A Preliminary Study Of Skin 3D Printing Using Human Epidermal Stem Cells And Fibroblasts

The seed cells used in 3D printing of the epidermal layer of skin include keratinyocyte cell lines and keratinocytes isolated from the skin tissues.However,the keratinocyte cell lines are transformed cells which may have different genetic characteritics from normal keratinocytes,and the expansion capacity of isolated keratinocytes is limited.Epidermal stem cells(EpSCs)can proliferate and differentiate into various types of cells in the epidermis.They are ideal seed cells for 3D printing and have good application prospects.At present,there are few reports about skin tissues printing using EpSCs as seed cells.ObjectiveTo fabricate skin tissues through extrusion-based bioprinting using human EpSCs and fibroblasts as seed cells.Methods1.Epidermal cell suspension was obtained by digesting human skin tissues with neutral protease and trypsin.EpSCs were isolated from epidermal cell suspension by their rapid adherence.EpSCs were identified by examining the expression of EpSCs markers through immnunofluorescence staining and flow cytometry assay.Fibroblasts were isolated from human skin tissues by type ? collagenase digestion.Fibroblasts were identified by detecting the expression of vimentin through immunofluorescence staining.2.Fibroblasts were cultured in KGM2 and DMEM,respectively.Cell proliferation was evaluated through an MTT assay.The medium which is better for fibroblast growth was chosen for culture of 3 D printed skin tissues.3.The dermal and epidermal layers were sequential printed by extrusion-based bioprinting using EpSCs and fibroblasts as seed cells,and fibrinogen and gelatin as scaffold materials.Thrombin was used to polymerize fibrin scaffolds from fibrinogen.The optimal concentration of fibroblasts for printing the dermal layer,and the optimal concentration of fibrinogen in the scaffold material for printing the epidermal layer were determined.4.The viability of EpSCs and fibroblasts in 3D printed tissues were examined by LIVE/DEAD cell viability assay.5.The structure and cell distribution of the 3D printed skin tissues were examined by hematoxylin-eosin staining(HE)and immunofluorescence staining.6.The barrier function of 3D printed skin tissues was examined by toluidine blue penetration test.Results1.Immunofluorescence staining showed that more than 90%of the EpSCs isolated from human skin were ?1 integrin or CK19 positive.Flow cytometry assay showed that 90.73%of the cells expressed high level of ?6 integrin and low level of CD71.Immunofluorescence staining showed that more than 98%of the fibroblasts isolated from the dermis ofhuman skin tissues were vimentin positive.The EpSCs and fibroblasts with high purity could be used as seed cells.2.Fibroblasts cultured in KGM2 medium grew better than in DMEM medium containing 10%FBS.KGM2 medium was chosen to culture 3D printed skin tissues.3.Skin tissues were bioprinted with scaffold materials containing 5 mg/ml in the dermal layer and 10 mg/ml in the epidermal layer,and with fibroblasts and EpSCs at 4×106/ml and 5×106/ml,respectively.EpSCs and fibroblasts in 3D printed tissues grew well.HE and immunofluorescence staining showed that the epidermal and dermal layer were well stratified,and the thickness of the epidermis was 74.72±5.25 ?m,which is similar to that of normal human skin tissues.4.The toluidine blue penetration test showed that after 7 days of air-liquid culture,the 3D printed skin tissues had a similar barrier function to that of normal human skin tissues.ConclusionSkin tissues with epidermal and dermal layers were fabricated by extrusion-based bioprinting using human EpSCs and fibroblast as seed cells,and fibrinogen and gelatin as scaffold materials.After culture,the thickness of the epidermis layer and barrier function of the engineered skin tissues were similar to those of normal skin tissues.