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Cell‐based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability‐based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high‐throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products., To address the challenge of stem‐cell‐derived red blood cells purification, we propose a label‐free approach to separate cells at high throughput based on their morphological (size) and mechanical (deformability) properties. The process consists of two main steps: (a) a microfluidic separation using inertial focusing in spiral microchannel and (b) membrane filtration.

Correction for ‘Deformability-induced lift force in spiral microchannels for cell separation’ by Ewa Guzniczak et al., Lab Chip, 2020, 20, 614–625.

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

Cell sorting and isolation from a heterogeneous mixture is a crucial task in many aspects of cell biology, biotechnology and medicine. Recently, there has been an interest in methods allowing cell separation upon their intrinsic properties such as cell size and deformability, without the need for use of biochemical labels. Inertial focusing in spiral microchannels has been recognised as an attractive approach for high-throughput cell sorting for myriad point of care and clinical diagnostics. Particles of different sizes interact to a different degree with the fluid flow pattern generated within the spiral microchannel and that leads to particles ordering and separation based on size. However, the deformable nature of cells adds complexity to their ordering within the spiral channels. Herein, an additional force, deformability-induced lift force (FD), involved in the cell focusing mechanism within spiral microchannels has been identified, investigated and reported for the first time, using a cellular deformability model (where the deformability of cells is gradually altered using chemical treatments). Using this model, we demonstrated that spiral microchannels are capable of separating cells of the same size but different deformability properties, extending the capability of the previous method. We have developed a unique label-free approach for deformability-based purification through coupling the effect of FD with inertial focusing in spiral microchannels. This microfluidic-based purification strategy, free of the modifying immuno-labels, allowing cell processing at a large scale (millions of cells per min and mls of medium per minute), up to high purities and separation efficiency and without compromising cell quality.

The mechanical performance of cardiomyocytes (CMs) is an important indicator of their maturation state and of primary importance for the development of therapies based on cardiac stem cells. As the mechanical analysis of adherent cells at high-throughput remains challenging, we explore the applicability of real-time deformability cytometry (RT-DC) to probe cardiomyocytes in suspension. RT-DC is a microfluidic technology allowing for real-time mechanical analysis of thousands of cells with a throughput exceeding 1000 cells per second. For CMs derived from human-induced pluripotent stem cells, we determined a Young's modulus of 1.25 ± 0.08 kPa which is in close range to previous reports. Upon challenging the cytoskeleton with cytochalasin D (CytoD) to induce filamentous actin depolymerization, we distinguish three different regimes in cellular elasticity. Transitions are observed below 10 nM and above 10 3 nM and are characterized by a decrease in Young's modulus. These regimes can be linked to cytoskeletal and sarcomeric actin contributions by CM contractility measurements at varying CytoD concentrations, where we observe a significant reduction in pulse duration only above 10 3 nM while no change is found for compound exposure at lower concentrations. Comparing our results to mechanical cell measurements using atomic force microscopy, we demonstrate for the first time to our knowledge, the feasibility of using a microfluidic technique to measure mechanical properties of large samples of adherent cells while linking our results to the composition of the cytoskeletal network. This article is part of a discussion meeting issue ‘Single cell ecology'.

Advances in cellular therapies have led to the development of new approaches for cell product purification and formulation, e.g., utilizing cell endogenous properties such as size and deformability as a basis for separation from potentially harmful undesirable by-products. However, commonly used additives such as Pluronic F-68 and other poloxamer macromolecules can change the mechanical properties of cells and consequently alter their processing. In this paper, we quantified the short-term effect of Pluronic F-68 on the mechanotype of three different cell types (Jurkat cells, red blood cells, and human embryonic kidney cells) using real-time deformability cytometry. The impact of the additive concentration was assessed in terms of cell size and deformability. We observed that cells respond progressively to the presence of Pluronic F-68 within first 3 h of incubation and become significantly stiffer (p-value < 0.001) in comparison to a serum-free control and a control containing serum. We also observed that the short-term response manifested as cell stiffening is true (p-value < 0.001) for the concentration reaching 1% (w/v) of the poloxamer additive in tested buffers. Additionally, using flow cytometry, we assessed that changes in cell deformability triggered by addition of Pluronic F-68 are not accompanied by size or viability alterations.

Spiral microchannels have shown promising results for separation applications. Hydrodynamic particle-particle interactions are a known factor strongly influencing focussing behaviours in inertial devices, with recent work highlighting how the performance of bidisperse mixtures is altered when compared with pure components, in square channels. This phenomenon has not been previously investigated in detail for spiral channels. Here, we demonstrate that, in spiral channels, both the proportion and deformability of larger particles (13 μm diameter) impact upon the recovery (up to 47% decrease) of small rigid particles (4 μm). The effect, observed at low concentrations (volume fraction