Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume,hydraulic conductivity and permeability of CD34(+) cells to dimethyl sulphoxide

Umbilical cord blood (UCB) is an accepted treatment for the reconstitution of bone marrow function following myeloablative treatment predominantly in children and juveniles. Current cryopreservation protocols use methods established for bone marrow and peripheral blood progenitors cells that have largely been developed empirically. Such protocols can result in losses of up to 50% of the nucleated cell population: losses unacceptable for cord blood. The design of optimal cryopreservation regimes requires the development of addition and elution protocols for the chosen cryoprotectant; protocols that minimise damaging osmotic transients. The biophysical parameters necessary to model the addition and elution of dimethyl sulphoxide to and from cord blood CD34(+) cells have been established. An electronic particle counting method was used to establish the volumetric response of CD34(+) cells to changes in osmolality of the suspending medium. The non-osmotic volume of the cell was 0.27 of the cells isotonic volume. The permeation kinetics of CD34(+) cells to water and dimethyl sulphoxide were investigated at two temperatures, +1.5 and +20 degrees C. Values for the hydraulic conductivity were 3.2 x 10(-8) and 2.8 x 10(-7)cm/atm/s, respectively. Values for the permeability of dimethyl sulphoxide at these temperatures were 4.2 x 10(-7) and 7.4 x 10(-6)cm/s, respectively. Clonogenic assays indicated that the ability of CD34(+) cells to grow and differentiate was significantly impaired outside the limits 0.6-4x isotonic. Based on the Boyle van't Hoff plot, the tolerable limits for cell volume excursion were therefore 45-140% of isotonic volume. The addition and elution of cryoprotectant was modelled using a two-parameter model. Current protocols for the addition of cryoprotectant based on exposure at +4 degrees C would require additional time for complete equilibration of the cryoprotectant. During the elution phase current protocols are likely to cause CD34(+) cells to exceed tolerable limits. The addition of a short holding period during elution reduces the likelihood of this occurring.     

Fundamental cryobiology of rat immature and mature oocytes: hydraulic conductivity in the presence of Me(2)SO, Me(2)SO permeability, and their activation energies

The hydraulic conductivity in the presence of dimethyl sulfoxide Me(2)SO (L(p)(Me(2)SO)), Me(2)SO (P(Me(2)SO)) permeability and reflection coefficient (sigma) of immature (germinal vesicle; GV) and mature (metaphase II; MII) rat oocytes were determined at various temperatures. A temperature controlled micropipette perfusion technique was used to conduct experiments at five different temperatures (30, 20, 10, 4, and -3 degrees C). Kedem and Katchalsky membrane transport theory was used to describe the cell volume kinetics. The cell volumetric changes of oocytes were calculated from the measurement of two oocyte diameters, assuming a spherical shape. The activation energies (E(a)) of L(p)(Me(2)SO) and P(Me(2)SO) were calculated using the Arrhenius equation. Activation energies of L(p)(Me(2)SO) for GV and MII oocytes were 34.30 Kcal/mol and 16.29 Kcal/mol, respectively; while the corresponding E(a)s of P(Me(2)SO) were 19.87 Kcal/mol and 21.85 Kcal/mol, respectively. These permeability parameters were then used to calculate cell water loss in rat oocytes during cooling at subzero temperatures. Based on these values, the predicted optimal cooling rate required to maintain extra- and intracellular water in near equilibrium for rat GV stage oocytes was found to be between 0.05 degrees C/min and 0. 025; while for rat MII oocytes, the corresponding cooling rate was 1 degrees C/min. These data suggest that standard cooling rates used for mouse oocytes (e.g., 0.5-1 degrees C/min) can also be employed to cryopreserve rat MII oocytes. However, the corresponding cooling rate required to avoid damage must be significantly slower for the GV stage rat oocyte. J. Exp. Zool. 286:523-533, 2000.     

Ultrastructure of human mature oocytes after vitrification

Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morpho-functional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microsco py has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.Key words: oocyte, MII, vitrification, ultrastructure, TEM, human.     

Gallbladder epithelial cell hydraulic water permeability and volume regulation

The hydraulic water permeability (Lp) of the cell membranes of Necturus gallbladder epithelial cells was estimated from the rate of change of cell volume after a change in the osmolality of the bathing solution. Cell volume was calculated from computer reconstruction of light microscopic images of epithelial cells obtained by the "optical slice" technique. The tissue was mounted in a miniature Ussing chamber designed to achieve optimal optical properties, rapid bath exchange, and negligible unstirred layer thickness. The control solution contained only 80% of the normal NaCl concentration, the remainder of the osmolality was made up by mannitol, a condition that did not significantly decrease the fluid absorption rate in gallbladder sac preparations. The osmotic gradient ranged from 11.5 to 41 mosmol and was achieved by the addition or removal of mannitol from the perfusion solutions. The Lp of the apical membrane of the cell was 1.0 X 10(-3) cm/s . osmol (Posm = 0.055 cm/s) and that of the basolateral membrane was 2.2 X 10(-3) cm/s . osmol (Posm = 0.12 cm/s). These values were sufficiently high so that normal fluid absorption by Necturus gallbladder could be accomplished by a 2.4-mosmol solute gradient across the apical membrane and a 1.1-mosmol gradient across the basolateral membrane. After the initial cell shrinkage or swelling resulting from the anisotonic mucosal or serosal medium, cell volume returned rapidly toward the control value despite the fact that one bathing solution remained anisotonic. This volume regulatory response was not influenced by serosal ouabain or reduction of bath NaCl concentration to 10 mM. Complete removal of mucosal perfusate NaCl abolished volume regulation after cell shrinkage. Estimates were also made of the reflection coefficient for NaCl and urea at the apical cell membrane and of the velocity of water flow across the cytoplasm.     

Recovery of functional capacity of human granulocytes after freeze-preservation with dimethyl sulphoxide.

Huma peripheral blood leucocytes (neutrophil rich) were collected either with preservative-free heparin (PFH) or acid citrate dextrose (ACD), frozen with dimethyl sulphoxide (DMSO) at a controlled rate, stored in liquid nitrogen at ?196 °C and reconstituted in a solution containing dextran polymer 70.A battery of tests including nitroblue tetrazolium (NBT) reduction, Candida phagocytic and candidacidal capacity was used to compare anticoagulants and reconstitution methods as they affect functional capacity of freeze-thawed neutrophils during a short post-thaw period.Heparin showed an overall advantage over acid citrate dextrose. A slow titration reconstitution method did not improve cell yield or functional capacity compared with a rapid dilution method and was a more cumbersome technique. The presence of complement greatly improved the capacity of reconstituted cells to reduct NBT and synthesize formazan. Freeze-thawed cells showed a selective response to stimulation as judged by the quantitative NBT test, responding strongly to zymosan in comparison with E. coli endotoxin. Lignocaine hydrochloride added to the reconstituent medium in concentrations up to 20 mmol/l did not have additional protective effect on post-thawed leucocytes as assessed by agglutination and leucocyte yields when compared with reconstitutent solutions containing only dextran. Reducing pH did not significantly slow the rate of gelling and leuco-agglutination or improve cell yields.Using these findings to optimize conditions reconstituted neutrophils retained 31.7% of fresh NBT activity, 27.6% of fresh phagocytic, and 22.3% of fresh candidacidal capacity.     

The permeability to water and cryoprotectants of immature and mature oocytes in the zebrafish (Danio rerio)

To identify a stage feasible for the cryopreservation of zebrafish oocytes, we investigated the permeability to water and cryoprotectants of immature (stage III) and mature (stage V) oocytes. The permeability to water (microm/min/atm) of immature oocytes at 25 degrees C (0.37) was significantly higher than that of mature oocytes (0.10). The permeability (x10(-3)cm/min) of immature oocytes to ethylene glycol, propylene glycol, and Me(2)SO (1.49-3.03) at 25 degrees C was substantially higher than that of mature oocytes approximately 0. The permeability of immature oocytes to glycerol was also high (1.75), although the permeability could not be measured in mature oocytes. Immature oocytes would be more suitable than mature oocytes for conservation of the zebrafish.     

Effects of salinity on growth,membrane permeability and root hydraulic conductivity in three saltbush species

The species of the genus Atriplex have been introduced in West Asia and North Africa to determine their adaptability for use as fodder species. These halophytes are well adapted to extreme environmental conditions and may possess interesting properties for soil rehabilitation. The effect of NaCl stress on growth, water relation and mineral nutrition were investigated in three xero-halophyte species of Atriplex used for rehabilitation of arid steppe in Algeria. Atriplex halimus, Atriplex canescens and Atriplex nummularia, were cultivated in hydroponic conditions and treated with increasing doses of NaCl (0–300 mM). All species showed positive plant growth for low and moderate levels of salinity. A. halimus had higher dry weight production than A. nummularia and A. canescens in high salinity concentration. Increasing concentration of salinity induced decrease in chlorophyll content (Chl a and b) and root hydraulic conductivity (L₀) in all species, especially in A. canescens. All three species showed marked increase in electrolyte leakage across the salinity gradient. In addition all species were able to accumulate a large quantity of sodium (Na), chloride (Cl) and proline and to maintain higher relative water content, which was probably associated with a greater capacity for osmotic adjustment, whereas potassium (K) and calcium (Ca) decreased with increase salinity. The data suggest that salt tolerance strategies in all Atriplex species could involve a delicate balance among ion accumulation, osmotic adjustment, production of osmotica and maintenance of relative water content and growth.     

Membrane permeability of the human granulocyte to water,dimethyl sulfoxide,glycerol, propylene glycol and ethylene glycol

Granulocytes are currently transfused as soon as possible after collection because they rapidly deteriorate after being removed from the body. This short shelf life complicates the logistics of granulocyte collection, banking, and safety testing. Cryopreservation has the potential to significantly increase shelf life; however, cryopreservation of granulocytes has proven to be difficult. In this study, we investigate the membrane permeability properties of human granulocytes, with the ultimate goal of using membrane transport modeling to facilitate development of improved cryopreservation methods. We first measured the equilibrium volume of human granulocytes in a range of hypo- and hypertonic solutions and fit the resulting data using a Boyle-van’t Hoff model. This yielded an isotonic cell volume of 378 μm³ and an osmotically inactive volume of 165 μm³. To determine the permeability of the granulocyte membrane to water and cryoprotectant (CPA), cells were injected into well-mixed CPA solution while collecting volume measurements using a Coulter Counter. These experiments were performed at temperatures ranging from 4 to 37 °C for exposure to dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol. The best-fit water permeability was similar in the presence of all of the CPAs, with an average value at 21 °C of 0.18 μm atm⁻¹ min⁻¹. The activation energy for water transport ranged from 41 to 61 kJ/mol. The CPA permeability at 21 °C was 6.4, 1.0, 8.4, and 4.0 μm/min for dimethyl sulfoxide, glycerol, ethylene glycol, and propylene glycol, respectively, and the activation energy for CPA transport ranged between 59 and 68 kJ/mol.     

Effect of dimethyl sulphoxide and some antibiotics on cultured human T-lymphoma cells as measured by microcalorimetry

The influence of dimethyl sulphoxide (I), penicillin/streptomycin (II), gentamicin (III), and amphotericin B (IV) on growing human T-lymphoma cells was measured by microcalorimetry. There was a dose-dependent decrease in the heat production rate of the cells after 24 h of incubation with I in concentrations ranging from 0-2% (v/v). At 3.6%, about half of the cells died. II and III had no effect on the cells after incubation for 6 days, at concentrations from 1 to 10 times that of the normal (50-500 IU/ml; 50-500 micrograms/ml). IV was used in combination with II (50 IU/ml; 50 micrograms/ml) and III (50 micrograms/ml), respectively, at concentrations between 0.25 and 7.5 micrograms/ml. After 6 days of incubation, the results were similar to those obtained with II and III separately.     

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle–van’t Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15 ± 0.01 using the new method, which was significantly smaller than the estimate of 0.37 ± 0.02 obtained using the conventional method (p < 0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.     

Effects of dimethyl sulphoxide on ATP content and protein synthesis inTetrahymena

Summary The content of ATP in cells exposed for 1 hour to 2.5% and 7.5% dimethyl sulphoxide (DMSO) was 90% and about 80%, respectively, of that in control cells. This difference of about 10% in the ATP content cannot explain the previously reported cessation of food vacuole formation in 7.5% DMSO and the uninhibited function in 2.5% DMSO (Nilsson 1974). However, DMSO had a dose-dependent effect on the rate of turnover of ATP in cell extracts, thus the amount of ATP expended per unit time in 7.5% DMSO is only 60% of that expended by extracts of control cells. The rate of protein synthesis was studied by electron microscope autoradiography which revealed considerably less labelled material in DMSO-treated cells than in control cells. Semi-quantitative estimates showed that cells in 2.5%, 5%, and 7.5% DMSO had a rate of incorporation of the labelled amino acid corresponding to 38%, 31%, and 51%, respectively, of that of control cells; the seemingly high rate of incorporation in 7.5% DMSO may reflect a low internal pool of amino acids in these cells. An additional fine structural detail is the induction of intranuclear fibrous bundles in all concentrations of DMSO. The findings are in accord with a random interference of DMSO, presumably by inducing conformational changes in some macromolecules which affect their cellular function.     

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. I. Osmotically inactive volume

We have investigated the confounding effects of dynamic range limitations on measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and propose an improved approach to parameter estimation. The conventional approach for analysis of cell size distributions measured by such particle sizing instruments requires data truncation: the mean cell volume is computed after exclusion of data below a specified lower bound (typically chosen to remove artifacts due to small-volume noise) and above an upper bound (typically governed by instrument limitations). The osmotically inactive volume is then estimated from a Boyle–van’t Hoff plot of the averaged volume data obtained after exposure to various solution osmolalities. We demonstrate that systematic exclusion of data in the conventional approach introduces bias that results in erroneously high estimates of the osmotically inactive volume fraction. To minimize this source of error, we have devised a new algorithm based on fitting a bimodal distribution model to the non-truncated volume data. In experiments with mouse insulinoma (MIN6) cells, the osmotically inactive volume fraction was estimated to be 0.15 ± 0.01 using the new method, which was significantly smaller than the estimate of 0.37 ± 0.02 obtained using the conventional method (p < 0.05). In silico experiments indicated that the parameter estimate obtained by the new method was accurate within 5%, whereas the error associated with the conventional approach was approximately 150%. Parametric analysis was used to elucidate the sensitivity of errors to variations in instrument dynamic range and cell volume distribution width.     

The effect of dimethyl sulphoxide on the structure and phase behaviour of palmitoleoylphosphatidylethanolamine

The thermotropic phase behaviour and structure of a nonbilayer-forming lipid, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, dispersed in water and in aqueous solutions of up to 50 wt% dimethyl sulphoxide (DMSO) have been characterised using synchrotron X-ray diffraction methods. It was found that the presence of DMSO in the solvent induced an increase in the temperature of lamellar-gel to lamellar-liquid-crystal phase transition and a decrease in the temperature of the lamellar-liquid-crystal to inverted-hexagonal phase transition of the phospholipid. The presence of DMSO also caused a decrease in the X-ray repeat spacings of all the phases studied. Electron density profiles of the phospholipid dispersed in water and 50 wt% DMSO in the bilayer gel state were calculated. The presence of 50 wt% DMSO caused the apparent disappearance of the solvent layer separating phospholipid bilayers in the gel state. The results suggest that DMSO contributes to the bilayer electron density profile and that the amphiphilic solvent molecules partition into the interfacial region.     

The effects of acute-phase inducers and dimethyl sulphoxide on delta-aminolaevulinate synthase activity in human HepG2 hepatoma cells.

The effects of acute-phase inducers and dimethyl sulphoxide (Me2SO) on delta-aminolaevulinate (ALA) synthase in HepG2 cells were examined. Treatment of cells with Me2SO resulted in a significant increase in ALA synthase activity. Interleukin-6 increased ALA synthase activity only slightly, but it substantially potentiated the induction of ALA synthase by Me2SO. These data suggest that ALA synthase activity in liver is altered during acute-phase reactions.