Cultured keratinocytes (Modern Cell and Tissue Technology)

Full-thickness skin biopsies (1-2 cm²) are obtained from the patients for culturing. The collected skin tissues are washed at least 8 times with phosphate buffered saline to remove coagulated blood and other contaminants.  Then, they are cut into small pieces. Tissue fragments are placed in 10 ml trypsin (0.00125%) and EDTA (0.01%), for 30 minutes with magnetic stirring at 100 rpm, to isolate cells. When a sufficient number of keratinocytes are isolated, trypsinization is terminated with a trypsin inhibitor, followed by centrifugation. The keratinocytes are seeded into a fetal bovine serum (FBS)-free culture medium and cultured at 37 °C in a 5% CO₂ incubator.

The cell culture medium is changed every two to three days. If cells in colonies overgrow to confluence, the cells are sub-cultured for 16 days. In order to prevent the differentiation thereof, keratinocytes at 70–80% confluence are detached from the culture dish using trypsin-EDTA, and obtained as the final product.