Department of Biomedical Engineering, Cleveland Clinic, Cleveland, Ohio, United States of America.
PLoS One. 2012;7(8):e39491. doi: 10.1371/journal.pone.0039491. Epub 2012 Aug 27.
Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.
利用新型的媒体配方，已经证明人类胎盘和脐带血来源的 CD34+细胞可以被高效地扩增和分化为红细胞。然而，从不成熟的细胞培养物中以高效率获得成熟且功能正常的红细胞仍然是一个重大的挑战。网织红细胞和成熟红细胞的一个显著特征是血红蛋白浓度的增加和细胞体积的减小，这导致在缺氧条件下暴露于强磁场和梯度中时，细胞的磁泳动性（MM）增加。利用这些初步观察结果，我们研究了一种非侵入性（无标记）的磁分离和分析过程，以富集和鉴定培养的功能性红细胞。除了在磁场中的磁性细胞分离和细胞运动分析之外，还通过相差显微镜对细胞沉降率和细胞体积分布进行了特征描述，使用免疫表型（血型糖蛋白 A）、血红蛋白浓度和剪切诱导的变形性（通过 ektacytometry 测量伸长指数，EI）来测试成熟红细胞的属性。使用商业的、填充柱高梯度磁分离器（HGMS）进行磁性分离。磁性富集部分包含 80%的成熟细胞（主要是网织红细胞），其 EI 与参考脐带血来源的 RBC 之间的重叠接近 70%，与成人供体 RBC 的重叠超过 50%。结果表明，基于成熟红细胞中存在顺磁性血红蛋白，可以实现基于 CD34+祖细胞衍生培养物的红细胞部分的无标记磁性富集。
