Influence of medium components on ex vivo megakaryocyte expansion

Reinfusion of ex vivo-expanded autologous megakaryocytes together with a st em cell transplantation may be useful to prevent or reduce the period of ch emotherapy-induced thrombocytopenia. In this study, we analyzed several ser um-containing and serum-free media to identify the most suitable medium for megakaryocyte expansion. Moreover, two thrombopoietin (Tpo)-mimetic peptid es were tested to evaluate whether they could replace Tpo in an expansion p rotocol. To analyze the effects of different media on megakaryocyte expansi on, we used an in vitro liquid culture system. For this purpose, CD34(+) ce lls were isolated from peripheral blood and cultured for 8 days in the pres ence of Tpo and interleukin-3 (IL-3). The presence of megakaryocytes was an alyzed by flow cytometric analysis after staining for CD41 expression. For our standard culture procedure, megakaryocyte medium (MK medium) supplement ed with 10% AB plasma was used. Addition of 5% or 2.5% AB plasma yielded hi gher numbers of megakaryocytes, implying the presence of inhibitory factors in plasma. However, some plasma components are required for optimal megaka ryocyte expansion because addition of less than 1% AB plasma or addition of human serum albumin instead of AB plasma resulted in the formation of lowe r numbers of megakaryocytes. Two commercially available serum-free media we re also tested: Cellgro and Stemspan. If CD34(+) cells were cultured in Cel lgro medium similar numbers of megakaryocytes were obtained as when CD34(+) cells were cultured in MK medium supplemented with 10% AB plasma. In MK me dium with 2.5% AB plasma, higher numbers of megakaryocytes were cultured th an in MK medium supplemented with 10% AB plasma. Therefore, Cellgro medium is not the best alternative medium. In cultures with Stemspan medium, highe r 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 obtaine d as in the presence of Tpo, but in cultures with PK1M the number of megaka ryocytes was lower. This study shows that high concentrations of plasma in medium inhibits megakaryocyte formation, but some plasma components are req uired for optimal megakaryocyte expansion. For an ex vivo expansion protoco l, it is worthwhile to test several media, because the number of megakaryoc ytes differs widely with the medium used.