Automated Microfluidic System with Active Mixing Enables Rapid Analysis of Biomarkers in 5 µL of Whole Blood.

We developed a microfluidic device for the rapid analysis of biomarkers in small volumes of whole blood. This device includes an onboard plasma separation module connected to a downstream bioanalysis module in which plasma mixes with reagents and the results of a colorimetric assay are recorded. Actuation of onboard microvalves within a bioanalysis module creates active mixing conditions that allowed us to achieve solution homogeneity within 5 min. To demonstrate utility, we carried out glucose detection in our device. With 5 µL of whole blood as an input, our microfluidic device enabled a time-to-answer of 10 min with a limit of detection of 0.21 ± 0.04 mM for glucose. This device has immediate applications for rapid and sensitive monitoring of hypoglycemia at the point of care (POC). Furthermore, our automated microfluidic device represents a platform technology that may be used to detect other biomarkers in whole blood.

Liquid refractive index measured through a refractometer based on diffraction gratings.

We developed two versions of refractometers to measure the refractive index of liquids. One refractometer comprises a glass cell with a surface relief grating on the inner face of one of its walls, while the other one is a microfluidic channel in the form of serpentine that behaves as a grating. Measurements of the liquid refractive index were performed by sensing the first order intensity. Several liquids have been used including an organic one. Calibration plots are shown.

Dynamic Generation of Concentration- and Temporal-Dependent Chemical Signals in an Integrated Microfluidic Device for Single-Cell Analysis.

Intracellular signaling pathways are affected by the temporal nature of external chemical signaling molecules such as neurotransmitters or hormones. Developing high-throughput technologies to mimic these time-varying chemical signals and to analyze the response of single cells would deepen our understanding of signaling networks. In this work, we introduce a microfluidic platform to stimulate hundreds of single cells with chemical waveforms of tunable frequency and amplitude. Our device produces a linear gradient of 9 concentrations that are delivered to an equal number of chambers, each containing 492 microwells, where individual cells are captured. The device can alternate between the different stimuli concentrations and a control buffer, with a maximum operating frequency of 33 mHz that can be adjusted from a computer. Fluorescent time-lapse microscopy enables to obtain hundreds of thousands of data points from one experiment. We characterized the gradient performance and stability by staining hundreds of cells with calcein AM. We also assessed the capacity of our device to introduce periodic chemical stimuli of different amplitudes and frequencies. To demonstrate our device performance, we studied the dynamics of intracellular Ca2+ release from intracellular stores of HEK cells when stimulated with carbachol at 4.5 and 20 mHz. Our work opens the possibility of characterizing the dynamic responses in real time of signaling molecules to time-varying chemical stimuli with single cell resolution.

Portable platform for leukocyte extraction from blood using sheath-free microfluidic DLD.

Leukocyte count is routinely performed for diagnostic purposes and is rapidly emerging as a significant biomarker for a wide array of diseases. Additionally, leukocytes have demonstrated considerable promise in novel cell-based immunotherapies. However, the direct retrieval of leukocytes from whole blood is a significant challenge due to their low abundance compared to erythrocytes. Here, we introduce a microfluidic-based platform that isolates and recovers leukocytes from diluted whole blood in a single step. Our platform utilizes a novel, sheathless method to initially sediment and focus blood cells into a dense stream while flowing through a tubing before entering the microfluidic device. A hexagonal-shaped structure, patterned at the device's inlet, directs all the blood cells against the channel's outer walls. The focused cells are then separated based on their size using the deterministic lateral displacement (DLD) microfluidic technique. We evaluated various parameters that could influence leukocyte separation, including different focusing structures (assessed both computationally and experimentally), the orientation of the tubing-chip interface, the effects of blood sample hematocrit (dilution), and flow rate. Our device demonstrated the ability to isolate leukocytes from diluted blood with a separation efficiency of 100%, a recovery rate of 76%, and a purity of 80%, while maintaining a cell viability of 98%. The device operates for over 30 min at a flow rate of 2 µL min-1. Furthermore, we developed a handheld pressure controller to drive fluid flow, enhancing the operability of our platform outside of central laboratories and enabling near-patient testing. Our platform can be integrated with downstream cell-based assays and analytical methods that require high leukocyte purity (80%), ranging from cell counting to diagnostics and cell culture applications.

Microfluidic 3D hepatic cultures integrated with a droplet-based bioanalysis unit.

A common challenge in microfluidic cell cultures has to do with analysis of cell function without replacing a significant fraction of the culture volume and disturbing local concentration gradients of signals. To address this challenge, we developed a microfluidic cell culture device with an integrated bioanalysis unit to enable on-chip analysis of picoliter volumes of cell-conditioned media. The culture module consisted of an array of 140 microwells with a diameter of 300 m which were made low-binding to promote organization of cells into 3D spheroids. The bioanalysis module contained a droplet generator unit, 15 micromechanical valves and reservoirs loaded with reagents. Each 0.8 nL droplet contained an aliquot of conditioned media mixed with assay reagents. The use of microvalves allowed us to load enzymatic assay and immunoassay into sequentially generated droplets for detection of glucose and albumin, respectively. As a biological application of the microfluidic device, we evaluated hormonal stimulation and glucose consumption of hepatic spheroids. To mimic physiological processes occurring during feeding and fasting, hepatic spheroids were exposed to pancreatic hormones, insulin or glucagon. The droplet-based bioanalysis module was used to measure uptake or release of glucose upon hormonal stimulation. In the future, we intend to use this microfluidic device to mimic and measure pathophysiological processes associated with hepatic insulin resistance and diabetes in the context of metabolic syndrome.

Microfluidic Organoid Cultures Derived from Pancreatic Cancer Biopsies for Personalized Testing of Chemotherapy and Immunotherapy.

Patient-derived cancer organoids (PDOs) hold considerable promise for personalizing therapy selection and improving patient outcomes. However, it is challenging to generate PDOs in sufficient numbers to test therapies in standard culture platforms. This challenge is particularly acute for pancreatic ductal adenocarcinoma (PDAC) where most patients are diagnosed at an advanced stage with non-resectable tumors and where patient tissue is in the form of needle biopsies. Here the development and characterization of microfluidic devices for testing therapies using a limited amount of tissue or PDOs available from PDAC biopsies is described. It is demonstrated that microfluidic PDOs are phenotypically and genotypically similar to the gold-standard Matrigel organoids with the advantages of 1) spheroid uniformity, 2) minimal cell number requirement, and 3) not relying on Matrigel. The utility of microfluidic PDOs is proven by testing PDO responses to several chemotherapies, including an inhibitor of glycogen synthase kinase (GSKI). In addition, microfluidic organoid cultures are used to test effectiveness of immunotherapy comprised of NK cells in combination with a novel biologic. In summary, our microfluidic device offers considerable benefits for personalizing oncology based on cancer biopsies and may, in the future, be developed into a companion diagnostic for chemotherapy or immunotherapy treatments.

Using Microfluidic Hepatic Spheroid Cultures to Assess Liver Toxicity of T-2 Mycotoxin.

The Fusarium fungi is found in cereals and feedstuffs and may produce mycotoxins, which are secondary metabolites, such as the T-2 toxin (T-2). In this work, we explored the hepatotoxicity of T-2 using microfluidic 3D hepatic cultures. The objectives were: (i) exploring the benefits of microfluidic 3D cultures compared to conventional 3D cultures available commercially (Aggrewell plates), (ii) establishing 3D co-cultures of hepatic cells (HepG2) and stellate cells (LX2) and assessing T-2 exposure in this model, (iii) characterizing the induction of metabolizing enzymes, and (iv) evaluating inflammatory markers upon T-2 exposure in microfluidic hepatic cultures. Our results demonstrated that, in comparison to commercial (large-volume) 3D cultures, spheroids formed faster and were more functional in microfluidic devices. The viability and hepatic function decreased with increasing T-2 concentrations in both monoculture and co-cultures. The RT-PCR analysis revealed that exposure to T-2 upregulates the expression of multiple Phase I and Phase II hepatic enzymes. In addition, several pro- and anti-inflammatory proteins were increased in co-cultures after exposure to T-2.

Patient-Specific Microfluidic Cancer Spheroid Cultures for Testing Cancer Therapies.

The field of oncology increasingly focuses on strategies to predict effectiveness of a given therapy on a patient-by-patient basis. Such precision or personalized oncology has the potential of significantly extending patient survival time. Patient-derived organoids are seen as the main source of patient tumor tissue that may be used for therapy testing in personalized oncology. The gold standard approach for culturing cancer organoids is in standard multi-well plates coated with Matrigel. Despite their effectiveness, these standard organoid cultures have drawbacks, namely, requirement of a large starting cell population and polydispersity of cancer organoid sizes. The latter drawback makes it challenging to monitor and quantify changes in organoid size in response to therapy. Microfluidic devices with integrated arrays of microwells may be used to both decrease the amount of starting cellular material required to form organoids and to standardize organoid size to make therapy assessment easier. Herein, we describe methodology for making microfluidic device as well as for seeding patient-derived cancer cells, culturing organoids, and testing therapies using these devices.

Using Electrochemical Immunoassay in a Novel Microtiter Plate to Detect Surface Markers of Preeclampsia on Urinary Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid bilayer nanovesicles secreted by cells. EVs contain biological information related to parental cells and provide biomarkers for disease diagnosis. We have previously shown that the levels of podocin and nephrin expression on urinary EVs may be used to diagnose renal injury associated with preeclampsia. This paper describes a nanoparticle-enabled immunoassay integrated with an electrochemical plate for quantifying podocin and nephrin expression in urinary EVs. The strategy entailed capturing EVs on an electrode surface and then labeling EVs with gold nanoparticles that are both functionalized with antibodies for target specificity and impregnated with redox-active metal ions for electrochemical detection. These immunoprobes produced an electrochemical redox signal proportional to the expression level of EV surface markers. Electrochemical immunoassays were carried out in a novel microtiter plate that contained 16 wells with working electrodes connected to onboard counter/reference electrodes via capillary valves. Upon validation with recombinant proteins, a microtiter plate was used for analysis of urinary EVs from healthy and preeclamptic pregnant women. This analysis revealed a higher podocin to nephrin ratio for preeclamptic women compared to healthy controls (4.31 vs 1.69) suggesting that this ratio may be used for disease diagnosis.

Guiding Hepatic Differentiation of Pluripotent Stem Cells Using 3D Microfluidic Co-Cultures with Human Hepatocytes.

Human pluripotent stem cells (hPSCs) are capable of unlimited proliferation and can undergo differentiation to give rise to cells and tissues of the three primary germ layers. While directing lineage selection of hPSCs has been an active area of research, improving the efficiency of differentiation remains an important objective. In this study, we describe a two-compartment microfluidic device for co-cultivation of adult human hepatocytes and stem cells. Both cell types were cultured in a 3D or spheroid format. Adult hepatocytes remained highly functional in the microfluidic device over the course of 4 weeks and served as a source of instructive paracrine cues to drive hepatic differentiation of stem cells cultured in the neighboring compartment. The differentiation of stem cells was more pronounced in microfluidic co-cultures compared to a standard hepatic differentiation protocol. In addition to improving stem cell differentiation outcomes, the microfluidic co-culture system described here may be used for parsing signals and mechanisms controlling hepatic cell fate.

Function of hepatocyte spheroids in bioactive microcapsules is enhanced by endogenous and exogenous hepatocyte growth factor.

The ability to maintain functional hepatocytes has important implications for bioartificial liver development, cell-based therapies, drug screening, and tissue engineering. Several approaches can be used to restore hepatocyte function in vitro, including coating a culture substrate with extracellular matrix (ECM), encapsulating cells within biomimetic gels (Collagen- or Matrigel-based), or co-cultivation with other cells. This paper describes the use of bioactive heparin-based core-shell microcapsules to form and cultivate hepatocyte spheroids. These microcapsules are comprised of an aqueous core that facilitates hepatocyte aggregation into spheroids and a heparin hydrogel shell that binds and releases growth factors. We demonstrate that bioactive microcapsules retain and release endogenous signals thus enhancing the function of encapsulated hepatocytes. We also demonstrate that hepatic function may be further enhanced by loading exogenous hepatocyte growth factor (HGF) into microcapsules and inhibiting transforming growth factor (TGF)-ß1 signaling. Overall, bioactive microcapsules described here represent a promising new strategy for the encapsulation and maintenance of primary hepatocytes and will be beneficial for liver tissue engineering, regenerative medicine, and drug testing applications.

Modeling gut neuro-epithelial connections in a novel microfluidic device.

The intestinal lumen is filled with diverse chemical and physical stimuli. Intestinal epithelial cells sense these stimuli and signal to enteric neurons which coordinate a range of physiologic processes required for normal digestive tract function. Yet, the neuro-epithelial connections remain poorly resolved, in part because the tools for orchestrating interactions between these cellular compartments are lacking. We describe the development of a two-compartment microfluidic device for co-culturing enteric neurons with intestinal epithelial cells. The device contains epithelial and neuronal compartments connected by microgrooves. The epithelial compartment was designed for cell seeding via injection and confinement of intestinal epithelial cells derived from human intestinal organoids. We demonstrated that organoids planarized effectively and retained epithelial phenotype for over a week. In the second chamber we dissociated and cultured intestinal myenteric neurons including intrinsic primary afferent neurons (IPANs) from transgenic mice that expressed the fluorescent protein tdTomato. IPANs extended projections into microgrooves, surrounded and frequently made contacts with epithelial cells. The density and directionality of neuronal projections were enhanced by the presence of epithelial cells in the adjacent compartment. Our microfluidic device represents a platform that may, in the future, be used to dissect structure and function of neuro-epithelial connections in the gut and other organs (skin, lung, bladder, and others) in health and disease.

A Compact Control System to Enable Automated Operation of Microfluidic Bioanalytical Assays.

We describe a control system for operating valve-enabled microfluidic devices and leverage this control system to carry out a complex workflow of plasma separation from 8 µL of whole blood followed by on-chip mixing of plasma with assay reagents for biomarker detection. The control system incorporates pumps, digital pressure sensors, a microcontroller, solenoid valves and off-the-shelf components to deliver high and low air pressure in the desired temporal sequence to meter fluid flow and actuate microvalves. Importantly, our control system is portable, which is suitable for operating the microvalve-enabled microfluidic devices in the point-of-care setting.

Coating Bioactive Microcapsules with Tannic Acid Enhances the Phenotype of the Encapsulated Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) may be differentiated into any adult cell type and therefore hold incredible promise for cell therapeutics and disease modeling. There is increasing interest in three-dimensional (3D) hPSC culture because of improved differentiation outcomes and potential for scale up. Our team has recently described bioactive heparin (Hep)-containing core-shell microcapsules that promote rapid aggregation of stem cells into spheroids and may also be loaded with growth factors for the local and sustained delivery to the encapsulated cells. In this study, we explored the possibility of further modulating bioactivity of microcapsules through the use of an ultrathin coating composed of tannic acid (TA). Deposition of the TA film onto model substrates functionalized with Hep and poly(ethylene glycol) was characterized by ellipsometry and atomic force microscopy. Furthermore, the presence of the TA coating was observed to increase the amount of basic fibroblast growth factor (bFGF) incorporation by up to twofold and to extend its release from 5 to 7 days. Most significantly, TA-microcapsules loaded with bFGF induced higher levels of pluripotency expression compared to uncoated microcapsules containing bFGF. Engineered microcapsules described here represent a new stem cell culture approach that enables 3D cultivation and relies on local delivery of inductive cues.

Bioactive hydrogel microcapsules for guiding stem cell fate decisions by release and reloading of growth factors.

Human pluripotent stem cells (hPSC) hold considerable promise as a source of adult cells for treatment of diseases ranging from diabetes to liver failure. Some of the challenges that limit the clinical/translational impact of hPSCs are high cost and difficulty in scaling-up of existing differentiation protocols. In this paper, we sought to address these challenges through the development of bioactive microcapsules. A co-axial flow focusing microfluidic device was used to encapsulate hPSCs in microcapsules comprised of an aqueous core and a hydrogel shell. Importantly, the shell contained heparin moieties for growth factor (GF) binding and release. The aqueous core enabled rapid aggregation of hPSCs into 3D spheroids while the bioactive hydrogel shell was used to load inductive cues driving pluripotency maintenance and endodermal differentiation. Specifically, we demonstrated that one-time, 1 h long loading of pluripotency signals, fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-ß1, into bioactive microcapsules was sufficient to induce and maintain pluripotency of hPSCs over the course of 5 days at levels similar to or better than a standard protocol with soluble GFs. Furthermore, stem cell-carrying microcapsules that previously contained pluripotency signals could be reloaded with an endodermal cue, Nodal, resulting in higher levels of endodermal markers compared to stem cells differentiated in a standard protocol. Overall, bioactive heparin-containing core-shell microcapsules decreased GF usage five-fold while improving stem cell phenotype and are well suited for 3D cultivation of hPSCs.

Rejuvenation of the aged brain immune cell landscape in mice through p16-positive senescent cell clearance.

Cellular senescence is a plausible mediator of inflammation-related tissue dysfunction. In the aged brain, senescent cell identities and the mechanisms by which they exert adverse influence are unclear. Here we used high-dimensional molecular profiling, coupled with mechanistic experiments, to study the properties of senescent cells in the aged mouse brain. We show that senescence and inflammatory expression profiles increase with age and are brain region- and sex-specific. p16-positive myeloid cells exhibiting senescent and disease-associated activation signatures, including upregulation of chemoattractant factors, accumulate in the aged mouse brain. Senescent brain myeloid cells promote peripheral immune cell chemotaxis in vitro. Activated resident and infiltrating immune cells increase in the aged brain and are partially restored to youthful levels through p16-positive senescent cell clearance in female p16-InkAttac mice, which is associated with preservation of cognitive function. Our study reveals dynamic remodeling of the brain immune cell landscape in aging and suggests senescent cell targeting as a strategy to counter inflammatory changes and cognitive decline.

Prospects and Opportunities for Microsystems and Microfluidic Devices in the Field of Otorhinolaryngology.

Microfluidic systems can be used to control picoliter to microliter volumes in ways not possible with other methods of fluid handling. In recent years, the field of microfluidics has grown rapidly, with microfluidic devices offering possibilities to impact biology and medicine. Microfluidic devices populated with human cells have the potential to mimic the physiological functions of tissues and organs in a three-dimensional microenvironment and enable the study of mechanisms of human diseases, drug discovery and the practice of personalized medicine. In the field of otorhinolaryngology, various types of microfluidic systems have already been introduced to study organ physiology, diagnose diseases, and evaluate therapeutic efficacy. Therefore, microfluidic technologies can be implemented at all levels of otorhinolaryngology. This review is intended to promote understanding of microfluidic properties and introduce the recent literature on application of microfluidic-related devices in the field of otorhinolaryngology.

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids.

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 µm diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with >95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had <50% viability 3 days after encapsulation. Spheroid formation of hPSCs inside core-shell microcapsules occurred within 48 h after encapsulation, with the spheroid diameter being a function of cell inoculation density. Overall, the microfluidic encapsulation technology described in this protocol was well-suited for hPSCs encapsulation and spheroid formation.

Core-shell hydrogel microcapsules enable formation of human pluripotent stem cell spheroids and their cultivation in a stirred bioreactor.

Cellular therapies based on human pluripotent stem cells (hPSCs) offer considerable promise for treating numerous diseases including diabetes and end stage liver failure. Stem cell spheroids may be cultured in stirred bioreactors to scale up cell production to cell numbers relevant for use in humans. Despite significant progress in bioreactor culture of stem cells, areas for improvement remain. In this study, we demonstrate that microfluidic encapsulation of hPSCs and formation of spheroids. A co-axial droplet microfluidic device was used to fabricate 400 µm diameter capsules with a poly(ethylene glycol) hydrogel shell and an aqueous core. Spheroid formation was demonstrated for three hPSC lines to highlight broad utility of this encapsulation technology. In-capsule differentiation of stem cell spheroids into pancreatic ß-cells in suspension culture was also demonstrated.

A versatile microfluidic device for multiple ex vivo/in vitro tissue assays unrestrained from tissue topography.

Precision-cut tissue slices are an important in vitro system to study organ function because they preserve most of the native cellular microenvironments of organs, including complex intercellular connections. However, during sample manipulation or slicing, some of the natural surface topology and structure of these tissues is lost or damaged. Here, we introduce a microfluidic platform to perform multiple assays on the surface of a tissue section, unhindered by surface topography. The device consists of a valve on one side and eight open microchannels located on the opposite side, with the tissue section sandwiched between these two structures. When the valve is actuated, eight independent microfluidic channels are formed over a tissue section. This strategy prevents cross-contamination when performing assays and enables parallelization. Using irregular tissues such as an aorta, we conducted multiple in vitro and ex vivo assays on tissue sections, including short-term culturing, a drug toxicity assay, a fluorescence immunohistochemistry staining assay, and an immune cell assay, in which we observed the interaction of neutrophils with lipopolysaccharide (LPS)-stimulated endothelium. Our microfluidic platform can be employed in other disciplines, such as tissue physiology and pathophysiology, morphogenesis, drug toxicity and efficiency, metabolism studies, and diagnostics, enabling the conduction of several assays with a single biopsy sample.