In vitro expansion of human mesenchymal stem cells in animal serum-free media

doi:10.3969/j.issn.2095-4344.2014.28.026

Abstract

Abstract:

BACKGROUND: Mesenchymal stem cells have been an important source of seed cells for tissue engineering, regenerative medicine and immunosuppressive therapy areas. The large expansion is the key to the clinical application of mesenchymal stem cells.
OBJECTIVE: To summarize the research progress in the in vitro expansion of human mesenchymal stem cells in animal serum-free media.
METHODS: A computer-based online retrieval of CNKI and PubMed databases was performed to search papers published between 2004 and 2014 using the key words of “mesenchymal (stromal) stem cells, animal serum-free media, humanized media, human serum, umbilical cord blood serum, platelet rich plasma, platelet lysate, defined medium” in English and Chinese, respectively. Finally, 41 articles were retained.
RESULTS AND CONCLUSION: Human serum, umbilical cord blood serum, platelet rich plasma, platelet lysate and defined medium can replace fetal bovine serum for large-scale expansion of mesenchymal stem cells in vitro. These media or additives have their advantages and disadvantages; therefore, it is urgent to guarantee the functional effectiveness and transplantation safety before large-scale, systematic clinical application of mesenchymal stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: mesenchymal stem cells, culture media, serum, platelet-rich plasma

CLC Number:

R318

Wu Di, Wu Xiao-yun, Wu Yan. In vitro expansion of human mesenchymal stem cells in animal serum-free media[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(28): 4583-4587.

Figures/Tables 1

2.1 人源性成分的培养基 2.1.1 人血清的培养基越来越多的研究报道指出使用人血清可替代胎牛血清扩增人骨髓间充质干细胞、脂肪干细胞、滑膜间充质干细胞等[11-12, 14]。人血清中含有间充质干细胞生长所需的生长激素、必需蛋白(转铁蛋白，血清白蛋白和粘连蛋白等)、生长刺激因子、氨基酸和维生素等必要成分。将人自体血清作为培养基的添加物，培养出的间充质干细胞的形态与添加胎牛血清相似，并且明显缩短了群体倍增时间，添加胎牛血清培养间充质干细胞的群体倍增时间是76-89 h，添加人血清培养间充质干细胞的群体倍增时间是41-54 h[17]。在间充质干细胞短期和长期培养中，使用人血清和胎牛血清培养的间充质干细胞生物学特性无明显差异，并表达间充质干细胞所有表面标记物，具有在体外诱导间充质干细胞成骨和成脂的分化能力。有研究报道，用人血清扩增培养其自体骨髓间充质干细胞，比胎牛血清培养的间充质干细胞扩增速度更快，脑卒中患者回输间充质干细胞后无明显不良反应[18]。另报道用人血清扩增培养的间充质干细胞回输治疗急慢性移植物抗宿主病取得成功[19]。但是，使用人血清也存在一些问题需要解决：第一，人血清中可能含有病原体，在间充质干细胞移植过程中发生病原体感染，因此，无论是自体或异体血清在使用之前，都应进行严格的检测。第二，血清捐献者的年龄直接影响其培养间充质干细胞的生长和分化能力[20]。 2.1.2 含脐血清的培养基人脐血具有来源广泛，采集方便，免疫原性低，不涉及伦理限制等优势。人脐血清富含干细胞生长、存活、增殖及分化所需的各种细胞因子，还含有丰富的刺激造血因子，因此可选用添加人脐血清的培养基对间充质干细胞进行培养。此外，人脐血清中已经确定的蛋白质有61种，尤以血清白蛋白和转铁蛋白的含量高[21]，人脐血清利于间充质干细胞的生长和增殖可能是因为这些蛋白质的促进作用。分别用添加体积分数为10%人脐血清和体积分数为10%胎牛血清的培养基进行间充质干细胞培养，细胞生长的倍增时间分别为31.3 h和44.2 h，这可能由于人脐血清高表达细胞周期调控分子D2，从而加快细胞周期活动，促进间充质干细胞的增殖[22]。经添加人脐血清的培养基培养的间充质干细胞仍具有维持自我更新和持续复制的能力，可得到近2 000倍扩增量的细胞[20]，而添加胎牛血清的培养基培养的间充质干细胞，在体外扩增后期就失去了细胞集落形成能力，显示出细胞衰老的情况。人脐血清被认为是较好的胎牛血清替代物，但是由于其成分的不确定性，使其用于临床规模扩增间充质干细胞受到一定限制，因此需要寻找一种成分明确的培养基扩增间充质干细胞。 2.1.3 富血小板血浆富血小板血浆是通过离心人全血而得到的含高体积分数血小板的血浆，与CaCl2、凝血酶混合后被激活，其中α颗粒释放多种生长因子，如血小板源性生长因子、表皮生长因子、转化生长因子β、血管内皮生长因子、成纤维细胞生长因子和类胰岛素生长因子等，各因子间发挥协同作用，但机制尚不清。一些研究者尝试用贫血小板血浆培养扩增人间充质干细胞，结果发现贫血小板血浆并不支持间充质干细胞的扩增，添加表皮生长因子、血小板源性生长因子和碱性成纤维细胞生长因子后的贫血小板血浆完全可以支持间充质干细胞的扩增和分化，且不影响间充质干细胞的细胞表型等生物学特性，进一步证明了表皮生长因子、血小板源性生长因子和碱性成纤维细胞生长因子是维持间充质干细胞生长的必需因子[23]。采用改良的富血小板血浆制备方法，促进血小板中生长因子的释放，即将加入肝素钠的全血通过液氮反复冻融，用于对人骨髓间充质干细胞的体外培养扩增，发现5%-10%的改良富血小板血浆对体外培养人骨髓间充质干细胞的增殖具有促进作用，其对细胞的增殖促进作用具有浓度依赖性，尤以10%浓度为佳，该浓度可促进人骨髓间充质干细胞群落中的Stro-1+细胞亚群的增殖，从而增强细胞群落的免疫抑制效应、均一性和原始性[24]。一些临床研究者倾向使用自体富血小板血浆扩增以获得临床数量的人间充质干细胞，这样更安全。人脐血可成为富血小板血浆的理想来源，脐血富血小板血浆中的血小板源性生长因子AB/BB和碱性成纤维细胞生长因子的含量明显高于外周血富血小板血浆，更有利于体外扩增间充质干细胞[15]。 2.1.4 人血小板裂解液血小板裂解液是富血小板血浆调整血小板浓度后，经反复冻融裂解血小板后获得富含各种生长和免疫相关因子的裂解液。制备过程中未加入凝血酶、CaCl2等激活剂引入异源成分，不仅降低了免疫原性，也降低了制备成本。用ELISA方法检测裂解后血浆各种因子浓度都明显高于裂解前[25]。血小板源性生长因子和碱性成纤维细胞生长因子是维持间充质干细胞生长的必需因子，但增加这两个因子浓度并不能高效地促进间充质干细胞的扩增，说明这两个因子为间充质干细胞扩增的必需因子，但不是高效因子[25]。有研究报道，血小板源性生长因子、表皮生长因子、转化生长因子β1和碱性成纤维细胞生长因子的浓度较高，约为全血中的17倍，其活性可持续5-8 d，在调节细胞增殖过程中起重要作用[26-30]。研究已经证实血小板裂解液可以替代动物血清实现实验室级别或者大规模临床级别扩增人骨髓间充质干细胞、脂肪干细胞、脐带间充质干细胞、牙髓干细胞等[11, 25,31-35]，是目前临床应用规模扩增间充质干细胞最合适的胎牛血清替代品。在细胞形态方面，与胎牛血清比较，5%血小板裂解液扩增的间充质干细胞形态更显细长，折光性更好，细胞直径更小，而直径更小的优点是可增加单位面积的间充质干细胞的数量，减少细胞培养耗材的使用量，节省培养成本，更有利于细胞进入人体后通过各种屏障。在扩增能力方面，血小板裂解液能使间充质干细胞增殖更快，汇合时间更短。血小板裂解液扩增的间充质干细胞有更强的成骨、软骨和脂肪分化的能力[31]。应用浓度为7%血小板裂解液和体积分数为10%胎牛血清扩增羊膜来源间充质干细胞，结果显示两种介质培养的羊膜来源间充质干细胞均为成纤维细胞样，但是含7%血小板裂解液较含体积分数为10%胎牛血清的培养基扩增羊膜来源间充质干细胞所用传代时间更短，这两种培养液培养的羊膜来源间充质干细胞均表达CD90、CD73和CD105，不表达CD45、CD34、CD14、CD19和HLA-DR，细胞分化的结果显示7%血小板裂解液培养的羊膜来源间充质干细胞具备向成骨细胞、成软骨细胞和成脂肪细胞分化的能力，均符合间充质干细胞的特征[36]。使用血小板裂解液可以有效地替代胎牛血清，实现临床级间充质干细胞的扩增培养[33, 35]。但一些研究发现血小板裂解液扩增的间充质干细胞对T、NK细胞增殖和细胞毒性功能抑制能力下降，下调前列腺素E2分泌，上调白细胞介素6、白细胞介素8和RANTES表达，显示了血小板裂解液扩增的间充质干细胞在临床应用于免疫调节剂方面具有局限性[37]。 2.2 化学成分明确无血清培养基人血清、人脐血清、富血小板血浆和血小板裂解液的主要作用是给细胞提供生长增殖所需的激素、生长因子、转移蛋白和其他营养物质，但存在缺点：①成分不明确、血液制品具有批间差异，不稳定，且受供者年龄等影响。②血液制品存在传染病、未知疾病检测等质量控制难点，使其具有潜在的风险。③血液来源有限。④人血清、人脐血清、富血小板血浆和血小板裂解液扩增效应的内在分子机制，及其对间充质干细胞的分子学改变都有待更多的研究来揭示。⑤血小板相关膜抗原引发的体内免疫学反应也应引起重视。为避免上述缺点，研究者提出了化学成分明确、无动物源成分和无血清的培养基，通过像鸡尾酒式的添加激素、无机盐、维生素、微量元素、氨基酸、白蛋白和生长因子(如碱性成纤维细胞生长因子、血小板源性生长因子和转化生长因子β)等成分明确的培养基，在体外培养的间充质干细胞具有较长时间生长增殖，可维持间充质干细胞的主要细胞表型、分化和免疫调节功能特征[38-39]。在原材料的选取上采用具有足够纯度的化学合成或者生物重组的原料(重组人胰岛素、重组人血清白蛋白和重组人生长因子等)，既能做到严格意义上的化学成分明确，又能避免使用血液制品提取物成分。一些实验室自行配制化学成分明确的无血清培养基，它由基础培养基IMDM或DMEM/F12和替代血清的添加物组成。主要成分为亚硒酸钠、转铁蛋白、牛血清白蛋白、胰岛素、氢化可的松、重组人表皮生长因子和重组人碱性成纤维细胞生长因子等。为保证间充质干细胞的贴壁，采用了明胶包被细胞培养瓶，成功培养出了人骨髓间充质干细胞，但因为有牛血清白蛋白和牛来源明胶，没有实现不含动物源成分的真正目的[40]。化学组分明确的培养基也有一些不足：①需要在高密度下接种。②价格较昂贵。③间充质干细胞在培养过程中易受机械和化学因素的影响。目前，化学成分明确的无血清培养基还处于初级阶段，这种培养基扩增后的间充质干细胞的迁移能力、免疫原性、免疫调节能力和细胞因子的表达还需要许多研究来进行探索。"

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