Nematic Fibrin Fibers Enabling Vascularized Thrombus Implants Facilitate Scarless Cutaneous Wound Healing.

Autologous implantable scaffolds that induce vasculogenesis have shown great potential in tissue regeneration; however, previous attempts mainly relied on cell-laden hydrogel patches using fat tissues or platelet-rich plasma, which are insufficient for generating a uniform vasculature in a scalable manner. Here, implantable vascularized engineered thrombi (IVETs) are presented using autologous whole blood, which potentiate effective skin wound healing by constructing robust microcapillary vessel networks at the wound site. Microfluidic shear stresses enable the alignment of bundled fibrin fibers along the direction of the blood flow streamlines and the activation of platelets, both of which offer moderate stiffness of the microenvironment optimal for facilitating endothelial cell maturation and vascularization. Rodent dorsal skin wounds patched with IVET present superior wound closure rates (96.08 ± 1.58%), epidermis thickness, collagen deposition, hair follicle numbers, and neutrophil infiltration, which are permitted by enhanced microvascular circulation. Moreover, IVET treatment accelerates wound healing by recruiting M2 phenotype macrophages.

Human Cell-Camouflaged Nanomagnetic Scavengers Restore Immune Homeostasis in a Rodent Model with Bacteremia.

Bloodstream infection caused by antimicrobial resistance pathogens is a global concern because it is difficult to treat with conventional therapy. Here, scavenger magnetic nanoparticles enveloped by nanovesicles derived from blood cells (MNVs) are reported, which magnetically eradicate an extreme range of pathogens in an extracorporeal circuit. It is quantitatively revealed that glycophorin A and complement receptor (CR) 1 on red blood cell (RBC)-MNVs predominantly capture human fecal bacteria, carbapenem-resistant (CR) Escherichia  coli, and extended-spectrum beta-lactamases-positive (ESBL-positive) E. coli, vancomycin-intermediate Staphylococcus aureus (VISA), endotoxins, and proinflammatory cytokines in human blood. Additionally, CR3 and CR1 on white blood cell-MNVs mainly contribute to depleting the virus envelope proteins of Zika, SARS-CoV-2, and their variants in human blood. Supplementing opsonins into the blood significantly augments the pathogen removal efficiency due to its combinatorial interactions between pathogens and CR1 and CR3 on MNVs. The extracorporeal blood cleansing enables full recovery of lethally infected rodent animals within 7 days by treating them twice in series. It is also validated that parameters reflecting immune homeostasis, such as blood cell counts, cytokine levels, and transcriptomics changes, are restored in blood of the fatally infected rats after treatment.

Enhanced Diamagnetic Repulsion of Blood Cells Enables Versatile Plasma Separation for Biomarker Analysis in Blood.

A hemolysis-free and highly efficient plasma separation platform enabled by enhanced diamagnetic repulsion of blood cells in undiluted whole blood is reported. Complete removal of blood cells from blood plasma is achieved by supplementing blood with superparamagnetic iron oxide nanoparticles (SPIONs), which turns the blood plasma into a paramagnetic condition, and thus, all blood cells are repelled by magnets. The blood plasma is successfully collected from 4 mL of blood at flow rates up to 100 µL min-1 without losing plasma proteins, platelets, or exosomes with 83.3±1.64% of plasma volume recovery, which is superior over the conventional microfluidic methods. The theoretical model elucidates the diamagnetic repulsion of blood cells considering hematocrit-dependent viscosity, which allows to determine a range of optimal flow rates to harvest platelet-rich plasma and platelet-free plasma. For clinical validations, it is demonstrated that the method enables the greater recovery of bacterial DNA from the infected blood than centrifugation and the immunoassay in whole blood without prior plasma separation.

An inflammatory vascular endothelium-mimicking microfluidic device to enable leukocyte rolling and adhesion for rapid infection diagnosis.

Recruitment of circulating leukocytes to sites of infection is of utmost importance in the development, propagation, and outcome of sepsis. These multi-step processes are mediated by interactions between adhesion receptors of leukocytes and cell adhesion molecules (CAMs) of endothelial cells, such as P-selectin, E-selectin and ICAM-1. However, the potential utility of the CAMs-facilitated leukocyte capture has not been thoroughly investigated as an index of the host response to infection for diagnostic purposes. Here, we report that the systemic infection affects the expression of CAMs ligands on leukocytes, upregulating the expression of P-selectin ligand-1 (PSGL-1) and increasing the number of PSGL-1- and E-selectin ligand-1 (ESL-1)-expressing leukocyte levels in septic blood. We leveraged this finding to determine infection by measuring the increased adhesion of leukocytes to an inflammatory vascular endothelium-mimicking microchannel coated with CAMs. We successfully validated that the proposed method can significantly differentiate infection in bacteremia and endotoxemia models in rats as early as an hour post-infection using a finger-prick volume of blood (50 µL), which were unachievable with the conventional diagnostic methods.

Measurement of the magnetic susceptibility of subtle paramagnetic solutions using the diamagnetic repulsion of polymer microparticles.

Herein, we described a microfluidic device that enabled the measurement of magnetic susceptibility of a subtle paramagnetic solution that could not be determined by conventional magnetic susceptibility measurement methods such as a superconducting quantum interference device (SQUID). We measured the diamagnetic repelling velocity of polystyrene microparticles suspended in a weak paramagnetic solution including 50-150 mM of gadolinium-diethylenetriamine penta-acetic acid (GD-DTPA) and superparamagnetic iron oxide nanoparticles (SPION) (10 nm in diameter) dispersed in distilled water at various concentrations. The measured diamagnetic repelling velocities correlated with our prediction and also with the magnetic susceptibility of the SPION solution measured by a SQUID magnetometer. Via this approach, we could determine the magnetic susceptibility of the subtle paramagnetic solution of GD-DTPA that could not be measured via the conventional method. This study provides us with the new capability of analyzing weakly paramagnetic solutions such as paramagnetic components or metal contaminants present in environmental liquid samples at low concentrations.

Vertically sheathing laminar flow-based immunoassay using simultaneous diffusion-driven immune reactions.

We present that enhanced simultaneous incubation of multiple antibodies (Abs) can be achieved by exploiting microfluidic laminar flows and difference in diffusivity between primary Ab (pAb) and secondary Ab (sAb). We demonstrate that injecting Ab of larger and smaller diffusivity (D Ab) in an upper and lower laminar flow over the analyte-coated bottom surface, respectively, would result in enhanced signal intensity in the given reaction time. To prove this, we simultaneously infused anti-prostate specific antigen (PSA) pAb (upper laminar flow) and quantum dot (QD) labeled secondary Ab (QD-sAb) (lower laminar flow) to generate two Ab laminar flows vertically sheathing each other in the microfluidic device in which PSA was immobilized on the glass bottom surface. Because of the larger D Ab of pAb than that of QD-sAb due to the heavy metal components of QD, anti-PSA pAb diffuses more rapidly toward the bottom surface where the immune reaction between PSA, pAb, and QD-sAb instantaneously occurs. We corroborated our principle by switching the position of the two Ab laminar flows (QD-sAb in upper and pAb in lower laminar flows) in the channel, which resulted in significantly lower intensity of QD signals than the previous method. Moreover, when we adjusted the interface of pAb and QD-sAb in upper and lower laminar flows, respectively, closer toward the bottom surface, the fluorescence signal was even more intensified. This is attributed to the increased flux of anti-PSA pAb more adjacent to the reaction site, which, in turn, enhances the binding efficiency of pAb to PSA on the surface.

Microfluidic on-chip immunohistochemistry directly from a paraffin-embedded section.

We present here a novel microfluidic platform that can perform microfluidic on-chip immunohistochemistry (IHC) processes on a formalin-fixed paraffin-embedded section slide. Unlike previous microfluidic IHC studies, our microfluidic chip made of organic solvent-resistant polyurethane acrylate (PUA) is capable of conducting on-chip IHC processes consecutively. A narrow channel wall structure of the PUA chip shows effective sealing by pressure-based reversible assembly with a section slide. We performed both on-chip IHC and conventional IHC processes and compared the IHC results based on the immunostaining intensity. The result showed that the effects of the on-chip deparaffinization, antigen retrieval, and immunoreaction processes on the IHC result were equivalent to conventional methods while reducing the total process time to less than 1/2. The experiment with breast cancer tissue shows that human epidermal growth factor receptor 2 (HER2) classification can be performed by obtaining a clearly distinguishable immunostaining intensity according to the HER2 expression level. We expect our on-chip microfluidic platform to provide a facile technique suitable for miniaturized, automated, and precise diagnostic devices, including a point-of-care device.

Advection Flows-Enhanced Magnetic Separation for High-Throughput Bacteria Separation from Undiluted Whole Blood.

A major challenge to scale up a microfluidic magnetic separator for extracorporeal blood cleansing applications is to overcome low magnetic drag velocity caused by viscous blood components interfering with magnetophoresis. Therefore, there is an unmet need to develop an effective method to position magnetic particles to the area of augmented magnetic flux density gradients while retaining clinically applicable throughput. Here, a magnetophoretic cell separation device, integrated with slanted ridge-arrays in a microfluidic channel, is reported. The slanted ridges patterned in the microfluidic channels generate spiral flows along the microfluidic channel. The cells bound with magnetic particles follow trajectories of the spiral streamlines and are repeatedly transferred in a transverse direction toward the area adjacent to a ferromagnetic nickel structure, where they are exposed to a highly augmented magnetic force of 7.68 µN that is much greater than the force (0.35 pN) at the side of the channel furthest from the nickel structure. With this approach, 91.68% ± 2.18% of Escherichia coli (E. coli) bound with magnetic nanoparticles are successfully separated from undiluted whole blood at a flow rate of 0.6 mL h-1 in a single microfluidic channel, whereas only 23.98% ± 6.59% of E. coli are depleted in the conventional microfluidic device.

A Microfluidic Immunostaining System Enables Quality Assured and Standardized Immunohistochemical Biomarker Analysis.

Immunohistochemistry (IHC) plays an important role in biomarker-driven cancer therapy. Although there has been a high demand for standardized and quality assured IHC, it has rarely been achieved due to the complexity of IHC testing and the subjective validation-based process flow of IHC quality control. We present here a microfluidic immunostaining system for the standardization of IHC by creating a microfluidic linearly graded antibody (Ab)-staining device and a reference cell microarray. Unlike conventional efforts, our system deals primarily with the screening of biomarker staining conditions for quantitative quality assurance testing in IHC. We characterized the microfluidic matching of Ab staining intensity using three HER2 Abs produced by different manufacturers. The quality of HER2 Ab was also validated using tissues of breast cancer patients, demonstrating that our system is an efficient and powerful tool for the standardization and quality assurance of IHC.

Pipetting-driven microfluidic immunohistochemistry to facilitate enhanced immunoreaction and effective use of antibodies.

Immunohistochemistry (IHC), which has been used to detect antigens in cells of a tissue section using an immunoreaction between an antibody and an antigen, is a practical tool for identifying the type and stage of diseases in cancer diagnosis and scientific research. However, conventional IHC requires long, laborious process times and high costs. Although microfluidic IHC platforms have been developed to overcome these limitations, the application of microfluidic IHC in real-world environments is still limited due to the additional equipment needed to operate the microfluidic systems. In addition, continuous flow in a microfluidic channel leads to a waste of unbound antibodies. In this study, we demonstrate a novel and easy-to-use microfluidic IHC platform operated only using a manual pipette that is commonly available in research laboratories or hospitals. No other device such as a pump or a controller is required to operate our system. Bidirectional flows of the antibody solution in a microfluidic device are induced by repetitive manual pipetting which facilitates the enhanced antigen-antibody reaction and enables the effective use of a limited amount of antibody. When breast cancer cell and tissue sections are reacted with antibodies using our platform, pipetting for less than 2 min is sufficient to obtain immunostaining results without damaging the sample. The staining intensity by our method is similar to that of the sample stained for 1 h by a conventional batch process. We believe that this pipetting-based approach to the operation of a microfluidic system allows end users to use microfluidic IHC more conveniently and easily in real-world environments.

Automated measurement of multiple cancer biomarkers using quantum-dot-based microfluidic immunohistochemistry.

We report an automated multiple biomarker measurement method for tissue from cancer patients using quantum dot (QD)-based protein detection combined with reference-based protein quantification and autofluorescence (AF) removal. For multiplexed detection of biomarkers in tissue samples, visualization of QDs on cytokeratin was performed to create a multichannel microfluidic device on sites with dense populations of tumor cells. Three major breast cancer biomarkers (i.e., estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) were labeled using QDs successively on cancer cells in tissue sections. For the automated measurement of biomarkers, a cytokeratin-based biomarker normalization method was used to measure the averaged expression of proteins. A novel AF-removal algorithm was developed, which normalizes the reference AF spectra reconstructed from unknown AF spectra based on random sampling. For accurate quantification of QDs, we automatically and accurately removed the AF signal from 344 spots of QD-labeled tissue samples using 240 reference AF spectra. Using analytical data with 10 tissue samples from breast cancer patients, the measured biomarker intensities were in good agreement with the results of conventional analyses.

User-friendly 3D bioassays with cell-containing hydrogel modules: narrowing the gap between microfluidic bioassays and clinical end-users' needs.

Cell-containing hydrogel modules as cell-hydrogel microunits for creating a physiologically relevant 3D in vivo-like microenvironment with multiple cell types and unique extracellular matrix (ECM) compositions facilitate long-term cell maintenance and bioassays. To date, there have been many important advances in microfluidic bioassays, which incorporate hydrogel scaffolds into surface-accessible microchambers, driven by the strong demand for the application of spatiotemporally defined biochemical stimuli to construct in vivo-like conditions and perform real-time imaging of cell-matrix interactions. In keeping with the trend of fostering collaborations among biologists, clinicians, and microfluidic engineers, it is essential to create a simpler approach for coupling cell-containing hydrogel modules and an automated bioassay platform in a user-friendly format. In this article, we review recent progress in hydrogel-incorporated microfluidics for long-term cell maintenance and discuss some of the simpler and user-friendly 3D bioassay techniques combined with cell-containing hydrogel modules that can be applied to mutually beneficial collaborations with non-engineers. We anticipate that this modular and user-friendly format interfaced with existing laboratory infrastructure will help address several clinical questions in ways that extend well beyond the current 2D cell-culture systems.

A quantum dot-based microfluidic multi-window platform for quantifying the biomarkers of breast cancer cells.

Conventional molecular profiling methods using immunochemical assays have limits in terms of multiplexity and the quantification of biomarkers in investigation of cancer cells. In this paper, we demonstrate a quantum dot (QD)-based microfluidic multiple biomarker quantification (QD-MMBQ) method that enables labeling of more than eight proteins immunochemically on cell blocks within 1 h, in a quantitative manner. An internal reference, ß-actin, was used as a loading control to compensate for differences in not only the cell number but also in staining quality among specimens. Furthermore, the microfluidic blocking method exhibited less nonspecific binding of QDs than the conventional static blocking method.

Quantum dot-based immunoassay enhanced by high-density vertical ZnO nanowire array.

In this paper, we report an efficient and high-performance immunoassay platform by combining high-density vertical ZnO nanowire array with photostable quantum dot (QD) labeling. The ZnO nanowire array provides a large surface area for the immobilization of biomolecules, which makes it an efficient substrate for the immunoreaction of biomolecules. When a sandwich immunoassay with QD label was conducted on various substrates, the ZnO nanowire substrate showed stronger fluorescence signal than ZnO thin film and bare glass substrates by 3.8 and 8.5 times, respectively. We found that the fluorescence resonance energy transfer (FRET) from QD to ZnO nanowire could be suppressed by extending their distance with multilayer biotin-streptavidin complex. In addition, we demonstrated the QD-based immunoassay of carcinoembryonic antigen (CEA) on a ZnO nanowire substrate, showing an excellent immunoassay performance with a very low detection limit (0.001 ng/mL) and a large detection range up to 100 ng/mL.

Breast cancer diagnostics using microfluidic multiplexed immunohistochemistry.

A quantitative, reproducible, fast and inexpensive multiplexed immunohistochemistry (IHC) system might play a locomotive role in drug screening and personalized medicine. Currently, fully automated IHC machines and sequential multiplexed IHC methods based upon multiple color reagents have been developed, with the evolution of such methods having revealed novel biological findings over the conventional IHC method, which is time consuming and labor intensive. We describe a novel parallel multiplexed IHC method using a microfluidic multiplexed immunohistochemistry (MMIHC) device for quantitative pathological diagnosis of breast cancer. The key factors for success of parallel multiplexed IHC are the fabrication of a robust microfluidic device, the interface between the device and a tissue slide, and an accurate fluidic control for multiple IHC reagents. In order to apply conventional thin-section tissues into on-chip systems without any additional modification process, a tissue slide-compatible assembler was developed for optimal compatibility of conventional IHC methods. With this approach, a perfect fluid control for various solutions was demonstrated without any leakage, bubble formation or cross-contamination. The results presented in this chapter indicate that the microfluidic IHC protocol developed can provide the possibility of tailored cancer treatments as well as precise histopathological diagnosis using numerous specific biomarkers.

Cellular hydrogel biopaper for patterned 3D cell culture and modular tissue reconstruction.

Microarchitectured freestanding cellular hydrogel biopaper as a novel 3D cell culture or tissue reconstruction module is reported. New harvesting, transfer, and assembly techniques are used to construct laminated tissue composites of the biopaper, such as hepatic hydrogel sheet modules with augmented liver function for stratified 3D hepatic tissue reconstruction.

Quantitative proteomic profiling of breast cancers using a multiplexed microfluidic platform for immunohistochemistry and immunocytochemistry.

This paper describes a multiplexed microfluidic immunohistochemistry (IHC)/immunocytochemistry (ICC) platform for quantitative proteomic profiling in breast cancer samples. Proteomic profiling via ICC was examined for four breast cancer cell lines (AU-565, HCC70, MCF-7, and SK-BR-3). The microfluidic device enabled 20 ICC assays on a biological specimen at the same time and a 16-fold decrease in time consumption, and could be used to quantitatively compare the expression level of each biomarker. The immunohistochemical staining from the microfluidic system showed an accurate localization of protein and comparable quality to that of the conventional IHC method. Although AU-565 and SK-BR-3 cell lines were classified by luminal subtype and adenocarcinomas and were derived from the same patient, weak p63 expression was seen only in SK-BR-3. The HCC70 cell line showed a triple-negative (estrogen receptor-negative/progesterone receptor-negative/human epidermal growth factor receptor 2-negative) phenotype and showed only cytokeratin 5 expression, a representative basal/myoepithelial cell marker. To demonstrate the applicability of the system to clinical samples for proteomic profiling, we were also able to apply this platform to human breast cancer tissue. This result indicates that the microfluidic IHC/ICC platform is useful for accurate histopathological diagnoses using numerous specific biomarkers simultaneously, facilitating the individualization of cancer therapy.