A novel, rapid method for a noncultured autologous skin cell suspension that preserves cell viability and wound healing capacity

Abstract

Objective: Noncultured autologous keratinocyte suspensions have been used for wound healing in burned patients. However, this process is time-consuming and potentially harmful due to the use of chemical agents. Here, we aimed to prepare noncultured autologous keratinocyte suspensions using a tissue homogenizer instead of chemical agents. Methods: Cell number and viability were measured using trypan blue exclusion assays of homogenized skin tissues from burn patients. Cell types were identified by analysis of cell markers using reverse transcription polymerase chain reaction and by scanning electron microscopy (SEM). Immunofluorescence assays were performed to distinguish cell types in suspension. Wound healing capacity was analyzed by measuring growth factor expression by enzyme-linked immunosorbent assay. The potential clinical applicability of noncultured skin cell suspensions was analyzed using a nude rat model. Results: After optimization of the homogenizer settings, cell viabilities ranged from 52% to 89%. Keratinocytes, fibroblasts, and melanocytes were identified in the suspension, and SEM images showed evidence of keratinocyte-like cell morphology. Several growth factors, including epidermal growth factor and basic fibroblast growth factor, were present in the cell suspensions. The rat model showed that these cell suspensions accelerated epithelialization and skin adnexa regeneration. Conclusions: Noncultured autologous keratinocyte suspensions could be generated using a tissue homogenizer without suppressing cell viability in vitro or wound healing potential in vivo. Additionally, this process was rapid and did not require the use of chemical agents