Ion Conductance-Based Perfusability Assay of Vascular Vessel Models in Microfluidic Devices

We present a novel methodology based on ion conductance to evaluate the perfusability of vascular vessels in microfluidic devices without microscopic imaging. The devices consisted of five channels, with the center channel filled with fibrin/collagen gel containing human umbilical vein endothelial cells (HUVECs). Fibroblasts were cultured in the other channels to improve the vascular network formation. To form vessel structures bridging the center channel, HUVEC monolayers were prepared on both side walls of the gel. During the culture, the HUVECs migrated from the monolayer and connected to the HUVECs in the gel, and vascular vessels formed, resulting in successful perfusion between the channels after culturing for 3–5 d. To evaluate perfusion without microscopic imaging, Ag/AgCl wires were inserted into the channels, and ion currents were obtained to measure the ion conductance between the channels separated by the HUVEC monolayers. As the HUVEC monolayers blocked the ion current flow, the ion currents were low before vessel formation. In contrast, ion currents increased after vessel formation because of creation of ion current paths. Thus, the observed ion currents were correlated with the perfusability of the vessels, indicating that they can be used as indicators of perfusion during vessel formation in microfluidic devices. The developed methodology will be used for drug screening using organs-on-a-chip containing vascular vessels.

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MiR-323 Inhibits Prostate Cancer Vascularization Through Adiponectin Receptor

Cellular Physiology and Biochemistry ◽

10.1159/000430313 ◽

2015 ◽

Vol 36 (4) ◽

pp. 1491-1498 ◽

Cited By ~ 17

Author(s):

Qiruo Gao ◽

Xudong Yao ◽

Junhua Zheng

Keyword(s):

Umbilical Vein ◽

Collagen Gel ◽

Adiponectin Receptor ◽

Luciferase Reporter ◽

Normal Prostate ◽

Vessel Formation ◽

Protein Levels ◽

Normal Prostate Tissue ◽

Prostate Cancers ◽

Umbilical Vein Endothelial Cells

Background/Aims: The current treatments fail to provide satisfactory cure for aggressive prostate cancers (PCs). Hence, further comprehension of PC metastasis is highly appreciated for improving the levels of therapy. We have previously shown that Adiponectin reduces the levels of vascular endothelial growth factor A (VEGF-A) in PCs to suppress tumor-associated neovascularization, possibly through AMPK/mTor signaling. Here, we studied the regulation of Adiponectin signaling in PCs. Methods: We analyzed the levels and correlation of Adiponectin receptor 1 (AdipoR1) and microRNA-323 (miR-323) in the PC specimen, compared to the paired normal prostate tissue. We analyzed the binding of miR-323 to the 3'UTR of AdipoR1 mRNA and its effects on AdipoR1 translation by bioinformatics analysis and by luciferase-reporter assay, respectively. We modified miR-323 levels in PC cells, and examined the effects on the expression of AdipoR1 and VEGF-A, as well as on vessel formation in a human umbilical vein endothelial cells (HUVECs) transwell collagen gel assay. Results: We detected significantly lower levels of AdipoR1 and significantly higher levels of miR-323 in PC specimen. Moreover, the levels of AdipoR1 and miR-323 are inversely correlated. Moreover, miR-323 was found to bind to the 3'UTR of AdipoR1 mRNA to inhibit its translation. Overexpression of miR-323 in PC cells decreased AdipoR1 protein levels, whereas inhibition of miR-323 increased AdipoR1 protein levels, without affecting AdipoR1 transcripts. Moreover, overexpression of miR-323 increased the levels of VEGF-A and the vessel formation by HUVECs, while inhibition of miR-323 decreased the levels of VEGF-A and the vessel formation by HUVECs. Conclusion: Our data demonstrate that miR-323 may increase VEGF-A-mediated cancer vascularization in PC cells through AdipoR1 suppression.

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Acoustic Cell Patterning in Hydrogel for Three-Dimensional Cell Network Formation

Micromachines ◽

10.3390/mi12010003 ◽

2020 ◽

Vol 12 (1) ◽

pp. 3

Author(s):

Kyo-in Koo ◽

Andreas Lenshof ◽

Le Thi Huong ◽

Thomas Laurell

Keyword(s):

Network Formation ◽

Umbilical Vein ◽

Three Dimensional ◽

Extrusion Process ◽

Fibroblast Cells ◽

Glass Capillary ◽

Cell Network ◽

Significant Difference ◽

The One ◽

Umbilical Vein Endothelial Cells

In the field of engineered organ and drug development, three-dimensional network-structured tissue has been a long-sought goal. This paper presents a direct hydrogel extrusion process exposed to an ultrasound standing wave that aligns fibroblast cells to form a network structure. The frequency-shifted (2 MHz to 4 MHz) ultrasound actuation of a 400-micrometer square-shaped glass capillary that was continuously perfused by fibroblast cells suspended in sodium alginate generated a hydrogel string, with the fibroblasts aligned in single or quadruple streams. In the transition from the one-cell stream to the four-cell streams, the aligned fibroblast cells were continuously interconnected in the form of a branch and a junction. The ultrasound-exposed fibroblast cells displayed over 95% viability up to day 10 in culture medium without any significant difference from the unexposed fibroblast cells. This acoustofluidic method will be further applied to create a vascularized network by replacing fibroblast cells with human umbilical vein endothelial cells.

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Rapid Fabrication of Microfluidic Devices for Biological Mimicking: A Survey of Materials and Biocompatibility

Micromachines ◽

10.3390/mi12030346 ◽

2021 ◽

Vol 12 (3) ◽

pp. 346

Author(s):

Hui Ling Ma ◽

Ana Carolina Urbaczek ◽

Fayene Zeferino Ribeiro de Souza ◽

Paulo Augusto Gomes Garrido Carneiro Leão ◽

Janice Rodrigues Perussi ◽

...

Keyword(s):

Shear Stress ◽

Cell Viability ◽

Cellular Response ◽

Fluid Shear Stress ◽

Umbilical Vein ◽

Microfluidic Devices ◽

In Vitro Assays ◽

Stress Effect

Microfluidics is an essential technique used in the development of in vitro models for mimicking complex biological systems. The microchip with microfluidic flows offers the precise control of the microenvironment where the cells can grow and structure inside channels to resemble in vivo conditions allowing a proper cellular response investigation. Hence, this study aimed to develop low-cost, simple microchips to simulate the shear stress effect on the human umbilical vein endothelial cells (HUVEC). Differentially from other biological microfluidic devices described in the literature, we used readily available tools like heat-lamination, toner printer, laser cutter and biocompatible double-sided adhesive tapes to bind different layers of materials together, forming a designed composite with a microchannel. In addition, we screened alternative substrates, including polyester-toner, polyester-vinyl, glass, Permanox® and polystyrene to compose the microchips for optimizing cell adhesion, then enabling these microdevices when coupled to a syringe pump, the cells can withstand the fluid shear stress range from 1 to 4 dyne cm2. The cell viability was monitored by acridine orange/ethidium bromide (AO/EB) staining to detect live and dead cells. As a result, our fabrication processes were cost-effective and straightforward. The materials investigated in the assembling of the microchips exhibited good cell viability and biocompatibility, providing a dynamic microenvironment for cell proliferation. Therefore, we suggest that these microchips could be available everywhere, allowing in vitro assays for daily laboratory experiments and further developing the organ-on-a-chip concept.

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Syndecan-1 Promotes Angiogenesis in Triple-Negative Breast Cancer through the Prognostically Relevant Tissue Factor Pathway and Additional Angiogenic Routes

Cancers ◽

10.3390/cancers13102318 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2318

Author(s):

Eyyad Nassar ◽

Nourhan Hassan ◽

Eslam A. El-Ghonaimy ◽

Hebatallah Hassan ◽

Mahmoud Salah Abdullah ◽

...

Keyword(s):

Breast Cancer ◽

Tissue Factor ◽

Cell Lines ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Network Formation ◽

Umbilical Vein ◽

Poor Survival ◽

Altered Expression ◽

Basal Breast Cancer

Triple-negative breast cancer (TNBC) is characterized by increased angiogenesis, metastasis, and poor survival. Dysregulation of the cell surface heparan sulfate proteoglycan and signaling co-receptor Syndecan-1 is linked to poor prognosis. To study its role in angiogenesis, we silenced Syndecan-1 in TNBC cell lines using a 3D human umbilical vein endothelial cell (HUVEC) co-culture system. Syndecan-1 siRNA depletion in SUM-149, MDA-MB-468, and MDA-MB-231 cells decreased HUVEC tubule network formation. Angiogenesis array revealed reduced VEGF-A and tissue factor (TF) in the Syndecan-1-silenced secretome. qPCR independently confirmed altered expression of F3, F7, F2R/PAR1, F2RL1/PAR2, VEGF-A, EDN1, IGFBP1, and IGFBP2 in SUM-149, MDA-MB-231, and MDA-MB-468 cells. ELISA revealed reduced secreted endothelin-1 (SUM-149, MDA-MB-468) and TF (all cell lines) upon Syndecan-1 depletion, while TF pathway inhibitor treatment impaired angiogenesis. Survival analysis of 3951 patients demonstrated that high expression of F3 and F7 are associated with better relapse-free survival, whereas poor survival was observed in TNBC and p53 mutant basal breast cancer (F3) and in ER-negative and HER2-positive breast cancer (F2R, F2RL1). STRING protein network analysis revealed associations of Syndecan-1 with VEGF-A and IGFBP1, further associated with the TF and ET-1 pathways. Our study suggests that TNBC Syndecan-1 regulates angiogenesis via the TF and additional angiogenic pathways and marks its constituents as novel prognostic markers and therapeutic targets.

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Simultaneous scanning ion conductance and atomic force microscopy with a nanopore: Effect of the aperture edge on the ion current images

Journal of Applied Physics ◽

10.1063/1.5053879 ◽

2018 ◽

Vol 124 (17) ◽

pp. 174902 ◽

Cited By ~ 8

Author(s):

Livie Dorwling-Carter ◽

Morteza Aramesh ◽

Csaba Forró ◽

Raphael F. Tiefenauer ◽

Ivan Shorubalko ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Ion Current ◽

Ion Conductance ◽

Force Microscopy ◽

Atomic Force

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The selective binding and transmigration of monocytes through the junctional complexes of human endothelium.

Journal of Experimental Medicine ◽

10.1084/jem.168.5.1865 ◽

1988 ◽

Vol 168 (5) ◽

pp. 1865-1882 ◽

Cited By ~ 43

Author(s):

N A Pawlowski ◽

G Kaplan ◽

E Abraham ◽

Z A Cohn

Keyword(s):

Silver Staining ◽

Umbilical Vein ◽

Collagen Gel ◽

Collagen Gels ◽

Silver Stain ◽

Term Survival ◽

Topographical Distribution ◽

Junctional Complexes

Human monocytes show a high affinity for vascular endothelium both in vitro and in vivo. To explore monocyte-endothelial interaction in greater detail, we have developed a new in vitro model for growth of human endothelial cells (EC). Human umbilical vein EC (HUVEC) cultured upon collagen gels form confluent monolayers of EC that bind silver at their intercellular border similar to cells in situ. Intercellular junctional structures, both adherens and tight junctions, were identified. In contrast, HUVEC grown on plastic surfaces did not stain with silver. The silver-staining characteristic of EC-collagen monolayers was reversible and related to their in vitro maturation and senescence. Silver staining of EC borders provided a grid by which the location of monocyte binding to the luminal surface of individual EC could be assessed. Using this technique, we found that monocytes preferentially bound to the margins of EC, in approximation to the silver-staining junctions. These results suggest that EC determinants recognized by monocytes occur in a unique topographical distribution on the apical face of EC. After binding, monocytes migrated through the EC monolayers at high basal rates. The lack of penetration of collagen gels in the absence of an EC monolayer suggested the generation of EC-specific chemotactic signal(s). Monocytes were observed to pass between EC without evidence of disruption of the monolayer. Silver stain remained present during all phases of migration, and under transmission electron microscopy, junctional complexes were found proximal to monocytes that had just completed their passage through the monolayer. After orientation to the basal surface of the EC monolayer, monocytes migrated randomly into the underlying collagen gel. Monocyte adherence, penetration, migration, and long term survival can be studied under these conditions.

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Sinusoidal voltage protocols for rapid characterisation of ion channel kinetics

10.1101/100677 ◽

2017 ◽

Cited By ~ 3

Author(s):

Kylie A. Beattie ◽

Adam P. Hill ◽

Rémi Bardenet ◽

Yi Cui ◽

Jamie I. Vandenberg ◽

...

Keyword(s):

Ion Channel ◽

Voltage Clamp ◽

Ion Current ◽

Ion Currents ◽

Channel Kinetics ◽

Sinusoidal Voltage ◽

Square Wave ◽

Voltage Clamps ◽

Ion Channel Kinetics ◽

Cell Cell

AbstractUnderstanding the roles of ion currents is crucial to predict the action of pharmaceuticals and mutations in different scenarios, and thereby to guide clinical interventions in the heart, brain and other electrophysiological systems. Our ability to predict how ion currents contribute to cellular electrophysiology is in turn critically dependent on our characterisation of ion channel kinetics — the voltage-dependent rates of transition between open, closed and inactivated channel states. We present a new method for rapidly exploring and characterising ion channel kinetics, applying it to the hERG potassium channel as an example, with the aim of generating a quantitatively predictive representation of the ion current. We fit a mathematical model to currents evoked by a novel 8 second sinusoidal voltage clamp in CHO cells over-expressing hERG1a. The model is then used to predict over 5 minutes of recordings in the same cell in response to further protocols: a series of traditional square step voltage clamps, and also a novel voltage clamp comprised of a collection of physiologically-relevant action potentials. We demonstrate that we can make predictive cell-specific models that outperform the use of averaged data from a number of different cells, and thereby examine which changes in gating are responsible for cell-cell variability in current kinetics. Our technique allows rapid collection of consistent and high quality data, from single cells, and produces more predictive mathematical ion channel models than traditional approaches.Table of Contents CategoryTechniques for Physiology1Key PointsIon current kinetics are commonly represented by current-voltage relationships, time-constant voltage relationships, and subsequently mathematical models fitted to these. These experiments take substantial time which means they are rarely performed in the same cell.Rather than traditional square-wave voltage clamps, we fit a model to the current evoked by a novel sum-of-sinusoids voltage clamp that is only 8 seconds long.Short protocols that can be performed multiple times within a single cell will offer many new opportunities to measure how ion current kinetics are affected by changing conditions.The new model predicts the current under traditional square-wave protocols well, with better predictions of underlying currents than literature models. The current under a novel physiologically-relevant series of action potential clamps is predicted extremely well.The short sinusoidal protocols allow a model to be fully fitted to individual cells, allowing us to examine cell-cell variability in current kinetics for the first time.

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Measurement of Ion Currents through Porous Membranes with Scanning Ion Conductance Microscopy

Analytical Chemistry ◽

10.1021/ac900065p ◽

2009 ◽

Vol 81 (12) ◽

pp. 4742-4751 ◽

Cited By ~ 48

Author(s):

Chiao-Chen Chen ◽

Maksymilian A. Derylo ◽

Lane A. Baker

Keyword(s):

Porous Membranes ◽

Ion Currents ◽

Ion Conductance ◽

Scanning Ion Conductance Microscopy

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Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00400.2003 ◽

2004 ◽

Vol 287 (3) ◽

pp. H994-H1002 ◽

Cited By ~ 47

Author(s):

Akinori Ueda ◽

Masaki Koga ◽

Mariko Ikeda ◽

Susumu Kudo ◽

Kazuo Tanishita

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Network Formation ◽

Three Dimensional ◽

Collagen Gel ◽

Flow Chamber ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Collagen Gels

Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after ∼10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (±0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (±0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.

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