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In JoVE (1)
Other Publications (7)
Articles by Jeongyun Kim in JoVE
JoVE General
Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions
1McFerrin Department of Chemical Engineering, Texas A&M University, 2Department of Biomedical Engineering, Texas A&M University
This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.
Other articles by Jeongyun Kim on PubMed
Hydrodynamic Microfabrication Via"on the Fly" Photopolymerization of Microscale Fibers and Tubes
A microfluidic apparatus capable of creating continuous microscale cylindrical polymeric structures has been developed. This system is able to produce microstructures (e.g. fibers, tubes) by employing 3D multiple stream laminar flow and "on the fly"in-situ photopolymerization. The details of the fabrication process and the characterization of the produced microfibers are described. The apparatus is constructed by merging pulled glass pipettes with PDMS molding technology and used to manufacture the fibers and tubes. By controlling the sample and sheath volume flow rates, the dimensions of the microstructures produced can be altered without re-tooling. The fiber properties including elasticity, stimuli responsiveness, and biosensing are characterized. Responsive woven fabric and biosensing fibers are demonstrated. The fabrication process is simple, cost effective and flexible in materials, geometries, and scales.
Photopolymerized Check Valve and Its Integration into a Pneumatic Pumping System for Biocompatible Sample Delivery
In this paper, we present a simple check valve whose operation mimics that of venous valves. Our check valve has a mono-leaflet and is constructed via an in situ fabrication method inside the PDMS platform. For the smooth operation of the valve's leaflet, the elasticity and the shape of the leaflet and the lubrication between the leaflet and the channel surface are important. We used 4-hydroxybutyl acrylate (4-HBA) as an elastic and photopolymerizable leaflet material. We mixed the triton X-100 with the 4-HBA pre-polymer solution for the adequate lubrication of the leaflet. We constructed the micro-pumping system by combining two venous-like check valves with an oscillating polymeric diaphragm driven by pneumatic force, and measured the flow rate according to the change of pumping frequency. We also investigated the pump's feasibility as a delivery system of biocompatible materials by using mouse embryo fibroblast cells.
Co-culture of Epithelial Cells and Bacteria for Investigating Host-pathogen Interactions
The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria co-exist. This equilibrium is perturbed by the entry of pathogens into the GI tract. A key step in the infection process is the navigation of the pathogen through the commensal bacterial layer to attach to epithelial cells. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer plays a significant role in determining the extent of attachment and colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we report the development of a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. A pneumatically-actuated system was developed to form reversible islands that allow development of bacterial biofilm along with culture of an epithelial cell monolayer. The co-culture model used to develop a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection. Using wild-type E. coli and a tnaA mutant (lacks the signal indole) as the commensal bacteria, we demonstrate that the commensal biofilm microenvironment is a key determinant of EHEC infectivity and virulence. Our model has the potential to be used in fundamental studies investigating the effect of GI tract signals on EHEC virulence as well as for screening of different probiotic strains for modulating pathogen infectivity in the GI tract.
Evaluation of Dextran-flufenamic Acid Ester As a Polymeric Colon-specific Prodrug of Flufenamic Acid, an Anti-inflammatory Drug, for Chronotherapy
Dextran-flufenamic acid ester (Dex-FFA) with varied degree of substitution (DS) was prepared by imidazolide method. Dex-FFA was stable in pH 1.2 or pH 6.8 buffer. The depolymerization degree of Dex-FFA by dextranase decreased as DS increased. Dex-FFA with DS of 13 or 20 released FFA up to 70% or 21% of the dose, respectively, on 24 h-incubation with the 10% cecal contents. FFA was liberated up to 29% of the dose on 24 h-incubation of dextranase pre-treated Dex-FFA with the homogenates of the upper intestine, whereas no FFA was detected devoid of dextranse-pretreatment. Upon oral administration of Dex-FFA (DS 13, 20 mg equivalent of FFA/kg) or FFA (10 mg/kg) to rats, t(max) for FFA with Dex-FFA administration delayed approximately 6 h compared with that of free FFA administration, while C(max) for FFA was similar. The plasma level for FFA became greater around 6 h after administration of Dex-FFA than free FFA and it was maintained throughout the period of 24 h-experiment. Dex-FFA markedly attenuated gastric ulcerogenicity of FFA. Taken together, Dex-FFA could be useful as a colon-specific prodrug which possesses anti-inflammatory properties and offers opportunities as a chronotherapeutic approach for the treatment of arthritis.
Synthetic Quorum-sensing Circuit to Control Consortial Biofilm Formation and Dispersal in a Microfluidic Device
To utilize biofilms for chemical transformations in biorefineries they need to be controlled and replaced. Previously, we engineered the global regulator Hha and cyclic diguanylate-binding BdcA to create proteins that enable biofilm dispersal. Here we report a biofilm circuit that utilizes these two dispersal proteins along with a population-driven quorum-sensing switch. With this synthetic circuit, in a novel microfluidic device, we form an initial colonizer biofilm, introduce a second cell type (dispersers) into this existing biofilm, form a robust dual-species biofilm and displace the initial colonizer cells in the biofilm with an extracellular signal from the disperser cells. We also remove the disperser biofilm with a chemically induced switch, and the consortial population could tune. Therefore, for the first time, cells have been engineered that are able to displace an existing biofilm and then be removed on command allowing one to control consortial biofilm formation for various applications.
A Microfluidic Device for High Throughput Bacterial Biofilm Studies
Bacteria are almost always found in ecological niches as matrix-encased, surface-associated, multi-species communities known as biofilms. It is well established that soluble chemical signals produced by the bacteria influence the organization and structure of the biofilm; therefore, there is significant interest in understanding how different chemical signals are coordinately utilized for community development. Conventional methods for investigating biofilm formation such as macro-scale flow cells are low-throughput, require large volumes, and do not allow spatial and temporal control of biofilm community formation. Here, we describe the development of a PDMS-based two-layer microfluidic flow cell (μFC) device for investigating bacterial biofilm formation and organization in response to different concentrations of soluble signals. The μFC device contains eight separate microchambers for cultivating biofilms exposed to eight different concentrations of signals through a single diffusive mixing-based concentration gradient generator. The presence of pneumatic valves and a separate cell seeding port that is independent from gradient-mixing channels offers complete isolation of the biofilm microchamber from the gradient mixer, and also performs well under continuous, batch or semi-batch conditions. We demonstrate the utility of the μFC by studying the effect of different concentrations of indole-like biofilm signals (7-hydroxyindole and isatin), either individually or in combination, on biofilm development of pathogenic E. coli. This model can be used for developing a fundamental understanding of events leading to bacterial attachment to surfaces that are important in infections and chemicals that influence the biofilm formation or inhibition.
N-succinylaspart-1-yl Celecoxib is a Potential Colon-specific Prodrug of Celecoxib with Improved Therapeutic Properties
To develop a colon-specific prodrug of celecoxib, a cyclooxygenase-2 selective inhibitor, which could improve cardiovascular toxicity and therapeutic effectiveness for chemoprevention of colorectal cancer, aspart-1-yl celecoxib (A1C) or aspart-4-yl celecoxib (A4C), succinyl celecoxib (SC), and N-succinylaspart-1-yl celecoxib (SA1C) or N-succinylaspart-4-yl celecoxib (SA4C) were prepared and evaluated as a prodrug with such beneficial properties. On incubation with the small intestinal contents while SC, SA1C, and SA4C were stable, A1C and A4C were degraded to liberate celecoxib. In the cecal contents, the other conjugates except for SC and SA4C were cleaved to release celecoxib. These results suggest the colon-specific delivery and activation of SA1C. On oral administration of SA1C or celecoxib, no SA1C was detected in the blood and urine, indicating the limited absorption of SA1C. SA1C delivered a much greater amount of celecoxib to the large intestine while keeping the plasma concentration of celecoxib at much lower level, which is consistent with no change of the serum level of 6-ketoprostaglandin F(1α) whose decrease is associated with the cardiovascular toxicity of celecoxib. Moreover, SA1C administered orally supplied a greater concentration of celecoxib for the whole colonic tissue. Taken together, SA1C may be a colon-specific prodrug of celecoxib with improved therapeutic properties. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
