两种方法培养人胎盘间充质干细胞的生物特性比较
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摘要
背景:胎盘间充质干细胞能够为细胞治疗提供理想的种子细胞,但其培养未曾使用过胰酶冷消化法,故对此方法进行探索,并与组织块法进行比较,从中筛选出一种方便易行、培养成功率更高的方法。目的:观察胰酶冷消化法与组织块法分离培养的人胎盘间充质干细胞的生物学特性。方法:采用胰酶冷消化法和组织块法从人胎盘中分离、纯化和传代培养人胎盘间充质干细胞(n=6)。记录胎盘间充质干细胞首次出现时间及原代培养周期,绘制第3代细胞生长曲线,流式细胞仪分析第3代细胞表面标志,检测其向成神经及成脂方向诱导分化能力。结果与结论:(1)使用上述2种培养方法均可获得胎盘间充质干细胞,胰酶冷消化法首次出现贴壁细胞时间早于组织块法(P <0.05),原代培养周期短于组织块法(P <0.05);(2)生长曲线提示在进入平台期后的各个时间点胰酶冷消化法培养的细胞数量明显多于组织块法(P<0.05);(3)2种细胞具有均一的细胞表型,均表达CD29,CD105,不表达CD34,CD45;(4)2种方法培养的胎盘间充质干细胞经诱导后均具有成神经及成脂分化潜能;(5)上述结果表明,对于胎盘间充质干细胞的培养而言,胰酶冷消化法具有明显优势。
BACKGROUND: Placental mesenchymal stem cells can provide ideal seed cells for cell therapy, but trypsin cold digestion has not been used for cell culture. Therefore, we explored this method and compared it with the tissue explant method to screen a more convenient and more successful culture method. OBJECTIVE: To observe the biological features of human placental mesenchymal stem cells isolated by trypsincold digestion and tissue explant method. METHODS: We isolated, purified, and subcultured mesenchymal stem cells(n=6) from human placenta by using trypsin cold digestion method and tissue explant method. The first appearance time and primary culture period of placental mesenchymal stem cells isolated using two methods were recorded. The growth curves of passage 3 cells from two culture methods were generated. Flow cytometry was used to analyze the expression of surface markers of passage 3 cells isolated using the two culture methods, and to study the neural and adipogenic differentiation of the cells. RESULTS AND CONCLUSION: Placental mesenchymal stem cells could be obtained using both culture methods. Adherent cells in the trypsin cold digestion group appeared significantly earlier than those in the tissue explant group(P < 0.05), and the primary culture period was also significantly shorter in the trypsin cold digestion method(P < 0.05). The growth curve of passage 3 cells showed a higher cell number at plateau growth using trypsin cold digestion than tissue explant method(P < 0.05). The cells isolated using two culture methods were positive for CD29 and CD105, but negative for CD34 and CD45, indicating the same cell phenotypes. After the induction, placental mesenchymal stem cells cultured by both methods had the potential to differentiate into nerve cells and adipocytes. Therefore, both culture methods can produce placental mesenchymal stem cells, but trypsin cold digestion method is more suitable for the culture of this kind of cells as compared with the tissue explant method.
BACKGROUND: Placental mesenchymal stem cells can provide ideal seed cells for cell therapy, but trypsin cold digestion has not been used for cell culture. Therefore, we explored this method and compared it with the tissue explant method to screen a more convenient and more successful culture method. OBJECTIVE: To observe the biological features of human placental mesenchymal stem cells isolated by trypsincold digestion and tissue explant method. METHODS: We isolated, purified, and subcultured mesenchymal stem cells(n=6) from human placenta by using trypsin cold digestion method and tissue explant method. The first appearance time and primary culture period of placental mesenchymal stem cells isolated using two methods were recorded. The growth curves of passage 3 cells from two culture methods were generated. Flow cytometry was used to analyze the expression of surface markers of passage 3 cells isolated using the two culture methods, and to study the neural and adipogenic differentiation of the cells. RESULTS AND CONCLUSION: Placental mesenchymal stem cells could be obtained using both culture methods. Adherent cells in the trypsin cold digestion group appeared significantly earlier than those in the tissue explant group(P < 0.05), and the primary culture period was also significantly shorter in the trypsin cold digestion method(P < 0.05). The growth curve of passage 3 cells showed a higher cell number at plateau growth using trypsin cold digestion than tissue explant method(P < 0.05). The cells isolated using two culture methods were positive for CD29 and CD105, but negative for CD34 and CD45, indicating the same cell phenotypes. After the induction, placental mesenchymal stem cells cultured by both methods had the potential to differentiate into nerve cells and adipocytes. Therefore, both culture methods can produce placental mesenchymal stem cells, but trypsin cold digestion method is more suitable for the culture of this kind of cells as compared with the tissue explant method.
引文
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[11]刘巨超,张兰,张博峰.新生大鼠肝细胞分离及体外培养方法[J].中国组织工程研究与临床康复,2009,13(53):10465-10468.
[12]王绮雯,吴启端,陈奕芝.温胰蛋白酶消化法和冷消化法培养乳鼠心肌细胞的比较[J].中国医药指南,2013,11(20):401-403.
[13]张飞,王一雄,武忠炎,等.人脐带间充质干细胞生物特性比较:胰酶冷消化和组织块法体外培养[J].中国组织工程研究, 2014,18(41):6614-6619.
[14]刘芳,杨辉,汪兆艳.自体骨髓间充质干细胞移植治疗唐氏综合征临床观察[J].转化医学杂志,2016,5(5):288-290.
[15]Li J, Wei Y, Yan L, et al. Multiplacenta derived stem cell/cytokine treatment increases survival time in a mouse model with radiationinduced bone marrow damage. Cytotechnology. 2016; 68(6):2677-2686.
[16]高舒平,曲春辉,周瑞.干细胞与转化医学研究进展浅析[J].中国实验动物学报,2016,24(4):440-442.
[17]Du WJ, Chi Y, Yang ZX, et al. Heterogeneity of proangiogenic features in mesenchymal stem cells derived from bone marrow, adipose tissue,umbilical cord, and placenta.Stem Cell Res Ther. 2016;7(1):163.
[18]Wegmeyer H, Br?ske AM, Leddin M, et al. Mesenchymal stromal cell characteristics vary depending on their origin.Stem Cells Dev. 2013;22(19):2606-2618.
[19]Gucciardo L, Ochsenbein-K?lble N, Ozog Y, et al. A comparative study on culture conditions and routine expansion of amniotic fluid-derived mesenchymal progenitor cells.Fetal DiagnTher. 2013;34(4):225-235.
[20]Beane OS, Fonseca VC, Cooper LL, et al. Impact of aging on the regenerative properties of bone marrow-, muscle-, and adiposederived mesenchymal stem/stromal cells.PLoS One. 2014;9(12):e115963.
[21]Bryzek A, Czekaj P, Plewka D, et al. Expression and co-expression of surface markers of pluripotency on human amniotic cells cultured in different growth media.Ginekol Pol. 2013;84(12):1012-1024.
[22]姚旺祥,裴国献,刘勇,等.兔胎盘源性间充质干祖细胞的分离方法比较[J].生物医学工程与临床,2007,11(2):85-87.
[23]马树立,李芳,徐彪,等.胎盘各组织来源间充质干细胞生物学特性比较[J].国际免疫学杂志,2013,36(5):397-401.
[2]Nitkin CR, Bonfield TL.Concise Review:Mesenchymal Stem Cell Therapy for Pediatric Disease:Perspectives on Success and Potential Improvements.Stem Cells Transl Med. 2017;6(2):539-565.
[3]Caplan AI.All MSCs are pericytes?Cell Stem Cell. 2008;3(3):229-230.
[4]Parekkadan B, Milwid JM.Mesenchymal stem cells as therapeutics.Annu Rev Biomed Eng. 2010;12:87-117.
[5]Wegmeyer H, Br?ske AM, Leddin M, et al. Mesenchymal stromal cell characteristics vary depending on their origin.Stem Cells Dev. 2013;22(19):2606-2618.
[6]Gucciardo L, Ochsenbein-K?lble N, Ozog Y, et al. A comparative study on culture conditions and routine expansion of amniotic fluid-derived mesenchymal progenitor cells.Fetal DiagnTher. 2013;34(4):225-235.
[7]Sun B, Meng XH, Liu R, et al. Mechanism study for hypoxia induced differentiation of insulin-producing cells from umbilical cord bloodderived mesenchymal stem cells.BiochemBiophys Res Commun.2015;466(3):444-4449.
[8]牛婷,李爱斌,曹景云,等.胎盘间充质干细胞的应用研究[J].中国组织工程研究,2015,19(32):5236-5242.
[9]郝白露,杨瑞峰,彭祥炽,等.改良的人脐带间充质干细胞培养方法[J].中国计划生育学杂志,2013,21(1):44-49.
[10]庞荣清,何洁,李福兵,等.一种简单的人脐带间充质干细胞分离培养方法[J].中华细胞与干细胞杂志(电子版),2011, 1(2):30-33.
[11]刘巨超,张兰,张博峰.新生大鼠肝细胞分离及体外培养方法[J].中国组织工程研究与临床康复,2009,13(53):10465-10468.
[12]王绮雯,吴启端,陈奕芝.温胰蛋白酶消化法和冷消化法培养乳鼠心肌细胞的比较[J].中国医药指南,2013,11(20):401-403.
[13]张飞,王一雄,武忠炎,等.人脐带间充质干细胞生物特性比较:胰酶冷消化和组织块法体外培养[J].中国组织工程研究, 2014,18(41):6614-6619.
[14]刘芳,杨辉,汪兆艳.自体骨髓间充质干细胞移植治疗唐氏综合征临床观察[J].转化医学杂志,2016,5(5):288-290.
[15]Li J, Wei Y, Yan L, et al. Multiplacenta derived stem cell/cytokine treatment increases survival time in a mouse model with radiationinduced bone marrow damage. Cytotechnology. 2016; 68(6):2677-2686.
[16]高舒平,曲春辉,周瑞.干细胞与转化医学研究进展浅析[J].中国实验动物学报,2016,24(4):440-442.
[17]Du WJ, Chi Y, Yang ZX, et al. Heterogeneity of proangiogenic features in mesenchymal stem cells derived from bone marrow, adipose tissue,umbilical cord, and placenta.Stem Cell Res Ther. 2016;7(1):163.
[18]Wegmeyer H, Br?ske AM, Leddin M, et al. Mesenchymal stromal cell characteristics vary depending on their origin.Stem Cells Dev. 2013;22(19):2606-2618.
[19]Gucciardo L, Ochsenbein-K?lble N, Ozog Y, et al. A comparative study on culture conditions and routine expansion of amniotic fluid-derived mesenchymal progenitor cells.Fetal DiagnTher. 2013;34(4):225-235.
[20]Beane OS, Fonseca VC, Cooper LL, et al. Impact of aging on the regenerative properties of bone marrow-, muscle-, and adiposederived mesenchymal stem/stromal cells.PLoS One. 2014;9(12):e115963.
[21]Bryzek A, Czekaj P, Plewka D, et al. Expression and co-expression of surface markers of pluripotency on human amniotic cells cultured in different growth media.Ginekol Pol. 2013;84(12):1012-1024.
[22]姚旺祥,裴国献,刘勇,等.兔胎盘源性间充质干祖细胞的分离方法比较[J].生物医学工程与临床,2007,11(2):85-87.
[23]马树立,李芳,徐彪,等.胎盘各组织来源间充质干细胞生物学特性比较[J].国际免疫学杂志,2013,36(5):397-401.