A novel bioreactor and culture method drives high yields of platelets from stem cells
A novel bioreactor and culture method drives high yields of platelets from stem cells
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DOI：
10.1111/trf.13375
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发表时间：
2016-01-01
期刊：
影响因子：
2.9
通讯作者：
Mitchell, W. Beau
中科院分区：
文献类型：
--
作者：
Avanzi, Mauro P.;Oluwadara, Oluwasijibomi E.;Mitchell, W. Beau
背景血小板（PLT）输注是治疗血小板减少症的主要方法。PLT只能从志愿者捐献者处获得，而PLT产品只有5天的保质期，这可能会限制PLT的供应，导致PLT短缺。干细胞衍生的plt可以帮助满足这一临床需求。然而，目前的培养方法产生的plt太少，无法用于临床应用。为了满足这一需求，使用新型生物反应器设计了一种明确的无血清培养方法，以提高干细胞来源的巨核细胞的plt产量。研究设计与方法从脐带血中分离scd34细胞，用多种试剂和无血清培养基在纳米纤维膜上扩增。然后在无血清条件下，用血小板生成素和干细胞因子培养这些细胞分化为巨核细胞谱系。在CD41细胞中加入Rho激酶抑制剂或肌动蛋白聚合抑制剂诱导多倍体。开发了一种新的生物反应器，再现了骨髓血管生态位的各个方面。经光学显微镜、荧光成像和流式细胞术证实，在生物反应器中流动的多倍体巨核细胞延长了plt并脱落了plt。结果scd34细胞扩增100倍。CD41细胞扩增100倍。每个输入巨核细胞从生物反应器中产生多达100个plt，每个输入CD34细胞的总产量为10(6)个plt。plt在激活后外化p -选择素。功能性plt可以在临床相关的规模上体外生产，使用无血清培养条件，采用新颖的逐步方法和创新的生物反应器。
BACKGROUNDPlatelet (PLT) transfusion is the primary treatment for thrombocytopenia. PLTs are obtained exclusively from volunteer donors, and the PLT product has only a 5-day shelf life, which can limit supply and result in PLT shortages. PLTs derived from stem cells could help to fill this clinical need. However, current culture methods yield far too few PLTs for clinical application. To address this need, a defined, serum-free culture method was designed using a novel bioreactor to increase the yield of PLTs from stem cell-derived megakaryocytes.STUDY DESIGN AND METHODSCD34 cells isolated from umbilical cord blood were expanded with a variety of reagents and on a nanofiber membrane using serum-free medium. These cells were then differentiated into megakaryocytic lineage by culturing with thrombopoietin and stem cell factor in serum-free conditions. Polyploidy was induced by addition of Rho kinase inhibitor or actin polymerization inhibitor to the CD41 cells. A novel bioreactor was developed that recapitulated aspects of the marrow vascular niche. Polyploid megakaryocytes that were subjected to flow in the bioreactor extended proPLTs and shed PLTs, as confirmed by light microscopy, fluorescence imaging, and flow cytometry.RESULTSCD34 cells were expanded 100-fold. CD41 cells were expanded 100-fold. Up to 100 PLTs per input megakaryocyte were produced from the bioreactor, for an overall yield of 10(6) PLTs per input CD34 cell. The PLTs externalized P-selectin after activation.DISCUSSIONFunctional PLTs can be produced ex vivo on a clinically relevant scale using serum-free culture conditions with a novel stepwise approach and an innovative bioreactor.