Dynamics of red blood cells in microporous membranes

We have performed microfluidic experiments with erythrocytes passing through a network of microchannels of 20–25 μm width and 5 μm of height. Red blood cells (RBCs) were flowing in countercurrent directions through microchannels connected by μm pores. Thereby, we have observed interesting flow dynamics. All pores were blocked by erythrocytes. Some erythrocytes have passed through pores, depending on the channel size and cell elasticity. Many RBCs split into two or more smaller parts. Two types of splits were observed. In one type, the lipid bilayer and spectrin network were cut at the same time. In the second type, the lipid bilayer reconnected, but the part of spectrin network stayed outside the cell forming a rope like structure, which could eventually break. The microporous membrane results in multiple breakups of the cells, which can have various clinical implications, e.g., glomerulus hematuria and anemia of patients undergoing dialysis. The cell breakup procedure is similar to the one observed in the droplet breakage of viscoelastic liquids in confinement.     

Coalescing drops in microfluidic parking networks: A multifunctional platform for drop-based microfluidics

Multiwell plate and pipette systems have revolutionized modern biological analysis; however, they have disadvantages because testing in the submicroliter range is challenging, and increasing the number of samples is expensive. We propose a new microfluidic methodology that delivers the functionality of multiwell plates and pipettes at the nanoliter scale by utilizing drop coalescence and confinement-guided breakup in microfluidic parking networks (MPNs). Highly monodisperse arrays of drops obtained using a hydrodynamic self-rectification process are parked at prescribed locations in the device, and our method allows subsequent drop manipulations such as fine-gradation dilutions, reactant addition, and fluid replacement while retaining microparticles contained in the sample. Our devices operate in a quasistatic regime where drop shapes are determined primarily by the channel geometry. Thus, the behavior of parked drops is insensitive to flow conditions. This insensitivity enables highly parallelized manipulation of drop arrays of different composition, without a need for fine-tuning the flow conditions and other system parameters. We also find that drop coalescence can be switched off above a critical capillary number, enabling individual addressability of drops in complex MPNs. The platform demonstrated here is a promising candidate for conducting multistep biological assays in a highly multiplexed manner, using thousands of submicroliter samples.     

Deformation of red blood cells using acoustic radiation forces

Acoustic radiation forces have been used to manipulate cells and bacteria in a number of recent microfluidic applications. The net force on a cell has been subject to careful investigation over a number of decades. We demonstrate that the radiation forces also act to deform cells. An ultrasonic standing wave field is created in a 0.1 mm glass capillary at a frequency of 7.9 MHz. Using osmotically swollen red-blood cells, we show observable deformations up to an aspect ratio of 1.35, comparable to deformations created by optical tweezing. In contrast to optical technologies, ultrasonic devices are potentially capable of deforming thousands of cells simultaneously. We create a finite element model that includes both the acoustic environment of the cell, and a model of the cell membrane subject to forces resulting from the non-linear aspects of the acoustic field. The model is found to give reasonable agreement with the experimental results, and shows that the deformation is the result of variation in an acoustic force that is directed outwards at all points on the cell membrane. We foresee applications in diagnostic devices, and in the possibility of mechanically stimulating cells to promote differentiation and physiological effects.     

Simulation of malaria-infected red blood cells in microfluidic channels: Passage and blockage

Malaria-infected red blood cells (iRBCs) become less deformable with the progression of infection and tend to occlude microcapillaries. This process has been investigated in vitro using microfluidic channels. The objective of this paper is to provide a quantitative basis for interpreting the experimental observations of iRBC occlusion of microfluidic channels. Using a particle-based model for the iRBC, we simulate the traverse of iRBCs through a converging microfluidic channel and explore the progressive loss of cell deformability due to three factors: the stiffening of the membrane, the reduction of the cell''s surface-volume ratio, and the growing solid parasites inside the cell. When examined individually, each factor tends to hinder the passage of the iRBC and lengthen the transit time. Moreover, at sufficient magnitude, each may lead to obstruction of narrow microfluidic channels. We then integrate the three factors into a series of simulations that mimic the development of malaria infection through the ring, trophozoite, and schizont stages. These simulations successfully reproduce the experimental observation that with progression of infection, the iRBC transitions from passage to blockage in larger and larger channels. The numerical results suggest a scheme for quantifying iRBC rigidification through microfluidic measurements of the critical pressure required for passage.     

The mechanical properties of stored red blood cells measured by a convenient microfluidic approach combining with mathematic model

The mechanical properties of red blood cells (RBCs) are critical to the rheological and hemodynamic behavior of blood. Although measurements of the mechanical properties of RBCs have been studied for many years, the existing methods, such as ektacytometry, micropipette aspiration, and microfluidic approaches, still have limitations. Mechanical changes to RBCs during storage play an important role in transfusions, and so need to be evaluated pre-transfusion, which demands a convenient and rapid detection method. We present a microfluidic approach that focuses on the mechanical properties of single cell under physiological shear flow and does not require any high-end equipment, like a high-speed camera. Using this method, the images of stretched RBCs under physical shear can be obtained. The subsequent analysis, combined with mathematic models, gives the deformability distribution, the morphology distribution, the normalized curvature, and the Young''s modulus (E) of the stored RBCs. The deformability index and the morphology distribution show that the deformability of RBCs decreases significantly with storage time. The normalized curvature, which is defined as the curvature of the cell tail during stretching in flow, suggests that the surface charge of the stored RBCs decreases significantly. According to the mathematic model, which derives from the relation between shear stress and the adherent cells'' extension ratio, the Young''s moduli of the stored RBCs are also calculated and show significant increase with storage. Therefore, the present method is capable of representing the mechanical properties and can distinguish the mechanical changes of the RBCs during storage. The advantages of this method are the small sample needed, high-throughput, and easy-use, which make it promising for the quality monitoring of RBCs.     

The role of band 3 protein in oxygen delivery by red blood cells

The synergistic effects of hemoglobin, carbonic anhydrase and the band 3 protein make red blood cells the ideal vehicle for oxygen delivering to the tissues. As long as oxygen is supplied by these ideal vehicles, oxygen intoxication of the tissues is precluded. Band 3 protein mediates the “Chloride-Shift”, i.e., the anion exchange of Cl⁻/HCO₃ ⁻. Because of the Chloride-Shift, red blood cells are able to recognize metabolically active tissues and to supply the minimum amount of oxygen to the tissues. Investigation into the molecular mechanisms of the anion exchange mediated by the band 3 protein was introduced.     

Sorting of circulating tumor cells (MV3-melanoma) and red blood cells using non-inertial lift

We demonstrate the method of non-inertial lift induced cell sorting (NILICS), a continuous, passive, and label-free cell sorting approach in a simple single layer microfluidic device at low Reynolds number flow conditions. In the experiments, we exploit the non-inertial lift effect to sort circulating MV3-melanoma cells from red blood cell suspensions at different hematocrits as high as 9%. We analyze the separation process and the influence of hematocrit and volume flow rates. We achieve sorting efficiencies for MV3-cells up to E_MV3 = 100% at Hct = 9% and demonstrate cell viability by recultivation of the sorted cells.     

Continuous removal of glycerol from frozen-thawed red blood cells in a microfluidic membrane device

Cryopreservation of human red blood cells (RBCs) in the presence of 40% glycerol allows a shelf-life of 10 years, as opposed to only 6 weeks for refrigerated RBCs. Nonetheless, cryopreserved blood is rarely used in clinical therapy, in part because of the requirement for a time-consuming (∼1 h) post-thaw wash process to remove glycerol before the product can be used for transfusion. The current deglycerolization process involves a series of saline washes in an automated centrifuge, which gradually removes glycerol from the cells in order to prevent osmotic damage. We recently demonstrated that glycerol can be extracted in as little as 3 min without excessive osmotic damage if the composition of the extracellular solution is precisely controlled. Here, we explore the potential for carrying out rapid glycerol extraction using a membrane-based microfluidic device, with the ultimate goal of enabling inline washing of cryopreserved blood. To assist in experimental design and device optimization, we developed a mass transfer model that allows prediction of glycerol removal, as well as the resulting cell volume changes. Experimental measurements of solution composition and hemolysis at the device outlet are in reasonable agreement with model predictions, and our results demonstrate that it is possible to reduce the glycerol concentration by more than 50% in a single device without excessive hemolysis. Based on these promising results, we present a design for a multistage process that is predicted to safely remove glycerol from cryopreserved blood in less than 3 min.     

Spatio-temporal analysis of tamoxifen-induced bystander effects in breast cancer cells using microfluidics

The bystander effect in cancer therapy is the inhibition or killing of tumor cells that are adjacent to those directly affected by the agent used for treatment. In the case of chemotherapy, little is known as to how much and by which mechanisms bystander effects contribute to the elimination of tumor cells. This is mainly due to the difficulty to distinguish between targeted and bystander cells since both are exposed to the pharmaceutical compound. We here studied the interaction of tamoxifen-treated human breast cancer MCF-7 cells with their neighboring counterparts by exploiting laminar flow patterning in a microfluidic chip to ensure selective drug delivery. The spatio-temporal evolution of the bystander response in non-targeted cells was analyzed by measuring the mitochondrial membrane potential under conditions of free diffusion. Our data show that the bystander response is detectable as early as 1 hour after drug treatment and reached effective distances of at least 2.8 mm. Furthermore, the bystander effect was merely dependent on diffusible factors rather than cell contact-dependent signaling. Taken together, our study illustrates that this microfluidic approach is a promising tool for screening and optimization of putative chemotherapeutic drugs to maximize the bystander response in cancer therapy.     

Frequency sweep rate dependence on the dielectrophoretic response of polystyrene beads and red blood cells

Alternating current (AC) dielectrophoresis (DEP) experiments for biological particles in microdevices are typically done at a fixed frequency. Reconstructing the DEP response curve from static frequency experiments is laborious, but essential to ascertain differences in dielectric properties of biological particles. Our lab explored the concept of sweeping the frequency as a function of time to rapidly determine the DEP response curve from fewer experiments. For the purpose of determining an ideal sweep rate, homogeneous 6.08 μm polystyrene (PS) beads were used as a model system. Translatability of the sweep rate approach to ∼7 μm red blood cells (RBC) was then verified. An Au/Ti quadrapole electrode microfluidic device was used to separately subject particles and cells to 10Vpp AC electric fields at frequencies ranging from 0.010 to 2.0 MHz over sweep rates from 0.00080 to 0.17 MHz/s. PS beads exhibited negative DEP assembly over the frequencies explored due to Maxwell-Wagner interfacial polarizations. Results demonstrate that frequency sweep rates must be slower than particle polarization timescales to achieve reliable incremental polarizations; sweep rates near 0.00080 MHz/s yielded DEP behaviors very consistent with static frequency DEP responses for both PS beads and RBCs.     

Characterizing the dielectric properties of human mesenchymal stem cells and the effects of charged elastin-like polypeptide copolymer treatment

Human mesenchymal stem cells (hMSCs) have three key properties that make them desirable for stem cell therapeutics: differentiation capacity, trophic activity, and ability to self-renew. However, current separation techniques are inefficient, time consuming, expensive, and, in some cases, alter hMSCs cellular function and viability. Dielectrophoresis (DEP) is a technique that uses alternating current electric fields to spatially separate biological cells based on the dielectric properties of their membrane and cytoplasm. This work implements the first steps toward the development of a continuous cell sorting microfluidic device by characterizing native hMSCs dielectric signatures and comparing them to hMSCs morphologically standardized with a polymer. A quadrapole Ti-Au electrode microdevice was used to observe hMSC DEP behaviors, and quantify frequency spectra and cross-over frequency of hMSCs from 0.010–35 MHz in dextrose buffer solutions (0.030 S/m and 0.10 S/m). This combined approach included a systematic parametric study to fit a core-shell model to the DEP spectra over the entire tested frequency range, adding robustness to the analysis technique. The membrane capacitance and permittivity were found to be 2.2 pF and 2.0 in 0.030 S/m and 4.5 pF and 4.1 in 0.10 S/m, respectively. Elastin-like polypeptide (ELP-) polyethyleneimine (PEI) copolymer was used to control hMSCs morphology to spheroidal cells and aggregates. Results demonstrated that ELP-PEI treatment controlled hMSCs morphology, increased experiment reproducibility, and concurrently increased hMSCs membrane permittivity to shift the cross-over frequency above 35 MHz. Therefore, ELP-PEI treatment may serve as a tool for the eventual determination of biosurface marker-dependent DEP signatures and hMSCs purification.     

Drop-to-drop liquid–liquid extraction of DNA in an electrowetting-on-dielectric digital microfluidics

Recent advancements in microfluidics and lab-on-a-chip technologies enabled miniaturization and automation of many downstream nucleic acid analysis steps such as PCR. However, DNA extraction/isolation protocol remains a stand-alone sample preparation step. For a quick sample-to-result solution, downstream protocols and sample preparation protocols need to be seamlessly integrated into a single lab-on-a-chip platform. As a step toward such integration, this paper introduces microfluidic DNA isolation using the liquid–liquid extraction (LLE) method in the drop-to-drop (DTD) format. The electrowetting-on-dielectric digital microfluidic platform is capable of handling a two-phase liquid system easily, which enables DTD LLE. In this study, the extraction of plasmid DNA (pDNA) from an aqueous sample to an ionic liquid is demonstrated. Prior to pDNA extraction study, the DTD LLE protocol was developed and optimized using organic dyes as solutes. The selective extraction of pDNA in the presence of proteins as interfering molecules is also demonstrated. This work implies that DTD LLE can substitute for magnetic beads steps in standard DNA isolation protocols.     

Changes in velocity profile according to blood viscosity in a microchannel

Red blood cells (RBCs) are important to dictate hemorheological properties of blood. The shear-thinning effect of blood is mainly attributed to the characteristics of the RBCs. Variations in hemorheological properties alter flow resistance and wall shear stress in blood vessels. Therefore, detailed understanding of the relationship between the hemorheological and hemodynamic properties is of great importance. In this study, blood viscosity and blood flow were simultaneously measured in the same microfluidic device by monitoring the flow-switching phenomenon. To investigate blood flows according to hemorheological variations, the flow rate of blood samples (RBCs suspended in autologous plasma, dextran-treated plasma, and in phosphate buffered saline solution) was precisely controlled with a syringe pump. Velocity profiles of blood flows were measured by using a micro-particle image velocimetry technique. The shape of velocity profiles was quantified by using a curve-fitting equation. It is found that the shape of the velocity profiles is highly correlated with blood viscosity. To demonstrate the relationship under ex vivo conditions, biophysical properties and velocity profiles were measured in an extracorporeal rat bypass loop. Experimental results show that increased blood viscosity seems to induce blunt velocity profile with high velocity component at the wall of the microchannel. Simultaneous measurement of blood viscosity and velocity profile would be useful for understanding the effects of hemorheological features on the hemodynamic characteristics in capillary blood vessels.     

Hydrodynamic mechanisms of cell and particle trapping in microfluidics

Focusing and sorting cells and particles utilizing microfluidic phenomena have been flourishing areas of development in recent years. These processes are largely beneficial in biomedical applications and fundamental studies of cell biology as they provide cost-effective and point-of-care miniaturized diagnostic devices and rare cell enrichment techniques. Due to inherent problems of isolation methods based on the biomarkers and antigens, separation approaches exploiting physical characteristics of cells of interest, such as size, deformability, and electric and magnetic properties, have gained currency in many medical assays. Here, we present an overview of the cell/particle sorting techniques by harnessing intrinsic hydrodynamic effects in microchannels. Our emphasis is on the underlying fluid dynamical mechanisms causing cross stream migration of objects in shear and vortical flows. We also highlight the advantages and drawbacks of each method in terms of throughput, separation efficiency, and cell viability. Finally, we discuss the future research areas for extending the scope of hydrodynamic mechanisms and exploring new physical directions for microfluidic applications.     

Quantitative detection of cells expressing BlaC using droplet-based microfluidics for use in the diagnosis of tuberculosis

This paper describes a method for the quantitative detection of cells expressing BlaC, a β-lactamase naturally expressed by Mycobacterium tuberculosis, intended for the diagnosis of tuberculosis. The method is based on the compartmentalization of bacteria in picoliter droplets at limiting dilutions such that each drop contains one or no cells. The co-encapsulation of a fluorogenic substrate probe for BlaC allows the quantification of bacteria by enumerating the number of fluorescent drops. Quantification of 10 colony forming units per milliliter is demonstrated. Furthermore, the encapsulation of single cell in drops maintains the specificity of the detection scheme even when the concentration of bacteria that do not express BlaC exceeds that expressing BlaC by one million-fold.     

Nutriceuticals in health and disease prevention

Conclusion  Available evidence suggests that we can not dismiss the potential value of nutriceuticals in disease and inhibition of atherosclerosis. Epidemiologic data suggest that antioxidant supplementation may be associated with a reduced risk of clinical events from atherosclerosis; howere, interventional trials only support a role for vitamin E in this regard. Many studies suggest that a link between fruit and vegetables in diet or the amounts of plasma antioxidant vitamins (ascorbic acid, tocopherols and carotenoids) and risk of death from cancer or coronary heart disease. The usefulness of antioxidant for prevention of cardiovascular disease is yet to be proven. However, studies offer important insights that together with the development of methods to identify individuals most likely to benefit, provide hope to clinicians seeking to use antioxidant vitamins with safety and efficacy for the treatment and prevention of cardiovascular disease. Only continued investigation into the mechanism (s) of action of candidate agents will determine whether they hold promise as a therapeutic intervention and only then, they can be recommended routinely to the patients. Thus, nutriceuticals are becoming more widely accepted as an adjunct to conventional therapies.     

The effect of red blood cell aggregation on velocity and cell-depleted layer characteristics of blood in a bifurcating microchannel

Red blood cell (RBC) aggregation is a multifaceted phenomenon, and whether it is generally beneficial or deleterious remains unclear. In order to better understand its effect on microvascular blood flow, the phenomenon must be studied in complex geometries, as it is strongly dependent on time, flow, and geometry. The cell-depleted layer (CDL) which forms at the walls of microvessels has been observed to be enhanced by aggregation; however, details of the characteristics of the CDL in complex regions, such as bifurcations, require further investigation. In the present study, a microchannel with a T-junction was used to analyze the influence of aggregation on the flow field and the CDL. Micro-PIV using RBCs as tracers provided high resolution cell velocity data. CDL characteristics were measured from the same data using a newly developed technique based on motion detection. Skewed and sharpened velocity profiles in the daughter branches were observed, contrary to the behavior of a continuous Newtonian fluid. RBC aggregation was observed to increase the skewness, but decrease the sharpening, of the velocity profiles in the daughter branches. The CDL width was found to be significantly greater, with a wider distribution, in the presence of aggregation and the mean width increased proportionally with the reciprocal of the fraction of flow entering the daughter branch. Aggregation also significantly increased the roughness of the interface between the CDL and the RBC core. The present results provide further insight into how RBC aggregation may affect the flow in complex geometries, which is of importance in both understanding its functions invivo, and utilizing it as a tool in microfluidic devices.     

Regulation of DNA conformations and dynamics in flows with hybrid field microfluidics

Visualizing single DNA dynamics in flow provides a wealth of physical insights in biophysics and complex flow study. However, large signal fluctuations, generated from diversified conformations, deformation history dependent dynamics and flow induced stochastic tumbling, often frustrate its wide adoption in single molecule and polymer flow study. We use a hybrid field microfluidic (HFM) approach, in which an electric field is imposed at desired locations and appropriate moments to balance the flow stress on charged molecules, to effectively regulate the initial conformations and the deformation dynamics of macromolecules in flow. With λ-DNA and a steady laminar shear flow as the model system, we herein studied the performance of HFM on regulating DNA trapping, relaxation, coil-stretch transition, and accumulation. DNA molecules were found to get captured in the focused planes when motions caused by flow, and the electric field were balanced. The trapped macromolecules relaxed in two different routes while eventually became more uniform in size and globule conformations. When removing the electric field, the sudden stretching dynamics of DNA molecules exhibited a more pronounced extension overshoot in their transient response under a true step function of flow stress while similar behaviors to what other pioneering work in steady shear flow. Such regulation strategies could be useful to control the conformations of other important macromolecules (e.g., proteins) and help better reveal their molecular dynamics.