Lankford Lee, Chen Y Julia, Saenz Zoe, Kumar Priyadarsini, Long Connor, Farmer Diana, Wang Aijun
Surgical Bioengineering Laboratory, Department of Surgery, University of California, Davis School of Medicine, Sacramento, California, USA.
Surgical Bioengineering Laboratory, Department of Surgery, University of California, Davis School of Medicine, Sacramento, California, USA.
Cytotherapy. 2017 Jun;19(6):680-688. doi: 10.1016/j.jcyt.2017.03.003. Epub 2017 Apr 21.
In this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech).
PMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA).
PMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3-5 × 10 cells/cm, and seeded cells were >95% viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots.
PMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities.
在本研究中，我们描述了一种符合现行药品生产质量管理规范（CGMP）的流程，用于分离、扩增和保存胎盘来源的间充质基质细胞（PMSC），以用作干细胞治疗。我们对接种于临床级猪小肠黏膜下层细胞外基质（SIS - ECM；库克生物技术公司）上的PMSC的活力、增殖和神经保护分泌谱进行了表征。
通过组织块培养从早期妊娠胎盘绒毛膜绒毛组织中分离PMSC。对细胞进行扩增、保存和筛选。使用流式细胞术测定多能性标志物的纯度和表达。分别使用3 -（4,5 - 二甲基噻唑 - 2 - 基）- 5 -（3 - 羧甲氧基苯基）- 2 -（4 - 磺基苯基）- 2H - 四唑（MTS）细胞增殖试验和荧光活/死试验测定PMSC在SIS - ECM上的最佳接种密度和活力。使用酶联免疫吸附测定（ELISA）分析生长因子分泌情况。
PMSC迅速扩增并保存。主细胞库和工作细胞库在6个月时活力稳定且活力下降最小。所有PMSC均无菌、无支原体、核型正常且内毒素水平低。通过免疫表型分析，PMSC具有同质性，几乎不表达或不表达多能性标志物。在SIS - ECM上的最佳接种密度为3 - 5×10个细胞/cm，接种的细胞活力>95%。在塑料和SIS - ECM上接种的PMSC可检测到神经营养因子分泌，不同供体批次之间存在差异。
来自早期妊娠胎盘组织的PMSC可以在稳定、有活力且无污染、基因正常和纯净的细胞库中迅速扩增和保存。PMSC的递送可以通过使用SIS - ECM来实现，SIS - ECM可维持细胞活力和蛋白质分泌。未来有必要进行体内研究以优化细胞接种和移植，从而最大限度地提高治疗能力。
