PIEZO1-mediated mechanosensing governs NK-cell killing efficiency and infiltration in three-dimensional matrices.

Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments are critical for their functions under physiological conditions. However, the role of mechanosensing in regulating NK-cell killing efficiency in physiologically relevant scenarios is poorly understood. Here, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK-cell killing efficiency in 3D is impaired against softened tumor cells, whereas it is enhanced against stiffened tumor cells. Notably, the durations required for NK-cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel among the examined candidates in NK cells. Perturbation of PIEZO1 abolishes stiffness-dependent NK-cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK-cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK-cell killing efficiency under 3D physiological conditions and the influence of environmental physical cues on NK-cell functions.

The Nanomechanical Properties of CLL Cells Are Linked to the Actin Cytoskeleton and Are a Potential Target of BTK Inhibitors.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. It is known that the ability of lymphocytes to recirculate strongly depends on their capability to rapidly rearrange their cytoskeleton and adapt to external cues; however, little is known about the differences occurring between CLL and healthy B cells during these processes. To investigate this point, we applied a single-cell optical (super resolution microscopy) and nanomechanical approaches (atomic force microscopy, real-time deformability cytometry) to both CLL and healthy B lymphocytes and compared their behavior. We demonstrated that CLL cells have a specific actomyosin complex organization and altered mechanical properties in comparison to their healthy counterpart. To evaluate the clinical relevance of our findings, we treated the cells in vitro with the Bruton's tyrosine kinase inhibitors and we found for the first time that the drug restores the CLL cells mechanical properties to a healthy phenotype and activates the actomyosin complex. We further validated these results in vivo on CLL cells isolated from patients undergoing ibrutinib treatment. Our results suggest that CLL cells' mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance, thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells.

Cell-surface contacts determine volume and mechanical properties of human embryonic kidney 293 T cells.

Alterations in the organization of the cytoskeleton precede the escape of adherent cells from the framework of cell-cell and cell-matrix interactions into suspension. With cytoskeletal dynamics being linked to cell mechanical properties, many studies elucidated this relationship under either native adherent or suspended conditions. In contrast, tethered cells that mimic the transition between both states have not been the focus of recent research. Using human embryonic kidney 293 T cells we investigated all three conditions in the light of alterations in cellular shape, volume, as well as mechanical properties and relate these findings to the level, structure, and intracellular localization of filamentous actin (F-actin). For cells adhered to a substrate, our data shows that seeding density affects cell size but does not alter their elastic properties. Removing surface contacts leads to cell stiffening that is accompanied by changes in cell shape, and a reduction in cellular volume but no alterations in F-actin density. Instead, we observe changes in the organization of F-actin indicated by the appearance of blebs in the semi-adherent state. In summary, our work reveals an interplay between molecular and mechanical alterations when cells detach from a surface that is mainly dominated by cell morphology.

Mechanical characterization of isolated mitochondria under conditions of oxidative stress.

Mechanical properties have been proven to be a pivotal parameter to enhance our understanding of living systems. While research during the last decades focused on cells and tissues, little is known about the role of organelle mechanics in cell function. Here, mitochondria are of specific interest due to their involvement in numerous physiological and pathological processes, e.g., in the production and homeostasis of reactive oxygen species (ROS). Using real-time fluorescence and deformability cytometry, we present a microfluidic technology that is capable to determine the mechanical properties of individual mitochondria at a throughput exceeding 100 organelles per second. Our data on several thousands of viable mitochondria isolated from rat C6 glial cells yield a homogenous population with a median deformation that scales with the applied hydrodynamic stress. In two proof-of-principle studies, we investigated the impact of exogenously and endogenously produced ROS on mitochondria mechanics. Exposing C6 cells to hydrogen peroxide (H2O2) triggers superoxide production and leads to a reduction in mitochondria size while deformation is increased. In a second study, we focused on the knockout of tafazzin, which has been associated with impaired remodeling of the mitochondrial membrane and elevated levels of ROS. Interestingly, our results reveal the same mechanical alterations as observed after the exposure to H2O2, which points to a unified biophysical mechanism of how mitochondria respond to the presence of oxidative stress. In summary, we introduce high-throughput mechanical phenotyping into the field of organelle biology with potential applications for understanding sub-cellular dynamics that have not been accessible before.

Mobilization of CD11b+/Ly6chi monocytes causes multi organ dysfunction syndrome in acute pancreatitis.

Objective: Acute pancreatitis (AP) is an inflammatory disorder, the severe form of which is burdened with multi-organ dysfunction and high mortality. The pathogenesis of life -threatening organ complications, such as respiratory and renal failure, is unknown. Design: Organ dysfunction was investigated in a mouse model of AP. The influence of monocytes and neutrophils on multi organ dysfunction syndrome (MODS) was investigated in vivo by antibody depletion. Using real-time-fluorescence and deformability-cytometry (RT-DC) analysis we determined the mechanical properties of neutrophils and monocytes during AP. Furthermore, blood samples of pancreatitis patients were used to characterize severity-dependent chemokine profiles according to the revised Atlanta classification. Results: Similar to AP in humans, severe disease in the mouse model associates with organ dysfunction mainly of lung and kidney, which is triggered by a mobilisation of Ly6g-/CD11b+/Ly6c hi monocytes, but not of Ly6g+/CD11b+ neutrophils. Monocyte depletion by anti-CCR2 antibody treatment ameliorated lung function (oxygen consumption) without interfering with the systemic immune response. RT-DC analysis of circulation monocytes showed a significant increase in cell size during SAP, but without a compensatory increase in elasticity. Patient chemokine profiles show a correlation of AP severity with monocyte attracting chemokines like MCP-1 or MIG and with leukocyte mobilisation. Conclusion: In AP, the physical properties of mobilized monocytes, especially their large size, result in an obstruction of the fine capillary systems of the lung and of the kidney glomeruli. A selective depletion of monocytes may represent a treatment strategy for pancreatitis as well as for other inflammation-related disorders.

The impact of cell culture media on the interaction of biopolymer-functionalized gold nanoparticles with cells: mechanical and toxicological properties.

Understanding the nanoparticle-cell interactions in physiological media is vital in determining the biological fate of the nanoparticles (NPs). These interactions depend on the physicochemical properties of the NPs and their colloidal behavior in cell culture media (CCM). Furthermore, the impact of the bioconjugates made by nanoparticle with proteins from CCM on the mechanical properties of cells upon interaction is unknown. Here, we analyzed the time dependent stability of gold nanoparticles (AuNPs) functionalized with citrate, dextran-10, dextrin and chitosan polymers in protein poor- and protein rich CCM. Further, we implemented the high-throughput technology real-time deformability cytometry (RT-DC) to investigate the impact of AuNP-bioconjugates on the cell mechanics of HL60 suspension cells. We found that dextrin-AuNPs form stable bioconjugates in both CCM and have a little impact on cell mechanics, ROS production and cell viability. In contrast, positively charged chitosan-AuNPs were observed to form spherical and non-spherical aggregated conjugates in both CCM and to induce increased cytotoxicity. Citrate- and dextran-10-AuNPs formed spherical and non-spherical aggregated conjugates in protein rich- and protein poor CCM and induced at short incubation times cell stiffening. We anticipate based on our results that dextrin-AuNPs can be used for therapeutic purposes as they show lower cytotoxicity and insignificant changes in cell physiology.

Interpretation of cell mechanical experiments in microfluidic systems depend on the choice of cellular shape descriptors.

The capability to parameterize shapes is of essential importance in biomechanics to identify cells, to track their motion, and to quantify deformation. While various shape descriptors have already been investigated to study the morphology and migration of adherent cells, little is known of how the mathematical definition of a contour impacts the outcome of rheological experiments on cells in suspension. In microfluidic systems, hydrodynamic stress distributions induce time-dependent cell deformation that needs to be quantified to determine viscoelastic properties. Here, we compared nine different shape descriptors to characterize the deformation of suspended cells in an extensional as well as shear flow using dynamic real-time deformability cytometry. While stress relaxation depends on the amplitude and duration of stress, our results demonstrate that steady-state deformation can be predicted from single cell traces even for translocation times shorter than their characteristic time. Implementing an analytical simulation, performing experiments, and testing various data analysis strategies, we compared single cell and ensemble studies to address the question of computational costs vs experimental accuracy. Results indicate that high-throughput viscoelastic measurements of cells in suspension can be performed on an ensemble scale as long as the characteristic time matches the dimensions of the microfluidic system. Finally, we introduced a score to evaluate the shape descriptor-dependent effect size for cell deformation after cytoskeletal modifications. We provide evidence that single cell analysis in an extensional flow provides the highest sensitivity independent of shape parametrization, while inverse Haralick's circularity is mostly applicable to study cells in shear flow.

Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease.

MYH9-related disease patients with mutations in the contractile protein nonmuscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild-to-moderate bleeding tendency. In this study, we used three mouse lines, each with one point mutation in the Myh9 gene at positions 702, 1424, or 1841, to investigate mechanisms underlying the increased bleeding risk. Agonist-induced activation of Myh9 mutant platelets was comparable to controls. However, myosin light chain phosphorylation after activation was reduced in mutant platelets, which displayed altered biophysical characteristics and generated lower adhesion, interaction, and traction forces. Treatment with tranexamic acid restored clot retraction in the presence of tPA and reduced bleeding. We verified our findings from the mutant mice with platelets from patients with the respective mutation. These data suggest that reduced platelet forces lead to an increased bleeding tendency in patients with MYH9-related disease, and treatment with tranexamic acid can improve the hemostatic function.

Divalent magnesium restores cytoskeletal storage lesions in cold-stored platelet concentrates.

Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2-10 mM Mg2+ on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4 °C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4 °C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p < 0.01; n = 6, day 7). These results were confirmed by immunofluorescence microscopy, showing that Mg2+ ≥ 4 mM prevents 4 °C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was not significantly different between RT stored (56.38 ± 29.36%) and cold-stored platelets with (55.22 ± 11.16%) or without magnesium (45.65 ± 11.59%; p = 0.042, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4 °C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4 °C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets.

Switching in the expression pattern of actin isoforms marks the onset of contractility and distinct mechanodynamic behavior during cardiomyocyte differentiation.

Differentiation of cardiac progenitor cells (CPC) into cardiomyocytes is a fundamental step in cardiogenesis, which is marked by changes in gene expression responsible for remodeling of the cytoskeleton and in altering the mechanical properties of cells. Here we have induced the differentiation of CPC derived from human pluripotent stem cells into immature cardiomyocytes (iCM) which we compare with more differentiated cardiomyocytes (mCM). Using atomic force microscopy and real-time deformability cytometry, we describe the mechanodynamic changes that occur during the differentiation process and link our findings to protein expression data of cytoskeletal proteins. Increased levels of cardiac-specific markers as well as evolution of cytoskeletal morphology and contractility parameters correlated with the expected extent of cell differentiation that was accompanied by hypertrophic growth of cells. These changes were associated with switching in the balance of the different actin isoforms where ß-actin is predominantly found in CPC, smooth muscle α-actin is dominant in iCM cells and sarcomeric α-actin is found in significantly higher levels in mCM. We link these cytoskeletal changes to differences in mechano-dynamic behavior of cells that translate to changes in Young's modulus that depend on the cell adherence. Our results demonstrate that the intracellular balance of actin isoform expression can be used as a sensitive ruler to determine the stage of differentiation during early phases of cardiomyocyte differentiation that correlates with an increased expression of sarcomeric proteins and is accompanied by changes in cellular elasticity.

Ex vivo anticoagulants affect human blood platelet biomechanics with implications for high-throughput functional mechanophenotyping.

Inherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used during collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by real-time deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size, and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.

Machine learning assisted real-time deformability cytometry of CD34+ cells allows to identify patients with myelodysplastic syndromes.

Diagnosis of myelodysplastic syndrome (MDS) mainly relies on a manual assessment of the peripheral blood and bone marrow cell morphology. The WHO guidelines suggest a visual screening of 200 to 500 cells which inevitably turns the assessor blind to rare cell populations and leads to low reproducibility. Moreover, the human eye is not suited to detect shifts of cellular properties of entire populations. Hence, quantitative image analysis could improve the accuracy and reproducibility of MDS diagnosis. We used real-time deformability cytometry (RT-DC) to measure bone marrow biopsy samples of MDS patients and age-matched healthy individuals. RT-DC is a high-throughput (1000 cells/s) imaging flow cytometer capable of recording morphological and mechanical properties of single cells. Properties of single cells were quantified using automated image analysis, and machine learning was employed to discover morpho-mechanical patterns in thousands of individual cells that allow to distinguish healthy vs. MDS samples. We found that distribution properties of cell sizes differ between healthy and MDS, with MDS showing a narrower distribution of cell sizes. Furthermore, we found a strong correlation between the mechanical properties of cells and the number of disease-determining mutations, inaccessible with current diagnostic approaches. Hence, machine-learning assisted RT-DC could be a promising tool to automate sample analysis to assist experts during diagnosis or provide a scalable solution for MDS diagnosis to regions lacking sufficient medical experts.

Inhibition of the NLRP3/IL-1ß axis protects against sepsis-induced cardiomyopathy.

BACKGROUND: Septic cardiomyopathy worsens the prognosis of critically ill patients. Clinical data suggest that interleukin-1ß (IL-1ß), activated by the NLRP3 inflammasome, compromises cardiac function. Whether or not deleting Nlrp3 would prevent cardiac atrophy and improve diastolic cardiac function in sepsis was unclear. Here, we investigated the role of NLRP3/IL-1ß in sepsis-induced cardiomyopathy and cardiac atrophy. METHODS: Male Nlrp3 knockout (KO) and wild-type (WT) mice were exposed to polymicrobial sepsis by caecal ligation and puncture (CLP) surgery (KO, n = 27; WT, n = 33) to induce septic cardiomyopathy. Sham-treated mice served as controls (KO, n = 11; WT, n = 16). Heart weights and morphology, echocardiography and analyses of gene and protein expression were used to evaluate septic cardiomyopathy and cardiac atrophy. IL-1ß effects on primary and immortalized cardiomyocytes were investigated by morphological and molecular analyses. IonOptix and real-time deformability cytometry (RT-DC) analysis were used to investigate functional and mechanical effects of IL-1ß on cardiomyocytes. RESULTS: Heart morphology and echocardiography revealed preserved systolic (stroke volume: WT sham vs. WT CLP: 33.1 ± 7.2 µL vs. 24.6 ± 8.7 µL, P < 0.05; KO sham vs. KO CLP: 28.3 ± 8.1 µL vs. 29.9 ± 9.9 µL, n.s.; P < 0.05 vs. WT CLP) and diastolic (peak E wave velocity: WT sham vs. WT CLP: 750 ± 132 vs. 522 ± 200 mm/s, P < 0.001; KO sham vs. KO CLP: 709 ± 152 vs. 639 ± 165 mm/s, n.s.; P < 0.05 vs. WT CLP) cardiac function and attenuated cardiac (heart weight-tibia length ratio: WT CLP vs. WT sham: -26.6%, P < 0.05; KO CLP vs. KO sham: -3.3%, n.s.; P < 0.05 vs. WT CLP) and cardiomyocyte atrophy in KO mice during sepsis. IonOptix measurements showed that IL-1ß decreased contractility (cell shortening: IL-1ß: -15.4 ± 2.3%, P < 0.001 vs. vehicle, IL-1RA: -6.1 ± 3.3%, P < 0.05 vs. IL-1ß) and relaxation of adult rat ventricular cardiomyocytes (time-to-50% relengthening: IL-1ß: 2071 ± 225 ms, P < 0.001 vs. vehicle, IL-1RA: 564 ± 247 ms, P < 0.001 vs. IL-1ß), which was attenuated by an IL-1 receptor antagonist (IL-1RA). RT-DC analysis indicated that IL-1ß reduced cardiomyocyte size (P < 0.001) and deformation (P < 0.05). RNA sequencing showed that genes involved in NF-κB signalling, autophagy and lysosomal protein degradation were enriched in hearts of septic WT but not in septic KO mice. Western blotting and qPCR disclosed that IL-1ß activated NF-κB and its target genes, caused atrophy and decreased myosin protein in myocytes, which was accompanied by an increased autophagy gene expression. These effects were attenuated by IL-1RA. CONCLUSIONS: IL-1ß causes atrophy, impairs contractility and relaxation and decreases deformation of cardiomyocytes. Because NLRP3/IL-1ß pathway inhibition attenuates cardiac atrophy and cardiomyopathy in sepsis, it could be useful to prevent septic cardiomyopathy.

Inactivation of Cerebral Cavernous Malformation Genes Results in Accumulation of von Willebrand Factor and Redistribution of Weibel-Palade Bodies in Endothelial Cells.

Cerebral cavernous malformations are slow-flow thrombi-containing vessels induced by two-step inactivation of the CCM1, CCM2 or CCM3 gene within endothelial cells. They predispose to intracerebral bleedings and focal neurological deficits. Our understanding of the cellular and molecular mechanisms that trigger endothelial dysfunction in cavernous malformations is still incomplete. To model both, hereditary and sporadic CCM disease, blood outgrowth endothelial cells (BOECs) with a heterozygous CCM1 germline mutation and immortalized wild-type human umbilical vein endothelial cells were subjected to CRISPR/Cas9-mediated CCM1 gene disruption. CCM1 -/- BOECs demonstrated alterations in cell morphology, actin cytoskeleton dynamics, tube formation, and expression of the transcription factors KLF2 and KLF4. Furthermore, high VWF immunoreactivity was observed in CCM1 -/- BOECs, in immortalized umbilical vein endothelial cells upon CRISPR/Cas9-induced inactivation of either CCM1, CCM2 or CCM3 as well as in CCM tissue samples of familial cases. Observer-independent high-content imaging revealed a striking reduction of perinuclear Weibel-Palade bodies in unstimulated CCM1 -/- BOECs which was observed in CCM1 +/- BOECs only after stimulation with PMA or histamine. Our results demonstrate that CRISPR/Cas9 genome editing is a powerful tool to model different aspects of CCM disease in vitro and that CCM1 inactivation induces high-level expression of VWF and redistribution of Weibel-Palade bodies within endothelial cells.

AQP8 is a crucial H2O2 transporter in insulin-producing RINm5F cells.

Peroxiporins are distinct aquaporins (AQP) which, beside water, also facilitate the bidirectional transport of hydrogen peroxide (H2O2) across cellular membranes. H2O2 serves as the major reactive oxygen species that mediates essential cell signaling events. In pancreatic ß-cells, H2O2 has been associated with the regulation of cell growth but in excess it leads to failure of insulin secretion, making it important for diabetes mellitus (DM) pathogenesis. In the present study, the role of aquaporin-8 (AQP8) as a peroxiporin was investigated in RINm5F cells. The role of AQP8 was studied in an insulin-producing cell model, on the basis of stable AQP8 overexpression (AQP8↑) and CRISPR/Cas9-mediated AQP8 knockdown (KD). A complete AQP8 knock-out was found to result in cell death, however we demonstrate that mild lentiviral re-expression through a Tet-On-regulated genetically modified AQP8 leads to cell survival, enabling functional characterization. Proliferation and insulin content were found to be increased in AQP8↑ cells underlining the importance of AQP8 in the regulation of H2O2 homeostasis in pancreatic ß-cells. Colocalization analyses of V5-tagged AQP8 proteins based on confocal microscopic imaging revealed its membrane targeting to both the mitochondria and the plasma membrane, but not to the ER, the Golgi apparatus, insulin vesicles, or peroxisomes. By using the fluorescence H2O2 specific biosensor HyPer together with endogenous generation of H2O2 using d-amino acid oxidase, live cell imaging revealed enhanced H2O2 flux to the same subcellular regions in AQP8 overexpressing cells pointing to its importance in the development of type-1 DM. Moreover, the novel ultrasensitive H2O2 sensor HyPer7.2 clearly unveiled AQP8 as a H2O2 transporter in RINm5F cells. In summary, these studies establish that AQP8 is an important H2O2 pore in insulin-producing RINm5F cells involved in the transport of H2O2 through the mitochondria and cell membrane and may help to explain the H2O2 transport and toxicity in pancreatic ß-cells.

Microfluidic Fabrication of Click Chemistry-Mediated Hyaluronic Acid Microgels: A Bottom-Up Material Guide to Tailor a Microgel's Physicochemical and Mechanical Properties.

The demand for tailored, micrometer-scaled biomaterials in cell biology and (cell-free) biotechnology has led to the development of tunable microgel systems based on natural polymers, such as hyaluronic acid (HA). To precisely tailor their physicochemical and mechanical properties and thus to address the need for well-defined microgel systems, in this study, a bottom-up material guide is presented that highlights the synergy between highly selective bio-orthogonal click chemistry strategies and the versatility of a droplet microfluidics (MF)-assisted microgel design. By employing MF, microgels based on modified HA-derivates and homobifunctional poly(ethylene glycol) (PEG)-crosslinkers are prepared via three different types of click reaction: Diels-Alder [4 + 2] cycloaddition, strain-promoted azide-alkyne cycloaddition (SPAAC), and UV-initiated thiol-ene reaction. First, chemical modification strategies of HA are screened in-depth. Beyond the microfluidic processing of HA-derivates yielding monodisperse microgels, in an analytical study, we show that their physicochemical and mechanical properties-e.g., permeability, (thermo)stability, and elasticity-can be systematically adapted with respect to the type of click reaction and PEG-crosslinker concentration. In addition, we highlight the versatility of our HA-microgel design by preparing non-spherical microgels and introduce, for the first time, a selective, hetero-trifunctional HA-based microgel system with multiple binding sites. As a result, a holistic material guide is provided to tailor fundamental properties of HA-microgels for their potential application in cell biology and (cell-free) biotechnology.

High-throughput cell and spheroid mechanics in virtual fluidic channels.

Microfluidics by soft lithography has proven to be of key importance for biophysics and life science research. While being based on replicating structures of a master mold using benchtop devices, design modifications are time consuming and require sophisticated cleanroom equipment. Here, we introduce virtual fluidic channels as a flexible and robust alternative to microfluidic devices made by soft lithography. Virtual channels are liquid-bound fluidic systems that can be created in glass cuvettes and tailored in three dimensions within seconds for rheological studies on a wide size range of biological samples. We demonstrate that the liquid-liquid interface imposes a hydrodynamic stress on confined samples, and the resulting strain can be used to calculate rheological parameters from simple linear models. In proof-of-principle experiments, we perform high-throughput rheology inside a flow cytometer cuvette and show the Young's modulus of isolated cells exceeds the one of the corresponding tissue by one order of magnitude.

Label-free on chip quality assessment of cellular blood products using real-time deformability cytometry.

Without cellular blood products such as platelet concentrates (PC), red blood cell concentrates (RCC), and hematopoietic stem cells (HPSC) modern treatments in medicine would not be possible. An unresolved challenge is the assessment of their quality with minimal cell manipulation. Minor changes in production, storage conditions, or blood bag composition may impact cell function, which can have important consequences on product integrity. This is especially relevant for personalized medicine, such as autologous T-cell therapy. Today a robust methodology that globally determines cell status directly before transfusion or transplantation is lacking. We demonstrate that measuring viscoelastic characteristics of peripheral blood cells using real-time deformability cytometry (RT-DC) provides comprehensive information on product quality, which is not accessible using conventional quality control tests. In addition, RT-DC requires few cells, a minimal sample volume and has a rapid turnaround time. We compared RT-DC to standard in vitro quality assays assessing: i) PC after storage at 4 °C and room temperature; ii) magnetic nanoparticle labeled platelets; iii) RCC stored in blood bags with different plasticizers; iv) RCC after gamma irradiation; and v) HPSC after cryopreservation with 5% or 10% dimethyl sulfoxide, respectively. Additionally, we evaluated the engraftment time of patients' platelets and leukocytes after transplantation of HPSC products. Our results demonstrate that label-free mechano-phenotyping can be used as a potential biomarker for quality assessment of cell-based pharmaceutical products.

A comparison of microfluidic methods for high-throughput cell deformability measurements.

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms.