Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vessel
Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vessel
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DOI：
10.1016/j.jbiotec.2006.01.020
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发表时间：
2006-07-25
影响因子：
4.1
通讯作者：
Cui, Zhanfeng
中科院分区：
文献类型：
--
作者：
Liu, Yang;Liu, Tianqing;Cui, Zhanfeng
脐带血单核细胞 (UCB MNC) 的扩增在旋转壁容器 (RWV) 生物反应器和组织培养瓶 (T 型瓶) 中进行，培养基中添加了相对低剂量的纯化重组人细胞因子（5.33 ng/ml IL-3、16 ng/ml SCF、3.33 ng/ml G-CSF、2.13 ng/ml） GM-CSF、7.47 ng/ml FL 和 7.47 ng/ml TPO)，持续 8 天。每 24 It 测量两个培养系统中培养基的细胞密度、pH 和渗透压。在0、144和197小时进行CD34(+)细胞的流式细胞术测定，在0、72、144和197小时进行甲基纤维素集落测定。两个培养系统中培养基的 pH 值和渗透压保持在适合造血干细胞 (HSC) 和祖细胞培养的范围内。 RWV 生物反应器与细胞稀释补料方案相结合，可有效扩展 UCB MNC。培养200小时后，总细胞数增加了435.5±87.6倍，CD34(+)细胞增加了32.7±15.6倍，粒细胞-巨噬细胞集落形成单位(CFU-GM)增加了21.7±4.9倍。然而，在T瓶中，总细胞密度发生轻微变化，CD34(+)细胞和CFU-GM数量减少。结果表明，RWV生物反应器可以为UCB MNCs扩增提供更好的环境，增强HSCs与辅助细胞之间的接触，使细胞因子的利用比T-flask更有效。 (c) 2006 Elsevier B.V. 保留所有权利。
Expansion of umbilical cord blood mononuclear cells (UCB MNCs) was carried out in a rotating wall vessel (RWV) bioreactor and tissue culture flasks (T-flasks) in serum-containing medium supplemented with relatively low doses of purified recombinant human cytokines (5.33 ng/ml IL-3, 16 ng/ml SCF, 3.33 ng/ml G-CSF, 2.13 ng/ml GM-CSF, 7.47 ng/ml FL and 7.47 ng/ml TPO) for 8 days. The cell density, pH and osmolality of the culture medium in the two culture systems were measured every 24 It. Flow cytometric assay for CD34(+) cells was carried out at 0, 144 and 197 h and methylcellulose colony assays were performed at 0, 72, 144 and 197 It. The pH and osmolality of the medium in the two culture systems were maintained in the proper ranges for hematopoietic stem cells (HSCs) and progenitors culture. The RWV bioreactor, combined with a cell-dilution feeding protocol, was efficient to expand UCB MNCs. At the end of 200 h culture, the total cell number was multiplied by 435.5 +/- 87.6 times, and CD34(+) cells 32.7 +/- 15.6 times, and colony-forming units of granulocyte-macrophage (CFU-GM) 21.7 +/- 4.9 times. While in T-flasks, however, total cells density changed mildly, CD34(+) cells and CFU-GM decreased in number. It is demonstrated that the RWV bioreactor can provide a better environment for UCB MNCs expansion, enhance the contact between HSCs and accessory cells and make the utilization of cytokines more effective than T-flask. (c) 2006 Elsevier B.V. All rights reserved.