Blázquez-Prunera Arantxa, Díez José María, Gajardo Rodrigo, Grancha Salvador
Research and Development, Bioscience Industrial Group, Grifols, Parets del Vallès, Barcelona, Spain.
Faculdade de Engenharia, Universidade do Porto, Porto, Portugal.
Stem Cell Res Ther. 2017 Apr 27;8(1):103. doi: 10.1186/s13287-017-0552-z.
Mesenchymal stem cells (MSCs) show promising characteristics for their use in advanced therapy medicinal products. However, there are some unresolved concerns, such as the use of animal components for their expansion. In this study we assessed the suitability of a xeno-free supplement for cell culture (SCC) derived from human plasma, to culture and expand human MSCs (hMSCs) from different origins. Characteristics of viable cultured hMSCs such as genetic stability, phenotype and multipotentiality were qualitatively evaluated.
hMSCs from adipose tissue (AT), bone marrow (BM) and umbilical cord (UC) and supplier sources (commercial/non-commercial) were used. After hMSCs expansion in a xeno-free medium, classical hMSCs markers were studied by immunocytochemistry, and genetic stability was tested by classic karyotyping. The capacity of hMSCs to differentiate into adipogenic, osteogenic, and chondrogenic cells in differentiation media was assessed using different staining. Different lots of SCC were used to assure consistency between batches.
All hMSCs tested maintained their morphology and adherence to plastic during their expansion, and preserved their genetic stability, phenotype and differentiation potential. No differences were observed when using different lots of SCC. Moreover, the proliferation rate, evaluated as population doubling time (PDT) of commercial BM and AT hMSCs, was higher in the xeno-free medium than in the control media provided by the suppliers of the cells (PDT of 4.6 for BM-hMSC and 6.4 for AT-hMSC in xeno-free medium, and 7.0 and 14.7 respectively in the commercial media). UC-hMSCs PDT was similar in all the media tested. When using non-commercial BM-hMSCs, PDT was lower in the xeno-free medium, but reverted to the control level with the addition of growth factors.
SCC-containing medium can be a feasible xeno-free alternative to expand hMSCs for advanced therapies.
间充质干细胞（MSCs）在先进治疗药物产品中的应用显示出有前景的特性。然而，仍存在一些未解决的问题，例如在其扩增过程中使用动物成分。在本研究中，我们评估了一种源自人血浆的无动物成分细胞培养补充剂（SCC）用于培养和扩增不同来源的人MSCs（hMSCs）的适用性。对培养的活hMSCs的遗传稳定性、表型和多能性等特性进行了定性评估。
使用来自脂肪组织（AT）、骨髓（BM）和脐带（UC）以及供应商来源（商业/非商业）的hMSCs。在无动物成分培养基中扩增hMSCs后，通过免疫细胞化学研究经典的hMSCs标志物，并通过经典核型分析测试遗传稳定性。使用不同的染色方法评估hMSCs在分化培养基中分化为脂肪细胞、成骨细胞和软骨细胞的能力。使用不同批次的SCC以确保批次间的一致性。
所有测试的hMSCs在扩增过程中均保持其形态并贴附于塑料培养皿，同时保持其遗传稳定性、表型和分化潜能。使用不同批次的SCC时未观察到差异。此外，以商业BM和AT hMSCs的群体倍增时间（PDT）评估的增殖率，在无动物成分培养基中高于细胞供应商提供的对照培养基（无动物成分培养基中BM-hMSC的PDT为4.6，AT-hMSC为6.4，商业培养基中分别为7.0和14.7）。UC-hMSCs的PDT在所有测试培养基中相似。使用非商业BM-hMSCs时，无动物成分培养基中的PDT较低，但添加生长因子后恢复到对照水平。
含SCC的培养基可作为一种可行的无动物成分替代品，用于扩增hMSCs以进行先进治疗。
