Procedures for Cryopreserving Cells

Techniques are available for the cryopreservation of most isolated cell types, microorganisms, and small embryos. These procedures allow banking of cells to prevent any genetic drift of a culture line, to renew a culture line if it becomes contaminated, for autologous or allogeneic transplantation, for in vitro fertility treatments and animal husbandry, and many other applications. Although the mechanisms of cryoinjury are not yet fully understood, cryopreservation techniques have been optimized for many cell types experimentally.

Preparation of Cells

Maintenance of a culture line requires strict attention be paid to minimizing the number of passages of the seed stock. This is a collection of frozen samples that are as close to the original culture as possible to minimize genetic changes that usually accumulate with each passage in a culture. The seed stock is maintained separately from the working stock, and is only used to renew the culture when problems arise or there is a loss of confidence in the fidelity of the culture. One the seed stock becomes depleted, the last seed vial is used to prepare a second seed stock, again with minimal passages after thawing. In the long run, careful maintenance of a seed stock is much less expensive that starting a research program over from scratch, so it is worth doing well.

Cryopreservation of animal cells invariably requires a cryoprotective additive for appreciable survival after thawing. Glycerol and dimethyl sulfoxide (DMSO) are the most commonly used and are probably effective enough for most cell types. Although both glycerol and DMSO are relatively non-toxic (prolonged bathing in DMSO has been implicated in irreversible changes to the shape of the lens of the eye in dogs), glycerol is usually preferred. DMSO penetrates cells more quickly than glycerol but it smells bad; perhaps that casts a suspicion over its toxicity. For larger cells, especially protozoans, glycerol may be impractical due to its lower permeability.

The cryoprotective agent (CPA) is diluted to the proper concentration in the medium being used to suspend the cells. Since CPA's like DMSO and glycerol serve as a solvent for sodium chloride, it is important to keep the molar concentration of NaCl at the required level in the CPA solution. For example, 100 ml of a 1M solution of DMSO in MEM might be made as follows:

7.1 ml DMSO
1.9 ml dH2O
1.0 ml 10x PBS
90.0 ml 1x MEM

Since the molecular weight of DMSO is 78 g/mol and the density is 1.1 g/ml, we use 7.1 ml in 100 ml to get 1 mol/l. Diluting the 1 ml of 10x PBS to 10 ml by adding DMSO and distilled water (dH2O) makes 10 ml of solution with isotonic salt. Adding this to MEM to make up the remainder of the 100 ml keeps the salt concentration constant (and isotonic) while diluting the DMSO to 1 M.

Usually, the CPA is specified as a % (either weight/weight or volume/volume) that falls into the range of 5-10% and the resulting dilution of salt is ignored (most people don't consider this rocket science, so good enough is good enough). The CPA can be sterilized by passing it through a sterile 0.2 ml syringe filter (washed with CPA to maintain concentrations). The CPA must be allowed to equilibrate within the cells before freezing begins. If the CPA is added at room temperature or higher, then 15 minutes is long enough for most cells (DMSO penetrates roughly ten times faster than glycerol). With large cells and/or low temperatures, equilibration periods of up to an hour may be required. With CPA concentrations greater than 2M, both the rate of addition and the time of exposure can lead to cell death. To reach a final concentration of 3M DMSO, for example, at least two additions should be done, separated by several minutes (first to 1.5M, then to 3M). The cells should not be left in this solution for more than a couple of hours as there may be toxic effects that manifest themselves with time.

Most viruses can be frozen or freeze-dried as cell-free preparations. Some require the host cell to be viable, in which case cryopreservation must be tailored to the cell.

Microbes grown under aerated conditions show greater recovery following cryopreservation than those grown under non-aerated conditions. Cells should be suspended in the growth medium with CPA at a concentration of 107 cells/ml or greater. Spore forming fungi should have the spores suspended in growth medium + CPA and non-spore forming fungi should have mycelia suspended in growth medium.

Tissue culture cells should be suspended between 106 and 107 cells/ml in the medium with CPA. The harvesting procedure prior to cryopreservation should be as gentle as possible and use of a medium with methyl red will give an indication of changes to the pH. The cell line should be free of contaminants before cryopreservation.

Freezing and Storage Vessels

Freezing is usually carried out in small plastic vials with screw-caps or snap-lids. Plastic straws are also used for sperm cells. The plastic vials can be rapidly cooled or warmed without the lids coming loose or any damage accruing to the vial. For cells that require very rapid warming, flame-sealed glass ampules are sometimes used. Because of the danger of liquid nitrogen leaking into cryovials during storage, and the subsequent rapid expansion upon warming, appropriate safety measures should be taken when thawing vials stored in liquid nitrogen.

For most animal cells and protozoans, controlled rate cooling is required for survival. Just as nobody ever got fired for buying IBM (at least up until the mid 80's), the unwritten rule of cryopreservation is to cool at 1C/min. This works pretty well because the addition of a cryoprotectant spreads the optimal cooling rate out over a fairly wide range and a lot of animal cells have optimal cooling rates between 0.1 and 10C/min. Without any further optimization, however, the recovery is likely to be sub-optimal. If there are properties affecting cell survival that are expressed as a continuum within a cell population, sub-optimal recovery may alter the phenotype of the population, thus it is better to find the optimal cooling rate for a particular cell type when practical.


Optimal Cooling Rates for 3 Mammalian Cell Types.

The optimal cooling rate for most mammalian cells occurs closer to 1C/min than ova or red cells, and higher concentrations of cryoprotectant can spread the range out so that appreciable recovery can be had at cooling rates that are far away from the optimum.

Methods for freezing at controlled cooling rates:

Ice formation in a sample of bulk solution occurs through nucleation, a stochastic process. Without any nucleation sites, the cell sample might supercool dramatically before nucleation occurs. If this happens, the osmotic gradient produced during crystallization will cause intracellular freezing and lead to near-zero recovery. In order to avoid supercooling, some seeding process is usually required. If sterile conditions don't need to be maintained, then adding a bit of frost to the sample once it cools below the melting point is adequate. If the sample is sealed, however, then either the outside of the vial can be contacted by a cold surface (e.g. forceps cooled in liquid nitrogen), or the cooling apparatus can give a blast of chilling to ensure nucleation (e.g. a liquid nitrogen freezer reaches -5C and then quickly chills to -25C and then goes back up to -5C to continue controlled rate cooling).

Once ice forms, continued crystallization will produce the latent heat of fusion, quickly warming the sample to the melting point. The freezing apparatus should be programmed to hold the temperature after seeding so that the latent heat is allowed to dissipate fully. This can be monitored by putting a thermocouple in a sample (minus the cells) and finding out how long it takes for the sample to equilibrate with the freezer (i.e. if the freezer is set at -5C and the melting point of the sample is -1C, the sample will remain at -1C for several minutes while latent heat is still being produced).

Maintaining the sample at the ideal cooling rate becomes less important as the temperature drops. Usually, below -60C, the sample can simply be plunged in liquid nitrogen (or transferred to a storage freezer) with no further loss of viability.

Storage and Thawing

The temperature at which samples are stored affects the length of time that they can be effectively kept. The lower the storage temperature, the longer they may be kept. For liquid nitrogen storage (-196C), the limiting factor is the background radiation that may cause an accumulation of DNA alterations, thus a suitably shielded freezer provides indefinite storage (in the real-world, however, the limiting factor is the trustworthiness of the technician who is responsible for filling the nitrogen tanks over the weekend). Dry ice (-80C) or mechanical freezers that maintain temperatures of -80C are suitable for storage on a time scale of months, but some biochemistry can occur at these temperatures.

The packing system that is employed within the freezer determines how often samples are exposed to warm temperatures (and thereby putting the sample at risk of damage). Stacked boxes that are poorly labelled usually mean that someone will take out a whole pile of boxes and place them at room temperature while they sort through them looking for a particular sample. If this occurs on a daily basis, the integrity of all the samples becomes suspect even though the temperature alarm on the freezer never goes off. Within liquid nitrogen dewars, plastic cryovials can be snapped into aluminum canes, increasing the storage capacity as well as providing a convenient method for organizing samples. Care should be taken to prevent putting seed stock vials with working stock, so that the seed stock canes are only removed when retrieval is necessary.

Thawing should be performed as rapidly as possible, without letting the temperature exceed 37C. Microwave warming is usually a poor idea as there is a positive feedback loop between warm water and heating efficiency (warm water tends to absorb more microwave radiation and hence get even warmer) leading to local hot spots. This is amplified when most of the sample is in the frozen state. The best way to warm samples is by immersion in a 37C water bath with gentle agitation (or mixing of the water bath). To increase the rate of warming, the sample should be frozen in a vial with a high surface area to volume ratio (such as straws). Care should be taken to prevent contamination of the sample when opening the vial and this should be done in a biosafety cabinet. If the concentration of CPA is high, then the sample must be either diluted stepwise, or with a non-penetrating solute such as sucrose present, to prevent expansion induced lysis. It is customary to measure the cell recovery after thawing.

Handling after thawing should also be performed with care. There is always a strong possiblity that some or all of the cells have sustained a non-lethal injury during freezing and thawing. After thawing, the cells will be directing their energy toward the repair of this injury and may be more susceptible to rough handling. Procedures such as vigorous centrifugation and chemical stress should be minimized.

Safety

Besides optimizing the preservation of biological material, these procedures must also maintain the highest possible standards of safety for those carrying out the work. If the entire inventory of a freezer isn't known, then it should be assumed that pathogens are present. Handling of samples in the freezer should be done according to techniques suitable for handling biohazardous material. This is in addition to those precautions that are necessary to prevent frostbite when working with liquid nitrogen or dry ice. Removing vials from liquid nitrogen also presents the additional danger of explosion, if nitrogen has leaked into the vial. Some sort of shielding may be required when exposing these vials to warm temperatures.

Play safe!


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Document last updated Jan. 27, 1999.
Copyright © 1999, Ken Muldrew.