High-throughput assessment of mechanical properties of stem cell derived red blood cells, toward cellular downstream processing.
High-throughput assessment of mechanical properties of stem cell derived red blood cells, toward cellular downstream processing.
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DOI：
10.1038/s41598-017-14958-w
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发表时间：
2017-10-31
影响因子：
4.6
通讯作者：
Bridle H
中科院分区：
文献类型：
--
作者：
Guzniczak E;Mohammad Zadeh M;Dempsey F;Jimenez M;Bock H;Whyte G;Willoughby N;Bridle H
干细胞产品，包括人造红细胞，需要有效的分选和纯化方法，以去除可能对临床应用有害的成分。然而，蜂窝下游处理的标准方法依赖于特定且昂贵的标签的使用(例如，FAC或MACs)。依靠细胞固有的机械和物理属性的技术提供了高通量、可扩展的替代方案，但需要了解机械表型。在这里，我们首次利用实时可变形性细胞术(RT-DC)和原子力显微镜(AFM)研究了CD34+细胞在11、14、18和21天分化为红细胞的过程中的变形性和大小变化，并排除了核。我们发现有核细胞和去核细胞的变形性有显著差异(p <； 0.0001；标准化混合模型)，但它们保持在相同的大小范围内。排出的原子核较小，因此可以通过基于大小的分离来去除。测得有核细胞、去核细胞和核(14天)的平均杨氏弹性模量分别为1.04 ± 0.47kPa、0.53 ± 0.12kPa和7.06 ± 4.07kPa.我们鉴定和量化了CD34+细胞在分化过程中机械特性的显著差异(p <； 0.0001；ANOVA)，这有助于开发新的人造红细胞纯化方法。
Stem cell products, including manufactured red blood cells, require efficient sorting and purification methods to remove components potentially harmful for clinical application. However, standard approaches for cellular downstream processing rely on the use of specific and expensive labels (e.g. FACS or MACS). Techniques relying on inherent mechanical and physical properties of cells offer high-throughput scalable alternatives but knowledge of the mechanical phenotype is required. Here, we characterized for the first time deformability and size changes in CD34+ cells, and expelled nuclei, during their differentiation process into red blood cells at days 11, 14, 18 and 21, using Real-Time Deformability Cytometry (RT-DC) and Atomic Force Microscopy (AFM). We found significant differences (p < 0.0001; standardised mixed model) between the deformability of nucleated and enucleated cells, while they remain within the same size range. Expelled nuclei are smaller thus could be removed by size-based separation. An average Young’s elastic modulus was measured for nucleated cells, enucleated cells and nuclei (day 14) of 1.04 ± 0.47 kPa, 0.53 ± 0.12 kPa and 7.06 ± 4.07 kPa respectively. Our identification and quantification of significant differences (p < 0.0001; ANOVA) in CD34+ cells mechanical properties throughout the differentiation process could enable development of new routes for purification of manufactured red blood cells.
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