中国组织工程研究 ›› 2015, Vol. 19 ›› Issue (19): 2973-2980.doi: 10.3969/j.issn.2095-4344.2015.19.004

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

自然沉降速度法分选骨髓间充质干细胞

蔡金宏1,林春博2,杨  渊2   

  1. 1广西医科大学创伤骨科手外科,广西壮族自治区南宁市  530021;2广西骨伤医院,广西壮族自治区南宁市  530001
  • 出版日期:2015-05-06 发布日期:2015-05-06
  • 通讯作者: 通讯作者:杨渊,教授,广西骨伤医院,广西壮族自治区南宁市 530001
  • 作者简介:蔡金宏,男,1986年生,广西壮族自治区北海市人,广西医科大学在读硕士,主要从事骨髓间充质干细胞研究。

Isolation of bone marrow mesenchymal stem cells by natural sedimentation velocity method

Cai Jin-hong1, Lin Chun-bo2, Yang Yuan2   

  1. 1Department of Hand Surgery, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China; 2Guangxi Orthopedics Hospital, Nanning 530001, Guangxi Zhuang Autonomous Region, China
  • Online:2015-05-06 Published:2015-05-06
  • Contact: Corresponding author: Yang Yuan, Professor, Guangxi Orthopedics Hospital, Nanning 530001, Guangxi Zhuang Autonomous Region, China
  • About author:Cai Jin-hong, Studying for master’s degree, Department of Hand Surgery, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China

摘要:

背景:在前期研究低渗结合自然沉降分选兔骨髓间充质干细胞的基础上,以工程学关于颗粒流体力学为指导,求出兔骨髓间充质干细胞在培养液中的沉降速度。通过自然沉降速度法分选及鉴定细胞,建立一个简单易行的分离、纯化、增殖间充质干细胞的方法。
目的:探讨自然沉降法分选骨髓间充质干细胞的可行性。
方法:梯度密度离心法确定兔骨髓间充质干细胞的密度区间(ρ1),扫描电镜测量出兔骨髓间充质干细胞的直径(d),液体密度计测算出培养液的密度(ρ2),黏度计测算出培养液的黏度(μ),选择合适的公式计算出骨髓间充质干细胞的沉降速度(Vt)。以此沉降速度从兔的骨髓细胞中分选兔骨髓间充质干细胞,体外培养并观察其增殖能力、细胞纯度、细胞分化潜能,同时测定兔骨髓间充质干细胞克隆集落形成率,记录原代培养时间。
结果与结论:①通过电镜测量得出兔骨髓间充质干细胞的直径为(20.37±4.58) μm,进一步确定兔骨髓间充质干细胞在培养液中的沉降速度为50-55 mm/h。②通过自然速度沉降法可成功从兔骨髓组织中分选出骨髓间充质干细胞,分选出的骨髓间充质干细胞原代培养呈成纤维样集落生长,经诱导后具有成骨、成脂分化潜能。③与梯度密度离心法比较,自然速度沉降法分离出的间充质干细胞具有原代培养时间短,细胞集落形成率高的特点。④自然速度沉降法在间充质干细胞分选过程中不采取任何干预措施,也不添加任何分离介质,仅通过物理原理自然沉降,可能对细胞损伤小,使细胞原有的生物学特性得到保留。

关键词: 干细胞, 骨髓干细胞, 自然沉降, 骨髓间充质干细胞, 沉降速度, 细胞分选

Abstract:

BACKGROUND: In previous studies, it is satisfied to sorting bone marrow mesenchymal stem cells based on natural sedimentation combined with low permeation. Then, based on particle hydromechanics theory, the settling velocity of bone marrow mesenchymal stem cells in culture liquid is calculated. A simple and easy method of separation, purification and proliferation of bone marrow mesenchymal stem cells is established by natural sedimentation velocity.
OBJECTIVE: To explore the feasibility of sorting bone marrow mesenchymal stem cells by natural sedimentation velocity method.
METHODS: The density interval of rabbit bone marrow mesenchymal stem cells (ρ1) was determined by density gradient centrifugation method. The diameter of rabbit bone marrow mesenchymal stem cells (d) was measured by scanning electron microscope. The density of culture liquid (ρ2) was measured by liquid density meter. The viscosity of the culture liquid (μ) was measured by viscosity meter. The settling velocity of bone marrow mesenchymal stem cells (Vt) was derived from the above four numerical values with the appropriate formula. Bone marrow mesenchymal stem cells were sorted from bone marrow tissue by natural sedimentation velocity method. Cell proliferation, purity and differentiation were observed. Meanwhile, the primary culture time of three 
different cell sorting methods was recorded; the colony formation rate of rabbit bone marrow mesenchymal stem cells was determined.
RESULTS AND CONCLUSION: (1) The diameter of rabbit bone marrow mesenchymal stem cells was (20.37±4.58) μm, and the setting velocity of rabbit bone marrow mesenchymal stem cells in the culture liquid was 50-55 mm/h. (2) Bone marrow mesenchymal stem cells could be successfully isolated from bone marrow tissue of rabbits by the natural sedimentation velocity method, which could be induced into osteoblasts and adipocytes. (3) The natural sedimentation velocity group cost less time than the other two density gradient centrifugation groups in the primary culture stage. The colony formation rate of the natural sedimentation velocity group was higher than that of the other two groups. (4) Natural sedimentation velocity method did not impose any intervention measures for sorting cells, which maybe maximally maintain cell viability and biological characteristics. The whole separation process was simple and safe, which may have little damage to the cells.

Key words: Bone Marrow, Mesenchymal Stem Cells, Cell Separation

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