Protocol optimization for obtaining the culture of rat dermal fibroblasts

Abstract

Introduction. Obtaining primary cultures of fibroblasts is necessary for conducting experimental studies to investigate the basic cell response mechanisms to various stimuli. Despite the widespread use of fibroblast cultures, methods for obtaining them from the skin are not standardized. The aim of the studywas to find the optimal conditions to obtain a fibroblast culture from rat skin using enzymatic tissue disaggregation. Materials and methods. The fibroblast culture was obtained from the dermis of 18 male Wistar rats (N=12, 5–6-week-old, and 60–65 g body weight; N=6, 8–10-week-old rats, and 160–180 g body weight). A series of experiments was carried out to obtain a fibroblast culture with selecting the collagenase type and defining its concentration and exposure time. Results.An optimized protocol to obtain rat dermis fibroblasts is presented. Moreover, the problems of obtaining a culture and practical aspects of its use are discussed. Conclusion. When obtaining a primary culture of rat skin fibroblasts we should consider the following fac-tors: the type of the enzyme, its concentration, and exposure time; the age of the animals; the area of skin graft collection. In young rats, the optimal result was achieved when the cells were isolated from the axillary zone using collagenase type II at a 1 mg/ml concentration during a 90-minute exposure. In adult animals, the enzymatic effect of collagenase type II on the skin graft obtained from the back was optimal at a 5 mg/ml concentration during a 120-minute exposure. Keywords: fibroblast culture, dermis, preparation protocols, rats