Objective To investigate the three-dimensional (3D) culture and morphology of human eccrine sweat gland cells. Methods The human eccrine sweat gland cells were isolated from normal abdominal full thickness skin by digestion of type II collagenase, and cultured in defined-keratinocyte serum free medium supplemented with 5 ng/mL recombinant human epidermal growth factor, 25 mg/mL bovine pituitary extract, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37℃ in a humidified atmosphere of 5%CO₂/95% air incubator. When the cell fusion reached above 80%, the cells were harvested and the concentration was adjusted to 1×10⁵ cells/mL. The mixture of 0.3 mL cell suspension and 0.3 mL Matrigel basement-membrane matrix was cultured in 12-well plate. The cell growth was observed under an inverted phase contrast microscope. At 14 days after culture, frozen sections were prepared and were stained with HE to observe the cells morphology, and immunohistochemical analysis was used to detect the antigen expressions of cytokeratin 7 (CK7) and CK19. Results Inverted phase contrast microscope observation showed that many free eccrine sweat gland tissues were seen after digestion of type II collagenase; eccrine sweat gland cells grew adhering to the wall at 3-5 days and continued division for 2-3 weeks to form single ring around the block sweat glands; cellular senescence were observed after 3-4 weeks. During the process of 3D culture, the single eccrine sweat gland cell divided into 2-4 cells after 2-3 days, and these cells subsequently formed small cell clusters, tubular-like structures and finally spheric-like shapes. After cultured for about 2 weeks, there was crack in part of the gelled mixture or liquefaction occurred. HE staining of frozen sections of the 3D cultures showed some of the tubular-like structures composed of 1-2 layers of epithelial cells, which were similar to the secretion part and the duct part of the eccrine sweat gland. Immunohistochemical analysis showed that CK7 and CK19 antigens expressed positively in the cells. Conclusion Human eccrine sweat gland cells cultured in Matrigel can form the 3D structures which simulate the morphology of eccrine sweat glands in vivo.

Citation： HUShenyou, CHENLu, LIXuexue, LIHaihong. THREE-DIMENSIONAL CULTURE AND MORPHOLOGICAL OBSERVATION OF HUMAN ECCRINE SWEAT GLAND CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(2): 162-166. doi: 10.7507/1002-1892.20140036 Copy