scholarly journals An Easy-to-Fabricate Cell Stretcher Reveals Density-Dependent Mechanical Regulation of Collective Cell Movements in Epithelia

2021 ◽  
Author(s):  
Kevin C. Hart ◽  
Joo Yong Sim ◽  
Matthew A. Hopcroft ◽  
Daniel J. Cohen ◽  
Jiongyi Tan ◽  
...  
Keyword(s):  
Low Cost ◽  
Cell Movement ◽  
Time Lapse ◽  
Adherent Cell ◽  
Dependent Manner ◽  
Cell Dynamics ◽  
Density Dependent ◽  
Cell Movements ◽  
Tissue Biology ◽  
Mechanical Regulation

Abstract Introduction Mechanical forces regulate many facets of cell and tissue biology. Studying the effects of forces on cells requires real-time observations of single- and multi-cell dynamics in tissue models during controlled external mechanical input. Many of the existing devices used to conduct these studies are costly and complicated to fabricate, which reduces the availability of these devices to many laboratories. Methods We show how to fabricate a simple, low-cost, uniaxial stretching device, with readily available materials and instruments that is compatible with high-resolution time-lapse microscopy of adherent cell monolayers. In addition, we show how to construct a pressure controller that induces a repeatable degree of stretch in monolayers, as well as a custom MATLAB code to quantify individual cell strains. Results As an application note using this device, we show that uniaxial stretch slows down cellular movements in a mammalian epithelial monolayer in a cell density-dependent manner. We demonstrate that the effect on cell movement involves the relocalization of myosin downstream of Rho-associated protein kinase (ROCK). Conclusions This mechanical device provides a platform for broader involvement of engineers and biologists in this important area of cell and tissue biology. We used this device to demonstrate the mechanical regulation of collective cell movements in epithelia.

scholarly journals An Easy-to-Fabricate Cell Stretching System Reveals Density-Dependent Mechanical Regulation of Collective Cell Movements in Epithelial Homeostasis

2020 ◽  
Author(s):  
Kevin C. Hart ◽  
Joo Yong Sim ◽  
Matthew A. Hopcroft ◽  
Daniel J. Cohen ◽  
Jiongyi Tan ◽  
...  
Keyword(s):  
Low Cost ◽  
Time Lapse ◽  
Adherent Cell ◽  
Dependent Manner ◽  
Cell Dynamics ◽  
Density Dependent ◽  
Cell Monolayers ◽  
Epithelial Homeostasis ◽  
Tissue Biology ◽  
A Cell

AbstractMechanical forces regulate many facets of tissue biology. Studying the effects of forces on cells requires real-time observations of single- and multi-cell dynamics in tissue models during controlled external mechanical input. Many of the existing devices used to conduct these studies are costly and complicated to fabricate, which reduces the availability of these devices to many laboratories. In this report, we show how to fabricate a simple, low-cost uniaxial stretching device with readily available materials and instruments that is compatible with high-resolution time-lapse microscopy of adherent cell monolayers. In addition, we show how to construct a pressure controller that induces a repeatable degree of stretch in monolayers, and a custom MATLAB code to quantify individual cell strains. Finally, as an application note using this device, we show that uniaxial stretch slows down cellular movements in an epithelial monolayer in a cell density-dependent manner that involves the relocalization of myosin downstream of Rho-associated protein kinase (ROCK). This mechanical device provides a platform for broader involvement of engineers and biologists in this important area of tissue biology.

scholarly journals Chemotactic Responses of Jurkat Cells in Microfluidic Flow-Free Gradient Chambers

Micromachines ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 384 ◽  
Author(s):  
Utku M. Sonmez ◽  
Adam Wood ◽  
Kyle Justus ◽  
Weijian Jiang ◽  
Fatima Syed-Picard ◽  
...  
Keyword(s):  
Immune Response ◽  
Cancer Progression ◽  
Soft Lithography ◽  
Cell Movement ◽  
Population Level ◽  
Jurkat Cells ◽  
Dependent Manner ◽  
Diverse Range ◽  
Microfluidic Flow ◽  
Cell Motion

Gradients of soluble molecules coordinate cellular communication in a diverse range of multicellular systems. Chemokine-driven chemotaxis is a key orchestrator of cell movement during organ development, immune response and cancer progression. Chemotaxis assays capable of examining cell responses to different chemokines in the context of various extracellular matrices will be crucial to characterize directed cell motion in conditions which mimic whole tissue conditions. Here, a microfluidic device which can generate different chemokine patterns in flow-free gradient chambers while controlling surface extracellular matrix (ECM) to study chemotaxis either at the population level or at the single cell level with high resolution imaging is presented. The device is produced by combining additive manufacturing (AM) and soft lithography. Generation of concentration gradients in the device were simulated and experimentally validated. Then, stable gradients were applied to modulate chemotaxis and chemokinetic response of Jurkat cells as a model for T lymphocyte motility. Live imaging of the gradient chambers allowed to track and quantify Jurkat cell migration patterns. Using this system, it has been found that the strength of the chemotactic response of Jurkat cells to CXCL12 gradient was reduced by increasing surface fibronectin in a dose-dependent manner. The chemotaxis of the Jurkat cells was also found to be governed not only by the CXCL12 gradient but also by the average CXCL12 concentration. Distinct migratory behaviors in response to chemokine gradients in different contexts may be physiologically relevant for shaping the host immune response and may serve to optimize the targeting and accumulation of immune cells to the inflammation site. Our approach demonstrates the feasibility of using a flow-free gradient chamber for evaluating cross-regulation of cell motility by multiple factors in different biologic processes.

scholarly journals Development of single-cell-level microfluidic technology for long-term growth visualization of living cultures of Mycobacterium smegmatis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Han Wang ◽  
Gloria M. Conover ◽  
Song-I Han ◽  
James C. Sacchettini ◽  
Arum Han
Keyword(s):  
Drug Treatment ◽  
Single Cell ◽  
Mycobacterium Smegmatis ◽  
Culture Media ◽  
Low Cost ◽  
Bacterial Pathogen ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Cell Phenotype

AbstractAnalysis of growth and death kinetics at single-cell resolution is a key step in understanding the complexity of the nonreplicating growth phenotype of the bacterial pathogen Mycobacterium tuberculosis. Here, we developed a single-cell-resolution microfluidic mycobacterial culture device that allows time-lapse microscopy-based long-term phenotypic visualization of the live replication dynamics of mycobacteria. This technology was successfully applied to monitor the real-time growth dynamics of the fast-growing model strain Mycobacterium smegmatis (M. smegmatis) while subjected to drug treatment regimens during continuous culture for 48 h inside the microfluidic device. A clear morphological change leading to significant swelling at the poles of the bacterial membrane was observed during drug treatment. In addition, a small subpopulation of cells surviving treatment by frontline antibiotics was observed to recover and achieve robust replicative growth once regular culture media was provided, suggesting the possibility of identifying and isolating nonreplicative mycobacteria. This device is a simple, easy-to-use, and low-cost solution for studying the single-cell phenotype and growth dynamics of mycobacteria, especially during drug treatment.

scholarly journals Density-dependent nerve growth factor regulation of Gs-alpha RNA in pheochromocytoma 12 cells.

1990 ◽  
Vol 10 (6) ◽  
pp. 3277-3279 ◽  
Author(s):  
G Tjaden ◽  
A Aguanno ◽  
R Kumar ◽  
D Benincasa ◽  
R M Gubits ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
G Protein ◽  
Alpha Subunit ◽  
Low Density ◽  
Dependent Manner ◽  
Nerve Growth ◽  
Density Dependent ◽  
Stimulatory G Protein ◽  
Gs Alpha

Nerve growth factor (NGF) affects levels of the alpha subunit of the stimulatory G protein (Gs-alpha) in pheochromocytoma 12 cells in a bidirectional, density-dependent manner. Cells grown at high density responded to NGF treatment with increased levels of Gs-alpha mRNA and protein. Conversely, in cells grown in low-density cultures, levels of this mRNA were lowered by NGF treatment.

scholarly journals An In Vitro Model for Assessing Corneal Keratocyte Spreading and Migration on Aligned Fibrillar Collagen

2018 ◽  
Vol 9 (4) ◽  
pp. 54 ◽  
Author(s):  
Pouriska Kivanany ◽  
Kyle Grose ◽  
Nihan Yonet-Tanyeri ◽  
Sujal Manohar ◽  
Yukta Sunkara ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Cell Movement ◽  
Time Lapse ◽  
Collagen Fibrils ◽  
Fibrillar Collagen ◽  
Freeze Injury

Background: Corneal stromal cells (keratocytes) are responsible for developing and maintaining normal corneal structure and transparency, and for repairing the tissue after injury. Corneal keratocytes reside between highly aligned collagen lamellae in vivo. In addition to growth factors and other soluble biochemical factors, feedback from the extracellular matrix (ECM) itself has been shown to modulate corneal keratocyte behavior. Methods: In this study, we fabricate aligned collagen substrates using a microfluidics approach and assess their impact on corneal keratocyte morphology, cytoskeletal organization, and patterning after stimulation with platelet derived growth factor (PDGF) or transforming growth factor beta 1 (TGFβ). We also use time-lapse imaging to visualize the dynamic interactions between cells and fibrillar collagen during wound repopulation following an in vitro freeze injury. Results: Significant co-alignment between keratocytes and aligned collagen fibrils was detected, and the degree of cell/ECM co-alignment further increased in the presence of PDGF or TGFβ. Freeze injury produced an area of cell death without disrupting the collagen. High magnification, time-lapse differential interference contrast (DIC) imaging allowed cell movement and subcellular interactions with the underlying collagen fibrils to be directly visualized. Conclusions: With continued development, this experimental model could be an important tool for accessing how the integration of multiple biophysical and biochemical signals regulate corneal keratocyte differentiation.

scholarly journals Time-lapse cross-hole electrical resistivity tomography (CHERT) for monitoring seawater intrusion dynamics in a Mediterranean aquifer

2019 ◽  
Author(s):  
Andrea Palacios ◽  
Juan José Ledo ◽  
Niklas Linde ◽  
Linda Luquot ◽  
Fabian Bellmunt ◽  
...  
Keyword(s):  
Electrical Conductivity ◽  
Electrical Resistivity ◽  
Seawater Intrusion ◽  
Electrical Resistivity Tomography ◽  
Low Cost ◽  
Heavy Rain ◽  
Time Lapse ◽  
Resistivity Tomography ◽  
Bulk Electrical Conductivity ◽  
Spatial Coverage

Abstract. Surface electrical resistivity tomography (ERT) is a widely used tool to study seawater intrusion (SWI). It is noninvasive and offers a high spatial coverage at a low cost, but it is strongly affected by decreasing resolution with depth. We conjecture that the use of CHERT (cross-hole ERT) can partly overcome these resolution limitations since the electrodes are placed at depth, which implies that the model resolution does not decrease in the zone of interest. The objective of this study is to evaluate the CHERT for imaging the SWI and monitoring its dynamics at the Argentona site, a well-instrumented field site of a coastal alluvial aquifer located 40 km NE of Barcelona. To do so, we installed permanent electrodes around boreholes attached to the PVC pipes to perform time-lapse monitoring of the SWI on a transect perpendicular to the coastline. After two years of monitoring, we observe variability of SWI at different time scales: (1) natural seasonal variations and aquifer salinization that we attribute to long-term drought and (2) short-term fluctuations due to sea storms or flooding in the nearby stream during heavy rain events. The spatial imaging of bulk electrical conductivity allows us to explain non-trivial salinity profiles in open boreholes (step-wise profiles really reflect the presence of fresh water at depth). By comparing CHERT results with traditional in situ measurements such as electrical conductivity of water samples and bulk electrical conductivity from induction logs, we conclude that CHERT is a reliable and cost-effective imaging tool for monitoring SWI dynamics.

scholarly journals 1289. Pravibismane is a Potent, Broad Spectrum Anti-Infective Small Molecule that Rapidly Disrupts Bacterial Bioenergetics and Halts Bacterial Growth

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S659-S660
Author(s):  
Brett Baker
Keyword(s):  
Membrane Potential ◽  
Cell Growth ◽  
Protein Expression ◽  
Broad Spectrum ◽  
Time Lapse ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Time Lapse Microscopy ◽  
Atp Levels

Abstract Background The rise in resistance to existing antimicrobials has prompted a need for the development of novel antibiotics. Microbion has identified a novel compound, pravibismane, with potent broad spectrum anti-infective and anti-biofilm activity. Methods Here we used a variety of assays, including Bacterial Cytological Profiling (BCP), to analyze pravibismane in E.coli to gain insight into its likely mechanism of action (MOA). The BCP profile of pravibismane suggested it rapidly shut down cell growth, potentially by turning off cellular gene or protein expression. This was confirmed using a plasmid based GFP induction assay in E.coli tolC that showed pravibismane strongly reduced expression of GFP. The kinetics, reversibility and MOA of pravibismane was further characterized by using time-lapse microscopy, wash out experiments and measurements of both membrane potential and relative intracellular ATP levels. Results We found that pravibismane acts rapidly (within 30 mins) to completely halt cell growth rather than causing immediate cell lysis such as that observed with non-specific cell damaging agents bleach or detergent. Inhibitor wash out experiments in which cells were exposed to pravibismane for 2 hours, washed to remove the compound, and then observed using time-lapse microscopy revealed that the effect of pravibismane is reversible and that cells recovered 8-12 hrs after removing the compound. Wash out experiments with an E.coli tolC strain carrying a plasmid with an IPTG inducible GFP demonstrated that transcription and translation ultimately resumed in most cells after washout. The bioenergetics of the membrane was measured using DiBAC 4(5), a membrane potential sensitive dye which can enter depolarized cells, which revealed that pravibismane caused depolarization of the membrane within 30 mins of exposure in a concentration dependent manner. Finally, a luciferase assay determined pravibismane reduced ATP levels (resulting in decreased luminescence) within 15 mins of exposure in a concentration dependent manner unlike antibiotic controls that had modest or no effect on luminescence. Conclusion Our results suggest that pravibismane acts rapidly to disrupt cellular bioenergetics, resulting in the immediate cessation of cell growth and protein expression. Disclosures Brett Baker, M.Sc., D.C., Microbion Corporation (Board Member, Employee)

scholarly journals Decrease in Membrane Fluidity and Traction Force Induced by Silica-Coated Magnetic Nanoparticles

2020 ◽  
Author(s):  
Tae Hwan Shin ◽  
Abdurazak Aman Ketebo ◽  
Da Yeon Lee ◽  
Seungah Lee ◽  
Seong Ho Kang ◽  
...  
Keyword(s):  
Magnetic Nanoparticles ◽  
Side Effects ◽  
Membrane Fluidity ◽  
Focal Adhesion ◽  
Membrane Damage ◽  
Cell Movement ◽  
Chemical Properties ◽  
Traction Force ◽  
Hek293 Cells ◽  
Dependent Manner

Abstract Background Nanoparticles are being used increasingly due to their unique physical and chemical properties and small size. It is well-known that nanoparticles cause side effects, however their biophysical assessment remains challenging. We addressed this issue by investigating the effects of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate [MNPs@SiO2(RITC)] on the biophysical aspects, such as membrane fluidity and traction force of human embryonic kidney 293 (HEK293) cells. We further extended our understanding on the biophysical effects of nanoparticles on cells using a combination of metabolic profiling and transcriptomic network analysis. Results Overdose (1.0 μg/µl) treatment of MNPs@SiO2(RITC) induced lipid peroxidation and decreased membrane fluidity in HEK293 cells. During membrane damage, HEK293 cells were morphologically shrunk and aspect ratio of the cells were significantly decreased upon MNPs@SiO2(RITC) treatment. Each of traction force (measured in micropillar) was found to be increased, thereby increasing the total traction force in MNPs@SiO2(RITC)-treated HEK293 cells. Due to the reduction in membrane fluidity and elevation of traction force, velocity of the cell movement was significantly decreased in MNPs@SiO2(RITC)-treated HEK293 cells. Moreover, intracellular ATP also decreased in a dose dependent manner upon MNPs@SiO2(RITC) treatment. To understand the biophysical changes in cells, we analysed transcriptome and metabolic profiles and generated metabotranscriptomics network. The network showed relationships among peroxidation of lipid, focal adhesion, cell movement, and related genes and metabolites. Furthermore, in silico prediction of the network showed increment in the peroxidation of lipid and suppression of focal adhesion and cell movement.Conclusion Taken together, our results demonstrate that overdosage of MNPs@SiO2(RITC) impairs cellular movement, followed by changes in the biophysical properties of cells, thus highlighting the need for biophysical assessment of nanoparticle-induced side effects.

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