Comparison Of The Performance Of Keratinocytes Under Different Culture Conditions And Optimization Of Human 3D Skin Substitute

Objective: The three-dimensional(3D)skin constructed by tissue engineering can simulate the biological function in vivo which has broad clinical application prospects.The use of heterologous ingredients greatly limits the clinical application of threedimensional skin in the future.Therefore,this study intends to Screen keratinocyte culture conditions and use an xeno-free system-cultured keratinocytes(KCs),to coculture with fibroblasts(HDFs),and human umbilical vein endothelial cells(HUVECs)for construction of a three-dimensional(3D)skin substitute.Method: First,we compared the vitro characterization of KCs cultured under three different conditions(3T3-J2 feeder layer,laminin LN-511 and recombinant human collagen I).Then,KCs cultured under three conditions were co-cultured with HDFs on Alvetex Scaffold scaffolds to construct a 3D skin substitute,and we simulated the epidermal keratinization by air-lifting.We identified the 3D skin substitute by HE staining,tissue immunofluorescence,and qPCR.Finally,an attempt was made to construct a three-dimensional skin substitute with blood vessels by adding umbilical vein endothelial cells,and the effect of co-cultivation of the three cells on epidermal growth was observed.Result:1.Keratinocytes show different growth patterns under three culture conditions,among which clone-like proliferation on the 3T3-J2 feeder layer,and non-clonal-like on LN-511 and collagen I.The results of qPCR and immunofluorescence staining showed that there were no significant differences in the expression of KRT14,KRT5,KRT15,P63,KRT1,KRT10,INV in three kinds of keratinocytes.2.Using keratinocytes and fibroblasts can construct a 3D skin substitute with epidermal and dermal structures and functions.qPCR showed that it expressed keratinocyte stemness markers similar to normal skin levels.Immunofluorescence staining suggested that the 3D skin substitute expressed specific markers of the epidermal layer(KRT14?KRT15?KRT10?LOR),basement membrane(Collagen IV?Collagen VII?Laminin?CD49f),and dermal layer(Collagen I?Vimentin V9).3.In the two-dimensional co-culture of HDFs and HUVECs,it was observed that the two cells can grow together.The expressions of interstitial cell marker CD44 and vascular endothelial cell marker CD31 were observed by immunofluorescence staining.The results of qPCR showed that the expression of keratinocyte stemness genes were found significantly higher in 3D skin substitutes with HUVECs than in 3D skin substitutes without HUVECs.conclusion:Both laminin 511 and collagen I give strong support to the long-term culture of keratinocytes,similar to feeder cells,and the stemness of keratinocytes can be maintained for a long time under these three culture conditions.The xeno-free system-cultured keratinocytes and fibroblasts can be used to construct a three-dimensional skin substitute highly similar to normal skin structure and function in vitro.It provides a reference for the removal of heterologous cells in future production.Human umbilical vein endothelial cells can grow together with fibroblasts,and co-culture of the three cells can promote the growth of epidermal layer.