Adhesion-based high-throughput label-free cell sorting using ridged microfluidic channels.

Numerous applications in medical diagnostics, cell engineering therapy, and biotechnology require the identification and sorting of cells that express desired molecular surface markers. We developed a microfluidic method for high-throughput and label-free sorting of biological cells by their affinity of molecular surface markers to target ligands. Our approach consists of a microfluidic channel decorated with periodic skewed ridges and coated with adhesive molecules. The periodic ridges form gaps with the opposing channel wall that are smaller than the cell diameter, thereby ensuring cell contact with the adhesive surfaces. Using three-dimensional computer simulations, we examine trajectories of adhesive cells in the ridged microchannels. The simulations reveal that cell trajectories are sensitive to the cell adhesion strength. Thus, the differential cell trajectories can be leveraged for adhesion-based cell separation. We probe the effect of cell elasticity on the adhesion-based sorting and show that cell elasticity can be utilized to enhance the resolution of the sorting. Furthermore, we investigate how the microchannel ridge angle can be tuned to achieve an efficient adhesion-based sorting of cells with different compliance.

Label-free microfluidic isolation of functional and viable lymphocytes from peripheral blood mononuclear cells.

The separation of peripheral blood mononuclear cells (PBMCs) into constituent blood cell types is a vital step to obtain immune cells for autologous cell therapies. The ability to separate PBMCs using label-free microfluidic techniques, based on differences in biomechanical properties, can have a number of benefits over other conventional techniques, including lower cost, ease of use, and avoidance of animal-derived labeling antibodies. Here, we report a microfluidic device that uses compressive diagonal ridges to separate PBMCs into highly pure samples of viable and functional lymphocytes. The technique utilizes the differences in the biophysical properties of PBMC sub-populations to direct the lymphocytes and monocytes into separate outlets. The biophysical properties of the monocytes and lymphocytes from healthy donors were first characterized using atomic force microscopy. Lymphocytes were found to be significantly stiffer than monocytes, with a mean cell stiffness of 1495 and 931 Pa, respectively. The differences in biophysical properties resulted in distinct trajectories through the microchannel terminating at different outlets, resulting in a lymphocyte sample with purity and viability both greater than 96% with no effect on the cells' ability to produce interferon gamma, a cytokine crucial for innate and adaptive immunity.

Probing interactions of red blood cells and contracting fibrin platelet clots.

Contraction of blood clots plays an important role in blood clotting, a natural process that restores hemostasis and regulates thrombosis in the body. Upon injury, a chain of events culminate in the formation of a soft plug of cells and fibrin fibers attaching to wound edges. Platelets become activated and apply contractile forces to shrink the overall clot size, modify clot structure, and mechanically stabilize the clot. Impaired blood clot contraction results in unhealthy volumetric, mechanical, and structural properties of blood clots associated with a range of severe medical conditions for patients with bleeding and thrombotic disorders. Due to the inherent mechanical complexity of blood clots and a confluence of multiple interdependent factors governing clot contraction, the mechanics and dynamics of clot contraction and the interactions with red blood cells (RBCs) remain elusive. Using an experimentally informed, physics-based mesoscale computational model, we probe the dynamic interactions among platelets, fibrin polymers, and RBCs, and examine the properties of contracted blood clots. Our simulations confirm that RBCs strongly affect clot contraction. We find that RBC retention and compaction in thrombi can be solely a result of mechanistic contraction of fibrin mesh due to platelet activity. Retention of RBCs hinders clot contraction and reduces clot contractility. Expulsion of RBCs located closer to clot outer surface results in the development of a dense fibrin shell in thrombus clots commonly observed in experiments. Our simulations identify the essential parameters and interactions that control blood clot contraction process, highlighting its dependence on platelet concentration and the initial clot size. Furthermore, our computational model can serve as a useful tool in clinically relevant studies of hemostasis and thrombosis disorders, and post thrombotic clot lysis, deformation, and breaking.

Effect of valve spacing on peristaltic pumping.

Peristaltic fluid pumping due to a periodically propagating contraction wave in a vessel fitted with one-way elastic valves is investigated numerically. It is concluded that the valve spacing within the vessel relative to the contraction wavelength plays a critical role in providing efficient pumping. When the valve spacing does not match the wavelength, the valves open asynchronously and the volume of the vessel segments bounded by two consecutive valves changes periodically, thereby inducing volumetric fluid pumping. The volumetric pumping leads to higher pumping flowrate and efficiency against an adverse pressure gradient. The optimum pumping occurs when the ratio of valve spacing to contraction wavelength is about2/3. This pumping regime is characterized by a longer period during which the valves are open. The results are useful for further understanding the pumping features of lymphatic system and provide insight into the design of biomimetic pumping devices.

Resolving the missing link between single platelet force and clot contractile force.

Blood clot contraction plays an important role in wound healing and hemostasis. Although clot contraction is known to be driven by platelets, how single platelet forces relate to the forces generated by macroscopic clots remains largely unknown. Using our microfabricated high-throughput platelet contraction cytometer, we find that single platelets have an average force of 34 nN ( n = 10 healthy individuals). However, multiple bulk clot experiments predict a mean single platelet force lower than 0.5 nN. To resolve this discrepancy, we use a mesoscale computational model to probe the mechanism by which individual platelets induce forces in macroscopic clots. Our experimentally informed model shows that the number of platelets in the clot cross-section defines the net clot force. We provide a relationship between single platelet force and the clot force that is useful for better understanding of blood disorders associated with bleeding and thrombosis, and facilitates the development of platelet-based and platelet-mimetic biomaterials.

Microfluidic transfection of mRNA into human primary lymphocytes and hematopoietic stem and progenitor cells using ultra-fast physical deformations.

Messenger RNA (mRNA) delivery provides gene therapy with the potential to achieve transient therapeutic efficacy without risk of insertional mutagenesis. Amongst other applications, mRNA can be employed as a platform to deliver gene editing molecules, to achieve protein expression as an alternative to enzyme replacement therapies, and to express chimeric antigen receptors (CARs) on immune cells for the treatment of cancer. We designed a novel microfluidic device that allows for efficient mRNA delivery via volume exchange for convective transfection (VECT). In the device, cells flow through a ridged channel that enforces a series of ultra-fast and large intensity deformations able to transiently open pores and induce convective transport of mRNA into the cell. Here, we describe efficient delivery of mRNA into T cells, natural killer (NK) cells and hematopoietic stem and progenitor cells (HSPCs), three human primary cell types widely used for ex vivo gene therapy applications. Results demonstrate that the device can operate at a wide range of cell and payload concentrations and that ultra-fast compressions do not have a negative impact on T cell function, making this a novel and competitive platform for the development of ex vivo mRNA-based gene therapies and other cell products engineered with mRNA.

Microfluidic Platform to Transduce Cell Viability to Distinct Flow Pathways for High-Accuracy Sensing.

Mechanical properties of cells such as stiffness can act as biomarkers to sort or detect cell functional properties such as viability. In this study, we report the use of a microfluidic device as a high-sensitivity sensor that transduces cell biomechanics to cell separation to accurately detect viability. Cell populations are flowed and deflected at a number of skew ridges such that deflection per ridge, cell-ridge interaction time, and cell size can all be used as sensor inputs to accurately determine the cell state. The angle of the ridges was evaluated to optimize the differences in cell translation between viable and nonviable cells while allowing continuous flow. In the first mode of operation, we flowed viable and nonviable cells through the device and conducted a sensitivity analysis by recording the cell's total deflection as a binary classifier that differentiates viable from nonviable cells. The performance of the sensor was assessed using an area under the curve (AUC) analysis to be 0.97. By including additional sensor inputs in the second mode of operation, we conducted a principal component analysis (PCA) to further improve the identification of the cell state by clustering populations with little overlap between viable and nonviable cells. We therefore found that microfluidic separation devices can be used to efficiently sort cells and accurately sense viability in a label-free manner.

Label-free microfluidic enrichment of cancer cells from non-cancer cells in ascites.

The isolation of a patient's metastatic cancer cells is the first, enabling step toward treatment of that patient using modern personalized medicine techniques. Whereas traditional standard-of-care approaches select treatments for cancer patients based on the histological classification of cancerous tissue at the time of diagnosis, personalized medicine techniques leverage molecular and functional analysis of a patient's own cancer cells to select treatments with the highest likelihood of being effective. Unfortunately, the pure populations of cancer cells required for these analyses can be difficult to acquire, given that metastatic cancer cells typically reside in fluid containing many different cell populations. Detection and analyses of cancer cells therefore require separation from these contaminating cells. Conventional cell sorting approaches such as Fluorescence Activated Cell Sorting or Magnetic Activated Cell Sorting rely on the presence of distinct surface markers on cells of interest which may not be known nor exist for cancer applications. In this work, we present a microfluidic platform capable of label-free enrichment of tumor cells from the ascites fluid of ovarian cancer patients. This approach sorts cells based on differences in biomechanical properties, and therefore does not require any labeling or other pre-sort interference with the cells. The method is also useful in the cases when specific surface markers do not exist for cells of interest. In model ovarian cancer cell lines, the method was used to separate invasive subtypes from less invasive subtypes with an enrichment of ~ sixfold. In ascites specimens from ovarian cancer patients, we found the enrichment protocol resulted in an improved purity of P53 mutant cells indicative of the presence of ovarian cancer cells. We believe that this technology could enable the application of personalized medicine based on analysis of liquid biopsy patient specimens, such as ascites from ovarian cancer patients, for quick evaluation of metastatic disease progression and determination of patient-specific treatment.

Fluid pumping of peristaltic vessel fitted with elastic valves.

Using numerical simulations, we probe the fluid flow in an axisymmetric peristaltic vessel fitted with elastic bi-leaflet valves. In this biomimetic system that mimics the flow generated in lymphatic vessels, we investigate the effects of the valve and vessel properties on pumping performance of the valved peristaltic vessel. The results indicate that valves significantly increase pumping by reducing backflow. The presence of valves, however, increases the viscous resistance therefore requiring greater work compared to valveless vessels. The benefit of the valves is the most significant when the fluid is pumped against an adverse pressure gradient and for low vessel contraction wave speeds. We identify the optimum vessel and valve parameters leading to the maximum pumping efficiency. We show that the optimum valve elasticity maximizes the pumping flow rate by allowing the valve to block more effectively the backflow while maintaining low resistance during the forward flow. We also examine the pumping in vessels where the vessel contraction amplitude is a function of the adverse pressure gradient as found in lymphatic vessels. We find that in this case the flow is limited by the work generated by the contracting vessel, suggesting that the pumping in lymphatic vessels is constrained by the performance of lymphatic muscle. Given the regional heterogeneity of valve morphology observed throughout the lymphatic vasculature, these results provide insight into how these variations might facilitate efficient lymphatic transport in the vessel's local physiologic context.

Platelet heterogeneity enhances blood clot volumetric contraction: An example of asynchrono-mechanical amplification.

Physiological processes such as blood clotting and wound healing as well as pathologies such as fibroses and musculoskeletal contractures, all involve biological materials composed of a contracting cellular population within a fibrous matrix, yet how the microscale interactions among the cells and the matrix lead to the resultant emergent behavior at the macroscale tissue level remains poorly understood. Platelets, the anucleate cell fragments that do not divide nor synthesize extracellular matrix, represent an ideal model to study such systems. During blood clot contraction, microscopic platelets actively pull fibers to shrink the macroscale clot to less than 10% of its initial volume. We discovered that platelets utilize a new emergent behavior, asynchrono-mechanical amplification, to enhanced volumetric material contraction and to magnify contractile forces. This behavior is triggered by the heterogeneity in the timing of a population of actuators. This result indicates that cell heterogeneity, often attributed to stochastic cell-to-cell variability, can carry an essential biophysical function, thereby highlighting the importance of considering 4 dimensions (space + time) in cell-matrix biomaterials. This concept of amplification via heterogeneity can be harnessed to increase mechanical efficiency in diverse systems including implantable biomaterials, swarm robotics, and active polymer composites.

The Benefits and Costs of Cybersecurity Risk Reduction: A Dynamic Extension of the Gordon and Loeb Model.

This article develops a dynamic extension of the classic model of cybersecurity investment formulated by Gordon and Loeb. In this dynamic model, results are influenced by the rate at which cybersecurity assets depreciate and the rate of return on investment. Depreciation costs are lower in the dynamic model than is implicitly assumed in the classic model, while the rate-of-return threshold is higher. On balance, the user cost of cybersecurity assets is lower in the dynamic model than is implicitly assumed in the classic model. This difference increases the economically efficient size of the cybersecurity system in value terms, increasing the efficient level of risk reduction.

Behavior and mechanics of dense microgel suspensions.

Suspensions of soft and highly deformable microgels can be concentrated far more than suspensions of hard colloids, leading to their unusual mechanical properties. Microgels can accommodate compression in suspensions in a variety of ways such as interpenetration, deformation, and shrinking. Previous experiments have offered insightful, but somewhat conflicting, accounts of the behavior of individual microgels in compressed suspensions. We develop a mesoscale computational model to probe the behavior of compressed suspensions consisting of microgels with different architectures at a variety of packing fractions and solvent conditions. We find that microgels predominantly change shape and mildly shrink above random close packing. Interpenetration is only appreciable above space filling, remaining small relative to the mean distance between cross-links. At even higher packing fractions, microgels solely shrink. Remarkably, irrespective of the single-microgel properties, and whether the suspension concentration is changed via changing the particle number density or the swelling state of the particles, which can even result in colloidal gelation, the mechanics of the suspension can be quantified in terms of the single-microgel bulk modulus, which thus emerges as the correct mechanical measure for these type of soft-colloidal suspensions. Our results rationalize the many and varied experimental results, providing insights into the relative importance of effects defining the mechanics of suspensions comprising soft particles.

Metachronal Actuation of Microscale Magnetic Artificial Cilia.

Biological cells often interact with the environment through carpets of microscopic hair-like cilia. These elastic structures are known to beat in a synchronized wavy fashion called metachronal motion to produce fluid transport. Metachronal motion emerges due to a phase difference between beating cycles of neighboring cilia and appears as traveling waves propagating along the ciliary carpet. We demonstrate submerged in water microscale magnetic cilia that are externally actuated to beat in a metachronal fashion. Two approaches are used to induce coordinated phase differences among the beating cilia. In the first case, we fabricate cilia with an imposed gradient of geometrical properties that are subject to a rotating uniform magnetic field. In the second scenario, a ciliary array is composed of identical cilia that experience a magnetic field that varies spatiotemporally. We demonstrate that magnetic cilia can achieve symplectic, antiplectic, and leoplectic metachrony.

Stiffness based enrichment of leukemia cells using microfluidics.

To improve the survival rate of cancer patients, new diagnosis strategies are necessary to detect lower levels of cancer cells before and after treatment regimens. The scarcity of diseased cells, particularly in residual disease after treatment, demands highly sensitive detection approaches or the ability to enrich the diseased cells in relation to normal cells. We report a label-free microfluidic approach to enrich leukemia cells from healthy cells using inherent differences in cell biophysical properties. The microfluidic device consists of a channel with an array of diagonal ridges that recurrently compress and translate flowing cells in proportion to cell stiffness. Using devices optimized for acute T cell leukemia model Jurkat, the stiffer white blood cells were translated orthogonally to the channel length, while softer leukemia cells followed hydrodynamic flow. The device enriched Jurkat leukemia cells from white blood cells with an enrichment factor of over 760. The sensitivity, specificity, and accuracy of the device were found to be > 0.8 . The values of sensitivity and specificity could be adjusted by selecting one or multiple outlets for analysis. We demonstrate that low levels of Jurkat leukemia cells (1 in 10 4 white blood cells) could be more quickly detected using flow cytometry by using the stiffness sorting pre-enrichment. In a second mode of operation, the device was implemented to sort resistive leukemia cells from both drug-sensitive leukemia cells and normal white blood cells. Therefore, microfluidic biomechanical sorting can be a useful tool to enrich leukemia cells that may improve downstream analyses.

Cell Mechanical and Physiological Behavior in the Regime of Rapid Mechanical Compressions that Lead to Cell Volume Change.

Cells respond to mechanical forces by deforming in accordance with viscoelastic solid behavior. Studies of microscale cell deformation observed by high speed video microscopy have elucidated a new cell behavior in which sufficiently rapid mechanical compression of cells can lead to transient cell volume loss and then recovery. This work has discovered that the resulting volume exchange between the cell interior and the surrounding fluid can be utilized for efficient, convective delivery of large macromolecules (2000 kDa) to the cell interior. However, many fundamental questions remain about this cell behavior, including the range of deformation time scales that result in cell volume loss and the physiological effects experienced by the cell. In this study, a relationship is established between cell viscoelastic properties and the inertial forces imposed on the cell that serves as a predictor of cell volume loss across human cell types. It is determined that cells maintain nuclear envelope integrity and demonstrate low protein loss after the volume exchange process. These results define a highly controlled cell volume exchange mechanism for intracellular delivery of large macromolecules that maintains cell viability and function for invaluable downstream research and clinical applications.

Phagocyte-Inspired Smart Microcapsules.

Phagocytes protect the organism by ingesting harmful foreign particles and cells. We use mesoscale computer simulations to design a phagocyte-inspired active microcapsule that is capable of selectively capturing nanoparticles dispersed in solvent. Our fully synthetic microdevice is actuated by a temperature-sensitive microgel enclosed inside a perforated spherical shell. The shell pores are decorated with a copolymer brush that regulates the transport of solutes into the capsule interior. When exposed to an external stimulus, the microgel swells, expanding through the shell pores to make contact with the nanoparticle-rich solution surrounding the capsule. Upon removal of the external stimulus, the gel retracts back into the shell, bringing along with it captured nanoparticles. We probe how periodic application of the stimulus combined with nanoparticle-microgel adhesion enable selectivity and enhance capturing efficiency of our nature-inspired microdevice.

Microfluidic generation of transient cell volume exchange for convectively driven intracellular delivery of large macromolecules.

Efficient intracellular delivery of target macromolecules remains a major obstacle in cell engineering and other biomedical applications. We discovered a unique cell biophysical phenomenon of transient cell volume exchange by using microfluidics to rapidly and repeatedly compress cells. This behavior consists of brief, mechanically induced cell volume loss followed by rapid volume recovery. We harness this behavior for high-throughput, convective intracellular delivery of large polysaccharides (2000 kDa), particles (100 nm), and plasmids while maintaining high cell viability. Successful proof of concept experiments in transfection and intracellular labeling demonstrated potential to overcome the most prohibitive challenges in intracellular delivery for cell engineering.

Extreme thermodynamics with polymer gel tori: Harnessing thermodynamic instabilities to induce large-scale deformations.

When a swollen, thermoresponsive polymer gel is heated in a solvent bath, it expels solvent and deswells. When this heating is slow, deswelling proceeds homogeneously, as observed in a toroid-shaped gel that changes volume while maintaining its toroidal shape. By contrast, if the gel is heated quickly, an impermeable layer of collapsed polymer forms and traps solvent within the gel, arresting the volume change. The ensuing evolution of the gel then happens at fixed volume, leading to phase separation and the development of inhomogeneous stress that deforms the toroidal shape. We observe that this stress can cause the torus to buckle out of the plane, via a mechanism analogous to the bending of bimetallic strips upon heating. Our results demonstrate that thermodynamic instabilities, i.e., phase transitions, can be used to actuate mechanical deformation in an extreme thermodynamics of materials.

Probing the effect of morphology on lymphatic valve dynamic function.

The lymphatic system is vital to the circulatory and immune systems, performing a range of important functions such as transport of interstitial fluid, fatty acid, and immune cells. Lymphatic vessels are composed of contractile walls and lymphatic valves, allowing them to pump lymph against adverse pressure gradients and to prevent backflow. Despite the importance of the lymphatic system, the contribution of mechanical and geometric changes of lymphatic valves and vessels in pathologies of lymphatic dysfunction, such as lymphedema, is not well understood. We develop a fully coupled fluid-solid, three-dimensional computational model to interrogate the various parameters thought to influence valve behavior and the consequences of these changes to overall lymphatic function. A lattice Boltzmann model is used to simulate the lymph, while a lattice spring model is used to model the mechanics of lymphatic valves. Lymphatic valve functions such as enabling lymph flow and preventing backflow under varied lymphatic valve geometries and mechanical properties are investigated to provide an understanding of the function of lymphatic vessels and valves. The simulations indicate that lymphatic valve function is optimized when valves are of low aspect ratio and bending stiffness, so long as these parameters are maintained at high enough values to allow for proper valve closing. This suggests that valve stiffening could have a profound effect on overall lymphatic pumping performance. Furthermore, dynamic valve simulations showed that this model captures the delayed response of lymphatic valves to dynamic flow conditions, which is an essential feature of valve operation. Thus, our model enhances our understanding of how lymphatic pathologies, specifically those exhibiting abnormal valve morphologies such as has been suggested to occur in cases of primary lymphedema, can lead to lymphatic dysfunctions.

Metachronal motion of artificial magnetic cilia.

Organisms use hair-like cilia that beat in a metachronal fashion to actively transport fluid and suspended particles. Metachronal motion emerges due to a phase difference between beating cycles of neighboring cilia and appears as traveling waves propagating along ciliary carpet. In this work, we demonstrate biomimetic artificial cilia capable of metachronal motion. The cilia are micromachined magnetic thin filaments attached at one end to a substrate and actuated by a uniform rotating magnetic field. We show that the difference in magnetic cilium length controls the phase of the beating motion. We use this property to induce metachronal waves within a ciliary array and explore the effect of operation parameters on the wave motion. The metachronal motion in our artificial system is shown to depend on the magnetic and elastic properties of the filaments, unlike natural cilia, where metachronal motion arises due to fluid coupling. Our approach enables an easy integration of metachronal magnetic cilia in lab-on-a-chip devices for enhanced fluid and particle manipulations.