Showing papers by "Mehmet Toner published in 2004"

Dynamic gene expression profiling using a microfabricated living cell array.

Abstract: 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.

Continuous flow microfluidic device for rapid erythrocyte lysis.

Abstract: Leukocyte isolation from whole blood to study inflammation requires the removal of contaminating erythrocytes. Leukocytes, however, are sensitive to prolonged exposure to hyper/hypoosmotic solutions, temperature changes, mechanical manipulation, and gradient centrifugation. Even though care is taken to minimize leukocyte activation and cell loss during erythrocyte lysis, it is often not possible to completely avoid it. Most procedures for removal of contaminating erythrocytes from leukocyte preparations are designed for bulk processing of blood, where the sample is manipulated for longer periods of time than necessary at the single-cell level. Ammonium chloride-mediated lysis is the most commonly used method to obtain enriched leukocyte populations but has been shown to cause some activation and selective loss of certain cell types. The leukocyte yield and subsequent activation status of residual leukocytes after NH4Cl-mediated lysis have been shown to depend on the time of exposure to the lysis buffer. W...

Effect of flow and surface conditions on human lymphocyte isolation using microfluidic chambers.

Abstract: Phenotypically pure subpopulations of lymphocytes can provide valuable insights into the immune response to injury and disease. The isolation of these subpopulations presents unique challenges, particularly when preprocessing incubation to attach fluorescent or antibody tags is to be minimized. This paper examines the separation of T and B lymphocytes from mixtures using microfluidic chambers coated with antibodies, focusing on flow conditions and surface chemistry. The adhesion of both cell types decreases as shear stress increases irrespective of the surface chemistry. The incorporation of poly(ethylene glycol) chains along with the antibodies on the chamber surface is shown to significantly improve the reproducibility of cell adhesion and is thus an important part of the overall system design. Furthermore, this technique is shown to be an effective way of isolating highly pure subpopulations of lymphocytes from model mixtures, even when the target cell concentration is low.

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

Abstract: 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.

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

Abstract: 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-cultur...

Single-cell chemical lysis in picoliter-scale closed volumes using a microfabricated device.

Abstract: Investigating the intracellular contents of single cells is essential for understanding physiologic and pathologic processes at the cellular level. While existing protocols for cell lysis and sample preparation work well for larger samples, scaling to a single-cell level is challenging because of unavoidable analyte dilution and losses. Thus, we are proposing a microfabricated device for the controlled handling and mixing of picoliter cell suspension and lysis solution volumes. Cells and fluids are independently isolated in two microchambers of 25-pL volumes using the geometry of the microchannels and the coordinated action of four on-chip thermopneumatic actuators. Virtual walls formed by liquid−air interfaces in the hydrophobic capillary separate the two volumes, which are subsequently allowed to mix after drawing the air out of the capillary connecting the two microchambers. Following cell lysis, a limited and stable dilution of intracellular components is achieved, simplifying the requirements for sub...

Microfluidic systems for size based removal of red blood cells and platelets from blood

Abstract: The invention features devices and methods for enriching a sample in one or more desired particles. An exemplary use of these devices and methods is for the enrichment of cells, e.g., white blood cells in a blood sample. In general, the methods of the invention employ a device that contains at least one sieve through which particles of a given size, shape, or deformability can pass. Devices of the invention have at least two outlets, and the sieve is placed such that a continuous flow of fluid can pass through the device without passing through the sieve. The devices also include a force generator for directing selected particles through the sieve. Such force generators employ, for example, diffusion, electrophoresis, dielectrophoresis, centrifugal force, or pressure-driven flow.

Use of sugars in cryopreserving human oocytes

Abstract: 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.

Isothermal desiccation and vitrification kinetics of trehalose-dextran solutions.

Abstract: 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.

Device and method for contacting picoliter volumes of fluid

Abstract: The invention features devices for mixing fluids, e.g., for lysing cells, and methods of use thereof. One device is based on the ability to control the flow of fluids, e.g., by contact angle and channel size. Fluids in this device can be divided to form segments of controlled volume, which are then brought together to initiate mixing. An exemplary use of the device is for the lysis of single cells. Another device is based on the ability to two mix two fluids in a channel and affinity capture of analytes. The devices can be integrated on the same chip with other devices, for example, for cell handling or analysis of DNA, mRNA, and proteins released from the lysis of a cell.

Cell sorting apparatus and methods for manipulating cells using the same

Abstract: A cell analysis and sorting apparatus is capable of monitoring over time the behavior of each cell in a large population of cells The cell analysis and sorting apparatus contains individually addressable cell locations Each location is capable of capturing and holding a single cell, and selectively releasing that cell from that particular location In one aspect of the invention, the cells are captured and held in wells, and released using vapor bubbles as a means of cell actuation In another aspect of the invention, the cells are captured, held and released using electric field traps

Preservation of biomaterials with transported preservation agents

Abstract: Biomaterial are preserved by exposing them to a preservation agent having preservation properties. The biomaterial has at least one transporter that allows uptake of the preservation agent into the biomaterial for loading the biomaterial with the preservation agent to an intracellular concentration sufficient for preserving the biomaterial. The preservation agent loaded biomaterial can then be prepared for storage, for example, by freezing, freeze drying, or drying.

Induction of Apoptosis in Response to Anhydrobiotic Conditions in Mammalian Cells

Abstract: Within many biomedical engineering subspecialties, there is a need for the reversible developmental arrest of cells and tissues in support of global distribution followed by on-demand restoration of cellular function (biopreservation). Recently, the field of anhydrobiotic (dry-state) preservation has emerged as a potential approach to achieve this goal. To date, investigations have focused on different approaches utilizing stabilizing molecules, such as trehalose, to achieve anhydrobiotic preservation, yet there remains a void in the understanding of the molecular-based physiological and biochemical responses of biologics to the desiccation process. Accordingly, we formulated and tested the hypothesis that apoptosis contributes to cell death following anhydrobiotic preservation through the activation of multiple pathways by the complex array of stressors associated with the desiccation process. In addition, we investigated the modulation of cell death following desiccation, hypothesizing that through inhi...

Effect of Cell Substrate Interactions on the Desiccation Behavior of Human Fibroblasts

Abstract: The biopreservation sciences have typically relied on the utilization of low temperature to suppress or arrest cellular physiological function in an attempt to extend biological time. Recently, however, several reports have detailed the successful anhydrobiotic (dry-state) preservation of cellular systems through the utilization of stabilizing molecules, such as trehalose. In this study, we investigated the influence of cell substrate interaction during and following desiccation on cell survival in a human fibroblast model. Cells were dried on differing culture surfaces (extra-cellular matrices, including poly-L-lysine, collagen, laminin, fibronectin, and standard plasma treated tissue culture ware) after allowing 10 min for loose cell attachment. Desiccated samples (13% residual moisture) were rehydrated and then cultured on the various differing extra-cellular matrices. Samples cultured on biologically active matrices (collagen, fibronectin, and laminin) following desiccation resulted in a significant i...

Device and method for contacting pocoliter volumes of fluids

Abstract: The invention features devices for mixing fluids, e.g., for lysing cells, and methods of use thereof. One device is based on the ability to control the flow of fluids, e.g., by contact angle and channel size. Fluids in this device can be divided to form segments of controlled volume, which are then brought together to initiate mixing. An exemplary use of the device is for the lysis of single cells. Another device is based on the ability to two mix two fluids in a channel and affinity capture of analytes. The devices can be integrated on the same chip with other devices, for example, for cell handling or analysis of DNA, mRNA, and proteins released from the lysis of a cell.

Conservation de matieres biologiques a l'aide d'agents de conservation absorbes

Abstract: L'invention concerne un procede de conservation de matieres biologiques, consistant a exposer ces matieres biologiques a un agent de conservation possedant des proprietes de conservation. Les matieres biologiques contiennent au moins un substrat permettant l'absorption de l'agent de conservation dans les matieres biologiques jusqu'a une concentration intracellulaire suffisante pour conserver les matieres biologiques. Les matieres biologiques contenant cet agent de conservation peuvent ensuite etre preparees pour le stockage, par congelation, lyophilisation ou sechage, par exemple.