Cell Squeezing Research Articles

Live-cell protein labelling with nanometre precision by cell squeezing.

Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy.

Modulation of collective cell behaviour by geometrical constraints.

Intracellular and extracellular mechanical forces play a crucial role during tissue growth, modulating nuclear shape and function and resulting in complex collective cell behaviour. However, the mechanistic understanding of how the orientation, shape, symmetry and homogeneity of cells are affected by environmental geometry is still lacking. Here we investigate cooperative cell behaviour and patterns under geometric constraints created by topographically patterned substrates. We show how cells cooperatively adopt their geometry, shape, positioning of the nucleus and subsequent proliferation activity. Our findings indicate that geometric constraints induce significant squeezing of cells and nuclei, cytoskeleton reorganization, drastic condensation of chromatin resulting in a change in the cell proliferation rate and the anisotropic growth of cultures. Altogether, this work not only demonstrates complex non-trivial collective cellular responses to geometrical constraints but also provides a tentative explanation of the observed cell culture patterns grown on different topographically patterned substrates. These findings provide important fundamental knowledge, which could serve as a basis for better controlled tissue growth and cell-engineering applications.

Cumulative Brain Injury from Motor Vehicle-Induced Whole-Body Vibration and Prevention by Human Apolipoprotein A-I Molecule Mimetic (4F) Peptide (an Apo A-I Mimetic)

Insidious cumulative brain injury from motor vehicle-induced whole-body vibration (MV-WBV) has not yet been studied. The objective of the present study is to validate whether whole-body vibration for long periods causes cumulative brain injury and impairment of the cerebral function. We also explored a preventive method for MV-WBV injury. A study simulating whole-body vibration was conducted in 72 male Sprague-Dawley rats divided into 9 groups (N = 8): (1) 2-week normal control; (2) 2-week sham control (in the tube without vibration); (3) 2-week vibration (exposed to whole-body vibration at 30 Hz and .5 G acceleration for 4 hours/day, 5 days/week for 2 weeks; vibration parameters in the present study are similar to the most common driving conditions); (4) 4-week sham control; (5) 4-week vibration; (6) 4-week vibration with human apolipoprotein A-I molecule mimetic (4F)-preconditioning; (7) 8-week sham control; (8) 8-week vibration; and (9) 8-week 4F-preconditioning group. All the rats were evaluated by behavioral, physiological, and histological studies of the brain. Brain injury from vibration is a cumulative process starting with cerebral vasoconstriction, squeezing of the endothelial cells, increased free radicals, decreased nitric oxide, insufficient blood supply to the brain, and repeated reperfusion injury to brain neurons. In the 8-week vibration group, which indicated chronic brain edema, shrunken neuron numbers increased and whole neurons atrophied, which strongly correlated with neural functional impairment. There was no prominent brain neuronal injury in the 4F groups. The present study demonstrated cumulative brain injury from MV-WBV and validated the preventive effects of 4F preconditioning.

Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines.

B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to “professional” APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale “cell squeezing” process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8+T-cells, and not CD4+T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8+T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8+T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8+T-cells, and decoupling of antigen uptake from B-cell activation.

J0250201 PDMS薄膜を用いた細胞表層の直接圧縮と細胞の力学特性評価

This paper presents a measurement method for Young's modulus of cells based on directly squeezing cells. A microfluidic cell squeezing device is used for measurement of the Young's modulus of cell. This device is designed so as to control the magnitude of pressure to cells. This device is made of transparent materials which permit in-situ monitoring of cellular deformation behavior by an optical microscope. Mechanical stress is directly applied to the cell surface with the deflection of the PDMS film above the cell culture chamber. Cells were gradually deformed within an elastic deformation range when the mechanical stress was applied. After eliminating the applied pressure, these cells were returned to the initial shape. The osteoblastic cells (MC3T3-E1) were used as target cell to evaluate the Young's modulus. Young's modulus of the osteoblastic cell was determined based on the cell deformation and the applied pressure. The result of Young's modulus of osteoblastic cells obtained from directly squeezing experiment was similar to the published values.

Neural systemic impairment from whole-body vibration.

Insidious brain microinjury from motor vehicle-induced whole-body vibration (WBV) has not yet been investigated. For a long time we have believed that WBV would cause cumulative brain microinjury and impair cerebral function, which suggests an important risk factor for motor vehicle accidents and secondary cerebral vascular diseases. Fifty-six Sprague-Dawley rats were divided into seven groups (n = 8): 1) 2-week normal control group, 2) 2-week sham control group (restrained in the tube without vibration), 3) 2-week vibration group (exposed to whole-body vibration at 30 Hz and 0.5g acceleration for 4 hr/day, 5 days/week, for 2 weeks), 4) 4-week sham control group, 5) 4-week vibration group, 6) 8-week sham control group, and 7) 8-week vibration group. At the end point, all rats were evaluated in behavior, physiological, and brain histopathological studies. The cerebral injury from WBV is a cumulative process starting with vasospasm squeezing of the endothelial cells, followed by constriction of the cerebral arteries. After the 4-week vibration, brain neuron apoptosis started. After the 8-week vibration, vacuoles increased further in the brain arteries. Brain capillary walls thickened, mean neuron size was obviously reduced, neuron necrosis became prominent, and wide-ranging chronic cerebral edema was seen. These pathological findings are strongly correlated with neural functional impairments.

Giant eccrine hidradenoma mimicking a malignant tumor of the salivary gland.

Giant eccrine hidradenoma mimicking a malignant tumor of the salivary gland.

Nuclear Deformability is Critically Dependent on Lamin A/B

During metastasis, single cells or clusters of cells leave an initial tumor, intravasate the blood system and form secondary metastasis at distinct locations of the body. Thereby, they migrate through the extra cellular matrix and squeeze through tiny pores in the collagen mesh. That is why the cells' ability to penetrate the collagen mesh plays a crucial role for healing and survival prognosis. Here, we investigate the link between cell rheology, i.e. cell stiffness and deformability, and the intermediate filament lamin. It has been suspected previously that high lamin A levels limit nuclear deformability [1,2] and increase cell viscosity, which decreases nuclear remodeling under stress [3]. We created stably transfected lamin-A-GFP and lamin-B2-mCherry U2OS and HEK293 cell lines of a wide range of lamin expression levels. We use a series of micron-scale constrictions (2-10 μm) to test the dependence of their nuclear and cytoskeletal rheology on lamin levels by measuring the cells' size and transit time through the channels. We find an interesting dependence of the cells' transit properties on lamin A and B2 expression, underlining the importance of nuclear lamins for cell migration and squeezing.[1] Lammerding et al.: Lamins A and C, but not Lamin B1 regulate Nuclear Mechanics, J. Biol. Chem. 281, 25768-25780 (2006).[2] Rowat et al.: Nuclear Envelope Composition Determines the Ability of Neutrophil-type Cells to Passage through Micron-scale Constrictions, J. Biol. Chem. 288, 288:8610-8618 (2013).[3] Swift et al.: Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation, Science 341, 1240104 (2013).

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells.

Sphingosylphosphorylcholine (SPC) is a naturally occurring bioactive lipid that is present in high density lipoproteins (HDL) particles and found at increased levels in blood and malignant ascites of patients with ovarian cancer. Here, we show that incubation of human epithelial tumour cells with SPC induces a perinuclear reorganization of intact keratin 8-18 filaments. This effect is specific for SPC, largely independent of F-actin and microtubules, and is accompanied by keratin phosphorylation. In vivo visco-elastic probing of single cancer cells demonstrates that SPC increases cellular elasticity. Accordingly, SPC stimulates migration of cells through size-limited pores in a more potent manner than lysophosphatidic acid (LPA). LPA induces actin stress fibre formation, but does not reorganize keratins in cancer cells and hence increases cellular stiffness. We propose that reorganization of keratin by SPC may facilitate biological phenomena that require a high degree of elasticity, such as squeezing of cells through membranous pores during metastasis.

Motion of red blood cells in a capillary with an endothelial surface layer: effect of flow velocity.

Interior surfaces of capillaries are lined with macromolecules forming an endothelial surface layer (ESL). A theoretical model is used to investigate effects of flow velocity on motion and axisymmetric deformation of red blood cells in a capillary with an ESL. Cell deformation is analyzed, including effects of membrane shear and bending elasticity. Plasma flow around the cell and through the ESL is computed using lubrication theory. The ESL is represented as a porous layer that exerts compressive forces on red blood cells that penetrate it. According to the model, hydrodynamic pressures generated by plasma flow around the cell squeeze moving red blood cells into narrow elongated shapes. If the ESL is 0.7 microm wide, with hydraulic resistivity of 2 x 10(8) dyn x s x cm(-4), and exerts a force of 20 dyn/cm2, predicted variation with flow velocity of the gap width between red blood cell and capillary wall agrees well with observations. Predicted gap at a velocity of 0.1 mm/s is approximately 0.6 microm vs. approximately 0.2 microm with no ESL. Predicted flow resistance increases markedly at low velocities. The model shows that exclusion of red blood cells from the ESL in flowing capillaries can result from hydrodynamic forces generated by plasma flow through the ESL.

Divertor target heat load reduction by electrical biasing, and application to COMPASS-D

A toroidally asymmetric potential structure in the scrape-off layer (SOL) plasma may be formed by toroidally distributed electrical biasing of the divertor target tiles. The resulting E× B convective motions should increase the plasma radial transport in the SOL and thereby reduce the heat load at the divertor [R.H. Cohen, D.D. Ryutov, Nucl. Fus. 37 (1997) 621]. In this paper, we develop theoretical modelling and describe the implementation of this concept to the COMPASS-D divertor. We show that a strong magnetic shear near the X-point should cause significant squeezing of the convective cells preventing convection from penetrating above the X-point. This should result in reduced heat load at the divertor target without increasing the radial transport in the portion of the SOL in direct contact with the core plasma, potentially avoiding any confinement degradation. Implementation of divertor biasing is in hand on COMPASS-D involving insulation of, and modifications to, the present divertor tiles. Calculations based on measured edge parameters suggest that modest currents ∼8 A/tile are required, at up to 150 V, to drive the convection. A technical test is preceding full bias experiments.

In vitro characterization of the occurrence of hemolysis during extracorporeal blood circulation using a mini hemodialyzer.

A minimodule dialyzer was made of polyacrylonitrile (PAN) hollow fibers and tested in an in vitro circuit under varying flow rates and blood volumes. Hemolysis occurred in the open circuit in the inlet and outlet chambers. The module was measured to investigate the hemolysis effect during dialysis. The results show that hemolysis ratio (HR) increased with blood flow rate. When the blood speed was increased from 27 to 70 cm/sec, the damaged fraction of red blood cells increased from 0.8% to 1.5%. Shearing in the hollow fiber contributed approximately 13% of the overall HR. Approximately 55% of the overall HR occurred at the inlet and outlet chambers. With increasing blood volume, the contacting surface area per unit blood volume (S/V) and the frequency of pump squeezing of red cells were decreased; hence the damage to the red blood cells and thus reduction of HR. When S/V was 2 cm(-1), the damaged fraction of red cells was approximately 0.7%. In addition, the tubing and connectors of the circuit caused further damage to the red blood cells. By referring to the linear velocity and blood volume, we can predict the HR in an actual protocol from the results of this experiment. This should improve the quality of hemodialysis and benefit the patient.

Cell squeezing

Plants contain many useful substances — sugar, nicotine and steroids, for example — that are often tricky to extract, so Daedalus is developing supercritical cell culture. Because supercritical fluids are wonderful solvents, product molecules will diffuse out through the plant cell walls at an incredible rate. The technique could have applications in herbal medicine and even in genetic engineering.

The squeezing of red blood cells through parallel-sided channels with near-minimal widths

An analysis of the motion and deformation of red blood cells between two parallel flat plates is presented. The motion is driven by an imposed pressure gradient in the surrounding fluid. Mammalian red cells are highly flexible, but deform at constant volume because the contents of the cell are incompressible, and at nearly constant surface area because the membrane strongly resists dilatation. Consequently, a minimum spacing between the plates exists, below which passage of intact cells is not possible. We consider spacings slightly larger than this minimum. The shape of the cell in this case is a disk with a rounded edge. The flow of the surrounding fluid is described using lubrication theory. Under the approximation that the distance between the plates is small compared with the cell diameter, cell shapes, pressure distributions, membrane stresses and cell velocities are deduced as functions of geometrical parameters. It is found that the narrow gaps between the cell and the plate are not uniform in width, and that as a result, membrane shear stresses are generated which increase in proportion to flow velocity. This contrasts with axisymmetric configurations, in which membrane shear stress remains bounded as cell velocity increases. The variation of cell velocity with spacing of the plates is similar to that previously demonstrated for rigid disk-shaped particles of corresponding dimensions.

Viscous motion of disk-shaped particles through parallel-sided channels with near-minimal widths

The motion of a rigid disk-shaped particle with rounded edges, which fits closely in the space between two parallel flat plates, and which is suspended in a viscous fluid subject to an imposed pressure gradient, is analysed. This problem is relevant to the squeezing of red blood cells through narrow slot-like channels which are found in certain tissues. Mammalian red cells, although highly flexible, conserve volume and surface area as they deform. Consequently, a red cell cannot pass intact through a channel which is narrower than some minimum width. In channels that are just wide enough to permit cell passage, the cell is deformed into its ‘critical’ shape: a disk with rounded edges. In this paper, the fluid mechanical aspects of such motions are considered, and the particle is assumed to be rigid with the critical shape. The channel cross-section is assumed to be rectangular. The flow of the suspending fluid is described using lubrication theory. Use of lubrication theory is justified by considering the motion of a circular cylinder between parallel plates. For disk-shaped particles, approximate solutions are obtained by applying lubrication equations throughout the flow domain. In the region beyond the particle, this is equivalent to assuming a Hele-Shaw flow. More accurate solutions are obtained by including effects of boundary layers around the particle and at the sides of the channel. Pressure distributions and particle velocities are computed as functions of geometrical parameters, and it is shown that the particle may move faster or slower than the mean velocity of the surrounding fluid, depending on the channel dimensions.

Organelle rearrangement and cell volume changes during squeezing invasion of peritoneal elastic lamina by targeted murine breast carcinoma cells

Murine breast cancer cell lines were developed to selectively invade the peritoneum while they proliferated in ascites form in the abdominal cavity. In a dominant form of invasion, tumor cells showed special affinity for elastin fibers and squeezed through narrow gaps in the elastic fiber meshwork of the stroma. Even in fixed tissue, such cells could be recognized as being in the process of invasive migration because of their dumbbell shape. This appearance was similar to that of diapedetic blood cells traversing bone marrow sinus endothelium. Three-dimensional STERECON graphics reconstruction from serial thick sections of 44 such cells was carried out. The reconstructions showed that, in mid-penetration, the cells spread extensively over the exterior surface of the elastic fiber meshwork. The cell surface contact of these forward projections was mainly with the elastic fiber outer coat of microfibrils, but small areas of the cell surface also fused directly to inner-core elastin. The morphological rearrangement of the cytoskeleton was minimal in both types of attachment areas. The location of these forward facing attachments is consistent with mechanisms for pulling the invasive cell through the gap. Lamellopodia formation and clustering of cytoplasmic organdies occurred more commonly at the forward-facing part of the cell. Morphometry of the reconstructions showed that a contraction of the whole cell occurred during the squeezing/migration process suggestive of an additional pushing process. However, our invasive cell lines showed marked differences in the degree of cell shrinkage. The process of adhesion and squeezing of tumor cells through elastin meshworks in vivo is clearly a complex phenomenon. Changes in cell surface activity appear to play a significant role in establishing the necessary ‘foothold’ component of invasion and, possibly, in the generation of tractive force as well.

Intramural hemorrhage and endothelial changes in atherosclerotic coronary artery after repetitive episodes of spasm in x-ray-irradiated hypercholesterolemic pigs.

To assess whether coronary spasm affects the progression of atherosclerosis and results in evolution of myocardial infarction, the role of coronary spasm on the fine structure of conduit coronary arteries was studied morphologically. Göttingen miniature pigs were fed a semisynthetic diet containing 2% cholesterol and 1.1% sodium cholate. One month after being on this diet, the pigs were anesthetized and the endothelium of a branch of the left coronary artery was denuded using a balloon catheter. X-ray irradiation in a dose of 1,500 rad was given twice selectively to the area denuded, after 4 and 5 months of cholesterol feeding. Five months after endothelial denudation, transient (group A) and repetitive episodes (group B) of coronary spasm were provoked by single and periodic (five times every 5 minutes) intracoronary injections of serotonin (10 micrograms/kg/injection), respectively. The extent of spasm by serotonin at the previously denuded site was 84 +/- 4% (n = 4) and 90 +/- 5% (n = 6) narrowing in groups A and B (p = NS between groups), respectively. Forty minutes after the final administration of serotonin, the left coronary artery was relaxed by nitroglycerin, and the heart was isolated and perfuse-fixed under physiological pressure. Intramural hemorrhage was noted at the spastic site in six pigs of group B but not in group A. The average percent luminal narrowing, on cross sections at the spastic site in group B, was significantly greater than in group A (56 +/- 7% vs. 23 +/- 5%, p less than 0.01). Scanning electron micrographs revealed that the endothelial lining was intact at the nonspastic site in both groups. In addition to the appearance of intercellular bridges at the spastic site in both groups, squeezing of endothelial cells and adhesion of white blood cells were present at the spastic site exclusively in group B. These findings are consistent with the hypothesis that repetitive spasm may have an important role in the progression of atherosclerosis and/or myocardial infarction.

The squeezing of red blood cells through capillaries with near-minimal diameters

An analysis is presented of the mechanics of red blood cells flowing in very narrow tubes. Mammalian red cells are highly flexible, but their deformations satisfy two significant constraints. They must deform at constant volume, because the contents of the cell are incompressible, and also at nearly constant surface area, because the red cell membrane strongly resists dilation. Consequently, there exists a minimal tube diameter below which passage of intact cells is not possible. A cell in a tube with this diameter has its critical shape: a cylinder with hemispherical ends. Here, flow of red cells in tubes with near-minimal diameters is analysed using lubrication theory. When the tube diameter is slightly larger than the minimal value, the cell shape is close to its shape in the critical case. However, the rear end of the cell becomes flattened and then concave with a relatively small further increase in the diameter. The changes in cell shape and the resulting rheological parameters are analysed using matched asymptotic expansions for the high-velocity limit and using numerical solutions. Predictions of rheological parameters are also obtained using the assumption that the cell is effectively rigid with its critical shape, yielding very similar results. A rapid decrease in the apparent viscosity of red cell suspensions with increasing tube diameter is predicted over the range of diameters considered. The red cell velocity is found to exceed the mean bulk velocity by an amount that increases with increasing tube diameter.