Osmotic response of oocytes using a microscope diffusion chamber: a preliminary study comparing murine and human ova

The hydraulic conductivity (Lp) of the plasma membrane determines how cells respond to the stresses of dehydration encountered during cryopreservation. We have used a microscope diffusion chamber which allows for direct real-time observation of the dynamic osmotic response of individual cells in microvolume suspension to compare the Lp of murine and human unfertilized ova. In this system, the response of an individual cell to the induced osmotic imbalance is documented via a series of photomicrographs or videotape; from these data the Lp can be computed. Donated human preovulatory oocytes were compared with macroscopically normal human ova, 43 hr after insemination, which had failed to fertilize (Ff) and with murine ova collected 13 hr post human chorionic gonadotropin injection. The permeability coefficients were 0.65 +/- 0.43, 0.84 +/- 0.39, and 0.36 +/- 0.07 micron3/micron2/atm/min. The results suggest that it may be possible to use Ff ova for experiments to design suitable cryopreservation procedures.     

Determination of diffusion and permeability coefficients in muscle

A theory is presented for the study of diffusion in heterogeneous tissue-like structures. It is applicable to a common type of measurement in which the change of the amount of substance remaining in the tissue is determined as the substance diffuses from the tissue into an adjacent medium, for instance, Ringer's solution. The main objective of this paper is to obtain a method for the calculation of the diffusion coefficient in the intercellular space and of the permeability coefficients between this space and the cells, based on the type of measurement mentioned above. Although the fundamental ideas upon which the theory is based are applicable to any type of tissue, the formulae derived are limited to the case in which the cells form a flat bundle of parallel fibers. The theory is applied to the experimental results of E. J. Harris and G. P. Burn on diffusion of sodium in the sartorius muscle of the frog. We find that if we know the ratio of the cellular and intercellular volumes of the muscle the ratio of the equilibrium concentrations of sodium outside and inside the cells can be determined. A very simple mathematical analysis of the experimental relation between the amount of substance diffusing out of the muscle and the time of diffusion gives us this ratio. The ratio of the equilibrium sodium concentrations in the case of the sartorius frog muscle is between about 10 and 30, depending on the muscle used. The same mathematical analysis makes it possible to obtain the permeability coefficients of muscle fibers through simple calculations, if their sizes are known. The permeability coefficients for the experimental work mentioned above using sodium are 1.25 to 11.5×10⁻⁸ cm/sec for the flow into the fibers and 3.2 to 16×10⁻⁷ cm/sec for the flow in the opposite direction. The determination of the diffusion coefficient in the intercellular space is more laborious and yields only an order of magnitude: 10⁻⁶ cm²/sec.     

The permeability of human lymphocytes to chloride

The normal amount of Cl in human lymphocytes is 82 mmoles/kg wet weight. Half of this undergoes rapid self-exchange with a half-time of 3 minutes, while the remainder exchanges slowly with a half-time of 180 minutes. The fast fraction of self-exchange of Cl also undergoes a rapid net loss into medium with a low concentration of Cl. Thus, exchange of Cl in lymphocytes has properties like that of K and Na with permeability constants on the order of 10(-6) cm/sec. The results are compatible with a simple model in which the fast fractions are dissolved within ordered cellular water at concentrations less than in the external medium and the slow fractions are adsorbed onto intracellular macromolecules.     

Determination of diffusion and permeability coefficients in nerve trunks

A mathematical theory is developed which permits the determination of certain parameters of an inhomogenous tissue, such as a nerve trunk without its epineurium. The parameters are the permeability coefficients for entrance into an exit of a substance from the nerve fibers, and the diffusion coefficient of the interstitial material. The experimental data required are the dimensions of the cross-section, the average diameter of the fibers, and the ratio of the cross-sectional are of the fibers to the total cross-section, as well as the time course of the decrease of the fraction of the substance left in the nerve trunk, when the trunk is immersed in a bathing solution containing none of it.     

Volume-induced increase of anion permeability in human lymphocytes

Peripheral blood mononuclear cells (PBM) readjust their volumes after swelling in hypotonic media. This regulatory volume decrease (RVD) is associated with a loss of cellular K+ and is thought to be promoted by an increased permeability to this ion. In contrast, no change in volume was observed when K+ permeability of PBM in isotonic media was increased to comparable or higher levels using valinomycin. Moreover, valinomycin-induced 86Rb+ loss in K+-free medium was considerably slower than in K+-rich medium. These results suggest that anion conductance limits net salt loss in isotonic media. Direct measurements of relative conductance confirmed that in volume-static cells, anion conductance is lower than that of K+. In volume-regulating cells depolarization occurred presumably as a result of increased anion conductance. Accordingly, the efflux of 36Cl from PBM was markedly increased by hypotonic stress. Since both membrane potential and intracellular 36Cl concentration are reduced in hypotonically swollen cells, the increased efflux is probably due to a change in Cl- permeability. Anions and cations seem to move independently through the volume-induced pathways: the initial rate of 86Rb uptake in swollen cells was not affected by replacement of external Cl- by SO=4; conversely, 36Cl fluxes were unaffected by substitution of K+ by Na+. The data indicate that anion conductance is rate-determining in salt and water loss from PBM. An increase in anion conductance is suggested to be the critical step of RVD of human PBM.     

Enumerating phytoplankton with an upright compound microscope using a modified settling chamber

A modified settling chamber is described which permits the use of an upright compound microscope for phytoplankton enumerations. The chamber is composed of a 75 × 51 mm rectangular or 70 mm round-glass microslide base with a 130 m thick piece of sheet styrene attached to the upper surface. A circular cell is cut into the styrene material making a 26 mm diameter chamber which is approximately 130–150 m deep. Settling procedures follow Utermöhl's technique, with the use of a 0.13–0.15 mm thick coverslip (50 × 45 mm) to cover the chamber. The overall thickness of the settling slide is 1.13 mm which does not impact on the optical requirements for most objectives, including oil immersion objectives. The chamber encloses a total volume of 69–80 mm³. No statistical differences are observed in cell, filament or colony counts between the new and traditional chambers. Furthermore, count comparisons at different cell concentrations using the new chambers give consistent results. Thus, the resolution and availability of an upright microscope makes the use of the new settling chamber an attractive method for phytoplankton counting, especially in teaching situations.     

Water diffusion permeability of human erythrocytes studied by a pulsed gradient NMR technique

Water diffusion permeability of human erythrocytes has been measured by NMR using a pulsed magnetic field gradient technique. The measurement of exchange rates was based on restricted diffusion of water molecules within red blood cells. This method avoids addition of paramagnetic ions, such as Mn²⁺ and is used in vivo.The mean lifetime of water inside human erythrocytes was found to be 17 ms at 24°C. A sulfhydryl reagent, known to inhibit water osmotic permeability, reduced significantly water diffusion across the red cell membrane.     

Increased anion permeability during volume regulation in human lymphocytes

Peripheral blood lymphocytes (p.b.ls) readjust their volumes after swelling in hypotonic media. An essential component of the regulatory response is an increase in K+ and Cl- permeability. No evidence was found for a tightly coupled co-transport of K+ and Cl-. The flux of either ion proceeds normally in the virtual absence of the transported counterion. Furthermore, alterations in membrane potential recorded during the phase of volume readjustment can be qualitatively accounted for by an increase in Cl- conductance. In tonsillar lymphocytes, a failure of the K+-permeability is nevertheless increased upon swelling. This further suggests that K+ and Cl- are transported during volume regulation through independent pathways. Cytoplasmic free Ca2+ appears to be involved in regulatory volume decrease. K+ and Cl-. Moreover, swelling and shrinking can be induced in isotonic K+-rich and K+-free media, respectively, by the Ca2+ ionophore. The ion flux and volume changes produced by either swelling or internal Ca2+ can be inhibited by similar concentrations of quinine and phenothiazines. The inhibitory activity of the latter drugs, which are powerful antagonists of calmodulin, suggests the participation of this Ca2+-regulator protein in volume regulation.     

Determination of LD7a structures on human lymphocytes]

The non-stimulation of DNA synthesis in human lymphocytes in the one way mixed lymphocyte culture conditions allows the typing for LD determinants. The authors describe their technique together with their cryo-preservation procedure and the results obtained in 24 control population and 26 multiple sclerosis patients tested for the presence of the LD7a determinant.     

Human normal B lymphocytes produce a growth-promoting activity for granulocyte progenitors in diffusion chamber culture after stimulation with pokeweed mitogen.

We have measured the effect of normal B lymphocytes on more primitive granulocyte progenitors (CFU-dG) clonal growth in double-diffusion chamber culture in vivo. It was found that pokeweed mitogen (PWM) stimulated B cells produce a growth-promoting activity which augments the CFU-dG--derived myeloid colony formation in a dose-dependent fashion. All colonies formed under the experimental conditions were composed exclusively of granulocytes at different stages of maturation. Unstimulated B lymphocytes did not effect the CFU-dG clonal proliferation.     

A new diffusion chamber system for the determination of drug permeability coefficients across the human intestinal epithelium that are independent of the unstirred water layer.

A new method for determining permeability coefficients, that are independent of the unstirred water layer (UWL), has been developed. The method was used to determine the cellular permeability coefficient of the rapidly absorbed drug testosterone in monolayers of the human intestinal epithelial cell line, Caco-2. Using a new diffusion cell with an effective stirring system based on a gas lift, the cellular permeability coefficient for testosterone was (1.98 +/- 0.13).10(-4) cm/s which is 3.5-times higher than the permeability coefficient obtained in the unstirred system. The thickness of the UWL obtained with the well stirred diffusion cell was 52 +/- 4 microns. This value is much lower than those previously reported in various well stirred in vitro models. The calculated cellular permeability of testosterone was 13-23-times lower than that for an UWL of the same thickness as the epithelial cell (17-30 microns). We conclude that the permeability of the epithelial monolayer must be included in calculations of the thickness of the UWL.     

Water diffusion permeability of human erythrocytes studied by a pulsed gradient NMR technique.

Water diffusion permeability of human erythrocytes has been measured by NMR using a pulsed magnetic field gradient technique. The measurement of exchange rates was based on restricted diffusion of water molecules within red blood cells. This method avoids addition of paramagnetic ions, such as Mn2+, and is used in vivo. The mean lifetime of water insed human erythrocytes was found to be 17 ms at 24 degrees C. A sulfhydryl reagent, known to inhibit water osmotic permeability, reduced significantly water diffusion across the red cell membrane.     

Modeling microbial chemotaxis in a diffusion gradient chamber

The diffusion gradient chamber (DGC) has proven to be a useful experimental tool for studying population-level microbial growth and chemotaxis. A mathematical model capable of reproducing the population-level patterns formed as a result of cellular growth and chemotaxis in the DGC has been developed. The model consists of coupled partial differential balance equations for cells, chemoattractants, and a nutrient, which are solved simultaneously by the alternating direction implicit method. Modeling simulation results were compared with population-level migration patterns of Escherichia coli growing on glycerol and responding to a gradient of the chemoattractant aspartate for two different initial conditions. To accurately reproduce the experimental results, a second chemoattractant equation was necessary. The second chemoattractant has been identified as oxygen by directly measuring oxygen gradients in the DGC. Important trends observed experimentally and reproduced by the model include the formation of a chemotactic wave, a reduction in the wave velocity as it encounters higher chemoattractant concentrations, and chemotaxis in response to two different chemoattractants simultaneously. The model was also used to study the relative magnitude of cell fluxes due to random motility and chemotaxis, and the suppression of chemotaxis due to receptor saturation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 191-205, 1997.     

Determination of catecholamine permeability coefficients for passive diffusion across phospholipid vesicle membranes

Summary A convenient catecholamine transport assay has been developed which permits continuous, instantaneous monitoring of transmembrane flux. Epinephrine transport has been examined by spectrophotometrically monitoring adrenochrome formation resulting from the passive diffusion of catecholamine into unilamellar phospholipid vesicles containing entrapped potassium ferricyanide. Ferricyanide oxidation of epinephrine under the conditions employed is fast compared to membrane transport, which obviates the need for intravesicular concentration or volume determinations. Epinephrine transport data over a pH 6 to 7 range have been fitted to an integrated rate equation from which a permeability coefficient for neutral epinephrine of 2.7±1.5×10^–6 cm/sec has been obtained.     

Non-steady-state O(2) diffusion in metmyoglobin solutions studied in a diffusion chamber

In this study, we studied the "passive" diffusion through myoglobin solutions by determining the oxygen diffusion coefficient (DO(2)) and the oxygen permeability (permeability O(2)) of metmyoglobin (metMb) solutions (3-33 g. 100 mL(-1)) at 25 degrees C. These oxygen diffusion parameters were determined in a diffusion chamber using a non-steady-state method and were also determined of albumin solutions (4-32 g. 100 mL(-1)) and distilled water for comparison. From these parameters, the oxygen solubility (alphaO(2)) could be calculated, because alphaO(2) = permeabilityO(2)/DO(2). Both DO(2) and permeabilityO(2) decreased with increasing metMb and albumin concentration. The values of DO(2), permeabilityO(2) and alphaO(2) of both metMb solutions and albumin solutions were comparable with literature values of methemoglobin (metHb) and serum protein solutions. The values of the metMb solutions can be used in following studies of facilitated oxygen diffusion through myoglobin solutions.