Developing a label-free microfluidic strategy for downstream processing of stem cell-derived red blood cells

Stem cell-originated therapeutic products, such as in vitro manufactured red blood cells (mRBC), offer a novel route to treating disease by administration of a viable somatic cells that have been selected and manipulated outside the body. Cell-based therapeutics are different to traditional biopharmaceutical products and that presents a challenge of developing robust and economically feasible manufacturing processes, especially in sample purification. To address this challenge, I investigated label-free separation methods based on cell endogenous properties such as size and deformability as sorting parameters. In this study the mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were characterised to identifying the best route for mRBC purification. For the first time it has been demonstrated how deformability-induced lift force affects and contributes to particles separation in spiral microchannels. These findings were translated and incorporated into a new route for high-throughput (processing millions of cells /min and mls of medium/ min) continuous purification strategy for separating mRBC from contaminant by-products (purity >99%). This work is anticipated to bring the benefits of mRBC to a wide range of patients by enabling their manufacture as a reliable, safe and controlled supply of red blood cells for transfusion

Limitation of spiral microchannels for particle separation in heterogeneous mixtures: impact of particles’ size and deformability

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 <0.0012), is attributed to the hydrodynamic capture of beads by larger cells. These changes in particles focussing behaviour directly impede the efficiency of the separation – diverting beads from locations expected from measurements with pure populations to co-collection with larger cells – and could hamper deployment of the technology for certain applications. Similar focussing behaviour alterations were noted when working with purification of stem cell end products

Deformability-induced lift force in spiral microchannels for cell separation

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

Impact of poloxamer 188 (Pluronic F-68) additive on cell mechanical properties, quantification by real-time deformability cytometry

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

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

Cell-based therapeutics, such as in vitro manufactured red blood cells (mRBC), 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 immuno-labelling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterised mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop a mRBC purification strategy. The approach consists of two main stages: (1) a microfluidic separation using inertial focusing for deformability-based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (2) 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 cells 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

Crimes adolescents et genre

Étude de genre ou critique des médias ? Cet article d’Arthur Vuattoux joue sur les deux registres. En effet, l’auteur se « focalise sur la mise en mots de deux affaires criminelles » et démontre, très clairement, que le traitement médiatique de ces deux évènements est passé au travers du prisme -déformant- des normes de genre. Dans le premier crime, un lycéen est en cause alors que dans l’autre, il s’agit de rendre compte des exactions d’un groupe d’adolescentes. Ces deux articles sont traité..

Gentrification et mixité

La gentrification est ce phénomène qui consiste, pour les personnes issues des classes moyennes-supérieures, à s’installer dans d’anciens quartiers populaires. La question centrale de cet article est de savoir si cette appropriation d’un territoire s’accompagne d’une volonté – et d’une pratique – de la mixité pour les enfants des ces familles. Grâce à une étude réalisée à la fois à Paris (quartier des Batignolles) et à San Francisco (Noe Valley), les auteurs de cet article en sont arrivés à m..

Gentrification et mixité

La gentrification est ce phénomène qui consiste, pour les personnes issues des classes moyennes-supérieures, à s’installer dans d’anciens quartiers populaires. La question centrale de cet article est de savoir si cette appropriation d’un territoire s’accompagne d’une volonté – et d’une pratique – de la mixité pour les enfants des ces familles. Grâce à une étude réalisée à la fois à Paris (quartier des Batignolles) et à San Francisco (Noe Valley), les auteurs de cet article en sont arrivés à m..