Physiological Fluid Flow Research Articles

Measurement of Covalent Bond Formation in Light-Curing Hydrogels Predicts Physical Stability under Flow.

Photo-crosslinking hydrogels are promising for tissue engineering and regenerative medicine, but challenges in reaction monitoring often leave their optimization subject to trial and error. The stability of crosslinked gels under fluid flow, as in the case of a microfluidic device, is particularly challenging to predict, both because of obstacles inherent to solid-state macromolecular analysis that prevent accurate chemical monitoring and because stability is dependent on size of the patterned features. To solve both problems, we obtained 1H NMR spectra of cured hydrogels which were enzymatically degraded. This allowed us to take advantage of the high-resolution that solution NMR provides. This unique approach enabled the measurement of degree of cross-linking (DoC) and prediction of material stability under physiological fluid flow. We showed that NMR spectra of enzyme-digested gels successfully reported on DoC as a function of light exposure and wavelength within two classes of photo-cross-linkable hydrogels: methacryloyl-modified gelatin and a composite of thiol-modified gelatin and norbornene-terminated polyethylene glycol. This approach revealed that a threshold DoC was required for patterned features in each material to become stable and that smaller features required a higher DoC for stability. Finally, we demonstrated that DoC was predictive of the stability of architecturally complex features when photopatterning, underscoring the value of monitoring DoC when using light-reactive gels. We anticipate that the ability to quantify chemical cross-links will accelerate the design of advanced hydrogel materials for structurally demanding applications such as photopatterning and bioprinting.

Retinal cytoarchitecture is preserved in an organotypic perfused human and porcine eye model

Pathobiology of the intact human retina has been challenging to study due to its relative inaccessibility and limited sample availability. Thus, there is a great need for new translational models that can maintain human retinal integrity and cytoarchitecture. The role of physiologic intraocular pressure (IOP) and fluid flow on retinal tissue has not been well studied. Here, we present an ex vivo organotypic model to assess the impact of physiological intraocular perfusion on retinal cytoarchitecture and cell survival. We demonstrate that retinal cytoarchitecture is remarkably well preserved following re-establishment of physiological IOP and aqueous humor dynamics for up to 24 h in ex vivo whole globe porcine and human eyes, comparable to freshly preserved control eyes. Accordingly, cell death was minimized in the perfused retinas, which also displayed normal markers of cellular metabolism and astrogliosis. These results are in marked contrast to contralateral control eyes without active perfusion, which displayed excessive cell death and disrupted cytoarchitecture at the same time point. These experiments demonstrate the critical impact that physiological pressure and fluid flow have on retinal tissue, and introduce a new pre-clinical model to study human and porcine retinal health and degeneration in a relevant biomechanical setting.

Mathematical modeling of electroosmotically driven peristaltic propulsion due to transverse deflections of two periodically deformable curved tubes of unequal wavelengths

The present study aims to investigate the viscid fluid propulsion due to the electroosmosis and transverse deflections of the sinusoidally deformable tubes of unequal wavelengths in the presence of electro-kinetic forces. This situation is estimated from the physical model of physiological fluid flow through a tubular structure in which an artificial flexible tube is being inserted. In this model, both peristaltically deforming tubes are taken in a curved configuration. The flow-governing momentum equations are simplified by the approximation of the long wavelength as compared to the outer tube's radius, whereas the Debye–Hückel approximation is used to simplify the equations that govern the electric potential distribution. Here, the authors have used the DSolve command in the scientific computing software MATHEMATICA 14 to obtain the expressions for electric potential and axial velocity of viscid fluid. In this work, the authors have analyzed the impact of various controlling parameters, such as the electro-physical parameters, curvature parameter, radius ratio, wavelength ratio, and amplitude ratios, on the various flow quantities graphically during the transport of viscid fluid through a curved endoscope. Here, contour plots are also drawn to visualize the streamlines and to observe the impacts of the control parameters on fluid trapping. During the analysis of the results, a noteworthy outcome extracted from the present model is that an increment in electro-physical parameters, such as Helmholtz–Smoluchowski velocity and the Debye–Hückel parameter, are responsible for enhancement in the shear stress at the inner tube's wall and the axial velocity under the influence of electro-kinetic forces. This is because of the electric double layer (EDL) thickness, which gets reduced on strengthening the Debye–Hückel parameter. This reduced EDL thickness is responsible for the enhancement in the axial velocity of the transporting viscid fluid. The present model also suggests that the axial velocity of viscid fluid can be reduced by enhancing the ratio of wavelengths of waves that travel down the walls of the outer curved tube and the inner curved tube. The above-mentioned results can play a significant role in developing and advancing the endoscopes that will be useful in many biomedical processes, such as gastroscopy, colonoscopy, and laparoscopy.

Perfusion Bioreactor Conditioning of Small-diameter Plant-based Vascular Grafts

Background:Vascular grafts are mainly composed of synthetic materials, but are prone to thrombosis and intimal hyperplasia at small diameters. Decellularized plant scaffolds have emerged that provide promising alternatives for tissue engineering. We previously developed robust, endothelialized small-diameter vessels from decellularized leatherleaf viburnum. This is the first study to precondition and analyze plant-based vessels under physiological fluid flow and pressure waveforms. Using decellularized leatherleaf viburnum as tissue-engineered grafts for implantation can have profound impacts on healthcare due to their biocompatibility and cost-effective production.Methods:A novel perfusion bioreactor was designed, capable of accurately controlling fluid flow rate and pressure waveforms for preconditioning of small-diameter vascular grafts. A closed-loop system controlled pressure waveforms, mimicking physiological values of 50–120 mmHg at a frequency of 8.75 Hz for fluid flow reaching 5 mL/min. Plant-based vascular grafts were recellularized with endothelial and vascular smooth muscle cells and cultured for up to 3 weeks in this bioreactor. Cell density, scaffold structure and mechanics, thrombogenicity, and immunogenicity of grafts were evaluated.Results:Bioreactor treatment with fluid flow significantly increased luminal endothelial cell density, while pressure waveforms reduced thrombus formation and maintained viable vascular smooth muscle cells within inner layers of grafts compared to static controls. Suture retention of grafts met transplantation standards and white cell viability was suitable for vascular remodeling.Conclusion:Low thrombogenicity of endothelialized leatherleaf viburnum holds great potential for vascular repair. This study provides insight into benefits of conditioning plant-based materials with hemodynamic forces at higher frequencies that have not previously been investigated.

A 3D bioreactor model to study osteocyte differentiation and mechanobiology under perfusion and compressive mechanical loading

Osteocytes perceive and process mechanical stimuli in the lacuno-canalicular network in bone. As a result, they secrete signaling molecules that mediate bone formation and resorption. To date, few three-dimensional (3D) models exist to study the response of mature osteocytes to biophysical stimuli that mimic fluid shear stress and substrate strain in a mineralized, biomimetic bone-like environment. Here we established a biomimetic 3D bone model by utilizing a state-of-art perfusion bioreactor platform where immortomouse/Dmp1-GFP-derived osteoblastic IDG-SW3 cells were differentiated into mature osteocytes. We evaluated proliferation and differentiation properties of the cells on 3D microporous scaffolds of decellularized bone (dBone), poly(L-lactide-co-trimethylene carbonate) lactide (LTMC), and beta-tricalcium phosphate (β-TCP) under physiological fluid flow conditions over 21 days. Osteocyte viability and proliferation were similar on the scaffolds with equal distribution of IDG-SW3 cells on dBone and LTMC scaffolds. After seven days, the differentiation marker alkaline phosphatase (Alpl), dentin matrix acidic phosphoprotein 1 (Dmp1), and sclerostin (Sost) were significantly upregulated in IDG-SW3 cells (p = 0.05) on LTMC scaffolds under fluid flow conditions at 1.7 ml/min, indicating rapid and efficient maturation into osteocytes. Osteocytes responded by inducing the mechanoresponsive genes FBJ osteosarcoma oncogene (Fos) and prostaglandin-endoperoxide synthase 2 (Ptgs2) under perfusion and dynamic compressive loading at 1 Hz with 5 % strain. Together, we successfully created a 3D biomimetic platform as a robust tool to evaluate osteocyte differentiation and mechanobiology in vitro while recapitulating in vivo mechanical cues such as fluid flow within the lacuno-canalicular network. Statement of significanceThis study highlights the importance of creating a three-dimensional (3D) in vitro model to study osteocyte differentiation and mechanobiology, as cellular functions are limited in two-dimensional (2D) models lacking in vivo tissue organization. By using a perfusion bioreactor platform, physiological conditions of fluid flow and compressive loading were mimicked to which osteocytes are exposed in vivo. Microporous poly(L-lactide-co-trimethylene carbonate) lactide (LTMC) scaffolds in 3D are identified as a valuable tool to create a favorable environment for osteocyte differentiation and to enable mechanical stimulation of osteocytes by perfusion and compressive loading. The LTMC platform imitates the mechanical bone environment of osteocytes, allowing the analysis of the interaction with other cell types in bone under in vivo biophysical stimuli.

Measurement of covalent bond formation in light-curing hydrogels predicts physical stability under flow.

Photocrosslinking hydrogels are promising for tissue engineering and regenerative medicine, but challenges in reaction monitoring often leave their optimization subject to trial and error. The stability of crosslinked gels under fluid flow, as in the case of a microfluidic device, is particularly challenging to predict, both because of obstacles inherent to solid-state macromolecular analysis that prevent accurate chemical monitoring, and because stability is dependent on size of the patterned features. To solve both problems, we obtained 1H NMR spectra of cured hydrogels which were enzymatically degraded. This allowed us to take advantage of the high-resolution that solution NMR provides. This unique approach enabled the measurement of degree of crosslinking (DoC) and prediction of material stability under physiological fluid flow. We showed that NMR spectra of enzyme-digested gels successfully reported on DoC as a function of light exposure and wavelength within two classes of photocrosslinkable hydrogels: methacryloyl-modified gelatin and a composite of thiol-modified gelatin and norbornene-terminated polyethylene glycol. This approach revealed that a threshold DoC was required for patterned features in each material to become stable, and that smaller features required a higher DoC for stability. Finally, we demonstrated that DoC was predictive of the stability of architecturally complex features when photopatterning, underscoring the value of monitoring DoC when using light-reactive gels. We anticipate that the ability to quantify chemical crosslinks will accelerate the design of advanced hydrogel materials for structurally demanding applications such as photopatterning and bioprinting.

Investigation for mixed convection flow of physiological fluid due to non-uniform vertical complex channel with entropy generation effects

Investigation for mixed convection flow of physiological fluid due to non-uniform vertical complex channel with entropy generation effects

Single-cell adhesive profiling in an optofluidic device elucidates CD8+ T lymphocyte phenotypes in inflamed vasculature-like microenvironments

Tissue infiltration by circulating leukocytes occurs via adhesive interactions with the local vasculature, but how the adhesive quality of circulating cells guides the homing of specific phenotypes to different vascular microenvironments remains undefined. We developed an optofluidic system enabling fluorescent labeling of photoactivatable cells based on their adhesive rolling velocity in an inflamed vasculature-mimicking microfluidic device under physiological fluid flow. In so doing, single-cell level multidimensional profiling of cellular characteristics could be characterized and related to the associated adhesive phenotype. When applied to CD8+ Tcells, ligand/receptor expression profiles and subtypes associated with adhesion were revealed, providing insight into inflamed tissue infiltration capabilities of specific CD8+ T lymphocyte subsets and how local vascular microenvironmental features may regulate the quality of cellular infiltration. This methodology facilitates rapid screening of cell populations for enhanced homing capabilities under defined biochemical and biophysical microenvironments, relevant to leukocyte homing modulation in multiple pathologies.

Abstract 6764: Vascularized tumor-on-chip model for evaluating chemotherapeutic-mediated damage to adjacent healthy tissue

Abstract Introduction: Systemic chemotherapy is an effective anticancer treatment for all stages of breast cancer, with intravenous (IV) infusion being the principal method of administration. In addition to targeting cancer, systemic chemotherapy can impact the viability and function of surrounding normal tissues and vasculature. Early-discovery research tools comparing novel and existing systemic treatment strategies are urgently needed to support predictive efficacy and toxicity efforts. Microfluidic systems have the potential to fill this need while simultaneously reducing animal use. The overall goal of the study is to use an advanced microfluidic tumor-on-chip system to simultaneously evaluate the therapeutic and toxic effects of IV chemotherapy on normal and cancerous tissues. Materials and Methods: Synthetic Tumor Networks, which are comprised of primary and secondary tissue sites separated by an interconnected vasculature, were developed using in vivo images and fabricated using soft lithography. Primary vascular endothelial cells were used to establish microvasculature. A normal breast cell line (MCF 10A), or a GFP-labeled metastatic human breast cancer cell line (MDA-MB-231/GFP), were cultured in the tissue sites in a three-dimensional (3D) environment using a human-derived hydrogel. The vascular networks were perfused with endothelial cell media under physiological fluid flow conditions to establish the model. An EZH2 methyltransferase inhibitor (EZH2 inhibitor III) was delivered by IV infusion to the model tissues. Real-time monitoring of cellular growth, tumor invasion and extravasation, and cellular viability were performed using fluorescence microscopy over a two-week period. Results and Discussion: Metastatic MDA-MB-231/GFP breast cancer cells proliferated rapidly in the primary tumor site. Tumor intravasation into the vascular channels was observed in models that received no anticancer treatment, as well as extravasation into the secondary tissue site and invasion of the healthy breast tissue. Differences in tumor viability and migratory behavior were observed between untreated models or models receiving IV infusions of the EZH2 inhibitor through the vascular networks. Vascular barrier integrity and normal breast tissue viability were monitored at early (24-72 hr) and late (7-14 days) timepoints after anticancer treatment to measure short- and long-term adverse drug impacts. These results provide a unique perspective on the in vivo realism of an in vitro system for monitoring both therapeutic and toxic effects of systemic chemotherapies. Conclusions: We have developed a 3D vascularized tumor-on-chip model for monitoring the impacts of systemic chemotherapeutic agents on tumor and healthy tissues. This model can be used to investigate therapeutic and toxic effects on multiple tissue types simultaneously using real-time imaging techniques. Citation Format: Deborah Ramsey, Jenna Rosano, Cristabel Gordon, Gwen Fewell. Vascularized tumor-on-chip model for evaluating chemotherapeutic-mediated damage to adjacent healthy tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6764.

Vascularized microfluidic models of major organ structures and cancerous tissues.

Organ-on-a-chip devices are powerful modeling systems that allow researchers to recapitulate the in vivo structures of organs as well as the physiological conditions those tissues are subject to. These devices are useful tools in modeling not only the behavior of a healthy organ but also in modeling disease pathology or the effects of specific drugs. The incorporation of fluidic flow is of great significance in these devices due to the important roles of physiological fluid flows in vivo. Recent developments in the field have led to the production of vascularized organ-on-a-chip devices, which can more accurately reproduce the conditions observed in vivo by recapitulating the vasculature of the organ concerned. This review paper will provide a brief overview of the history of organ-on-a-chip devices, before discussing developments in the production of vascularized organs-on-chips, and the implications these developments hold for the future of the field.

Convection theory on thermally radiative peristaltic flow of Prandtl tilted magneto nanofluid in an asymmetric channel with effects of partial slip and viscous dissipation

A theoretical investigation has been conducted to analyze the peristaltic motion of an asymmetric channel carrying magneto-Prandtl nanofluid under the influence of double-diffusive convection, which includes both thermal and concentration gradients. Moreover, the effects of thermal radiation, viscous dissipation, and partial slip have also been taken into consideration. The mathematical expressions for mixed convection and viscous dissipation have been derived and the influential dynamic parameters have been depicted as geographical flow patterns with a focus on double-diffusive convection and partial slip. NDSolve in Mathematica, the computational technique, has been used to obtain the results. The outcomes for the classic Newtonian fluid have been taken as a special parameter of the study. The findings of this investigations can be beneficial in improving gastrointestinal movements and pumping in various engineering devices. Such analysis also provides a provision to flow of physiological fluids within vessels and arteries for transportation of food nutrients, blood circulation, carrying oxygen, waste excretion, heat, and other nutrients in the human body.

3D-Printed Impedance Micropump for Continuous Perfusion of the Sample and Nutrient Medium Integrated with a Liver-On-Chip Prototype.

In recent decades, organ-on-chip devices have gained substantial interest as an alternative for studying the pathophysiological processes relevant to drug screening. Micropumps are being utilized to simulate the in vivo physiological fluid flow more realistically in these organ-on-chip devices. Micropumps play a crucial role in pumping, perfusion, and circulation of fluids in various microdevices such as on-chip PCR, DNA microarrays, miniature bioreactor cell separation, and lab-on-chip biosensing platforms. With the rapid growth in technology, efficient pumping for proper circulation of media and nutrients has become imperative. In this study, we have described the design and development of an open-source impedance micropump for continuous perfusion of nutrient medium in a liver-on-chip prototype. This micropump is controlled via an integrated microcontroller board, with an observed flow rate ranging from 0.2 to 2 mL/min. Google Sketchup 2020 and DLP 3D printing were used to fabricate small precise parts of the impedance micropump. The flow rate was measured to characterize the actuating performance of the micropump. The poly-dimethyl siloxane-based liver-on-chip prototype has been fabricated using a soft photolithography procedure. Further, a study of continuous perfusion of culture medium through the liver-on-chip containing the Hepg2 cell line was successfully performed by integrating it with the impedance micropump. Hoechst staining and Alamar Blue observed cell viability to confirm the healthy cell growth inside the liver-on-chip microfluidic chip. The compactness of the overall setup allows it to fit in a Petri plate, eliminating chances of contamination while cell handling.

The Modular µSiM Reconfigured: Integration of Microfluidic Capabilities to Study In Vitro Barrier Tissue Models under Flow.

Microfluidic tissue barrier models have emerged to address the lack of physiological fluid flow in conventional "open-well" Transwell-like devices. However, microfluidic techniques have not achieved widespread usage in bioscience laboratories because they are not fully compatible with traditional experimental protocols. To advance barrier tissue research, there is a need for a platform that combines the key advantages of both conventional open-well and microfluidic systems. Here, a plug-and-play flow module is developed to introduce on-demand microfluidic flow capabilities to an open-well device that features a nanoporous membrane and live-cell imaging capabilities. The magnetic latching assembly of this design enables bi-directional reconfiguration and allows users to conduct an experiment in an open-well format with established protocols and then add or remove microfluidic capabilities as desired. This work also provides an experimentally-validated flow model to select flow conditions based on the experimental needs. As a proof-of-concept, flow-induced alignment of endothelial cells and the expression of shear-sensitive gene targets are demonstrated, and the different phases of neutrophil transmigration across a chemically stimulated endothelial monolayer under flow conditions are visualized. With these experimental capabilities, it is anticipated that both engineering and bioscience laboratories will adopt this reconfigurable design due to the compatibility with standard open-well protocols.

A multi-organ-on-chip to recapitulate the infiltration and the cytotoxic activity of circulating NK cells in 3D matrix-based tumor model.

The success of immunotherapeutic approaches strictly depends on the immune cells interaction with cancer cells. While conventional in vitro cell cultures under-represent the complexity and dynamic crosstalk of the tumor microenvironment, animal models do not allow deciphering the anti-tumor activity of the human immune system. Therefore, the development of reliable and predictive preclinical models has become crucial for the screening of immune-therapeutic approaches. We here present an organ-on-chip organ on chips (OOC)-based approach for recapitulating the immune cell Natural Killer (NK) migration under physiological fluid flow, infiltration within a 3D tumor matrix, and activation against neuroblastoma cancer cells in a humanized, fluid-dynamic environment. Circulating NK cells actively initiate a spontaneous “extravasation” process toward the physically separated tumor niche, retaining their ability to interact with matrix-embedded tumor cells, and to display a cytotoxic effect (tumor cell apoptosis). Since NK cells infiltration and phenotype is correlated with prognosis and response to immunotherapy, their phenotype is also investigated: most importantly, a clear decrease in CD16-positive NK cells within the migrated and infiltrated population is observed. The proposed immune-tumor OOC-based model represents a promising approach for faithfully recapitulating the human pathology and efficiently employing the immunotherapies testing, eventually in a personalized perspective. An immune-organ on chip to recapitulate the tumor-mediated infiltration of circulating immune cells within 3D tumor model.

Comparison Between Franz Diffusion Cell and a novel Micro-physiological System for In Vitro Penetration Assay Using Different Skin Models.

In vitro diffusive models are an important tool to screen the penetration ability of active ingredients in various formulations. A reliable assessment of skin penetration enhancing properties, mechanism of action of carrier systems, and an estimation of a bioavailability are essential for transdermal delivery. Given the importance of testing the penetration kinetics of different compounds across the skin barrier, several in vitro models have been developedThe aim of this study was to compare the Franz Diffusion Cell (FDC) with a novel fluid-dynamic platform (MIVO) by evaluating penetration ability of caffeine, a widely used reference substance, and LIP1, a testing molecule having the same molecular weight but a different lipophilicity in the two diffusion chamber systems. A 0.7% caffeine or LIP1 formulation in either water or propylene glycol (PG) containing oleic acid (OA) was topically applied on the Strat-M® membrane or pig ear skin, according to the infinite-dose experimental condition (780 ul/cm2). The profile of the penetration kinetics was determined by quantify the amount of molecule absorbed at different time-points (1, 2, 4, 6, 8 hours), by means of HPLC analysis. Both diffusive systems show a similar trend for caffeine and LIP1 penetration kinetics. The Strat-M® skin model shows a lower barrier function than the pig skin biopsies, whereby the PGOA vehicle exhibits a higher penetration, enhancing the effect for both diffusive chambers and skin surrogates. Most interestingly, MIVO diffusive system better predicts the lipophilic molecules (i.e. LIP1) permeation through highly physiological fluid flows resembled below the skin models.

Electrochemical Approach to Measure Physiological Fluid Flow Rates

In vivo measurement of the flow rate of physiological fluids such as the blood flow rate in the heart is vital in critically ill patients and for those undergoing surgical procedures. The reliability of these measurements is therefore quite crucial. However, current methods in practice for measuring flow rates of physiological fluids suffer from poor repeatability and reliability. Here, we assessed the feasibility of a flow rate measurement method that leverages time transient electrochemical behavior of a tracer that is injected directly into a medium (the electrochemical signal caused due to the tracer injectate will be diluted by the continued flow of the medium and the time response of the current—the electrodilution curve—will depend on the flow rate of the medium). In an experimental flow loop apparatus equipped with an electrochemical cell, we used the AC voltammetry technique and tested the feasibility of electrodilution-based measurement of the flow rate using two mediums—pure water and anticoagulated blood—with 0.9 wt% saline as the injectate. The electrodilution curve was quantified using three metrics—change in current amplitude, total time, and change in the total charge for a range of AC voltammetry settings (peak voltages and frequencies). All three metrics showed an inverse relationship with the flow rate of water and blood, with the strongest negative correlation obtained for change in current amplitude. The findings are a proof of concept for the electrodilution method of the flow rate measurement and offer the potential for physiological fluid flow rate measurement in vivo.

Experimental liver models: From cell culture techniques to microfluidic organs-on-chip.

The liver is one of the most studied organs of the human body owing to its central role in xenobiotic and drug metabolism. In recent decades, extensive research has aimed at developing in vitro liver models able to mimic liver functions to study pathophysiological clues in high-throughput and reproducible environments. Two-dimensional (2D) models have been widely used in screening potential toxic compounds but have failed to accurately reproduce the three-dimensionality (3D) of the liver milieu. To overcome these limitations, improved 3D culture techniques have been developed to recapitulate the hepatic native microenvironment. These models focus on reproducing the liver architecture, representing both parenchymal and nonparenchymal cells, as well as cell interactions. More recently, Liver-on-Chip (LoC) models have been developed with the aim of providing physiological fluid flow and thus achieving essential hepatic functions. Given their unprecedented ability to recapitulate critical features of the liver cellular environments, LoC have been extensively adopted in pathophysiological modelling and currently represent a promising tool for tissue engineering and drug screening applications. In this review, we discuss the evolution of experimental liver models, from the ancient 2D hepatocyte models, widely used for liver toxicity screening, to 3D and LoC culture strategies adopted for mirroring a more physiological microenvironment for the study of liver diseases.

Efficient Portable Urea Biosensor Based on Urease Immobilized Membrane for Monitoring of Physiological Fluids.

Numerous studies have addressed the utilization of glutaraldehyde (GA) as a homobifunctional cross-linker. However, its applicability has been impeded due to several issues, including the tendency of GA molecules to undergo polymerization. Herein, a portable urea biosensor was developed for the real-time monitoring of the flow of physiological fluids; this was achieved by using disuccinimidyl cross-linker-based urease immobilization. Urease was immobilized on a porous polytetrafluoroethylene (PTFE) solid support using different disuccinimidyl cross-linkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and bis-N-succinimidyl-(pentaethylene glycol) ester (BS(PEG)5). A urease activity test revealed that DSS exhibited the highest urease immobilizing efficiency, whereas FT-IR analysis confirmed that urease was immobilized on the PTFE membrane via DSS cross-linking. The membrane was inserted in a polydimethylsiloxane (PDMS) fluidic chamber that generated an electrochemical signal in the presence of a flowing fluid containing urea. Urea samples were allowed to flow into the urea biosensor (1.0 mL/min) and the signal was measured using chronoamperometry. The sensitivity of the DSS urea biosensor was the highest of all the trialed biosensors and was found to be superior to the more commonly used GA cross-linker. To simulate real-time monitoring in a human patient, flowing urea-spiked human serum was measured and the effective urease immobilization of the DSS urea biosensor was confirmed. The repeatability and interference of the urea biosensor were suitable for monitoring urea concentrations typically found in human patients.

SARS-CoV-2 induced intestinal responses with a biomimetic human gut-on-chip

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic. Clinical evidence suggests that the intestine is another high-risk organ for SARS-CoV-2 infection besides the lungs. However, a model that can accurately reflect the response of the human intestine to the virus is still lacking. Here, we created an intestinal infection model on a chip that allows the recapitulation of human relevant intestinal pathophysiology induced by SARS-CoV-2 at organ level. This microengineered gut-on-chip reconstitutes the key features of the intestinal epithelium-vascular endothelium barrier through the three-dimensional (3D) co-culture of human intestinal epithelial, mucin-secreting, and vascular endothelial cells under physiological fluid flow. The intestinal epithelium showed permissiveness for viral infection and obvious morphological changes with injury of intestinal villi, dispersed distribution of mucus-secreting cells, and reduced expression of tight junction (E-cadherin), indicating the destruction of the intestinal barrier integrity caused by virus. Moreover, the vascular endothelium exhibited abnormal cell morphology, with disrupted adherent junctions. Transcriptional analysis revealed abnormal RNA and protein metabolism, as well as activated immune responses in both epithelial and endothelial cells after viral infection (e.g., upregulated cytokine genes), which may contribute to the injury of the intestinal barrier associated with gastrointestinal symptoms. This human organ system can partially mirror intestinal barrier injury and the human response to viral infection, which is not possible in existing in vitro culture models. It provides a unique and rapid platform to accelerate COVID-19 research and develop novel therapies.

Ultrasound in the presence of microbubbles in a dynamic invitro microfluidic blood-brain barrier model

Controllable, localized permeability of the blood-brain barrier (BBB) has been a goal of health professionals for enhanced delivery of drugs and therapeutic compounds for cancer and other neurological medical conditions. The transient permeability induced by transcranial ultrasound exposure in the presence of microbubbles has been shown to be a promising option, but little is known about the exact mechanism and response on a cellular level. A microfluidic device developed to recreate the in vivo microenvironment, including physiological fluid flow, was seeded with human cerebral microvessel endothelial cells surrounded by a matrix of astrocytes. The devices were exposed to ultrasound with and without the presence of microbubbles. The ultrasound parameters ranged in frequency from 0.5 to 1.55 MHz, duty factor from 0.0001–0.001 and pulse repetition frequency of 1 Hz–10 Hz. Microbubble size, composition, and concentration within the pumped fluid were also varied, along with varied flow rate. The transepithelial electrical resistance (TEER) and cell morphology were monitored throughout and post ultrasound exposure to understand the effects on permeability and cell viability. With this controlled variation of the ultrasound and microbubble parameters, a greater understanding of the induced permeability could lead to more success for clinical utilization.