Biomedical subjects

Mehmet Toner

Publications and source records attributed to Mehmet Toner.

At least 55 records · Page 3Linked to original sources

PubMed2004

Dynamic gene expression profiling using a microfabricated living cell array.

We describe the development of a microfluidic platform for continuous monitoring of gene expression in live cells. This optically transparent microfluidic device integrates high-throughput molecular stimulation with nondestructive monitoring of expression events in individual living cells, hence, a living cell array (LCA). Several concentrations of a soluble molecular stimulus are generated in an upstream microfluidic network and used to stimulate downstream reporter cells, each containing a green fluorescence reporter plasmid for a gene of interest. Cellular fluorescence is continuously monitored and quantified to infer the expression dynamics of the gene being studied. We demonstrate this approach by profiling the activation of the transcription factor NF-kappaB in HeLa S3 cells in response to varying doses of the inflammatory cytokine TNF-alpha. The LCA platform offers a unique opportunity to simultaneously control dynamic inputs and measure dynamic outputs from adherent mammalian cells in a high-throughput fashion. This approach to profiling expression dynamics, in conjunction with complementary techniques such as DNA microarrays, will help provide a more complete picture of the dynamic cellular response to diverse soluble stimuli.

Gene Expression Profiling↗

PubMed2004

Isothermal desiccation and vitrification kinetics of trehalose-dextran solutions.

The promise of dried state preservation is based on the hypothesis that lowering molecular mobility to halt chemical reaction and deterioration rates is the primary factor for the long-term stability of the dried specimen. In this research, the feasibility of utilizing isothermal, isobaric vitrification as an economical alternative to the preservation technologies currently in use (mainly, cryopreservation and lyophilization) is explored. Desiccation and vitrification kinetics of model trehalose and trehalose-dextran systems were examined using gravimetric analysis, modulated differential scanning calorimetry, and X-ray crystallography. It was shown that vitrification can be achieved isothermally without crystallization and that vitrification of trehalose solutions can be significantly accelerated by incorporating high-molecular-weight dextrans. Additionally, it was shown that, for the same water content, the glass transition temperature of the trehalose-dextran solution is significantly higher than that of the binary trehalose solution, making the glassy state achievable and storage feasible.

Calorimetry, Differential Scanning↗

PubMed2004

Designing a hepatocellular microenvironment with protein microarraying and poly(ethylene glycol) photolithography.

In this study, robotic protein printing was employed as a method for designing a cellular microenvironment. Protein printing proved to be an effective strategy for creating micropatterned co-cultures of primary rat hepatocytes and 3T3 fibroblasts. Collagen spots (ca. 170 microm in diameter) were printed onto amino-silane- and glutaraldehyde-modified glass slides. Groups of 15-20 hepatocytes attached to collagen regions in a highly selective manner forming cell clusters corresponding in size to the printed collagen domains. Fibroblasts, seeded onto the same surface, adhered and spread around arrays of hepatocyte islands creating a heterotypic environment. The co-cultured hepatocytes produced and maintained high levels of liver-specific biomarkers, albumin and urea, over the course of 2 weeks. In addition, protein printing was combined with poly(ethylene glycol) photolithography to define intercellular contacts within the clusters of hepatocytes residing on individual collagen islands. Glass slides, treated with 3-acryloxypropyl trichlorosilane and imprinted with 170 m diameter collagen spots, were micropatterned with a high-density array of 30 microm x 30 microm poly(ethylene glycol) (PEG) wells. As a result, discrete groups of ca. 9 PEG microwells became functionalized with the cell-adhesive ligand. When exposed to micropatterned surfaces, hepatocytes interacted exclusively with collagen-modified regions, attaching and becoming confined at a single-cell level within the hydrogel wells. Micropatterning strategies proposed here will lead to greater insights into hepatocellular behavior and will benefit the fields of hepatic tissue engineering and liver biology.

3T3 Cells↗

PubMed2004

Hepatic tissue engineering for adjunct and temporary liver support: critical technologies.

The severe donor liver shortage, high cost, and complexity of orthotopic liver transplantation have prompted the search for alternative treatment strategies for end-stage liver disease, which would require less donor material, be cheaper, and less invasive. Hepatic tissue engineering encompasses several approaches to develop adjunct internal liver support methods, such as hepatocyte transplantation and implantable hepatocyte-based devices, as well as temporary extracorporeal liver support techniques, such as bioartificial liver assist devices. Many tissue engineered liver support systems have passed the "proof of principle" test in preclinical and clinical studies; however, they have not yet been found sufficiently reliably effective for routine clinical use. In this review we describe, from an engineering perspective, the progress and remaining challenges that must be resolved in order to develop the next generation of implantable and extracorporeal devices for adjunct or temporary liver assist.

Biomedical Technology↗

PubMed2004

Use of sugars in cryopreserving human oocytes.

In the last 20 years, a worldwide effort to cryopreserve oocytes has resulted in 40 infants and approximately 50 ongoing pregnancies being reported. While the ability to freeze human embryos has become a standard of practice in assisted reproductive technologies, obtaining reliable techniques for oocyte cryopreservation has been more difficult. The unique properties of the mature oocyte, such as the meiotic stage with sensitive spindle structure as well as the large cell volume, are responsible for the limited success obtained to date. There have been two approaches to cryopreserving the oocyte: (i) slow freeze-rapid thaw, and (ii) vitrification protocols with rapid cooling-rapid warming. Both methods have incorporated sugars (sucrose) as a beneficial non-permeating extracellular cryoprotectant. Studies of organisms that survive extreme conditions of freezing/dehydration have demonstrated the ability to accumulate intracellular sugars to afford protection and survival. A novel technique using microinjection of sugars into the oocyte for cryopreservation has been developed as an alternative approach to external addition of sugars. Freezing the human oocyte has been a challenging goal; however, developing research and efforts will, in the near future, provide women with an important option for their reproductive health.

Cell Survival↗

PubMed2004

Cryopreservation of stem cells using trehalose: evaluation of the method using a human hematopoietic cell line.

While stem cell cryopreservation methods have been optimized using dimethylsulfoxide (DMSO), the established techniques are not optimal when applied to unfertilized human embryonic cells. In addition, important questions remain regarding the toxicity and characteristics of DMSO for treatment of stem cells for clinical use. The objective of this study was to establish an optimal method for cryopreservation of stem cells using low concentrations (0.2 M) of trehalose, a nontoxic disaccharide of glucose, which possesses excellent protective characteristics, in place of current methods utilizing high concentrations (1-2 M) of DMSO. A human hematopoietic cell line was used in this investigation as a surrogate for human stem cells. Trehalose was loaded into cells using a genetically engineered mutant of the pore-forming protein alpha-hemolysin from Staphylococcus aureus. This method results in a nonselective pore equipped with a metal-actuated switch that is sensitive to extracellular zinc concentrations, thus permitting controlled loading of trehalose. Preliminary experiments characterized the effects of poration on TF-1 cells and established optimal conditions for trehalose loading and cell survival. TF-1 cells were frozen at 1 degrees C/min to -80 degrees C with and without intra- and extracellular trehalose. Following storage at -80 degrees C for 1 week, cells were thawed and evaluated for viability, differentiation capacity, and clonogenic activity in comparison to cells frozen with DMSO. Predictably, cells frozen without any protective agent did not survive freezing. Colony-forming units (CFU) generated from cells frozen with intra- and extracellular trehalose, however, were comparable in size, morphology, and number to those generated by cells frozen in DMSO. There was no observable alteration in phenotypic markers of differentiation in either trehalose- or DMSO-treated cells. These data demonstrate that low concentrations of trehalose can protect hematopoietic progenitors from freezing injury and support the concept that trehalose may be useful for freezing embryonic stem cells and other primitive stem cells for therapeutic and investigational use.

Animals↗

PubMed2004

Long-term function of cryopreserved rat hepatocytes in a coculture system.

The goal of this study was to investigate postpreservation long-term function of cryopreserved primary rat hepatocytes using the hepatocyte/3T3-J2 fibroblast coculture system. The long-term function of thawed hepatocytes cocultured with fibroblasts was evaluated and compared with hepatocytes cultured without fibroblasts. Fresh isolated primary rat hepatocytes were frozen at a controlled rate (-1 degrees C/min) up to -80 degrees C, and then stored in liquid nitrogen for up to 90 days. Thawed hepatocytes were thereafter cocultured with 3T3-J2 murine fibroblasts and cocultivation was monitored for 14 days. The viability of fresh isolated hepatocytes was 91.4%, and that of cryopreserved hepatocytes was 82.1%. Cellular morphology and polarity, which were determined by the localization of actin filaments and connexin-32, were successfully maintained in cryopreserved hepatocytes following cryopreservation. Albumin and urea synthesis reached the maximum level and became stable after day 7 in coculture in both fresh and cryopreserved hepatocytes. Urea synthesis of cryopreserved hepatocytes was maintained 89.0% of nonfrozen fresh control, and albumin production of cryopreserved hepatocytes was 63.7% of control in coculture. Cytochrome P450 activity, which was measured by deethylation of ethoxyresorufin, was also maintained in cryopreserved hepatocytes at 88.6% of nonfrozen fresh control in coculture. The retention of synthetic and detoxification activities was verified to be well preserved during extended low-temperature storage (90 days). Both fresh control and cryopreserved hepatocytes cultured without fibroblast did not retain their synthetic and detoxification functions in long-term culture. These data illustrate that, through the utilization of our cryopreservation procedure, primary hepatocyte function was successfully maintained when placed into coculture configuration following thawing.

Actin Cytoskeleton↗

PubMed2003

Measurement of trehalose loading of mammalian cells porated with a metal-actuated switchable pore.

Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that sugars have in protecting cells from lethal injury. Among the key determinants of desiccation tolerance is the intracellular trehalose concentration, and thus quantifying the amount and rate of trehalose accumulation has now become very critical to the success of these desiccation approaches. We introduced trehalose into 3T3 fibroblasts, human keratinocytes, and rat hepatocytes using a genetically engineered mutant of the pore-forming alpha-hemolysin from Staphylococcus aureus. Manipulating the extracellular Zn(2+) concentration selectively opens and closes this pore ( approximately 2 nm) and enables controlled loading of cells with sugars. We quantified intracellular trehalose using gas chromatography-mass spectroscopy (GC-MS) to examine the trimethylsilyl derivative of intracellular trehalose. Using the GC-MS method, we demonstrate that the switchable characteristics of H5 alpha-hemolysin permit controlled loading of the high concentrations of trehalose (up to 0.5 M) necessary for engineering desiccation tolerance in mammalian cells.

3T3 Cells↗

PubMed2003

Efficacy of an extracorporeal flat-plate bioartificial liver in treating fulminant hepatic failure.

BACKGROUND: Fulminant hepatic failure is associated with a high mortality rate. Orthotopic liver transplantation is the only established treatment for patients who do not respond to medical management. A major limitation of this treatment is a shortage of donor organs, resulting in many patients dying while waiting for a transplant. An extracorporeal bioartificial liver (BAL) has the potential to provide temporary support for patients with fulminant hepatic failure (FHF) and for patients awaiting orthotopic liver transplantation. We developed a flat-plate BAL with an internal membrane oxygenator in which porcine hepatocytes were cultured as a monolayer. MATERIALS AND METHODS: Twenty-four hours after cannulation of the left carotid artery and right jugular vein, FHF was induced in rats by administering 2 intraperitoneal injections of D-galactosamine (GalN) (1.2 g/kg) at a 12-h interval. The rats were connected to a BAL device 24 h after the first GalN injection and underwent extracorporeal perfusion for a duration of 10 h. Liver histology, liver-specific markers, and animal survival up to 168 h (7 days) were examined. RESULTS: Histologically, liver damage was reduced in the animal group treated with the hepatocyte-based BAL device. Significant reductions occurred in the plasma ammonia levels and prothrombin times in the group treated with the seeded BAL device. Animal survival in the group treated with the seeded BAL device was significantly higher (50.0%) than in the control animal group treated with an unseeded BAL device (11.1%). CONCLUSIONS: This flat-plate BAL with an internal membrane oxygenator and cultured porcine hepatocytes has yielded encouraging results in the treatment of rats with GalN-induced FHF.

Animals↗

PubMed2003

Quantitative microinjection of trehalose into mouse oocytes and zygotes, and its effect on development.

Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. The objective of this study was first to establish a method for quantitative delivery of trehalose as a model sugar into oocytes, and then to evaluate its effect on development of mouse zygotes. To this end, a quantitative microinjection technique was developed using volumetric response of microdroplets suspended in dimethylpolysilaxene. To verify accuracy of this technique, both microdroplets and oocytes were microinjected with fluorophore-labeled dextran. Thereafter, injection volumes were calculated from fluorescence intensity, and volumetric responses of both microdroplets and oocytes. Comparison of calculated injection volumes revealed that this technique reflects microinjection into oocytes with pL-accuracy. The next series of experiments focused on toxicity of injection buffers (i.e., 10mM Tris and 15mM Hepes) and trehalose. Microinjection of Hepes and Tris buffer in the presence of 0.1M trehalose resulted in blastocyst rates of 86 and 72%, respectively, without a significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of zygotes was studied as a function of intracellular trehalose concentrations. Microinjection of trehalose up to 0.15M resulted in development to blastocyst stage similar to controls (85 and 87%, respectively) while the blastocyst rate was significantly decreased (43%) in the presence of 0.20M intracellular trehalose. When transferred to foster mothers, trehalose-injected zygotes (0.1M) implanted and developed to day 16 fetuses similar to controls, healthy pups were born. The findings of this study suggest that trehalose at effective intracellular concentrations does not impair development of mouse zygotes.

Animals↗

PubMed2003

Influence of insulin therapy on burn wound healing in rats.

BACKGROUND: Insulin is proposed as a therapy for suppressing muscle wasting after burn trauma although the long-term effects of this therapy on wound healing are not yet known. The present study was designed to investigate the effect of systemically administered insulin therapy on burn wound healing. MATERIALS AND METHODS: Young rats weighing 80-150 g were subjected to 15-20% total body surface area burn injury on their shaved dorsum. The insulin dosage was increased over the first 3 days in each rat from 0.25 U (Day 1), 0.5 U (Day 2), and 1.0 U (Day 3) per 100 g body wt. The rats were euthanized at the fourth or fifteenth day postinjury. Skin sections were analyzed by histochemistry and quantitative polarization microscopy. RESULTS: Histology showed a decreased number of inflammatory cells and increased vasodilation in the insulin-treated animals at Day 4 relative to untreated rats; at Day 15 there was increased reepithelialization. Quantitative analysis using polarization microscopy and picrosirius red staining showed an increased collagen deposition in wounds by Day 4 in insulin-treated rats relative to untreated burn controls. CONCLUSION: These results indicate that insulin induces accelerated wound healing associated with diminished inflammation and increased collagen deposition.

Animals↗

PubMed2002

Overview of the Alliance for Cellular Signaling.

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells--B lymphocytes (the cells of the immune system) and cardiac myocytes--to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

B-Lymphocytes↗

PubMed2002

Desiccation tolerance of spermatozoa dried at ambient temperature: production of fetal mice.

Long-term preservation of mouse sperm by desiccation is economically and logistically attractive. The current investigation is a feasibility study of the preservation of mouse sperm by convective drying in an inert gas (nitrogen). Mouse sperm from the B6D2F1 strain isolated in an EGTA-supplemented Tris-HCl buffer were dried using three different drying rates and were stored for 18-24 h at 4 degrees C. The mean final moisture content was <5% for all the protocols. After intracytoplasmic sperm injection (ICSI), the mean blastocyst formation rates were 64%, 58%, and 35% using the rapid-, moderate-, and slow-drying protocols, respectively. The slow-drying protocol resulted in a rate of development significantly lower than that observed using rapid- and moderate-drying protocols and indicated that a slower drying rate may be detrimental to the DNA integrity of mouse sperm. The transfer of 85 two- or four-cell embryos that were produced using rapidly desiccated sperm resulted in 11 fetuses (13%) on Day 15 compared with the production of 34 fetuses (40%) produced using the transfer of 86 two- or four-cell embryos that were produced using fresh sperm (P < 0.05). The results demonstrate the feasibility of using a convective drying protocol for the successful desiccation of mouse sperm and identifies some of the important parameters required for optimization of the procedure.

Animals↗

PubMed2002

A microfabrication-based dynamic array cytometer.

We have developed a microfabricated device for use in parallel luminescent single-cell assays that can sort populations upon the basis of dynamic functional responses to stimuli. This device is composed of a regular array of noncontact single-cell traps. These traps use dielectrophoresis to stably confine cells and hold them against disrupting fluid flows. Using quantitative modeling, we have designed traps with a novel asymmetric extruded-quadrupole geometry. This new trap can be physically arrayed and electrically addressed, enabling our cytometer. Situating an array of these traps in a microchannel, we have introduced cells into the array and demonstrated observation of fluorescent dynamic responses followed by sorting. Such a device has potential for use in investigating functional processes, as revealed by temporal behavior, in large numbers of single cells.

Cell Separation↗

PubMed2002

Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device.

Although a wealth of knowledge about chemotaxis has accumulated in the past 40 years, these studies have been hampered by the inability of researchers to generate simple linear gradients instantaneously and to maintain them at steady state. Here we describe a device microfabricated by soft lithography and consisting of a network of microfluidic channels that can generate spatially and temporally controlled gradients of chemotactic factors. When human neutrophils are positioned within a microchannel, their migration in simple and complex interleukin-8 (IL-8) gradients can be tested. The cells exhibit strong directional migration toward increasing concentrations of IL-8 in linear gradients. Neutrophil migration halts abruptly when cells encounter a sudden drop in the chemoattractant concentration to zero ("cliff" gradient). When neutrophils are challenged with a gradual increase and decrease in chemoattractant ("hill" gradient), however, the cells traverse the crest of maximum concentration and migrate further before reversing direction. The technique described in this paper provides a robust method to investigate migratory cells under a variety of conditions not accessible to study by earlier techniques.

Biological Assay↗

PubMed2002

Metabolic pre-conditioning of cultured cells in physiological levels of insulin: generating resistance to the lipid-accumulating effects of plasma in hepatocytes.

Understanding the regulation of hepatocyte lipid metabolism is important for several biotechnological applications involving liver cells. During exposure of hepatocytes to plasma, as is the case in extracorporeal bioartificial liver assist devices, it has been reported that hepatic-specific functions, e.g., albumin and urea synthesis and diazepam removal, are dramatically compromised and hepatocytes progressively accumulate cytoplasmic lipid droplets. We hypothesized that the composition of hepatocyte culture medium significantly affects lipid metabolism during subsequent plasma exposure. Rat hepatocytes were cultured in medium containing either physiological (50 microU/mL) or supra-physiological (500 mU/mL) insulin levels for 1 week and then exposed to human plasma supplemented with or without amino acids. We found that insulin's anabolic effects, such as stimulation of triglyceride storage, were carried over from the pre-conditioning to the plasma exposure period. While hepatocytes cultured in high insulin medium accumulated large quantities of triglycerides during subsequent plasma exposure, culture in low insulin medium largely prevented lipid accumulation. Urea and albumin secretion, as well as the ammonia removal rate, were largely unaffected by insulin but increased with amino acid supplementation. Thus, hepatocyte metabolism during plasma exposure can be modulated by medium pre-conditioning and supplements added to plasma.

Albumins↗

PubMed2002

Antiproteolytic action of insulin in burn-injured rats.

BACKGROUND: Negative nitrogen balance is a typical metabolic response to burn injury resulting in decreased muscle mass and activity. Since insulin is an anabolic hormone, using insulin as a prophylactic agent in burned patients has received some attention. The present study was carried out to investigate the systemic effect of insulin on burn injury-induced muscle wasting. PATIENTS AND METHODS: A 15-20% total body surface area (TBSA) scald burn injury was inflicted on the shaved dorsum of rats. Rats were treated with a daily subcutaneous insulin injection for 3 days (0.25-1.0 U/day). After the treatment, a variety of insulin-dependent physiological parameters were monitored. Overall body protein degradation rates were determined by measuring the urinary tyrosine. Also, protein degradations were measured in diaphragm muscles, splenocytes, and peripheral blood mononuclear cells to directly confirm the antiproteolytic activity of insulin. RESULTS: Administration of insulin to burn-injured rats restored body weight primarily by reducing accelerated protein degradation and regaining the intracellular protein content in individual skeletal muscle. The measured physiological parameters showed no possible side effects. Protein degradation in immune cells was also suppressed after the therapy. CONCLUSION: Results indicate that lower dose insulin particularly suppresses protein degradation without causing secondary effects. It may be a useful approach to preventing burn injury-induced muscle wasting and also has a potential to improve immune response.

Animals↗

PubMed2002

Bioengineering of liver assist devices.

Over 30 000 patients die annually in the United States from liver failure. In fulminant hepatic failure, a clinical syndrome associated with high mortality, orthotopic liver transplantation is the primary therapeutic option for patients not responding to supportive therapy. However, the persistent scarcity of donor organs has limited this therapeutic modality, resulting in a continued increase in the number of patients who die waiting for a donor liver. An extracorporeal bioartificial liver device could provide vital support to a liver failure patient until a donor liver was available or until the patient's own liver regenerated. Although it is unclear which liver-specific functions must be provided by such a device to be effective, a constant challenge has been to obtain stable, well-differentiated, and normally functioning hepatocytes that can be cultured at high cell densities. Many of the devices currently undergoing clinical trials are limited by designs which are prone to substrate limitations, resulting in compromised hepatocyte function. In devices that avoid substrate limitations, hepatocyte functions can be optimized, thereby leading to increased device efficiency. In this overview, the authors describe the critical issues involved in bioartificial liver development and discuss their experiences in hepatocyte culture optimization within the context of a microchannel, flat-plate bioartificial liver device with an internal membrane oxygenator.

Animals↗