Peter Mazur

Survival of Mouse Embryos Frozen to -196° and -269°C

Mouse embryos survived freezing to -196�C. Survival required slow cooling (0.3� to 2�C per minute) and slow warming (4� to 25�C per minute). Depending on the specific rates used, 50 to 70 percent of more than 2500 frozen and thawed early embryos developed into blastocysts in culture after storage at -196�C for up to 8 days. When approximately 1000 of the survivors, including some frozen to -269�C (4�K), were transferred into foster mothers, 65 percent of the recipients became pregnant. More than 40 percent of the embryos in these pregnant mice gave rise to normal, living full-term fetuses or newborn mice.

A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells.

Abstract When Chinese hamster tissue-culture cells are frozen in a variety of suspending media, the percentage of cells surviving is maximal at optimum cooling rates, rates that are 2–4 orders of magnitude lower than those used to freeze cells for subsequent processing by the electron microscopy techniques of freeze-cleaving and freeze-substitution. The existence of such optima suggests that at least two factors dependent on cooling rate interact to determine the ultimate survival of a frozen-thawed cell. Other data are consistent with the view that the causes of injury in rapidly and slowly frozen cells are different. First, cells frozen rapidly in 0.4 M solutions of sucrose, glycerol, and dimethyl sulfoxide, or in 0.004 M polyvinylpyrrolidone, are inactivated to a much greater extent by slow warming than are cells frozen slowly in those solutions; that is, cells frozen at rates greater than the optimum are considerably more sensitive to slow warming. Second, the inactivation rate of cells frozen rapidly in glycerol is greater at −40 °C than that of cells frozen slowly. Third, the temperatures at which cells are killed as they are slowly frozen are very different from those observed during the slow warming of rapidly frozen cells. The precise nature of the two factors remains uncertain, but indirect evidence suggests that cells cooled slower than optimum are killed by alterations in the properties of the extracellular and intracellular solution induced by ice formation (e.g., high solute concentrations), and that cells cooled faster than optimum are killed by the formation of intracellular ice and its subsequent recrystallization during warming. Such intracellular recrystallization may be a potentially serious source of artifacts in frozen material processed for electron microscopy at temperatures above −60 °C, and perhaps even above −100 °C.

Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos

The first successful freezing of early embryos to −196°C in 1972 required that they be cooled slowly at ∼1°C/min to about −70°C. Subsequent observations and physical/chemical analyses indicate that embryos cooled at that rate dehydrate sufficiently to maintain the chemical potential of their intracellular water close to that of the water in the partly frozen extracellular solution. Consequently, such slow freezing is referred to as equilibrium freezing. In 1972 and since, a number of investigators have studied the responses of embryos to departures from equilibrium freezing. When disequilibrium is achieved by the use of higher constant cooling rates to −70°C, the result is usually intracellular ice formation and embryo death. That result is quantitatively in accord with the predictions of the physical/chemical analysis of the kinetics of water loss as a function of cooling rate. However, other procedures involving rapid nonequilibrium cooling do not result in high mortality. One common element in these other nonequilibrium procedures is that, before the temperature has dropped to a level that permits intracellular ice formation, the embryo water content is reduced to the point at which the subsequent rapid nonequilibrium cooling results in either the formation of small innocuous intracellular ice crystals or the conversion of the intracellular solution into a glass. In both cases, high survival requires that subsequent warming be rapid, to prevent recrystallization or devitrification. The physical/ chemical analysis developed for initially nondehydrated cells appears generally applicable to these other nonequilibrium procedures as well.

Effects of preezing on marrow stem cell suspensions: Interactions of cooling and warming rates in the presence of pvp, sucrose, or glycerol*†

Summary Based on results with microorganisms, one theory of freezing damage in cells states that the survival of a frozen-thawed cell is critically dependent on the rate at which it is cooled to subzero temperatures. The hypothesis suggests that the ultimate survival of a frozen and thawed cell is a function of the interaction of two factors that are oppositely dependent on cooling rate: 1) exposure to solution effects such as increased electrolyte concentration, pH changes, and dehydration and 2) the formation of intracellular ice during cooling and the extent of its recrystallization during warming. Using the colony-forming ability of the stem cells of mouse marrow as an assay of viability, we have sought to test the applicability of this theory to freezing damage in a nucleated mammalian cell. Stem cells suspended in each of three concentrations of glycerol, two of polyvinylpyrrolidone (mol wt, 40,000), or one of sucrose were frozen at rates varying from 0.3 to 500°C per min to −196°C, held for about 1 hr, and then thawed rapidly at about 900°C per min. In a second type of experiment, cells cooled at various rates were warmed either rapidly or slowly. Under all conditions, the survival of the colony-forming ability of the stem cells varied as a function of cooling rate, showing a distinct maximum at one rate. The optimum rate, however, varied with the specific additive and its concentration. For example, maximum survival was obtained for cells suspended in 1.25 m , 0.8 m , and 0.4 m glycerol and cooled at 1.5°C per min, 15°C per min, and 100°C per min, respectively. If cells in 1.25 m glycerol were cooled at 1.5°C per min, survival was about 65%, regardless of whether the suspensions were thawed at 2°C per min or 900°C per min, but if these cells were cooled at 200°C per min survival was 25% when the cells were thawed at 900°C per min and only 5% when they were thawed at 2°C per min. Good survival was also obtained in two additives that we believe to be nonpermeating, namely PVP and sucrose. Here, too, survivals showed distinct maxima as a function of cooling rate. Moreover, since the maximum survival was about 55% for cells suspended in 0.35 m sucrose but only 20% for cells in 0.4 m glycerol, it appears as if a nonpermeating additive is more effective on a molar basis in preventing freezing damage than is glycerol which has been assumed to permeate nucleated cells. All of these results are consistent with the “two-factor” theory of freezing damage developed from findings with microorganisms.

Interactions of cooling velocity, temperature, and warming velocity on the survival of frozen and thawed yeast

Summary The survival of cells of the yeast Saccharomyces cerevisiae after freezing and thawing is markedly dependent on the cooling and warming velocity and on the minimum temperature to which the cells are cooled. However, survival is not affected by variations in the time the cells are held at a given temperature, at least not by variations between 10 and 110 min. When cells are cooled to −196°C, survival after warming is almost always higher than 10% when cooling is at 7°C per min or slower, but drops to as low as 10−6% when cooling is 45 to 4700°C per min. The extent of the drop depends both on the particular cooling rate and warming rate used. For a given cooling rate, its extent varies inversely with warming rate, and no drop occurs when warming is 48,000°C per min, the highest velocity attained. Slow warming is also more adverse to slowly cooled cells, but only when the cells are cooled to below −60°C; and even then the effect is much less than after rapid cooling. Most of the protective effect of slow cooling is evident by the time the cells have cooled to −20°C; most of the adverse effects of the slow warming of slowly cooled cells occur below −40°C. The data support the hypothesis that the major cause of injury occurs when intracellular ice forms in the cells and recrystallizes during slow warming. The lethality of rapid cooling, then, is due to its increasing the probability of intracellular freezing. The resulting crystals can, however, be innocuous if warming is fast enough to prevent changes in crystal shape prior to melting. There are suggestions that some intracellular ice may even form in cells cooled slowly to below −60°C. On the other hand, injury to cells cooled slowly to temperatures above −60°C does not appear aseribable to intracellular ice; however, the present data do not indicate to what factor it is ascribable.

THE ROLE OF CELL MEMBRANES IN THE FREEZING OF YEAST AND OTHER SINGLE CELLS

Much of the damage in cells tha t a re subjected to subzero temperatures appears ascribable to two physical factors : the concentration of solutes tha t accompanies ice formation, and the formation of large ice crystals within the cell (Mazur, 1965). However, low-temperature exposure is by no means invariably lethal, for a wide variety of cells can survive cooling to -190°C. and below under appropriate conditions. Whether or not damage occurs under a particular se t of conditions depends on a number of parameters, among the most important of which a re the permeability properties of the plasma membrane. The properties of the membrane strongly influence the likelihood of intracellular freezing. Furthermore, when cells a r e killed by freezing and thawing, their permeability barriers a re almost invariably disrupted. There a re suggestions tha t this damage is a direct consequence of freezing, and not jus t a postmortem effect.

Fundamental Cryobiology of Mammalian Spermatozoa

Publisher Summary This chapter discusses the fundamental cryobiology of mammalian spermatozoa. The low motility of cryopreserved mammalian spermatozoa and the often lower conception rates may be due to the fact that procedures for cryopreservation of many mammalian cell types, including sperm, have evolved empirically. The primary assay of sperm function is the use of insemination and measurement of pregnancy initiation. The approaches to measuring sperm plasma membrane integrity include supervital staining and hyposmotic swelling. The other general approach to evaluating plasma membrane integrity is to assay the maintenance of membrane semipermeability by testing the cells ability to change its volume when exposed to anisomotic conditions. Survival of cells subjected to cryopreservation depends not only on the presence of a permeating cryoprotective agent (CPA) but also on the concentration of the CPA. Viability of mammalian sperm is very sensitive to osmotic stress and the associated cell volume excursion. The optimization of CPA addition and removal procedures is also elaborated.

Cryopreservation of the Germplasm of Animals Used in Biological and Medical Research: Importance, Impact, Status, and Future Directions

Abstract Molecular genetics and developmental biology have created thousands of new strains of laboratory animals, including rodents, Drosophila, and zebrafish. This process will accelerate. A decreasing fraction can be maintained as breeding colonies; hence, the others will be lost irretrievably unless their germplasm can be cryopreserved. Because of the increasingly critical role of cryopreservation, and because of wide differences in the success with which various forms of germplasm can be cryopreserved in various species, the National Institutes of Health National Center for Research Resources held a workshop on April 10–11, 2007, titled “Achieving High-Throughput Repositories for Biomedical Germplasm Preservation.” The species of concern were mouse, rat, domestic swine, rhesus monkey, and zebrafish. Our review/commentary has several purposes. The first is to summarize the status of the cryopreservation of germplasm from these species as assessed in the workshop. The second is to discuss the nature of the major underlying problems when survivals are poor or highly variable and possible ways of addressing them. Third is to emphasize the importance of a balance between fundamental and applied research in the process. Finally, we assess and comment on the factors to be considered in transferring from a base of scientific information to maximally cost-effective processes for the preservation of this germplasm in repositories. With respect to the first purpose, we discuss the three methods of preservation in use: slow equilibrium freezing, rapid nonequilibrium vitrification, and the use of intracytoplasmic sperm injection to achieve fertilization with sperm rendered nonviable by other preservation treatments. With respect to the last purpose, we comment on and concur with the workshops recommendations that cryopreservation largely be conducted by large, centralized repositories, and that both sperm (low front-end but high rederivation costs) and embryos (high front-end but modest rederivation costs) be preserved.

Kinetics of Water Loss and the Likelihood of Intracellular Freezing in Mouse Ova Influence of the Method of Calculating the Temperature Dependence of Water Permeability

To avoid intracellular freezing and its usually lethal consequences, cells must lose their freezable water before reaching their ice-nucleation temperature. One major factor determining the rate of water loss in the temperature dependence of the water permeability,Lp (hydraulic conductivity). Because of the paucity of water permeability measurements at subzero temperatures, that temperature dependence has usually been extrapolated from above-zero measurements. The extrapolation has often been based on an exponential dependence ofLp on temperature. This paper compares the kinetics of water loss based on that extrapolation with that based on an Arrhenius relation betweenLp and temperature, and finds substantial differences below −20 to −25°C. Since the ice-nucleation temperature of mouse ova in the cryoprotectants DMSO and glycerol is usually below −30°C, the Arrhenius form of the water-loss equation was used to compute the extent of supercooling in ova cooled at rates between 1 and 8°C/min and the consequent likelihood of intracellular freezing. The predicted likelihood agrees well with that previously observed. The water-loss equation was also used to compute the volumes of ova as a function of cooling rate and temperature. The computed cell volumes agree qualitatively with previously observed volumes, but differ quantitatively.

Osmotic consequences of cryoprotectant permeability and its relation to the survival of frozen-thawed embryos

Abstract The freezing of a living cell involves a complex physicochemical process of heat and water transport between the cell and its surrounding medium. Embryos survive cryopreservation only in the presence of a cryoprotectant in concentrations between 1 and 2M. During the addition and dilution of a permeating cryoprotectant, the cell undergoes osmotic changes in cell size. As a consequence, if the addition or particularly the dilution are carried out inappropriately, the viability of cells can be affected. Equations which model the influx and efflux of cryoprotectants in cells can be used to calculate the optimum and most practical addition and removal method. However, the equations require the permeability coefficient of the cryoprotectant, a quantity that has only experimentally determined for a few of the developmental stages of two species.