A preliminary study on the method of single micro extrusion-based 3D printing of skin tissue

Abstract

Objective To explore the feasibility of single micro extrusion 3D printing method for skin tissue. Methods Fibroblasts were isolated and cultured from human dermis and identified by immunofluorescence staining. Fibroblasts and HaCaT cells were used as seed cells. Gelatin, hyaluronic acid and fibrinogen were used to form scaffolds. HE staining and immunofluorescence staining were used to observe the cell distribution of 3D printed skin. The viability of cells in 3D printed skin tissue was detected by living/dead cell staining kit. Toluidine blue penetration assay was used to detect the barrier function of printed skin. Results More than 98% of the cultured fibroblasts isolated from skin dermis were vimentin positive. When the concentration of fibrinogen in the dermis and epidermis was 5 mg/ml and 1 mg/ml respectively, fibroblasts (4×106 cells/ml) printed 8 layers, and HaCaT cells (5×106 cells/ml) printed 1 layer, the prepared 3D printed skin tissue was cultured in air-liquid interface for 7 days and HE staining and immunofluorescence detection showed that the epidermis and dermis were well stratified with the thickness of epidermis being (63.5±3.5) μm. The viability of cells in 3D printed skin tissue was (90.2±0.9)% and (95.3±0.8)%, respectively, after submerge culture for 2 days and air-liquid interface culture for 7 days. After air-liquid interface culture for 7 days, the 3D printed skin tissue had similar barrier function to normal skin. Conclusion We have established a single micro extrusion 3D printing method for skin tissue, and successfully prepared tissue-engineered skin with structure, epidermis thickness and barrier function similar to normal human skin tissue, which may become an effective method for mass production of tissue-engineered skin in the future. Key words: Skin; Fibroblasts; HaCaT cells; Micro extrusion printing; Tissue structure