An inexpensive "do-it-yourself" device for rapid generation of uniform tumor spheroids.

Three-dimensional (3D) cancer cell culture models such as tumor spheroids better recapitulate in vivo tumors than conventional two-dimensional (2D) models. However, two major challenges limit the routine use of 3D tumor spheroids. Firstly, most existing methods of generating tumor spheroids are not high-throughput. Secondly, tumor spheroids generated using current methods are highly variable in dimension. Here, we describe a simple 'Do-It-Yourself (DIY)' device that can be assembled for less than $7 of parts and generate uniform tumor spheroids in a high-throughput manner. We used a simple phone coin vibrating motor to superimpose the vibration for breaking a laminar jet of cell-loaded alginate solution into equally sized spherical beads. We generated 3,970 tumor spheroids/min, which exhibited a hypoxic core recapitulating in vivo tumors and could be used to test the diffusion efficacy of anticancer drugs. Such low-cost, easy-to-fabricate, simple-to-operate systems with high-throughput outcomes are essential to democratize and standardize cancer research.

An architecturally rational hemostat for rapid stopping of massive bleeding on anticoagulation therapy.

Hemostatic devices are critical for managing emergent severe bleeding. With the increased use of anticoagulant therapy, there is a need for next-generation hemostats. We rationalized that a hemostat with an architecture designed to increase contact with blood, and engineered from a material that activates a distinct and undrugged coagulation pathway can address the emerging need. Inspired by lung alveolar architecture, here, we describe the engineering of a next-generation single-phase chitosan hemostat with a tortuous spherical microporous design that enables rapid blood absorption and concentrated platelets and fibrin microthrombi in localized regions, a phenomenon less observed with other classical hemostats without structural optimization. The interaction between blood components and the porous hemostat was further amplified based on the charged surface of chitosan. Contrary to the dogma that chitosan does not directly affect physiological clotting mechanism, the hemostat induced coagulation via a direct activation of platelet Toll-like receptor 2. Our engineered porous hemostat effectively stopped the bleeding from murine liver wounds, swine liver and carotid artery injuries, and the human radial artery puncture site within a few minutes with significantly reduced blood loss, even under the anticoagulant treatment. The integration of engineering design principles with an understanding of the molecular mechanisms can lead to hemostats with improved functions to address emerging medical needs.

Engineered cell-laden alginate microparticles for 3D culture.

Advanced microfabrication technologies and biocompatible hydrogel materials facilitate the modeling of 3D tissue microenvironment. Encapsulation of cells in hydrogel microparticles offers an excellent high-throughput platform for investigating multicellular interaction with their surrounding microenvironment. Compartmentalized microparticles support formation of various unique cellular structures. Alginate has emerged as one of the most dominant hydrogel materials for cell encapsulation owing to its cytocompatibility, ease of gelation, and biocompatibility. Alginate hydrogel provides a permeable physical boundary to the encapsulated cells and develops an easily manageable 3D cellular structure. The interior structure of alginate hydrogel can further regulate the spatiotemporal distribution of the embedded cells. This review provides a specific overview of the representative engineering approaches to generate various structures of cell-laden alginate microparticles in a uniform and reproducible manner. Capillary nozzle systems, microfluidic droplet systems, and non-chip based high-throughput microfluidic systems are highlighted for developing well-regulated cellular structure in alginate microparticles to realize potential drug screening platform and cell-based therapy. We conclude with the discussion of current limitations and future directions for realizing the translation of this technology to the clinic.

Assessment of transient changes in oxygen diffusion of single red blood cells using a microfluidic analytical platform.

Red blood cells (RBCs) capability to deliver oxygen (O2) has been routinely measured by P50. Although this defines the ability of RBCs to carry O2 under equilibrium states, it cannot determine the efficacy of O2 delivery in dynamic blood flow. Here, we developed a microfluidic analytical platform (MAP) that isolates single RBCs for assessing transient changes in their O2 release rate. We found that in vivo (biological) and in vitro (blood storage) aging of RBC could lead to an increase in the O2 release rate, despite a decrease in P50. Rejuvenation of stored RBCs (Day 42), though increased the P50, failed to restore the O2 release rate to basal level (Day 0). The temporal dimension provided at the single-cell level by MAP could shed new insights into the dynamics of O2 delivery in both physiological and pathological conditions.

Human Nonalcoholic Steatohepatitis on a Chip.

Nonalcoholic steatohepatitis (NASH), an advanced stage of nonalcoholic fatty liver disease (NAFLD), is a rapidly growing and global health problem compounded by the current absence of specific treatments. A major limiting factor in the development of new NASH therapies is the absence of models that capture the unique cellular structure of the liver microenvironment and recapitulate the complexities of NAFLD progression to NASH. Organ-on-a-chip platforms have emerged as a powerful approach to dynamically model diseases and test drugs. Herein, we describe a NASH-on-a-chip platform. Four main types of human primary liver cells (hepatocytes [HCs], Kupffer cells, liver sinusoidal endothelial cells, and hepatic stellate cells [HSCs]) were cocultured under microfluidic dynamics. Our chip-based model successfully recapitulated a functional liver cellular microenvironment with stable albumin and urea secretion for at least 2 weeks. Exposing liver chips to a lipotoxic environment led to gradual development of NASH phenotypic characteristics, including intracellular lipid accumulation, hepatocellular ballooning, HSC activation, and elevation of inflammatory and profibrotic markers. Further, exposure of the chip to elafibranor, a drug under study for the therapy of NASH, inhibited the development of NASH-specific hallmarks, causing an ~8-fold decrease in intracellular lipids, a 3-fold reduction in number of ballooned HCs, a significant reduction in HSC activation, and a significant decrease in the levels of inflammatory and profibrotic markers compared with controls. Conclusion: We have successfully developed a microfluidic NASH-on-a-chip platform that recapitulates the main NASH histologic endpoints in a single chip and that can emerge as a powerful noninvasive, human-relevant, in vitro platform to study disease pathogenesis and develop novel anti-NASH drugs.

Engineering in Medicine To Address the Challenge of Cancer Drug Resistance: From Micro- and Nanotechnologies to Computational and Mathematical Modeling.

Drug resistance has profoundly limited the success of cancer treatment, driving relapse, metastasis, and mortality. Nearly all anticancer drugs and even novel immunotherapies, which recalibrate the immune system for tumor recognition and destruction, have succumbed to resistance development. Engineers have emerged across mechanical, physical, chemical, mathematical, and biological disciplines to address the challenge of drug resistance using a combination of interdisciplinary tools and skill sets. This review explores the developing, complex, and under-recognized role of engineering in medicine to address the multitude of challenges in cancer drug resistance. Looking through the "lens" of intrinsic, extrinsic, and drug-induced resistance (also referred to as "tolerance"), we will discuss three specific areas where active innovation is driving novel treatment paradigms: (1) nanotechnology, which has revolutionized drug delivery in desmoplastic tissues, harnessing physiochemical characteristics to destroy tumors through photothermal therapy and rationally designed nanostructures to circumvent cancer immunotherapy failures, (2) bioengineered tumor models, which have benefitted from microfluidics and mechanical engineering, creating a paradigm shift in physiologically relevant environments to predict clinical refractoriness and enabling platforms for screening drug combinations to thwart resistance at the individual patient level, and (3) computational and mathematical modeling, which blends in silico simulations with molecular and evolutionary principles to map mutational patterns and model interactions between cells that promote resistance. On the basis that engineering in medicine has resulted in discoveries in resistance biology and successfully translated to clinical strategies that improve outcomes, we suggest the proliferation of multidisciplinary science that embraces engineering.

Vibration motor-integrated low-cost, miniaturized system for rapid quantification of red blood cell aggregation.

Human red blood cells (RBCs) aggregate under low shear conditions, which significantly modulates flow resistance and tissue perfusion. A higher aggregation tendency in blood thus serves as an important clinical indicator for the screening of cardiovascular disorders. Conventional ways of measuring RBC aggregation still require large sample volumes, cumbersome manual procedures, and expensive benchtop systems. These inconvenient and high-cost measurement methods hamper their clinical applicability. Here, we propose a low-cost, miniaturized system to overcome the limitations of these methods. Our system utilizes a coin vibration motor (CVM) to generate a localized vortex for disaggregating RBCs in a disposable fluidic chip. The design of the chip was optimized with fluid dynamics simulations to ensure sufficient shear flow in the localized vortex for RBC disaggregation. The time-dependent increase in light transmittance from an LED light source through the plasma gap while the RBCs re-aggregate is captured with a CMOS camera under stasis conditions to quantify the level of RBC aggregation. Our CVM-based aggregometer was validated against a commercial benchtop system for human blood samples under physiological and pathological conditions, and showed an excellent performance with a high intraclass correlation coefficient of 0.995. In addition, we were able to achieve a rapid measurement (<4 min) with the CVM-based aggregometer, requiring only a 6 µl blood sample. These illustrate the potential of our CVM-based aggregometer for low-cost point-of-care diagnostics without compromising the measurement sensitivity.

Quantum biological tunnel junction for electron transfer imaging in live cells.

Quantum biological electron transfer (ET) essentially involves in virtually all important biological processes such as photosynthesis, cellular respiration, DNA repair, cellular homeostasis, and cell death. However, there is no real-time imaging method to capture biological electron tunnelling in live cells to date. Here, we report a quantum biological electron tunnelling (QBET) junction and its application in real-time optical detection of QBET and the dynamics of ET in mitochondrial cytochrome c during cell life and death process. QBET junctions permit to see the behaviours of electron tunnelling through barrier molecules with different barrier widths. Using QBET spectroscopy, we optically capture real-time ET in cytochrome c redox dynamics during cellular apoptosis and necrosis in living cells. The non-invasive real-time QBET spectroscopic imaging of ET in live cell open a new era in life sciences and medicine by providing a way to capture spatiotemporal ET dynamics and to reveal the quantum biological mechanisms.

Near-Wall Migration Dynamics of Erythrocytes in Vivo: Effects of Cell Deformability and Arteriolar Bifurcation.

Red blood cell (RBC) deformability has a significant impact on microcirculation by affecting cell dynamics. Despite previous studies that have demonstrated the margination of rigid cells and particles in vitro, little information is available on the in vivo margination of deformability-impaired RBCs under physiological flow and hematocrit conditions. Thus, in this study, we examined how the deformability-dependent, RBC migration alters the cell distribution under physiological conditions, particularly in arteriolar network flows. The hardened RBCs (hRBCs) were found to preferentially flow near the vessel walls of small arterioles (diameter = 47.1-93.3 µm). The majority of the hRBCs (63%) were marginated within the range of 0.7R-0.9R (R: radial position normalized by vessel radius), indicating that the hRBCs preferentially accumulated near the vessel walls. The laterally marginated hRBCs maintained their lateral positions near the walls while traversing downstream with attenuated radial dispersion. In addition, the immediate displacement of RBCs while traversing a bifurcation also contributes to the near-wall accumulation of hRBCs. The notable difference in the inward migration between the marginated nRBCs and hRBCs after bifurcations further supports the potential role of bifurcations in the accumulation of hRBCs near the walls.

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle.

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

Biomimetic Precapillary Flow Patterns for Enhancing Blood Plasma Separation: A Preliminary Study.

In this study, a biomimetic microfluidic plasma separation device is discussed. The design of the device drew inspiration from in vivo observations of enhanced cell-free layer (CFL) formation downstream of vascular bifurcations. The working principle for the plasma separation was based on the plasma skimming effect in an arteriolar bifurcation, which is modulated by CFL formation. The enhancement of the CFL width was achieved by a local hematocrit reduction near the collection channel by creating an uneven hematocrit distribution at the bifurcation of the channel. The device demonstrated a high purity of separation (~99.9%) at physiological levels of hematocrit (~40%).

Symmetry recovery of cell-free layer after bifurcations of small arterioles in reduced flow conditions: effect of RBC aggregation.

Heterogeneous distribution of red blood cells (RBCs) in downstream vessels of arteriolar bifurcations can be promoted by an asymmetric formation of cell-free layer (CFL) in upstream vessels. Consequently, the CFL widths in subsequent downstream vessels become an important determinant for tissue oxygenation (O2) and vascular tone change by varying nitric oxide (NO) availability. To extend our previous understanding on the formation of CFL in arteriolar bifurcations, this study investigated the formation of CFL widths from 2 to 6 vessel-diameter (2D-6D) downstream of arteriolar bifurcations in the rat cremaster muscle (D = 51.5 ± 1.3 µm). As the CFL widths are highly influenced by RBC aggregation, the degree of aggregation was adjusted to simulate levels seen during physiological and pathological states. Our in vivo experimental results showed that the asymmetry of CFL widths persists along downstream vessels up to 6D from the bifurcating point. Moreover, elevated levels of RBC aggregation appeared to retard the recovery of CFL width symmetry. The required length of complete symmetry recovery was estimated to be greater than 11D under reduced flow conditions, which is relatively longer than interbifurcation distances of arterioles for vessel diameter of ∼50 µm. In addition, our numerical prediction showed that the persistent asymmetry of CFL widths could potentially result in a heterogeneous vasoactivity over the entire arteriolar network in such abnormal flow conditions.

Erythrocyte aggregation may promote uneven spatial distribution of NO/O2 in the downstream vessel of arteriolar bifurcations.

This study examined the effect of red blood cell (RBC) aggregation on nitric oxide (NO) and oxygen (O2) distributions in the downstream vessels of arteriolar bifurcations. Particular attention was paid to the inherent formation of asymmetric cell-free layer (CFL) widths in the downstream vessels and its consequential impact on the NO/O2 bioavailability after the bifurcations. A microscopic image-based two-dimensional transient model was used to predict the NO/O2 distribution by utilizing the in vivo CFL width data obtained under non-, normal- and hyper-aggregating conditions at the pseudoshear rate of 15.6±2.0s(-1). In vivo experimental result showed that the asymmetry of CFL widths was enhanced by the elevation in RBC aggregation level. The model demonstrated that NO bioavailability was regulated by the dynamic fluctuation of the local CFL widths, which is corollary to its modulation of wall shear stress. Accordingly, the uneven distribution of NO/O2 was prominent at opposite sides of the arterioles up to six vessel-diameter (6D) away from the bifurcating point, and this was further enhanced by increasing the levels of RBC aggregation. Our findings suggested that RBC aggregation potentially augments both the formation of asymmetric CFL widths and its influence on the uneven distribution of NO/O2 in the downstream flow of an arteriolar bifurcation. The extended heterogeneity of NO/O2 downstream (2D-6D) also implied its potential propagation throughout the entire arteriolar microvasculature.

Alteration of Blood Flow in a Venular Network by Infusion of Dextran 500: Evaluation with a Laser Speckle Contrast Imaging System.

This study examined the effect of dextran-induced RBC aggregation on the venular flow in microvasculature. We utilized the laser speckle contrast imaging (LSCI) as a wide-field imaging technique to visualize the flow distribution in venules influenced by abnormally elevated levels of RBC aggregation at a network-scale level, which was unprecedented in previous studies. RBC aggregation in rats was induced by infusing Dextran 500. To elucidate the impact of RBC aggregation on microvascular perfusion, blood flow in the venular network of a rat cremaster muscle was analyzed with a stepwise reduction of the arterial pressure (100 → 30 mmHg). The LSCI analysis revealed a substantial decrease in the functional vascular density after the infusion of dextran. The relative decrease in flow velocity after dextran infusion was notably pronounced at low arterial pressures. Whole blood viscosity measurements implied that the reduction in venular flow with dextran infusion could be due to the elevation of medium viscosity in high shear conditions (> 45 s-1). In contrast, further augmentation to the flow reduction at low arterial pressures could be attributed to the formation of RBC aggregates (< 45 s-1). This study confirmed that RBC aggregation could play a dominant role in modulating microvascular perfusion, particularly in the venular networks.

Microfluidic device for sheathless particle focusing and separation using a viscoelastic fluid.

Continuous sheathless particle separation with high efficiency is essential for various applications such as biochemical analyses and clinical diagnosis. Here, a novel microfluidic device for highly efficient, sheathless particle separation using an elasticity-dominant non-Newtonian fluid is proposed. Our device consists of two stages: sheathless three-dimensional focusing (1 st stage) and separation (2nd stage). It is designed based on the principle of a viscoelasticity-induced particle lateral migration, which promises precise separation of particles in a microfluidic device. Particles of 5- and 10-µm diameters were all focused at the centerline of a circular channel at the 1st stage and successfully separated at the 2nd stage with an efficiency of ∼99.9% using size-based lateral migration of particles induced by the viscoelasticity of the medium. We also demonstrated the capability of our device for separation of blood cells into multiple fractions. The tunability of separable particle size could be achieved by changing the viscoelastic property of the medium and flow rate.

Two-dimensional transient model for prediction of arteriolar NO/O2 modulation by spatiotemporal variations in cell-free layer width.

Despite the significant roles of the cell-free layer (CFL) in balancing nitric oxide (NO) and oxygen (O2) bioavailability in arteriolar tissue, many previous numerical approaches have relied on a one-dimensional (1-D) steady-state model for simplicity. However, these models are unable to demonstrate the influence of spatiotemporal variations in the CFL on the NO/O2 transport under dynamic flow conditions. Therefore, the present study proposes a new two-dimensional (2-D) transient model capable of predicting NO/O2 transport modulated by the spatiotemporal variations in the CFL width. Our model predicted that NO bioavailability was inversely related to the CFL width as expected. The enhancement of NO production by greater wall shear stress with a thinner CFL could dominate the diffusion barrier role of the CFL. In addition, NO/O2 availability along the vascular wall was inhomogeneous and highly regulated by dynamic changes of local CFL width variation. The spatial variations of CFL widths on opposite sides of the arteriole exhibited a significant inverse relation. This asymmetric formation of CFL resulted in a significantly imbalanced NO/O2 bioavailability on opposite sides of the arteriole. The novel integrative methodology presented here substantially highlighted the significance of spatiotemporal variations of the CFL in regulating the bioavailability of NO/O2, and provided further insight about the opposing effects of the CFL on arteriolar NO production.

Influence of erythrocyte aggregation at pathological levels on cell-free marginal layer in a narrow circular tube.

Human red blood cells (RBCs) were perfused in a circular micro-tube (inner diameter of 25 µm) to examine the dynamic changes of cell-free marginal region at both physiological (normal) and pathophysiological (hyper) levels of RBC aggregation. The cell-free area (CFA) was measured to provide additional information on the cell-free layer (CFL) width changes in space and time domains. A prominent enhancement in the mean CFL width was found in hyper-aggregating conditions as compared to that in non-aggregating conditions (P <  0.001). The frequent contacts between RBC and the tube wall were observed and the contact frequency was greatly decreased when the aggregation level was increased from none to normal (P <  0.05) and to hyper (P <  0.001) levels. In addition, the enhanced aggregation from none to hyper levels significantly enlarged the CFA (P <  0.01). We concluded that the RBC aggregation at pathophysiological levels could promote not only the CFL width (one-dimensional parameter) but also the spatiotemporal variation of CFA (two-dimensional parameter).

Effect of erythrocyte aggregation at pathological levels on NO/O2 transport in small arterioles.

This study examined the effects of red blood cell (RBC) aggregation at pathological levels on NO/O2 transport in small arterioles. Transient gas diffusion simulations were performed with in vivo cell-free layer (CFL) widths data obtained from arteriolar flows in the rat cremaster muscle. The CFL data were measured at physiological and pathological levels of aggregation under reduced flow conditions (pseudoshear rate = 31.4 ± 10.5 s-1). Our results showed that the mean peak NO concentration significantly decreased with increasing the aggregation level from non-aggregating to normal-aggregating (P < 0.05) and to hyper-aggregating (P < 0.01) conditions. In contrast, the partial O2 pressure (PO2) in pathological aggregating conditions significantly increased from those under non-aggregating (P < 0.001) and normal-aggregating (P < 0.05) conditions. Although the NO scavenging by RBCs could be impaired with a thicker CFL at higher levels of aggregation, the overall decrease in NO production due to reduction of wall shear stress with the thicker CFL dominantly limited the NO availability in tissue. On the other hand, the O2 availability in tissue increased due to the relatively high core hematocrit in the blood lumen with the thicker CFL.

A review of numerical methods for red blood cell flow simulation.

In this review, we provide an overview of the simulation techniques employed for modelling the flow of red blood cells (RBCs) in blood plasma. The scope of this review omits the fluid modelling aspect while focusing on other key components in the RBC-plasma model such as (1) describing the RBC deformation with shell-based and spring-based RBC models, (2) constitutive models for RBC aggregation based on bridging theory and depletion theory and (3) additional strategies required for completing the RBC-plasma flow model. These include topics such as modelling fluid-structure interaction with the immersed boundary method and boundary integral method, and updating the variations in multiphase fluid property through the employment of index field methods. Lastly, we summarily discuss the current state and aims of RBC modelling and suggest some research directions for the further development of this field of modelling.

Hybrid capillary-inserted microfluidic device for sheathless particle focusing and separation in viscoelastic flow.

A novel microfluidic device which consists of two stages for particle focusing and separation using a viscoelastic fluid has been developed. A circular capillary tube was used for three-dimensional particle pre-alignment before the separation process, which was inserted in a polydimethylsiloxane microchannel. Particles with diameters of 5 and 10 µm were focused at the centerline in the capillary tube, and the location of particles was initialized at the first bifurcation. Then, 5 and 10 µm particles were successfully separated in the expansion region based on size-dependent lateral migration, with ∼99% separation efficiency. The proposed device was further applied to separation of MCF-7 cells from leukocytes. Based on the cell size distribution, an approximate size cutoff for separation was determined to be 16 µm. At 200 µl/min, 94% of MCF-7 cells were separated with the purity of ∼97%. According to the trypan blue exclusion assay, high viability (∼90%) could be achieved for the separated MCF-7 cells. The use of a commercially available capillary tube enables the device to be highly versatile in dealing with particles in a wide size range by using capillary tubes with different inner diameters.