PDF(Pubmed)
Abstract：
Muscle-derived mesenchymal stromal cells (mdMSCs) hold great promise in regenerative medicine due to their immunomodulatory properties, multipotent differentiation capacity and ease of collection. However, traditional in vitro expansion methods use fetal bovine serum (FBS) and have numerous limitations including ethical concerns, batch-to-batch variability, immunogenicity, xenogenic contamination and regulatory compliance issues. This study investigates the use of 10% equine platelet lysate (ePL) obtained by plasmapheresis as a substitute for FBS in the culture of mdMSCs in innovative 2D and 3D models. Using muscle microbiopsies as the primary cell source in both models showed promising results. Initial investigations indicated that small variations in heparin concentration in 2D cultures strongly influenced medium coagulation with an optimal proliferation observed at final heparin concentrations of 1.44 IU/mL. The two novel models investigated showed that expansion of mdMSCs is achievable. At the end of expansion, the 3D model revealed a higher total number of cells harvested (64.60 ± 5.32 million) compared to the 2D culture (57.20 ± 7.66 million). Trilineage differentiation assays confirmed the multipotency (osteoblasts, chondroblasts and adipocytes) of the mdMSCs generated in both models with no significant difference observed. Immunophenotyping confirmed the expression of the mesenchymal stem cell (MSC) markers CD-90 and CD-44, with low expression of CD-45 and MHCII markers for mdMSCs derived from the two models. The generated mdMSCs also had great immunomodulatory properties. Specific immunological extraction followed by enzymatic detection (SIEFED) analysis demonstrated that mdMSCs from both models inhibited myeloperoxidase (MPO) activity in a strong dose-dependent manner. Moreover, they were also able to reduce reactive oxygen species (ROS) activity, with mdMSCs from the 3D model showing significantly higher dose-dependent inhibition compared to the 2D model. These results highlighted for the first time the feasibility and efficacy of using 10% ePL for mdMSC expansion in novel 2D and 3D approaches and also that mdMSCs have strong immunomodulatory properties that can be exploited to advance the field of regenerative medicine and cell therapy instead of using FBS with all its drawbacks.
摘要：
肌肉来源的间充质基质细胞（mdMSCs）由于其免疫调节特性而在再生医学中具有广阔的前景，多能分化能力和易于收集。然而,传统的体外扩增方法使用胎牛血清（FBS），并且有许多局限性，包括伦理问题，批次间的可变性，免疫原性,异种污染和监管合规问题。这项研究调查了通过血浆置换获得的10％马血小板裂解物（ePL）在创新的2D和3D模型中的mdMSC培养中作为FBS的替代品的使用。在两种模型中使用肌肉微活检作为主要细胞来源均显示出有希望的结果。初步研究表明，2D培养物中肝素浓度的微小变化强烈影响培养基的凝血，在肝素终浓度为1.44IU/mL时观察到最佳增殖。所研究的两个新模型表明mdMSC的扩增是可实现的。在扩张结束时,3D模型显示，与2D培养物（57.20±766万）相比，收获的细胞总数（64.60±532万）更高。三系分化试验证实了多能性(成骨细胞，在两种模型中产生的mdMSC的成软骨细胞和脂肪细胞），没有观察到显着差异。免疫分型证实了间充质干细胞(MSC)标志物CD-90和CD-44的表达,来自两个模型的mdMSC的CD-45和MHCII标志物的低表达。产生的mdMSC也具有良好的免疫调节特性。特异性免疫提取，然后进行酶检测（SIEFED）分析表明，来自两个模型的mdMSC以强剂量依赖性方式抑制髓过氧化物酶（MPO）活性。此外，它们还能够降低活性氧（ROS）活性，与2D模型相比,来自3D模型的mdMSC显示出显著更高的剂量依赖性抑制。这些结果首次强调了在新型2D和3D方法中使用10％ePL进行mdMSC扩增的可行性和有效性，并且mdMSC具有强大的免疫调节特性，可用于推进再生医学和细胞治疗领域，而不是使用FBS及其所有缺点。
