Wu Jie-Ying, Lu Yan, Chen Jin-Song, Wu Shao-Qing, Tang Xue-Wei, Li Yan
Guangzhou Cord Blood Bank, Guangzhou Women and Children's Medical Center, Guangzhou 510623, Guangdong Province, China. E-mail:
Guangzhou Cord Blood Bank, Guangzhou Women and Children's Medical Center, Guangzhou 510623, Guangdong Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2015 Aug;23(4):1112-9. doi: 10.7534/j.issn.1009-2137.2015.04.040.
To investigate the feasibility of umbilical cord blood plasma (UCP) as a replacement for fetal bovine serum (FBS) for culturing mesenchymal stem cells (MSC) derived from umbilical cord, and to observe the supporting effects of these cells (served as a feeder layer) on ex vivo expanding of human umbilical cord blood CD34(+) cells.
Umbilical cord blood (UCB) units were suitable if the Guangzhou cord blood bank donor selection criteria strictly were fulfilled. UCP were ready to use after the collection from the plasma depletion/reduction during the processing and pooling of suitable UCB units (at least 30 units were screened for pathogens and microorganisms, and qualified). Umbilical cord mesenchymal stem cells (UCMSC) were harvested from the umbilical cord tissue of health full-term newborns after delivery by enzyme digestion and divided into 3 groups: group 1 and 2 were cultured in the presence of DMEM/F12 containing either FBS or UCP; and group 3 was cultured in serum-free medium (StemPro® MSC SFM CTS™). Morphology, proliferation and surface marker expression were examined by flow cytometry, and the differentiation toward adipogenic and osteogenic lineages was used for investigating the effect of media on UCMSC after 3-5 passages. Next, the cells cultured in the three different media were cryopreserved and thawed, then prepared as feeder layers with the name of UCMSC(FBS), UCMSC(UCP), and UCMSC(SFM), respectively. The CD34⁺ cells were separated from UCB by magnetic activated cell sorting (MACS) and divided into 4 groups cultured in StemPro(-34) SFM medium added with hematopoietic cytokine combination (StemSpan® CC100). The control group included only CD34⁺ cells as group A (blank control) and experimental groups included UCMSC(FBS) + CD34⁺ cells as group B, UCMSC(UCP) + CD34⁺ cells as group C, UCMSC(SFM) + CD34⁺ cells as group D, and cells in all groups were cultured ex vivo for 7 days. The nucleated cell (NC) number was counted by cell counter, CD34⁺ cells were measured by flow cytometry, and clonogenic assay was conducted at day 0 and 7 of culture. The expansion efficiency was assessed.
The morphology (spindle-shaped and plastic-adherent), the immunophenotype (high positive percentage of CD73, CD90, CD105 and CD166) and the differentiation potential (osteogenic and adipogenic) were almost indistinguishable among the cells cultured in any of these three media except for the expression of CD105 in group 3 (serum-free medium) was lower than that in other 2 groups (P < 0.05). UCMSC grown in UCP medium demonstrated significantly higher proliferation rates than that in media containing FBS or commercial serum-free supplement (P < 0.05). After co-culture for 7 days, the CD34⁺ cell percentage decreased in all the groups, while NC were amplified effectively and the CD34⁺ cell number increased with the same order as group C or D group B or A (control group) (P < 0.05). As compared with the colony-forming unit (CFU) number at day 0, there was no significant difference in the expansion multiple between group C and D, while the expansion of CFU in group C were higher than that in group B and A.
The UCP can be used as a better animal-free serum supplement for growth, maintenance and differentiation of UCMSC, thus would be a safe choice for clinical-scale production of human MSC.
探讨脐带血血浆（UCP）替代胎牛血清（FBS）培养脐带间充质干细胞（MSC）的可行性，并观察这些细胞（作为饲养层）对人脐带血CD34(+)细胞体外扩增的支持作用。
严格符合广州脐带血库供者选择标准的脐带血（UCB）单位才适用。在处理和汇集合适的UCB单位（至少筛选30个单位的病原体和微生物且合格）过程中，经血浆去除/减少收集后即可使用UCP。分娩后从健康足月新生儿的脐带组织中通过酶消化法获取脐带间充质干细胞（UCMSC），并分为3组：第1组和第2组分别在含FBS或UCP的DMEM/F12培养基中培养；第3组在无血清培养基（StemPro® MSC SFM CTS™）中培养。通过流式细胞术检测形态、增殖和表面标志物表达，并在传代3 - 5次后利用向脂肪和成骨谱系的分化来研究培养基对UCMSC的影响。接下来，将在三种不同培养基中培养的细胞冻存并复苏，然后分别制备成名为UCMSC(FBS)、UCMSC(UCP)和UCMSC(SFM)的饲养层。通过磁珠分选法（MACS）从UCB中分离出CD34⁺细胞，并分为4组，在添加造血细胞因子组合（StemSpan® CC100）的StemPro(-34) SFM培养基中培养。对照组仅含CD34⁺细胞作为A组（空白对照），实验组包括UCMSC(FBS) + CD34⁺细胞作为B组、UCMSC(UCP) + CD34⁺细胞作为C组、UCMSC(SFM) + CD34⁺细胞作为D组，所有组的细胞均体外培养7天。用细胞计数仪计数有核细胞（NC）数量，通过流式细胞术检测CD34⁺细胞，并在培养第0天和第7天进行集落形成试验。评估扩增效率。
除第3组（无血清培养基）中CD105的表达低于其他2组（P < 0.05）外，在这三种培养基中培养的细胞在形态（纺锤形且贴壁）、免疫表型（CD73、CD90、CD105和CD166高阳性率）和分化潜能（成骨和成脂）方面几乎无差异。在UCP培养基中生长的UCMSC增殖率显著高于含FBS或市售无血清补充剂的培养基（P < 0.05）。共培养7天后，所有组的CD34⁺细胞百分比均下降，而NC有效扩增，且CD34⁺细胞数量按C组或D组＞B组＞A组（对照组）的顺序增加（P < 0.05）。与第0天的集落形成单位（CFU）数量相比，C组和D组的扩增倍数无显著差异，而C组的CFU扩增高于B组和A组。
UCP可作为更好的无动物血清补充剂用于UCMSC的生长、维持和分化，因此将是临床规模生产人MSC的安全选择。
