Large-scale in vitro production of red blood cells from human peripheral blood mononuclear cells
Large-scale in vitro production of red blood cells from human peripheral blood mononuclear cells
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DOI：
10.1182/bloodadvances.2019000689
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发表时间：
2019-11-12
期刊：
影响因子：
7.5
通讯作者：
van den Akker, Emile
中科院分区：
文献类型：
--
作者：
Heshusius, Steven;Heideveld, Esther;van den Akker, Emile
输注供体来源的红细胞 (RBC) 是最常见的细胞治疗形式。然而，捐助者的可用性以及同种免疫和其他输血相关并发症的潜在风险可能会限制输血单位的可用性，特别是对于长期输血的患者。体外培养、可定制的红细胞将消除这些担忧并进一步提高精准医疗。大规模、具有成本效益的生产取决于培养条件的优化。我们开发了一种成分确定的培养基，并根据良好生产规范 (GMP) 培养要求调整了我们的方案，该培养基可重复地从外周血单核细胞中提供纯红系培养物，无需事先进行 CD34(+) 分离，并且在 2S 天内红细胞数量增加 3 x 10(7) 倍（或从 1 亿个外周血单核细胞中，可以产生 2 至 4 mL 浓缩红细胞）。扩增的成红细胞培养物可在 12 天内分化为 CD71(dim) CD235a(+)CD44(+)CD117(-)DRAQ5(-)RBC。超过 90% 的细胞去核并表达成人血红蛋白以及正确的血型抗原。培养红细胞的变形能力和氧结合能力与体内网织红细胞相当。分化期间每日进行 RNA 采样，然后进行 RNA 测序，提供了终末红细胞生成过程中发生的变化的高分辨率图/资源。该培养过程与使用每个反应器容量为 1 L 的 G-Rex 生物反应器进行升级兼容，从而可以过渡到临床研究和小规模应用。
Transfusion of donor-derived red blood cells (RBC) is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units, especially for chronically transfused patients. In vitro cultured, customizable RBC would negate these concerns and further increase precision medicine. Large-scale, cost-effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to good manufacturing practice (GMP) culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD34(+) isolation, and a 3 x 10(7)-fold increase in erythroblasts in 2S days (or from 100 million peripheral blood mononuclear cells, 2 to 4 mL packed red cells can be produced). Expanded erythroblast cultures could be differentiated to CD71(dim) CD235a(+)CD44(+)CD117(-)DRAQ5(-)RBC in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen-binding capacity of cultured RBC was comparable to in vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-sequencing provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1 L per reactor, allowing transition toward clinical studies and small-scale applications.