Avanzi Mauro P, Oluwadara Oluwasijibomi E, Cushing Melissa M, Mitchell Maxwell L, Fischer Stephen, Mitchell W Beau
New York Blood Center, New York, New York.
Weill Cornell Medical College, New York, New York.
Transfusion. 2016 Jan;56(1):170-8. doi: 10.1111/trf.13375. Epub 2015 Oct 15.
Platelet (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.
CD34 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.
CD34 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.
Functional 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.
血小板（PLT）输注是治疗血小板减少症的主要方法。血小板仅从志愿捐献者处获得，且血小板制品的保质期仅为5天，这可能会限制供应并导致血小板短缺。源自干细胞的血小板有助于满足这一临床需求。然而，目前的培养方法产生的血小板数量远远不足以用于临床应用。为满足这一需求，设计了一种明确的无血清培养方法，使用新型生物反应器来提高干细胞来源的巨核细胞产生的血小板产量。
从脐带血中分离出的CD34细胞使用无血清培养基，在多种试剂作用下并在纳米纤维膜上进行扩增。然后在无血清条件下，将这些细胞与血小板生成素和干细胞因子一起培养，使其分化为巨核细胞系。通过向CD41细胞中添加Rho激酶抑制剂或肌动蛋白聚合抑制剂来诱导多倍体形成。开发了一种新型生物反应器，该反应器模拟了骨髓血管微环境的某些方面。经光镜、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伸出前血小板并释放血小板。
CD34细胞扩增了100倍。CD41细胞扩增了100倍。每个输入的巨核细胞可从生物反应器中产生多达100个血小板，每个输入的CD34细胞的总产量为10⁶个血小板。激活后，血小板外化P-选择素。
使用无血清培养条件、新颖的分步方法和创新的生物反应器，可以在临床相关规模上体外产生功能性血小板。
