Cell Processing Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, 10 Center Drive-MSC-1184, Building 10, Room 3C720, Bethesda, MD, 20892-1184, USA.
Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD, 20892, USA.
J Transl Med. 2018 Mar 14;16(1):65. doi: 10.1186/s12967-018-1400-3.
Bone marrow stromal cells (BMSCs) have classically been cultured in media supplemented with fetal bovine serum (FBS). As an alternative to FBS, pooled solvent detergent apheresis platelets, HPGF-C18, was evaluated for BMSC culture.
A comparison of passage 2 BMSC growth revealed that 10% HPGF-C18 produced similar cell numbers as 20% FBS. Marrow aspirates from 5 healthy subjects were cultured for 4 passages in 10% HPGF-C18 or 20% FBS and were analyzed for proliferation, colony formation efficiency (CFE), surface marker expression, suppression of mixed lymphocyte reactions (MLRs), global gene and microRNA expression analysis. BMSC supernatant cytokine and growth factor concentrations were also compared.
Primary cultures of marrow aspirates in 10% HPGF-C18 and 20% FBS yielded similar numbers and CFE. After 4 passages, 10% HPGF-C18 and 20% FBS yielded similar numbers of BMSCs, surface marker expression patterns and immunosuppression effects. Gene and microRNA expression analysis revealed that BMSCs cultured under the two conditions had distinct expression profiles. Gene Set Enrichment Analysis (GSEA) revealed HPGF-C18-cultured BMSCs were enriched in metabolic processing and biosynthetic pathways, cell proliferation and cell cycle pathways, and immune response pathways. FBS-cultured BMSCs were enriched in MAPK signaling, TGF-beta signaling, cell adhesion and extracellular matrix pathways. Differently expressed microRNAs were related to the osteogenesis of BMSCs. The supernatant of HPGF-C18 BMSCs had higher levels of PEDF and TGFB1 and lower levels of IL6, VEGF, SDF1 and PLGF.
Traditional measures, expansion, surface marker expression and inhibition of MLRs suggest that BMSC cultured in HPGF-C18 and FBS were similar, but analysis at the molecular level revealed many differences. BMSCs cultured in HPGF-C18 should be assessed in specific functional assays that reflect application-specific potency before substituting FBS with HPGF-C18.
骨髓基质细胞（BMSCs）传统上在含有胎牛血清（FBS）的培养基中培养。作为 FBS 的替代品，已评估了混合溶剂洗涤剂血小板、HPGF-C18 用于 BMSC 培养。
比较第 2 代 BMSC 的生长情况发现，10%的 HPGF-C18 产生的细胞数量与 20%的 FBS 相似。从 5 名健康受试者的骨髓抽吸物中，在 10%的 HPGF-C18 或 20%的 FBS 中培养 4 代，并分析增殖、集落形成效率（CFE）、表面标志物表达、混合淋巴细胞反应（MLR）抑制、全基因组和 microRNA 表达分析。还比较了 BMSC 上清液细胞因子和生长因子浓度。
骨髓抽吸物的原代培养在 10%的 HPGF-C18 和 20%的 FBS 中产生相似数量和 CFE。经过 4 代培养，10%的 HPGF-C18 和 20%的 FBS 产生相似数量的 BMSCs、表面标志物表达模式和免疫抑制作用。基因和 microRNA 表达分析显示，在两种条件下培养的 BMSCs 具有明显不同的表达谱。基因集富集分析（GSEA）显示，HPGF-C18 培养的 BMSCs 在代谢加工和生物合成途径、细胞增殖和细胞周期途径以及免疫反应途径中富集。FBS 培养的 BMSCs 在 MAPK 信号、TGF-β信号、细胞黏附和细胞外基质途径中富集。差异表达的 microRNAs 与 BMSCs 的成骨作用有关。HPGF-C18 BMSCs 的上清液中 PEDF 和 TGFB1 的水平较高，而 IL6、VEGF、SDF1 和 PLGF 的水平较低。
传统的测量方法、扩增、表面标志物表达和 MLR 抑制表明，在 HPGF-C18 和 FBS 中培养的 BMSC 相似，但在分子水平上的分析显示出许多差异。在替代 FBS 之前，HPGF-C18 培养的 BMSCs 应在特定的功能测定中进行评估，这些测定反映了特定应用的效力。
