Scalable Platform for Human Embryonic Stem Cell Differentiation to Cardiomyocytes in Suspended Microcarrier Cultures
Scalable Platform for Human Embryonic Stem Cell Differentiation to Cardiomyocytes in Suspended Microcarrier Cultures
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DOI：
10.1089/ten.tec.2010.0104
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发表时间：
2010-12-01
影响因子：
3
通讯作者：
Oh, Steve
中科院分区：
文献类型：
--
作者：
Lecina, Marti;Ting, Sherwin;Oh, Steve
用于人胚胎干细胞（hESC）衍生的心肌细胞（CM）生产的可扩展平台可以为细胞治疗、药物筛选和心脏毒性测试提供容易获得的CM来源。我们设计并优化了一个可扩展的平台，该平台使用无血清培养基中的微载体培养物，补充有丝分裂原活化蛋白激酶抑制剂SB 203580。使用不同的微载体（DE-53、Cytodex-1和3、FACT和TOSOH-10）来研究类型、大小、形状和微载体浓度对分化效率的影响。在浓度为0.125 mg/mL的TOSOH-10（鱼精蛋白衍生的10 μ m珠）上增殖的hESC产生80%的跳动聚集体、三倍的细胞扩增和20%的CM（通过荧光激活细胞分选肌球蛋白重链和α-辅肌动蛋白表达来测定）。接种在该系统中的CM/hESC的比率为0.62，相比之下，在胚状体对照培养物中为0.22。已使用HES-3和H1细胞系测试了平台稳健性，并在悬浮旋转培养中证明了其可扩展性。然而，转瓶培养物产量下降至0.33 CM/hESC，可能是由于剪切应力导致一些细胞死亡。从分化的聚集体解离的细胞显示心脏特异性标志物如α-辅肌动蛋白、肌球蛋白重链和轻链、肌钙蛋白I、结蛋白和emilin-2的阳性染色。最后，还通过有/无阿司咪唑的QT延长（QTempo）试验显示了CM功能。本研究代表了一种新的可扩展的生物处理系统，用于使用符合药品生产质量管理规范的试剂进行CM生产。
A scalable platform for human embryonic stem cell (hESC)-derived cardiomyocyte (CM) production can provide a readily available source of CMs for cell therapy, drug screening, and cardiotoxicity tests. We have designed and optimized a scalable platform using microcarrier cultures in serum-free media supplemented with SB203580 mitogen-activated protein kinase-inhibitor. Different microcarriers (DE-53, Cytodex-1 and 3, FACT, and TOSOH-10) were used to investigate the effects of type, size, shape, and microcarrier concentrations on the differentiation efficiency. hESCs propagated on TOSOH-10 (protamine derivatized 10-mu m beads) at the concentration of 0.125 mg/mL produced 80% beating aggregates, threefold cell expansion, and 20% of CMs (determined by fluorescence-activated cell sorting for myosin heavy chain and a-actinin expression). The ratio of CM/hESC seeded in this system was 0.62 compared to 0.22 in the embryoid body control cultures. The platform robustness has been tested with HES-3 and H1 cell lines, and its scalability was demonstrated in suspended spinner cultures. However, spinner culture yields dropped to 0.33 CM/hESC probably due to shear stress causing some cell death. Cells dissociated from differentiated aggregates showed positive staining for cardio-specific markers such as alpha-actinin, myosin heavy and light chain, troponin I, desmin, and emilin-2. Finally, CM functionality was also shown by QT-prolongation (QTempo) assay with/without Astemizole. This study represents a new scalable bioprocessing system for CM production using reagents that can comply with Good Manufacturing Practice.