Mechanical diagnosis of human erythrocytes by ultra-high speed manipulation unraveled critical time window for global cytoskeletal remodeling

Large deformability of erythrocytes in microvasculature is a prerequisite to realize smooth circulation. We develop a novel tool for the three-step “Catch-Load-Launch” manipulation of a human erythrocyte based on an ultra-high speed position control by a microfluidic “robotic pump”. Quantification of the erythrocyte shape recovery as a function of loading time uncovered the critical time window for the transition between fast and slow recoveries. The comparison with erythrocytes under depletion of adenosine triphosphate revealed that the cytoskeletal remodeling over a whole cell occurs in 3 orders of magnitude longer timescale than the local dissociation-reassociation of a single spectrin node. Finally, we modeled septic conditions by incubating erythrocytes with endotoxin, and found that the exposure to endotoxin results in a significant delay in the characteristic transition time for cytoskeletal remodeling. The high speed manipulation of erythrocytes with a robotic pump technique allows for high throughput mechanical diagnosis of blood-related diseases.

[1]  H Schmid-Schönbein,et al.  The red cell as a fluid droplet: tank tread-like motion of the human erythrocyte membrane in shear flow. , 1978, Science.

[2]  J. Käs,et al.  Optical deformability of soft biological dielectrics. , 2000, Physical review letters.

[3]  H Schmid-Schönbein,et al.  A counter-rotating "rheoscope chamber" for the study of the microrheology of blood cell aggregation by microscopic observation and microphotometry. , 1973, Microvascular research.

[4]  R. Johnsen,et al.  Theory and Experiment , 2010 .

[5]  R M Hochmuth,et al.  Membrane viscoelasticity. , 1976, Biophysical journal.

[6]  O. Linderkamp,et al.  Effect of lipid A on the deformability, membrane rigidity and geometry of human adult red blood cells , 1992, European journal of clinical investigation.

[7]  R. Waugh,et al.  Elastic area compressibility modulus of red cell membrane. , 1976, Biophysical journal.

[8]  R. Skalak,et al.  Deformation of Red Blood Cells in Capillaries , 1969, Science.

[9]  M. Ichikawa,et al.  Quantification of the Influence of Endotoxins on the Mechanics of Adult and Neonatal Red Blood Cells. , 2015, The journal of physical chemistry. B.

[10]  M. Tao,et al.  Phosphorylation of ankyrin decreases its affinity for spectrin tetramer. , 1985, The Journal of biological chemistry.

[11]  Jochen Guck,et al.  Mechanics Meets Medicine , 2013, Science Translational Medicine.

[12]  E. Sackmann,et al.  Measurement of erythrocyte membrane elasticity by flicker eigenmode decomposition. , 1995, Biophysical journal.

[13]  A. Cowman,et al.  Contribution of parasite proteins to altered mechanical properties of malaria-infected red blood cells. , 2002, Blood.

[14]  Gerhard Gompper,et al.  Equilibrium physics breakdown reveals the active nature of red blood cell flickering , 2015, Nature Physics.

[15]  Howard A Stone,et al.  Dynamics of shear-induced ATP release from red blood cells , 2008, Proceedings of the National Academy of Sciences.

[16]  Stefan Schinkinger,et al.  Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. , 2005, Biophysical journal.

[17]  G. Karniadakis,et al.  Combined Simulation and Experimental Study of Large Deformation of Red Blood Cells in Microfluidic Systems , 2010, Annals of Biomedical Engineering.

[18]  E. Evans,et al.  Intrinsic material properties of the erythrocyte membrane indicated by mechanical analysis of deformation. , 1975, Blood.

[19]  H. Noguchi,et al.  Shape transitions of fluid vesicles and red blood cells in capillary flows. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[20]  N. Gov,et al.  Red blood cell membrane fluctuations and shape controlled by ATP-induced cytoskeletal defects. , 2005, Biophysical journal.

[21]  Subra Suresh,et al.  Large deformation of living cells using laser traps , 2004 .

[22]  O. Linderkamp,et al.  Endotoxin binding to erythrocyte membrane and erythrocyte deformability in human sepsis and in vitro , 2003, Critical care medicine.

[23]  Thomas M Fischer,et al.  Shape memory of human red blood cells. , 2004, Biophysical journal.

[24]  Kamolrat Silamut,et al.  The deformability of red blood cells parasitized by Plasmodium falciparum and P. vivax. , 2004, The Journal of infectious diseases.

[25]  H J Meiselman,et al.  Determination of red blood cell shape recovery time constant in a Couette system by the analysis of light reflectance and ektacytometry. , 1996, Biorheology.

[26]  Chwee Teck Lim,et al.  Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria. , 2005, Acta biomaterialia.

[27]  N. Mohandas,et al.  Modulation of Erythrocyte Membrane Mechanical Function by Protein 4.1 Phosphorylation* , 2005, Journal of Biological Chemistry.

[28]  Subra Suresh,et al.  Cytoskeletal dynamics of human erythrocyte , 2007, Proceedings of the National Academy of Sciences.

[29]  Nonequilibrium fluctuations of mechanically stretched single red blood cells detected by optical tweezers , 2013, European Biophysics Journal.

[30]  R. Hochmuth,et al.  Red cell extensional recovery and the determination of membrane viscosity. , 1979, Biophysical journal.

[31]  G J Streekstra,et al.  A new method to study shape recovery of red blood cells using multiple optical trapping. , 1995, Biophysical journal.

[32]  Subra Suresh,et al.  Biomechanics of red blood cells in human spleen and consequences for physiology and disease , 2016, Proceedings of the National Academy of Sciences.

[33]  Erich Sackmann,et al.  Hydrodynamic deformation reveals two coupled modes/time scales of red blood cell relaxation , 2012 .

[34]  N. Mohandas,et al.  Modulation of Erythrocyte Membrane Mechanical Function by β-Spectrin Phosphorylation and Dephosphorylation (*) , 1995, The Journal of Biological Chemistry.

[35]  R. Waugh,et al.  Viscoelastic properties of erythrocyte membranes of different vertebrate animals. , 1976, Microvascular research.

[36]  Thomas M Fischer,et al.  Creep and stress relaxation of human red cell membrane , 2017, Biomechanics and modeling in mechanobiology.

[37]  J. Simeon,et al.  Direct measurement of the area expansion and shear moduli of the human red blood cell membrane skeleton. , 2001, Biophysical journal.

[38]  F. Brochard,et al.  Frequency spectrum of the flicker phenomenon in erythrocytes , 1975 .

[39]  Cell Mechanics: Combining Speed with Precision. , 2015, Biophysical journal.

[40]  R. Hochmuth,et al.  Force relaxation and permanent deformation of erythrocyte membrane. , 1983, Biophysical journal.

[41]  Fumihito Arai,et al.  Catch, load and launch toward on-chip active cell evaluation , 2016, 2016 IEEE International Conference on Robotics and Automation (ICRA).

[42]  A. Zilman,et al.  Cytoskeleton confinement and tension of red blood cell membranes. , 2003, Physical review letters.

[43]  K. Jacobson,et al.  Local measurements of viscoelastic parameters of adherent cell surfaces by magnetic bead microrheometry. , 1998, Biophysical journal.

[44]  Dino Di Carlo,et al.  Hydrodynamic stretching of single cells for large population mechanical phenotyping , 2012, Proceedings of the National Academy of Sciences.

[45]  Dong Sun,et al.  Mechanical modeling of red blood cells during optical stretching. , 2010, Journal of biomechanical engineering.

[46]  Martin Lenz,et al.  ATP-dependent mechanics of red blood cells , 2009, Proceedings of the National Academy of Sciences.

[47]  Fumihito Arai,et al.  Red blood cell fatigue evaluation based on the close-encountering point between extensibility and recoverability. , 2014, Lab on a chip.

[48]  Philip S Low,et al.  Regulation of membrane-cytoskeletal interactions by tyrosine phosphorylation of erythrocyte band 3. , 2011, Blood.

[49]  U. Keyser,et al.  Real-time deformability cytometry: on-the-fly cell mechanical phenotyping , 2015, Nature Methods.

[50]  N. Gov,et al.  Direct Cytoskeleton Forces Cause Membrane Softening in Red Blood Cells. , 2015, Biophysical journal.

[51]  P. Low,et al.  Role of band 3 tyrosine phosphorylation in the regulation of erythrocyte glycolysis. , 1991, The Journal of biological chemistry.

[52]  Rafi Korenstein,et al.  Mechanical Fluctuations of the Membrane–Skeleton Are Dependent on F-Actin ATPase in Human Erythrocytes , 1998, The Journal of cell biology.

[53]  Yu Sun,et al.  High-throughput biophysical measurement of human red blood cells. , 2012, Lab on a chip.

[54]  Pietro Cicuta,et al.  Flickering analysis of erythrocyte mechanical properties: dependence on oxygenation level, cell shape, and hydration level. , 2009, Biophysical journal.

[55]  Oliver Otto,et al.  Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment , 2015, Biophysical journal.

[56]  N. Mohandas,et al.  Shear-Response of the Spectrin Dimer-Tetramer Equilibrium in the Red Blood Cell Membrane* , 2002, The Journal of Biological Chemistry.

[57]  E. Evans,et al.  Molecular maps of red cell deformation: hidden elasticity and in situ connectivity. , 1994, Science.

[58]  Stefan Schinkinger,et al.  Reconfigurable microfluidic integration of a dual-beam laser trap with biomedical applications , 2007, Biomedical microdevices.

[59]  J. Lippincott-Schwartz,et al.  Current Topics in Membranes and Transport , 2012 .

[60]  D. Discher,et al.  Kinematics of red cell aspiration by fluorescence-imaged microdeformation. , 1996, Biophysical journal.