Changes In Cell Deformability Research Articles

Three-Dimensional Shapes and Cell Deformability of Rat Red Blood Cells during and after Asphyxial Cardiac Arrest.

Changes in microcirculation are believed to perform an important role after cardiac arrest. In particular, rheological changes in red blood cells (RBCs) have been observed during and after ischemic-reperfusion injury. Employing three-dimensional laser interferometric microscopy, we investigated three-dimensional shapes and deformability of RBCs during and after asphyxial cardiac arrest in rats at the individual cell level. Rat cardiac arrest was induced by asphyxia. Five rats were maintained for 7 min of no-flow time, and then, cardiopulmonary resuscitation (CPR) was started. Blood samples were obtained before cardiac arrest, during CPR, and 60 min after return of spontaneous circulation (ROSC). Quantitative phase imaging (QPI) techniques based on laser interferometry were used to measure the three-dimensional refractive index (RI) tomograms of the RBC, from which structural and biochemical properties were retrieved. Dynamic membrane fluctuations in the cell membrane were also quantitatively and sensitively measured in order to investigate cell deformability. Mean corpuscular hemoglobin, mean cell volume, mean corpuscular hemoglobin concentration, and red blood cell distribution width remained unchanged during CPR and after ROSC compared with those before cardiac arrest. QPI results revealed that RBC membrane fluctuations, sphericity, and surface area did not change significantly during CPR or after ROSC compared with initial values. In conclusion, no three-dimensional shapes and cell deformability changes in RBCs were detected.

Sharp-edged geometric obstacles in microfluidics promote deformability-based sorting of cells

Sorting cells based on their intrinsic properties is a highly desirable objective, since changes in cell deformability are often associated with various stress conditions and diseases. Deterministic lateral displacement (DLD) devices offer high precision for rigid spherical particles, while their success in sorting deformable particles remains limited due to the complexity of cell traversal in DLDs. We employ mesoscopic hydrodynamics simulations and demonstrate prominent advantages of sharp-edged DLD obstacles for probing deformability properties of red blood cells (RBCs). By consecutive sharpening of the pillar shape from circular to diamond to triangular geometry, a pronounced cell bending around an edge is achieved, serving as a deformability sensor. Bending around the edge is the primary mechanism, which governs the traversal of RBCs through such DLD device. This strategy requires an appropriate degree of cell bending by fluid stresses, which can be controlled by the flow rate, and exhibits good sensitivity to moderate changes in cell deformability. We expect that similar mechanisms should be applicable for the development of novel DLD devices that target intrinsic properties of many other cells.

A scalable filtration method for high throughput screening based on cell deformability.

Cell deformability is a label-free biomarker of cell state in physiological and disease contexts ranging from stem cell differentiation to cancer progression. Harnessing deformability as a phenotype for screening applications requires a method that can simultaneously measure the deformability of hundreds of cell samples and can interface with existing high throughput facilities. Here we present a scalable cell filtration device, which relies on the pressure-driven deformation of cells through a series of pillars that are separated by micron-scale gaps on the timescale of seconds: less deformable cells occlude the gaps more readily than more deformable cells, resulting in decreased filtrate volume which is measured using a plate reader. The key innovation in this method is that we design customized arrays of individual filtration devices in a standard 96-well format using soft lithography, which enables multiwell input samples and filtrate outputs to be processed with higher throughput using automated pipette arrays and plate readers. To validate high throughput filtration to detect changes in cell deformability, we show the differential filtration of human ovarian cancer cells that have acquired cisplatin-resistance, which is corroborated with cell stiffness measurements using quantitative deformability cytometry. We also demonstrate differences in the filtration of human cancer cell lines, including ovarian cancer cells that overexpress transcription factors (Snail, Slug), which are implicated in epithelial-to-mesenchymal transition; breast cancer cells (malignant versus benign); and prostate cancer cells (highly versus weekly metastatic). We additionally show how the filtration of ovarian cancer cells is affected by treatment with drugs known to perturb the cytoskeleton and the nucleus. Our results across multiple cancer cell types with both genetic and pharmacologic manipulations demonstrate the potential of this scalable filtration device to screen cells based on their deformability.

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.

Cancer cells become less deformable and more invasive with activation of β-adrenergic signaling.

Invasion by cancer cells is a crucial step in metastasis. An oversimplified view in the literature is that cancer cells become more deformable as they become more invasive. β-adrenergic receptor (βAR) signaling drives invasion and metastasis, but the effects on cell deformability are not known. Here, we show that activation of β-adrenergic signaling by βAR agonists reduces the deformability of highly metastatic human breast cancer cells, and that these stiffer cells are more invasive in vitro We find that βAR activation also reduces the deformability of ovarian, prostate, melanoma and leukemia cells. Mechanistically, we show that βAR-mediated cell stiffening depends on the actin cytoskeleton and myosin II activity. These changes in cell deformability can be prevented by pharmacological β-blockade or genetic knockout of the β2-adrenergic receptor. Our results identify a β2-adrenergic-Ca2+-actin axis as a new regulator of cell deformability, and suggest that the relationship between cell mechanical properties and invasion might be dependent on context.

Role molecular signaling pathways in changes of red blood cell deformability

This study was designed to investigate the dependency of the red blood cell deformability upon activation of extra- and intracellular signaling pathways. Exposures of red blood cells (RBCs) to catecholamines and to insulin led to positive change in the RBC deformability. When forskolin, a stimulator of adenylyl cyclase (AC), was added to RBC suspension, the RBC deformability was increased. Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP. The inhibitors of phosphodiesterase (PDE) activity increased red cell deformability. These results revealed a considerable role of the AC-cAMP signaling system in the regulation of red blood cell deformability. The rise of the red blood cell Ca(2+) influx, stimulated by mechanical loading or A23187 was accompanied by a marked lowering of RBC deformability. At the same time blocking of Ca(2+) entry into RBC by verapamil or Ca(2+) chelating by EGTA led to significant deformability rise. The comparison of the effect of the different protein kinases on the red blood cell deformability showed that it was altered more considerable under PKA activation by forskolin or dB-cAMP than by other protein kinases. There was a lesser but quite statistically significant effect of tyrosine protein kinase (TPK) on RBC microrheology. Whereas the microrheological effect of PKC was not so considerable. The problem of the short-term regulation of red blood cell microrheology is examined. The latter includes: the modes of activation of extra- and intracellular molecular signaling pathways, ligand - receptor interaction, second messengers, membrane protein phosphorylation. On the whole the total data clearly show that the red cell deformability changes are connected with activation of different extra - and intracellular signaling pathways. It seems reasonable to suppose that red blood cell deformability changes were mainly associated with activation of the AC-cAMP-PKA pathway, and with decrease of Ca(2+) entry into cells.

Macro- and microrheological parameters of blood in patients with cerebral and peripheral atherosclerosis: The molecular change mechanisms after pentoxifylline treatment

This study was designed to evaluate hemorheological changes in patients with cerebrovascular disease (CVD) and peripheral arterial disease (PAD) after 4 weeks of pentoxifylline therapy as well as to study red blood cell microrheological variables after the cell incubation with pentoxifylline and some phosphodiesterase (PDE) activity inhibitors. The patients with CVD (n = 50) and PAD (n = 33) were treated with pentoxifylline (400 mg, thrice a day) for 4 weeks. Before and after drug therapy the hemorheological measurements including plasma and whole blood viscosity, red blood cell aggregation (RBCA) and deformability (RBCD) were completed. In vitro study RBCs were incubated with: 1) Vinpocetine--inhibitor PDE-1, 10 μM; 2) Rolipram--PDE-4, 10 μM; 3) Isobutyl-methylxanthine (IBMX)--nonselective PDE inhibitor, 100 μM and with pentoxifylline, 10 μM The cell incubation was performed at 37 °C for 15 min. There were the positive changes of hemorheological profile after 4 weeks of the pentoxifylline therapy both in CVD and PAD patients. The marked RBCD changes were observed after the in vitro cell pentoxifylline treatment as well. Perhaps it is connected with the inhibition of the phosphodiesterase activity in RBCs. An application of drugs and chemicals that can inhibit the PDE activity resulted in RBCD rise and RBCA decrease. The experiments with the use of selective PDE inhibitors have revealed the similar red cell deformability changes. Vinpocetine increased RBCD significantly (p < 0.05). PDE-4 inhibitor--Rolipram stimulated RBCD by 15% (p < 0.05). Some more effective was IBMX. After cell incubation with it a significant rise of the deformability (by 27%; p < 0.05) was found. All drugs, having PDE activity decreased RBCA, but the most pronounced effect had Vinpocetine (50%; p < 0.05). Thus, administered pentoxifylline, daily (1200 mg), during four weeks improves hemorheological profile and especially its microrheological part as well as the blood transport capacity in subjects with cerebral and peripheral vascular disorders. It is most probably red cell microrheological control mechanisms may be associated with the phosphodiesterase activity alterations.

The role of nitric oxide in reducing deformability of Lewis lung tumor cell stimulated by inflammatory cytokines

Highly metastatic cells, especially in the lungs, are known to be resistant to nitric oxide (NO)-mediated cytotoxicity, compared with poorly or non-metastatic cells. However, the precise mechanisms connecting NO and metastasis remain to be determined. To clarify the role of NO in the characteristic changes in NO-resistant cells in response to inflammatory cytokines, we used Lewis lung tumor (LLT) cells, which are known to be highly metastatic NO-resistant cells, and determined the changes in cell deformability and the gene expression profile after the cells were stimulated using cytokine mixture or an NO donor. Both exogenous NO and endogenous NO via inducible NO synthase produced by cytokines decreased cell deformability by enhancing actin polymerization. The expression of several genes associated with actin polymerization was changed so as to increase actin filaments in the cells by enhancing actin polymerization and by suppressing actin depolymerization, actin filament severing, and barbed-end actin filament capping. In conclusion, inflammatory cytokine stimulation reduces deformability of LLT cells and enhances actin polymerization which is mainly controlled by the same genes induced by NO.

Biomechanics and biophysics of cancer cells

The past decade has seen substantial growth in research into how changes in the biomechanical and biophysical properties of cells and subcellular structures influence, and are influenced by, the onset and progression of human diseases. This paper presents an overview of the rapidly expanding, nascent field of research that deals with the biomechanics and biophysics of cancer cells. The review begins with some key observations on the biology of cancer cells and on the role of actin microfilaments, intermediate filaments and microtubule biopolymer cytoskeletal components in influencing cell mechanics, locomotion, differentiation and neoplastic transformation. In order to set the scene for mechanistic discussions of the connections among alterations to subcellular structures, attendant changes in cell deformability, cytoadherence, migration, invasion and tumor metastasis, a survey is presented of the various quantitative mechanical and physical assays to extract the elastic and viscoelastic deformability of cancer cells. Results available in the literature on cell mechanics for different types of cancer are then reviewed. Representative case studies are presented next to illustrate how chemically induced cytoskeletal changes, biomechanical responses and signals from the intracellular regions act in concert with the chemomechanical environment of the extracellular matrix and the molecular tumorigenic signaling pathways to effect malignant transformations. Results are presented to illustrate how changes to cytoskeletal architecture induced by cancer drugs and chemotherapy regimens can significantly influence cell mechanics and disease state. It is reasoned through experimental evidence that greater understanding of the mechanics of cancer cell deformability and its interactions with the extracellular physical, chemical and biological environments offers enormous potential for significant new developments in disease diagnostics, prophylactics, therapeutics and drug efficacy assays.

Biomechanics and biophysics of cancer cells

The past decade has seen substantial growth in research into how changes in the biomechanical and biophysical properties of cells and subcellular structures influence, and are influenced by, the onset and progression of human diseases. This paper presents an overview of the rapidly expanding, nascent field of research that deals with the biomechanics and biophysics of cancer cells. The review begins with some key observations on the biology of cancer cells and on the role of actin microfilaments, intermediate filaments and microtubule biopolymer cytoskeletal components in influencing cell mechanics, locomotion, differentiation and neoplastic transformation. In order to set the scene for mechanistic discussions of the connections among alterations to subcellular structures, attendant changes in cell deformability, cytoadherence, migration, invasion and tumor metastasis, a survey is presented of the various quantitative mechanical and physical assays to extract the elastic and viscoelastic deformability of cancer cells. Results available in the literature on cell mechanics for different types of cancer are then reviewed. Representative case studies are presented next to illustrate how chemically induced cytoskeletal changes, biomechanical responses and signals from the intracellular regions act in concert with the chemomechanical environment of the extracellular matrix and the molecular tumorigenic signaling pathways to effect malignant transformations. Results are presented to illustrate how changes to cytoskeletal architecture induced by cancer drugs and chemotherapy regimens can significantly influence cell mechanics and disease state. It is reasoned through experimental evidence that greater understanding of the mechanics of cancer cell deformability and its interactions with the extracellular physical, chemical and biological environments offers enormous potential for significant new developments in disease diagnostics, prophylactics, therapeutics and drug efficacy assays.

In vivo neutrophil sequestration within lungs of humans is determined by in vitro "filterability"

Neutrophils are normally delayed in transit through the lung microcirculation, relative to the passage of erythrocytes. This sequestration contributes to a pulmonary pool of neutrophils that may relate to the relative inability of neutrophils to deform compared with erythrocytes when in transit in the pulmonary capillaries. A micropore membrane was used to model the human pulmonary microcirculation, in which cell deformability was measured as the pressure developed during filtration of the cells through the membrane at a constant flow. We demonstrated a significant correlation between in vitro deformability and in vivo lung sequestration of indium-111-labeled neutrophils in 10 normal subjects (r = 0.69, P less than 0.02). In eight patients with stable chronic obstructive pulmonary disease, this relationship was not significant (r = -0.2, P greater than 0.05). Furthermore, in a subject with microscopic pulmonary telangiectasia known to allow significant passage of 30-microns microspheres, neutrophils passed through the lungs without delay. Moreover, neutrophils from patients studied acutely with an exacerbation of chronic obstructive pulmonary disease were temporarily less deformable (P less than 0.01). These studies confirm that cell deformability is an important determinant of the normal neutrophil sequestration within the lungs. Changes in cell deformability may alter the extent of this sequestration.

Red Cell Deformability Changes as a Prognostic Indicator of Postoperative Morbidity in Cardiac Surgery Using Cariopulmonary Bypass

Preoperative and intraoperative red cell deformability assessed by a standard microfiltration technique was used to study the gross rheologic changes of red cells due to disease and mechanical trauma in relation to postoperative compli cations following the use of cardiopulmonary bypass (CPB) for elective heart surgery in 142 patients in NYHA (New York Heart Association) class III. The red cell filtration rate (RCFR) in μL/s was determined before surgery and grouped rheologically into four functional classes (GTCA = Gothenborg Cardio-Thoracic Association). Compared with their laboratory's normal reference for 200 healthy individuals (mainly blood donors) (61 ± 9 μL/s ± 2 SD), the RCFR was signifi cantly reduced by 21% (p &lt; 0.05) to GCTA class I (1-25% reduction) in 18 pa tients, by 34% (p&lt;0.01) to class II (26-50% reduction) in 99, and by 54% (p&lt;0.001) to class III (51-75% reduction) in 25. Significant RCFR reductions during CPB were seen in all the groups, where the mean reduction was lowest in class I (15% p&lt;0.001) and highest in class III (76% p&lt;0.001) as compared with normal subjects. The analyses of complications showed that significantly higher frequencies of bleeding, blood transfusion, respirator usage, and arrhythmia were found in class III than in class I (1.5 vs 0.8 L, p&lt;0.05; 3 vs 1.5 units, p &lt; 0.05; 14 vs 6 hours, p&lt;0.05 respectively). The authors conclude that preoperative red blood cell deformability testing and grading of the patients' microrheologic status of red blood cells can be a complement to the NYHA function classification and can also be useful for study ing the morbid effects of surgical trauma associated with CPB.

Time-Dependent Alterations in the Deformability of Human Neutrophils in Response to Chemotactic Activation

AbstractTransit times of individual human neutrophils through single capillary-sized pores were measured to determine the time-dependent changes in the mechanical behavior of the cells during activation by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (FMLP) and in response to cytochalasin B (CTB) and colchicine. FMLP elicited a two-phase response consisting of a rapid increase in cell stiffness, which peaked between 2 and 3 minutes, followed by a partial recovery of deformability to a level significantly above that of control after 5 minutes. The mechanical changes closely followed changes in F-actin content, although the peak in cell stiffness appeared to lag the F-actin response. Treatment with 3 µmol/L CTB produced a transient decrease in cell rigidity followed by a return to control level in 10 minutes, whereas treatment with 30 µmol/L CTB resulted in a sustained decrease in cell transit times to a level 60% of control. Addition of 3 µmol/L CTB to cells prestimulated with FMLP produced a rapid (1 to 2 minutes) cessation of changes in cellular deformability produced by the FMLP. Colchicine treatment did not decrease cell rigidity, but produced a delayed increase in F-actin content accompanied by increased stiffness of the cells. These results implicate actin as the major determinant of the mechanical behavior of the neutrophil, as measured by whole cell deformability tests. The significant changes in cell deformability that occur in response to fractional changes in F-actin content suggest that changes in the structure of the actin network occur during these processes.

Time-dependent alterations in the deformability of human neutrophils in response to chemotactic activation

Transit times of individual human neutrophils through single capillary- sized pores were measured to determine the time-dependent changes in the mechanical behavior of the cells during activation by the chemotactic agent formyl-methionyl-leucyl-phenylalanine (FMLP) and in response to cytochalasin B (CTB) and colchicine. FMLP elicited a two- phase response consisting of a rapid increase in cell stiffness, which peaked between 2 and 3 minutes, followed by a partial recovery of deformability to a level significantly above that of control after 5 minutes. The mechanical changes closely followed changes in F-actin content, although the peak in cell stiffness appeared to lag the F- actin response. Treatment with 3 mumols/L CTB produced a transient decrease in cell rigidity followed by a return to control level in 10 minutes, whereas treatment with 30 mumols/L CTB resulted in a sustained decrease in cell transit times to a level 60% of control. Addition of 3 mumols/L CTB to cells prestimulated with FMLP produced a rapid (1 to 2 minutes) cessation of changes in cellular deformability produced by the FMLP. Colchicine treatment did not decrease cell rigidity, but produced a delayed increase in F-actin content accompanied by increased stiffness of the cells. These results implicate actin as the major determinant of the mechanical behavior of the neutrophil, as measured by whole cell deformability tests. The significant changes in cell deformability that occur in response to fractional changes in F-actin content suggest that changes in the structure of the actin network occur during these processes.

Postoperative morbidity following red cell deformability changes during cardiopulmonary bypass using bubble and membrane oxygenators : Belboul A, Al Khaja N, Bergman P, et al: Surg 23:258–263, 1989.

Postoperative morbidity following red cell deformability changes during cardiopulmonary bypass using bubble and membrane oxygenators : Belboul A, Al Khaja N, Bergman P, et al: Surg 23:258–263, 1989.

Postoperative Morbidity Following Red Cell Deformability Changes During Cardiopulmonary Bypass Using Bubble and Membrane Oxygenators

The preoperative blood cell trauma and postoperative morbidity were com pared in a prospective randomized study in two groups of patients undergoing cardiac surgery with extracorporeal circulation (ECC). One group was oxygen ated during ECC with a membrane oxygenator (MO) and the other with a bub ble oxygenator (BO). Red cell trauma was ascertained by a standard microfiltration technique that expressed the rheologic properties of standard ized red cell suspensions. The filtrability of the cell suspension was significantly reduced during ECC for both membrane (MO) and bubble (BO) oxygenators. However, the bubble type proved to be more traumatic, and the difference was statistically signifi cant after thirty minutes of ECC. The filtrability was reduced by 20% in the MO group and by 35% in the BO group at the end of ECC (p &lt; 0.01). A significantly higher frequency of postoperative bleeding (p &lt; 0.05), blood transfusion (p &lt; 0.05), and arrhythmias was found in the BO group (p &lt; 0.01). This study demonstrates that the microrheology of red blood cells is better preserved with the membrane oxygenator, which is associated with a smoother postoperative course, owing probably to a better preserved microcirculation. The membrane oxygenator can be recommended even for short perfusions and routine procedures requiring cardiopulmonary bypass (CPB) and should be used whenever possible.

The development of a filtration system for evaluating flow characteristics of erythrocytes

A complete description of the pathophysiology of sickle cell disease requires a physiologically meaningful measurement of red cell deformability. We have designed and built a system which allows one to determine filtration characteristics of erythrocytes. A dilute red cell suspension is forced through a 3.0-μm polycarbonate Nuclepore membrane with a constant positive pressure of 20 mm Hg. Under these conditions blockage of the pores in the polycarbonate membrane is insignificant and flow is linear. We use the relative number of cells filtered through the membrane as a means of approximating the mean deformability of cells in the suspension. Using this system we have compared erythrocytes from various mammals and shown that our technique is sensitive in detecting not only differences in cell deformabilities between mammalian species but also changes in cell deformability of human red cells due to exchange transfusion and application of drugs. There was a positive correlation between cell filtrability and percentage cell recovery (coefficient of correlation, 0.65) and a negative correlation between cell size and filtrability (coefficient of correlation, −0.61). The filtrabilities of normal volunteers and sickle cell disease patients were found to be 71.8 ± 6.6 and 53.6 ± 5.0%, respectively. This system is sensitive and reliable, and should be useful in evaluating both the contribution of filtrability to the viability of red cells in vivo and potential therapeutic agents for sickle cell disease.