The use of human-derived feeder layers for the cultivation of transplantable human epidermal cell sheet to repair second degree burn wounds

Abstract

Background and objectives: Human epidermal cell sheet (human-ECS) is a feasible treatment option for wound injury. Traditionally, researchers often use murine 3T3 fibroblast cells as feeder layer to support human epidermal cell sheet grafts, thus increase risk to deliver animal-borne infection. To overcome the potential risks involved with xenotransplantation, we develop human foreskin fibroblast cell as feeder layer culture system and investigate the effects of human-ECS on second-degree burn wound healing in mini-pig in order to develop more effective and safer therapies to enhance wound healing in human.

Materials and methods: Human epidermal keratinocytes and fibroblasts were isolated from foreskin tissue and were co-cultured to manufacture human-ECS. The cell morphology was monitored with phase-contrast microscopy, the stem cell markers were assessed by flow cytometry, and by colony-forming efficiency (CFE) assay. The structure of human-ECS was observed by hematoxylin and eosin staining. Expression of cytokines in human-ECS was confirmed by enzyme-linked immunosorbent assay. Second-degree burn wounds were created on the dorsal of miniature pig to evaluate the effect of oil gauze, oil gauze combined with commercial epidermal growth factor (EGF) cream, and oil gauze combined with human-ECS. Wound healing rate, histological examination, and Masson staining were measured to observe the wound repair efficacy. Real-time PCR and Western blot were utilized to detect the expression level of EGF and interleukin 6 (IL-6).

Results: Stratified human-ECS with 6-7 layers of epidermal cells was successfully cultivated with human-derived feeder cells, in which epidermal cell highly expressed CD49f and CFE was 3% ± 0.45%. Application of human-ECS induced a higher wound healing rate than commerical EGF cream and oil gauze control. The expression of EGF in human-ECS group was higher than those in the other groups; however, the expression of IL-6 was significantly decreased at day 14 by human-ECS treatment group.

Conclusions: Human-derived feeder cells are suitable for cultivation of human-ECS, avoiding pathogen transmission. Human-ECS could enhance second-degree burn wound healing, and its promoting effect involved secreting a variety of cytokines to regulate tissue reparative process.

Keywords: cytokines; epidermal cell sheet; human fibroblast cells; wound healing.

FIGURE 1

Generation of human cultured epidermal cell sheet and application.

FIGURE 2

The biological characteristics of human fibroblast cells. (A) Morphology of human foreskin‐derived fibroblast cells. (B) Population doubling time was estimated across passages for fibroblast cell. (C) Flow cytometry analysis of cells for expression of the fibroblast marker (CD90).

FIGURE 3

Characteristics of the human‐epidermal cell sheet (ECS). Confluent keartinocytes are well‐differentiated with clonal growth. Photographs showed the cells of “paving stone” appearance. (B) Flow cytometry analysis of cells for expression of the specific markers. (C) Representative pictures of colony formation. (D) The expression of factors was detected in the supernatant of human‐ECS. (E) Morphology of human epidermal cells grown on human‐derived feeder layers.

FIGURE 4

Assessment of the wound healing rate. (A) Photographs by visual monitoring of the wound size among different treated groups at days 3, 7, and 14 after wounding. (B) Percentages of wound healing rate in all groups. The results showed that human‐epidermal cell sheet (ECS) group had significant improvement in wound healing compared with the oil gauze only group in 14 days after grafting (p < 0.01). Data are expressed as the mean ± standard deviation of three experiments. Statistical analysis was performed using one‐way analysis of variance (ANOVA) test, *p < 0.05; **p < 0.01.

FIGURE 5

Histopathological evaluation of wound healing on day 3. (A) The first row represents the pictures of hematoxylin and eosin (H&E) staining , the second row shows the re‐epithelization site, and the third row highlights infiltrating inflammatory cells. The black dash shows the migrating epithelium. Original magnification: ×4 ( top row); ×20 (middle row); ×40 (bottom row). (B) Quantification of infiltration of inflammatory cells. No significant difference was found among groups. Values are presented as the mean ± standard deviation. Statistical analysis was performed using one‐way analysis of variance (ANOVA).

FIGURE 6

Histopathological evaluation of wound healing on day 7. (A) The first row represents the pictures of hematoxylin and eosin (H&E) staining , the second row shows the re‐epithelization and granulation tissue, the third row highlights infiltrating inflammatory cells. The black dash shows epithelial layer and granular layer. Original magnification: ×4 ( top row); ×20 (middle row); ×40 (bottom row). The epithelial thickness (B) granulation tissue thickness (C), and immune cell infiltration (D) were evaluated in a quantitative manner. No significant difference was found among groups. Values are presented as the mean ± standard deviation. Statistical analysis was performed using one‐way analysis of variance (ANOVA).

FIGURE 7

Histopathological evaluation of wound healing on day 14. The first row represents the pictures of hematoxylin and eosin (H&E) staining , the second row shows the re‐epithelization and granulation tissue, the third row highlights infiltrating inflammatory cells, and the last row indicates blood vessel. The black dash shows epithelial layer and granular layer. Black arrowheads mark the vascular structures. From the first to the fourth row, the original magnification: ×4, ×20, ×40, ×20. The epithelial thickness (B), granulation tissue thickness (C), immune cell infiltration (D) and the number of new blood vessels were evaluated in a quantitative manner. There was a significant difference in epithelial thickness and inflammation in human‐epidermal cell sheet (ECS) group at day 14. Values are presented as the mean ± standard deviation. Statistical analysis was performed using one‐way analysis of variance (ANOVA), *p < 0.05, **p < 0.01.

FIGURE 8

Collagen accumulation in skin wounds of three groups. (A) Masson's trichrome staining images of sections after treatment with three groups at different time points. (B) The collagen deposition was quantified using the ImageJ software. There was a significant difference in collagen accumulation in human‐epidermal cell sheet (ECS) and commercial EGF groups at day 14. Values are presented as the mean ± standard deviation. Statistical analysis was performed using one‐way analysis of variance (ANOVA). *p < 0.05, **p < 0.01, scale bar = 200 µm.

FIGURE 9

The expression of EGF and IL‐6 in skin wounds of three groups. Results were measured by Western blot (A, B, and C) and real‐time PCR (D and E) in the mini‐pig model. Values are presented as the mean ± standard deviation. Statistical analysis was performed using one‐way analysis of variance (ANOVA).*p < 0.05; **p < 0.01; ***p < 0.001.