Using Reconstructed Tissue Engineering Skin Models To Test Skin Irritation

Tissue-engineered skin models have been used in many studies, often with the goal of replacing animal experiments. Most of current skin models reconstructed with primary keratinocytes, however, cell resources were limited and each cell passage was varying conditions, so, the consistency and reproducibility of skin models were weaken. The objective of this study was to reconstruct 2 kinds of skin models to test skin irritation. 1 kind reconstructed with the human keratinocyte cell line (HaCaT), the other reconstructed with human primary keratinocyte. We selected 20 chemicals described in the Performance Standards of the European Center for the Validation of Alternative Methods (ECVAM) as test chemicals to test skin irritation in the 2 kinds of skin models. After exposure to the chemicals, cell viability was measured by a methyl tetrazolium (MTT) assay, and condition medium was collected to provide an additional evaluation of skin irritation through the detection of release of interleukin (IL)-1αin the medium. In addition, we compared the consistency and reproducibility of the 2 kinds of skin models. The 2 kinds of skin models had a similar morphology. Results of the testing using the 20 chemicals showed that 2 kinds of skin models were both applicable to skin irritation testing protocol set out in the ECVAM performance standards. Furthermore, skin model containing HaCaT cells, the sensitivity and specificity were higher than skin model containing primary keratinocytes. The consistency and reproducibility of the comparison results showed that skin model containing HaCaT cells could continuously and correctly predict skin irritation. In conclusion, skin models containing HaCaT cells and primary keratinocytes were all suitable to test skin irritation. Moreover, the fronter had better consistency and reproducibility than the later in testing of skin irritation. Thus, skin model containing HaCaT cells may be better to test skin irritation and replace animal experiments.The study was composed of three sections as follows:PartⅠ. Cell proliferation capacity of comparision keratinocytes and immortalization keratinocyte cell line HaCaTTo compare proliferation capacity of human primary keratinocytes and immortalized keratinocyte cell line HaCaT. We compared cell cycle cell apoptosis and growth curve. Conclusion: Immortalized keratinocyte cell line HaCat proliferation capacity better than primary keratinocytes.PartП. Using primary keratinocytes and immortalized keratinocyte cell line to reconstruct skin models to test skin irritationOne kind of skin model was reconstructed with the normal human fibroblasts and collagenⅠ. The collagen solution was then placed onto a 75 mm nylon mesh, incubated at 37°C for 30 min in the CO2 incubator, and allowed to gel. The collagen gel with the fibroblasts was then incubated for 10 days in the CO2 incubator. We selected the 3rd passage of the keratinocytes and collected them gently by disintegrating them into a single cell suspension at 1.0×106 cells/cm2 and then seeded them onto the collagen gel containing fibroblasts. The collagen gel containing the cells was incubated for 7 days. It was then raised onto the liquid-air surface for 7 days until ready for use. We used the same way to reconstruct skin model containing keratinocyte cell line HaCaT. We compared morphology and immunochemical and barrier function. Conclusion: two kinds of skin models are all suitable to test skin irritation.PartШ. Using two kinds of skin models to test skin irritationThe 20 reference test chemicals described in the ECVAM performance standards, which test criteria according episkin test criteria. We test the sensitivity and specifity and IL-1α. Conclusion: the skin model containing the keratinocyte cell line exhibited better sensitivity and specificity.