scholarly journals A microfluidic dual gradient generator for conducting cell-based drug combination assays

2016 ◽  
Vol 8 (1) ◽  
pp. 39-49 ◽  
Author(s):  
Devrim Kilinc ◽  
Jefrem Schwab ◽  
Stefano Rampini ◽  
Oshoke W. Ikpekha ◽  
Ashwin Thampi ◽  
...  
Keyword(s):  
Cell Motility ◽  
Drug Combination ◽  
Cultured Cells ◽  
Proof Of Concept ◽  
Concentration Gradients ◽  
Gradient Generator ◽  
Cell Assays ◽  
Dual Gradient ◽  
Conducting Cell ◽  
Linear Concentration

We present a microfluidic gradient generator that exposes cultured cells to orthogonally-aligned linear concentration gradients of two molecules. Live-cell assays quantifying apoptotic signaling and cell motility are provided as proof-of-concept.

Electrokinetic Concentration Gradient Generation Using a Converging-Diverging Microchannel

2005 ◽  
Author(s):  
Jacky S. H. Lee ◽  
Yandong Hu ◽  
Dongqing Li
Keyword(s):  
Concentration Gradient ◽  
Electroosmotic Flow ◽  
Channel Length ◽  
Concentration Profiles ◽  
Concentration Gradients ◽  
Simple Method ◽  
Gradient Generator ◽  
Gradient Generation ◽  
Diffusive Mixing ◽  
Linear Concentration

Creation of concentration gradients is important in the study of biological and chemical processes that are sensitive to concentration variations. This paper presents a simple method to generate a linear concentration gradient in electroosmotic flow in microchannels with converging and diverging geometries. The method is based on the enhanced diffusive mixing inside the microchannel. By varying the converging-diverging geometries, the degree of diffusive mixing can be controlled. Different concentration gradients can be obtained by varying the applied potential and the geometry. Concentration profiles with minimal axial variations can be achieved with a deviation of 7% and 3% over a channel length of 3mm and 1mm, respectively, for a 400μm wide microchannel. Although the underlying physics and mechanisms for creating concentration profiles in a converging-diverging microchannel are the same as a T-shaped micromixer, the converging-diverging microchannel can produce desired concentration profiles in a much shorter distance (shorter by a factor of 2∼3.5 compared to a T-shape mixer). A serially connected concentration gradient generator is also realized with the ability to generate two concentration gradient ranges in the same microchannel. Numerical simulations and experiments were carried out to investigate the factors contributed to the generation of the concentration gradients.

scholarly journals TLK1-MK5 axis drives prostate cancer cell motility and pathologic features of aggressiveness

2021 ◽  
Author(s):  
Md Imtiaz Khalil ◽  
Vibha Singh ◽  
Judy King ◽  
Arrigo De Benedetti
Keyword(s):  
Prostate Cancer ◽  
Wound Healing ◽  
Cell Motility ◽  
Cell Lines ◽  
Cultured Cells ◽  
Cellular Migration ◽  
Dependent Manner ◽  
Lncap Cells ◽  
Pathologic Features ◽  
Pharmacologic Inhibition

Abstract Background: Majority of prostate cancer (PCa) related fatalities occur due to metastasis of cancer cells to adjacent and distal organs. We identified the novel interaction between two kinases (TLK1-MK5) that in part may initiate a signaling cascade promoting PCa metastasis. In PCa, TLK1-MK5 signaling might be crucial as androgen deprivation therapy leads to increased expression of TLK1 and compensatory activation of MK5 in metastatic castration-resistant prostate cancer patients. Methods: We performed scratch wound repair and 3D chemotactic migration assays to determine the motility rates of different TLK1 and MK5 perturbed cells. Co-IP, His, and GST pull down, in vitro kinase (IVK) assays and mass spectrometry (MS) were conducted to determine TLK1-MK5 interaction and phosphorylation. Western blotting (WB), immunohistochemistry (IHC) and bioinformatic analysis were used to examine TLK1 and pMK5 levels in PCa cell lines, mice prostate tumors and PCa tissue microarray (TMA). Results: Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 can significantly reduce wound healing rate in MEF and LNCaP cells. However, TLK1 overexpression alone in the MK5 −/− MEF cells did not increase the wound healing which suggested that TLK1 cannot enhance cellular migration in absence of MK5. Our reciprocal co-IPs, His- and GST pull down assays confirmed TLK1-MK5 interaction in cultured cells. Incubation of purified recombinant TLK1B and MK5 increases the phosphorylation of MK5 and its kinase activity. MS analysis identified three unique phosphorylation sites in MK5 (S160, S354, S386) by TLK1B. While our WB detected substantial amount of pMK5 S354 and TLK1 in all major PCa cell lines, anti-androgen treatment increased pMK5 S354 level in a dose-dependent manner and pharmacologic inhibition of TLK1 reduces pMK5 S354 level in LNCaP cells. IHC staining of TRAMP mice prostate tissues also exhibited increased pMK5 S354 level in aggressive tumor compared to benign regions. Finally, IHC analysis of PCa TMA indicated a correlation between elevated pMK5 S354 level and generally higher Gleason scores as well as nodal metastatic status of the tumors. Conclusion: Our data support that TLK1-MK5 signaling is functionally involved in driving PCa cell motility and clinical aggressiveness, hence, disruption of this axis may inhibit the metastasis of PCa.

scholarly journals A Concentration Gradients Tunable Generator with Adjustable Position of the Acoustically Oscillating Bubbles

Micromachines ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 827
Author(s):  
Bendong Liu ◽  
Zhigao Ma ◽  
Jiahui Yang ◽  
Guohua Gao ◽  
Haibin Liu
Keyword(s):  
Concentration Gradient ◽  
Acoustic Waves ◽  
Gas Permeability ◽  
Liquid Interface ◽  
Biological Research ◽  
Concentration Gradients ◽  
New Device ◽  
Gradient Generator ◽  
Oscillating Bubbles ◽  
Pdms Chip

It is essential to control concentration gradients at specific locations for many biochemical experiments. This paper proposes a tunable concentration gradient generator actuated by acoustically oscillating bubbles trapped in the bubble channels using a controllable position based on the gas permeability of polydimethylsiloxane (PDMS). The gradient generator consists of a glass substrate, a PDMS chip, and a piezoelectric transducer. When the trapped bubbles are activated by acoustic waves, the solution near the gas–liquid interface is mixed. The volume of the bubbles and the position of the gas–liquid interface are regulated through the permeability of the PDMS wall. The tunable concentration gradient can be realized by changing the numbers and positions of the bubbles that enable the mixing of fluids in the main channel, and the amplitude of the applied voltage. This new device is easy to fabricate, responsive, and biocompatible, and therefore has great application prospects. In particular, it is suitable for biological research with high requirements for temporal controllability.

Numerical and Experimental Study on a Microfluidic Concentration Gradient Generator for Arbitrary Approximate Linear and Quadratic Concentration Curve Output

2017 ◽  
Vol 16 (1) ◽  
Author(s):  
Xueye Chen ◽  
Zengliang Hu ◽  
Lei Zhang ◽  
Zhen Yao ◽  
Xiaodong Chen ◽  
...  
Keyword(s):  
Finite Element Method ◽  
Concentration Gradient ◽  
Fluid Dynamic ◽  
Milling Machine ◽  
Microfluidic Chips ◽  
The Finite Element Method ◽  
Pmma Substrate ◽  
Concentration Gradients ◽  
Biochemical Engineering ◽  
Gradient Generator

AbstractThis work introduces a simple and versatile method for researching the concentration gradient generator (CGG) which can present the arbitrary approximate linear and quadratic concentration gradient curves output. The concentration gradients of arbitrary approximate linear curves with two inlets and arbitrary quadratic curves in the CGG with three inlets are obtained with the corresponding flow velocities. The CGG was simulated basing on the finite element method (FEM). The fluid-dynamic and mass-transport about the CGG was studied. Moreover, the feasibility of simulation was clearly verified by an experiment which two microfluidic chips of CGG on the PMMA substrate were processed using CNC engraving and milling machine. The paper successfully demonstrates the controllability of concentration gradient profiles in CGG with two inlets and three inlets. The study on the CGG can help the trends study of cell and molecule in different samples in the biochemical engineering.

scholarly journals Rho and Rab Small G Proteins Coordinately Reorganize Stress Fibers and Focal Adhesions in MDCK Cells

1998 ◽  
Vol 9 (9) ◽  
pp. 2561-2575 ◽  
Author(s):  
Hiroshi Imamura ◽  
Kenji Takaishi ◽  
Katsutoshi Nakano ◽  
Atsuko Kodama ◽  
Hideto Oishi ◽  
...  
Keyword(s):  
Cell Motility ◽  
G Protein ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Family Members ◽  
Focal Adhesions ◽  
Protein Family ◽  
Stress Fibers ◽  
Small G Protein ◽  
Rab Family

The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility.

scholarly journals A Proof-of-Concept Study Using Numerical Simulations of an Acoustic Spheroid-on-a-Chip Platform for Improving 3D Cell Culture

Sensors ◽  
2021 ◽  
Vol 21 (16) ◽  
pp. 5529
Author(s):  
Arash Yahyazadeh Shourabi ◽  
Roozbeh Salajeghe ◽  
Maryam Barisam ◽  
Navid Kashaninejad
Keyword(s):  
Flow Rate ◽  
Cultured Cells ◽  
3D Cell Culture ◽  
Potential Candidate ◽  
Proof Of Concept ◽  
Multicellular Spheroids ◽  
Lab On Chip ◽  
Main Challenge ◽  
Biological Studies ◽  
On Chip

Microfluidic lab-on-chip devices are widely being developed for chemical and biological studies. One of the most commonly used types of these chips is perfusion microwells for culturing multicellular spheroids. The main challenge in such systems is the formation of substantial necrotic and quiescent zones within the cultured spheroids. Herein, we propose a novel acoustofluidic integrated platform to tackle this bottleneck problem. It will be shown numerically that such an approach is a potential candidate to be implemented to enhance cell viability and shrinks necrotic and quiescent zones without the need to increase the flow rate, leading to a significant reduction in costly reagents’ consumption in conventional spheroid-on-a-chip platforms. Proof-of-concept, designing procedures and numerical simulation are discussed in detail. Additionally, the effects of acoustic and hydrodynamic parameters on the cultured cells are investigated. The results show that by increasing acoustic boundary displacement amplitude (d0), the spheroid’s proliferating zone enlarges greatly. Moreover, it is shown that by implementing d0  = 0.5 nm, the required flow rate to maintain the necrotic zone below 13% will be decreased 12 times compared to non-acoustic chips.

A Simple Device for Tissue Dehydration: Application of Concentration Gradient Generator

1976 ◽  
Vol 34 ◽  
pp. 338-339
Author(s):  
D. Goldfarb ◽  
K. Miyai ◽  
J. Hegenauer
Keyword(s):  
Electron Microscopy ◽  
Concentration Gradient ◽  
Chromatographic Separation ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Simple Device ◽  
Concentration Gradients ◽  
Gradient Generator ◽  
Mechanical Methods ◽  
General Configuration

We describe a simple device for dehydrating tissue for electron microscopy. Devices of the same general configuration are widely applied in chromatographic separation and density gradient centrifugation, which requires the generation of simple concentration gradients in a continuous rather than stepwise fashion. The application of this technique to tissue dehydration saves time and expense compared to manual and commercially available mechanical methods.The device consists of two parts: I. a gradient generator, and II. a tissue tray (Fig. 1). The upper part of the device consists of the concentration gradient generator, composed of reservoirs A, B, C and Delrin plate D. Torus A and cylinder B are connected by a channel E. Stopcock F permits independent filling of the reservoirs.

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