Fibach E, Manor D, Treves A, Rachmilewitz E A
Department of Hematology, Hadassah University Hospital, Jerusalem, Israel.
Int J Cell Cloning. 1991 Jan;9(1):57-64. doi: 10.1002/stem.5530090108.
Most studies of erythropoiesis in vitro have employed cloning methods in semisolid medium. We have recently described a two-step liquid culture procedure that supports the proliferation and differentiation of human erythroid progenitors. In the present study, we have modified the procedure to allow large-scale cultures of erythroid cells derived from normal donors. The culture is divided into two phases. In the first phase, which is erythropoietin (Epo) independent, the early erythroid progenitors multiply and differentiate. In the second, Epo-dependent phase, they mature into orthochromatic normoblasts and enucleated erythrocytes. Using this procedure, erythroid cell yield reached 7.5 x 10(6)/ml and a total of 7 x 10(8) cells could be harvested per blood unit. A comparison of the growth of erythroid cells in liquid culture to their colony growth in semisolid culture indicated that cell growth was superior: 1) in liquid culture in terms of cell yield per originally cultured mononuclear cell, 2) per ml culture and per culture surface area and in the purity of the resultant erythroid cell population. In addition, it permits easier manipulation of the culture condition and components and sampling of greater than 1 x 10(7) cells at each maturation stage subsequent to the proerythroblast stage. This liquid culture procedure might provide an important experimental tool for studying erythroid cell development.
大多数体外红细胞生成研究采用半固体培养基中的克隆方法。我们最近描述了一种两步液体培养程序，该程序支持人类红系祖细胞的增殖和分化。在本研究中，我们对该程序进行了改进，以实现对正常供体来源的红系细胞进行大规模培养。培养分为两个阶段。在第一个阶段，即不依赖促红细胞生成素（Epo）的阶段，早期红系祖细胞增殖并分化。在第二个依赖Epo的阶段，它们成熟为正染性晚幼红细胞和去核红细胞。使用该程序，红系细胞产量达到7.5×10⁶/ml，每单位血液可收获总计7×10⁸个细胞。将液体培养中红系细胞的生长与其在半固体培养中的集落生长进行比较表明，细胞生长在以下方面更具优势：1）在液体培养中，就每个最初培养的单个核细胞的细胞产量而言；2）每毫升培养物、每培养表面积以及所得红系细胞群体的纯度。此外，它允许更轻松地控制培养条件和成分，并在早幼红细胞阶段之后的每个成熟阶段对超过1×10⁷个细胞进行采样。这种液体培养程序可能为研究红系细胞发育提供重要的实验工具。
