Ultrarapidly Frozen Research Articles

Inhibition of extracellular ice crystals growth for testing the cryodamaging effect of intracellular ice in a model of ram sperm ultra-rapid freezing

ABSTRACT We aimed to test the common belief, that as the sperm cell is the smallest cell in an organism and, therefore, it contains a small amount of intracellular water, the intracellular ice should not strongly affect the sperm cell during the cryopreservation. To investigate the effect of intracellular ice, we developed a novel approach of ram spermatozoa ultra-rapid freezing with the inhibition of the growth of extracellular ice crystals during freezing. In our approach, ram sperm was supplemented with cell-impermeable synthetic ice blocker 1,4-Cyclohexanediol, leaving a sample unsupplemented as a control. Sperm in cryostraws were ultra-rapidly frozen, and then thawed. We hypothesized, that only in the case when the intracellular ice plays the role in the cryodamage, the post-freezing intactness of spermatozoa frozen in the presence of extracellular ice inhibitors should be equal (or even less) to the intactness of spermatozoa frozen without extracellular inhibitors. No statistically significant difference (p = 0.98) was observed between the post-freezing intactness of sperm ultra-rapidly frozen in the presence of 1,4-Cyclohexanediol and the intactness of sperm frozen without 1,4-Cyclohexanediol. We concluded that the intracellular ice plays the role in sperm cryodamage, at least in the model of ram sperm ultra-rapid freezing. Highlights Here, we investigated the cryodamaging factors during ram sperm ultra-rapid freezing; Optimized ultra-rapid freezing will provide positive economic implications for the sheep industry worldwide.

Endocrine function and lymphocyte infiltration of newborn rat ovaries after ultrarapid freezing and allotransplantation

To evaluate the endocrine function and lymphocyte infiltration of ultrarapidly frozen newborn rat ovaries in adult recipients. Animal study. Reproductive Medicine Laboratory of Ningxia Medical College. Newborn rats within 24 hours after birth and Sprague-Dawley female adult rats. Newborn or adult rat ovary tissues were cryopreserved with ultrarapid freezing method, using 1.5 M propylene glycol and 0.1 M sucrose as a cryoprotectant agent. After thawing, they were allotransplanted under the kidney capsule of ovariectomized adult female rats to assess the function and the microstructure. Vaginal smear, serum E2 level in recipients, grafts histologic observation, 3beta-hydroxy steroid dehydrogenase histochemistry staining, and CD4(+)/CD8(+) T cell immunohistochemistry staining. Frozen-thawed newborn rat ovaries survived and established a vascular network with the kidney of recipients. More growing follicles were found in these survival grafts. No significant differences were found in both resumption rates, the day of initiating estrous cycles, and E2 level between the frozen adult and frozen newborn rat ovaries transplant group. Among all the groups, the number of lymphocyte in the frozen newborn rat ovary transplant group is the lowest. This is the first report for ultrarapid cryopreservation that can preserve the development potential of immature ovaries in ovariectomized adult recipients and further reduce their immunogenicity successfully.

Computational Fluid Dynamics Analysis of Cell Cooling Process

Vitrification is the solidification of a solution without crystallization, caused by an increase of viscosity. It results from a balance between the highest realizable cooling rates and the highest concentrations of cryoprotectants (CPA) not toxic for the cells for the period of exposure. To vitrify with reduced CPA concentrations the cooling rates have to be increased. The objective of our study was to verify the thermal feasibility of a vitrification process using a computational fluid dynamics (CFD) model. The first step in our stepwise approach to our project is focused on an ultrarapid freezing process in absence of cryoprotectants. The main parameters which influence the cooldown and increased cooling rate inside the biologic material (e.g., the thermophysical parameters, the size and the shape of the specimen) have been investigated. In our test cases the living material is considered frozen by direct contact with a surface at cryogenic temperature. Both numerical values and false color images of the thermal fields are available. It is confirmed that higher cooling rates can be obtained by decreasing the thickness of the material placed on the cold surface or changing the cryogen. Variations of the other thermophysical parameters had little influence on the freezing time. From the results it appears that it is impossible to define only one cooling rate for specimen vitrification and that large quantities of biological material could be ultrarapidly frozen on wide cold surfaces with low material thickness.

In vitro maturation, fertilization and embryo development after ultrarapid freezing of immature human oocytes

The purpose of this study was to determine the rates of maturation, fertilization and embryo development of ultrarapidly frozen immature oocytes (immature cumulus-oocyte complexes; COCs) obtained from antral follicles in ovaries of patients with chocolate ovarian cysts. The COCs were cryopreserved by a vitrification method using 5.5 mol ethylene glycol l (-1) plus 1.0 mol sucrose l (-1) in Dulbecco's PBS (DPBS). The survival, maturation and fertilization rates, and the percentage of embryos developing to the two-cell stage were 59, 64, 70 and 71%, respectively. No significant differences were noted in the rates of maturation, fertilization and embryo development between control and cryopreserved oocytes. Two embryos that developed from cryopreserved oocytes of the oocyte donor programme were selected for transfer into the uterus of a recipient with premature ovarian failure, after the recipient had received steroid replacement. A biochemical pregnancy occurred in the recipient after embryo transfer. These results indicate that immature oocytes can survive after cryopreservation and subsequently can be cultured to mature oocytes that are capable of undergoing fertilization in vitro and developing into embryos.

In vitro maturation, fertilization and embryo development after ultrarapid freezing of immature human oocytes.

The purpose of this study was to determine the rates of maturation, fertilization and embryo development of ultrarapidly frozen immature oocytes (immature cumulus-oocyte complexes; COCs) obtained from antral follicles in ovaries of patients with chocolate ovarian cysts. The COCs were cryopreserved by a vitrification method using 5.5 mol ethylene glycol l (-1) plus 1.0 mol sucrose l (-1) in Dulbecco's PBS (DPBS). The survival, maturation and fertilization rates, and the percentage of embryos developing to the two-cell stage were 59, 64, 70 and 71%, respectively. No significant differences were noted in the rates of maturation, fertilization and embryo development between control and cryopreserved oocytes. Two embryos that developed from cryopreserved oocytes of the oocyte donor programme were selected for transfer into the uterus of a recipient with premature ovarian failure, after the recipient had received steroid replacement. A biochemical pregnancy occurred in the recipient after embryo transfer. These results indicate that immature oocytes can survive after cryopreservation and subsequently can be cultured to mature oocytes that are capable of undergoing fertilization in vitro and developing into embryos.

Ultra-rapid freezing of mouse oocytes lowers the cell number in the inner cell mass of 5 day old in-vitro cultured blastocysts.

We demonstrated previously that ultra-rapid freezing of mouse oocytes with 3.5 M dimethylsulphoxide (DMSO) decreased cell numbers in day 5 in-vitro cultured blastocysts. In the present study we counted cell numbers of trophectoderm (TE) and inner cell mass (ICM) separately following differential labelling of TE with propidium iodide (red) and ICM with bisbenzimide (blue). Blastocysts were from four groups of oocytes: (i) cumulus-enclosed; (ii) hyaluronidase-treated cumulus-free; (iii) cumulus-free and exposed to 3.5 M DMSO; and (iv) cumulus-free and ultrarapidly frozen with 3.5 M DMSO. Mean (+/-SD) blastocyst cell numbers were 54.7 +/- 22.0, 51.1 +/- 17.3, 52.3 +/- 13.1 and 40.4 +/- 18.4, respectively. Mean TE cell numbers were 31.7 +/- 18.2, 28.9 +/- 13.3, 31.2 +/- 13.3 and 26.2 +/- 16.5 while mean ICM cell numbers were 23.0 +/- 9.4, 22.2 +/- 9.4, 21.1 +/- 7.3 and 14.2 +/- 7.3, respectively. Blastocyst and ICM cell numbers were significantly lower in the group derived from ultra-rapidly frozen oocytes compared with all other groups. Significantly more blastocysts had < or = 32 cells and in blastocysts with > 64 cells a lower mean percentage of ICM was found. Ultra-rapid freezing of mouse oocytes with 3.5 M DMSO can thus lead to day 5 in-vitro cultured blastocysts with significantly decreased ICM cell numbers. The residual ICM cell number in affected blastocysts may not reach a critical mass sufficient for successful postimplantation development.

Pregnancies after transfer of ultrarapidly frozen human embryos.

We report our experience of freezing human embryos using an ultrarapid freezing method. The patients were superovulated. Oocytes were inseminated and cultured in HTF + 10% serum. A maximum of three embryos was transferred and the rest of the embryos were frozen ultrarapidly after a 3-min equilibration period in PB1 + 3.5 M DMSO + 0.25 M sucrose. Embryos were thawed in a 37 degrees C water bath for 6 sec, then cultured in PB1 + 20% serum for 10 min. The surviving embryos were transferred into patients on the same day of thawing. Sixty-three embryos were thawed, of which 52 embryos (83%) survived with at least one intact blastomere. Nineteen frozen-thawed embryo transfers were made. The mean embryos per transfer was 2.7. Three pregnancies (16%/transfer) were established. One miscarriage occurred in the eighth week of pregnancy. Two pregnancies went to term and three healthy infants were born. The present data demonstrate that ultrarapid freezing is a method worth consideration in the area of human embryo freezing.

The Structure of the Contractile Apparatus in Ultrarapidly Frozen Smooth Muscle: Freeze-Fracture, Deep-Etch, and Freeze-Substitution Studies

The structure of the smooth muscle contractile apparatus was studied using ultrarapid freezing followed by freeze substitution or by longitudinal freeze-fracture, deep-etch, and platinum-carbon replication. Freeze substitution minimises the detrimental effects of chemical fixation and freeze fracture eliminates them entirely whilst revealing the ultrastructure in three dimensions. Unidirectionally shadowed freeze-fracture replicas of ultra-rapidly frozen, relaxed, intact smooth muscle showed a well-preserved actin filament structure; the 5.5-nm repeat of the actin subunits was clearly observed. In transversely fractured tissue the thick filaments were revealed, with a distribution comparable to that seen in transverse sections of freeze-substituted muscle. Relaxed muscle permeabilised using Triton X-100 showed a similar structure to that of intact tissue after ultrarapid freezing and examination both by freeze fracture and by freeze substitution; the ratios of actin to myosin were also comparable. In permeabilised, rigorised tissue the structure of the actomyosin complex was revealed in detail; this was especially clear in freeze-substituted muscle. A cross-bridge spacing of 38 nm was measured in freeze-fractured, deep-etched tissue. The structural detail revealed is compatible with a side polar model of the actomyosin interaction and with the sliding filament mechanism of muscle contraction.

Influence of the developmental stage and the equilibration time on the outcome of ultrarapid cryopreservation of mouse embryos.

Mouse embryos of different developmental stages from 1- to 8-cell inclusive were ultrarapidly frozen in 0.25 ml French straws after various periods of equilibration (1, 3, 5 and 9 min) in freezing-buffer containing 3.5 M dimethylsulphoxide (DMSO), 0.25 M sucrose, and 20% fetal calf serum (FCS) in phosphate buffered saline (PBS). After thawing in a 37 degrees C waterbath and dilution for 5 min in 0.25 M sucrose in PBS/FCS the embryos were cultured in Ham's F10 medium with 10% FCS (37 degrees C, 5% CO2, 95% humidity) for 4-6 days. The rates of expanded and hatching blastocysts were then evaluated and compared to the corresponding rates of non-frozen controls. Thus, for each cleavage stage, the optimal equilibration time was evaluated. It was 1 min for the 1-cell and 8-cell stages (32 and 81% blastocysts, respectively), and 3-5 min for the 2-cell (76 and 73%, respectively) and the 4-cell stage (88 and 87%, respectively). It is concluded that the ultrarapid freezing method described provides satisfactory results for all tested cleavage stages, but not for the 1-cell stage.

Association of ultrarapid freezing of mouse oocytes with increased polyploidy at the pronucleate stage, reduced cell numbers in blastocysts and impaired fetal development

Mature mouse oocytes were frozen ultrarapidly with a cryoprotectant solution consisting of 3.5 mol dimethylsulfoxide l-1 and 0.5 mol sucrose l-1 or exposed to the freezing solution and recovered. Freshly collected oocytes were used as controls. After ultrarapid freezing and thawing, a high mean percentage of oocytes (78%) survived. The incidence of parthenogenetic activation in frozen-thawed oocytes was not increased. After insemination in vitro, the rate of two-cell formation was decreased (59% versus 69%). Examination of the chromosome complement of first-cleavage embryos revealed that the incidence of polyploid embryos was four times that of the control group (23% versus 6%). Fewer fertilized eggs progressed to the blastocyst stage (49% versus 81%) and the mean number of cells per blastocyst was decreased. Furthermore, the capacity of transferred blastocysts to develop in vivo was reduced. Autopsy at day 17 of gestation revealed that the proportion of embryos implanting was lower in the embryos derived from ultrarapidly frozen-thawed oocytes. Furthermore, some of the living fetuses were abnormal, but our sample size is too small for the effect to be significant. In the solution control group, no differences were found compared with the control group. Although the study needs further results to draw definite conclusions, our findings question whether the applied protocol is suitable for mouse oocytes.

Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein. A deep-etch and immunolocalization study of ultrarapidly frozen tissue.

The intima from aortas of normal New Zealand White rabbits was studied 2 hours after infusion of 320 mg human low density lipoprotein (LDL), resulting in a plasma concentration of at least five times and maximally 20 times the values found in normal rabbit serum. The following techniques were used: 1) ultrarapid freezing without chemical fixation, followed by freeze-etching; 2) immunofluorescence microscopy; and 3) postembedding immunogold-labeling electron microscopy. In the latter two methods MB47, a murine monoclonal antibody against human apolipoprotein B, was used as the primary antibody. Control rabbits were infused with the same volume of buffer only. Rotary-shadowed replicas of samples from the LDL-injected rabbits showed the deposition of lipidlike particles in the subendothelial-intimal space that were the size of the injected LDL (23 nm). In focal areas of the intima, groups of 23-nm-sized lipidlike particles and larger lipidlike structures were found enmeshed in the extracellular matrix. Control replicas were essentially free of lipid deposition. Immunofluorescence microscopy of frozen aortic cross sections showed an overall increase in apolipoprotein B in the intima of the LDL-injected rabbits. The presence of apolipoprotein B in the intima was also confirmed by immunoelectron microscopy. These in vivo results show that clustering of LDL-sized particles occurs in the intima within 2 hours of excessive LDL uptake. It also demonstrates the interaction of these LDL-sized particles with the filaments of the extracellular matrix. The clustering of the LDL-sized particles supports the possibility that LDL self-aggregation may occur in vivo and that components of the extracellular matrix are involved in this process.

Fluorescein diacetate assessment of embryo viability after ultrarapid freezing of human multipronucleate embryos

To assess embryo viability after ultrarapid freezing-thawing. We studied the fluorescence pattern of 35 ultrarapidly frozen-thawed multipronucleate human embryos exposed to fluorescein diacetate. All the embryos were obtained from the Medical Center for Fertility Diagnostics and In Vitro Fertilization and Embryo Transfer at Leuven (Belgium), a private care center. None. None. The fluorescence pattern was evaluated at room temperature after a 1-minute incubation in fluorescein diacetate solution, 4 hours and 24 hours after thawing. Healthy human multipronucleate embryos, when exposed to fluorescein diacetate, accumulated intracellular fluorescein and fluoresced brightly under ultraviolet light. On the other hand, embryos presenting submicroscopic cell membranes damage caused by different processes (e.g., high or low temperatures) lost the ability to accumulate intracellular fluorescein. All the ultrarapidly frozen embryos with normal looking blastomeres fluoresced brightly after a short exposure to fluorescein diacetate. Our experiments indicate an intact cell membrane permeability and an integrity of the intracytoplasmatic esterase enzyme activity of human embryos ultrarapidly frozen.

Ultrarapid embryo freezing: effect of dissolved gas and pH of the freezing solutions and straw irradiation.

While optimizing the ultrarapid embryo freezing procedure we noted that embryo survival was lowest when gas bubbles formed in the straws. Here we report the influences of gassing the freezing solutions with 5% CO2 in air, degassing the solution, the pH of the medium and straw irradiation on the survival and development in vitro of 2-cell mouse embryos. Embryos were ultrarapidly frozen in medium M2 containing 3 M dimethyl sulphoxide and 0.25 M sucrose. Significantly fewer embryos survived freezing and thawing in gassed solutions. The subsequent development of intact embryos to blastocysts was similar in gassed and non-gassed solutions. Survival and development of embryos frozen in solutions of pH 7.0, 7.6 or 8.0 was similar, but fewer embryos developed to blastocysts after freezing in media at pH 6.0. In straws sterilized by gamma irradiation before use, embryo survival after freezing and thawing was dramatically reduced compared with that seen when non-irradiated straws were used. With non-gassed solutions significantly more gas bubbles formed in irradiated straws than in non-irradiated straws. Although bubble formation was significantly reduced in all straws by using degassed freezing solutions, this did not improve embryo survival. We conclude that under conditions normally used for ultrarapid freezing, dissolved gas and pH have less effect on embryo viability than the use of irradiated straws.

Ultrarapid freezing: a new low-cost and effective method of embryo cryopreservation

The authors describe a rapid freezing method (ultrarapid freezing) that has been developed for cryopreservation of early cleavage stage embryos. In the present experiments, 2-cell mouse embryos were frozen under a wide range of conditions in an attempt to optimize their survival and viability in vitro and in vivo. The experiments show that embryos exposed briefly (2 to 2.5 minutes) to relatively high concentrations of dimethyl sulfoxide (3 to 4 M) and 0.25 M sucrose survive and develop when plunged directly into liquid nitrogen and thawed in a 37 degrees C waterbath when sealed in 0.25-ml plastic pailettes. Survival and viability rates of ultrarapidly frozen embryos after thawing were comparable to those obtained with conventional slow-freezing techniques. The authors believe that this freezing technique can be further improved and that the speed, ease, and low cost of the method make it a very attractive alternative to more conventional methods for freezing early cleavage stage embryos.

Membrane structure in ultrarapidly frozen, unpretreated, freeze-fractured myocardium.

Ultrarapid freezing has been applied to monitor the structure of the freeze-fractured myocardial sarcolemma. Our two goals were to demonstrate that large areas of membrane can be preserved free of visible ice crystal damage and, thus, be amenable to quantitative analysis and to compare the structure of directly frozen myocardial membranes with conventionally prepared tissue. The E face was most affected by lack of chemical pretreatment. First, our laboratory reported an increase in E face particle density from 379 +/- 30/micron 2 in conventional fixed tissue to 489 +/- 18/micron 2 in unpretreated tissue. Discrete arrays of 12-15 nm particles on the E face were a striking feature of the unfixed sarcolemma. However, P face intramembrane particle (IMP) density remained unchanged from previous estimates in fixed tissue. Specialized regions of the sarcolemma were enhanced in ultrarapidly frozen tissue. Particle domains of the adherens junctions were very prominent in forming a cap alongside the gap junctions. Both the P and E faces of the gap junctions were highly ordered into hexagonal arrays. Caveolae in the membrane were infrequent in both P and E faces.

Secretion and membrane recycling in plant cells: novel intermediary structures visualized in ultrarapidly frozen sycamore and carrot suspension-culture cells

Freeze-fracture electron microscopy of propane-jet-frozen samples has been employed to investigate vesicle-mediated secretion and membrane recycling events in carrot (Daucus carota L.) and sycamore maple (Acer pseudoplatanus L.) suspension-culture cells. Stabilization of the cells by means of ultrarapid freezing has enabled us to preserve the cells in a turgid state and to visualize new intermediate membrane configurations related to these events. Indeed, many of the observed membrane configurations, such as flattened membrane vesicles with slit-shaped membrane fusion sites and horseshoe-shaped membrane infoldings, appear to result from the action of turgor forces on the plasma membrane. Individual cells exhibited great variations in numbers and types of membrane configurations postulated to be related to secretion and membrane-recycling events. In the majority of cells, the different membrane profiles displayed a patchy distribution, and within each patch the membrane configurations tended to be of the same stage. This result indicates that secretory events are triggered in domains measuring from 0.1 to about 10 μm in diameter. Based on an extensive analysis of the different membrane configurations seen in our samples, we have formulated the following model of vesicle-mediated secretion in plant cells: Fusion of a secretory vesicle with the plasma membrane leads to the formation of a single, narrow-necked pore that increases in diameter up to about 60 nm. During discharge, the vesicle is flattened, forming a disc-shaped structure perpendicular to the plane of the plasma membrane. As the vesicle is flattened, the pore is converted to a slit, the maximum length of which coincides with the diameter of the flattened vesicle. The flattened vesicle then tips over and concomitantly the plasma-membrane slit becomes curved into a horseshoe-shaped configuration as it extends along the outer margins of the tipped-over vesicle. Some coated pits are present interspersed between the above-mentioned structures, but their numbers appear insufficient to account for an exclusively endocytotic mechanism of membrane recycling. Instead, our micrographs are more consistent with a mixed mode of recycling of membrane components to the cortical endoplamic reticulum and to Golgi cisternae that involves both internalization of membrane by endocytosis and of individual lippid molecules by unknown mechanisms (lipid exchange proteins?). To this end, overall flattening out of the horseshoe-shaped membrane infoldings is accompanied by a retraction and reduction in size of their central, tongue-like structure.

Advances in ultrarapid freezing for the preservation of cellular ultrastructure

AbstractMost of our current knowledge of cellular ultrastructure is derived from studies of chemically fixed and chemically cryoprotected preparations. In the first part of this review, we document the many artifacts associated with chemical techniques that render them unsuitable for further refinement of our understanding of cellular ultrastructure. The best method currently available for the preservation of cellular ultrastructure is ultrarapid freezing. The second part of this review is a consideration of the physics of ice crystal formation in biological systems, which suggests that ice crystals will be present in any frozen, uncryoprotected specimen. We define an ultrarapidly frozen preparation as one in which the ice crystals are so small as to be invisible at the electron microscopic level. Improvements in the ease of application and reliability of ultrarapid freezing techniques have reached the point that these techniques can be used by anyone requiring the best achievable preservation of cellular ultrastructure. In the third part of this review, we describe and critique the five methods of ultrarapid freezing in current use.

A novel model for fluid secretion by the trypanosomatid contractile vacuole apparatus.

We have studied fluid secretion by the contractile vacuole apparatuss of the trypanosomatid flagellate Leptomonas collosoma with thin sections and freeze-fracture replicas of cells stabilized by ultrarapid freezing without prior fixation or cryoprotection. The ultrarapid freezing has revealed membrane specializations related to fluid segregation and transport as well as membrane rearrangements which may accompany water expulsion at systole. This osmoregulatory apparatu consists of the spongiome, the contractile vacuole, and the fluid discharge site. The coated tubules of the spongiome converge on the contractile vacuole from all directions. These 60- to 70-nm tubules contain characteristic double rows of 11-nm intramembrane particles in a helical configuration which fracture predominantly with the E face. Short double rows of similar particles are also frequently found on both faces of the contractile vacuole itself, in addition to many smaller particles on the P face. The spongiome tubules fuse with the vacuole during the filling stage of each cycle and then detach before secretion. The contractile vacuole membrane is permanently attached to the plasma membrane of the flagellar pocket by a dense adhesion plaque. In some ultrarapidly frozen cells, 20- to 40-nm perforations can be visualized within the plaque and the adjacent membranes during the presumptive time of discharge. The formation of the plaque perforations and the membrane channels occurs without fusion of the vacuole and the plasma membrane and does not require extracellular calcium. On the basis of our results, we have developed a model for water secretion which suggests that the adhesion plaque may induce pore formation in the adjoining lipid bilayers, thereby allowing bulk expulsion of the fluid.