A Dynamic Culture Method to Produce Ovarian Cancer Spheroids under Physiologically-Relevant Shear Stress

The transcoelomic metastasis pathway is an alternative to traditional lymphatic/hematogenic metastasis. It is most frequently observed in ovarian cancer, though it has been documented in colon and gastric cancers as well. In transcoelomic metastasis, primary tumor cells are released into the abdominal cavity and form cell aggregates known as spheroids. These spheroids travel through the peritoneal fluid and implant at secondary sites, leading to the formation of new tumor lesions in the peritoneal lining and the organs in the cavity. Models of this process that incorporate the fluid shear stress (FSS) experienced by these spheroids are few, and most have not been fully characterized. Proposed herein is the adaption of a known dynamic cell culture system, the orbital shaker, to create an environment with physiologically-relevant FSS for spheroid formation. Experimental conditions (rotation speed, well size and cell density) were optimized to achieve physiologically-relevant FSS while facilitating the formation of spheroids that are also of a physiologically-relevant size. The FSS improves the roundness and size consistency of spheroids versus equivalent static methods and are even comparable to established high-throughput arrays, while maintaining nearly equivalent viability. This effect was seen in both highly metastatic and modestly metastatic cell lines. The spheroids generated using this technique were fully amenable to functional assays and will allow for better characterization of FSS’s effects on metastatic behavior and serve as a drug screening platform. This model can also be built upon in the future by adding more aspects of the peritoneal microenvironment, further enhancing its in vivo relevance.

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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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Proximal tubule apical endocytosis is modulated by fluid shear stress via an mTOR-dependent pathway

Molecular Biology of the Cell ◽

10.1091/mbc.e17-04-0211 ◽

2017 ◽

Vol 28 (19) ◽

pp. 2508-2517 ◽

Cited By ~ 19

Author(s):

Kimberly R. Long ◽

Katherine E. Shipman ◽

Youssef Rbaibi ◽

Elizabeth V. Menshikova ◽

Vladimir B. Ritov ◽

...

Keyword(s):

Shear Stress ◽

Ion Transport ◽

Proximal Tubule ◽

Fluid Shear Stress ◽

Kidney Development ◽

Endocytic Pathway ◽

Fluid Shear ◽

Intermediate Phenotypes ◽

Glomerulotubular Balance

Cells lining the proximal tubule (PT) have unique membrane specializations that are required to maintain the high-capacity ion transport and endocytic functions of this nephron segment. PT cells in vivo acutely regulate ion transport in response to changes in glomerular filtration rate (GFR) to maintain glomerulotubular balance. PT cells in culture up-regulate endocytic capacity in response to acute changes in fluid shear stress (FSS); however, it is not known whether GFR modulates PT endocytosis to enable maximally efficient uptake of filtered proteins in vivo. Here, we show that cells cultured under continuous FSS develop an expanded apical endocytic pathway and increased endocytic capacity and lysosomal biogenesis. Furthermore, endocytic capacity in fully differentiated cells is rapidly modulated by changes in FSS. PT cells exposed to continuous FSS also acquired an extensive brush border and basolateral membrane invaginations resembling those observed in vivo. Culture under suboptimal levels of FSS led to intermediate phenotypes, suggesting a threshold effect. Cells exposed to FSS expressed higher levels of key proteins necessary for PT function, including ion transporters, receptors, and membrane-trafficking machinery, and increased adenine nucleotide levels. Inhibition of the mechanistic target of rapamycin (mTOR) using rapamycin prevented the increase in cellular energy levels, lysosomal biogenesis, and endocytic uptake, suggesting that these represent a coordinated differentiation program. In contrast, rapamycin did not prevent the FSS-induced increase in Na+/K+-ATPase levels. Our data suggest that rapid tuning of the endocytic response by changes in FSS may contribute to glomerulotubular balance in vivo. Moreover, FSS provides an essential stimulus in the differentiation of PT cells via separate pathways that up-regulate endocytosis and ion transport capacity. Variations in FSS may also contribute to the maturation of PT cells during kidney development and during repair after kidney injury.

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ShearFAST: a user-friendly in vitro toolset for high throughput, inexpensive fluid shear stress experiments.

10.1101/2020.01.31.929513 ◽

2020 ◽

Author(s):

Thomas Brendan Smith ◽

Alessandro Marco De Nunzio ◽

Kamlesh Patel ◽

Haydn Munford ◽

Tabeer Alam ◽

...

Keyword(s):

Shear Stress ◽

High Throughput ◽

Fluid Shear Stress ◽

Tracking System ◽

Frequency Measurement ◽

Camera Motion ◽

Fluid Shear ◽

Mean Frequency

Fluid shear stress is a key modulator of cellular physiology in vitro and in vivo, but its effects are under-investigated due to requirements for complicated induction methods. Herein we report the validation of ShearFAST; a smartphone application that measures the rocking profile on a standard laboratory cell rocker and calculates the resulting shear stress arising in tissue culture plates. The accuracy with which this novel approach measured rocking profiles was validated against a graphical analysis, and also against measures reported by an 8-camera motion tracking system. ShearFASTs angle assessments correlated well with both analyses (r ≥0.99, p ≤0.001) with no significant differences in pitch detected across the range of rocking angles tested. Rocking frequency assessment by ShearFAST also correlated well when compared to the two independent validatory techniques (r ≥0.99, p ≤0.0001), with excellent reproducibility between ShearFAST and video analysis (mean frequency measurement difference of 0.006 ± 0.005Hz) and motion capture analysis (mean frequency measurement difference of 0.008 ± 0.012Hz). These data make the ShearFAST assisted cell rocker model make it an attractive approach for economical, high throughput fluid shear stress experiments. Proof of concept data presented reveals a protective effect of low-level shear stress on renal proximal tubule cells submitted to simulations of pretransplant storage.

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Duration of exposure to high fluid shear stress is critical in shear-induced platelet activation-aggregation

Thrombosis and Haemostasis ◽

10.1160/th03-03-0145 ◽

2003 ◽

Vol 90 (10) ◽

pp. 672-678 ◽

Cited By ~ 17

Author(s):

Zhang Jian-ning ◽

Angela Bergeron ◽

Qinghua Yu ◽

Carol Sun ◽

Latresha McBride ◽

...

Keyword(s):

Shear Stress ◽

Platelet Aggregation ◽

Platelet Activation ◽

Whole Blood ◽

Fluid Shear Stress ◽

High Shear ◽

Platelet Response ◽

Short Pulses ◽

Hydrodynamic Effects

SummaryPlatelet functions are increasingly measured under flow conditions to account for blood hydrodynamic effects. Typically, these studies involve exposing platelets to high shear stress for periods significantly longer than would occur in vivo. In the current study, we demonstrate that the platelet response to high shear depends on the duration of shear exposure. In response to a 100 dyn/cm2 shear stress for periods less than 10-20 sec, platelets in PRP or washed platelets were aggregated, but minimally activated as demonstrated by P-selectin expression and binding of the activation-dependent αIIbβ3 antibody PAC-1 to sheared platelets. Furthermore, platelet aggregation under such short pulses of high shear was subjected to rapid disaggregation. The disaggregated platelets could be re-aggregated by ADP in a pattern similar to unsheared platelets. In comparison, platelets that are exposed to high shear for longer than 20 sec are activated and aggregated irreversibly. In contrast, platelet activation and aggregation were significantly greater in whole blood with significantly less disaggregation. The enhancement is likely via increased collision frequency of platelet-platelet interaction and duration of platelet-platelet association due to high cell density. It may also be attributed to the ADP release from other cells such as red blood cells because increased platelet aggregation in whole blood was partially inhibited by ADP blockage. These studies demonstrate that platelets have a higher threshold for shear stress than previously believed. In a pathologically relevant timeframe, high shear alone is likely to be insufficient in inducing platelet activation and aggregation, but acts synergistically with other stimuli.

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Nanoparticle localization in blood vessels: dependence on fluid shear stress, flow disturbances, and flow-induced changes in endothelial physiology

Nanoscale ◽

10.1039/c8nr03440k ◽

2018 ◽

Vol 10 (32) ◽

pp. 15249-15261 ◽

Cited By ~ 21

Author(s):

M. Juliana Gomez-Garcia ◽

Amber L. Doiron ◽

Robyn R. M. Steele ◽

Hagar I. Labouta ◽

Bahareh Vafadar ◽

...

Keyword(s):

Shear Stress ◽

Fluid Shear Stress ◽

Fluid Shear ◽

Nanoparticle Distribution ◽

Flow Models ◽

Flow Disturbances ◽

Induced Changes ◽

Stress Flow

Hemodynamic factors drive nanoparticle distribution in vivo and in vitro in cell-based flow models.

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CD44 regulates blood-brain barrier integrity in response to fluid shear stress

10.1101/2020.01.28.924043 ◽

2020 ◽

Author(s):

Brandon J. DeOre ◽

Paul P. Partyka ◽

Fan Fan ◽

Peter A. Galie

Keyword(s):

Blood Flow ◽

Shear Stress ◽

Blood Brain Barrier ◽

Fluid Shear Stress ◽

Small Gtpase ◽

Brain Barrier ◽

Fluid Shear ◽

Blood Brain

AbstractFluid shear stress is an important mediator of vascular permeability, yet the molecular mechanisms underlying the response of the blood-brain barrier to shear have yet to be studied in cerebral vasculature despite its importance for brain homeostasis. The goal of this study is to probe components of shear mechanotransduction within the blood-brain barrier to gain a better understanding of pathologies associated with changes in cerebral blood flow including ischemic stroke. Interrogating the effects of shear stress in vivo is complicated by the complexity of factors in the brain parenchyma and the difficulty associated with modulating blood flow regimes. Recent advances in the ability to mimic the in vivo microenvironment using three-dimensional in vitro models provide a controlled setting to study the response of the blood-brain barrier to shear stress. The in vitro model used in this study is compatible with real-time measurement of barrier function using transendothelial electrical resistance as well as immunocytochemistry and dextran permeability assays. These experiments reveal that there is a threshold level of shear stress required for barrier formation and that the composition of the extracellular matrix, specifically the presence of hyaluronan, dictates the flow response. Gene editing to modulate the expression of CD44, a receptor for hyaluronan that previous studies have identified to be mechanosensitive, demonstrates that the receptor is required for the endothelial response to shear stress. Manipulation of small GTPase activity reveals CD44 activates Rac1 while inhibiting RhoA activation. Additionally, adducin-γ localizes to tight junctions in response to shear stress and RhoA inhibition and is required to maintain the barrier. This study identifies specific components of the mechanosensing complex associated with the blood-brain barrier response to fluid shear stress, and therefore illuminates potential targets for barrier manipulation in vivo.

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PGC1α Regulates the Endothelial Response to Fluid Shear Stress via Telomerase Reverse Transcriptase Control of Heme Oxygenase-1

10.1101/2021.05.26.445830 ◽

2021 ◽

Author(s):

Shashi Kant ◽

Khanh-Van Tran ◽

Miroslava Kvandova ◽

Amada D. Caliz ◽

Hyung-Jin Yoo ◽

...

Keyword(s):

Shear Stress ◽

Reverse Transcriptase ◽

Heme Oxygenase ◽

Fluid Shear Stress ◽

Heme Oxygenase 1 ◽

Telomerase Reverse Transcriptase ◽

Fluid Shear ◽

Flow Alignment

Fluid shear stress (FSS) is known to mediate multiple phenotypic changes in the endothelium. Laminar FSS (undisturbed flow) is known to promote endothelial alignment to flow that is key to stabilizing the endothelium and rendering it resistant to atherosclerosis and thrombosis. The molecular pathways responsible for endothelial responses to FSS are only partially understood. Here we have identified peroxisome proliferator gamma coactivator-1α (PGC-1α) as a flow-responsive gene required for endothelial flow alignment in vitro and in vivo. Compared to oscillatory FSS (disturbed flow) or static conditions, laminar FSS (undisturbed flow) increased PGC-1α expression and its transcriptional co-activation. PGC-1α was required for laminar FSS-induced expression of telomerase reverse transcriptase (TERT) in vitro and in vivo via its association with ERRα and KLF4 on the TERT promoter. We found that TERT inhibition attenuated endothelial flow alignment, elongation, and nuclear polarization in response to laminar FSS in vitro and in vivo. Among the flow-responsive genes sensitive to TERT status was heme oxygenase-1 (HMOX1), a gene required for endothelial alignment to laminar FSS. Thus, these data suggest an important role for a PGC-1α-TERT-HMOX1 axis in the endothelial stabilization response to laminar FSS.

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Differential Responses of Endothelial Cells to Positive and Negative Wall Shear Stress Gradients

ASME 2010 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2010-19535 ◽

2010 ◽

Author(s):

Jennifer Dolan ◽

Sukhjinder Singh ◽

Hui Meng ◽

John Kolega

Keyword(s):

Shear Stress ◽

Wall Shear Stress ◽

Vessel Wall ◽

Fluid Shear Stress ◽

Cerebral Aneurysms ◽

In Vivo Studies ◽

Outer Side ◽

Fluid Shear ◽

Wall Shear

Cerebral aneurysms tend to develop at bifurcation apices or the outer side of curved vessels where the blood vessel wall experiences complex hemodynamics. In vivo studies have recently revealed that the initiation of cerebral aneurysms is confined to a well-defined hemodynamic microenvironment. Specifically aneurysms form where the vessel wall experiences high fluid shear stress (wall shear stress, WSS) and flow is accelerating, so that the wall is exposed to a positive spatial gradient in the fluid shear stress (wall shear stress gradient, WSSG)[1,2]. Closer examination of such in vivo studies reveals that exposure of the vessel wall to equally high WSS in the presence of decelerating flow, that is, negative WSSG, does not result in aneurysm-like remodeling.

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