A stable long-term hepatocyte culture system for studies of physiologic processes: cytokine stimulation of the acute phase response in rat and human hepatocytes.

Prior studies on the in vitro hepatic acute phase response have involved either hepatoma cell lines or conventional short-term cultures of primary hepatocytes. No data are available on the response of primary hepatocytes in stable long-term culture systems. In this study, the acute phase response of rat and human hepatocytes in a new long-term culture system was examined in response to interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF-alpha). The cultured cells were sandwiched between two layers of collagen in a (double-gel) configuration which has been shown to preserve both hepatocyte function and morphology over prolonged periods of time. The stability of this culture configuration enabled us to investigate, for the first time, the temporal aspects of the response in addition to the effects of the mediators on protein secretion. Exposure of rat hepatocytes to IL-6 after culture for 16 days resulted in a 2-fold reduction of albumin secretion and a 15-fold increase in the secretion rates of fibrinogen and alpha 2-macroglobulin. In all instances, the peak response occurred at 48 h after IL-6 exposure, and all protein secretion rates returned to pretreatment values within 5 days posttreatment. Changes in the mRNA levels of these proteins in response to IL-6 corresponded with those changes seen with the secreted products, indicating pretranslational regulation. Administration of IL-1 beta to rat hepatocyte produced a similar decline of albumin secretion and a 5-fold increase of fibrinogen secretion, whereas alpha 2-macroglobulin secretion remained undisturbed.(ABSTRACT TRUNCATED AT 250 WORDS)     

T-cell therapies for T-cell lymphoma

T-cell lymphomas represent a subpopulation of non-Hodgkin lymphomas with poor outcomes when treated with conventional chemotherapy. A variety of novel agents have been introduced as new treatment strategies either as first-line treatment or in conjunction with chemotherapy. Immunotherapy has been demonstrated to be a promising area for new therapeutics, including monoclonal antibodies and adoptive cellular therapeutics. T-cell therapeutics have been shown to have significant success in the treatment of B-cell malignancies and are rapidly expanding as potential treatment options for other cancers including T-cell lymphomas. Although treating T-cell lymphomas with T-cell therapeutics has unique challenges, multiple targets are currently being studied both preclinically and in clinical trials.     

Oxygen uptake rates and liver-specific functions of hepatocyte and 3T3 fibroblast co-cultures

Bioartificial liver (BAL) devices have been developed to treat patients undergoing acute liver failure. One of the most important parameters to consider in designing these devices is the oxygen consumption rate of the seeded hepatocytes which are known to have oxygen consumption rates 10 times higher than most other cell types. Hepatocytes in various culture configurations have been tested in BAL devices including those formats that involve co-culture of hepatocytes with other cell types. In this study, we investigated, for the first time, oxygen uptake rates (OUR)s of hepatocytes co-cultured with 3T3-J2 fibroblasts at various hepatocyte to fibroblast seeding ratios. OURs were determined by measuring the rate of oxygen disappearance using a ruthenium-coated optical probe after closing and sealing the culture dish. Albumin and urea production rates were measured to assess hepatocyte function. Lower hepatocyte density co-cultures demonstrated significantly higher OURs (2 to 3.5-fold) and liver- specific functions (1.6-fold for albumin and 4.5-fold for urea production) on a per cell basis than those seeded at higher densities. Increases in OUR correlated well with increased liver-specific functions. OURs (V(m)) were modeled by fitting Michaelis-Menten kinetics and the model predictions closely correlated with the experimental data. This study provides useful information for predicting BAL design parameters that will avoid oxygen limitations, as well as maximize metabolic functions.     

Thermal performance of quartz capillaries for vitrification

In this paper we report the thermal behavior of a new approach for vitrification. Thermal performance of traditional open pulled straws is compared with a new technique based on the combined use of quartz capillaries with slush nitrogen. This new method of vitrification achieved ultrafast cooling rates of 250,000 °C/min. As a result, a much lower concentration of cryoprotectant was needed to reach vitrification. In fact, a cryoprotectant solution typically used in oocyte slow freezing protocols was shown to remain transparent after cooling to liquid nitrogen temperatures indicating apparent “vitrification”. This approach offers a new and very promising technique for vitrification of cells using low levels of cryoprotectants.     

Starfish oocytes form intracellular ice at unusually high temperatures

Starfish oocytes, eggs, and embryos are popular models for studying meiotic maturation, fertilization, and embryonic development. Their large (170- to 200-microm) oocytes are obtainable in copious amounts and are amenable to manipulations that mammalian oocytes are not. The most formidable obstacle to working with marine oocytes is their seasonal availability, yet a successful means of preserving them for use during the nonreproductive season has not been reported. The aim of this study was to investigate the response of starfish oocytes to freezing with rapid and slow cooling rates under a variety of conditions to develop a cryopreservation protocol for these cells. Cryomicroscopic observation revealed that starfish oocytes in isotonic medium undergo intracellular ice formation (IIF) at very high subzero temperatures, such that the mean difference between the temperature of extracellular ice formation (T(EIF)) and IIF (TI(IF)) was less than 3 degrees C and the average T(IIF) was approximately between -4 and -6 degrees C. Neither partial cellular dehydration nor addition of the cryopreservative dimethyl sulfoxide significantly depressed the T(IIF). Under some conditions, we observed ice nucleation at multiple locations within the cytoplasm, suggesting that several factors contribute to the unusually high T(IIF) during controlled-rate freezing and thus vitrification may be a more suitable method for cryopreserving these cells.     

Beneficial effect of intracellular trehalose on the membrane integrity of dried mammalian cells

Recently, there has been much interest in using trehalose and other small carbohydrates to preserve mammalian cells in the dried state as an alternative to cryopreservation. Here, we report on the successful preservation of plasma membrane integrity after drying, as a first step toward full preservation of mammalian cells. Trehalose was introduced into cells using a genetically engineered version of alpha-hemolysin, a pore-forming protein; the cells were then dried and stored for weeks at different temperatures with approximately 90% recovery of the intact plasma membrane. We show that protection of the plasma membrane by internal trehalose is dose dependent and estimate the amount of internal trehalose required for adequate protection to be approximately 10(10) molecules/cell. In addition, a minimal amount of water (approximately 15 wt%) appears to be necessary. These results show that a key component of mammalian cells can be preserved in a dried state for weeks under mild conditions (-20 degrees C and 5% relative humidity) and thereby suggest new approaches to preserving mammalian cells.     

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.     

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.