Showing papers by "Mehmet Toner published in 1998"

Microfabrication of hepatocyte/fibroblast co-cultures: role of homotypic cell interactions.

Abstract: Cell-cell interactions are important in embryogenesis, in adult physiology and pathophysiology of many disease processes. Co-cultivation of parenchymal and mesenchymal cells has been widely utilized as a paradigm for the study of cell-cell interactions in vitro. In addition, co-cultures of two cell types provide highly functional tissue constructs for use in therapeutic or investigational applications. The inherent complexity of such co-cultures creates difficulty in characterization of cell-cell interactions and their effects on function. In the present study, we utilize conventional "randomly distributed" co-cultures of primary rat hepatocytes and murine 3T3-J2 fibroblasts to investigate the role of increasing fibroblast density on hepatic function. In addition, we utilize microfabrication techniques to localize both cell populations in patterned configurations on rigid substrates. This technique allowed the isolation of fibroblast number as an independent variable in hepatic function. Notably, homotypic hepatocyte interactions were held constant by utilization of similar hepatocyte patterns in all conditions, and the heterotypic interface (region of contact between cell populations) was also held constant. Co-cultures were probed for synthetic and metabolic markers of liver-specific function. The data suggest that fibroblast number plays a role in modulation of hepatocellular response through homotypic fibroblast interactions. The response to changes in fibroblast number are distinct from those attributed to increased contact between hepatocytes and fibroblasts. This approach will allow further elucidation of the complex interplay between two cell types as they form a functional model tissue in vitro or as they interact in vivo to form a functional organ.

Cellular micropatterns on biocompatible materials.

Abstract: We present a method to produce micropatterns of cells on tissue culture substrates. A network of deep elastomeric microchannels defining the desired pattern is sealed onto the surface of interest, and a protein template is created by injecting sub-milliliter quantities of protein solution into the microchannels. Protein adsorbs only on the areas that were exposed to the microflow. After the channels are flushed and the elastomer is removed, cells attach only on the protein template. Micropatterns of collagen or fibronectin were used to selectively adhere cells on various biomedical polymers and on heterogeneous or microtextured substrates. Since the bare substrate areas remain apt for seeding other, more adhesive cell types such as fibroblasts, we were able to create micropatterned co-cultures. Our method allows for inexpensive patterning of a rich assortment of biomolecules, cells, and surfaces under physiological conditions.

Probing heterotypic cell interactions: Hepatocyte function in microfabricated co-cultures

Abstract: Replacement of liver function using extracorporeal bioartificial systems has been attempted with limited success. The instability of the hepatocyte phenotype in vitro has restricted the useful lifetime of these devices. Co-cultivation of hepatocytes with mesenchymal cells is one method that has been widely utilized to stabilize the liver-specific function of isolated cells; however, co-culture has yet to be successfully incorporated in a bioreactor setting. In this study, we probed heterotypic cell interactions in co-cultures of hepatocytes and 3T3 in order to better understand the cellular microenvironment necessary to induce and stabilize liver-specific functions. Using microfabrication and conventional techniques to control the heterotypic interface, the effects of varying degrees of heterotypic interaction on tissue function (albumin and urea synthesis) were examined. Our data indicated maximal induction of liver-specific functions in cultures with maximal initial heterotypic interaction, and that ind...

Stabilization of active recombinant retroviruses in an amorphous dry state with trehalose.

Abstract: The disaccharide trehalose is found to be effective for stabilization of active recombinant retroviruses in an amorphous dry state achieved through ambient-temperature vacuum dehydration of retroviral supernatants. Studies revealed that trehalose is a significantly better desiccation protectant than sucrose, glucose, and dextran: dextran has essentially no protective effect on retroviral survival after drying and rehydration. X-ray diffractometry of the retroviral supernatant dried with trehalose demonstrated its amorphous nature. The ability to dehydrate retroviral stocks at ambient temperatures into a stable glassy state will have a profound effect for researchers and commercial biotechnology companies which supply retroviral vectors for human gene therapy and basic research.

Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle—stage mouse oocytes

Abstract: Purpose Our purpose was to assess the effect of cryopreservation on cytoskeleton of germinal vesicle (GV) mouse oocytes and determine whether irreversible spindle damage and related digyny associated with cryopreservation of metaphase II (MII) oocytes can be avoided.

Prevention of Hemolysis in Rapidly Frozen Erythrocytes by Using a Laser Pulse

Abstract: This paper reports on the successful recovery of rapidly frozen unprotected erythrocytes by vitrification of the intracellular solution with a laser pulse prior to thawing. Erythrocytes that were frozen at 10,000 degrees C/min exhibited 100% hemolysis when thawed unless they were first irradiated by a 7 ns. laser pulse that selectively targeted the intracellular ice so that it was melted and resolidified into a glass phase. Up to 80% of the cells treated in this way remained intact after thawing. Wright's staining confirmed a healthy cell morphology and the retention of hemoglobin in the laser treated cells. While it is well known that small amounts of intracellular ice can be tolerated by cells, the findings of this study are the first to indicate that intracellular ice may be innocuous even when formed in substantial quantities provided that crystal growth and coalescence can somehow be avoided during warming.

Correlation between electrical accident parameters and injury

Abstract: Management of electrical hazards is a multidisciplinary challenge. Injury from electrical hazards affects working adults in a majority of the reported cases. Electrical arcs occur frequently in the electrical trauma setting, reportedly in as many as 75% of accidents. It is nearly impossible to precisely determine the tissue exposure to current in most cases. Reports have been unable to identify relationships between exposure parameters in electrical trauma and clinical outcome. If such a relationship exists, its knowledge would be of great value to treating physicians, therapists and safety engineers. In this article, the thermoacoustic wave effects or "blast explosion" from arcs are reviewed. The authors estimate the magnitude of pressure that would be created as a result of the thermal acoustic energy released from hypothetical electrical arc events. The published thresholds for injury from blast are presented. Finally, the relationships between how an electrical accident occurs, the exposures at the accident scene and the potential for injuries are addressed.

Intracellular calcium dynamics during photolysis.

Abstract: The objective of this investigation was to gain a deeper understanding of the intracellular events that precede photolysis of cells. A model system, consisting of malignant melanoma cells pretreated with the calcium sensitive fluorescent dye, Fluo-3, was used to examine the intracellular calcium dynamics in single-cell photolysis experiments. Exposure of the cells to 632 nm laser light in the presence of photosensitizer, tin chlorin e6, resulted in a rise in intracellular calcium. The increase in intracellular calcium was blocked using a variety of calcium channel blocking agents, including verapamil, nifedipine, and nickel. Treatment with the channel blockers was also effective in either decreasing or eliminating cell death despite the presence of lethal doses of photosensitizer and irradiation. These results show that intracellular calcium rises prior to plasma membrane lysis, and that this early rise in intracellular calcium is necessary for membrane rupture.

Microfabrication in Biology and Medicine

Abstract: During the last 30 years, the semiconductor industry has had a significant influence on our society through advances in computers, communications, energy, and transportation. Semiconductor technology exploded following the development of exquisitely precise and versatile miniaturization techniques that allowed features on computer chips to be reduced to submicron dimensions, while substantially improving processing speed and drastically reducing cost. At the heart of this revolution was the creation of three-dimensional integrated circuit elements by a series of processes collectively known as “microfabrication”. During the past few years, the application of microfabrication techniques has entered the medical field and has initiated the development of powerful new diagnostic devices used for cancer, AIDS, and genetic diseases. In addition, microfabrication tools developed for the microelectronics industry have also entered the basic science arena and are beginning to serve as a driving force for discovery in cell biology, neurobiology, pharmacology, and tissue engineering.

Method for cryopreserving oocytes

Abstract: A method for inhibiting the formation of intracellular ice formation in viable cells is disclosed. A method of increasing survival rates of the cells after freezing and thawing is also disclosed. A preparation of viable cells immersed in a solution having a cryoprotectant is prepared. The preparation is then supercooled to a temperature below its freezing point. Then the formation of extracellular ice is promoted at a temperature above that at which intracellular ice formation occurs in the cells. The preparation is then cooled to a final storage temperature.