Rapidly cooled human sperm: no evidence of intracellular ice formation

BACKGROUND: The cellular damage that human spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. Alternatively, the cell damage may be the result of an osmotic imbalance encountered during thawing. This article examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. METHODS: In this study, human spermatozoa were cooled at a range of cooling rates from 0.3 to 3000 degrees C/min. The ultrastructure of the samples was examined by cryo scanning electron microscopy and freeze substitution to determine whether intracellular ice formed during rapid cooling and to examine alternative mechanisms of cell injury during rapid cooling. RESULTS: No intracellular ice formation was detected at any cooling rate. Freeze substitution of cells that had been cooled at 3000 degrees C/min and then slowly warmed showed that the cells had become plasmolysed and had evidence of membrane damage. CONCLUSIONS: Cell damage to human spermatozoa, at cooling rates of up to 3000 degrees C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.     

The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing

A firm biophysical basis for the cryopreservation of human spermatozoa is limited by a lack of knowledge regarding the water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPA). Cryomicroscopy cannot be used to measure dehydration during freezing in human spermatozoa because of their highly non-spherical shape and their small dimensions which are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of human sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPA. Using previously published data, the human sperm cell was modelled as a cylinder of length 40.2 micrometer and a radius of 0.42 micrometer with an osmotically inactive cell volume, V(b), of 0.23V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The 'combined best fit' membrane permeability parameters at 5 and 10 degrees C/min for human sperm cells in modified media are: L(pg) = 2. 4x10(-14) m(3)/Ns (0.14 micrometer/min-atm) and E(Lp) = 357.7 kJ/mol (85. 5 kcal/mol) (R(2) = 0.98), and in CPA media (with 6% glycerol and 10% egg yolk) are L(pg)[cpa] = 0.67x10(-14) m(3)/Ns (0.04 micrometer/min-atm) and E(Lp)[cpa] = 138.9 kJ/mol (33.2 kcal/mol) (R(2) = 0.98). These parameters are significantly different from previously published parameters for human spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in human sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPA. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates (<100 degrees C/min) and the numerically predicted optimal cooling rates (>7000 degrees C/min) obtained using previously published suprazero human sperm permeability parameters which do not account for the presence of extracellular ice.     

Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes.

The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2 degrees C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by approximately 23 degrees C in mouse and only by approximately 6.5 degrees C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the incidence of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31(32%), 19/34 (56%) and 52/56 (93%) respectively. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5 degrees C.     

Fertilization and development of mouse oocytes cryopreserved using a theoretically optimized protocol

Rational design of a cryopreservation protocol was demonstratedby using theoretical models of the cryopreservation processto develop an optimal freezing protocol for mouse oocytes. Acoupled mechanistic model of the processes of freeze-inducedcell dehydration and intracellular ice formation was developed,and cryomicroscopical measurements of intracellular ice formationkinetics were used to determine biophysical parameters requiredby the model, and to test model predictions of the freezingbehaviour of mouse oocytes. A simple phenomenological modelfor oocyte damage resulting from exposure to concentrated electrolyteand cryoprotectant solutions during cryopreservation was obtainedby defining a cost function equal to the duration of the freezingprotocol. A two-step freezing protocol was theoretically optimizedby using a sequential simplex algorithm to minimize the costfunction, subject to the constraint that the predicted probabilityof intracellular ice formation remain below 5%, yielding a putativeoptimum at the cooling rate B = 0.59°C/min, and plunge temperatureTp = –67°C. By systematically varying B and Tp aboutthese values in experiments with mouse oocytes cryopreservedin 1.5 M dimethyl sulphoxide, the maximal recovery of intactoocytes with a normal morphology (82%) was obtained for B =0.59°C/min and Tp = –80°C. Further evaluationof the fertilizability and developmental capacity of oocytescryopreserved using the optimized protocol yielded cleavageto the 2-cell stage in 65% of oocytes inseminated, and blastocystformation in 50% of these 2-cell embryos.     

A novel approach to sperm cryopreservation

Human spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been achieved by the standard approaches of cryobiology. Conventional approaches to cryopreservation impose a linear change of temperature with time; however, the stresses that cells encounter during cryopreservation are all non-linear with time. In this paper it is shown that improved methods of cryopreservation may be developed by specifically manipulating the manner in which cells experience physical changes instead of imposing a linear temperature reduction. Several treatments were compared: control of solidification to achieve constant ice formation with time was more damaging than the standard linear reduction in temperature. However, treatments which followed a chosen non-linear concentration profile, referred to as 'controlled concentration' allowed recovery of almost all the cells which were motile before freezing. The biophysical basis of these different responses was examined using the cryostage of a scanning electron microscope and freeze substitution and it was found that, surprisingly, all samples of spermatozoa in the frozen state were neither osmotically dehydrated nor had any visible intracellular ice. Viability on thawing did not appear to correlate with conventional theories of cellular freezing injury, which suggests that for human spermatozoa other factors determine viability following freezing and thawing.     

Biotransport Phenomena in Freezing Mammalian Oocytes

Water transport across the cell plasma membrane and intracellular ice formation (IIF)—the two biophysical events that may cause cell injury during cryopreservation—were studied by cryomicroscopy and modeling using mammalian (Peromyscus) oocytes. Unusually high activation energy for water transport across the cell plasma membrane was identified indicating that the water transport process is unusually sensitive to temperature (and cooling rate). Although literally all studies on IIF were conducted using protocols with ice-seeding (seeding extracellular ice usually at ≥−7 °C), it is not used for cell cryopreservation by vitrification that is becoming increasingly popular today. In this article, we show that ice-seeding has a significant impact on IIF. With ice-seeding and cooling at 60 °C/min, IIF was observed to occur over a wide range from approximately −8 to −48 °C with a clear change of the ice nucleation mechanism (from surface- to volume-catalyzed nucleation) at approximately −43 °C. On the contrary, without ice-seeding, IIF occurred over a much narrower range from approximately −19 to −27 °C without a noticeable change of the nucleation mechanism. Moreover, the kinetics of IIF without ice-seeding was found to be strongly temperature (and cooling rate) dependent. These findings indicate the importance of quantifying the IIF kinetics in the absence of ice-seeding during cooling for development of optimal vitrification protocols of cell cryopreservation.     

Cryobiology of non-human primate oocytes

The responses to various stresses involved with cryopreserv-ationprotocols were investigated using non-human primate oocytes.Fluorescence microscopy was used to assess the status of theF-actin microfilament system of rhesus monkey oocytes afterexposure to different concentrations of glycerol. The F-actinorganization around the cortex and hi the transzonal processeswas modified by exposure to 1.0 or 2.0 M glycerol at ambienttemperature. These effects were reduced significantly when exposureto glycerol was combined with cooling to 0C. Cynomolgus monkeyoocytes were also subjected to hyperosmotic stress and observedfor morphological changes. An irregular shrinkage phenomenonwas observed with germinal vesicle or metaphase I but not metaphaseII (MIl) oocytes. The irregular shrinkage became uniform andspherical when the oocytes were pretreated with ethyleneglycol-bis-(3-aminoethylether)N, N, N', N' tetraacetic acid (EGTA) before exposure tohypertonic solution. Also, in-vitro-matured MIl oocytes fromcynomolgus monkeys were used to determine crucial biophysicalparameters for freezing primate oocytes. The permeability ofoocyte plasma membrane to water, Lpg, and its activation energy,E_LP, were determined between 0 and -12C in the absence of cryoprotectiveadditives. The Lpg was found to be 3.8 10–14 m3N/s andthe ELp was 141.5 kJ/mol. The pre-exponential kinetic and exponentialthermodynamic parameters of intracellular ice formation weredetermined to be 8108 m2/s and 2.2 109 K5 respectively. Bycombining models of water transport and intracellular ice formation,the cumulative fraction of oocytes with intracellular ice asa function of the cooling rate was also predicted, and it wasshown to correlate reasonably with experimental observations.     

海藻酸钠／壳聚糖微囊冻存的低温显微实验

目的研究海藻酸钠-壳聚糖-海藻酸钠微胶囊（ACA）的冻存机制，探索一种ACA微囊低温保存的降温方法。方法ACA微囊随机分为2组，分别加入生理盐水（对照组）与终浓度为10％的低温保护剂二甲基亚砜（DMSO）（DMSO组），每组ACA微囊悬液置于可控低温显微镜载物台中，分别以1、10、30、100℃／min的降温速率降温至-120℃左右时停止降温，在显微镜下观察ACA微囊的形态改变。将2组完全冷冻的ACA微囊以50℃／min复温速率复温至37℃，在倒置显微镜下观察其形态学改变，计算微囊的完好率、皱缩率与破损率。结果对照组以1、10℃／min的降温速率冻存ACA微囊时，生成的冰晶粗大，冰晶的生长可导致微囊发生形变；而DMSO组以30、100℃／min的降温速率冻存ACA微囊时，生成的冰晶较为细小，对微囊的损伤也更小，并且复温后的微囊形态保存良好，与对照组和同组以1、10℃／min降温速率冻存比较，其微囊完好率明显增高（P〈0．05）。结论微囊在低温保存过程中主要受到冰晶生长所导致的机械性损伤，提高降温速率与添加低温保护剂可以有效地抑制冰晶生长。     

血管冻存过程中的应力分析

为了预测血管组织低温保存过程中所受到的机械损伤,本文建立了一维对称具有同心孔的圆筒模型,模拟计算了降温过程壁面温度随时间的变化;冻结过程中体积膨胀和组织内温度分布不均引起的热应力随时间变化及其在壁面的分布．本文计算了热应力在径向、周向和轴向的分布,径向应力小于周向应力和轴向应力;由相变引起的应力大于由温度分布不均引起的应力;主要考虑了降温速率的影响,降温速率越高,组织内部所受热应力越大;当降温速率是１Ｋ／ｍｉｎ时最大周向应力为０．１２Ｍｐａ,降温速率为１２Ｋ／ｍｉｎ时最大周向应力为０．６Ｍｐａ;当热应力超过血管组织所能承受的极限值时,在血管壁上会产生裂纹;使组织受到损伤。     

Using multiple-electrode impedance measurements to monitor cryosurgery

We outfitted cryoprobes with electrodes and used them in conjunction with a multiple channel electrical impedance tomography (EIT) system to record data during freezing experiments in a shallow saline tank. We made measurements using electrodes mounted on the probes and the tank's periphery. Reconstructed images based on both sets of electrodes indicate a significant improvement in the appearance of the ice ball over using tank electrodes alone. The size of the ice balls was varied by deliberately altering the cooling rate. We found a positive correlation between the measured size of the ice ball and the sizes of isocontour lines in the reconstructed impedance maps. Similarly, the shape of the ice balls was altered by circulating the saline about the probe. Two-dimensional reconstructed impedance contours indicated a deformation in agreement with the shape of the ice ball during the experiments. These findings suggest that using multielectrode impedance sensing may constitute a means for monitoring cryosurgery.     

Exocytosis and Spreading of Normal and Aberrant α‐Synuclein

It is now established that α‐synuclein can be physiologically secreted to the extracellular space. In this sense, mechanisms that govern the secretion of the protein may be of importance in the initiation and progress of synucleinopathies. It is possible that increased secretion may aid the formation of toxic seeds extracellularly. Alternatively, reduced presence of extracellular α‐synuclein due to impaired secretion may increase the intracellular load and trigger intracellular seeding. Once outside, α‐synuclein can exert various paracrine actions on neighboring cells again by mechanisms that have not been fully elucidated. It has been demonstrated that, when applied extracellularly, α‐synuclein species can induce multiple neurotoxic and inflammatory responses, and aid the transmission of pathology between neurons. Still, the exact mechanism(s) by which secreted α‐synuclein affects the homeostasis of other neurons is still not well understood. A portion of α‐synuclein has been shown to be associated with the surface and lumen of exosomes which can transfer it to the surrounding cells, and potentially trigger seeding. Interestingly, increased exosome release has been linked to pathological situations of lysosomal dysfunction as observed in Parkinson's disease (PD). However, the possibility that the observed α‐synuclein pathology spread is attributable to the passive diffusion of the initial injected α‐synuclein strains cannot be excluded. Importantly, most of the studies that have so far addressed the role of extracellular α‐synuclein have not employed naturally secreted forms of the protein. It is plausible that deregulation in the normal processing of secreted α‐synuclein may aid the formation of “toxic” species and as such it may also be a causative risk factor for PD. In this capacity, elucidation of the underlying mechanisms that regulate the protein‐levels of extracellular α‐synuclein becomes essential. Such mechanisms could involve its proteolytic clearance from the extracellular milieu.     

Morphological study by an 'in vivo cryotechnique' of the shape of erythrocytes circulating in large blood vessels

Changes in the shape of erythrocytes circulating in large blood vessels of mice were examined by our 'in vivo cryotechnique'. The abdominal aorta and inferior vena cava (IVC) were cut vertically with a precooled knife and simultaneously an isopentane–propane mixture (−193°C) was poured over them for freezing. They were freeze-substituted in acetone containing 2% osmium tetroxide. Some specimens were embedded in Quetol-812, and thick or ultrathin sections were examined by light or transmission electron microscopy. Serial ultrathin sections were used to reconstruct 3-dimensional images of native erythrocytes. Others were transferred into t-butyl alcohol and freeze-dried for scanning electron microscopy. The tissue surfaces were sufficiently frozen to prevent large ice crystal formation, and erythrocyte shapes were also preserved. The shapes of circulating erythrocytes appeared to be varied in the abdominal aorta but typical biconcave discoid shapes were rarely observed. Conversely, erythrocytes were approximately biconcave discoid in shape in the IVC. Our in vivo cryotechnique was useful for clarifying the in vivo morphology of erythrocytes circulating in large blood vessels.     

The sweet thing about Type 1 diabetes: a cryoprotective evolutionary adaptation

The reasons for the uneven worldwide distribution of Type 1 diabetes mellitus have yet to be fully explained. Epidemiological studies have shown a higher prevalence of Type 1 diabetes in northern Europe, particularly in Scandinavian countries, and Sardinia. Recent animal research has uncovered the importance of the generation of elevated levels of glucose, glycerol and other sugar derivatives as a physiological means for cold adaptation. High concentrations of these substances depress the freezing point of body fluids and prevent the formation of ice crystals in cells through supercooling, thus acting as a cryoprotectant or antifreeze for vital organs as well as in their muscle tissue. In this paper, we hypothesize that factors predisposing to elevated levels of glucose, glycerol and other sugar derivatives may have been selected for, in part, as adaptive measures in exceedingly cold climates. This cryoprotective adaptation would have protected ancestral northern Europeans from the effects of suddenly increasingly colder climates, such as those believed to have arisen around 14,000 years ago and culminating in the Younger Dryas. When life expectancy was short, factors predisposing to Type 1 diabetes provided a survival advantage. However, deleterious consequences of this condition have become significant only in more modern times, as life expectancy has increased, thus outweighing their protective value. Examples of evolutionary adaptations conferring selection advantages against human pathogens that result in deleterious effects have been previously reported as epidemic pathogenic selection (EPS). Such proposed examples include the cystic fibrosis mutations in the CFTR gene bestowing resistance to Salmonella typhi and hemochromatosis mutations conferring protection against iron-seeking intracellular pathogens. This paper is one of the first accounts of a metabolic disorder providing a selection advantage not against a pathogenic stressor alone, but rather against a climatic change. We thus believe that the concept of EPS should now include environmental factors that may be nonorganismal in nature. In so doing we propose that factors resulting in Type 1 diabetes be considered a result of environmental pathogenic selection (EnPS).     

Preferential vitrification of water in small alginate microcapsules significantly augments cell cryopreservation by vitrification

The morphological changes of small (~100 μm) alginate microcapsules and the biophysical alterations of water in the microcapsules during cryopreservation were studied using cryomicroscopy and scanning calorimetry. It was found that water in the small microcapsules can be preferentially vitrified over water in the bulk solution in the presence of 10% (v/v) or more dimethylsulfoxide (DMSO, a cryoprotectant), which resulted in an intact morphology of the microcapsules post cryopreservation with a cooling rate of 100°C/min. A small amount of Ca²⁺ (up to 0.15 M) was also found to help maintain the microcapsule integrity during cryopreservation, which is attributed to the enhancement of the alginate matrix strength by Ca²⁺ rather than promoting vitrification of water in the microcapsules. The preferential vitrification of water in small microcapsules was further found to significantly augment cell cryopreservation by vitrification at a low concentration of cryoprotectants (i.e., 10% (v/v)) using a small quartz microcapillary (400 μm in diameter). Therefore, the small alginate microcapsule could be a great system for protecting living cells that are highly sensitive to stresses due to freezing (i.e., ice formation) and high concentration of cryoprotectants from injury during cryopreservation.     

Relationship between generation and plasma concentration of anorganic phosphorus. In vivo studies on dialysis patients and in vitro studies on erythrocytes

The plasma concentration of inorganic phosphorus (Pi) was determined before, during and after hemodialysis in 28 patients with chronic renal failure. Pi plasma concentration decreased rapidly when hemodialysis was started but did not fall below normal levels during continued dialysis. These changes of Pi concentration were fitted to a model of Pi kinetics in which Pi delivery to plasma is a nonlinear function of the extracellular Pi concentration. In separate in vitro studies, erythrocytes from six subjects with normal renal function and from 14 patients with chronic renal failure were incubated in homologous plasma with various amounts of Pi added. All other factors known to affect the Pi shift between intra- and extracellular fluid compartments (pH, calcium concentration) were kept constant. The relation between Pi concentration in plasma used for incubation and in red cells after 1h incubation suggested a mechanism in which a high plasma concentration results in movement of Pi into red cells where Pi is stored most probably in glucose esters. At low Pi plasma concentration Pi is delivered to plasma at a rate which cannot be explained solely by passive movement of intracellular Pi to plasma but requires additional generation from intracellular storage forms. The generation and delivery of Pi in patients and in their erythrocytes indicate a simple cell-mediated Pi homeostasis counter-acting abnormal fluctuations of plasma Pi.     

Fabrication of porous gelatin scaffolds for tissue engineering.

A novel method which employs water present in swollen hydrogels as a porogen for shape template was suggested for preparing porous materials. Biodegradable hydrogels were prepared through crosslinking of gelatin with glutaraldehyde in aqueous solution, followed by rinsing and washing. After freezing the swollen hydrogels, the ice formed within the hydrogel network was sublimated by freeze-drying. This simple method produced porous hydrogels. Irrespective of any rinsing and washing processes, water was homogeneously distributed into the hydrogel network, allowing the hydrogel network to uniformly enlarge and the ice to act as a porogen during the freezing process. Different porous structures were obtained by varying the freezing temperature. Hydrogels frozen in liquid nitrogen, had a two-dimensionally ordered structure, while the hydrogels prepared at freezing temperatures near -20 degrees C, showed a three-dimensional structure with interconnected pores. As the freezing temperature was lowered, the hydrogel structure gradually became more two-dimensionally ordered. These results suggest that the porosity of dried hydrogels can be controlled by the size of ice crystals formed during freezing. It was concluded that the present freeze-drying procedure is a bio-clean method for formulating biodegradable sponges of different pore structures without use of any additives and organic solvents.     

Erythrocyte freezing in the presence of hydroxyethyl starch]

Biochemical properties of erythrocytes frozen in the presence of hydroxyethyl starch (HES) as preserving substance produced in this country were assessed. Erythrocytes were frozen adding HES in 4 different concentrations. The most favourable results were obtained with erythrocytes subjected to the action of HES in 30% and 35% concentrations. The erythrocyte 2,3-DPG level during freezing with 35% HES was within normal limits while it decreased by 10% after defreezing when 30% HES had been used. The ATP concentration in erythrocytes fell by 50% in relation to the initial value. A slight fall of P50 was observed and erythrocyte loss after defreezing did not exceed 10%. The 35% HES solution in a 0.15 mol/l sodium chloride solution was most favourable for freezing erythrocytes in liquid nitrogen. A simple method of preparation of defrozen erythrocytes for transfusion was elaborated.     

The development of a cryoscopic apparatus

The development and use of a cryoscopic apparatus are described. Temperatures are simply measured by means of a thermistor and WHEATSTONE bridge. However, the thermal conditions within the cryoscopic cell during freezing were closely controlled with the result that a quick and accurate method for determining a series of freezing points without supercooling was made possible. The apparatus and method have been tested using solutes of molecular weights up to 2000.     

Hemolysis-in-gel (HIG) test for antibodies to influenza A, measles and mumps using liquid nitrogen freezed erythrocytes coupled with the respective viral antigen.

A drawback with the hemolysis-in-gel test is the constant need for fresh erythrocytes which must be treated with virus before incorporation in the gel. This problem was overcome by freezing small droplets containing erythrocytes to which antigen had been attached. The droplets were stored at -70 degrees C or in liquid nitrogen. This modification was applied in detecting antibodies to influenza, measles and mumps viruses and the results were shown to equal those obtained with fresh erythrocytes.     

A randomized comparison of the cryopreservation of one-cell human embryos with a slow controlled-rate cooling procedure or a rapid cooling procedure by direct plunging into liquid nitrogen

We conducted a randomized prospective study of the cryopreservation of one-cell human embryos, comparing a slow controlled-rate freezing procedure with a rapid cooling procedure by direct plunging into liquid nitrogen. We analysed the numbers of embryos that were recovered immediately after thawing (= recovery), the number of embryos morphologically intact after thawing and subsequent dilution of the cryoprotectants (= survival), the numbers of embryos undergoing further cleavage after 24 h of in-vitro culture (= cleavage) and the implantation of transferred embryos (= children born per frozen-thawed embryo transferred). We demonstrated that the recovery of embryos was greater after slow controlled-rate freezing. Survival was greater after rapid cooling and the number of embryos undergoing further cleavage was higher after slow controlled-rate freezing. Although the birth rate was twice as high after slow controlled-rate freezing as after rapid cooling, this difference was not statistically significant. In conclusion, our results show clearly that for the freezing of one-cell human embryos, slow controlled-rate freezing is more efficient than rapid cooling. Before rapid cooling is used routinely in clinical in- vitro fertilization programmes, its safety and reproducibility must be convincingly demonstrated.