Key Factors And Mechanism For The In Vitro Culture System Of Human Sweat Gland Cells

There are tens of millions of burn patients every year in our country, which mild burn patients is acceptable for skin repair, but for deep burn patients, sweat glands can’t by subcutaneous remaining normal tissue to later repair. In the resent years, human sweat gland cells have been used in tissue engineering skin construction and skin damage repair research has become a hot spot. The biological characteristics of Sweat gland cells and regeneration of sweat glands start late than other research, in order to effectively promote the proliferation of sweat gland cells in vitro, more exploration focus on maintaining biological characteristics and function of sweat gland cells. At present, there are two kinds of culture medium, which are used to culture the sweat gland cells: serum and serum free, but the system is not perfect. Our research aims to compare different culture medium for maintaining the biological characteristics and functions of sweat gland cell, and further explore mechanism of serum and sonic hedgehog in sweat gland in vitro culture conditions and optimize human sweat gland cells in vitro.Part I A novel culture medium for human sweat gland cells: Serum is an indispensable factor in the maintenance of biological characteristicsObjective: To formulate an ideal medium for the culture of human sweat gland cells(HSGCs) and to establish a method to induce HSGCs proliferation while maintaining the characteristics of sweat gland cells.Methods: Sweat gland cells isolated from human skin were cultured for two weeks in three different media, including normal sweat gland(SG) medium, keratinocyte growth medium-2(KGM-2) medium, and a mix named SG medium KGM-2 medium 1:1. HSGC morphology, proliferation, marker expression and sweat gland-like structure forming efficiency were compared using real-time PCR and three-dimensional tissue culture.Results: HSGCs cultured in SG medium KGM-2 medium 1:1 displayed a higher proliferation rate and a more stable morphology in comparison to those cultured in SG medium and KGM-2 medium. Real-time PCR results indicated that they also exhibited higher expression levels of SMA, K77, CEA, K8, K18, EDAR, C-MYC, KLF-4, and OCT-4 than cells cultured in SG medium and KGM-2 medium. Three-dimensional culture analysis showed that HSGCs cultured in SG medium KGM-2 medium 1:1 differentiated into sweat gland-like structures, while cells cultured in KGM-2 medium underwent cornification. We further showed that the maintenance of biological characteristics occurred due to the presence of serum. Cells cultured in medium in the absence of serum differentiated into keratinocytes.Conclusion: SG medium KGM-2 medium 1:1 appears to be a suitable culture medium for HSGCs.Part II Effect of Hedgehog Sonic on the biological characteristics and function of sweat gland cells cultured in vitroObjective: To formulate an ideal medium for the culture of human sweat gland cells(HSGCs) and to establish a method to induce HSGCs proliferation while maintaining the characteristics of sweat gland cells.Methods: Sweat gland cells isolated from human skin were cultured for two weeks in two different media, including a mix named SG medium KGM-2 medium 1:1 and a mix named SG medium KGM-2 medium 1:1 with Shh. HSGC morphology, proliferation, marker expression and sweat gland-like structure forming efficiency were compared using real-time PCR and three-dimensional tissue culture.Results: HSGCs cultured in a mix named SG medium KGM-2 medium 1:1 with Shh displayed a same proliferation rate in comparison to those cultured in a mix named SG medium KGM-2 medium 1:1. Real-time PCR results indicated that they also exhibited higher expression levels of SMA, K77, CEA, K8, K18, EDAR, C-MYC, KLF-4, and OCT-4 than cells cultured in SG medium but same as the original sweat gland cells. KGM-2 medium 1:1. Three-dimensional culture analysis showed that HSGCs cultured in SG medium KGM-2 medium 1:1 with Shh differentiated into more sweat gland-like structures than cells cultured in a mix named SG medium KGM-2 medium 1:1.Conclusion: Shh helps to improve the human sweat gland cells to form more sweat gland-like structures.Summary: Through the comparison of human sweat gland cells culture mediums, our study further optimize the human sweat gland cells culture system in vitro and found suitable culture conditions for sweat gland cells in a mix named SG medium KGM-2 medium 1:1. Besides, we proved serum is a important factor in maintaining the biological characteristics of human sweat gland cells. On this basis, we added the sonic hedgehog factor in the former mixed medium, we proved that sonic hedgehog factor can improve the human sweat gland cells in 3D culture in vitro in formation of sweat human gland like structure efficiency.