蛋白芯片筛选用于培养Muse细胞的纤维蛋白原和明胶组合基质  

Preparation of protein chips for screening matrix for Muse cell culture based on tibrinogen and gelatin

作　　者：李秀亚 盛扬 王圣依 黎悦 张汝芝[2] 潘艳 孙一新 邓林红 Mark Bradley 张嵘 Li Xiuya;Sheng Yang;Wang Shengyi;Li Yue;Zhang Ruzhi;Pan Yan;Sun Yixin;Deng Linhong;Mark Bradley;Zhang Rong(School of Materials Science and Engineering,Changzhou University,Changzhou 213164,Jiangsu Province,China;Department of Dermatology and Venereology,Third Affiliated Hospital of Soochow University,Changzhou 213000,Jiangsu Province,China;Institute of Biomedical Engineering and Health Sciences,Changzhou University,Changzhou 213164,Jiangsu Province,China;School of Chemistry,University of Edinburgh,Edinburgh EH93 JJ1,Scotland)

机构地区：[1]常州大学材料科学与工程学院,江苏省常州市213164 [2]苏州大学附属第三医院皮肤性病科,江苏省常州市213000 [3]常州大学生物医学工程与健康科学研究院,江苏省常州市213164 [4]爱丁堡大学化学学院,苏格兰爱丁堡市EH93 JJ1

出　　处：《中国组织工程研究》2022年第21期3312-3318,共7页Chinese Journal of Tissue Engineering Research

基　　金：江苏省六大人才高峰创新团队项目(SWYY-CXTD-001),项目负责人:张嵘;常州市科技局国际合作项目(CZ20190019),项目负责人:张嵘。

摘　　要：背景:Muse细胞在再生医学中具有巨大的医疗潜力,但目前其体外培养需要通过细胞簇悬浮的方法,不利于大规模扩增和应用。蛋白质是细胞外基质的重要组成部分之一,不同的蛋白质在细胞生长过程中起着不同的作用,因此可以通过高通量的方法快速筛选有利于Muse细胞在二维表面黏附扩增的蛋白质基质材料。目的:选出合适的蛋白质基材,用于Muse细胞的表面黏附培养和传代。方法:以牛血清白蛋白、牛血纤维蛋白原和明胶为原料,通过喷墨打印进行点样和蛋白质两两物理混合,制备成蛋白微点阵列芯片,利用高通量筛选方法选出适合Muse细胞黏附的最佳蛋白质组合。采用常规方法在蛋白基材表面进行Muse细胞传代培养并进行表征。结果与结论:(1)由高通量筛选出两种有利于原代Muse细胞黏附的蛋白组合,两种组合分别为:牛血清白蛋白与明胶、牛血纤维蛋白原与明胶,组合比例均为7/17;(2)利用CCK8法测试原代Muse细胞在两种组合蛋白包被板的生长状况,各组细胞都呈现正常的增殖情况;经过6 d的培养,在牛血纤维蛋白原与明胶组合包被板上培养的细胞呈现较好的增殖状态;(3)通过对在蛋白包被板上传代培养的1代及3代Muse细胞的免疫荧光染色结果进行分析,结果显示,由牛血纤维蛋白原和明胶按7/17比例混合制备的基材能支持Muse细胞至少3代的黏附培养与扩增,并且维持较好的干细胞特性。BACKGROUND:Stress-tolerant multilineage differentiated(Muse)cells have excellent medical potential for regenerative medicine.However,at present,Muse cells need to be cultured and proliferated in vitro by suspension of cell aggregation,which is not conducive to large-scale expansion and application.Protein is one of the important components of extracellular matrix.Different proteins play different roles in cell growth.Therefore,the high-throughput method can be used to rapidly screen protein matrix that is conducive to the adhesion and proliferation of Muse cells on a two-dimensional surface.OBJECTIVE:To identify suitable protein substrates for surface adhesion and passaging of Muse cells.METHODS:Bovine serum albumin,bovine blood fibrin,and gelatin were used as raw materials,and their solutions were printed on glass slides by an ink jet printer and mixed on site to prepare protein microarray chips.The optimal protein combination for the adhesion of Muse cells was identified by using highthroughput screening approach.The Muse cells were cultured and passaged on the identified protein substrates for characterization.RESULTS AND CONCLUSION:(1)Two protein combinations were obtained by high-throughput screening,which were beneficial to the adhesion of primary Muse cells.They were bovine serum albumin/gelatin and bovine blood fibrinogen/gelatin,with combination ratios of 7/17 for both.(2)CCK-8 assay showed that Muse cells proliferated well in all groups.Among them,cells cultured on plates coated with bovine blood fibrinogen/gelatin proliferated well in 6 days.(3)By analyzing the immunofluorescence staining results of passages 1 and 3 of Muse cells cultured on the protein-coated plate,it was found that the combination of bovine blood fibrinogen and gelatin with a ratio of 7/17 in weight supported the adhesion and proliferation of Muse cells for at least 3 passages,maintaining good stem cell characteristics.

关 键 词：Muse细胞 细胞医疗 细胞扩增 牛血清白蛋白 牛血纤维蛋白原 明胶 高通量筛选 干细胞特性 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]