To begin, take the skin biopsy tube with fibroblast medium. Remove the medium, and wash the skin punch biopsy thrice with five milliliters of sterile, pre-warmed PBS. Using sterile forceps, transfer the skin punch biopsy to a 10-centimeter cell culture plate, and cut it widthwise into three or four pieces with a sterile scalpel, leaving the epidermis and dermis.
Transfer each piece into a sterile, 35-millimeter, plastic cell culture dish, and press it gently into the culture dish. Allow it to dry for five to 10 minutes until the liquid evaporates and the biopsy is attached to the cell culture plastic. Using a 1, 000-microliter pipette, add one milliliter of fibroblast medium dropwise around the biopsy to a final volume of three milliliters.
Incubate the sample for seven days at 37 degrees Celsius and 5%carbon dioxide without feeding or moving the dish. At the end of the incubation, change the medium to fresh, pre-warmed fibroblast medium. Finally, use a phase contrast microscope to monitor the outgrown spindle-like fibroblasts around the skin biopsy, and freeze the selected cells after growing them.
