Mata Márcia F, Lopes João Pedro, Ishikawa Masakazu, Alaiti Mohamad A, Cabral Joaquim M S, da Silva Cláudia L, Costa Marco A
Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
Department of Medicine, Case Cardiovascular Research Institute, Harrington-McLaughlin Heart & Vascular Institute, University Hospitals Case Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
Cytotherapy. 2015 Dec;17(12):1777-84. doi: 10.1016/j.jcyt.2015.09.003. Epub 2015 Oct 21.
The therapeutic application of CD34+ circulating progenitor cells (which includes endothelial progenitor cells) has been hampered by the quantity and quality of isolated circulating CD34(+) cells from the patient's peripheral blood. Our group had previously established a suspension culture system for human CD34(+) cells, with increased quantity and quality (QQ) of the angiogenic cell product. We successfully scaled up the expansion process with the use of culture bags because there is the need to move toward a dynamic and fully controlled bioreactor system to meet Good Manufacturing Practice (GMP) standards and attain clinically meaningful cell doses in a time- and cost-effective way.
CD34(+) cells isolated from mobilized peripheral blood of healthy donors were expanded ex vivo for 7 days in QQ medium (serum-free) in cell culture bags (30 mL) and pre- and post-expansion cells were characterized by means of flow cytometry and quantitative polymerase chain reaction; angiogenic potential was assessed by use of the in vitro tube formation assay.
Our data show effective expansion of the cultured population (7-fold) while maintaining the stem/progenitor content and increasing the endothelial population. Moreover, post-expanded cells showed higher tube formation capacity compared with pre-expanded cells. In addition, an upregulation of the anti-inflammatory gene expression and a downregulation of pro-inflammatory genes were observed, which suggests that the increase in angiogenic potential is not paired with an increase in the inflammatory profile.
The QQ expansion method was successfully scaled up to cell culture bags and was able to meet GMP standards, with a higher in vitro angiogenic profile.
循环CD34+祖细胞（包括内皮祖细胞）的治疗应用受到从患者外周血中分离出的循环CD34+细胞数量和质量的限制。我们团队之前建立了一种人CD34+细胞的悬浮培养系统，可提高血管生成细胞产物的数量和质量（QQ）。我们成功地利用培养袋扩大了扩增过程，因为需要朝着动态且完全可控的生物反应器系统发展，以满足药品生产质量管理规范（GMP）标准，并以具有时间和成本效益的方式获得具有临床意义的细胞剂量。
从健康供体动员的外周血中分离出的CD34+细胞，在细胞培养袋（30 mL）中的QQ培养基（无血清）中进行体外扩增7天，扩增前后的细胞通过流式细胞术和定量聚合酶链反应进行表征；通过体外管形成试验评估血管生成潜力。
我们的数据显示培养群体有效扩增（7倍），同时保持了干/祖细胞含量并增加了内皮细胞群体。此外，扩增后的细胞与扩增前的细胞相比显示出更高的管形成能力。另外，观察到抗炎基因表达上调，促炎基因表达下调，这表明血管生成潜力的增加并未伴随着炎症特征的增加。
QQ扩增方法成功扩大到细胞培养袋，能够满足GMP标准，具有更高的体外血管生成特征。
