95 GATA E(3): 95-101,

Cryopreservation of Transgenic Mice KIMBALL 0. POMEROY

Advances in cryopreservation enable one to freeze embryos without the use of a programmable freezing machine or complex protocols. These methods achieve high rates of survival when mouse embryos are frozen. Understanding the factors that influence the survival of cryopreserved embryos can aid troubleshooting and in adapting freezing strategies from mice to other species. Frozen stocks of transgenics can be maintained indefinitely in liquid nitrogen. Cryopreservation of these valuable animals not only protects them from environmental catastrophes, but also is an economical method of storing lines for future detailed analysis.

Introduction Researchers spend thousands of dollars to produce lines of transgenic mice. Some transgenics express overt alterations of phenotype, but the vast majority either do not express the gene, express the gene without obvious phenotypic changes, or expression is not detected. Even when several transgenic lines are produced using the same DNA construct, usually only a few of the lines are characterized in detail. Because a large amount of time and money are invested in producing unique transgenics, surplus founder animals are often bred and caged for future analysis. Expenses for housing mice can run into thousands of dollars each month. A more prudent method to maintain valuable lines is cryopreservation. Cryopreservation of mouse embryos can not only reduce maintenance costs, but can safeguard highly valuable transgenic lines against eradication through disease, genetic contamination, faulty equipment, tire, or floods. New techniques now allow the freezing of all stages of mouse preimplantation embryos, inexpensively and with a minimum amount of equipment and skills. In fact, laboratories producing transgenic mice alFrom the Hurley

Medical

Center, Flint, Michigan, USA. Address correspondence to Kimball 0. Pomeroy, PhD, Department of Obstetrics and Gynecology, Mount Sinai Medical Center, Gumenick Building, 4300 Alton Road, Miami Beach. FL 33140, USA. Received 17 July 1990; revised November 12, 1990; accepted IO December 1990.

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ready possess most of the necessary skills and equipment for freezing mouse embryos. The purpose of this review is to (a) provide a basic understanding of cryopreservation principles, (b) describe how damage during cryopreservation occurs and how it can be avoided, (c) describe in detail two freezing protocols, and (d) discuss future technologies in cryopreservation that may affect transgenic animal production. Although this review deals primarily with freezing mouse embryos, the theories described apply equally well to other species.

An Overview Embry& from several species have been frozen successfully, including mouse, rabbit, sheep, goat, cattle, horse, cat, and human. Equipment to freeze embryos has traditionally included expensive, programmable freezing machines, liquid nitrogen storage tanks, a low-power stereoscopic microscope, freezing solutions, and either straws or ampules to freeze the embryos in. Recently, several simple methods have been developed to freeze embryos that do not require programmable freezing machines [ 1, 21. Freezing protocols usually involve several steps. First, embryos are exposed to cryoprotectants (substances that increase the survival of cells to cryopreservation). Next, embryos are placed into a straw and the straw is cooled to about -6°C. Ice formation is induced and then the embryo is cooled to lower temperatures. Finally, the straw is plunged into liquid nitrogen for storage. Embryos are usually thawed at room temperature or in a water bath. The cryoprotectant is then gradually removed and embryos are transferred into suitable recipients. An understanding of the factors that influence the survival of embryos during freezing and of basic principles of cryopreservation will aid in troubleshooting problems that may occur during freezing. They will also enable the evaluation of new freezing regimens.

Role of Cryoprotectants Embryos are frozen in culture medium or phosphate-buffered saline (PBS) supplemented with one or more cryoprotectants. The cryoprotective property of glycerol was serendipitously discovered in 1948 by Polge, Smith, and Parkes. A. S. Parkes describes the discovery: Publishing

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In the autumn of 1948 my colleagues, Dr. Audrey Smith and Mr. C. Polge, were attempting to repeat the results which Shaffner, Henderson and Card (1941) had obtained in the use of levulose solutions to protect fowl spermatozoa against the effects of freezing and thawing. Small success attended the efforts, and pending inspiration a number of the solutions were put away in the cold-store. Some months later work was resumed with the same material and negative results were again obtained with all of the solutions except one which almost completely preserved motility in fowl spermatozoa frozen to -79°C. This very curious result suggested that chemical changes in the levulose, possibly caused or assisted by the flourishing growth of mould which had taken place during storage, had produced a substance with surprising powers of protecting living cells against the effects of freezing and thawing. Tests, however, showed that the mysterious solution not only contained no unusual sugars, but in fact contained no sugar at all. Meanwhile, further biological tests had shown that not only was motility preserved after freezing and thawing, but also, to some extent, fertilizing power. At this point, with some trepidation, the small amount (lo- 15 ml.) of the miraculous solution remaining was handed over to our colleague Dr. D. Elliott for chemical analysis. He reported that the solution contained glycerol, water, and a fair amount of protein! It was then realized the Mayer’s albumin-the glycerol and albumen of the histologist-had been used in the course of morphological work on the spermatozoa at the same time as the levulose solutions were being tested, and with them had been put away in the cold-store. Obviously there had been some confusion with the various bottles, though we never found out exactly what had happened. Tests with new material very soon showed that the albumen played no part in the protective effect, and our low temperature work became concentrated on the effects of glycerol in protecting living cells

against the effects of low temperature [3]. Since the discovery of glycerol’s cryoprotective properties, other cryoprotectants, including dimethyl sulfoxide, 1,2-propanediol (propylene glycol), ethylene glycol, acetamide, methanol, sucrose, erythritol, raffinose, and trehalose have been used to freeze embryos. Serum, an ill-defined cryoprotectant, often makes up lo%-50% of the freezing solution. Although the mechanism of action of cryoprotectants is not understood, it appears that the ability to permeate the cell membrane to some degree may be necessary [4, 51. Cryoprotectants may act by stabilizing cell mem-

K. 0. Pomeroy

branes, decreasing the freezing point of solutions, lessening the effects of dehydration on cells, or promoting glass formation (vitrification) rather than ice crystal formation of intracellular solutions. Vitrification is the solidification of a solution without ice crystal formation. The solid, called a glass, behaves as an extremely viscous, supercooled liquid [6, 71. One should realize that most cryoprotectants can damage embryos either directly through chemical toxicity, or indirectly by exerting osmotic pressure on the cells resulting in severe dehydration. In order to reduce the possible deleterious effects of cryoprotectants, embryos should be exposed to them for as short a time as possible and at low temperatures. Toxicity and permeability of the cryoprotectant are important considerations in choosing a cryoprotectant for freezing embryos of species for which standard freezing protocols do not exist. When an embryo is exposed to cryoprotectant, it will initially shrink by losing its water. The water will rapidly move out of the cell to dilute out the “salty” extracellular environment (Figure 1A). This occurs because the cell membrane is more permeable to water than it is to the cryoprotectant. If the embryo loses too much water, the concentration of intracellular solutes may be toxic to the embryo. This phenomenon is known as solution effects. Sometimes, cells must be exposed gradually to increasing concentrations of the cryoprotectant to avoid solution effects. If the cell membrane is fairly permeable to the cryoprotectant, the need to expose embryos to slowly increasing concentrations of cryoprotectant can be eliminated.

Cooling and Dehydration Methods of cryopreservation can be divided into two general categories: equilibrium and nonequilibrium [8]. The major difference between these two types of cryopreservation is the rate of cooling and whether the cooling rate allows for osmotic equilibrium to become established between the extracellular and intracellular compartments with respect to cryoprotectants. In equilibrium cooling, embryos are exposed to moderate cryoprotectant concentrations and are cooled slowly (0.3”-2”Umin). Embryos are dehydrated during this slow cooling process. In nonequilibrium cooling, embryos are exposed to high molar concentrations of cryoprotectant and are cooled rap-

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of Transgenic Mice

DEHYDRATION DURING COOLING

A REHYDRATION DURING THAWING

2$igigwater _

6 Figure 1. (A) Diagrammatic representation of osmotic dehydrationduring cooling. Water freezes extracellulary. This increases the extracellular osmolality and so water flows out of the cell to dilute out the “salty” extracellular compartment. This removes water from the cell and increases the intracellular osmolality. (B) Diagram of rehydration during thawing. Water rapidly rushes into the dehydrated cell as cryoprotectant slowly diffuses into the extracellular compartment. (Thickness of arrows indicates relative permeability of solution to cell membrane.)

idly (IO”-50”Umin). Embryos are dehydrated rapidly, exposure times to cryoprotectants are reduced, and vitrification occurs soon after cooling begins.

Equilibrium

Cooling

In equilibrium methods of freezing, one is trying to cool the embryo slowly enough to allow it to dehydrate, and thus reduce large ice crystal formation, but not so slowly as to dehydrate the cells so that the embryo is damaged by high intracellular osmolality and the resulting solution effects. When embryos are cooled slowly, it is important that ice crystal formation begins early in the cooling cycle to initiate dehydration of the cell. This is usually accomplished by “seeding”forceps cooled in liquid nitrogen are touched to the straw containing the embryos. 0 1991 Elsevier

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Dehydration during cooling occurs through the following mechanism. If a solution is cooled slowly, say 0.3”C/min, extracellular ice crystals form outside the cell upon seeding (Figure 1A). As this extracellular water freezes, the concentration of extracellular solutes increases. Because the osmolality outside of the cell is greater than the osmolality inside the cell, water leaves the cell to dilute out the salty extracellular environment. The removal of water from the cell increases the osmolality of the intracellular compartment. At - 10°C there may be as much as a 20-fold increase in osmolality [9]. This increased osmolality further reduces the freezing point of the intracellular compartment and delays freezing so that further dehydration of the cell can occur. If the cooling rate is sufficiently low, intracellular water will flow out of the cells and freeze extracellularly, resulting in dehydration of the cells [lo]. If cells are cooled too rapidly, sufficient water is not removed, the cells do not dehydrate, and damage can occur through the formation of large intracellular ice crystals [l I]. The cooling rate must not, however, be so slow that the cells are severely dehydrated. This could result in extremely high concentrations of intracellular solutes that could damage the cell through solution effects. One method to aid dehydration is to expose embryos to a nonpermeable solute such as sucrose. The sucrose will increase the extracellular osmolality and water will leave the cells. Four factors determine the rate by which water crosses the cell membrane. The first is the type of cell-each cell type has its own characteristic permeability to water. Second is the surface to volume ratio of the cell-water will move out of a small cell faster than out of a large cell. Third is the temperature-water crosses membranes more rapidly at high temperatures than at low ones. Fourth is the osmotic pressure of the surrounding solution on the cell-water will cross a membrane more rapidly into a very salty solution than into a slightly salty solution. Seeding not only initiates dehydration, but also reduces supercooling. Although pure water freezes at O”C, the freezing point of other water solutions is determined by the concentration of particles it contains as solutes. The freezing point of a solution decreases by -2°C as the concentration of molecules in it increases by I M. The freezing point of most freezing solution is about -3°C. As one cools these solutions, freezing Publishing

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often occurs at >lo”C below this actual freezing point. This phenomenon is called supercooling. When a solution supercools and then undergoes a phase change from a liquid to a solid, a large amount of energy is released into the surrounding medium (Figure 2). The release of this energy, the latent heat of fusion, may rewarm the solution and induce the formation of large ice crystals upon recrystalization. It is thought that these large crystals can mechanically damage the zona pellucida or the cell membrane [ 121. To prevent supercooling, thereby decreasing the amount of energy released upon formation of the solid phase and reducing the size of intracellular ice crystals, an ice crystal is seeded into the freezing solution. As mentioned above, damage to embryos during cooling occurs either through cryoprotectant toxicity, severe dehydration of the cell (solution effects), or the shearing action due to the formation of large, intracellular ice crystals (usually from lack of dehydration).

Nonequilibrium

Cooling

In nonequilibrium cooling, exposure times to cryoprotectants and other harsh conditions of cooling are reduced. Mechanical damage from the shearing action of ice crystal formation is also decreased or eliminated. Embryos are dehydrated rapidly upon exposure to high concentrations of cryoprotectants and rapid cooling is begun soon after exposure of the embryos to cryoprotectant. Because of the high osmolality, the solution solidifies into a glasslike state without the formation of ice crystals [lo]. This vitritica-

SEEDING AT -5

tion allows for a more rapid and simpler technique for freezing cells.

Thawing The optimal thawing rate for an embryo is determined mainly by the rate at which the embryo was cooled. Embryos cooled slowly need to be thawed slowly, whereas those cooled rapidly must be thawed rapidly. Slowly cooled embryos usually contain very little intracellular water. If they are then thawed rapidly, water will rush into the “salty” intracellular milieu, damaging the cell through osmotic shock or lysis. When embryos are cooled rapidly, formation of large ice crystals may occur during slow thawing (recrystalization). This slow thawing may result in poor survival of the embryos [ 131. Thus embryonic survival is influenced by both the cooling rate and the rate of thawing. After thawing, the potentially toxic cryoprotectants must be removed. This is particularly important when high molar concentrations of cryoprotectant are used, as is often the case in nonequilibrium methods of cryopreservation. Because the cell membrane is more permeable to water than the cryoprotectants, an embryo placed into culture medium without stepwise removal of cryoprotectant will often lyse. Water will rapidly flow into the cell, while intracellular cryoprotectant exits more slowly (Figure 1B). This rapid change in osmolality can damage cells through what is known as osmotic shock. Damage during cryoprotectant removal can be reduced by either exposing the embryos to decreasing concentrations of cryoprotectant stepwise or by placing the embryo in a sucrose solution. Sucrose indirectly impedes the influx of water into the cell. Swelling of cells, which usually can be seen between steps in stepwise dilution, does not occur to as great an extent in sucrose solutions [5]. Exposure times to sucrose, however, are critical, as prolonged exposure can damage the embryo through severe dehydration and resulting solution effects.

Damage During Cryopreservation

TIME

Figure 2. Diagrammatic graph of temperature while freezing an aqueous solution in which seeding is performed (dotted line) and in which seeding is not performed (solid line).

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Embryos go through four stages during cryopreservation where damage may occur. These stages are (a) initial exposure and equilibration to the cryoprotectant, (b) cooling to subzero temperatures, (c) thawing and, finally, (d) dilution and removal of the cryoprotectant. Damage to the emNew York, NY 10010

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bryo can occur through one or more of the following mechanisms: cryoprotectant toxicity, osmotic shock, solution effects, and intracellular ice formation [14, 151. The specific mechanism responsible for embryonic damage is dependent upon the species of embryo, the developmental stage of the embryo, the cryoprotectant used, and the cooling and warming parameters. Most damage to embryos occurs during freezing or thawing, with intracellular ice formation and solution effects being the major culprits. Little if any damage occurs to embryos during storage in liquid nitrogen. At storage temperatures, usually around - 196”C, there is insufficient energy for Theoretically, the only known most reactions. danger at this temperature is DNA breakage caused by background radiation. For all intents, embryos can be stored in liquid nitrogen indefinitely.

Two Methods of Ctyopreservation Most cryopreservation regimens have been determined empirically. There are several methods to freeze mouse embryos, which vary in cryoprotectant used, cooling rates, seeding temperatures, plunging temperatures, thawing rates, and methods to add or remove the cryoprotectant. It is not the purpose of this review to present all the permutations that have been investigated; rather, two different methods for freezing mouse embryos will be discussed. One is an equilibrium method (slow cooling), which utilizes a programmable freezing machine. The second is a fast cooling method developed at the Salk Institute, which uses only a -70°C freezer. Both methods should be tried first on one of the hardy Fl strains (C57BL/6 x CBA or C57BLi6 x DBA/2) to ensure the methods work in your laboratory.

Slow Cooling The cryoprotectant solutions consist of a base solution of PBS with 10% fetal calf serum (FCS) to which is added glycerol to yield 5% and 10% glycerol (vol/vol) solutions. The solutions are filter sterilized through a 0.2-p,rn filter and can be stored frozen for future use. Mouse embryos @-cell to early blastocysts) are placed into PBS + 10% FCS just prior to cryoprotectant equilibration. Embryos float and shrink as they begin to equilibrate. A tissue culture dish is filled with 10% glycerol and the em0 1991 Elsevier

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bryos are transferred into this dish for another 10 min. While the embryos are equilibrating, the 0.25-ml straw is labeled with the reference number, date, and the name of the mouse line. To make loading of the straw easier, first rinse it with sterile PBS, being careful not to wet the cotton plug. After trimming the orifice of a tuberculin syringe, a 0.25-ml straw can be inserted into the orifice and the embryos can be loaded by slowly pulling back on the plunger of the syringe. Straws are loaded with IO-20 embryos per straw. About 3 cm of 10% glycerol is drawn up into the straw. Next, an air bubble (3-4 mm), 4 cm of 10% glycerol containing the embryos, another air bubble, and then more fluid is drawn up until the cotton wick is wet (Figure 3). The straw can now be sealed by a heat sealer or hematocrit cryosealer, or by dipping the wet end of the straw into PVA powder. (Supplies such as straws and sealing powder can be obtained from most veterinary supply houses.) The sealing system should be tried on a dummy straw first to ensure that the system works. A poor seal may allow liquid nitrogen to seep into the straw and cause the straw to explode during thawing. Straws should be laid horizontal prior to cooling to avoid trapping embryos near the meniscus. After the embryos have been in the 10% glycerol solution for a total of 10 min, the straws are cooled from room temperature to -6°C at 2”C/ min. The embryos are held at this temperature for 5 min and the straw is seeded by lightly grasping the straw with forceps cooled in liquid nitrogen. Seeding should be performed rapidly (1-2 s) and exposure to ambient air should be minimized. Straws should be held at - 6°C for an additional 10 min and then cooled to -30°C at O.S”C/min. When the temperature reaches -3O”C, straws are plunged quickly into liquid nitrogen. It is important that the straw is not warmed during seeding or plunging. To thaw embryos frozen using the above protocol, a solution of 0.6 M sucrose and 10% glycerol in PBS f 10% FCS is filter sterilized. Straws are rapidly removed from the liquid nitrogen and

EMBRYOS

PVA PLUG

SEED HERE

AIR BUBBLE

AIR BUBBLE

COlTON PLUG

Figure 3. Correct seeding location and placement of embryos %%Zi%ubbles in a freezing straw.

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placed into a 37°C water bath until the ice disappears. The sealed end of the straw is cut off with scissors, and is held over a dish of 0.6 M sucrose and 10% glycerol. Next, the cotton plugged end is snipped off. The solution will usually pour into the dish. If it fails to, the fluid can be expelled with a tuberculin syringe. The embryos are located through the microscope (19-20 x ) and are left in the sucrose-glycerol solution for 10 min. The embryos are placed into culture medium, rinsed twice, and then can be cultured or transferred into recipients. Fast Cooling This method of cryopreservation does not necessitate a programmable freezer, but is more sensitive to minor variations in protocol, especially time. It is therefore important to follow the protocol exactly. There is little room for error. The freezing solution contains glycerol (3.25 M; 24% vol/vol) and sucrose (0.5 M) in PBS + 10% FCS. Handling of embryos and straws is similar to the slow cooling method. Morula or g-cell mouse embryos (2.5 days after hCG) are placed into PBS + 10% FCS and are rinsed twice by serial passage through PBS + 10% FCS. Next, embryos are placed into 2 ml of freezing solution. The exposure time to this solution, prior to cooling, should be 20 min at room temperature. During this time, 0.25ml straws are loaded with the embryos positioned between two air bubbles as diagrammed in Figure 3. After 20 min, straws are placed horizontally on the bottom of a - 60” to - 80°C freezer and allowed to cool for 5-15 min. A small container filled with liquid nitrogen is placed in the freezer and using cooled forceps the straws are immediately plunged into the liquid nitrogen. Straws can now be transferred into a tank for storage. Straws are thawed by placing them into a 25°C water bath until the ice disappears. The embryos are then placed into 2-3 ml of a solution of 0.5 M sucrose in PBS + 10% FCS. After 12 min, the embryos are placed into PBS + 10% FCS, rinsed twice, and then cultured or transferred into recipients. Using these two techniques, ~80% of mouse embryos should develop to the blastocyst stage upon culture. Embryo transfer methods will not be mentioned here except to point out that the recipient strain will affect the pregnancy rate. Many researchers use C57BL/6 x CBA Fl recipFor a good methodology of embryo ients.

transfer, see Manipulating Hogan et al. 1161.

the Mouse Embryo by

A Transgenic Embryo Bank For economic reasons, only lines of mice that are currently being investigated should be maintained as live stock. All others should be frozen for future assaying and to protect them against environmental hazards. Ideally, embryos should be stored in two separate liquid nitrogen tanks; then if one bank is accidentally destroyed, the other is available to rescue the line. To ensure further that embryos are not accidentally lost due to failure to maintain proper liquid nitrogen levels, tanks can be equipped with liquid nitrogen level monitors for -$400. One should freeze - 100-200 embryos per line. Some lines, especially inbred lines, are diflicult to superovulate. In actuality, this may merely reflect a suboptimal dose of pregnant mare serum gonadotropin. Varying the pregnant mare serum gonadotropin dosage from 1 to 20 units with 2-unit increments may improve superovulation. There is also an age-dependent response to superovulation. Ideally, mice should be 4-6 weeks of age for superovulation. If lo-20 embryos are frozen per straw, one should be able to produce about live offspring per straw. All straws should be labeled with indelible ink. A colorcoding scheme can help in rapid identification of the straw. Besides pertinent information about the transgenic line, one should record the exact freezing protocol, as this will affect the thawing protocol used and freezing protocols may change over the years.

Future Developments The maintenance of large colonies of mice for the production of oocytes for microinjection is time consuming and expensive. It would be convenient for researchers if a commercial company could remove this burden by selling large quantities of frozen mouse zygotes ready for microinjection. Because of membrane changes that occur during freezing, it may be necessary to freeze zygotes prior to the pronuclear stage and then culture them in vitro to the pronuclear stage. Alternatively, researchers could freeze large batches of zygotes and thaw them for use later for microinjection. This would decrease the number of mice researchers would have to maintain and would decrease the amount of time the researcher would

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have to spend in superovulating mice and collecting zygotes. This would also allow researchers to inject zygotes independent of the mouse colony’s light-dark cycle. Cryopreservation of mouse embryos is a mature technology with a relatively high survival rate of -75%, but there are some species (such as pigs) in which survival after cryopreservation is quite low. Because some of these species may be the only ones suitable for certain transgenic studies, it is important that methods are devised to freeze these embryos. By better understanding theories of cryobiology we may be able not only to develop techniques to freeze embryos from difficult to freeze species, but we may also develop cryopreservation methods that are simpler and less time consuming.

Fast Cooling Protocol Cooling 1. Place 8-cell to morulae (2.5 days post-hCG) mouse embryos into PBS + 10% FCS. 2. Rinse embryos twice in 2 ml PBS + 10% FCS. 3. Place embryos into 3.25 M glycerol-O.5 M sucrose solution. 4. Load embryos into straws. 5. Keep embryos at room temperature so that total exposure to the freezing solution is for 20 min. 6. Place straws horizontally onto the bottom of - 60” to - 80°C freezer. 7. Cool straws in freezer for 5- 15 min. 8. Plunge straws into liquid nitrogen and store. Thawing 1. Place straws into 25°C water until ice melts. 2. Expel embryos into 2 ml 0.5 M sucrose for exactly 12 min. 3. Transfer embryos into culture medium and rinse twice. 4. Culture or transfer embryos into recipients.

Appendix Slow Cooling Protocol Cooling 1. Place &cell to early blastocyst (2.5-3 days post-hCG) mouse embryos into PBS + 10% FCS. Rinse embryos twice in 2 ml PBS + 10% 2. FCS. 3. Place embryos in 5% glycerol for 10 min. 4. Place embryos into 10% glycerol for 10 min. straws with embryos and 5. Load prelabeled seal. 6. Begin cooling within lo-30 min from when embryos were first exposed to 10% glycerol. 7. Cool straws 2Wmin to -6°C and hold at -6°C for 5 min. 8. Seed straws and hold for an additional 10 min. 9. Cool straws O.S”C/min to - 30°C. 10. Plunge straws into liquid nitrogen and store. Thawing 1. Place straws into 37°C water until ice melts. 2. Expel embryos into 10% glycerol + 0.6 M sucrose for exactly 10 min. 3. Transfer embryos into culture medium and rinse twice. 4. Culture or transfer embryos into recipients.

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