Abstract: Song Xiaojie, Shi Xiaoting, Yao Qifeng, Wu Yue Research and Development Center, JALA Group Corporation, Shanghai 200233, China Corresponding author: Wu Yue, Email: wuyue@jala.com.cn 【Abstract】 Objective To construct a collagen matrix-based skin model that can last a long time in vitro for potential application of subsequent tests and studies. Methods The porcine collagen and normal human skin fibroblasts were mixed and seeded into the culture dish, which were cultured for 3 - 4 days to obtain a dermal structure. Then, the third- to seventh-passage normal human keratinocytes were seeded onto the dermal surface and cultured for 14 days to obtain a double-layer skin model. Hematoxylin and eosin （HE）staining and Masson staining were performed to evaluate the morphology and structure of the skin model, and electron microscopy was conducted to observe the ultrastructure of the skin model. Immunohistochemical study and immunofluorescence staining were conducted to determine the of major markers in each layer of the skin model. Results HE and Masson staining showed that the skin model and normal human skin tissues showed very similar dermal and epidermal structures. After harvest of the skin model, it can be cultured in vitro for another 14 days with favorable and stable dermal and epidermal structures. Electron microscopy showed lipids in the stratum corneum, keratohyalin granules in the stratum granulosum, corneodesmosomes, desmosomes and basal membrane in the skin model. Immuno-histochemical and immunofluorescence staining showed the consistent of transglutaminase, filaggrin, keratin 10 and Ki67 in the epidermis in both the skin model and normal skin. Moreover, the of type Ⅳ collagen and laminin-5 in the basal membrane, as well as that of typeⅠ collagen, type Ⅲ collagen and fibrillin in the dermis were both consistent between the skin model and normal skin. Conclusion The constructed three-dimensional （3D） skin model is highly analogous to normal human skin in the aspect of tissue structure and various protein , and the skin model can be stably cultured in vitro for at least 14 days after harvest.

Key words: Skin, artificial, Collagen, Fibroblasts, Keratinocytes