High-throughput assessment of mechanical properties of stem cell derived red blood cells, toward cellular downstream processing

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.

Purifying stem cell‐derived red blood cells: a high‐throughput label‐free downstream processing strategy based on microfluidic spiral inertial separation and membrane filtration

“…The first step to develop the label‐free strategy for purifying mRBC from the contaminant nucleated cells and free‐floating nuclei was to identify if there was a unique set of label‐free markers, such as cell size and deformability that would allow characterization of each of the subsets within the final product. These findings are detailed for donor III in Guzniczak et al (2017) and for donors I and II in Table S3 (for the convenience of the reader, also, data on donor III are included in STable 3). The study was further conducted here for cells from three different donors (see Figure 3 for exemplary data of one replica from each donor).…”

“…However, this was achieved by coculture with macrophages, making the protocol challenging to scale‐up (Goers, Freemont, & Polizzi, 2014). With lower enucleation rates, the presence of residual nucleated cells and expelled nuclei constitute a potential danger if intended for transfusion into patients (Bouhassira, 2008; Guzniczak et al, 2017). Undifferentiated nucleated cells can give rise to teratomas (benign tumors of differentiating cells) and teratocarcinomas (malignant metastatic tumors composed of highly proliferative cells; McGowan, Campbell, & Mountford, 2018), thus they have to be removed from the sample and require adequate purification approaches.…”

Loss of clathrin heavy chain enhances actin-dependent stiffness of mouse embryonic stem cells

“…These include endocytic pathways and proteins, mechanical properties, in addition to transcription factors and epigenetic modifiers. Recent reports have described using mechanical phenotyping as an label-free and efficient technique for large-scale purification of cells [33]. However, a better understanding of the interplay between mechanical properties and expression of stem cell-specific transcription factors is required before mechanical phenotyping can be used for large-scale purification of cells.…”

Optical Tweezers in Studies of Red Blood Cells

“…In terms of erythropoiesis, the RBCs experience complete changes in cell composition and membrane mechanical properties during the journey of RBC production from the immature pronormoblasts to the mature biconcave discocytes [108]. Studies have shown that measurements of membrane mechanical attributes of the developing RBCs indicate the structural and functional maturation of the membrane skeleton [103].…”

“…Immature RBCs experience dramatic changes in shape, cellular composition, and membrane mechanical and physical properties throughout the erythropoietic process [103]. The quantitative evaluation of the stiffness of the nucleated and enucleated RBCs during maturation enables the direct evaluation of the development of membrane skeleton [108]. Mature human red blood cells (RBCs) are unique mammalian cells in biconcave shape (typically 6-8 µm in diameter and 2 µm in thickness) that contain no nucleus or subcellular metabolic structures [130].…”