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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