Abstract
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.
Highlights
Around 112.5 million blood donations are collected across 176 countries in over 13000 blood centres[1]
Manufactured red blood cells have been positively validated as a potential clinical product in 2011 by Giarratana et al.[7], with potentially transfusable RBCs already produced in vitro using embryonic stem cells[5], induced pluripotent stem cells[8], CD34+ cells sourced from bone marrow[4] or umbilical cord blood[9], and recently an immortalized adult human erythroid line (Bristol Erythroid Line Adult BEL-A)[10]
For the wide-spread adoption of Manufactured red blood cells (mRBCs), and other cell therapies, challenges related to cell source, maturation and viability need to be addressed by biologists, while advancements in cell processing technologies will be required to manufacture those cells in meaningful quantities and achieve satisfactory purity
Summary
Around 112.5 million blood donations are collected across 176 countries in over 13000 blood centres[1]. Passive sorting (such as inertial focusing[25], pinch flow fractionation[26], deterministic lateral displacement[27] and filtration28) exploits properties of the device design and, except for a liquid pumping system, they do not require any other external forces These systems present many potential advantages such as a reduced number of sample processing steps (e.g. by reducing staining/washing steps), relatively high-throughput (millilitres/min[29] and litres/min30) and efficiency (>90%)[31,32,33]. Sorting in this type of system is facilitated purely by endogenous cell properties such as size and deformability and further evidence is needed to quantify the cell mechanotype, to overcome the existing lack of knowledge on e.g. mRBCs mechanical properties[34], and determine the potential for mechanotype based sorting. Staining of the nucleus and cytoskeletal proteins was undertaken to investigate the potential contribution of these factors to the observed mechanotypical changes
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