A preliminary study of osmotic dehydration in zebrafish embryos: Implications for vitrification and ultra-fast laser warming.

To date, traditional cryopreservation techniques have not been amenable to zebrafish (Danio rerio) embryos, due in part to their large yolky eggs, which have a low surface to volume ratio and limited permeability to water and cryoprotectants. However, recent vitrification and ultra-rapid warming studies in mice have demonstrated successful preservation by dehydrating 85% or more of their total water content. We hypothesized that this approach may help overcome the barriers to embryo cryopreservation among D. rerio. The purpose of this study was to determine the osmotic tolerance limit of D. rerio embryos under conditions relevant to cryopreservation. We found that embryos undergoing gastrulation (30%-70% epiboly) were particularly sensitive to osmotic dehydration/rehydration. By contrast, a subset of embryos dehydrated during or after segmentation (20-22 somite, prim 5) survived 3 h in a 2 M sucrose solution but exhibit developmental delay, edema and trunk necrosis 2-4 days post-treatment.

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

Mazur and collaborators began examining the validity of initial views regarding mouse oocyte and embryo vitrification and found that most are partially or fully wrong. First, the relative effects of warming and cooling rates on the survival of mouse oocytes subjected to a vitrification procedure were determined. The high sensitivity to warming rate strongly suggests that the lethality of slow warming is a consequence of either the crystallisation of intracellular glassy water during warming or the recrystallisation during slow warming of small intracellular crystals that had formed during cooling. Warming rates of 107°C min-1 were achieved in 0.1-µL drops of ethylene glycol-acetamide-Ficoll-sucrose (EAFS) solution plus a small amount of India ink on Cryotops warmed using an infrared laser pulse. Under these conditions, survival rates of 90% were obtained even when mouse oocytes were suspended in 0.3× EAFS, a concentration that falls in the range that many cells can tolerate. A second important finding was that the survival of oocytes is more dependent on the osmotic withdrawal of much of the intracellular water before vitrification than it is on the penetration of cryoprotective solutes into the cells. Herein we review the roles of internal ice formation, vitrification and recrystallisation. It remains to be seen how widely these findings will be applicable to other types of cells and tissues from other species.

Intracellular ice formation in mouse zygotes and early morulae vs. cooling rate and temperature-experimental vs. theory.

In this study, mature female mice of the ICR strain were induced to superovultate, mated, and collected at either zygote or early morula stages. Embryos suspended in 1 M ethylene glycol in PBS containing 10 mg/L Snomax for 15 min, then transferred in sample holder to Linkam cryostage, cooled to and seeded at 7 °C, and then observed and photographed while being cooled to -70 °C at 0.5-20 °C/min. Intracellular ice formation (IIF) was observed as abrupt ''flashing''. Two types of flashing or IIF were observed in this study. Extracellular freezing occurred at a mean of -7.7 °C. In morulae, about 25% turned dark within ±1 °C of extracellular ice formation (EIF). These we refer to as "high temperature'' flashers. In zygotes, there were no high temperature flashers. All the zygotes flashed at temperatures well below the temperature for EIF. Presumably high temperature flashers were a consequence of membrane damage prior to EIF or damage from EIF. We shall not discuss them further. In the majority of cases, IIF occurred well below -7.7 °C; these we call ''low temperature'' flashers. None flashed with cooling rate (CR) of 0.5 °C/min in either zygotes or morulae. Nearly all flashed with CR of 4 °C/min or higher, but the distribution of temperatures is much broader with morulae than with zygotes. Also, the mean flashing temperature is much higher with morulae (-20.9 °C) than with zygotes (-40.3 °C). We computed the kinetics of water loss with respect to CR and temperature in both mouse zygotes and in morulae based on published estimates of Lp and it is Ea. The resulting dehydration curves combined with knowledge of the embryo nucleation temperature permits an estimate of the likelihood of IIF as a function of CR and subzero temperature. The agreement between these computed probabilities and the observed values are good.

Physical parameters, modeling, and methodological details in using IR laser pulses to warm frozen or vitrified cells ultra-rapidly.

We report additional details of the thermal modeling, selection of the laser, and construction of the Cryo Jig used for our ultra-rapid warming studies of mouse oocytes (Jin et al., 2014). A Nd:YAG laser operating at 1064 nm was selected to deliver short 1ms pulses of sufficient power to produce a warming rate of 1×10(7)°C/min from -190°C to 0°C. A special Cryo Jig was designed and built to rapidly remove the sample from LN2 and expose it to the laser pulse. India ink carbon black particles were required to increase the laser energy absorption of the sample. The thermal model reported here is more general than that previously reported. The modeling reveals that the maximum warming rate achievable via external warming across the cell membrane is proportional to (1/R(2)) where R is the cell radius.

Extreme rapid warming yields high functional survivals of vitrified 8-cell mouse embryos even when suspended in a half-strength vitrification solution and cooled at moderate rates to -196°C.

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to do so is to subject them to procedures that convert cell water into a non-crystalline glass. Current belief is that to achieve this vitrification, cells must be suspended in very high concentrations of glass-inducing solutes (i.e., ≥6 molal) and cooled at very high rates (i.e., ≫1000°C/min). We report here that both these beliefs are incorrect with respect to the vitrification of 8-cell mouse embryos. In this study, precompaction 8-cell embryos were vitrified in several dilutions of EAFS10/10 using various cooling rates and warming rates. Survival was based on morphology, osmotic functionality, and on the ability to develop to expanded blastocysts. With a warming rate of 117,500°C/min, the percentages of embryos vitrified in 1×, 0.75×, and 0.5× EAFS that developed to blastocysts were 93%, 92%, and 83%, respectively. And the percentages of morphological survivors that developed to expanded blastocysts were 100%, 92%, and 97%, respectively. Even when the solute concentration of the EAFS was reduced to 33% of normal, we obtained 40% functional survival of these 8-cell embryos.

Survivals of mouse oocytes approach 100% after vitrification in 3-fold diluted media and ultra-rapid warming by an IR laser pulse.

Vitrification is the most sought after route to the cryopreservation of animal embryos and oocytes and other cells of medical, genetic, and agricultural importance. Current thinking is that successful vitrification requires that cells be suspended in and permeated by high concentrations of protective solutes and that they be cooled at very high rates to below -100°C. We report here that neither of these beliefs holds for mouse oocytes. Rather, we find that if mouse oocytes are suspended in media that produce considerable osmotic dehydration before vitrification and are subsequently warmed at ultra high rates (10,000,000°C/min) achieved by a laser pulse, nearly 100% will survive even when cooled rather slowly and when the concentration of solutes in the medium is only 1/3rd of standard.

The survival of mouse oocytes shows little or no correlation with the vitrification or freezing of the external medium, but the ability of the medium to vitrify is affected by its solute concentration and by the cooling rate.

As survival of mouse oocytes subjected to vitrification depends far more on the warming rate than on the cooling rate, we wished to determine whether the lack of correlation between survival and cooling rate was mirrored by a lack of correlation between cooling rate and vitrification of the medium (EAFS), and between survival and the vitrification of the medium. The morphological and functional survival of the oocytes showed little or no relation to whether or not the EAFS medium vitrified or froze. We studied if the droplet size and the elapsed time (between placing the droplet on the Cryotop and the start of cooling) affects the result through modification of the cooling rate and solute concentration. Dehydration was rapid; consequently, the time between the placing the droplets into a Cryotop and cooling must be held to a minimum. The size of the EAFS droplet that is being cooled does not seem to affect vitrification. Finally, the degree to which samples of EAFS vitrify is firmly dependent on both its solute concentration and the cooling rate.

Regulatory volume decrease in COS-7 cells at 22 °C and its influence on the Boyle van't Hoff relation and the determination of the osmotically inactive volume.

Cryobiological analyses assume that the direction and rate of water movements across cell membranes and equilibrium cell volumes are determined solely by differences in the chemical potentials of intra- and extra-cellular water. A consequence of this assumption is that cells obey the Boyle van't Hoff (BvH) law which states that cell volumes are a linear function of reciprocal osmolality. Extrapolation of the BvH plot to infinite osmolality yields a quantity b, the fractional volume of the cell occupied by solids. In many cells, however, a cell volume excursion above the isotonic volume initiates an energy-requiring response that causes the swollen cells to shrink back to or towards isotonic volume. It is referred to as regulatory volume decrease (RVD). We have observed a strong RVD in COS-7 cells. If not eliminated by keeping exposure times short, this RVD produces a b that is 60% too high (0.48 vs. 0.30). These results indicate the importance of examining cells for volume regulatory mechanisms before performing measurements to determine their osmotic parameters.

Fully hydrated yeast cells imaged with electron microscopy.

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells.

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.

Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation.

The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0  M ethylene glycol in PBS for 15  min, cooled in a Linkam cryostage to -7.0  ° C, induced to freeze externally, and finally cooled at 20  ° C/min to -70  ° C. IIF that occurred during the 20  ° C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were -34, -40, -35, and -25  ° C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10-15  ° C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-stranded Aqp3 RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested.

Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification.

There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to -196°C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95 to 69,250°C/min to -196°C and for each cooling rate, subjecting them to five warming rates back above 0°C at rates ranging from 610 to 118,000°C/min. In samples warmed at the highest rate (118,000°C/min), survivals were 70% to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610°C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95 to 69,000°C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming.

The temperature and type of intracellular ice formation in preimplantation mouse embryos as a function of the developmental stage.

Our studies the past 5 yr have concentrated on intracellular ice formation (IIF) in mature mouse oocytes at the metaphase stage of meiosis II. Here we report an analogous investigation of the temperature of intracellular ice nucleation in preimplantation embryo stages from one-cell to early morula suspended in 1 M ethylene glycol/PBS and cooled at 20 degrees C/min to -70 degrees C. Physical modeling indicates that oocytes and preimplantation embryos undergo very little osmotic shrinkage at that cooling rate. As a consequence, their interior becomes increasingly supercooled until the supercooling is abruptly terminated by IIF. Four categories of IIF were observed. The first two were 1) those undergoing IIF at temperatures well below the temperature of external ice formation (EIF; -7.2 degrees C) vs. 2) those undergoing IIF within 1 degrees C of the EIF temperature. The other two categories were those multicellular stages in which 3) all the blastomeres underwent IIF simultaneously vs. 4) those in which blastomeres underwent IIF sequentially. Embryos in categories 1 and 3 constituted the majority (80-90%), and for them, the mean IIF temperatures of one-cell, two-cell, four- to six-cell, and early eight-cell ranged from -37 degrees C to -43 degrees C, temperatures that indicate that IIF is a consequence of homogeneous nucleation. However, the IIF nucleation temperature of early morulae in categories 1 and 3 was markedly higher; namely, -23.1 +/- 1.5 degrees C. This marked rise in nucleation temperature coincides with the appearance of aquaporin 3 and gap junctions in early morulae (compacted eight-cell), and is presumably causally related.

A biologist's view of the relevance of thermodynamics and physical chemistry to cryobiology.

Thermodynamics and physical chemistry have played powerful roles the past 45years in interpreting cryobiological problems and in predicting cryobiological outcomes. The author has been guided by a few core principles in using these concepts and tools and this paper discusses these core principles. They are (1) the importance of chemical potentials and of the difference between the chemical potentials of water and solutes inside the cell and outside in determining the direction and rate of fluxes of water and solutes. (2) The influence of the curvature of an ice crystal on its chemical potential and on the ability of ice to pass through pores in cell membranes, on the nucleation temperature of supercooled water, and on the recrystallization of ice. (3) The use of Le Chatalier's Principle in qualitatively predicting the direction of a reaction in response to variables like pressure. (4) The fact that the energy differences between State A and State B are independent of the path taken to go from A to B. (5) The importance of being aware of the assumptions underlying thermodynamic models of cryobiological events. And (6), the difficulties in obtaining experimental verification of thermodynamic and physical-chemical models.

Comparison between the temperatures of intracellular ice formation in fresh mouse oocytes and embryos and those previously subjected to a vitrification procedure.

When cells that have been subjected to supposedly innocuous freezing or vitrification procedures are used as the source material for subsequent experiments, it is important that they possess or exhibit the same relevant properties as fresh cells. In this study, we compared the temperatures of intracellular ice formation (IIF) in previously vitrified mouse oocytes/embryos with those in fresh intact ones. In the case of MII oocytes, 2-cell embryos, 4-6-cell embryos, and morulae, there are no significant differences (p>0.05); namely, -33.3 degrees C (fresh) vs. -35.4 degrees C (vitrified) with MII oocytes, -40.6 degrees C (fresh) vs. -38.7 degrees C (vitrified) with 2-cell embryos, -38.0 degrees C (fresh) vs. -39.4 degrees C (vitrified) with 4-6-cell embryos, -24.5 degrees C (fresh) vs. -24.2 degrees C (vitrified) with morulae. But, in 8-cell embryos, there is a significant difference (p<0.05) between fresh (-37.9 degrees C) and vitrified (-32.9 degrees C). If we include this significant difference, the overall IIF temperature of fresh cells is 0.74 degrees C lower than that of previously vitrified cells. If we exclude it, the IIF temperature for fresh cells is 0.32 degrees C higher than that for previously vitrified cells. Our conclusion then is that there is no difference between the IIF temperatures of fresh and previously vitrified cells.

Simple, inexpensive attainment and measurement of very high cooling and warming rates.

We have developed a simple, inexpensive system (<$300 US) for measuring cooling and warming rates of small (∼ 0.1µl) aqueous samples at rates as high as 10(5)°C/min. The measurement system itself, can track rates approaching one million°C/min. For temperature sensing, a Type T thermocouple with 50µm wire was used. The thermocouple output voltage was read with an inexpensive USB based digital oscilloscope interfaced to a laptop computer, and the raw data were processed with MS Excel.

The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure.

The formation of more than trace amounts of ice in cells is lethal. The two contrasting routes to avoiding it are slow equilibrium freezing and vitrification. The cryopreservation of mammalian oocytes by either method continues to be difficult, but there seems a slowly emerging consensus that vitrification procedures are somewhat better for mouse and human oocytes. The approach in these latter procedures is to load cells with high concentrations of glass-inducing solutes and cool them at rates high enough to induce the glassy state. Several devices have been developed to achieve very high cooling rates. Our study has been concerned with the relative influences of warming rate and cooling rate on the survival of mouse oocytes subjected to a vitrification procedure. Oocytes suspended in an ethylene glycol-acetamide-Ficoll-sucrose solution were cooled to -196 degrees C at rates ranging from 37 to 1827 degrees C/min between 20 and -120 degrees C, and for each cooling rate, warmed at rates ranging from 139 to 2950 degrees C/min between -70 and -35 degrees C. The results are unambiguous. If the samples were warmed at the highest rate, survivals were >80% over cooling rates of 187-1827 degrees C/min. If the samples were warmed at the lowest rate, survivals were near 0% regardless of the cooling rate. We interpret the lethality of slow warming to be a consequence of it allowing time for the growth of small intracellular ice crystals by recrystallization.

Determination of the water permeability (Lp) of mouse oocytes at -25 degrees C and its activation energy at subzero temperatures.

Typically, subzero permeability measurements are experimentally difficult and infrequently reported. Here we report an approach we have applied to mouse oocytes. Interrupted cooling involves rapidly cooling oocytes (50 degrees C/min) to an intermediate temperature above the intracellular nucleation zone, holding them for up to 40 min while they dehydrate, and then rapidly cooling them to -70 degrees C or below. If the intermediate holding temperature and holding time are well chosen, high post thaw survival of the oocytes is possible because the freezable water is removed during the hold. The length of time required for the exit of the freezable water allows the water permeability at that temperature to be determined. These experiments used 1.5M ethylene glycol in PBS and included a transient hold of 2 min for equilibration at -10 degrees C, just below the extracellar ice formation temperature. We obtain an Lp=1.8 x 10(-3)microm min(-1)atm(-1) at -25 degrees C based on a hold time of 30 min yielding 80% survival and the premise that most of the freezable water is removed during the 30 min hold. If we assume that the water permeability is a continuous function of temperature and that its Ea changes at 0 degrees C, we obtain a subzero Ea of 21 kcal/mol; higher than the suprazero value of 14 kcal/mol. A number of assumptions are required for these water loss calculations and the resulting value of Lp can vary by up to a factor of 2, depending on the choices make.

Intracellular ice formation in yeast cells vs. cooling rate: predictions from modeling vs. experimental observations by differential scanning calorimetry.

To survive freezing, cells must not undergo internal ice formation during cooling. One vital factor is the cooling rate. The faster cells are cooled, the more their contents supercool, and at some subzero temperature that supercooled cytoplasm will freeze. The question is at what temperature? The relation between cooling rate and cell supercooling can be computed. Two important parameters are the water permeability (Lp) and its temperature dependence. To avoid intracellular ice formation (IIF), the supercooling must be eliminated by dehydration before the cell cools to its ice nucleation temperature. With an observed nucleation temperature of -25 degrees C, the modeling predicts that IIF should not occur in yeast cooled at <20 degrees C/min and it should occur with near certainty in cells cooled at >or=30 degrees C/min. Experiments with differential scanning calorimetry (DSC) confirmed these predictions closely. The premise with the DSC is that if there is no IIF, one should see only a single exotherm representing the freezing of the external water. If IIF occurs, one should see a second, lower temperature exotherm. A further test of whether this second exotherm is IIF is whether it disappears on repeated freezing. IIF disrupts the plasma membrane; consequently, in a subsequent freeze cycle, the cell can no longer supercool and will not exhibit a second exotherm. This proved to be the case at cooling rates >20 degrees C/min.