van den Oudenrijn S, von dem Borne A E, de Haas M
Central Laboratory of the Netherlands Blood Transfusion Service (CLB) and Laboratory of Experimental and Clinical Immunology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
J Hematother Stem Cell Res. 2001 Feb;10(1):193-200. doi: 10.1089/152581601750098516.
Reinfusion of ex vivo-expanded autologous megakaryocytes together with a stem cell transplantation may be useful to prevent or reduce the period of chemotherapy-induced thrombocytopenia. In this study, we analyzed several serum-containing and serum-free media to identify the most suitable medium for megakaryocyte expansion. Moreover, two thrombopoietin (Tpo)-mimetic peptides were tested to evaluate whether they could replace Tpo in an expansion protocol. To analyze the effects of different media on megakaryocyte expansion, we used an in vitro liquid culture system. For this purpose, CD34(+) cells were isolated from peripheral blood and cultured for 8 days in the presence of Tpo and interleukin-3 (IL-3). The presence of megakaryocytes was analyzed by flow cytometric analysis after staining for CD41 expression. For our standard culture procedure, megakaryocyte medium (MK medium) supplemented with 10% AB plasma was used. Addition of 5% or 2.5% AB plasma yielded higher numbers of megakaryocytes, implying the presence of inhibitory factors in plasma. However, some plasma components are required for optimal megakaryocyte expansion because addition of less than 1% AB plasma or addition of human serum albumin instead of AB plasma resulted in the formation of lower numbers of megakaryocytes. Two commercially available serum-free media were also tested: Cellgro and Stemspan. If CD34(+) cells were cultured in Cellgro medium similar numbers of megakaryocytes were obtained as when CD34(+) cells were cultured in MK medium supplemented with 10% AB plasma. In MK medium with 2.5% AB plasma, higher numbers of megakaryocytes were cultured than in MK medium supplemented with 10% AB plasma. Therefore, Cellgro medium is not the best alternative medium. In cultures with Stemspan medium, higher numbers of megakaryocytes were obtained compared to MK medium with 10% AB plasma. Stemspan is thus a good alternative for MK medium. Two Tpo-mimetic peptides, AF13948 and PK1M, were tested for their ability to replace Tpo. In cultures with AF13948, comparable numbers of megakaryocytes were obtained as in the presence of Tpo, but in cultures with PK1M the number of megakaryocytes was lower. This study shows that high concentrations of plasma in medium inhibits megakaryocyte formation, but some plasma components are required for optimal megakaryocyte expansion. For an ex vivo expansion protocol, it is worthwhile to test several media, because the number of megakaryocytes differs widely with the medium used.
将体外扩增的自体巨核细胞与干细胞移植一起回输，可能有助于预防或缩短化疗引起的血小板减少期。在本研究中，我们分析了几种含血清和无血清培养基，以确定最适合巨核细胞扩增的培养基。此外，还测试了两种血小板生成素（Tpo）模拟肽，以评估它们是否可以在扩增方案中替代Tpo。为了分析不同培养基对巨核细胞扩增的影响，我们使用了体外液体培养系统。为此，从外周血中分离出CD34(+)细胞，并在Tpo和白细胞介素-3（IL-3）存在的情况下培养8天。通过对CD41表达进行染色后，用流式细胞术分析巨核细胞的存在情况。对于我们的标准培养程序，使用补充有10% AB血浆的巨核细胞培养基（MK培养基）。添加5%或2.5% AB血浆可产生更多数量的巨核细胞，这意味着血浆中存在抑制因子。然而，一些血浆成分对于最佳巨核细胞扩增是必需的，因为添加少于1% AB血浆或用人血清白蛋白代替AB血浆会导致形成的巨核细胞数量减少。还测试了两种市售的无血清培养基：Cellgro和Stemspan。如果将CD34(+)细胞培养在Cellgro培养基中，获得的巨核细胞数量与将CD34(+)细胞培养在补充有10% AB血浆的MK培养基中时相似。在含有2.5% AB血浆的MK培养基中培养的巨核细胞数量高于补充有
