Recrystallization Of Intracellular Ice Research Articles

Improving vitrification efficiency of human in vitro matured oocytes by the addition of LEA proteins.

Can the addition of late embryogenesis-abundant (LEA) proteins as a cryoprotective agent during the vitrification cryopreservation of in vitro matured oocytes enhance their developmental potential after fertilization? LEA proteins improve the developmental potential of human in vitro matured oocytes following cryopreservation, mostly by downregulating FOS genes, reducing oxidative stress, and inhibiting the formation of ice crystals. Various factors in the vitrification process, including cryoprotectant toxicity, osmotic stress, and ice crystal formation during rewarming, can cause fatal damage to oocytes, thereby affecting the oocytes developmental potential and subsequent clinical outcomes. Recent studies have shown that LEA proteins possess high hydrophilicity and inherent stress tolerance, and can reduce low-temperature damage, although the molecular mechanism it exerts protective effects is still unclear. Two LEA proteins extracted and purified by us were added to solutions for vitrification-warming of oocytes at concentrations of 10, 100, and 200 µg/mL, to determine the optimal protective concentration for each protein. Individual oocyte samples were collected for transcriptomic analysis, with each group consisting of three sample replicates. Immature oocytes were collected from patients who were undergoing combined in vitro fertilization (IVF) treatment and who had met the designated inclusion and exclusion criteria. These oocytes underwent in vitro maturation (IVM) culture for experimental research. A fluorescence microscope was used to detect the levels of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and calcium in the mitochondria of vitrified-warmed human oocytes treated with different concentrations of LEA proteins, and the protective effect of the protein on mitochondrial function was assessed. The levels of intracellular ice recrystallization inhibition (IRI) in human oocytes after vitrification-warming were characterized by the cryomicroscope, to determine the LEA proteins inhibitory effect on recrystallization. By analyzing transcriptome sequencing data to investigate the potential mechanism through which LEA proteins exert their cryoprotective effects. The secondary structures of AfrLEA2 and AfrLEA3m proteins were shown to consist of a large number of α-helices and the proteins were shown to be highly hydrophilic, in agreement with previous reports. Confocal microscopy results showed that the immunofluorescence of AfrLEA2-FITC and AfrLEA3m-FITC-labeled proteins appeared to be extracellular and did not penetrate the cell membrane compared with the fluorescein isothiocyanate (FITC) control group, indicating that both AfrLEA2 and AfrLEA3m proteins were extracellular. The group treated with 100 µg/mL AfrLEA2 or AfrLEA3mprotein had more uniform cytoplasmic particles and fewer vacuoles compared to the 10 and 200 µg/mL groups and were closest to the fresh group. In the 100 µg/mL groups, MMPs were significantly higher while ROS and calcium levels were significantly lower than those in the control group and were closer to the levels observed in fresh oocytes. Meanwhile, 100 µg/mL of AfrLEA2 or AfrLEA3m protein caused smaller ice crystal formation in the IRI assay compared to the control group treated with dimethylsulphoxide (DMSO) and ethylene glycol (EG); thus, the recrystallization inhibition was superior to that with the conventional cryoprotectants DMSO and EG. Further results revealed that the proteins improved the developmental potential of human oocytes following cryopreservation, likely by downregulating FOS genes and reducing oxidative stress. The in vitro-matured metaphase II (IVM-MII) oocytes used in the study, due to ethical constraints, may not accurately reflect the condition of MII oocytes in general. The AfrLEA2 and AfrLEA3m proteins are recombinant proteins and their synthetic stability needs to be further explored. LEA proteins, as a non-toxic and effective cryoprotectant, can reduce the cryoinjury of oocytes during cryopreservation. It provides a new promising method for cryopreservation of various cell types. This work was supported by the National Key Research and Development Program of China (2022YFC2703000) and the National Natural Science Foundation of China (52206064). The authors declare no competing interest. N/A.

Modulating intracellular ice recrystallization in hepatocytes using small carbohydrate-based ice recrystallization inhibitors

Modulating intracellular ice recrystallization in hepatocytes using small carbohydrate-based ice recrystallization inhibitors

Cryoprotectant-dependent control of intracellular ice recrystallization in hepatocytes using small molecule carbohydrate derivatives

To promote the recovery of cells that undergo intracellular ice formation (IIF), it is imperative that the recrystallization of intracellular ice is minimized. Hepatocytes are more prone to IIF than most mammalian cells, and thus we assessed the ability of novel small molecule carbohydrate-based ice recrystallization inhibitors (IRIs) to permeate and function within hepatocytes. HepG2 monolayers were treated with N-(4-chlorophenyl)-d-gluconamide (IRI 1), N-(2-fluorophenyl)-d-gluconamide (IRI 2), or para-methoxyphenyl-β-D-glycoside (IRI 3) and fluorescent cryomicroscopy was used for real time visualization of intracellular ice recrystallization. Both IRI 2 and IRI 3 reduced rates of intracellular recrystallization, whereas IRI 1 did not. IRI 2 and IRI 3, however, demonstrated a marked reduction in efficiency in the presence of the most frequently used permeating cryoprotectants (CPAs): glycerol, propylene glycol (PG), dimethyl sulfoxide (DMSO), and ethylene glycol (EG). Nevertheless, IRI 3 reduced rates of intracellular recrystallization relative to CPA-only controls in the presence of glycerol, PG, and DMSO. Interestingly, IRI preparation in trehalose, a commonly used non-permeating CPA, did not impact the activity of IRI 3. However, trehalose did increase the activity of IRI 1 while decreasing that of IRI 2. While this study suggests that each of these compounds could prove relevant in hepatocyte cryopreservation protocols where IIF would be prominent, CPA-mediated modulation of intracellular IRI activity is apparent and warrants further investigation.

Increased Productivity and Antifreeze Activity of Ice-binding Protein from Flavobacterium frigoris PS1 Produced using Escherichia coli as Bioreactor

Ice-binding proteins (IBPs) inhibit the growth and recrystallization of intracellular ice, enabling polar organisms to survive at subzero temperatures. IBPs are promising materials in biomedical applications such as cryopreservation and the hypothermic storage of cells, tissues, and organs. In this study, recombinant IBP from the antarctic bacterium Flavobacterium frigoris PS1 (FfIBP) was produced by Escherichia coli used as bioreactor, to examine the feasibility of scale-up. Oxygen transfer was the most important factor influencing cell growth and FfIBP production during pilot-scale fermentation. The final yield of recombinant FfIBP produced by E. coli harboring the pET28a-FfIBP vector system was 1.6 g/L, 3.8-fold higher than that from the previously published report using pCold I-FfIBP vector system, and its thermal hysteresis activity was 2.5°C at 9.7 µM. This study demonstrates the successful pilot-scale production of FfIBP.

Modulating Intracellular Ice Growth with Cell-Permeating Small-Molecule Ice Recrystallization Inhibitors.

Ice formation remains central to our understanding of the effects of low temperatures on the biological response of cells and tissues. The formation of ice inside of cells and the net increase in crystal size due to recrystallization during thawing is associated with a loss of cell viability during cryopreservation. Because small-molecule ice recrystallization inhibitors (IRIs) can control the growth of extracellular ice, we sought to investigate the ability of two aryl-glycoside-based IRIs to permeate into cells and control intracellular ice recrystallization. An interrupted graded freezing technique was used to evaluate the IRI permeation into human red blood cells (RBCs) and mitigate cell damage during freezing and thawing. The effect of IRIs on the intracellular growth of ice crystals in human umbilical vein endothelial cells (HUVECs) was visualized in real time under different thawing conditions using fluorescence cryomicroscopy. Adding an aryl glycoside to 15% glycerol significantly increased post-thaw RBC integrity by up to 55% during slow cooling compared with the 15%-glycerol-only control group. The characteristics of the cryobiological behavior of the RBCs subjected to the interrupted graded freezing suggest that the aryl-glycoside-based IRI is internalized into the RBCs. HUVECs treated with the IRIs were shown to retain a large number of small ice crystals during warming to high subzero temperatures and demonstrated a significant inhibition of intracellular ice recrystallization. Under slow thawing conditions, the aryl glycoside IRI p-bromophenyl-β-d-glucoside was shown to be most effective at inhibiting intracellular ice recrystallization. We demonstrate that IRIs are capable of internalizing into cells, altering the cryobiological response of cells to slow and rapid freezing and controlling intracellular ice recrystallization during freezing. We conclude that IRIs have tremendous potential as cryoprotectants for the preservation of cells and tissues at high subzero temperatures.

C-1015: Implications of the consequences of laser-induced ultra-rapid warming of mouse oocytes and embryos to vitrification theory and to the successful vitrification of other cell types

Jin, Kleinhans, and Mazur have reported in this meeting that up to 100% of oocytes and 2-cell embryos survive vitrification in a 3-fold dilution of EAFS medium if warmed at 10,000,000 °C/min by an IR laser pulse. The usual cooling rate was 69,000 °C/min, but 95% also survived when cooled at 10,000 °C/min. Thus, survival is far more dependent on the rate of warming than on the rate of cooling, the reason being that very high warming rates prevent or impede the recrystallization of intracellular ice. In all our experiments, the oocytes or embryos were held for 2.0 min in 1/3 × EAFS before vitrification That time was chosen because Pedro (1997) found it to produce the best survival after vitrification. EAFS media are strongly hyperosmotic so that oocytes/embryos placed in them, undergo an osmotic shrink-swell pattern with time. There is an initial rapid shrinkage to a minimum volume due to water loss, followed by a much slower re-swelling due to the slow influx of the permeating solutes. Based on Pedro’s data, that minimum occurs near 2 min, and osmotic theory argues that at that point, the cells have lost 85% of their original water and very little permeating solute has entered them. The strong inference, then, is that the high subsequent survivals are a consequence of the 85% osmotic dehydration and are not due to the intracellular presence of permeating solutes. Supporting that conclusion is Jin and Mazur’s report that 77% of oocytes and 95% of 2-cell and 8-cell embryos survive vitrification when suspended in media containing only the non-permeating solutes sucrose provided they are warmed at 1 × 107 °C/min by a pulse from our IR laser. None survived when warmed 100× more slowly in the absence of the laser. These findings are at odds with orthodox beliefs about the causes of injury from vitrification. They could possibly open the way to the successful vitrification of important cell types that to date have been partially or fully refractory to this approach.

037 Relative importance of cooling and warming rate in obtaining high survivals of mouse oocytes and embryos after vitrification

There is accelerating interest in the cryopreservation of human oocytes and preimplantation embryos, and this has prompted parallel research with mouse oocytes and embryos by us and others. The survival of a cell requires that no more than minimal amounts of small crystals of ice form inside the cell. One way to avoid intracellular ice is to vitrify cells; i.e., to convert cell water to a glass rather than to ice. The belief has been that this demands that both the cooling rate and the concentration of glass-inducing solutes in the cells be very high. But high solute concentrations can themselves be osmotically and chemically damaging. Our studies have shown that the first belief (that cooling needs to be extremely rapid) is not correct with respect to the vitrification of mouse oocytes. The important requirement is that the warming rate be extremely high. We subjected mouse oocytes in the vitrification solution EAFS 10/10 to vitrification procedures using a broad matrix of cooling and warming rates. Morphological survivals exceeded 80% when they were warmed at the highest rate (117,000 °C/min) even when the prior cooling rate was as low as 880 °C/min. Functional survival was >81% and 54% with the highest warming rate after cooling at 69,000 and 880 °C/min, respectively. We have also found the second belief to be erroneous. We show that a high percentage of mouse oocytes survive vitrification in EAFS media that contain only half the usual concentration of solutes, provided they are warmed extremely rapidly; i.e., >100,000 °C/min. Again, the cooling rate is of less consequence. This suggests that the major cause of death is the recrystallization of intracellular ice into large crystals during warming. We have cryomicroscope and thermodynamic evidence that supports that view. Source of funding: NIH R01OD011201. Conflict of interest: None declared. pmazur@utk.edu

2. Roles of intracellular ice formation, vitrification of cell water, and recrystallization of intracellular ice on the survival of mouse embryos and oocytes

Although sperm were successfully cryopreserved in 1950, another 22 years elapsed before the successful preservation of a mammalian embryo. In 1963, I had carried out physical–chemical analyses indicating that if cells were cooled too rapidly, they would undergo lethal intracellular ice formation (IIF), and defined what is meant by “too rapidly” for a particular cell. To avoid IIF, the cell had to dehydrate during cooling, and the required rate of cooling depended chiefly on the permeability of a particular cell to water and on the temperature coefficient of that permeability. Between 1968 and 1972, Stanley Leibo and I turned our attention to the survival of yeast, mouse marrow stem cells, and Chinese Hamster tissue culture cells as functions of cooling rate and temperature. We found that plots of survival vs. cooling rate formed an inverted “U”, with high survivals at a medium rate, and lower survivals after very slow and very rapid cooling. We proposed a “Two-Factor” hypothesis which stated that the right hand arm of the inverted U was a result of IIF at high cooling rates. Interestingly, the optimum cooling rate for maximum survival varied over a wide range for the different cells. This was chiefly because the cells differed widely in water permeability and the temperature coefficient of that permeability. In fact if the known values of these parameters were used to generate the theoretical curves, the predicted relation between IIF and cooling rate agreed quite closely with the observed drop in survival vs. cooling rate. In 1971 David Whittingham reported the successful cryopreservation of mouse 8-cell embryos cooled at 50 °C/min in PVP. Leibo and I were excited but perplexed about these results – perplexed because our computations indicated that mouse embryos would undergo lethal IIF when cooled at that rate. We calculated that the cooling rate would have to be about 1 °C/min.. That turned out to be the case. With that change and the substitution of Me 2 SO for PVP, David, Stanley and I obtained very good in vitro and in vivo survival of 8-cell embryos cooled to −196 °C (or −269 °C). The ability to cryopreserve mouse embryos quickly had important applied consequences and important fundamental consequences. Perhaps, the most important direct application was that it permitted the long-term maintenance of thousands of mutant lines of mice in the form of preimplantation embryos in a few liquid nitrogen tanks. The indirect applications stemmed from the fact that essentially the same procedures worked for early embryos of over 20 other mammals, including humans. In the last decade, clinical interest has been emphasizing the cryopreservation of human oocytes. They have proved more difficult to freeze and so interest has shifted somewhat to cryopreserving them by vitrification. As discussed, one way to avoid lethal IIF is to cool cells slowly enough so that the freezable water in the cell flows out of the cell and freezes externally. An alternative approach is to avoid intracellular ice by vitrifying cellular water. Vitrification involves converting water into a non-crystalline or amorphous glass. To achieve this conversion, it has been the strong belief that cells have to be in solutions containing a very high concentration of glass-inducing solutes and have to be cooled very rapidly. In recent work, however, we have found that neither of these is true for mouse oocytes. The important factor is not the cooling rate – it is the warming rate, and the warming rate has to be very high. Furthermore, if the warming rate is very high, one can cut in half the concentration of solutes in the vitrification solution and still obtain high survivals.

Stability of mouse oocytes at −80 °C: the role of the recrystallization of intracellular ice

The germplasm of mutant mice is stored as frozen oocytes/embryos in many facilities worldwide. Their transport to and from such facilities should be easy and inexpensive with dry ice at -79 °C. The purpose of our study was to determine the stability of mouse oocytes with time at that temperature. The metaphase II oocytes were cryopreserved with a vitrification solution (EAFS10/10) developed by M Kasai and colleagues. Two procedures were followed. In one, the samples were cooled at 187 °C/min to -196 °C, warmed to -80 °C, held at -80 °C for 1 h to 3 months, and warmed to 25 °C at one of three rates. With the highest warming rate (2950 °C/min), survival remained at 75% for the first month, but then slowly declined to 40% over the next 2 months. With the slowest warming (139 °C/min), survival was only ∼ 5% even at 0 time at -80 °C. In the second procedure, the samples were cooled at 294 °C/min to -80 °C (without cooling to -196 °C) and held for up to 3 months before warming at 2950 °C/min. Survival was ∼ 90% after 7 days and dropped slowly to 35% after 3 months. We believe that small non-lethal quantities of intracellular ice formed during the cooling and that the intracellular crystals increased to a damaging size by recrystallization during the 3 month's storage at -80 °C. From the practical point of view, this protocol yields sufficient stability to make it feasible to ship oocytes worldwide in dry ice.

43. Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice

43. Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice

40. Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling

40. Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling

Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling

Intracellular ice formation (IIF) is almost invariably lethal. In most cases, it results from the too rapid cooling of cells to below −40 °C, but in some cases it is manifested, not during cooling, but during warming when cell water that vitrified during cooling first devitrifies and then recrystallizes during warming. Recently, Mazur et al. [P. Mazur, I.L. Pinn, F.W. Kleinhans, Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling, Cryobiology 55 (2007) 158–166] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to −25 °C, holding them 10 min, rapidly cooling them to −70 °C, and warming them slowly until thawed. No IIF occurred during cooling but intracellular freezing, as evidenced by blackening of the cells, became detectable at −56 °C during warming and was complete by −46 °C. The present study differs in that the oocytes were warmed rapidly from −70 °C to temperatures between −65 and −50 °C and held for 3–60 min. This permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy ( E a) for the blackening process; namely, 27.5 kcal/mol. This translates to about a quadrupling of the blackening rate for every 5 °C rise in temperature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000 °C/min. A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8 °C. These findings have significant implications both for cryobiology and cryo-electron microscopy.

Enhanced survival of yeast expressing an antifreeze gene analogue after freezing

Yeast, like most organisms, survives poorly under freezing conditions. It has been proposed that after rapid cooling yeast suffers a loss in viability from the recrystallization of intracellular ice. Antifreeze proteins found in the blood of certain polar fishes have been shown to be potent inhibitors of ice recrystallization at very low concentrations. We have examined the feasibility of protecting rapidly cooled yeast cells from freezing damage by inhibiting the recrystallization of intracellular ice through in vivo expression of an antifreeze analogue gene. A chemically synthesized gene encoding a protein similar to but differing from the antifreeze proteins of the fish Pseudopleuronectes americanus (winter flounder) was genetically fused to the 3′ end of a truncated staphylococcal Protein A gene. When the fused gene was expressed in the budding yeast Saccharomyces cerevisiae, its cells were shown to produce a new chimeric protein that inhibited the recrystallization of ice in vitro. Yeast cells expressing the chimeric antifreeze protein showed a twofold increase in survival after rapid freezing (95 °C/min to −196 °C) and moderate rates of warming (26 to 64 °C/min) compared to cells lacking the chimeric protein.

The effect of cooling and warming rates on the survival of cryopreserved L-cells

A tissue culture assay has been used to measure the survival of murine lymphoma cells (L-cells) after freezing and thawing in the presence of 2 M glycerol or 1.6 M dimethyl sulfoxide. The effect of variations in cooling rate (0.1 to 10.0 °C/min) and warming rate (0.3 to 200 °C/min) were studied. It was found that survival exhibited a peak at the “conventional” combination of slow cooling and rapid warming (~1 and 200 °C/ min, respectively). It was also shown, however, that a second peak of similar magnitude occurred when the cells were cooled and rewarmed at 0.2-0.3 °C/min. These results are interpreted on the basis of current theories of freezing injury, stressing the importance of damage produced by the recrystallization of intracellular ice and by solute loading. The ultraslow rates of cooling and rewarming which produced the second survival peak are practicable for whole organs, and their potential importance for organ cryopreservation is apparent.

Survival of frozen-thawed human red cells as a function of cooling and warming velocities

Human red cells were equilibrated for 30 min at 20 °C in buffered saline containing 2 m glycerol and then frozen to −196 °C at 0.27, 1.7, 59, 180, 480, 600, and 1300 °C/ min and warmed at 0.47, 1, 26, 160, and 550 °C/min. Cells frozen at 600 and 1300 °C/min responded in the classical fashion for cells containing intracellular ice; i.e., survivals were low when warming was slow (<10%), but increased progressively with increasing warming rate. The sensitivity to slow warming presumably reflects the recrystallization of intracellular ice. Cells frozen at 59 and 180 °C/ min yielded high survivals at all warming rates. This response is also consistent with the findings for other cells cooled just slowly enough to preclude intracellular ice. Cells frozen very slowly at 0.27 and 1.7 °C/ min, however, responded differently; survivals were considerably higher when warming was slow (0.47 or 1 °C/min) than when it was 26, 160, or 550 °C/min. This response is analogous to that observed recently by others in mouse embryos and in higher plant tissue-culture cells and to that observed for many years in higher plants. It also confirms previous observations of Meryman in human red cells. It may reflect osmotic shock from rapid dilution but, if so, the basis of the osmotic shock is uncertain.

Visualization of freezing damage. II. Structural alterations during warming

There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from −20 ° to −50 °C and then rapidly cooled to −196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above −40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as −45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as −20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.