Showing papers by "Mehmet Toner published in 2005"

Blood-on-a-Chip

Abstract: ▪ Abstract Accurate, fast, and affordable analysis of the cellular component of blood is of prime interest for medicine and research. Yet, most often sample preparation procedures for blood analysis involve handling steps prone to introducing artifacts, whereas analysis methods commonly require skilled technicians and well-equipped, expensive laboratories. Developing more gentle protocols and affordable instruments for specific blood analysis tasks is becoming possible through the recent progress in the area of microfluidics and lab-on-a-chip-type devices. Precise control over the cell microenvironment during separation procedures and the ability to scale down the analysis to very small volumes of blood are among the most attractive capabilities of the new approaches. Here we review some of the emerging principles for manipulating blood cells at microscale and promising high-throughput approaches to blood cell separation using microdevices. Examples of specific single-purpose devices are described togethe...

Cryo-Injury and Biopreservation

Abstract: Mammalian cells appear to be naturally tolerant to cold temperatures, but the formation of ice when cells are cooled leads to a variety of damaging effects. The study of cryo-injury, therefore, becomes the study of when and how ice is formed both inside and outside the cell during cooling. Protectant chemicals are used to control or prevent ice formation in many preservation protocols, but these chemical themselves tend to be damaging. Cooling and warming rates also strongly affect the amount and location of ice that is formed. Through careful modification of these parameters successful cold preservation techniques for many cell types have been developed, but there are many more cell types that have defied preservation techniques, and the extension of cell-based techniques to tissues and whole organs has been very limited. There are many aspects to the damaging effects of ice in cells that are still poorly understood. In this brief article we review our current understanding of cellular injury and highlight the aspects of cellular injury during cryopreservation that are still poorly understood.

Development of a microfabricated cytometry platform for characterization and sorting of individual leukocytes

Abstract: Organizing leukocytes into high-density arrays makes these cells amenable to rapid optical characterization and subsequent sorting, pointing to clinical and basic science applications. The present paper describes development of a cytometry platform for creating high-density leukocyte arrays and demonstrates retrieval of single cells from the array. Poly(ethylene glycol) (PEG) photolithography was employed to fabricate arrays of microwells composed of PEG hydrogel walls and glass attachment pads 20 µm × 20 µm and 15 µm × 15 µm in size. PEG micropatterned glass surfaces were further modified with cell-adhesive ligands, poly-L-lysine, anti-CD5 and anti-CD19 antibodies, in order to engineer specific cell–surface interactions within the individual wells. Localization of the fluorescently-labeled proteins in the glass attachment pads of PEG microwells was visualized by fluorescence microscopy. Glass slides micropatterned with PEG and cell-adhesive ligands were exposed to T-lymphocytes for 30 min. These anchorage-independent cells became selectively captured in the ligand-modified microwells forming high-density cell arrays. Cell occupancy in the microwells was found to be antibody-dependent, reaching 94.6 ± 2.3% for microwells decorated with T-cell specific anti-CD5 antibodies. Laser capture microdissection (LCM) was investigated as a method for sorting cells from the array and retrieval of single selected cells was demonstrated.

Microfabricated grooved substrates as platforms for bioartificial liver reactors.

Abstract: An extracorporeal bioartificial liver device has the potential to provide temporary hepatic support for patients with liver failure. Our goal was to optimize the flow environment for the cultured hepatocytes in a flat-plate bioreactor, specifically focusing on oxygen delivery using high medium flow rates while reducing the detrimental effects of the resulting shear stresses. We used photolithographic techniques to fabricate microgrooves onto the underlying glass substrate. The microgrooves, perpendicular to the axial flow direction, protected the hepatocytes from the shear stress induced by the flowing medium. Using finite element analysis, we found that the velocity gradient change near the cell surface (i.e., bottom of the grooves) was smaller than that near the top surface of the flow channel, indicating that the grooves would provide protection to the attached cells from the mechanical effects of the flowing medium. We also determined that the shear stress at the cell surface could be reduced by as much as 30 times (channel height of 100 microm) in the grooved-substrate (0.5 dyn/cm(2)) bioreactor compared to the flat-substrate (15 dyn/cm(2)) bioreactor for a medium flow rate of 4.0 mL/min. Albumin and urea synthesis rates of hepatocytes cocultured with 3T3-J2 fibroblasts remained stable over 5 days of perfusion in the grooved-substrate bioreactor, whereas in the flat-substrate bioreactor they decreased over the same time period. These studies indicate that under "high" flow conditions the microgrooved-substrate in the bioreactor can decrease the detrimental effects of shear stress on the hepatocytes while providing adequate oxygenation, thereby resulting in stable liver-specific function.

Mouse Sperm Desiccated and Stored in Trehalose Medium Without Freezing

Abstract: Mouse sperm with and without trehalose were desiccated under nitrogen gas and stored at 4°C and 22°C. After rehydration, sperm were injected into oocytes using intracytoplasmic sperm injection and embryonic development was followed. Sperm were dried for 5.0, 6.25, or 7.5 min, stored at 22°C for 1 wk with and without trehalose. The percentages of blastocysts that developed from sperm with trehalose were 51%, 31%, and 20%, respectively, which was significantly higher than sperm without trehalose (10%, 3%, and 5%, respectively). Desiccation and storage in medium with trehalose significantly increased sperm developmental potential compared to medium without trehalose. Sperm dried for 5 min produced more blastocysts than sperm dried for 6.25 or 7.5 min. When sperm were dried in trehalose for 5 min and stored for 1 wk, 2 wk, 1 mo, or 3 mo at 4°C, the percentages of blastocysts were 73%, 84%, 63%, and 39%; whereas those stored at 22°C for 1 wk, 2 wk, or 1 mo were significantly lower (53%, 17%, and 6%, respectively). Embryos from sperm partially desiccated in trehalose for 5 min and stored at 4°C for 1 or 3 mo were transferred to 10 pseudopregnant recipients. Implantation rates were 81% and 48%; live fetuses were 26% and 5%, respectively. One of the recipients delivered three live fetuses. The results show that trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing.

Enrichment using antibody-coated microfluidic chambers in shear flow: model mixtures of human lymphocytes.

Abstract: Isolation of phenotypically-pure cell sub- populations from heterogeneous cell mixtures such as blood is a difficult yet fundamentally important task. Current techniques such as fluorescent activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) require pre-incubation with antibodies which lead to processing times of at least 15-60 min. In this study, we explored the use of antibody-coated micro- fluidic chambers to negative deplete undesired cell types, thus obtaining an enriched cell subpopulation at the outlet. We used human lymphocyte cell lines, MOLT-3 and Raji, as a model system to examine the dynamic cell binding behavior on antibody coated surfaces under shear flow. Shear stress ranging between 0.75 and 1.0 dyn/cm 2 was found to provide most efficient separa- tion.Celladhesionwasshowntofollowpseudo-firstorder kinetics, and an anti-CD19 coated (Raji-depletion) device with � 2.6 min residence time was demonstrated to produce 100% pure MOLT-3 cells from 50-50 MOLT-3/Raji mixture. We have developed a mathematical model of the separation device based on the experimentally deter- mined kinetic parameters that can be extended to design future separation modules for other cell mixtures. We conclude that we can design microfluidic devices that exploits the kinetics of dynamic cell adhesion to antibody coated surfaces to provide enriched cell subpopulations within minutes of total processing time. 2005 Wiley Periodicals, Inc.

Cryopreservation of Isolated Primary Rat Hepatocytes: Enhanced Survival and Long-term Hepatospecific Function

Abstract: Major steps have been taken recently in the development of treatments for hepatocellular disease including bioartificial liver (BAL) devices and hepatocyte transplantation. Reports on the utilization of BAL devices have detailed improvements in biochemical and neurologic parameters, as well as overall survival in several animal models.1,2 In addition to these reports, several systems have been evaluated for biocompatibility and safety in phase I clinical trials, and presently at least 1 BAL is involved in a large phase II/III clinical trial.3–5 In inherited metabolic diseases of the liver, hepatocyte transplantation offers another therapeutic option in attempt to restore liver function.6 Furthermore, transplantation of isolated hepatocytes may provide a vehicle for hepatocyte-directed ex vivo gene therapy.7–9 For both BAL and hepatocytes transplantation to fully reach their clinical potential, however, isolated hepatocytes need to be preserved for significant periods of time (months to years) so that they can be appropriately banked and distributed for on-demand utilization. In addition, cellular preservation would allow for extensive testing and validation of cell sources to assess the safety and efficacy of the bioproduct. Moreover, freshly isolated and cultured hepatocytes constitute suitable model systems for use in molecular biology, genetic studies, pharmacology, toxicology, cancer, and parasitology. Hepatocyte utilization in these areas would be enhanced if there were reliable, reproducible, and effective techniques for long-term storage of hepatocytes. Cryopreservation represents one tenable option for long-term hepatocyte preservation. Various strategies have been described over the last 25 years for cryopreserving hepatocytes. Although progress has been reported for cultured hepatocytes,10,11 attempts to cryopreserved isolated primary hepatocytes have resulted in limited success. Reports detailing cell viability are demonstrated high viability, ranging from 30% to 90% immediately postthaw12–17 and reveal a continuous decline in cell number within a few hours.18 Additionally, many investigators have reported that despite high viability, few hepatocytes were able to attach to culture surfaces and survive for extended periods of time.13,19 Reports detailing assessment of cellular function (protein secretion, urea synthesis, CP450 activity) have also focused on short-term (hours, 1–2 days) analysis. A recent report using University of Wisconsin solution as a carrier solution or cryopreservation showed high postthaw viability (85%), but the viability dropped to 62.5% after 7 days.20 The use of postthaw processing, such as centrifugation in Percoll gradients, has resulted in selected assessment of viable cell function and, when unquantified, fails to provide an accurate evaluation of the efficacy of a given cryopreservation protocol. Overall, studies that carefully detail the survival and function of entire populations of cryopreserved isolated hepatocytes indicate low cell recovery and greatly impaired metabolic activity. Recent reports that showed significant beneficial effects of an intracellular-like solution, HypoThermosol (HTS), during hypothermic storage at 4°C prompted us to evaluate this solution for the cryopreservation of primary hepatocytes.21,22 Based upon previous findings, we hypothesized the utilization of HTS would improve overall hepatocyte viability through a modulation of the cellular biochemical response to the cryopreservation process, as well as maintaining improved cellular function in comparison to nonpreserved cells. In the present study, we demonstrate that cryopreservation of isolated hepatocytes using HTS results in a yield and long-term survival that are significantly higher than reported in earlier studies. Further, concordant functional results bring the use of isolated cryopreserved hepatocytes in BAL devices within reach.

System for cell enrichment

Abstract: The present invention provides systems useful for the enrichment of analytes, for example, cells of selected types, including but not limited to blood cells, stem cells, and pathogens, in samples. The invention also provides methods for analyzing the condition of a patient based on characteristics identified through analysis of the analytes in case and control samples.

Kits for Prenatal Testing

Abstract: The invention relates to a kit for prenatal testing comprising a size-based separation module which enriches a first cell type from a maternal blood sample found in vivo in a pregnant female at a concentration of less than 1% of all blood cells, and a set of instructions for analyzing said one or more enriched cells to make a prenatal diagnosis. In some embodiments, the size-based separation module can comprise a plurality of obstacles to selectively direct the one or more cells of the first cell type in a first direction away from one or more cells of a second cell type.

Magnetic device for isolation of cells and biomolecules in a microfluidic environment

Abstract: The present invention features a new and useful magnetic device and methods of its use for isolation, enrichment, and purification of cells, proteins, DNA, and other molecules. In general the device includes magnetic regions or obstacles to which magnetic particles can bind. The chemical groups, i.e., capture moieties, on the surface of the magnetic particles may then be used to bind particles, e.g., cells, or molecules of interest from complex samples, and the bound species may then be selectively released for downstream collection or further analysis.

System for size based separation and analysis

Abstract: The invention relates to one or more size-based separation modules adapted to increase a concentration of a first analyte in a sample by at least 10,000 fold, wherein said first analyte has an initial concentration in said sample of less than 1×10−3 analytes/μL, and an analyzer for analyzing said first analytes in an enriched medium.

The influence of microtextured basal lamina analog topography on keratinocyte function and epidermal organization.

Abstract: The rational design of future bioengineered skin substitutes requires an understanding of the mechanisms by which the three-dimensional microarchitecture of tissue scaffolds modulates keratinocyte function. Microtextured basal lamina analogs were developed to investigate the relationship between the characteristic topography at the dermal-epidermal interface of native skin and keratinocyte function. Microfabrication techniques were used to create master patterns, negative replicates, and collagen membranes with ridges and channels of length scales (e.g., grooves of 50-200 microm in depth and width) similar to the invaginations found in basal lamina at the dermal-epidermal junction of native skin. Keratinocytes were seeded on the surfaces of basal lamina analogs, and histological analyses were performed after 7 days of tissue culture at the air-liquid interface. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microtextured membranes. Morphometric analyses of immunostained skin equivalents suggest that keratinocyte stratification and differentiation increases as channel depth increases and channel width decreases. This trend was most pronounced in channels with the highest depth-to-width ratios (i.e., 200 microm deep, 50 microm wide). It is anticipated that the findings from these studies will elucidate design parameters to enhance the performance of future bioengineered skin substitutes.

Business methods for prenatal Diagnosis

Abstract: The present invention relates to business methods in which screening services and diagnostics for the condition of a fetus are provided. Fetal abnormalities, include chromosomal and other genetic ies, are detected through the analysis of fetal cells obtained from maternal blood samples.

Progressive elimination of microinjected trehalose during mouse embryonic development

Abstract: Recently, sugars such as trehalose have been introduced into mammalian cells by overcoming the permeability barrier of cell membranes, and have provided improved tolerance against stresses associated with freezing and drying. However, the fate of the intracellular sugars has remained an open question. To address this issue, mouse oocytes were microinjected with 0.1 mol/l trehalose, and intracellular trehalose and glucose concentrations were determined during embryonic development using a high performance liquid chromatography and pulsed amperometric detection protocol. Trehalose was not detected in non-injected controls at any stage of development. In the microinjection group, the amount of intracellular trehalose progressively decreased as embryos developed. There was a corresponding increase in intracellular glucose concentration at the two-cell stage, suggesting cleavage of trehalose to two glucose molecules. In summary, this study presents a simple, highly sensitive protocol to determine intracellular sugars. The data reveal rapid elimination of microinjected trehalose during embryonic development. These findings have implications for designing osmolarity-optimized culture media for sugar-injected oocytes.

Analysis of desiccation and vitrification characteristics of carbohydrate films by shear-wave resonators.

Abstract: Desiccated state preservation of mammalian cells and tissues in the presence of carbohydrates has started to show promise in the last two decades. Certain carbohydrates play a major role in preservation by reducing molecular mobility in the desiccated state. In this communication, the feasibility of utilizing shear-wave resonators to collect real-time molecular mobility information during desiccation and vitrification of carbohydrate based thin films was demonstrated. Simultaneous quartz crystal microbalance experimentation and optical imaging were utilized to determine the conditions for thin film formation and the vitrification kinetics of certain carbohydrate solutions of biological importance. Using the technique presented here, it was possible to gain insight into the vitrification characteristics of carbohydrate solutions establishing the basics for future research with quantitative analysis of film properties and experimentation with live mammalian cells.

Selective enhancement of cytochrome p-450 activity in rat hepatocytes by in vitro heat shock.

Abstract: We investigated the effect of heat shock on cytochrome P-450 activity in rat hepatocytes and report a significant, selective, and time-dependent enhancement of cytochrome P-450 activity in heatshocked hepatocytes. Stable long-term cultures of rat hepatocytes were heat shocked (42.5°C) for 1 to 3 h and allowed to recover at 37°C. Cytochrome P-450-dependent ethoxyresorufin O-dealkylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were measured up to 48 h after heat shock treatment. In general, the optimal heat shock exposure time was between 2 and 3 h. BROD activity (induced by sodium phenobarbital) increased approximately 6-fold in hepatocytes heat shocked for 3 h in comparison with hepatocytes maintained at 37°C. EROD activity (induced by 3-methylcholanthrene) increased 2-fold on exposure to heat shock for 2 h. The expression of inducible heat shock proteins Hsp70 and Hsp32 was verified by Western immunoblot analyses. In the absence of the appropriate inducer, heat shock treatment did not ...

Erratum: A network-based analysis of systemic inflammation in humans (Nature (2005) 437 (1032-1037) DOI: 10.1038/nature03985)

Abstract: Nature 437, 1032–1037 (2005) doi:10.1038/nature03985 In this Letter, the affiliations of authors participating in the Inflammation and Host Response to Injury Large Scale Collaborative Research Program are incorrectly listed. The renumbered and amended footnote listing is given here.

Screening cellular responses to cytokine pulses in a microfluidic device

Abstract: The cellular response to a molecular stimulus depends on the concentration and duration of the stimulus Conventional assays often neglect timing because the experiments are prohibitively tedious and time consuming In this work, we describe a microfluidic living cell assay that allows efficient characterization of stimulus timing A microfluidic network is designed to allow controlled parallel delivery of different stimulus regimens to downstream linear arrays of living GFP reporter cells In contrast to conventional techniques that require a separate experiment for each exposure regimen and each response time point, this assay screens exposure durations and response dynamics in a single experiment We demonstrate the approach by studying the effect of 7 durations of TNF-alpha exposure on activation of NFKB-mediated transcription at 17 time points, equivalent to 114 conventional experiments This general approach to studying cell signaling dynamics has significant scaling potential and will be useful for high throughput drug development and investigation of dynamic signaling in basic cell biology