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Results: 1 to 10 of 16

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Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy.
Liv N, van Oosten Slingeland DS, Baudoin JP, Kruit P, Piston DW, Hoogenboom JP
(2016) ACS Nano 10: 265-73
MeSH Terms: Animals, Bioreactors, Cell Line, Cells, Immobilized, Cercopithecus aethiops, Endocytosis, Fibroblasts, Microscopy, Electron, Scanning, Optical Imaging, Quantum Dots
Show Abstract · Added February 4, 2016
We present an approach toward dynamic nanoimaging: live fluorescence of cells encapsulated in a bionanoreactor is complemented with in situ scanning electron microscopy (SEM) on an integrated microscope. This allows us to take SEM snapshots on-demand, that is, at a specific location in time, at a desired region of interest, guided by the dynamic fluorescence imaging. We show that this approach enables direct visualization, with EM resolution, of the distribution of bioconjugated quantum dots on cellular extensions during uptake and internalization.
0 Communities
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10 MeSH Terms
A dual-column solid phase extraction strategy for online collection and preparation of continuously flowing effluent streams for mass spectrometry.
Enders JR, Marasco CC, Wikswo JP, McLean JA
(2012) Anal Chem 84: 8467-74
MeSH Terms: Bioreactors, Equipment Design, Mass Spectrometry, Microfluidic Analytical Techniques, Salts, Solid Phase Extraction
Show Abstract · Added March 7, 2014
Current desalination techniques for mass spectrometry-based protocols are problematic for performing temporal response studies where increased temporal resolution requires small samples and faster sampling frequencies, which greatly increases the number of samples and sample preparation time. These challenges are pertinent to cellular dynamics experiments, where it is important to sample the biological system frequently and with as little sample waste as possible. To address these needs, we present a dual-column online solid phase extraction (SPE) approach capable of preconcentrating and preparing a constantly perfusing sample stream, with minimal to no sample loss. This strategy is evaluated for use in microfluidic bioreactor studies specifically aimed at characterizing suitable sample flow rates, temporal resolving power, and analyte concentrations. In this work, we demonstrate that this strategy may be used for flow rates as low as 500 nL/min, with temporal resolving power on the order of 3 min, with analyte loadings ranging from femtomoles to picomoles for metabolites. Under these conditions, recoveries of ca. 80% are obtained even at femtomole loadings.
1 Communities
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6 MeSH Terms
Thick-tissue bioreactor as a platform for long-term organotypic culture and drug delivery.
Markov DA, Lu JQ, Samson PC, Wikswo JP, McCawley LJ
(2012) Lab Chip 12: 4560-8
MeSH Terms: Bioreactors, Cell Culture Techniques, Cells, Cultured, Docetaxel, Drug Delivery Systems, Epithelial Cells, Equipment Design, Humans, Microfluidic Analytical Techniques, Peptide Hydrolases, Protease Inhibitors, Structure-Activity Relationship, Taxoids
Show Abstract · Added March 7, 2014
We have developed a novel, portable, gravity-fed, microfluidics-based platform suitable for optical interrogation of long-term organotypic cell culture. This system is designed to provide convenient control of cell maintenance, nutrients, and experimental reagent delivery to tissue-like cell densities housed in a transparent, low-volume microenvironment. To demonstrate the ability of our Thick-Tissue Bioreactor (TTB) to provide stable, long-term maintenance of high-density cellular arrays, we observed the morphogenic growth of human mammary epithelial cell lines, MCF-10A and their invasive variants, cultured under three-dimensional (3D) conditions inside our system. Over the course of 21 days, these cells typically develop into hollow "mammospheres" if cultured in standard 3D Matrigel. This complex morphogenic process requires alterations in a variety of cellular functions, including degradation of extracellular matrix that is regulated by cell-produced matrix proteinases. For our "drug" delivery testing and validation experiments we have introduced proteinase inhibitors into the fluid supply system, and we observed both reduced proteinase activity and inhibited cellular morphogenesis. The size inhibition results correlated well with the overall proteinase activities of the tested cells.
1 Communities
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13 MeSH Terms
Albumin handling by renal tubular epithelial cells in a microfluidic bioreactor.
Ferrell N, Ricci KB, Groszek J, Marmerstein JT, Fissell WH
(2012) Biotechnol Bioeng 109: 797-803
MeSH Terms: Albumins, Animals, Bioreactors, Cells, Cultured, Chromatography, Gel, Electrophoresis, Polyacrylamide Gel, Endocytosis, Epithelial Cells, Kidney Tubules, Microfluidics, Molecular Weight, Opossums, Protein Transport, Proteolysis
Show Abstract · Added August 21, 2013
Epithelial cells in the proximal tubule of the kidney reclaim and metabolize protein from the glomerular filtrate. Proteinuria, an overabundance of protein in the urine, affects tubular cell function and is a major factor in the progression of chronic kidney disease. By developing experimental systems to study tubular protein handling in a setting that simulates some of the environmental conditions of the kidney tubule in vivo, we can better understand how microenviromental conditions affect cellular protein handling to determine if these conditions are relevant in disease. To this end, we used two in vitro microfluidic models to evaluate albumin handling by renal proximal tubule cells. For the first system, cells were grown in a microfluidic channel and perfused with physiological levels of shear stress to evaluate the effect of mechanical stress on protein uptake. In the second system, a porous membrane was used to separate an apical and basolateral compartment to evaluate the fate of protein following cellular metabolism. Opossum kidney (OK) epithelial cells were exposed to fluorescently labeled albumin, and cellular uptake was determined by measuring the fluorescence of cell lysates. Confocal fluorescence microscopy was used to compare uptake in cells grown under flow and static conditions. Albumin processed by the cells was examined by size exclusion chromatography (SEC) and SDS-PAGE. Results showed that cellular uptake and/or degradation was significantly increased in cells exposed to flow compared to static conditions. This was confirmed by confocal microscopy. Size exclusion chromatography and SDS-PAGE showed that albumin was broken down into small molecular weight fragments and excreted by the cells. No trace of intact albumin was detectable by either SEC or SDS-PAGE. These results indicate that fluid shear stress is an important factor mediating cellular protein handling, and the microfluidic bioreactor provides a novel tool to investigate this process.
Copyright © 2011 Wiley Periodicals, Inc.
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14 MeSH Terms
Microfabricated scaffold-guided endothelial morphogenesis in three-dimensional culture.
Liu Y, Markov DA, Wikswo JP, McCawley LJ
(2011) Biomed Microdevices 13: 837-46
MeSH Terms: Basement Membrane, Bioreactors, Cell Culture Techniques, Collagen, Dimethylpolysiloxanes, Endothelium, Vascular, Fibroblasts, Humans, Microvessels, Morphogenesis, Reproducibility of Results, Surface Properties, Tissue Scaffolds
Show Abstract · Added March 27, 2014
Morphogenesis is a fundamental process by which new blood vessels are formed during angiogenesis. The ability to control angiogenesis would lead to improvements in tissue engineering constructions; indeed, the study of angiogenesis has numerous clinical applications, for example, in the investigation of metastatic cancer, peripheral and coronary vascular disease, and wound healing. Conventional in vitro organotypic cell culture approaches to these studies are limited primarily by their reliance on microvascular vessel formation through a random process of morphogenesis that lacks the spatial reproducibility and orientation needed for high-throughput drug testing. We have developed a bioreactor system for scaffold-guided tubulogenesis coupled with 3-D organotypic culture to spatially control vessel formation and its orientation. To create microchannels to guide microvessel formation, we fabricated rigid scaffolds using photolithography and light curing epoxy, and soft scaffolds formed by a polydimethylsiloxane (PDMS) stamp directly into collagen. Scaffolds seeded with dermal microvascular endothelial cells were placed between gelled layers of collagen containing dermal fibroblasts within a Transwell filter system and cultured for up to 2 weeks to allow for vessel maturation. Morphological analysis of thin tissue sections following standard histology and immunohistochemical detection of endothelial cells, fibroblasts, and basement membrane confirmed vessel formation along the microchannel walls with either scaffold. This system may also provide a means to explore revascularization within decellularized extracellular matrices, the culture of microvessel networks with controlled geometries, and possibly the spatial guidance of angiogenesis for interfacing with an external microfluidic supply network. As a new tool for guided angiogenesis, our approach introduces new possibilities for identification of anti-angiogenic therapeutics.
1 Communities
2 Members
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13 MeSH Terms
Luminescence as a continuous real-time reporter of promoter activity in yeast undergoing respiratory oscillations or cell division rhythms.
Robertson JB, Johnson CH
(2011) Methods Mol Biol 734: 63-79
MeSH Terms: Aerobiosis, Bioreactors, Cell Culture Techniques, Cell Division, Cells, Cultured, Genes, Reporter, Luminescence, Promoter Regions, Genetic, Saccharomyces cerevisiae, Time Factors
Show Abstract · Added February 12, 2015
This chapter describes a method for generating yeast respiratory oscillations in continuous culture and monitoring rhythmic promoter activity of the culture by automated real-time recording of luminescence. These techniques chiefly require the use of a strain of Saccharomyces cerevisiae that has been genetically modified to express firefly luciferase under the control of a promoter of interest and a continuous culture bioreactor that incorporates a photomultiplier apparatus for detecting light emission. Additionally, this chapter describes a method for observing rhythmic (cell cycle-related) promoter activity in small batch cultures of yeast through luminescence monitoring.
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10 MeSH Terms
A microfluidic bioreactor with integrated transepithelial electrical resistance (TEER) measurement electrodes for evaluation of renal epithelial cells.
Ferrell N, Desai RR, Fleischman AJ, Roy S, Humes HD, Fissell WH
(2010) Biotechnol Bioeng 107: 707-16
MeSH Terms: Animals, Bioreactors, Cell Proliferation, Dogs, Electric Impedance, Electrodes, Epithelial Cells, Fluorescent Antibody Technique, Humans, Membrane Proteins, Microfluidic Analytical Techniques, Phosphoproteins, Staining and Labeling, Tight Junctions, Tubulin, Zonula Occludens-1 Protein
Show Abstract · Added August 21, 2013
We have developed a bilayer microfluidic system with integrated transepithelial electrical resistance (TEER) measurement electrodes to evaluate kidney epithelial cells under physiologically relevant fluid flow conditions. The bioreactor consists of apical and basolateral fluidic chambers connected via a transparent microporous membrane. The top chamber contains microfluidic channels to perfuse the apical surface of the cells. The bottom chamber acts as a reservoir for transport across the cell layer and provides support for the membrane. TEER electrodes were integrated into the device to monitor cell growth and evaluate cell-cell tight junction integrity. Immunofluorescence staining was performed within the microchannels for ZO-1 tight junction protein and acetylated α-tubulin (primary cilia) using human renal epithelial cells (HREC) and MDCK cells. HREC were stained for cytoskeletal F-actin and exhibited disassembly of cytosolic F-actin stress fibers when exposed to shear stress. TEER was monitored over time under normal culture conditions and after disruption of the tight junctions using low Ca(2+) medium. The transport rate of a fluorescently labeled tracer molecule (FITC-inulin) was measured before and after Ca(2+) switch and a decrease in TEER corresponded with a large increase in paracellular inulin transport. This bioreactor design provides an instrumented platform with physiologically meaningful flow conditions to study various epithelial cell transport processes.
© 2010 Wiley Periodicals, Inc.
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16 MeSH Terms
The implantable artificial kidney.
Fissell WH, Roy S
(2009) Semin Dial 22: 665-70
MeSH Terms: Bioreactors, Dialysis Solutions, Hemodiafiltration, Humans, Kidney Failure, Chronic, Kidneys, Artificial, Membranes, Artificial, Permeability, Renal Replacement Therapy
Show Abstract · Added August 21, 2013
The confluence of an increasing prevalence of end-stage renal disease (ESRD), clinical trial data suggestive of benefit from quotidian dialysis, and ongoing cost/benefit reanalysis of healthcare spending have stimulated interest in technological improvements in provision of ESRD care. For the last decade, our group has focused on enabling technologies that would permit a paradigm shift in dialysis care similar to that brought by implantable defibrillators to arrhythmia management. Two significant barriers to wearable or implantable dialysis persist: package size of the dialyzer and water requirements for preparation of dialysate. Decades of independent research into highly efficient membranes and cell-based bioreactors culminated in a team effort to develop an implantable version of the University of Michigan Renal Assist Device. In this review, the rationale for the design of the implantable artificial kidney is described.
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9 MeSH Terms
Imaging analysis of the in vivo bioreactor: a preliminary study.
Holt GE, Halpern JL, Lynch CC, Devin CJ, Schwartz HS
(2008) Clin Orthop Relat Res 466: 1890-6
MeSH Terms: Animals, Area Under Curve, Bioreactors, Bone Morphogenetic Proteins, Imaging, Three-Dimensional, Magnetic Resonance Imaging, Male, Osteogenesis, Rats, Rats, Sprague-Dawley, Tissue Engineering, Tomography, X-Ray Computed
Show Abstract · Added March 5, 2014
The in vivo bioreactor is a hermetically sealed, acellular hydroxyapatite scaffold coated with growth factors that has a pulsating vascular pedicle leash threaded through its center. Tissue-engineered bone is created in weeks while the bioreactor remains embedded under the skin of an animal. The bioreactor also provides a model to study osteogenesis and pathologic scenarios such as tumor progression and metastasis by creating a controlled microenvironment that makes skeletogenesis amenable to genetic and physical manipulation. Animal euthanasia is required to quantitate bioreactor osteogenesis through histomorphometry. Nondestructive measures of new bone growth within the bioreactor are critical to future applications and are the primary questions posed in this study. We compared microcomputed tomography and micro-MRI assessments of bioreactor osteogenesis with conventional histomorphometric measurements in 24 bioreactors and asked if new bone formation could be calculated while the animal was alive. Microcomputed tomography visually, but not numerically, differentiated engineered new bone on its coral scaffold. Dynamic contrast-enhanced micro-MRI demonstrated augmented vascular flow through the bioreactor. Three-dimensional imaging can nondestructively detect tissue-engineered osteogenesis within the implanted bioreactor in vivo, furthering the usefulness of this unique model system.
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12 MeSH Terms
Modeling of effects of nutrient gradients on cell proliferation in microfluidic bioreactor.
Hu G, Quaranta V, Li D
(2007) Biotechnol Prog 23: 1347-54
MeSH Terms: Animals, Bioreactors, Cell Count, Cell Proliferation, Cells, Cultured, Epidermal Growth Factor, Humans, Microfluidic Analytical Techniques, Models, Theoretical, Oxygen
Show Abstract · Added February 18, 2013
Precise control of the microenvironment is highly desirable in cell culture to study the cell biology. Microfluidic based bioreactors provide a promising method for the spatial and temporal control of cell growth and stimuli. A three-dimensional nutrient transport model, incorporating the monolayer cell growth model, has been developed to investigate the influence of gradients of oxygen and epidermal growth factor (EGF) on the cell culture in the continuous-flow microchannel bioreactor. Our results demonstrate that applying inlet concentration gradients of oxygen and EGF can induce variations of cell density in the y-direction. It is further found that compared to the oxygen gradients the EGF concentration gradients are more efficient in regulating the cell growth.
2 Communities
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10 MeSH Terms