215 Immune Cell Model for Burn Immune Reactions Figure 3. Cytokine levels in medium of (burn-injured) FSEs after 3 or 7 days of culture with monocytes. Samples from biological duplicates were averaged per donor. Concentrations are reported in pg/mL medium. Experiments were performed in duplicate using keratinocytes and fibroblasts from 3 different donors and monocytes from 3 different donors. The dashed line indicates the lowest level of quantification. Statistically significant differences were calculated using Wilcoxon signed-rank test. No comparisons were made with monocytes in matrix only. Significant differences are indicated by asterisks: *: p < 0.05. T cells that migrated into full skin equivalents highly expressed Th1 and Th17 chemokine receptors, irrespective of burn injury To simulate an adaptive immune response to burn injury, CD3/CD28 bead pre-activated T cells were brought into (burn-injured) FSEs. About 2.5 × 105 T cells were placed between the transwell membrane and the dermal side of the FSEs, and cultured for 3 days (see Figure 6C for procedure), based on previous findings [35]. Pre-activated T cells cultured in suspension or in a matrix without skin cells served as controls. Using microscopy, we found that T cells actively migrated into the FSEs (Figure 4A). After 3 days of culture, cells were isolated from the FSEs to analyze the T cells by flow cytometry. Based on the flow cytometry analysis, only a small portion (2.8 × 103) of T cells migrated into the FSEs (Figure 4B). About 86.7 % of these T cells were CD4+ (Figure 4C). The majority of T cells in 7
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