人脐带来源间充质干细胞分离培养方法的优化初探
发布时间：2018-09-06 11:05
【摘要】：目的：通过对混合酶组分、培养基的选择、接种密度、首次换液时间等条件进行优化,探讨优选人脐带来源间充质干细胞(MSCs)体外培养纯化的最佳方法,为脐带MSCs的制备和在临床的广泛应用奠定研究基础。 方法： 1不同混合酶组分分离培养人脐带来源间充质干细胞 无菌条件下取正常足月剖腹产的脐带近胎几段；脐带组织均分为9等分,每一等分脐带长度为0.5cm,仔细剔除动静脉,剪碎组织呈1mm×1mm×lmm大小；按不同混合酶浓度比值划分为混合酶Ⅰ组,混合酶Ⅱ组和混合酶Ⅲ组,每组再按消化时间分为1、2、3h三个亚组：重悬细胞终体积均定位4ml并计数,每个亚组计数6次,采用含血清的DMEM培养液体外培养细胞。观察、计数并比较相同时间消化时间内细胞总数,活细胞比值以及对细胞增殖活性的影响。 2不同培养基、接种密度和首次换液时间培养人脐带来源间充质干细胞 将获得的单个核细胞分别以5×105、1×106、5×106、1×107密度接种于六孔板中,观察细胞贴壁延伸时间、原代培养时间；分别以间充质专用培养基(MesencultTM),L—DMEM、DMEM/F12均含有10%胎牛血清,对分离得到的细胞进行培养,观察细胞贴壁及生长状况。 3人脐带来源间充质干细胞的鉴定和定向诱导流式细胞仪检测人脐带来源间充质干细胞CD34、CD44、CD45、CD29、CD105的表达情况。Vonkassa方法和油红O染色分别检测人脐带来源间充质干细胞成骨、成脂的诱导。 结果： 1不同混合酶组分分离培养人脐带来源间充质干细胞：采用三种不同混合酶浓度进行消化,发现相同消化时间内,混合酶Ⅲ组消化细胞最多,细胞总数与其他组比较,具有显著性差异(P0.05)。在作用时间为3h时,混合酶Ⅲ组获取的细胞中活细胞比值显著低于混合酶Ⅰ组和混合酶Ⅱ组获取细胞的活细胞比值,差异具有统计学意义(P0.01)。 2比较4种接种密度,观察比较发现以5×105个/cm2密度接种细胞长势缓慢,一直无法传代,以5×106个/cm2密度接种细胞在出现延伸时间(92.0±6.3h)及原代培养时间(21.2±4.1d)方面均短于其余接种密度,差异具有显著性意义。 3不同培养基对人脐带来源间充质干细胞体外培养的影响：18份标本中其中用间充质专用培养基培养的有14份(77.8%)得到均一的间充质干细胞。其余4份得到的异质性贴壁细胞多,细胞不均一,形态多样。采用DMEM/F12培养的18份标本中,有11份(61.1%)得到均一的间充质干细胞,6份得到异质性贴壁细胞,1份细胞呈悬浮状态,未贴壁。用L-DMEM培养基的18份标本中,有9份(50.0%)最终得到均一的间充质干细胞,但生长速度缓慢,7份得到异质性贴壁细胞,2份细胞呈悬浮状态,未贴壁。 4流式细胞仪表面标志鉴定CD31、CD34、CD45呈阴性,CD44、CD29、CD105呈阳性,符合脐带来源间充质干细胞的生长特性。 5脐带来源间充质干细胞经成骨、成脂诱导后可表达骨细胞、脂肪细胞特征。 结论： 在其他条件不变的情况下,0.3%Ⅱ型胶原酶、0.1%胰酶、0.02%EDTA、0.1%透明质酸酶和0.1%DNA酶混合液消化脐带组织块2h获取间充质干细胞,采用间充质干细胞专用培养基体外培养所得细胞,5×106个/cm2为脐带MSCs培养的适宜接种密度。优化条件下培养的细胞符合间充质干细胞特征。我们优化了人脐带来源间充质干细胞高效的分离扩增体系,为将来成体干细胞的临床应用提供了可靠的MSCs新来源。
[Abstract]:OBJECTIVE: To explore the best method of culture and purification of human umbilical cord derived mesenchymal stem cells (MSCs) in vitro by optimizing the composition of mixed enzymes, the selection of medium, the density of inoculation, and the time of first fluid exchange, so as to lay a foundation for the preparation and clinical application of umbilical cord MSCs.
Method:
1 Isolation and culture of human umbilical cord derived mesenchymal stem cells with different mixed enzyme components
Umbilical cord tissues were divided into 9 equal parts, each equal length of umbilical cord was 0.5 cm, the arteries and veins were carefully removed, and the shredded tissues were 1 mm 65 The cells were divided into three subgroups at 1, 2, and 3 h: the final volume of suspended cells was positioned at 4 ml and counted. Each subgroup was counted 6 times. Cells were cultured in serum-containing DMEM medium in vitro.
2 culture of human umbilical cord derived mesenchymal stem cells with different culture medium, inoculation density and first exchange time.
Mononuclear cells were inoculated into six-well plate at the density of 5 105, 1 106, 5 106, 1 Condition.
The expression of CD34, CD44, CD45, CD29 and CD105 in human umbilical cord-derived mesenchymal stem cells was detected by flow cytometry. The osteogenesis and adipogenic induction of human umbilical cord-derived mesenchymal stem cells were detected by Vonkassa method and oil red O staining respectively.
Result:
1 Isolation and culture of human umbilical cord-derived mesenchymal stem cells with different enzyme mixtures: three different enzyme mixtures were used for digestion. It was found that the number of digestive cells in group III was the largest in the same digestive time, and the total number of cells in group III was significantly different from that in other groups (P 0.05). Cell ratio was significantly lower than that of mixed enzyme group I and mixed enzyme group II (P 0.01).
2. Comparing the four inoculation densities, it was found that the growth rate of the cells inoculated with 5 105 / cm 2 density was slower than that of the other inoculation densities. The cells inoculated with 5
3 Effects of different media on human umbilical cord-derived mesenchymal stem cells in vitro culture: 14 (77.8%) of the 18 specimens were cultured in special mesenchymal medium to obtain homogeneous mesenchymal stem cells. Homogeneous mesenchymal stem cells were obtained in 61.1%. Heterogeneous adherent cells were obtained in 6 samples. One cell was suspended and not adhered to the wall.
Flow cytometry showed that CD31, CD34 and CD45 were negative, CD44, CD29 and CD105 were positive, which accorded with the growth characteristics of umbilical cord derived mesenchymal stem cells.
5 umbilical cord derived mesenchymal stem cells can express osteoblasts and adipocytes after osteogenic and adipogenic induction.
Conclusion:
Under the same conditions, 0.3% collagenase type II, 0.1% trypsin, 0.02% EDTA, 0.1% hyaluronidase and 0.1% DNA enzyme mixture were used to digest umbilical cord tissue blocks for 2 hours to obtain mesenchymal stem cells. The cells were cultured in vitro using the special medium of mesenchymal stem cells, and 5 *106/cm 2 was the suitable density for umbilical cord MSCs culture. The cultured cells conform to the characteristics of mesenchymal stem cells. We optimized the efficient isolation and amplification system of human umbilical cord-derived mesenchymal stem cells and provided a reliable new source of MSCs for the clinical application of adult stem cells in the future.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2226159
[Abstract]:OBJECTIVE: To explore the best method of culture and purification of human umbilical cord derived mesenchymal stem cells (MSCs) in vitro by optimizing the composition of mixed enzymes, the selection of medium, the density of inoculation, and the time of first fluid exchange, so as to lay a foundation for the preparation and clinical application of umbilical cord MSCs.
Method:
1 Isolation and culture of human umbilical cord derived mesenchymal stem cells with different mixed enzyme components
Umbilical cord tissues were divided into 9 equal parts, each equal length of umbilical cord was 0.5 cm, the arteries and veins were carefully removed, and the shredded tissues were 1 mm 65 The cells were divided into three subgroups at 1, 2, and 3 h: the final volume of suspended cells was positioned at 4 ml and counted. Each subgroup was counted 6 times. Cells were cultured in serum-containing DMEM medium in vitro.
2 culture of human umbilical cord derived mesenchymal stem cells with different culture medium, inoculation density and first exchange time.
Mononuclear cells were inoculated into six-well plate at the density of 5 105, 1 106, 5 106, 1 Condition.
The expression of CD34, CD44, CD45, CD29 and CD105 in human umbilical cord-derived mesenchymal stem cells was detected by flow cytometry. The osteogenesis and adipogenic induction of human umbilical cord-derived mesenchymal stem cells were detected by Vonkassa method and oil red O staining respectively.
Result:
1 Isolation and culture of human umbilical cord-derived mesenchymal stem cells with different enzyme mixtures: three different enzyme mixtures were used for digestion. It was found that the number of digestive cells in group III was the largest in the same digestive time, and the total number of cells in group III was significantly different from that in other groups (P 0.05). Cell ratio was significantly lower than that of mixed enzyme group I and mixed enzyme group II (P 0.01).
2. Comparing the four inoculation densities, it was found that the growth rate of the cells inoculated with 5 105 / cm 2 density was slower than that of the other inoculation densities. The cells inoculated with 5
3 Effects of different media on human umbilical cord-derived mesenchymal stem cells in vitro culture: 14 (77.8%) of the 18 specimens were cultured in special mesenchymal medium to obtain homogeneous mesenchymal stem cells. Homogeneous mesenchymal stem cells were obtained in 61.1%. Heterogeneous adherent cells were obtained in 6 samples. One cell was suspended and not adhered to the wall.
Flow cytometry showed that CD31, CD34 and CD45 were negative, CD44, CD29 and CD105 were positive, which accorded with the growth characteristics of umbilical cord derived mesenchymal stem cells.
5 umbilical cord derived mesenchymal stem cells can express osteoblasts and adipocytes after osteogenic and adipogenic induction.
Conclusion:
Under the same conditions, 0.3% collagenase type II, 0.1% trypsin, 0.02% EDTA, 0.1% hyaluronidase and 0.1% DNA enzyme mixture were used to digest umbilical cord tissue blocks for 2 hours to obtain mesenchymal stem cells. The cells were cultured in vitro using the special medium of mesenchymal stem cells, and 5 *106/cm 2 was the suitable density for umbilical cord MSCs culture. The cultured cells conform to the characteristics of mesenchymal stem cells. We optimized the efficient isolation and amplification system of human umbilical cord-derived mesenchymal stem cells and provided a reliable new source of MSCs for the clinical application of adult stem cells in the future.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
【引证文献】
相关硕士学位论文 前1条
1 武爽;鲁西黄牛脐带间充质干细胞的分离培养及生物学特性研究[D];中国农业科学院;2012年
，本文编号：2226159
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