PubMed Health⌕ Search

Biomedical subjects

J K Critser

Publications and source records attributed to J K Critser.

At least 55 records · Page 3Linked to original sources
PubMed1998

Water and DMSO membrane permeability characteristics of in-vivo- and in-vitro-derived and cultured murine oocytes and embryos.

Although embryo cryopreservation is routine for many mammalian species, it is important to know how the fundamental cryobiology of these cells changes with development. Progressive cleavage divisions result in a reduction in the blastomere surface area available for water and cryoprotectant mass transport. Therefore, the membrane permeability of murine oocytes, zygotes, 2-cell, 4-cell, and 8-cell embryos to water (Lp), and dimethylsulphoxide (PDMSO), and the reflection coefficient, sigma (sigma) were determined. Oocytes or zygotes were recovered, cumulus cells removed, then cultured until use. Oocytes and embryos were immobilized and perfused with treatment solutions at 24 degrees C. Osmotically induced cell volume changes over time were videotaped followed by image analysis. The Lp values in the presence of dimethylsulphoxide (DMSO) were 0.77, 0.81, 0.94, 0.86, and 1.10 microm/min/atm, and the PDMSO values were 1.85, 2.04, 2.41, 1.95, and 1.25x10(-3) cm/min for oocytes, zygotes, 2, 4, and 8-cell embryos respectively. The Lp values in the presence of DMSO were significantly (P < 0.05) higher than those in the absence of DMSO. Treating the whole embryo as a single osmotic entity leads to significantly (P < 0.05) elevated PDMSO estimates relative to those based upon measurements of individual blastomeres. These data indicate that both Lp and PDMSO estimates are lower when predicted on an individual blastomere basis. The data also show that neither Lp nor PDMSO differ among oocytes, zygotes, 2-cell and 4-cell embryos. However, the significantly higher Lp and lower PDMSO of the 8-cell stage support the hypothesis that fundamental cryobiological differences may require developmental stage-specific embryo cryopreservation protocols.

Animals↗
PubMed1998

Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation.

The osmotic tolerance limits for boar spermatozoa were determined at 22 degrees C. These cells can swell to within 1.02 times and shrink to within 0.97 times their isosmotic volume and maintain > 70% motility. In the presence of an extender, cells can swell to within 1.1 times and shrink to within 0.97 times their isosmotic volume and maintain > 70% motility. Plasma membrane permeability coefficients were determined in the presence of 1 M dimethyl sulfoxide (DMSO), 1 M glycerol, and 2 M ethylene glycol (EG) at 22 degrees C. Hydraulic conductivity (Lp) was estimated to be 0.120+/-0.016 (mean+/-SEM), 0.138+/-0.006, and 0.204 +/-0.021 microm/min/atm in the presence of DMSO, glycerol, and EG, respectively, at 22 degrees C. Solute permeability (P[CPA]) was determined to be 0.930+/-0.118, 0.481+/-0.045, and 1.98+/-0.106 x 10(-3) cm/min, for DMSO, glycerol, and EG, respectively. Subsequent experiments were performed at 8 degrees C and 0 degrees C. Activation energies were calculated for Lp in the presence of glycerol and EG to be 7.20 and 11.51 Kcal/mol, respectively. The activation energies for P(CPA) were 4.06 and 7.48 Kcal/mol for glycerol and EG permeability, respectively. These membrane characteristics were used to calculate volume flux during addition and removal of cryoprotectant agents as well as during cooling and warming. In addition, the potential for intracellular ice formation during cooling and warming was calculated.

Animals↗
PubMed1997

Novel microwave technology for cryopreservation of biomaterials by suppression of apparent ice formation.

Ice formation inside or outside cells has been proposed to be a factor causing cryoinjury to cells/tissues during cryopreservation. How to control, reduce, or eliminate the ice formation has been an important research topic in fundamental cryobiology. The objective of this study was to test a hypothesis that the coupled interaction of microwave radiation and cryoprotectant concentration could significantly influence ice formation and enhance potential vitrification in cryopreservation media at a relative slow cooling rate. Test samples consisted of a series of solutions with ethylene glycol (a cryoprotectant) concentration ranging from 3 to 5.5 M. A specific microwave resonant cavity was built and utilized to provide an intense oscillating electric field. Solutions were simultaneously exposed to this electric field and cooled to -196 degrees C by rapid immersion in liquid nitrogen. Control samples were similarly submerged in liquid nitrogen but without the microwave field. The amount of ice formation was determined by analysis of digital images of the samples. The morphology of the solidified samples was observed by cryomicroscopy. It was found that ice formation was greatly influenced by microwave irradiation. For example, ice formation could be reduced by roughly 56% in 3.5 M ethylene glycol solutions. An average reduction of 66% was observed in 4.5 M solutions. Statistical analysis indicated that the main effects of microwave and ethylene glycol concentration as well as the interaction between these two factors significantly (P < 0.01) influenced ice formation amount, confirming the hypothesis. This preliminary study suggests that a combined use of microwave irradiation and cryoprotectant might be a potential approach to control ice formation in cells/tissues during the cooling process and to enhance vitrification of these biomaterials for long-term cryopreservation.

Cryopreservation↗
PubMed1997

The determination of membrane permeability coefficients of canine pancreatic islet cells and their application to islet cryopreservation.

Sufficient numbers of pancreatic islets for successful allotransplantation can be achieved by storing and then pooling islets from several donors. Optimal MHC matching and infectious disease screening also require long-term storage of islets, and cryopreservation is currently the only practical approach. Cryopreservation protocols may be optimized by modeling the changes in cell volume and the associated damage incurred during cryoprotectant addition and dilution and during cooling and warming. The objective of the present work was to determine the following biophysical parameters of canine islet cells; the osmotically inactive cell volume (Vb), hydraulic conductivity (Lp), cryoprotectant permeability coefficient (Ps), and the reflection coefficient sigma. A determination of these parameters allows the simulation of cell responses using computer models. Islets were isolated by collagenase digestion and Euro-Ficoll purification. After 24 h culture, islets were dissociated into single cells using trypsin and 2 mM EGTA. The kinetic change in cell volume as a function of time after exposure to 2 M dimethyl sulfoxide (Me2SO) was measured using an electronic particle counter at 22, 5, and -3 degrees C. At -11 degrees C, cells were preloaded with 1 M Me2SO and exposed to 4 M Me2SO to prevent the formation of ice in the working solution. Kedem-Katchalsky theory was used to describe the cell volume change kinetics, and a three-parameter curve fitting was performed using the Marquardt-Levenberg method to determine Lp, Ps, and sigma values. The Lp was determined to be 0.19 +/- 0.05, 0.037 +/- 0.005, 0.020 +/- 0.003, and 0.013 +/- 0.005 micron.min-1.atm-1 (mean +/- SD) at 22, 5, -3, and -11 degrees C, respectively. The Ps values were 1.05 +/- 0.50, 0.15 +/- 0.04, 0.096 +/- 0.028, and 0.067 +/- 0.029 x 10(-3) cm.min-1 at 22, 5, -3, and -11 degrees C, respectively. The sigma values were 0.81 +/- 0.16, 0.91 +/- 0.09, 0.80 +/- 0.21, and 0.98 +/- 0.04 at 22, 5, -3, and -11 degrees C, respectively. The temperature dependence or activation energy of Lp and Ps was calculated, using the Arrhenius equation, to be 12.7 and 13.5 kcal.mol-1, respectively. These permeability parameters were used to calculate cell water loss and the likelihood of lethal intracellular freezing during cooling, as well as both water flux and solute concentration gradients across the cell membrane during warming.

Animals↗
PubMed1997

Osmotic characteristics of isolated human and canine pancreatic islets.

Cryopreservation protocols for pancreatic islets may be optimized by modeling the changes in volume that occur during cryoprotectant addition/dilution and cooling/warming. Modeling may be facilitated if it can be demonstrated that isolated islets behave as ideal osmometers where the equilibrium volume is a linear function of the reciprocal of the osmolality of the extracellular medium (the Boyle van't Hoff relationship). The present study was performed to test the hypotheses that: (i) human and canine islets exhibit a linear osmotic response during exposure to hypo-, iso-, and hyperosmotic solutions and to determine the human and canine islets osmotically inactive fraction (Vbp); (ii) to determine if the ionic properties of a concentrated electrolyte solution have an effect on the osmotic response; and (iii) to determine if the osmotic response of pancreatic islets varies between species (human and canine). Islets were isolated from four human and four canine pancreases using standard collagenase digestion and EuroFicoll purification. After 12 h incubation at 37 degrees C, islets (n = 4 from each isolation) were exposed to varying salt or sucrose concentrations (75-1200 mOsm/kg). Islets were held by a micropipette system attached to an inverted microscope, and solutions were exchanged while videotaping. An imaging system was used to determine the cross-sectional area of the islet which was then used to estimate the spherical volume. The results of linear regression analysis indicated that both human and canine islets respond ideally to osmotic conditions within a range of 150-1200 mOsm/kg in both the electrolyte (salt) and the nonelectrolyte (sucrose) solutions. The Vbp value (mean +/- SD) of the four donors was 0.513 +/- 0.068 for canine islets in sucrose solutions, 0.474 +/- 0.068 for canine islets in salt solutions, 0. 425 +/- 0.073 for human islets in sucrose solutions, and 0.39 +/- 0. 073 for human islets in salt solutions. There was a significant difference between human and canine islets (P < 0.05) but not between experimental solutes (NaCl or sucrose) (P < 0.05). These data may be used in conjunction with osmotic tolerance data to develop improved methods for the cryopreservation of isolated islets.

Animals↗
PubMed1997

Live births after autologous transplant of cryopreserved mouse ovaries.

The fertility of mice after autologous transplantation of ovaries, before or after cryopreservation, was investigated in this study. Female mice were randomly assigned to either sham-operated (n = 14), ovariectomized (n = 11), fresh (n = 12) or cryopreserved (n = 11) ovarian transplant groups. Ovaries were cryopreserved in 1.4 M dimethyl sulphoxide (DMSO) by cooling to -55 degrees C at 0.5 degree C/min (ice nucleation at -7 degrees C), plunged in liquid nitrogen and then thawed at room temperature. Oestrous cyclicity was observed 7 days after sham operation or 15 days after fresh or cryopreserved ovarian transplant. Ovariectomized animals did not demonstrate oestrous cyclicity but were mated, and no pregnancies resulted. Live births were recorded from all sham-operated, all fresh transplant, and 8/11 (73%) cryopreserved transplant animals. Overall mean +/- SEM litter sizes from fresh (4.32 +/- 0.44) and cryopreserved (4.71 +/- 0.57) transplant groups were smaller (P < 0.05) than those of sham-operated animals (12.54 +/- 0.44), although the sizes were not significantly different (P > 0.05) from each other. Animals were mated at least four times, with four litters of live pups from 4/4 sham-operated, 1/10 fresh and 1/9 cryopreserved ovarian transplant animals. Litter sizes from pups of sham-operated and transplant animals were not significantly different from each other. Following autologous transplantation of mouse ovaries, before or after cryopreservation, offspring appeared normal, with high rates of fertility.

Animals↗
PubMed1997

Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa.

The objective was to test the hypothesis that the optimal cryoprotective agent for cryopreservation of human spermatozoa would be a solute for which cells have the highest plasma membrane permeability, resulting in the least amount of volume excursion during its addition and removal. To test this hypothesis, theoretical simulations were performed using membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were performed using data from four different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulations were applied to determine approaches for the addition and removal of 1 M and 2 M final concentrations of cryoprotectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol was predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested using glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a higher (P < 0.05) recovery of motile spermatozoa after cryopreservation when using 1 M ethylene glycol than with 1 M glycerol, supporting the hypothesis that use of the cryoprotectant for which the cell has the highest permeability will result in higher cell survival.

Cryopreservation↗
PubMed1997

Allogeneic and xenogeneic transplantation of cryopreserved ovarian tissue to athymic mice.

Cryopreserved ovarian tissue has major applications for female oncology patients and for the development of genome resource banks. The objective of the present study was to develop a bioassay of cryopreserved ovarian tissue function after allogeneic and xenogeneic transplantation to ovariectomized athymic nude (nu/nu) Balb/C mice. Transplant function was assessed by examination of vaginal smears, number of live births, and posttransplant histology. Animals were sham operated (group I; n = 4) or ovariectomized (group II; n = 5) or were given transplants of either fresh (group III; n = 3) or cryopreserved (group IV; n = 4) Institute of Cancer Research-strain mouse ovarian tissue or cryopreserved sheep ovarian tissue (group V; n = 7). Vaginal smears were examined 5-7 times per week; the number of days between visualizations of epithelial cells in smears was 4.3 +/- 0.6 for group I, 8.6 +/- 3.8 for group II, 3.4 +/- 0.4 for group III, 3.3 +/- 0.5 for group IV, and 4.6 +/- 0.6 for group V. Epithelial cells were seen for 1.2-1.7 consecutive days; this value was significantly different between groups III and V. Live births were recorded from 3 of 4 animals from group I, 0 of 5 animals from group II, 2 of 3 animals from group III, and 1 of 4 animals from group IV. In vivo function and long-term survival of cryopreserved ovarian tissue after allogeneic or xenogeneic transplant were confirmed by the examination of vaginal cytology, and offspring were derived from allografts.

Animals↗
PubMed1996

Development of a novel microperfusion chamber for determination of cell membrane transport properties.

A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 micron. min-1. atm-1 in the absence of DMSO and 0.65 microns. min-1. atm-1 in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 micron/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 microns. min-1. atm-1, which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.

Animals↗
PubMed1996

Sperm capacitation and the acrosome reaction are compromised in teratospermic domestic cats.

The efficiency of sperm capacitation and of the acrosome reaction was studied in the teratospermic domestic cat to evaluate further the etiology of compromised zona pellucida penetration and oocyte fertilization. Specific objectives were to compare normospermic and teratospermic cat ejaculates for 1) the kinetics and timing of sperm capacitation in vitro as determined by an ionophore-induced acrosome reaction; 2) the incidence of spontaneous acrosomal loss; 3) the ability of capacitated, swim-up processed sperm to acrosome-react in response to chemical (calcium ionophore) or physiological (solubilized zonae pellucidae) inducers; and 4) differences in acrosomal ultrastructure by use of transmission electron microscopy (TEM). Acrosomal status was determined with the fluorescent probe Arachis hypogaea (peanut) agglutinin. The timing of in vitro capacitation differed (p < 0.05) between cat populations. Normospermic samples were capacitated at 2.0 h postcentrifugation, whereas teratospermic samples required 2.5 h to become capacitated. At 2.5 h, sperm from teratospermic males were less capable (p < 0.05) of completing the acrosome reaction after ionophore exposure (49.3 +/- 8.0%) than sperm from normospermic males (73.3 +/- 3.8%). Levels of spontaneous acrosomal loss/reaction over time were similar (p > 0.05) between cat groups (range, 7.6-17.8%). In swim-up separated sperm from normospermic cats, ionophore A23187 was a more potent inducer (p < 0.05) of the acrosome reaction (70.1 +/- 6.5%) than solubilized zonae pellucidae (31.1 +/- 1.2%). Swim-up separated sperm from teratospermic cats, however, were compromised in the ability to acrosome react, regardless of inducer (ionophore, 23.9 +/- 3.3%; solubilized zonae pellucidae, 23.9 +/- 4.7%; p > 0.05). Sperm motility patterns over time indicated that differences in acrosomal status were not influenced by cell death. The frequency of abnormal acrosomes detected by TEM was higher (p < 0.05) in teratospermic (30.0 +/- 3.9%) than in normospermic (3.1 +/- 1.3%) samples. Swim-up separation failed to reduce (p > 0.05) the proportion of sperm cells with malformed acrosomes (swim-up, 33.5 +/- 3.5%; washed, 26.6 +/- 4.6%). These results indicate that sperm from teratospermic cats exhibit a high incidence of malformed acrosomes detectable only at the ultrastructural level. Nevertheless, acrosomal dysfunction is not related exclusively to structural defects because > 40.0% of swim-up separated sperm with structurally normal acrosomes still are incapable of completing the acrosome reaction. This suggests that compromised capacitation and acrosomal dysfunction may be responsible for low fertilization success in the teratospermic domestic cat.

Acrosome↗
PubMed1996

Osmotic tolerance limits and properties of murine spermatozoa.

Osmotic tolerance of spermatozoa is a critical determinant of functional survival after cryopreservation. This study first tested the hypothesis that mouse spermatozoa behave as linear osmometers, using an electronic particle counter to measure the change in sperm volume in response to anisosmotic solutions. The resulting Boyle-van't Hoff plot was linear (r2 = 0.99) from 75 to 1200 mOsmolal and indicates that 60.7% of the total cell volume is osmotically inactive. Next, mouse sperm tolerance to osmotic stress was determined by assessment of plasma membrane integrity, mitochondrial viability, and motility. Each functional endpoint was measured after exposure to anisosmotic solutions and again after return to isosmolality. The dual fluorescent stains-carboxyfluorescein diacetate with propidium iodide and Rhodamine 123 with propidium iodide-were used to determine membrane integrity and functional mitochondria, respectively. Motility was measured by video microscopy in the range of 1-2400 mOsmolal and was further analyzed from 140 to 600 mOsmolal using computer-assisted semen analysis. The data indicate that motility is substantially more sensitive to osmotic stress than either mitochondrial viability or membrane integrity and that mouse spermatozoa should be maintained within 76-124% of their isosmotic volume during cryopreservation in order to maintain > 80% of pretreatment motility.

Animals↗
PubMed1996

Nonluteal estrous cycles of 3-week duration are initiated by anovulatory luteinizing hormone peaks in African elephants.

Previous attempts to characterize the estrous cycle of elephants have yielded conflicting estimates of cycle length and LH profiles. In order to establish artificial breeding programs in this species, resolution of these issues is needed. Therefore, four female African elephants housed at the Indianapolis Zoo were studied for approximately 6 mo beginning in December 1994. Blood was collected weekly, and the serum was immediately analyzed for progesterone (P4). Whenever the weekly concentration of P4 was found to be low, blood was collected one or four times per day. All serum samples were assayed for LH, and the daily samples were assayed for P4 and estradiol. Transient increases of serum LH (designated as peaks) were observed four times in each of the four females. Of these 16 LH peaks, 8 were classified as ovulatory LH (ovLH) peaks and 8 were classified as anovulatory LH (anLH) peaks. Peaks designated ovLH averaged 3.60 +/- 0.67 ng/ml (mean +/- SEM); serum P4 measured during these peaks began to increase 2-3 days before each ovLH peak and continued to increase for several weeks thereafter, reaching a peak of 675 +/- 35 pg/ml. The eight other LH peaks, designated anLH peaks, were of similar (p > 0.05) magnitude averaging 3.07 +/- 0.72 ng/ml, but the serum concentration of P4 remained very low (< 80 pg/ml) during and for several weeks after these peaks. Six peaks designated anLH occurred an average of 12.2 +/- 1.4 days after serum P4 had declined below 80 pg/ml. In each elephant, there was a regular sequence in which each ovLH peak was followed by a luteal-active period lasting about 60 days and then about 12 days later by one anLH peak. Each anLH peak was followed 19-22 days later by one ovLH peak, but serum P4 remained at nonluteal levels throughout this interval between peaks. The authors propose to designate this interval after the anLH peak and before the next ovLH peak as a nonluteal (i.e., low P4) estrous cycle of only 3-wk duration. Following each short nonluteal estrous cycle, there was a single ovLH peak that initiated one luteal-active estrous cycle lasting 10-11 wk until terminated by the next anLH peak. The present results demonstrate that nonpregnant African elephants, housed in the absence of males, alternate between short nonluteal estrous cycles and long luteal-active estrous cycles. Daily measurements of serum P4 can be used to distinguish between the two types of estrous cycles and thereby provide a clinical prediction about the optimum time for artificial insemination.

Animals↗
PubMed1996

Osmotic properties of boar spermatozoa and their relevance to cryopreservation.

A series of six experiments was conducted to determine the fundamental cryobiological properties of boar spermatozoa to develop optimal approaches for cryopreserving this important cell type. In the first experiments, boar spermatozoa samples were diluted in various osmolalities of experimental solutions (185-900 mOsmol kg-1) to provide hypo-, iso-, and hyperosmotic conditions. Equilibrium cell volumes (Expts 1 and 2) were measured after exposure for 3 min and the change in cell volume was measured over time using an electronic particle counter (Expt 3). The isosmotic cell volume was found to be 26.3 +/- 0.39 microns 3 (mean +/- SEM; n = 5). Over this range of osmolalities, boar spermatozoa behaved as linear osmometers (a linear volume versus 1/osm plot, r2 = 0.99) with an osmotically inactive cell fraction of 67.4 +/- 4.5%. The rate of water permeability (Lp) was determined to be 1.03 +/- 0.05 microns min-1 atm-1, which was consistent within and among donors (P > 0.130). A second series of experiments was performed to determine the effect of temperature and osmolality on boar sperm motility (Expt 4), and the effect of osmolality on the integrity of the sperm plasma membrane and its temperature dependence. Plasma membrane integrity was measured before and after boar spermatozoa were returned to an isosmolality (Expt 6). Motility was not affected at 30 degrees C, relative to that at room temperature, but was significantly decreased (P < 0.05) at 8 degrees C and 0 degree C (yielding a relative reduction to 85% and 35% of original motility, respectively; n = 6). Sperm motility was not significantly decreased (P > 0.05) until the osmolality reached 210 mOsmol kg-1, at which time motility began to decrease from 95% to 10% of the original value at 90 mOsmol kg-1. The integrity of the plasma membrane of boar spermatozoa was found to be dependent on temperature, donor and osmolality, decreasing significantly (P < 0.05) below room temperature, and below 185 mOsmol kg-1 (P < 0.05). There was no significant difference (P > 0.10) in the integrity of the plasma membrane of the samples before and after returning to 290 mOsmol kg-1, indicating that osmotic damage occurs during the initial change from isosmotic to hyposmotic media. These osmotic characteristics could be used to determine optimal conditions for cryopreservation of boar spermatozoa.

Animals↗
PubMed1995

Fracture phenomena in an isotonic salt solution during freezing and their elimination using glycerol.

Thermal stress and consequent fracture in frozen organs or cell suspensions have been proposed to be two causes of cell cryoinjury. A specific device was developed to study the thermal stress and the fracture phenomena during a slow cooling process of isotonic NaCl solutions with different concentrations of glycerol (cryoprotectant) in a cylindrical tube. It was shown from the experimental results that glycerol significantly influenced the solidification process of the ternary solutions and reduced the thermal stress. The higher the initial glycerol concentration, the lower the thermal stress in the frozen solutions. Glycerol concentrations over 0.3 M were sufficient to eliminate the fracture of the frozen solutions under the present experimental conditions. To explain the action of glycerol in reducing the thermal stress and preventing the ice fracture, a further study on ice crystal formation and growth of ice in these solutions was undertaken using cryomicroscopy. It is known from previous studies that an increase of initial glycerol concentration reduced frozen fraction of water in the solution at any given low temperature due to colligative properties of solution, which reduced the total ice volume expansion during water solidification. The present cryomicroscopic investigation showed that under a fixed cooling condition the different initial glycerol concentrations induced the different microstructures of the frozen solutions at not only a given low temperature but also a given frozen fraction of water. It has been known that ice volume expansion during solidification is a major factor causing the thermal stress and the interior microstructure is critical for the mechanical strength of a solid. Therefore, functions of glycerol in reducing the total ice volume expansion during water solidification and in influencing interior microstructure of the ice may contribute to reduce the thermal stress and prevent the fracture in the frozen solutions.

Animals↗
PubMed1995

Water permeability and its activation energy for individual hamster pancreatic islet cells.

Coupled with the rapid development of clinical pancreatic islet transplantation, there is an increasing requirement for cryopreservation of viable islets. Fundamental cryobiology requires determination of several cryobiophysical parameters to predict optimal cryopreservation procedures. These include water permeability or hydraulic conductivity (Lp) and its activation energy (Ea), the permeability of the cell plasma membrane to a cryoprotectant(s) (Ps) and its Ea, the osmotically inactive fraction of cell volume (Vb), and the intracellular ice formation temperature. For islet cells, these parameters have not previously been reported. In the present studies, the Lp, its Ea, and Vb were determined for isolated individual golden hamster pancreatic islet cells. The Lp and Vb parameters were also measured for corresponding exocrine cells. Both islet and the exocrine cells appeared to be ideal osmometers over the experimental range when examined by the Boyle Van't-Hoff relationship (linear regression, r = 0.99 for both types of cells). Extrapolation of these plots generated Vb values of 0.40 for the islet cells and 0.45 for the pancreatic exocrine cells. To determine the Lp, kinetic changes of cell volume over time (dv/dt) in response to anisoosmotic conditions (ranging from 145 mOsm/kg to 1.35 Osm/kg) were measured using an electronic particle counter. The experimental data were fitted to generate the Lp values by least-squares curve fitting to a differential equation describing osmotic water movement across the plasma membrane. For pancreatic islet cells, the Lp was determined to be 0.25 +/- 0.03 microns/min/atm (mean +/- SD, n = 14) at 22 degrees C, 0.54 +/- 0.07 (n = 10), 0.06 +/- 0.008 (n = 9), and 0.01 +/- 0.001 (n = 9) at 37, 8 and 0 degrees C, respectively. The Ea for Lp was calculated from the slope of the Arrhenius plot based upon the mean Lp values at the four different temperatures. The Ea was 16.21 Kcal/mol between 0 and 37 degrees C. Based upon these values, an optimal cooling rate for cryopreserving pancreatic islet cells is predicted to be approximately 0.5 degrees C min. The Lp for the individual exocrine cells was determined to be 3.73 +/- 1.75 microns/min/atm (n = 13) at 22 degrees C, which was approximately 10 times the Lp value of the corresponding islet cells.

Animals↗
PubMed1995

Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol.

Use of a cryoprotective agent is indispensable to prevent injury to human spermatozoa during the cryopreservation process. However, addition of cryoprotective agents to spermatozoa before cooling and their removal after warming may create severe osmotic stress for the cells, resulting in injury. The objective of this study was to test the hypothesis that the degree (or magnitude) of human sperm volume excursion can be used as an independent indicator to evaluate and predict possible osmotic injury to spermatozoa during the addition and removal of cryoprotective agents. Glycerol was used as a model cryoprotective agent in the present study. To test this hypothesis, first the tolerance limits of spermatozoa to swelling in hypo-osmotic solutions (iso-osmotic medium diluted with water) and to shrinkage in hyperosmotic solutions (iso-osmotic medium with sucrose) were determined. Sperm plasma membrane integrity was measured by fluorescent staining, and sperm motility was assessed by computer-assisted semen analysis before, during and after the anisosomotic exposure. The result indicate firstly that motility was much more sensitive to anisosmotic conditions than membrane integrity, and secondly that motility was substantially more sensitive to hypotonic than to hypertonic conditions. Based on the experimental data, osmotic injury as a function of sperm volume excursion (swelling or shrinking) was determined. The second step, using these sperm volume excursion limits and previously measured glycerol and water permeability coefficients of human spermatozoa, was to predict, by computer simulation, the cell osmotic injury caused by different procedures for the addition and removal of glycerol. The predicted sperm injury was confirmed by experiment. Based on this study, an analytical methodology has been developed for predicting optimal protocols to reduce osmotic injury associated with the addition and removal of hypertonic concentrations of glycerol in human spermatozoa.

Buffers↗