Cryopreservation of murine cells

Last modified on June 3, 2010

This was the topic of a 2000 ILAR Journal. Cryopreservation of murine cells on a large scale would have great implications for the preservation of mice produced by genetic manipulation for which phenotyping has not been completed. There may be some parallels with the preservation of endangered species currently practiced by conservationists and zoos, as well as with human infertility treatment. In fact, the conservationists have guidelines for deciding how and when to preserve valuable biological materials that can be adapted for laboratory animal use. In general, good success has been achieved with cryopreservation of mouse embryos, moderate success with rat embryos and mouse ovarian tissue, little success with mouse spermatozoa and oocytes, and no success published so far with rat spermatozoa.

Major steps in cryopreservation are (1) addition of cryoprotective agents, (2) cooling, usually to -196°C, (3) warming, and (4) removal of cryoprotective agents. At -196°, cells will be stable for times measured geologically, so the real issues relate to cooling, cryoprotection, and warming.{4066}

With cooling, ice forms at between -5° and -15°C in the external medium, but the cell contents remain unfrozen. However, the cells shrink. Subsequent events depend upon the cooling rate. The freezing behavior of the cells can be modified by adding cryoprotectants, which affect the rates of water transport, nucleation and crystal growth. "Solution effects" are those that may cause cell injury as a result of concentration of solutes, and they are enhanced when the cooling rate is too slow. The two-factor hypothesis put forth by Mazur explains the cell responses to freezing: (1) at slow cooling rates, cryoinjury occurs due to solution effects, and (2) at fast cooling rates, cryoinjury occurs due to lethal intracellular ice formation. Experimentally, different cell types generate curves of cooling rate vs. survival that are upside-down U in shape.{4066}

Whether a cooling rate is too fast or too slow depends upon the ability of water to move across the cell membranes. Four simultaneous equations can be used to describe the rate at which water is transported across a cell membrane. The results can be used to calculate the extent of supercooling of cells as a function of the cooling rate. Cryobiological factors that must be known to make the calculations include the permeability coefficient to water, the temperature coefficient or activation energy, osmoles of solute initially inside the cell, and the surface: volume ratio. Numerous investigators have developed devices or models to determine cell membrane permeability. Ultimately, sets of curves are generated calculating the relative volume of cell water vs. the temperature. As temperature decreases from 0° to -60°, relative volume of cell water decreases.{4066}

Cryoprotectants have been around for 50 years, with glycerol being the oldest. DMSO, ethylene glycol, methanol, propylene glycol and dimethylacetamide are also used. The most important characteristic is that they can easily permeate cells and are relatively non-toxic in concentrations of 1M or more. They protect by decreasing the concentration of electrolytes during freezing, thereby decreasing osmotic shrinkage. Some non-permeating agents are also used to augment the effectiveness of permeating agents.{4066}

The response to warming is often dependent on what happened during freezing and has not been studied much. Warming techniques tend to be somewhat crude, such as putting in your pocket or thawing between the hands. For human sperm, warming in air at 20-35°C turns out the highest viability and motility.{4066}

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Cryopreservation of spermatozoa

Spermatozoa are especially good models for investigating the role of intracellular structures and the plasma membrane; this is because sperm have both motility and the acrosome reaction which depend on the function of organelles and the plasma membrane. Glycerol is toxic to many sperm such as bull, boar and human. Studies with bull sperm have indicated that very short exposure times yield higher post-thaw motility, but human sperm don't seem to care. Current investigation surrounds the question of whether sensitivity to glycerol depends on the osmotic consequences or chemical toxicity. Another reason sperm are important to study is that they are subject to cold shock in some species (bull, ram, boar and stallion), but not in others (human and rabbit). This refers to conditions between the time of collection and the time when supercooling is performed, i.e. from ambient temperature to +5°C.{4066}

The cryobiological factors of spermatozoa appear to vary greatly among species and among mouse strains. Mouse and boar sperm appear to have "exquisitely narrow" osmotic limits, while human sperm is more tolerant. Osmotic limits can be widened by the use of cryoprotective solutions based on glycerol, DMSO, ethylene glycol, egg yolk or skim milk. In fact, the authors claim that the vast majority of research over the last 50 years has been on these extenders rather than on the basic cryobiology of cell types, and that knowledge of spermatozoal cryobiology lags far behind that of other cell types. Relatively successful freezing of mouse spermatozoa can be obtained without using cryoprotectants at all.{4065}

Differences between murine sperm and that of other species may be based in part upon the lipid content of the cell membrane. One reason mouse sperm may be so sensitive to freezing damage is that the cytoskeleton anchors the plasma membrane to the internal structure of the cell. Mouse spermatozoa also appear to be very easily damaged by routine mechanical factors such as mixing, centrifugation and pipetting. {4065}

The success of cryopreservation can be measured in many ways (such as sperm viability, sperm motility, sperm fertility in vitro, and live births), and there is not a linear relationship among these measurements. For example, although the plasma membrane may remain intact in various osmotic concentrations, motility seems to be much more sensitive to osmotic changes. Strain differences are evident: for example, in vitro fertilization of B6C3F1 oocytes was successful in 61% if B6C3F1 sperm were used, 17% if 129/J sperm were used, and only 3% if C57BL/6J sperm were used. In general, spermatozoa from hybrids can more successfully be frozen than spermatozoa of inbred mice.{4065}

There has been even less research on rat spermatozoa, but unpublished work from these authors suggests that there are similarities between mouse and rat freezing success.{4065}

Two special techniques are not yet in common use. The first is a method of cell vitrification in which the freezing rate is so rapid that intracellular "glass" is formed rather than ice, and warming is rapid enough so that the cytoplasm remains vitreous. This has been used in mouse and Drosophila embryos, but not sperm. The second is the use of intracytoplasmic sperm injection, a special technique that can employ even freeze-dried or non-viable sperm. This has successfully produced live mice in one laboratory at the University of Hawaii.{4065}

It is possible to "rescue" a valuable mouse strain by simply collecting sperm post-mortem. Success (live births) have been reported from spermatozoa collected from mice that had been dead for 24 hours and kept at 22°C, as well as from mice that had been refrigerated for 7 days.{4497}

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Cryopreservation of ovarian tissues and oocytes

Although embryos have been successfully frozen on a practical basis from several species (including the mouse), freezing of oocytes and tissue pieces has been more difficult. Mouse oocytes have been successfully frozen, with more success than those of other species.{4067}

Adult mouse and rat ovaries are 2-5 mm³. The cortex contains the follicles and corpora lutea, while the medulla consists of blood vessels and lymphatics in connective tissue. A primordial follicle consists of an oocyte surrounded by one layer of flattened cells. As maturation progresses the diameter increases and steroid hormones are produced. The surrounding cells increase in number and vascularization. Stages include secondary and tertiary follicles and the mature Graafian follicle. Both metabolic and structural integrity must be preserved during freezing.{4067}

The oocyte is the largest mammalian cell. The oolemma (cell membrane) is surrounded by the zona pellucida and several layers of cumulus cells. Immature oocytes are arrested in prophase of the first meiotic division; they have a distinct large nucleus called the germinal vesicle. After gonadotropin stimulation the germinal vesicle breaks down, a metaphase plate forms, and the oocyte is arrested at metaphase of the second meiotic division (or MII) characterized by the presence of a polar body. Cytoskeletal elements such as microtubules and microfilaments and cortical granule vesicles in the cytoplasm are important in the maturation process. If the meiotic spindle is disrupted by the freezing process, chromosomal abnormalities often occur.{4067}

Ovarian tissue cryopreservation and subsequent transplantation have been studied since the 1950s, but it languished until the 1990s because other techniques had not yet been developed such as cryoprotective agents. Presently, most studies use 1.5M DMSO and slow cooling followed by storage in liquid nitrogen. Mice and rats have been used to study autograft and allograft transplantation of frozen ovarian tissue; in 1994 a lamb was born after a successful autograft. Xenotransplantation of frozen tissues has been studied in immunodeficient mice, and yields information on in vivo follicle development. Xenotransplants from sheep, cats, marmosets, elephants, humans and most recently cows have been studied. Problems that occur with tissue transplants include poor heat and mass transfer, poor cryoprotectant permeation, and poor post-transplant vascularization. A significant loss of viable follicles occurs after thawing and transplantation.{4067}

A satisfactory technique for preserving valuable mouse strains has been superovulation and freezing of embryos. However, since not all strains superovulate well, other methods are being studied. Both adult and fetal cryopreserved ovaries can be used to restore fertility in mice, although litter size is small. Advantages are that only a single step is required (freezing the ovary vs. embryo transfer or in vitro fertilization). Ovarian tissue freezing in mice is considered an adjunct for genome banking.{4067}

Whole ovaries have been successfully transplanted after death of the donors, when kept at room temperature for up to 2 hours. This was a controlled experiment in which the donors were the healthy homozygous transgenic littermates of the nontransgenic recipients, age 7 weeks. The purpose of the experiment was to determine how long after the death of a valuable mouse the ovaries could be surgically transplanted into a suitable recipient and still result in live births.{4497}

As usual, rat techniques for tissue cryopreservation lag behind. Survival of preserved ovaries is poor, but there have been successes. Current issues relate to development of better cryopreservation protocols and the use of immunodeficient rats such as Hsd:RH-rnu, athymic nude rats or New Zealand nude rats.{4067}

Oocyte preservation has been studied extensively in the mouse. Oocytes seem to be particularly sensitive to non-physiological conditions. They have a complex subcellular system of organelles that function in spindle morphogenesis and organelle movement, and there are important interactions with somatic cells that must be maintained. Two stages of maturation (the germinal vesicle and MII) have been tried. The germinal vesicle stage is usually better because damage to the spindle is avoided and aneuploidy is not as likely. Other factors to consider are zona hardening (which should be avoided prematurely and is related to cortical granule vesicles) and transient cytoplasmic calcium concentration changes.{4067}

Preservation of primordial follicles may be a good alternative to oocyte preservation. Primordial follicles are smaller, arrested in prophase I of meiosis and do not have a zona pellucida, cortical granules or spindle fibers which are all sensitive to the effects of freezing. Preliminary studies in mice are leading to the concept of complete in vitro development of primordial follicles, even to the stage of in vitro fertilization. This would have great implications for human medicine.{4067}

In humans, banking of ovarian tissue is important because human oocytes have poor survival rates. Xenotransplants of human ovarian tissue into SCID mice kidney capsules do better when FSH is given to stimulate follicular development.{4067}

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Cryopreservation of embryos for rederivation

NIH established the Animal Genetic Resource in 1979 as a stock center of laboratory animal models. The bank contains 300,000 rat, mouse and rabbit embryos, some of which serve as the sole repository of the preserved strains. To preserve all these embryos, the response of the animal to superovulation or estrous synchronization are critical. The animal's genotype affects its ovulation rate in response to gonadotropins, ability of the embryo to develop in vitro, and the rate of development and quality of the embryos. In order to preserve a strain, a bank of 500 embryos is generally needed. The assumption is that only 10% will be successfully turned into live births if and when needed, an assumption which is conservative but shown to work well.{4068}

Embryos are collected from rats and mice in different ways. Rats are estrous-synchronized using GnRH-a, then paired with a fertile male 2 days later. Embryos are flushed out of excised oviducts and uterine horns on the morning of the 10th day after GnRH, and those at the normal 8-cell to blastocyst stage are cryopreserved in straws. After thawing, the embryos are transferred to the oviducts of pseudopregnant or pregnant N:NIH recipients. An average of 8 embryos is transferred to each oviduct on the afternoon of the day mating was confirmed.{4068}

Mice are superovulated with eCG and hCG, then paired with the appropriate male. Embryos are flushed from excised oviducts and uterine horns 65-70 hours after hCG and judged sound if they are at the 8-16 blastomere stage. After freezing and thawing, they are transferred into pseudopregnant females, usually B6D2F1 or FVC3F1 strains that have been paired with similar vasectomized males. {4068}

A model is considered to have been successfully banked if (1) a total of 500 embryos carrying the gene of interest are cryopreserved, (2) pups have been rederived from the banked embryos, (3) rederived pups exhibit the desired genotype and (4) rederived pups have themselves been bred and produced live pups. {4068}

For rats, somewhere between 50-600 donor females are needed to successfully preserve the model. Most require <100 embryo donors. An average of 71% of the females yield embryos. The mean number of embryos recovered is 8 (4-10). Variation is due to differences in the efficiency of estrous synchronization, mating failure due to infertility or behavior, and anatomical abnormalities (i.e. ipsilateral gonadal agenesis of ACI/N).{4068}

For mutant mice, three strategies can be used: (1) homozygous male and female; (2) unaffected female x homozygous male; or (3) unaffected female x heterozygous male. In case (3), 1,000 embryos need to be banked to ensure that 500 will carry the gene. A range of 23-250 donor females is needed, with most requiring <75 donors to obtain the 500 embryos. Variation is due to the number of females yielding embryos, mean number of embryos per female, and genotype. Highest producing genotypes are C57BL/6N or FVB/N inbred backgrounds.{4068}

Successful births occur in approximately 27% of rats and 23% of mice.{4068}

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Comments? Send an email to janet.rodgers@vet.ox.ac.uk