旋转生物反应器内球形体培养对人胎盘间充质干细胞炎症因子分泌的影响  

Effects of spheroid culture in a rotating bioreactor on secretion of inflammatory factors by human placenta-derived mesenchymal stem cells

作　　者：张萍萍 梁婷婷 范明松 陈黎 张世昌 Zhang Pingping;Liang Tingting;Fan Mingsong;Chen Li;Zhang Shichang(Department of Laboratory Medicine,First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,Jiangsu Province,China;Department of Obstetrics,First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,Jiangsu Province,China;Department of Gynaecology and Obstetrics,Shenzhen Hospital of Southern Medical University,Shenzhen 518110,Guangdong Province,China;Department of Clinical Laboratory Medicine,Shenzhen Hospital of Southern Medical University,Shenzhen 518110,Guangdong Province,China)

机构地区：[1]南京医科大学第一附属医院检验学部,江苏省南京市210029 [2]南京医科大学第一附属医院产科,江苏省南京市210029 [3]南方医科大学深圳医院妇产科,广东省深圳市518110 [4]南方医科大学深圳医院检验科,广东省深圳市518110

出　　处：《中国组织工程研究》2024年第19期3012-3017,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81671836),项目负责人:张世昌。

摘　　要：背景:生物反应器内球形体培养是既能维持间充质干细胞干性又能大规模扩增的体外培养方法,明确其对干细胞免疫调节作用的影响有利于指导临床应用。目的:探讨旋转生物反应器内球形体培养对人胎盘间充质干细胞炎症因子分泌的影响。方法:从人胎盘组织中分离培养人胎盘间充质干细胞,分别进行二维培养和旋转生物反应器培养,利用倒置相差显微镜、免疫组化染色和CCK-8实验观察细胞形态与增殖能力,RT-qPCR和流式荧光法检测多种炎症因子基因表达与蛋白分泌量。结果与结论:①旋转生物反应器内培养的人胎盘间充质干细胞聚集成多细胞球形体,数量与体积逐渐增加;②苏木精-伊红染色可见旋转生物反应器内培养4 d的人胎盘间充质干细胞在球形体内分布均匀,形态正常;③免疫组化染色显示球形体内有大量人胎盘间充质干细胞的细胞核Ki-67阳性;④CCK-8实验结果显示,生物反应器内球形体培养的人胎盘间充质干细胞活力显著高于二维培养的细胞;⑤RT-qPCR和流式免疫荧光法检测结果显示,旋转生物反应器内球形体培养的人胎盘间充质干细胞中白细胞介素1β、白细胞介素4、白细胞介素6、白细胞介素8、白细胞介素10、白细胞介素17、肿瘤坏死因子α和干扰素α的基因表达量和蛋白分泌量显著高于二维培养的细胞;⑥结果表明,旋转生物反应器内球形体培养能显著提高人胎盘间充质干细胞分泌多种炎症因子的能力,加强人胎盘间充质干细胞的免疫调节作用。BACKGROUND:Spheroid culture of mesenchymal stem cells in bioreactors is an in vitro culture method to maintain their stemness properties and allow for large-scale expansion.Clarifying its effects on the immunoregulation effect of stem cells is beneficial for their clinical application.OBJECTIVE:To investigate the effects of spheroid culture in a rotating bioreactor on the secretion of inflammatory factors by human placenta-derived mesenchymal stem cells.METHODS:Placenta-derived mesenchymal stem cells isolated from human placenta tissue were cultured in two-dimensional culture or a rotating bioreactor culture.Cell morphology and proliferation ability were observed using inverted phase contrast microscopy,immunohistochemical staining,and CCK-8 assay.The gene expression and protein secretion of several inflammatory factors were detected by RT-qPCR and flow immunofluorescence assay.RESULTS AND CONCLUSION:(1)Mesenchymal stem cells cultured in a rotating bioreactor aggregated into multicellular spheroids,which gradually increased in number and volume.(2)The hematoxylin-eosin staining results showed that the mesenchymal stem cells cultured in the rotating bioreactor for 4 days were evenly distributed and had normal morphology in the spheroids.(3)Immunohistochemical staining results revealed many mesenchymal stem cells with Ki-67 positive in the spheroids.(4)The CCK-8 assay results exhibited that the viability of mesenchymal stem cells derived from spheroid culture was significantly higher than that of cells cultured in two-dimensional culture.(5)The results of RT-qPCR and flow immunofluorescence assay demonstrated that the gene expression and protein secretion(interleukin 1β,interleukin-4,interleukin-6,interleukin-8,interleukin-10,interleukin-17,tumor necrosis factorαand interferonα)of inflammatory factors derived from mesenchymal stem cells cultured in the rotating bioreactor were significantly higher than those in two-dimensional culture.(6)Our results indicate that spheroid culture in a rotating bioreactor can sig

关 键 词：胎盘间充质干细胞 生物反应器 球形体 炎症因子 免疫调节 

分 类 号：R459.9[医药卫生—治疗学] R318.1[医药卫生—临床医学] Q813.1[生物学—生物工程]