Mettananda Sachith, Clark Kevin, Fisher Chris A, Sloane-Stanley Jackie A, Gibbons Richard J, Higgs Douglas R
Medical Research Council (MRC) Molecular Hematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom; Department of Paediatrics, Faculty of Medicine, University of Kelaniya, Ragama, Sri Lanka.
Medical Research Council (MRC) Molecular Hematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom.
Exp Hematol. 2018 Apr;60:10-20. doi: 10.1016/j.exphem.2018.01.001. Epub 2018 Jan 9.
In vitro erythroid differentiation systems are used to study the mechanisms underlying normal and abnormal erythropoiesis and to test the effects of various extracellular factors on erythropoiesis. The use of serum or conditioned medium in liquid cultures and the seeding of cultures with heterogeneous peripheral blood mononuclear cells confound the reproducibility of these systems. Newer erythroid differentiation culture systems have overcome some of these limitations by using a fully defined, serum-free medium and initiating cultures using purified CD34 cells. Although widely used in bulk cultures, these protocols have not been rigorously tested in high-throughput or single-cell assays. Here, we describe a serum-free erythroid differentiation system suitable for small-scale and single-cell experiments. This system generates large numbers of terminally differentiated erythroid cells of very high purity. Here we have adapted this culture system to a 96-well format and have developed a protocol to grow erythroid colonies from single erythroid progenitors in minute culture volumes.
体外红系分化系统用于研究正常和异常红细胞生成的潜在机制，并测试各种细胞外因子对红细胞生成的影响。在液体培养中使用血清或条件培养基以及用异质性外周血单核细胞接种培养物会影响这些系统的可重复性。更新的红系分化培养系统通过使用完全确定的无血清培养基并使用纯化的CD34细胞启动培养，克服了其中一些局限性。尽管这些方案在批量培养中广泛使用，但尚未在高通量或单细胞分析中进行严格测试。在这里，我们描述了一种适用于小规模和单细胞实验的无血清红系分化系统。该系统可产生大量纯度非常高的终末分化红系细胞。在这里，我们已将此培养系统调整为96孔板形式，并开发了一种方案，以在微小培养体积中从单个红系祖细胞培养红系集落。
