Cryoprotective effect of polyols on rat embryos during two-step freezing.

The cryoprotective effect of polyols on rat embryos was measured after two-step freezing, and the mechanism of action of polyols on embryo survival was examined. Rat embryos frozen in solution of polyol by two-step method at the morula stage showed higher survival than that obtained using DMSO. As the number of hydroxyl groups increased, the cryoprotective effect of the polyol increased. However, this was true only when the additive could permeate the cell membrane. Of the additives tested, four or five carbon polyols were most effective at concentrations of 0.3 or 1.0 M than two, three, six, or seven carbon polyols. The highest survival rate was obtained with adonitol, which yielded 83% embryo survival at 1.0 M and 67% even at 0.3 M. Embryos frozen in 0.3 M adonitol and transferred directly into foster mothers without any dilution of the additive after thawing developed into live young. During slow cooling below -40 degrees C, embryonic blastomeres exhibited cell fusion only in the presence of adonitol. These findings suggest that one cryoprotective action of polyols is that the hydroxyl groups act both on the cell surface and the cytoplasm to stabilize the bound water on the embryonic membrane, and that the length of the C-chain determines the permeability of the membrane to the additive.     

The effect of the cryoprotectants,dimethylsulfoxide and glycerol on water transport in the human red blood cell

The response of human red blood cells to the cryoprotective agents, DMSO and glycerol, has been investigated using a pulsed NMR method. The experimentally determined parameters are: (1) the intracellular transverse relaxation time, T_2a; (2) the mean residence time of intracellular water, τ_a, which is effectively a reciprocal measure of the rate of water transport across the red blood cell membrane; and (3) the activation energy for this process. The quantitative data indicate that the observed effects are colligative rather than species-specific in origin.     

The acute-phase response of cultured rat hepatocytes. System characterization and the effect of human cytokines.

Hepatocytes were isolated from adult livers and cultured for periods of up to 5 days as monolayers at an initial density of 10(6) cells/10cm2 in Williams E medium containing insulin, dexamethasone and 5% foetal-calf serum. The daily production of 11 plasma proteins was measured by electroimmunoassay and compared with the concentrations of the same proteins in the plasma of normal rats and of those with experimental inflammation. Hepatocytes from normal rats synthesized proteins in relative amounts which were similar to the relative proportions of the same proteins in the plasma of turpentine-injected animals. The pattern changed only slowly during 5 days in culture, but it did so profoundly either when the medium was devoid of dexamethasone or when human cytokines (from endotoxin-stimulated monocytes or unstimulated human squamous-carcinoma cell line COLO-16) were added. The cytokines consistently increased the synthesis of alpha 2-macroglobulin and fibrinogen and depressed that of albumin; variable increases in the synthesis of alpha 1-acute-phase globulin, alpha 1-acid glycoprotein, haptoglobin and alpha 1-proteinase inhibitor, and variable decreases in transferrin synthesis, were seen, whereas the synthesis of antithrombin III, alpha 1-macroglobulin and prothrombin remained virtually unaffected. The cytokine effects on protein synthesis required the presence of dexamethasone. The hepatocyte-stimulating activity derived from monocytes chromatographed on Sephadex G-100 corresponding to 30 000 Da, as opposed to the lymphocyte-activating factor, which was eluted as a molecule of approx. 15 000 Da. This suggests that both activities probably reside with distinct molecular species in the preparations of human cytokines.     

The effect of dimethylsulfoxide and thiourea on the water diffusion permeability of the E. coli membrane

It is shown that diffusive water permeability of E. coli cell membranes at 4-24 degrees C is in the range from 16.6 to 35.0 microM.s-1, that is close to erythrocyte membrane water permeability, but higher than that of lipid bilayers. Cryoprotectants (DMSO and thiourea) at the concentration of 1.0 M considerably decrease water bacterial membrane permeability. 1.5- and 2-fold, respectively. The obtained results are discussed in relation to two possible water transport ways through pores of the protein nature or lipid bilayer damages.     

The effect of carbon tetrachloride on isolated rat hepatocytes

Isolated rat hepatocytes were incubated with carbon tetrachloride (CCl4) at a concentration of 0.2 mol CCl4/ml of incubation medium. The ultrastructural alterations and release of lactate dehydrogenase (LDH) and glutamate-oxaloacetate transaminase (GOT), were recorded after different periods of incubation. After 5 min incubation with CCl4, morphological changes observed by electron microscopy, involved the plasma membrane. The endoplasmic reticulum and mitochondria were altered later. These morphological alterations were accompanied by an early release of LDH and GOT into the incubation medium. It is concluded that, in contrast with its in vivo effects, in vitro CCl4 can induced an early morphological alteration of the hepatocyte plasma membrane before damaging the endoplasmic reticulum.     

Effect of intercellular junction protein expression on water transport during freezing of MIN6 cells

A mouse insulinoma (MIN6) strain in which connexin expression has been inhibited by antisense technology holds promise as an experimental model system for investigating the role of gap junctions in intercellular ice propagation. However, to properly interpret measurements of intracellular ice formation kinetics, the effects of cell dehydration on cytoplasmic supercooling must be determined. Thus, the cell membrane water permeability in monolayer cultures of the antisense-transfected MIN6 strain was measured using a fluorescence quenching method. By repeating the experiments at 4 °C, 12 °C, 21 °C, and 37 °C, the activation energy for water transport was determined to be E_a = 51 ± 3 kJ/mol. Although differences between membrane permeability measurements in theantisense and wild-type strains were not statistically significant, simulation of water transport during rapid freezing (130 °C/min) predicted that intracellular supercooling in the genetically modified MIN6 strain may become significantly larger than the supercooling in wild-type cells at temperatures below −15 °C.     

Epinephrine regulation of amino acid transport in rat hepatocytes isolated during development

The effect of epinephrine on the amino acid transport mediated by system A was investigated by determining the uptake of 2-amino [1-14C]isobutyric acid (AIB) in rat hepatocytes, freshly isolated at different stages of pre- and postnatal development. The data obtained show that the hormone increased AIB uptake, enhancing the Vmax, while Km was unchanged. This effect was evident in cells from adult, 18- to 20-day-old fetus, and neonate rat. Actinomycin D or cycloheximide abolished the hormone dependent increase. Experiments carried out with alpha- and beta-antagonists showed that the effect of epinephrine was beta-mediated in fetal life and alpha-mediated in adult life. Membrane binding experiments showed a higher value for epinephrine and beta-agonist dihydroalprenolol in the fetus versus the adult. The calcium depletion obtained after cell incubation with EGTA or calcium ionophore A23187 reduced the hormonal stimulation in the adult, and was ineffective in the prenatal period. An involvement of cAMP was present in the epinephrine modulation of AIB transport, both in adult and in fetal life.     

Effect of different freezing rates during cryopreservation of rat mesenchymal stem cells using combinations of hydroxyethyl starch and dimethylsulfoxide

ABSTRACT: BACKGROUND: Mesenchymal stem cells (MSCs) are increasingly used as therapeutic agents as well as research tools in regenerative medicine. Development of technologies which allow storing and banking of MSC with minimal loss of cell viability, differentiation capacity, and function is required for clinical and research applications. Cryopreservation is the most effective way to preserve cells long term, but it involves potentially cytotoxic compounds and processing steps. Here, we investigate the effect of decreasing dimethyl sulfoxide (DMSO) concentrations in cryosolution by substituting with hydroxyethyl starch (HES) of different molecular weights using different freezing rates. Post-thaw viability, phenotype and osteogenic differentiation capacity of MSCs were analysed. RESULTS: The study confirms that, for rat MSC, cryopreservation effects need to be assessed some time after, rather than immediately after thawing. MSCs cryopreserved with HES maintain their characteristic cell surface marker expression as well as the osteogenic, adipogenic and chondrogenic differentiation potential. HES alone does not provide sufficient cryoprotection for rat MSCs, but provides good cryoprotection in combination with DMSO, permitting the DMSO content to be reduced to 5%. There are indications that such a combination would seem useful not just for the clinical disadvantages of DMSO but also based on a tendency for reduced osteogenic differentiation capacity of rat MSC cryopreserved with high DMSO concentration. HES molecular weight appears to play only a minor role in its capacity to act as a cryopreservation solution for MSC. The use of a 'straight freeze' protocol is no less effective in maintaining post-thaw viability of MSC compared to controlled rate freezing methods. CONCLUSION: A 5% DMSO / 5% HES solution cryopreservation solution using a 'straight freeze' approach can be recommended for rat MSC.     

The inhibitory effect of choline and other quaternary ammonium compounds on thiamine transport in isolated rat hepatocytes

Quaternary ammonium compounds, such as choline and acetylcholine significantly inhibited thiamine uptake in isolated rat hepatocytes. Kinetic analysis using Lineweaver-Burk and Dixon plots of inhibition experiments revealed that choline and acetylcholine were purely competitive inhibitors for thiamine uptake with Ki values of 0.61 mM and 0.31 mM, respectively. Among quaternary ammonium compounds, hemicholinium-3 and curare were the strongest inhibitors, and kinetic studies showed that these compounds were also purely competitive inhibitors with Ki values of 12.5 microM and 4.3 microM, respectively. These results indicate that choline, acetylcholine and their structural analogs share a common binding site with thiamine in isolated rat hepatocytes. On the other hand, choline uptake by isolated rat hepatocytes occurred by a saturable mechanism with a Kt of 162 +/- 3.85 microM and Vmax of 80.1 +/- 1.30 pmol/10(5) cells per min as well as by a nonsaturable mechanism. Thiamine, pyrithiamine, oxythiamine, chloroethylthiamine and dimethialium inhibited choline uptake, while thiamine phosphates such as thiamine monophosphate and thiamine pyrophosphate insignificantly inhibited uptake. Although a Lineweaver-Burk plot of choline uptake in the presence of thiamine showed that thiamine also competitively inhibited choline uptake, a Dixon plot of the inhibition experiment was hyperbolic and indicated that the inhibition of choline uptake by thiamine was 'pseudo-competitive'. On the basis of these results, it is suggested that in isolated rat hepatocytes thiamine and choline do not share common transport sites.     

Kinetics of 3-O-methyl-D-glucose transport in isolated rat hepatocytes.

3-O-Methyl-D-glucose transport across the plasma membrane of isolated rat hepatocytes was followed for net entry of the sugar into sugar-free cells (zero trans entry), net exit of sugar into sugar-free medium (zero trans exit) and for unidirectional entry and exit fluxes when cells had been equilibrated with sugar in the extracellular medium (equilibrium exchange entry and exit). These measurements were performed at 20 degrees C and pH 7.4 by the use of simple manual methods. Initial rates of transport showed a Michaelis--Menten dependency on the sugar concentration at the cis side of the membrane over the range of concentrations tested (100 microM to 100 mM). Transport was found to be symmetrical with no evidence of substrate stimulation of transport from the trans side of the membrane. Parameters (mean values +/- S.E.M.) of transport were estimated as Vmax. 86.2 +/- 9.7 mmol/litre of cell water per min and Km 18.1 +/- 5.9 mM for exchange entry, Vmax. 78.8 +/- 5.3 mmol/litre of cell water per min and Km 17.6 +/- 3.5 mM for exchange exit, Vmax. 84.1 +/- 8.4 mmol/litre of cell water per min and Km 16.8 +/- 4.6 mM for zero trans exit.     

The production of free radicals during the autoxidation of cysteine and their effect on isolated rat hepatocytes

Autoxidizing cysteine has been shown to produce thiyl and hydroxyl radicals. Hydrogen peroxide increased the yield of both radicals which was inhibited by catalase but stimulated by copper / zinc superoxide dismutase. This effect is due to increased bydrogen peroxide production by copper / zinc superoxide dismutase as a result of superoxide dismutation. The production of superoxide radicals could not be detected probably because of its low reactivity, however, measurement of oxygen uptake and reduction of ferricytochrome c by autoxidizing cysteine clearly implicate the involvement of super oxide radicals. The production of hydroxyl radicals is postulated to proceed through a Fenton reaction, however, this may not necessarily be metal ion controlled. Autoxidizing cysteine disrupts the integrity of heptocytes causing release of glutathione, adenosine triphosphate and lactate dehydrogenase indicating that it is of little use as a therapeutic agent.     

The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na+-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of alpha-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of alpha-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na+ influx.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).     

Calorimetric measurement of water transport and intracellular ice formation during freezing in cell suspensions

The current study presents a new and novel analysis of heat release signatures measured by a differential scanning calorimeter (DSC) associated with water transport (WT), intracellular ice formation (IIF) and extracellular ice formation (EIF). Correlative cryomicroscopy experiments were also performed to validate the DSC data. The DSC and cryomicroscopy experiments were performed on human dermal fibroblast cells (HDFs) at various cytocrit values (0–0.8) at various cooling rates (0.5–250 °C/min). A comparison of the cryomicroscopy experiments with the DSC analysis show reasonable agreement in the water transport (cellular dehydration) and IIF characteristics between both the techniques with the caveat that IIF measured by DSC lagged that measured by cryomicroscopy. This was ascribed to differences in the techniques (i.e. cell vs. bulk measurement) and the possibility that not all IIF is associated with visual darkening. High and low rates of 0.5 °C/min and 250 °C/min were chosen as HDFs did not exhibit significant IIF or WT at each of these extremes respectively. Analysis of post-thaw viability data suggested that 10 °C/min was the presumptive optimal cooling rate for HDFs and was independent of the cytocrit value. The ratio of measured heat values associated with IIF (q_IIF) to the total heat released from both IIF and water transport or from the total cell water content in the sample (q_CW) was also found to increase as the cooling rate was increased from 10 to 250 °C/min and was independent of the sample cytocrit value. Taken together, these observations suggest that the proposed analysis is capable of deconvolving water transport and IIF data from the measured DSC latent heat thermograms in cell suspensions during freezing.