中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (19): 2997-3003.doi: 10.12307/2022.377

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

两种方法提取人脐带间充质干细胞三维培养生物学特性的比较

陈思铭，胡加伟，李丽丽，伍永强，彭维杰   

  1. 赣南医学院，江西省赣州市   341000
  • 收稿日期:2021-03-10 修回日期:2021-04-17 接受日期:2021-08-04 出版日期:2022-07-08 发布日期:2021-12-28
  • 通讯作者: 彭维杰，博士，教授，硕士、博士研究生导师，赣南医学院，江西省赣州市 341000
  • 作者简介:陈思铭，男，1996年生，江西省赣州市人，汉族，赣南医学院在读硕士，主要从事干细胞、组织工程、生物打印研究。
  • 基金资助:
    国家自然科学基金(81860327)，项目负责人：彭维杰

Comparison of biological characteristics of two kinds of primary isolated human umbilical cord mesenchymal stem cells under three-dimensional culture

Chen Siming, Hu Jiawei, Li Lili, Wu Yongqiang, Peng Weijie   

  1. Gannan Medical University, Ganzhou 341000, Jiangxi Province, China
  • Received:2021-03-10 Revised:2021-04-17 Accepted:2021-08-04 Online:2022-07-08 Published:2021-12-28
  • Contact: Peng Weijie, MD, Professor, Master, Doctoral supervisor, Gannan Medical University, Ganzhou 341000, Jiangxi Province, China
  • About author:Chen Siming, Master candidate, Gannan Medical University, Ganzhou 341000, Jiangxi Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81860327 (to PWJ)

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摘要：

文题释义：
人脐带间充质干细胞：是指存在于新生儿脐带组织中的一种围产期干细胞。这类细胞符合国际细胞治疗协会提出的鉴定间充质干细胞的最低标准：①贴壁塑料材质培养器皿生长能力；②细胞表型：阳性表达CD73、CD90、CD105，同时阴性表达CD34、CD45、CD14、CD19和HLA-DR；③具有多向分化潜能：包括向骨、软骨、脂肪分化的能力。近些年，人脐带间充质干细胞作为组织工程的种子细胞得到广泛的应用。
三维培养：是一种新型的模拟体内细胞生长微环境的培养方式。传统二维培养的关键局限性是无法模拟体内结构和微环境，与体内细胞相比，在形态特征、增殖、分化潜能、信号传导等方面具有差异。三维培养系统比二维培养能更有效地模仿组织样结构而得到研究者的关注，主要包括细胞球体、水凝胶包埋、生物反应器、生物支架等培养方式。

背景：人脐带间充质干细胞提取方法主要有酶消化法和组织块贴壁法，明确两种方法提取的细胞在二维、三维培养下的生物学特性，对组织工程中种子细胞提取方法及培养方式的选择具有重要意义。
目的：比较酶消化法和组织块贴壁法提取的人脐带间充质干细胞在二维、三维培养下的生物学特性。
方法：酶消化法和组织块贴壁法分离人脐带间充质干细胞，扩增培养至第3代进行二维、三维培养；扫描电镜观察细胞球表征；死活染色观察细胞球存活情况；Alamar Blue检测细胞增殖能力；流式细胞术检测免疫表型CD34、CD44、CD45、CD90、CD105的表达；免疫荧光、免疫印迹检测多能性转录因子Sox2、Nanog、Oct4表达。
结果与结论：①酶消化法比组织块贴壁法获取细胞时间更短，二维培养下组织块贴壁法的细胞增殖能力优于酶消化法，但在三维培养条件下细胞增殖缓慢且无差异。三维培养3 d后细胞成紧密球体，存活良好。②两种方法提取的细胞在二维、三维培养条件下均表达CD44、CD90、CD105，不表达CD34、CD45。二维培养下，酶消化法提取细胞的CD105表达高于组织块贴壁法；三维培养下，酶消化法提取细胞的CD90、CD105表达也高于组织块贴壁法。③二维和三维培养下两种方法提取细胞均表达多能性转录因子Sox2、Nanog、Oct4。二维培养下，酶消化法提取细胞3个转录因子表达均高于组织块贴壁法；但三维培养下，组织块贴壁法提取细胞Sox2的表达高于酶消化法，Nanog、Oct4的表达两组间无差异。

https://orcid.org/0000-0002-8904-1903 (陈思铭) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 干细胞, 脐带, 间充质干细胞, 三维培养, 细胞球, 生物学特性, 免疫表型, 转录因子

Abstract: BACKGROUND: The human umbilical cord mesenchymal stem cells isolation methods mainly include enzymatic digestion and tissue attachment. It is important to understand the biological characteristics of the two methods isolated cells in two-dimensional and three-dimensional culture conditions; it is of great significance to the selection of cell isolation methods and culture condition in tissue engineering.  
OBJECTIVE: To compare the biological characteristics of human umbilical cord mesenchymal stem cells from the enzymatic digestion and tissue attachment methods in two-dimensional and three-dimensional culture conditions.
METHODS:  Human umbilical cord mesenchymal stem cells were isolated by digestion method and tissue attachment method, amplified and cultured to passage 3 and divided into two-dimensional and three-dimensional culture. Scanning electron microscope was used to observe the cell spheroids. Live\dead staining was utilized to detect cell spheroid survival. Alamar Blue was applied to detect cell proliferation. Flow cytometry was used to detect the expression of immunophenotypes CD34, CD44, CD45, CD90, and CD105. Immunofluorescence and western blot assay were used to detect the expression of pluripotent transcription factors Sox2, Nanog, and Oct4.  
RESULTS AND CONCLUSION: (1) Enzymatic digestion method took shorter time to obtain cells than the attachment method. The cell proliferation ability of the attachment method was better than that of the enzyme digestion under two-dimensional culture, but the proliferation was slow and there was no difference under three-dimensional culture conditions. After 3 days of spheroid culture, the cells became compact spheroids and survived well. (2) The cells isolated by the two methods all expressed CD44, CD90, and CD105 under two-dimensional and three-dimensional culture conditions, but did not express CD34 and CD45. Under two-dimensional culture, the expression of CD105 cells isolated by enzymatic digestion was higher than that of tissue attachment method; under three-dimensional culture, the expression of CD90 and CD105 cells isolated by enzymatic digestion was also higher than that of tissue attachment method. (3) Both isolation methods under two-dimensional and three-dimensional culture expressed pluripotency transcription factors Sox2, Nanog, and Oct4. Under the two-dimensional culture, the expression of the three transcription factors of the cells extracted by the enzymatic digestion method was higher than that of the tissue attachment method. However, under the three-dimensional culture, the expression of Sox2 in the tissue attachment method was higher than that of the enzymatic digestion method, and the expression levels of Nanog and Oct4 were not different between the two groups.

Key words: stem cells, umbilical cord, mesenchymal stem cells, three-dimensional culture, cell pellets, biological characteristics, immunophenotype, transcription factors

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