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Evaluation of Culture Medium for Human Keratinocytes

Fabiana Regina Xavier Batista, Jussara Rehder and Maria Beatriz Puzzi

Human keratinocytes are needed for tissue engineering of skin applied in tissue repair and regeneration aimed at clinical application. The need to have high cell concentrations within a short time when performing cell proliferation is vital. The growth and maintenance of these cells commonly involve the use of MCDB 153 medium, which is supplemented with animal-derived components, such as growth factors and fetal bovine serum (FBS). The aim of this work was to evaluate different formulations based on MCDB 153 medium for keratinocyte proliferation. For that, several experiments were realized to define which supplements are important for skin cell culture using 24 factorial design. Skin samples were obtained from four consenting patients submitted to dermolipectomy, the plastic surgery operation. These fragments were treated with 0.25% trypsin-EDTA for four hours, at 37ºC. Furthermore, the epidermis was separated from the dermis, providing skin cells. Batch cultures were performed in 6 and 96-well plates, in addition to 25 cm2 flasks. Results concerning cell growth and metabolism showed that keratinocytes were full grown up in MCDB 153 medium containing insulin (7.5 µg.mL-1), bovine pituitary extract-BPE (80 µg.mL-1), epidermal growth factor-EGF (0.08 µg.mL-1), hydrocortisone (0.63 µg.mL-1) and glutamine (1 g.L-1). On the other hand, culture media proposed in the experimental design did not resulted in satisfactory cell growth. In addition, although, the initial glucose concentration was low in the most of cases, the lactate was strongly produced by cells reaching 1 g.L-1.