Search Results (17)

Hydraulic shear has been widely accepted as one of the essential factors modulating phytoplankton growth. Previous experimental studies of algal growth have been conducted at the macroscopic level, and direct observation at the cell scale has been lacking. In this study, an algal-cell dynamic continuous observation platform (ACDCOP) is proposed with a parallel-plate flow chamber (PPFC) to capture cellular growth images which are then used as input to a computer vision algorithm featuring a pre-trained backpropagation neural network to quantitatively evaluate the volumes and volumetric growth rates of individual cells. The platform was applied to investigate the growth of Scenedesmus quadricauda cells under different hydraulic shear stress conditions. The results indicated that the threshold shear stress for the development of Scenedesmus quadricauda cells was 270 µL min⁻¹ (5.62 × 10⁻⁵ m² s⁻³). Cellular growth was inhibited at very low and very high intensities of hydraulic shear. Among all the experimental groups, the longest growth period for a cell, from attachment to PPFC to cell division, was 5.7 days. Cells with larger initial volumes produced larger volumes at division. The proposed platform could provide a novel approach for algal research by enabling direct observation of algal growth at the cell scale, and could potentially be applied to investigate the impacts of various environmental stressors such as nutrient, temperature, and light on cellular growth in different algal species. Full article

The endothelial glycocalyx (GCX) plays a crucial role in vascular health and integrity and influences many biochemical activities through mechanotransduction, in which heparan sulfate (HS) plays a major role. Endothelin-1 (ET-1) is a potent vasoregulator that binds to the endothelin B receptor (ETB) on endothelial cells (ECs), stimulating vasodilation, and to the endothelin A receptor on smooth muscle cells, stimulating vasoconstriction. While the shear stress (SS) dependence of ET-1 and HS is well documented, there is limited research documenting the SS dependence of the ETB. Understanding the SS dependence of the ETB is crucial for clarifying the role of hemodynamic forces in the endothelin system. We hypothesize that GCX HS regulates the expression of the ETB on the EC surface in an SS-dependent manner. Human lung microvascular ECs were exposed to SS in a parallel-plate flow chamber for 12 h. Damage to the GCX was simulated by treatment with 15 mU/mL heparinase-III during SS exposure. Immunostaining and qPCR were used to evaluate changes in ET-1, ETB, and HS expression. Results indicate that ETB expression is SS sensitive, with at least a 1.3-fold increase in ETB protein expression and a 0.6 to 0.4-fold-change decrease in ETB mRNA expression under SS. This discrepancy suggests post-translational regulation. In some cases, enzymatic degradation of HS attenuated the SS-induced increase in ETB protein, reducing the fold-change difference to 1.1 relative to static controls. This implies that ETB expression may be partially dependent on HS-mediated mechanotransduction, though inconclusively. Furthermore, ET-1 mRNA levels were elevated two-fold under SS without a corresponding rise in ET-1 protein expression or significant impact from HS degradation, implying that post-translational regulation of ET-1 occurs independently of HS. Full article

The interaction between integrin α4β7 and mucosal vascular addressin cell-adhesion molecule-1 (MAdCAM-1) facilitates the adhesion of circulating lymphocytes to the surface of high endothelial venules in inflammatory bowel diseases (IBDs). Lymphocyte adhesion is a multistep cascade involving the tethering, rolling, stable adhesion, crawling, and migration of cells, with integrin α4β7 being involved in rolling and stable adhesions. Targeting the integrin α4β7–MAdCAM-1 interaction may help decrease inflammation in IBDs. This interaction is regulated by force; however, the underlying mechanism remains unknown. Here, we investigate this mechanism using a parallel plate flow chamber and atomic force microscopy. The results reveal an initial increase in the lifetime of the integrin α4β7–MAdCAM-1 interaction followed by a decrease with an increasing force. This was manifested in a two-state curve regulated via a catch-bond–slip-bond conversion regardless of Ca²⁺ and/or Mg²⁺ availability. In contrast, the mean rolling velocity of cells initially decreased and then increased with the increasing force, indicating the flow-enhanced adhesion. Longer tether lifetimes of single bonds and lower rolling velocities mediated by multiple bonds were observed in the presence of Mg²⁺ rather than Ca²⁺. Similar results were obtained when examining the adhesion to substrates co-coated with chemokine CC motif ligand 25 and MAdCAM-1, as opposed to substrates coated with MAdCAM-1 alone. In conclusion, the integrin α4β7–MAdCAM-1 interaction occurs via ion- and cytokine-dependent flow-enhanced adhesion processes and is regulated via a catch-bond mechanism. Full article

The use of three-dimensional (3D) cell cultures has become increasingly popular in the contexts of drug discovery, disease modelling, and tissue engineering, as they aim to replicate in vivo-like conditions. To achieve this, new hydrogels are being developed to mimic the extracellular matrix. Testing the ability of these hydrogels is crucial, and the presented 3D-printed microfluidic perfusion system offers a novel solution for the parallel cultivation and evaluation of four separate 3D cell cultures. This system enables easy microscopic monitoring of the hydrogel-embedded cells and significantly reduces the required volumes of hydrogel and cell suspension. This cultivation device is comprised of two 3D-printed parts, which provide four cell-containing hydrogel chambers and the associated perfusion medium chambers. An interfacing porous membrane ensures a defined hydrogel thickness and prevents flow-induced hydrogel detachment. Integrated microfluidic channels connect the perfusion chambers to the overall perfusion system, which can be operated in a standard CO₂-incubator. A 3D-printed adapter ensures the compatibility of the cultivation device with standard imaging systems. Cultivation and cell staining experiments with hydrogel-embedded murine fibroblasts confirmed that cell morphology, viability, and growth inside this cultivation device are comparable with those observed within standard 96-well plates. Due to the high degree of customization offered by additive manufacturing, this system has great potential to be used as a customizable platform for 3D cell culture applications. Full article

MAdCAM-1 binds to integrin α₄β₇, which mediates the rolling and arrest of circulating lymphocytes upon the vascular endothelia during lymphocytic homing. The calcium response by adhered lymphocytes is a critical event for lymphocyte activation and subsequent arrest and migration under flow. However, whether the interaction of integrin α₄β₇ /MAdCAM-1 can effectively trigger the calcium response of lymphocytes remains unclear, as well as whether the fluid force affects the calcium response. In this study, we explore the mechanical regulation of integrin α₄β₇-induced calcium signaling under flow. Flou-4 AM was used to examine the calcium response under real-time fluorescence microscopy when cells were firmly adhered to a parallel plate flow chamber. The interaction between integrin α₄β₇ and MAdCAM-1 was found to effectively trigger calcium signaling in firmly adhered RPMI 8226 cells. Meanwhile, increasing fluid shear stress accelerated the cytosolic calcium response and enhanced signaling intensity. Additionally, the calcium signaling of RPMI 8226 activated by integrin α₄β₇ originated from extracellular calcium influx instead of cytoplasmic calcium release, and the signaling transduction of integrin α₄β₇ was involved in Kindlin-3. These findings shed new light on the mechano-chemical mechanism of calcium signaling in RPMI 8226 cells induced by integrin α₄β_7. Full article

The vascular endothelial cells constitute the innermost layer. The cells are exposed to mechanical stress by the flow, causing them to express their functions. To elucidate the functions, methods involving seeding endothelial cells as a layer in a chamber were studied. The chambers are known as parallel plate, T-chamber, step, cone plate, and stretch. The stimulated functions or signals from endothelial cells by flows are extensively connected to other outer layers of arteries or organs. The coculture layer was developed in a chamber to investigate the interaction between smooth muscle cells in the middle layer of the blood vessel wall in vascular physiology and pathology. Additionally, the microfabrication technology used to create a chamber for a microfluidic device involves both mechanical and chemical stimulation of cells to show their dynamics in in vivo microenvironments. The purpose of this study is to summarize the blood flow (flow inducing) for the functions connecting to endothelial cells and blood vessels, and to find directions for future chamber and device developments for further understanding and application of vascular functions. The relationship between chamber design flow, cell layers, and microfluidics was studied. Full article

During mechanical stimulation-induced bone remodeling, interstitial fluid around microcracks may produce a flow field with gradient fluid shear stress (FSS). Osteoclast precursors can sense this gradient FSS and migrate toward the low FSS region. However, the local distribution of wall FSS on bone cells under a flow field with globally gradient FSS remains unknown. In this study, finite element models of a modified plate flow chamber with cells were constructed. The effect of oscillatory flow with different FSS levels and cell spacings or frequencies on the distribution of local wall FSS around cells was simulated by using a fluid–solid coupling method. Results showed that the polarization of wall FSS distribution in a cell decreased with the increase in cell spacing. At a low FSS level, the frequency of oscillatory flow had a minimal effect on the wall FSS distribution. At a high FSS level, the increase in flow frequency enhanced the fluctuation of local wall FSS distribution on cells. These results provide a basis for future research on the flow-induced migration of osteoclast precursors and clarify the mechanism of mechanical stimulation-induced bone resorption. Full article

Endothelial and epithelial cellular barriers play a vital role in the selective transport of solutes and other molecules. The properties and function of these barriers are often affected in case of inflammation and disease. Modelling cellular barriers in vitro can greatly facilitate studies of inflammation, disease mechanisms and progression, and in addition, can be exploited for drug screening and discovery. Here, we report on a parallelizable microfluidic platform in a multiwell plate format with ten independent cell culture chambers to support the modelling of cellular barriers co-cultured with 3D tumor spheroids. The microfluidic platform was fabricated by microinjection molding. Electrodes integrated into the chip in combination with a FT-impedance measurement system enabled transepithelial/transendothelial electrical resistance (TEER) measurements to rapidly assess real-time barrier tightness. The fluidic layout supports the tubeless and parallelized operation of up to ten distinct cultures under continuous unidirectional flow/perfusion. The capabilities of the system were demonstrated with a co-culture of 3D tumor spheroids and cellular barriers showing the growth and interaction of HT29 spheroids with a cellular barrier of MDCK cells. Full article

Cardiovascular disease is a major cause of death globally. This has led to significant efforts to develop new anti-thrombotic therapies or re-purpose existing drugs to treat cardiovascular diseases. Due to difficulties of obtaining healthy human blood vessel tissues to recreate in vivo conditions, pre-clinical testing of these drugs currently requires significant use of animal experimentation, however, the successful translation of drugs from animal tests to use in humans is poor. Developing humanised drug test models that better replicate the human vasculature will help to develop anti-thrombotic therapies more rapidly. Tissue-engineered human blood vessel (TEBV) models were fabricated with biomimetic matrix and cellular components. The pro- and anti-aggregatory properties of both intact and FeCl₃-injured TEBVs were assessed under physiological flow conditions using a modified parallel-plate flow chamber. These were perfused with fluorescently labelled human platelets and endothelial progenitor cells (EPCs), and their responses were monitored in real-time using fluorescent imaging. An endothelium-free TEBV exhibited the capacity to trigger platelet activation and aggregation in a shear stress-dependent manner, similar to the responses observed in vivo. Ketamine is commonly used as an anaesthetic in current in vivo models, but this drug significantly inhibited platelet aggregation on the injured TEBV. Atorvastatin was also shown to enhance EPC attachment on the injured TEBV. The TEBV, when perfused with human blood or blood components under physiological conditions, provides a powerful alternative to current in vivo drug testing models to assess their effects on thrombus formation and EPC recruitment. Full article

Despite the distant metastasis of cervical cancer cells being a prominent cause of mortality, neither the metastasis capacity nor the in vitro conditions mimicking adhesion of cervical cancer cells to endothelial cells have been fully elucidated. Circulating metastatic cancer cells undergo transendothelial migration and invade normal organs in distant metastasis; however, the putative molecular mechanism remains largely uncertain. In this study, we describe the use of an in vitro parallel-plate flow chamber to simulate the dynamic circulation stress on cervical cancer cells and elucidate their vascular adhesion and metastasis. We isolate the viable and shear stress-resistant (SSR) cervical cancer cells for mechanistic studies. Remarkably, the identified SSR-HeLa and SSR-CaSki exhibited high in vitro adhesive and metastatic activities. Hence, a consistently suppressed miR-128 level was revealed in SSR cell clones compared to those of parental wild-type (WT) cells. Overexpressed miR-128 attenuated SSR-HeLa cells’ adherence to human umbilical cord vein endothelial cells (HUVECs); in contrast, suppressed miR-128 efficiently augmented the static adhesion capacity in WT-HeLa and WT-CaSki cells. Hence, amplified miR-128 modestly abolished in vitro SSR-augmented HeLa and CaSki cell movement, whereas reduced miR-128 aggravated the migration speed in a time-lapse recording assay in WT groups. Consistently, the force expression of miR-128 alleviated the SSR-enhanced HeLa and CaSki cell mobility in a wound healing assay. Notably, miR-128 mediated SSR-enhanced HeLa and CaSki cells’ adhesion and metastasis through suppressed ITGA5, ITGB5, sLex, CEACAM-6, MMP9, and MMP23 transcript levels. Our data provide evidence suggesting that miR-128 is a promising microRNA that prevented endothelial cells’ adhesion and transendothelial migration to contribute to the SSR-enhanced adhesion and metastasis progression under a parallel-plate flow chamber system. This indicates that the nucleoid-based miR-128 strategy may be an attractive therapeutic strategy to eliminate tumor cells resistant to circulation shear flow, prevent vascular adhesion, and preclude subsequent transendothelial metastasis. Full article

Parallel plate flow chambers are widely used to expose cultured cells to physiological flows for the investigation of a variety of diseases. These applications usually involve the generation of continuous and steady fluid flow over cell monolayers for extended durations, usually a few days. Another technique is to generate a fast high-stress wave over the cells to see the immediate effect of flow-induced stresses. This can be achieved by propagating an air/liquid interface, in other words, a bubble, over cell monolayers. The approach is relevant to the reopening event of fluid-filled lung bronchioles and alveoli during mechanical ventilation therapy of Acute Respiratory Distress Syndrome. This article explains how we generate a stress wave using a parallel plate flow chamber and presents representative results of this wave on cultured lung epithelial cells. Full article

Different studies have shown that the incorporation of carbon nanotubes (CNTs) into poly(dimethylsiloxane) (PDMS) enables the production of composite materials with enhanced properties, which can find important applications in the biomedical field. In the present work, CNT/PDMS composite materials have been prepared to evaluate the effects of pristine and chemically functionalized CNT incorporation into PDMS on the composite’s thermal, electrical, and surface properties on bacterial adhesion in dynamic conditions. Initial bacterial adhesion was studied using a parallel-plate flow chamber assay performed in conditions prevailing in urinary tract devices (catheters and stents) using Escherichia coli as a model organism and PDMS as a control due to its relevance in these applications. The results indicated that the introduction of the CNTs in the PDMS matrix yielded, in general, less bacterial adhesion than the PDMS alone and that the reduction could be dependent on the surface chemistry of CNTs, with less adhesion obtained on the composites with pristine rather than functionalized CNTs. It was also shown CNT pre-treatment and incorporation by different methods affected the electrical properties of the composites when compared to PDMS. Composites enabling a 60% reduction in cell adhesion were obtained by CNT treatment by ball-milling, whereas an increase in electrical conductivity of seven orders of magnitude was obtained after solvent-mediated incorporation. The results suggest even at low CNT loading values (1%), these treatments may be beneficial for the production of CNT composites with application in biomedical devices for the urinary tract and for other applications where electrical conductance is required. Full article

Urinary tract infections are one of the most common hospital-acquired infections, and they are often associated with biofilm formation in indwelling medical devices such as catheters and stents. This study aims to investigate the antibiofilm performance of a polymer brush—poly[oligo(ethylene glycol) methyl ether methacrylate], poly(MeOEGMA)—and evaluate its effect on the antimicrobial susceptibility of Escherichia coli biofilms formed on that surface. Biofilms were formed in a parallel plate flow chamber (PPFC) for 24 h under the hydrodynamic conditions prevailing in urinary catheters and stents and challenged with ampicillin. Results obtained with the brush were compared to those obtained with two control surfaces, polydimethylsiloxane (PDMS) and glass. The polymer brush reduced by 57% the surface area covered by E. coli after 24 h, as well as the number of total adhered cells. The antibiotic treatment potentiated cell death and removal, and the total cell number was reduced by 88%. Biofilms adapted their architecture, and cell morphology changed to a more elongated form during that period. This work suggests that the poly(MeOEGMA) brush has potential to prevent bacterial adhesion in urinary tract devices like ureteral stents and catheters, as well as in eradicating biofilms developed in these biomedical devices. Full article

Immune thrombocytopenia (ITP) is an autoimmune condition primarily induced by the loss of immune tolerance to the platelet glycoproteins. Here we develop a novel flow cytometry approach to analyze integrin α_IIbβ₃ functioning in ITP in comparison with Glanzmann thrombasthenia (GT) (negative control) and healthy pediatric donors (positive control). Continuous flow cytometry of Fura-Red-loaded platelets from whole hirudinated blood was used for the characterization of platelet responses to conventional activators. Calcium levels and fibrinogen binding were normalized to ionomycin-induced responses. Ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Platelets from all ITP patients had significantly higher cytosolic calcium concentration in the quiescent state compared to healthy donors (15 ± 5 nM vs. 8 ± 5 nM), but calcium increases in response to all activators were normal. Clustering analysis revealed two subpopulations of ITP patients: the subgroup with high fibrinogen binding (HFB), and the subgroup with low fibrinogen binding (LFB) (8% ± 5% for LFB vs. 16% ± 3% for healthy donors in response to ADP). GT platelets had calcium mobilization (81 ± 23 nM), fibrinogen binding (5.1% ± 0.3%) and thrombus growth comparable to the LFB subgroup. Computational modeling suggested phospholipase C-dependent platelet pre-activation for the HFB subgroup and lower levels of functional integrin molecules for the LFB group. Full article

Background: Metabolic syndrome is associated with low-grade systemic inflammation, which is a key driver of premature atherosclerosis. We characterized immune cell behavior in metabolic syndrome, its consequences, and the potential involvement of the CX₃CL1/CX₃CR1 and CCL2/CCR2 chemokine axes. Methods: Whole blood from 18 patients with metabolic syndrome and 21 age-matched controls was analyzed by flow cytometry to determine the leukocyte immunophenotypes, activation, platelet-leukocyte aggregates, and CX₃CR1 expression. ELISA determined the plasma marker levels. Platelet-leukocyte aggregates adhesion to tumor necrosis factor-α (TNFα)-stimulated arterial endothelium and the role of CX₃CL1/CX₃CR1 and CCL2/CCR2 axes was investigated with the parallel-plate flow chamber. Results: When compared with the controls, the metabolic syndrome patients presented greater percentages of eosinophils, CD3⁺ T lymphocytes, Mon2/Mon3 monocytes, platelet-eosinophil and -lymphocyte aggregates, activated platelets, neutrophils, eosinophils, monocytes, and CD8⁺ T cells, but lower percentages of Mon1 monocytes. Patients had increased circulating interleukin-8 (IL-8) and TNFα levels and decreased IL-4. CX₃CR1 up-regulation in platelet-Mon1 monocyte aggregates in metabolic syndrome patients led to increased CX₃CR1/CCR2-dependent platelet-Mon1 monocyte adhesion to dysfunctional arterial endothelium. Conclusion: We provide evidence of generalized immune activation in metabolic syndrome. Additionally, CX₃CL1/CX₃CR1 or CCL2/CCR2 axes are potential candidates for therapeutic intervention in cardiovascular disorders in metabolic syndrome patients, as their blockade impairs the augmented arterial platelet-Mon1 monocyte aggregate adhesiveness, which is a key event in atherogenesis. Full article