利用微载体悬浮培养人脐带间充质干细胞

doi:10.16288/j.yczz.18-123

遗传 ›› 2018, Vol. 40 ›› Issue (12): 1120-1128.doi: 10.16288/j.yczz.18-123

利用微载体悬浮培养人脐带间充质干细胞

李夏^1,^2,³,滑慧娟^2,³,郝捷^2,³,王柳^2,³,刘忠华¹()

^1. 东北农业大学生命科学学院,哈尔滨 150030
^2. 北京干细胞库,北京 100190
^3. 中国科学院动物研究所,北京 100101

收稿日期:2018-06-15 修回日期:2018-09-03 出版日期:2018-12-20 发布日期:2018-09-03
通讯作者: 刘忠华 E-mail:liuzhonghua@neau.edu.cn
作者简介:李夏,硕士研究生,专业方向：发育生物学。E-mail: lixia_bjscb@foxmail.com
基金资助:
中国科学院重点部署项目(编号：ZDRW-ZS-2016-2)资助

Culturing of human umbilical cord mesenchymal stem cells via a 3D suspension culture system using the microcarrier

Li Xia^1,^2,³,Hua Huijuan^2,³,Hao Jie^2,³,Wang Liu^2,³,Liu Zhonghua¹()

^1. College of Life Science, Northeast Agricultural University, Harbin, 150030, China
^2. Beijing Stem Cell Bank, Chinese Academy of Sciences, Beijing 100190, China
^3. Institute of Zoology, Chinese Academy of Science, Beijing 100101, China

Received:2018-06-15 Revised:2018-09-03 Online:2018-12-20 Published:2018-09-03
Contact: Liu Zhonghua E-mail:liuzhonghua@neau.edu.cn
Supported by:
Supported by the Key Deployment Projects of CAS (No. ZDRW-ZS-2016-2)

摘要/Abstract

摘要：

随着干细胞研究的不断深入,干细胞功能分化研究和临床应用转化的需求日益提升。人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUCMSCs)来源广泛,不仅自我更新能力强、能够分化成多种类型的成体细胞,而且其自身具有免疫调节能力,不易引发免疫排斥反应,在干细胞功能分化研究和临床应用中具有巨大应用前景和应用潜力。目前,传统的细胞培养方式培养效率低、细胞活性较差,不能满足日益增长的研究和应用需求。本研究利用微载体结合旋转瓶的悬浮培养方法,通过优化细胞接种量及转速等影响因素,快速获得大量高质量的人脐带间充质干细胞。经悬浮培养总细胞量可高达到7×10 ⁸个细胞/L,而且细胞活性较高,MSC 特异性标记物表达良好,在恢复平面培养后仍能维持MSC的正常细胞形态和增殖能力。高效脐带间充质干细胞悬浮培养体系的初步建立,为未来的干细胞功能分化研究和临床应用奠定了基础。

关键词: 人脐带间充质干细胞, 3D悬浮培养体系, 平面(2D)培养体系, 微载体

Abstract:

With the rapid development of stem cell research, the demands for high quality stem cells in cell differentiation studies, cell therapy and clinical applications increase significantly. Human umbilical cord mesenchymal stem cells (hUCMSCs) hold great potentials in these applications due to its wide availability, self-renewal capacity, good pluripotency, and in particular, self-immunomodulation ability. However, the conventional adherent culture system remains challenging in mass production of high-quality stem cells. In this study, we set up a 3D suspension culture system by using spinner flasks added with microcarrier. By optimizing the seeding density and rotation speed, we achieved a cell density as higher as 7×10 ⁸cells/L per vessel. The cultured MSCs had good viability, and the expression levels of the MSC markers were similar to those of 2D-cultured MSCs. After being transferred back into a 2D culture system, the MSCs still maintained normal morphology and proliferation ability. These results indicated that the 3D suspension culture system established in this study provides a fundamental basis for mass production of high-quality MSCs for future research and clinical applications.

Key words: human umbilical cord mesenchymal stem cells (hUCMSCs), 3D Suspension culture system, Adherent (2D) culture system, Microcarrier

李夏, 滑慧娟, 郝捷, 王柳, 刘忠华. 利用微载体悬浮培养人脐带间充质干细胞[J]. 遗传, 2018, 40(12): 1120-1128.

Li Xia, Hua Huijuan, Hao Jie, Wang Liu, Liu Zhonghua. Culturing of human umbilical cord mesenchymal stem cells via a 3D suspension culture system using the microcarrier[J]. Hereditas(Beijing), 2018, 40(12): 1120-1128.

参考文献

[1]	Yen BL, Huang HI, Chien CC, Jui HY, Ko BS, Yao M, Shun CT, Yen ML, Lee MC, Chen YC . Isolation of multipotent cells from human term placenta. Stem Cells, 2005,23(1):3-9.
[2]	Kern S, Eichler H, Stoeve J, Klüter H, Bieback K . Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells, 2006,24(5):1294-1301.
[3]	Yang XF, He X, He J, Zhang LH, Su XJ, Dong ZY, Xu YJ, Li Y, Li YL . High efficient isolation and systematic identification of human adipose-derived mesenchymal stem cells. J Biomed Sci, 2011,18(1):59.
[4]	Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH . Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Blood, 2004,103(5):1669-1675.
[5]	Romanov YA, Svintsitskaya VA, Smirnov VN . Searching for alternative sources of postnatal human mesenchymal stem cells: Candidate MSC-like cells from umbilical cord. Stem Cell, 2003,21(1):105-110.
[6]	Caplan AI . Mesenchymal stem cells. J Orthop Res, 1991,9(5):641-650.
[7]	Kuroda Y, Kitada M, Wakao S, Nishikawa K, Tanimura Y, Makinoshima H, Goda M, Akashi H, Inutsuka A, Niwa A, Shigemoto T, Nabeshima Y, Nakahata T, Nabeshima Y, Fujiyoshi Y, Dezawa M . Unique multipotent cells in adult human mesenchymal cell populations. Proc Natl Acad Sci USA, 2010,107(19):8639-8643.
[8]	Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringden O . HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol, 2003,31(10):890-896.
[9]	Klyushnenkova E, Mosca JD, Zernetkina V, Majumdar MK, Beggs KJ, Simonetti DW, Deans RJ , McIntosh KR.T cell responses to allogeneic human mesenchymal stem cells: immunogenicity, tolerance, and suppression. J Biomed Sci, 2005,12(1):47-57.
[10]	Scheel C, Eaton EN, Li SH, Chaffer CL, Reinhardt F, Kah KJ, Bell G, Guo W, Rubin J, Richardson AL, Weinberg RA . Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast. Cell, 2011,145(6):926-940.
[11]	Otto WR, Wright NA . Mesenchymal stem cells: from experiment to clinic. Fibrogenesis Tissue Repair, 2011,4(1):20.
[12]	Caplan AI, Correa D . The MSC: an injury drugstore. Cell Stem Cell, 2011,9(1):11-15.
[13]	Aggarwal S, Pittenger MF . Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 2005,105(4):1815-1822.
[14]	Ohno T, Kaneda H, Nagai Y, Fukushima M . Regenerative medicine in critical limb ischemia. J Atheroscler Thromb, 2012,19(10):883-889.
[15]	Zhao Y, Tang F, Xiao Z, Han G, Wang N, Yin N, Chen B, Jiang X, Yun C, Han W, Zhao C, Cheng S, Zhang S, Dai J . Clinical study of neuroregen scaffold combined with human mesenchymal stem cells for the repair of chronic complete spinal cord injury. Cell Transplant, 2017,26(5):891-900.
[16]	Trounson A , McDonald C.Stem cell therapies in clinical trials: Progress and challenges. Cell Stem Cell, 2015,17(1):11-22.
[17]	Mehta J, Mehta J, Frankfurt O, Altman J, Evens A, Tallman M, Gordon L, Williams S, Winter J, Krishnamurthy J, Duffey S, Singh V, Meagher R, Grinblatt D, Kaminer L, Singhal S . Optimizing the CD34 + cell dose for reduced-intensity allogeneic hematopoietic stem cell transplantation. Leuk Lymphoma, 2009,50(9):1434-1441.
[18]	Kehoe DE, Jing D, Lock LT, Tzanakakis ES . Scalable stirred-suspension bioreactor culture of human pluripotent stem cells. Tissue Eng Part A, 2010,16(2):405-421.
[19]	Duijvestein M, Vos AC, Roelofs H, Wildenberg ME, Wendrich BB, Verspaget HW, Kooy-Winkelaar EM, Koning F, Zwaginga JJ, Fidder HH, Verhaar AP , Fibbe WE,van den Brink GR, Hommes DW. Autologous bone marrow-derived mesenchymal stromal cell treatment for refractory luminal Crohn's disease: results of a phase I study. Gut, 2010,59(12):1662-1669.
[20]	Serra M, Brito C, Correia C, Alves PM . Process engineeering of human pluripotent stem cells for clinical application. Trends Biotechnol, 2012,30(6):350-359.
[21]	Villa-Diaz LG, Ross AM, Lahann J, Krebsbach PH . Concise review: The evolution of human pluripotent stem cell culture: from feeder cells to synthetic coatings. Stem Cells, 2013,31(1):1-7.
[22]	Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E . Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy, 2006,8(4):315-317.
[23]	Heathman TR, Stolzing A, Fabian C, Rafiq QA, Coopman K, Nienow AW, Kara B, Hewitt CJ . Scalability and process transfer of mesenchymal stromal cell production from monolayer to microcarrier culture using human platelet lysate. Cytotherapy, 2016,18(4):523-535.
[24]	Carmelo JG, Fernandes-Platzgummer A , Diogo MM,da Silva CL, Cabral JM. A xeno-free microcarrier-based stirred culture system for the scalable expansion of human mesenchymal stem/stromal cells isolated from bone marrow and adipose tissue. Biotechnol J, 2015,10(8):1235-1247.
[25]	Sun LY, Hsieh DK, Syu WS, Li YS, Chiu HT, Chiou TW . Cell proliferation of human bone marrow mesenchymal stem cells on biodegradable microcarriers enhances in vitro differentiation potential. Cell Prolif, 2010,43(5):445-456.
[26]	Petry F, Smith JR, Leber J, Salzig D, Czermak P, Weiss ML . Manufacturing of human umbilical cord mesenchymal stromal cells on microcarriers in a dynamic system for clinical use. Stem Cells Int, 2016,2016:4834616.
[27]	Santos F, Andrade PZ, Abecasis MM, Gimble JM, Chase LG, Campbell AM, Boucher S, Vemuri MC, Silva CL, Cabral JM . Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier- based culture system under xeno-free conditions. Tissue Eng Part C Methods, 2011,17(12):1201-1210.
[28]	Goh TK, Zhang ZY, Chen AK, Reuveny S, Choolani M, Chan JK, Oh SK . Microcarrier culture for efficient expansion and osteogenic differentiation of human fetal mesenchymal stem cells. Biores Open Access, 2013,2(2):84-97.
[29]	Kobolak J, Dinnyes A, Memic A, Khademhosseini A, Mobasheri A . Mesenchymal stem cells: Identification, phenotypic characterization, biological properties and potential for regenerative medicine through biomaterial micro-engineering of their niche. Methods, 2016,99:62-68.
[30]	Jossen V, van den Bos C, Eibl R, Eibl D . Manufacturing human mesenchymal stem cells at clinical scale: process and regulatory challenges. Appl Microbiol Biotechnol, 2018,102(9):3981-3994.
[31]	Yang Y, Rossi FM, Putnins EE . Ex vivo expansion of rat bone marrow mesenchymal stromal cells on microcarrier beads in spin culture. Biomaterials, 2007,28(20):3110-3120.
[32]	Eibes G, dos Santos F, Andrade PZ, Boura JS, Abecasis MM, da Silva CL, Cabral JM . Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier-based stirred culture system. J Biotechnol, 2010,146(4):194-197.
[33]	Schop D, van Dijkhuizen-Radersma R, Borgart E, Janssen FW, Rozemuller H, Prins HJ, de Bruijn JD . Expansion of human mesenchymal stromal cells on microcarriers: growth and metabolism. J Tissue Eng Regen Med, 2010,4(2):131-140.
[34]	Heathman TR, Glyn VA, Picken A, Rafiq QA, Coopman K, Nienow AW, Kara B, Hewitt CJ . Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process. Biotechnol Bioeng, 2015,112(8):1696-1707.
[35]	Rafiq QA, Brosnan KM, Coopman K, Nienow AW, Hewitt CJ . Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor. Biotechnol Lett, 2013,35(8):1233-1245.
[36]	Wu QF, Wu CZ, Dong B, Wang LS . Cultivation of humanmesenchymal stem cells on macroporous CultiSpher G microcarriers.J Exp Hematol, 2003(1):15-21.
[36]	吴清法, 吴祖泽, 董波, 王立生 . 微载体悬浮培养成人骨髓间充质干细胞. 中国实验血液学杂志, 2003(1):15-21.
[37]	Han BS, Shen BY, Peng CH, Shi MM, Cheng DF, Wang XM, Wang ZH, Zhou GW, Li HW . Scale-up culture of adult human mesenchymal stem cells on microcarrier Cytodex 3.J Shanghai Jiao Tong Univ Med Sci, 2006(6):581-585.
[37]	韩宝三, 沈柏用, 彭承宏, 施敏敏, 程东峰, 王小明, 王兆海, 周光文, 李宏为 , 微载体cytodex 3大量扩增成人骨髓间充质干细胞的初步研究. 上海交通大学学报(医学版), 2006(6):581-585.

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

利用微载体悬浮培养人脐带间充质干细胞

Culturing of human umbilical cord mesenchymal stem cells via a 3D suspension culture system using the microcarrier

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics