Culturing keratinocytes from patients with burn injury

BACKGROUND: A burn injury, particularly when extensive and deep, leads not only to local tissue damage but also to systemic alterations that impair the skin’s regenerative capacity. Modern therapeutic approaches, including cell-based technologies, aim to accelerate skin repair in conditions where autologous tissue is insufficient. Despite using cells derived from unaffected skin regions, their viability and ability to form epithelial structures may be substantially reduced. This study investigates changes occurring in the epidermis of uninjured skin in patients with extensive burns and explores effective strategies to enhance the viability of cultured keratinocytes.

AIM: This study aimed to examine the systemic impact of burn injury on the viability of epidermal keratinocytes from uninjured skin and to develop approaches to enhance their viability during in vitro culture.

METHODS: Immunofluorescence analysis of specialized markers was performed on cryosections of skin obtained from healthy donors and patients with burn injuries. The proportions of viable cells and epidermal stem cells in keratinocyte cultures were assessed using flow cytometry. The adhesive capacity, colony-forming ability, and formation of confluent cell layers were evaluated in vitro for keratinocytes isolated from healthy donors and donors with burns.

RESULTS: Keratinocytes isolated from patients with burns exhibited reduced adhesion and viability during the first days of culture. Immunofluorescence analysis revealed abnormal expression of keratins 6 and 17 in the epidermis of uninjured skin from patients with burns, an increased proportion of cells with nuclear localization of YAP1, and decreased integrin expression, indicating the development of a pro-inflammatory phenotype. Flow cytometry demonstrated a reduction in epidermal stem cell fractions (ITGα6^high/CD71^low) in patients with burns. To enhance cell adhesion and survival, culture methods involving collagen coating of the culture surface and the addition of a Rho-associated kinase inhibitor to the culture medium were proposed, both of which promoted cell growth and rapid formation of a confluent layer.

CONCLUSION: A modified culture protocol incorporating type I collagen and a Rho-associated kinase inhibitor improves the efficiency of keratinocyte expansion from patients with burns. These findings may optimize the production of cell cultures for burn treatment, enhance the survival of transplanted cells, and ultimately improve the effectiveness of tissue engineering approaches in combustiology.