两种精原干细胞高效能纯化方法的比较研究

Comparative study of two high-efficiency methods for purifying spermatogonial stem cells

目的:比较贴壁分离法和免疫磁珠法纯化小鼠精原干细胞(mSSCs)的优缺点。方法:分别选取10只12-15日龄的雄性C57BL/6小鼠,颈椎脱臼法处死,摘取睾丸用酶消化法获得曲细精管单细胞悬液,分别用贴壁分离法和免疫磁珠法从单细胞悬液中分离纯化mSSCs,并针对两种方法在细胞数量、分离效率以及对细胞增殖生长的影响等方面进行比较。结果:两种纯化方法均可从小鼠曲细精管单细胞悬液中分离纯化得到干细胞,并可在体外培养后呈现出典型的精原干细胞特有的葡萄串状克隆,体外连续培养增殖超3个月。10只小鼠的睾丸经差异贴壁法纯化后可以得到3×10^(5)±0.4×10^(5)个mSSCs(n=5),细胞回收率(纯化后细胞数/曲细精管单细胞悬液细胞数)为1.5%±0.1%(n=5);经免疫磁珠法可以得到6×10^(5)±0.4×10^(5)个mSSCs(n=5),细胞回收率为3.0%±0.1%(n=5),免疫磁珠法得到的干细胞数量更高。差异贴壁法得到的干细胞更纯,因为体外培养5 d左右即得到干细胞集落,而免疫磁珠法得到的干细胞则约10 d才可以看到明显的细胞集落,但是两种纯化方法对细胞体外长期增殖生长没有明显的影响。结论:两种方法均可以纯化得到高质量的mSSCs,,但两种方法各有优缺点。差异贴壁法较免疫磁珠法经济、实用,无需购买专门的设备和抗体磁珠,但获得的细胞数量相对较低,用时也较长。

Objective:To compare the advantages and disadvantages of the differential attachment method and immunomagnetic bead method for purification of mouse spermatogonial stem cells(mSSCs).Methods:Ten male C57BL/6 mice aged 12~15 days were selected and sacrificed by cervical dislocation.Testes were collected and the seminiferous tubule single cell suspension was obtained by enzymatic digestion.mSSCs were purified by using the differential attachment method and immunomagnetic bead method respectively.Then a detailed comparison of the two methods in terms of cell number,separation efficiency,and impact on cell proliferation and growth was conducted.Results:Both of the methods could isolate and purify stem cells from single cell suspension of mouse seminiferous tubules.mSSCs showed typical grape cluster-like clones in vitro culture,which could be continuously cultured and proliferated for over 3 months in vitro.The testes of 10 mice could obtain 3×10^(5)±0.4×10^(5) mSSCs(n=5)by differential attachment method,cell recovery rate(the number of cells after purification/the number of cells of the single cell suspension of seminiferous tubules)was 1.5%±0.1%;6×10^(5)±0.4×10^(5) mSSCs(n=5)could be obtained by immunomagnetic bead method.The recovery rate was about 3%±0.1%,and the number of stem cells obtained by the immunomagnetic bead method was higher.The stem cells obtained by the differential attachment method were more pure,because the stem cell colonies were preferentially obtained after 5 days of in vitro culture,while the stem cells obtained by the immunomagnetic bead method needed to be cultured for about 10 days before the obvious cell colonies could be observed,but the two types of purification method had no obvious effect on the long-term growth of cells in vitro.Conclusion:Both methods can get high quality mSSCs,but both methods have their own advantages and disadvantages.The differential attachment method is more economical and practical than the other,it does not require special equipment,but the stem cell number obtained is relatively lower and the time needed is longer.