Red Blood Cell Stiffness and Adhesion Are Species-Specific Properties Strongly Affected by Temperature and Medium Changes in Single Cell Force Spectroscopy

Besides human red blood cells (RBC), a standard model used in AFM-single cell force spectroscopy (SCFS), little is known about apparent Young’s modulus (Ea) or adhesion of animal RBCs displaying distinct cellular features. To close this knowledge gap, we probed chicken, horse, camel, and human fetal RBCs and compared data with human adults serving as a repository for future studies. Additionally, we assessed how measurements are affected under physiological conditions (species-specific temperature in autologous plasma vs. 25 °C in aqueous NaCl solution). In all RBC types, Ea decreased with increasing temperature irrespective of the suspension medium. In mammalian RBCs, adhesion increased with elevated temperatures and scaled with reported membrane sialic acid concentrations. In chicken only adhesion decreased with higher temperature, which we attribute to the lower AE-1 concentration allowing more membrane undulations. Ea decreased further in plasma at every test temperature, and adhesion was completely abolished, pointing to functional cell enlargement by adsorption of plasma components. This halo elevated RBC size by several hundreds of nanometers, blunted the thermal input, and will affect the coupling of RBCs with the flowing plasma. The study evidences the presence of a RBC surface layer and discusses the tremendous effects when RBCs are probed at physiological conditions.     

A discrete-particle model of blood dynamics in capillary vessels

We investigate the mechanism of aggregation of red blood cells (RBC) in capillary vessels. We use a discrete-particle model in 3D to model the flow of plasma and RBCs within a capillary tube. This model can accurately capture the scales from 0.001 to 100 microm, far below the scales that can be modeled numerically with classical computational fluid dynamics. The flexible viscoelastic red blood cells and the walls of the elastic vessel are made up of solid particles held together by elastic harmonic forces. The plasma is represented by a system of dissipative fluid particles. Modeling has been carried out using 1 to 3 million solid and fluid particles. We have modeled the flow of cells with vastly different shapes, such as normal and "sickle" cells. The two situations involving a straight capillary and a pipe with a choking point have been considered. The cells can coagulate in spite of the absence of adhesive forces in the model. We conclude that aggregation of red blood cells in capillary vessels can be stimulated by depletion forces and hydrodynamic interactions. The cluster of "sickle" cells formed in the choking point of the capillary efficiently decelerates the flow, while normal cells can pass through. These qualitative results from our first numerical results accord well with the laboratory findings.     

Passive recruitment of circulating leukocytes into capillary sprouts from existing capillaries in a microfluidic system

Recent evidence implicating leukocytes in angiogenesis raises the question of whether leukocytes and other cells circulating with the blood in microvascular networks can home to capillary sprouts intraluminally. This study describes an investigation of leukocyte trafficking in sprouting capillaries fabricated using soft lithography. The leukocytes passing with whole blood through existing capillaries were able to enter microfabricated capillary sprouts of variable length and sprouting angle due to the mechanical interaction with red blood cells (RBCs) at the sprouting bifurcation, in spite of the complete absence of blood flow through the blind-ended sprouts or any chemoattractants. The RBCs formed "comet tails" (the densely packed cellular trains forming behind leukocytes as they move through narrow capillaries) and effectively pushed leukocytes into the microfabricated sprouts while bypassing them at the sprouting bifurcation. Individual sprouts filled with several leukocytes, as wells as RBCs and platelets, were observed. The results of this study suggest that (i) blood cells are likely present in capillary sprouts throughout their development, (ii) leukocytes and other circulating cells may use this mechanism to home to capillary sprouts intraluminally for direct engraftment, and (iii) tissues may use this phenomenon as another mechanism for local recruitment of leukocytes from the blood stream.     

Peptide-mediated selective adhesion of smooth muscle and endothelial cells in microfluidic shear flow

Microfluidic devices have recently emerged as effective tools for cell separation compared to traditional techniques. These devices offer the advantages of small sample volumes, low cost, and high purity. Adhesion-based separation of cells from heterogeneous suspensions can be achieved by taking advantage of specific ligand-receptor interactions. The peptide sequences Arg-Glu-Asp-Val (REDV) and Val-Ala-Pro-Gly (VAPG) are known to bind preferentially to endothelial cells (ECs) and smooth muscle cells (SMCs), respectively. This article examines the roles of REDV and VAPG and fluid shear stress in achieving selective capture of ECs and SMCs in microfluidic devices. The adhesion of ECs in REDV-coated devices and SMCs in VAPG-coated devices increases significantly compared to that of the nontargeted cells with decreasing shear stress. Furthermore, the adhesion of these cells is shown to be independent of whether these cells flow through the devices as suspensions of only one cell type or as a heterogeneous suspension containing ECs, SMCs, and fibroblasts. Whereas the overall adhesion of cells in the devices is determined mainly by shear stress, the selectivity of adhesion depends on the type of peptide and on the device surface as well as on the shear stress.     

Indoxyl sulfate- and P-cresol-induced monocyte adhesion and migration is mediated by integrin-linked kinase-dependent podosome formation

Cardiovascular disease is an important cause of death in patients with chronic kidney disease (CKD). Protein-bound uremic toxins, such as p-cresyl and indoxyl sulfate (IS), are poorly removed during hemodialysis, leading to vascular endothelial dysfunction and leukocyte extravasation. These processes can be related to dynamic adhesion structures called podosomes. Several studies have indicated the role of integrin-linked kinase (ILK) in the accumulation of integrin-associated proteins in podosomes. Here, we investigated the involvement of ILK and podosome formation in the adhesion and extravasation of monocytes under p-cresol (pc) and IS exposure. Incubation of THP-1 human monocyte cells with these toxins upregulated ILK kinase activity. Together, both toxins increased cell adhesion, podosome formation, extracellular matrix degradation, and migration of THP-1 cells, whereas ILK depletion with specific small interfering RNAs suppressed these processes. Interestingly, F-actin colocalized with cortactin in podosome cores, while ILK was colocalized in podosome rings under toxin stimulation. Podosome Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) and AKT protein depletion demonstrated that monocyte adhesion depends on podosome formation and that the ILK/AKT signaling pathway is involved in these processes. Ex vivo experiments showed that both toxins induced adhesion and podosome formation in leukocytes from wild-type mice, whereas these effects were not observed in leukocytes of conditional ILK-knockdown animals. In summary, under pc and IS stimulation, monocytes increase podosome formation and transmigratory capacity through an ILK/AKT signaling pathway-dependent mechanism, which could lead to vascular injury. Therefore, ILK could be a potential therapeutic target for the treatment of vascular damage associated with CKD.Subject terms: Experimental models of disease, Atherosclerosis, Mechanisms of disease, Integrins, End-stage renal disease     

ICAM-1/LFA-1 interaction contributes to the induction of endothelial cell-cell separation: implication for enhanced leukocyte diapedesis

The basic route and mechanism for diapedesis has not yet to be fully defined. Here we present evidence that "cell-cell separation" between endothelial cells (ECs) may provide a route for leukocyte diapedesis. We unexpectedly found that extensive interaction between peripheral blood leukocytes and ECs that were activated by TNF-α induced the opening of EC contacts and, surprisingly, resulted in cell-cell separation. This event was specific to the intercellular adhesion molecules-1 (ICAM-1)/leukocyte function-associated antigen-1 interaction, as demonstrated by the following: (1) ICAM-1 expression correlated with increased EC contraction; and (2) the blocking of ICAM-1 selectively inhibited EC separation. Thus, we suggest that "cell-cell separation" could be a mechanism for diapedesis in situations that may require massive leukocyte infiltration.     

Red blood cell rheology using single controlled laser-induced cavitation bubbles

The deformability of red blood cells (RBCs) is an important property that allows the cells to squeeze through small capillary vessels and can be used as an indicator for disease. We present a microfluidic based technique to quantify the deformability of RBCs by stretching a collection of RBCs on a timescale of tens of microseconds in a microfluidic chamber. This confinement constrains the motion of the cell to the imaging plane of the microscope during a transient cavitation bubble event generated with a focused and pulsed laser. We record and analyze the shape recovery of the cells with a high-speed camera and obtain a power law in time, consistent with other dynamic rheological results of RBCs. The extracted exponents are used to characterize the elastic properties of the cells. We obtain statistically significant differences of the exponents between populations of untreated RBCs and RBCs treated with two different reagents: neuraminidase reduces the cell rigidity, while wheat germ agglutinin stiffens the cell confirming previous experiments. This cavitation based technique is a candidate for high-throughput screening of elastic cell properties because many cells can be probed simultaneously in situ, thus with no pre-treatment.     

Bioadhesive control of plasma proteins and blood cells from umbilical cord blood onto the interface grafted with zwitterionic polymer brushes

In this work, bioadhesive behavior of plasma proteins and blood cells from umbilical cord blood (UCB) onto zwitterionic poly(sulfobetaine methacrylate) (polySBMA) polymer brushes was studied. The surface coverage of polySBMA brushes on a hydrophobic polystyrene (PS) well plate with surface grafting weights ranging from 0.02 mg/cm(2) to 0.69 mg/cm(2) can be effectively controlled using the ozone pretreatment and thermal-induced radical graft-polymerization. The chemical composition, grafting structure, surface hydrophilicity, and hydration capability of prepared polySBMA brushes were determined to illustrate the correlations between grafting properties and blood compatibility of zwitterionic-grafted surfaces in contact with human UCB. The protein adsorption of fibrinogen in single-protein solutions and at complex medium of 100% UCB plasma onto different polySBMA brushes with different grafting coverage was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The grafting density of the zwitterionic brushes greatly affects the PS surface, thus controlling the adsorption of fibrinogen, the adhesion of platelets, and the preservation of hematopoietic stem and progenitor cells (HSPCs) in UCB. The results showed that PS surfaces grafted with polySBMA brushes possess controllable hydration properties through the binding of water molecules, regulating the bioadhesive and bioinert characteristics of plasma proteins and blood platelets in UCB. Interestingly, it was found that the polySBMA brushes with an optimized grafting weight of approximately 0.1 mg/cm(2) at physiologic temperatures show significant hydrated chain flexibility and balanced hydrophilicity to provide the best preservation capacity for HSPCs stored in 100% UCB solution for 2 weeks. This work suggests that, through controlling grafting structures, the hemocompatible nature of grafted zwitterionic polymer brushes makes them well suited to the molecular design of regulated bioadhesive interfaces for use in the preservation of HSPCs from human UCB.     

Monitoring the status of T-cell activation in a microfluidic system

Leukocyte adhesion to the endothelium through surface molecules such as E-selectin and intercellular adhesion molecule-1 (ICAM-1) is a critical cellular event reflecting the physiological status of both cell types. Here we present a microfluidic system that can not only easily monitor the interaction between leukocytes and endothelial cells under physiological conditions, but also screen drug candidates for potential modulation of this interaction. Shear stress, which is an important factor for the binding of activated T cells to tumor necrosis factor-alpha (TNF-α)-treated human umbilical vein endothelial cells (HUVECs), was easily controlled by adjusting the flow rate in the microfluidic system. Whole blood of patients with systemic lupus erythematosus (SLE) who have auto-reactive T cells were infused into the activated HUVECs which subsequently showed a higher level of binding compared to a control blood sample from a person without SLE. When these autoreactive T cells were treated with immunosuppressors tacrolimus and cyclosporin A, the binding of the T cells to HUVECs was dramatically decreased. Therefore, this microfluidic system is capable of differentiating the physiological status of T cells or endothelial cells representing different disease conditions, as well as being useful for the identification of novel reagents that modulate the functions of leukocytes or endothelial cells.     

Linear Poly(methyl glycerol) and Linear Polyglycerol as Potent Protein and Cell Resistant Alternatives to Poly(ethylene glycol)

The nonspecific interaction of proteins with surfaces in contact with biofluids leads to adverse problems and is prevented by a biocompatible surface coating. The current benchmark material among such coatings is poly(ethylene glycol) (PEG). Herein, we report on the synthesis of linear polyglycerol derivatives as promising alternatives to PEG. Therefore, gold surfaces as a model system are functionalized with a self‐assembled monolayer (SAM) by a two‐step anhydride coupling and a direct thiol immobilization of linear poly(methyl glycerol) and polyglycerol. Surface plasmon resonance (SPR) spectroscopy reveals both types of functionalized surfaces to be as resistant as PEG towards the adsorption of the test proteins fibrinogen, pepsin, albumin, and lysozyme. Moreover, linear polyglycerols adsorb even less proteins from human plasma than a PEG‐modified surface. Additional cell adhesion experiments on linear poly(methyl glycerol) and polyglycerol‐modified surfaces show comparable cell resistance as for a PEG‐modified surface. Also, in the case of long‐term stability, high cell resistance is observed for all samples in medium. Additional in vitro cell‐toxicity tests add to the argument that linear poly(methyl glycerol) and polyglycerol are strong candidates for promising alternatives to PEG, which can easily be modified for biocompatible functionalization of other surfaces.     

Plasma Surface Modification of Biomedical Polymers: Influence on Cell-Material Interaction

Polymers are commonly used in industry because of their excellent bulk properties, such as strength and good resistance to chemicals. Their surface properties are for most application inadequate due to their low surface energy. A surface modification is often needed, and plasma surface modification is used with success the past decades. In the past few years, also plasma surface modification for biomedical polymers has been investigated. For biomedical polymers, the surface properties need to be altered to promote a good cell adhesion, growth and proliferation and to make them suitable for implants and tissue engineering scaffolds. This review gives an overview of the use of plasma surface modification of biomedical polymers and the influence on cell-material interactions. First, an introduction on cell-material interaction and on antibacterial and antifouling surfaces will be given. Also, different plasma modifying techniques used for polymer surface modification will be discussed. Then, an overview of literature on plasma surface modification of biopolymers and the resulting influence on cell-material interaction will be given. After an overview of plasma treatment for improved cell-material interaction, plasma polymerization and plasma grafting techniques will be discussed. Some more specialized applications will be also presented: the treatment of 3D scaffolds for tissue engineering and the spatial control of cell adhesion. Antibacterial and antifouling properties, obtained by plasma techniques, will be discussed. An overview of research dealing with antibacterial surfaces created by plasma techniques will be given, antifouling surfaces will be discussed, and how blood compatibility can be improved by preventing protein adhesion.     

Poly(N-vinylpyrrolidone)-modified surfaces repel plasma protein adsorption

The present work aimed to study the interaction between plasma proteins and PVP-modified surfaces under more complex protein conditions.In the competitive adsorption of fibrinogen(Fg) and human serum albumin(HSA),the modified surfaces showed preferential adsorption of HSA.In 100%plasma,the amount of Fg adsorbed onto PVP-modified surfaces was as low as 10 ng/cm~2,suggesting the excellent protein resistance properties of the modified surfaces.In addition, immunoblots of proteins eluted from the modified surfaces after plasma contact confirmed that PVP-modified surfaces can repel most plasma proteins,especially proteins that play important roles in the process of blood coagulation.     

Extraction methods of red blood cell membrane proteins for Multidimensional Protein Identification Technology (MudPIT) analysis

Since red blood cells (RBCs) lack nuclei and organelles, cell membrane is their main load-bearing component and, according to a dynamic interaction with the cytoskeleton compartment, plays a pivotal role in their functioning. Even if erythrocyte membranes are available in large quantities, the low abundance and the hydrophobic nature of cell membrane proteins complicate their purification and detection by conventional 2D gel-based proteomic approaches. So, in order to increase the efficiency of RBC membrane proteome identification, here we took advantage of a simple and reproducible membrane sub-fractionation method coupled to Multidimensional Protein Identification Technology (MudPIT). In addition, the adoption of a stringent RBC filtration strategy from the whole blood, permitted to remove exhaustively contaminants, such as platelets and white blood cells, and to identify a total of 275 proteins in the three RBC membrane fractions collected and analysed. Finally, by means of software for the elaboration of the great quantity of data obtained and programs for statistical analysis and protein classification, it was possible to determine the validity of the entire system workflow and to assign the proper sub-cellular localization and function for the greatest number of the identified proteins.     

An inexpensive thread-based system for simple and rapid blood grouping

This study investigates the use of thread as a flexible and low-cost substrate for the rapid grouping of blood. The use of a capillary substrate such as thread for blood grouping utilises the sensitivity of the flow resistance of large particles in narrow capillary channels to separate agglutinated red blood cells (RBCs) from plasma. Large and discrete particles formed in a continuous liquid phase do not provide capillary wicking driving force and fall behind the capillary wicking front, leading to their separation from the wicking liquid. The capillary substrate therefore provides a very promising but different mechanism for the separation of the agglutinated RBCs and the blood serum phase compared to most existing blood grouping methods. The principle of chromatographic separation is also exploited in this study via the use of suitable dyes to enhance the visual detection of the agglutinated RBCs and the serum phase; surprising and encouraging outcomes are obtained. Using a thread-based device, the ABO and Rh groups can be successfully determined with only 2 μL of whole blood from a pricked finger tip within 1 min and without pre-treatment of the blood sample. It is hoped that a new, inexpensive, rapid and simple method may provide an easy-to-use blood grouping platform well suited to those in developing or remote regions of the world.     

Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test

To elucidate the protein–protein interactions of hemoglobin (Hb) variants A and A₂, HbA was first shown to bind with HbA₂ in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA₂ outside the RBC was shown by cross electrophoresis. The starch–agarose gel electrophoresis of hemolysate, RBCs, freeze‐thawed RBCs and the supernatant of freeze‐thawed RBCs showed that the interaction between HbA and HbA₂ was affected by membrane integrity. To identify the proteins involved in the interaction, protein components located between HbA and HbA₂ in RBCs (RBC HbA‐HbA₂) and hemolysate (hemolysate HbA‐HbA₂) were isolated from the starch–agarose gel and separated by 5–12% SDS‐PAGE. The results showed that there was a ≈22 kDa protein band located in the RBC HbA‐HbA₂ but not in hemolysate HbA‐HbA₂. Sequencing by LC/MS/MS showed that this band was a protein complex that included mainly thioredoxin peroxidase B, α‐globin, δ‐globin and β‐globin. Thus, using our unique in vivo whole blood cell electrophoresis release test, Hbs were proven for the first time to interact with other proteins in the live RBC.     

A Commercial Nonbinding Surface Effectively Reduces Fibrinogen Adsorption but Does Not Prevent Platelet Adhesion to Fibrinogen

A commercial nonbinding surface effectively prevents protein adsorption; however, the platelet phenotype on this surface has yet to be defined. This study evaluates platelet adhesion and adsorption of several plasma/extracellular matrix (ECM) proteins to the nonbinding surface compared to other commonly used nontreated and high-binding surfaces. Platelet adhesion to uncoated microplates and those coated with fibrinogen or collagen is quantified by colorimetric assay. The binding capacity of the examined surfaces for plasma/ECM proteins is evaluated by measuring the relative and absolute protein adsorption. Compared to other surfaces, the nonbinding surface effectively prevents platelet adsorption, i.e. by 61-93% (Enzyme-Linked Immunosorbent Assay, ELISA), and reduces platelet adhesion, i.e. by 92%, when not coated with any protein. The nonbinding surface also decreases platelet deposition on collagen (up to 31%), but not fibrinogen. The nonbinding surface seems to be more of a low-fouling than nonfouling material, as it is able to reduce fibrinogen adsorption but not prevent platelet adhesion to fibrinogen. This feature should be considered when using the nonbinding surface for in vitro platelet testing.     

Recent Advances on Surface-modified Biomaterials Promoting Selective Adhesion and Directional Migration of Cells

The surfaces and interfaces of biomaterials interact with the biological systems in multi-scale levels, and thereby influence the biological functions and comprehensive performance in vitro and in vivo. In particular, a surface promoting the selective adhesion and directional migration of desired types of cells in complex environment is extremely important in the repair and regeneration of tissues such as peripheral nerve and blood vessel, and long-term application of intracorporal devices such as intravascular implants. Therefore, surface modification of biomaterials is a facile and effective method to achieve the desired cell-biomaterials interactions. In this short review, recent advances on the surface modification of biomaterials to regulate selective cell adhesion and migration are briefly summarized. In particular, the surface properties of biomaterials are manipulated via the convenient introduction of amino groups to the ester-based polymers, the formation of polyelectrolyte multilayers, and the fabrication of topology and gradient cues, etc., followed by the association of chemical and biological signals such as collagen, heparin, hyaluronic acid, peptides and cell growth factors. The selective adhesion and directional migration of various types of cells such as endothelial cells(ECs), smooth muscle cells(SMCs), hepatocytes and Schwann cells(SCs) are achieved over the competitive counterpart cells by the use of cell-resisting substances and cell-selective motifs on gradient substrates in most cases. Recent advances on cell behaviors in three-dimensional(3D) cell-extracellular matrix(ECM)-mimicking substrates are also reviewed.     

Heparin coating of poly(ethylene terephthalate) decreases hydrophobicity, monocyte/leukocyte interaction and tissue interaction

Woven poly(ethylene terephthalate) (PET) is widely used in implantable medical devices. Upon implantation, fibrinogen interacts with the PET and changes conformation, such that the fibrinogen P2 epitope may become exposed. This allows inflammatory cells to interact with the material. In this study we have coated PET with heparin and show that this decreases PET hydrophobicity and the presence of the fibrinogen P2 epitope on the material surface. In addition, we show that heparin-induced reduction of PET hydrophobicity correlates with decreased exposure of the fibrinogen P2 epitope and reduced adhesion of monocytes. Reduction of PET hydrophobicity was furthermore associated with reduced PMN elastase production and decreased interaction between PET and embryonic chicken tissue. We conclude that the heparin coating-induced decrease in PET hydrophobicity is associated with decreased interaction between PET and inflammatory cells. Independent of this interaction, the hydrophobic nature of the heparin coating is related to tissue interaction as demonstrated by a reduction in adhesion, growth and spreading of tissue on PET. The combination of these properties makes heparin coating a candidate for improving biocompatibility of PET.     

Single-molecule resolution of interfacial fibrinogen behavior: effects of oligomer populations and surface chemistry

Through the use of single-molecule total internal reflection fluorescence microscopy, the dynamic behavior of fibrinogen was observed at the interface between aqueous solution and various solid surfaces. Multiple populations of objects were observed, as characterized by surface residence times, interfacial diffusion, and fluorescence intensity. On all surfaces, populations exhibited direct links between surface residence time, rate of diffusion, and fluorescence intensity. In particular, longer-lived populations diffused more slowly and exhibited greater fluorescence intensity, leading to the conclusion that the objects represented fibrinogen monomers and discrete oligomer populations (dimers, trimers, etc.), and that these oligomer populations play an important role in the protein-surface interaction because of their long surface residence times. Two or three diffusive modes were observed for most populations, indicating that protein aggregates have multiple mechanisms for interaction with solid substrates. In addition, the fastest diffusive mode is believed to represent a hopping mode that often precedes desorption events. Surprisingly, a monolayer of 5000 Da poly(ethylene glycol) (PEG5000) increased surface residence time and slowed diffusion of fibrinogen relative to bare fused silica or hydrophobically modified fused silica, suggesting that the mechanism of PEG resistance to protein adhesion is more sophisticated than the simple repulsion of individual proteins.     

A microfabricated deformability-based flow cytometer with application to malaria

Malaria resulting from Plasmodium falciparum infection is a major cause of human suffering and mortality. Red blood cell (RBC) deformability plays a major role in the pathogenesis of malaria. Here we introduce an automated microfabricated "deformability cytometer" that measures dynamic mechanical responses of 10(3) to 10(4) individual RBCs in a cell population. Fluorescence measurements of each RBC are simultaneously acquired, resulting in a population-based correlation between biochemical properties, such as cell surface markers, and dynamic mechanical deformability. This device is especially applicable to heterogeneous cell populations. We demonstrate its ability to mechanically characterize a small number of P. falciparum-infected (ring stage) RBCs in a large population of uninfected RBCs. Furthermore, we are able to infer quantitative mechanical properties of individual RBCs from the observed dynamic behavior through a dissipative particle dynamics (DPD) model. These methods collectively provide a systematic approach to characterize the biomechanical properties of cells in a high-throughput manner.