5-10 mm in diameter. Collect the tissue sample in sterile BME fibroblast medium. Procedure 1 Rapidly wash the skin biopsy in PBS in a Petri dish, cut into small fragments and transfer these to a flask. Using a sterile Pasteur pipette with flame-rounded tip, distribute the small tissue fragments over the bottom surface of the culture flask. Pass the flask rapidly and carefully through the Bunsen flame in order to evaporate the medium so that the minced tissue pieces adhere to the plastic surface, but so as not to heat-damage the minced tissue. Take care not to cook the tissue! Carefully add BME medium for fibroblast growth, firmly close the lid of the flask and place in CO2 incubator. The next day, slightly unscrew the lid of the flask so that the tissue can “breathe.” Replace the culture medium after two days and, from this point on, replace it three times a week. The fibroblasts will start to grow from the minced fragments in 2-3 days. When there are sufficient cells, they are detached enzymatically and plated in Petri dishes, or 75 cm2 culture flasks, for proliferation see next