Effect of different irradiation doses on human and murine fibroblasts used as feeder layer for keratinocytes’ culture

Abstract

Background & Aim The establishment of human keratinocytes’ culture (KTs) to obtain human Tissue Engineered Skin Substitutes is under research nowadays. Best results have been achieved culturing KTs on a feeder layer of irradiated 3T3 murine fibroblasts (i3T3s). Nevertheless, molecular effects of murine cells on KTs is unknown and for this reason, irradiated human fibroblasts (iHFs) have been studied. The objective of this study is to evaluate the effect of different irradiation doses on cell reducing power and cell proliferation of both fibroblasts. Methods, Results & Conclusion Methods 3T3s were obtained from a commercial vial of 3T3-Swiss Albino. HFs were isolated from human skin biopsies. After culturing and final recovery, both cell types were irradiated to doses of 50 and 100 Gy to constitute the feeder. Non-irradiated cells were used as control. Each condition was cultured 15 days in two 24-well plates (2 cm2/well). In each plate, cell densities cultured in triplicate were: 7,500 cells/cm2 and 10,000 cells/cm2. One plate was used for reducing power analysis; using a dilution of PrestoBlue™ (10%). The other plate was used for cell proliferation analysis; using Trypan Blue solution. N for each plate was 3 and a value of p Results Analysis of each type of feeder (Figs. 1-2) revealed significant differences at Days 10 and 15, between both irradiation doses and non-irradiated cells. In the case of iHFs, irradiation dose had a significant effect on cell reducing power, mainly at Day 15. Analysis of i3T3s revealed that the effect of irradiation on reducing power was extremely significant from Day 3 to 15, but no dose-dependent. A comparative analysis (Fig. 3) indicated that the number of non-irradiated cells after 15 days was higher in the case of 3T3s than HFs (644,620 cells vs 143,740 cells), but cell reducing power was lower in the case of murine fibroblasts (72.33% vs 80.67%). In the case of irradiated cells, iHFs were more resistant to irradiation in both, cell reducing power (27.5% vs 4.92%) and cell proliferation (5,962 cells vs 1,351 cells) at Day 15. Conclusion No differences exist considering initial cell density or irradiation dose in both feeders. Comparative analysis revealed that irradiation process is more effective in 3T3s than in HFs. This study needs to be completed with the incorporation of KTs on the different feeder conditions analyzed. Supported by PI17/02083, SAS PI-0458-2016 and PhD fellowship FI18/00269–Alvaro Sierra-Sanchez).