General Guide for Cryogenically Storing Animal Cell Cultures

Introduction

Maintaining healthy, growing cell cultures is a demanding task made more difficult by the ever-present risk of their loss through accidents or contamination. In addition, actively growing cell cultures are not static but, like all populations of microorganisms, subject to age-related or environmentally-induced changes which can result in their ongoing evolution and potential loss.

These problems are reduced by using cryogenic preservation to stop biological time for cell cultures, effectively putting them into true suspended animation. This concept, long a favorite ploy of science fiction writers and movie producers, has been a reality since the important discovery by Polge, Smith and Parkes (11) in 1949 that glycerol prevents injury to cells caused by freezing. Many cook book-style protocols are now available for freezing cells and these procedures usually perform well ( 3,6, 13,14,15,16 ). It is essential, however, when problems arise or protocol adaptations and improvements must be made, that the underlying concepts on which they are based are well understood. This guide examines both the basic theoretical concepts and practical aspects necessary for successfully freezing animal cells and managing a cell repository.

Advantages of Freezing Cell Cultures

Once successfully frozen and stored, cell cultures require little time and effort for their maintenance. The only real cost is the expense of maintaining an ultracold (-130� or lower) mechanical freezer or liquid nitrogen supply. This limited expense compares very favorably with the time, effort and substantial cost of the media and supplies necessary for maintaining actively growing cultures, or for the cost of obtaining a new culture from a repository. Frozen cultures provide an important backup supply for replenishing occasional losses due to contamination or accidents and provide the assurance of a homogeneous culture supply. Cellular changes or alterations occur in all actively growing populations. These changes often result in the loss of important characteristics during evolution of the cultures thereby introducing unwanted variables into long-term experiments. Cryogenically preserved cultures apparently do not undergo any detectable changes once they are stored below -130� (1,8) . Therefore, the biological effects of in vitro cellular aging and evolution may be minimized by frequently returning to frozen stock cultures, allowing ongoing long-term culture experiments to be successfully completed without these unwanted variables. Frozen cultures also provide a valuable baseline against which future experimentally-induced changes may be compared or measured.

General Events During Cell Freezing

To understand why freezing protocols work, it is necessary to examine both the intracellular and extracellular events occurring in animal cell cultures during the freezing process (2,4,8) . Initial cooling from room temperature to 0� slows cellular metabolism, rapidly disrupting active transport and ionic pumping. Usually this disruption does not result in cellular damage if the culture medium is osmotically balanced. As cooling continues (0?to -20�) ice crystals begin to form in the extracellular environment which increases the solute concentration of the culture medium. As a result, water begins to move out of the cells and into the partially frozen extracellular medium, beginning the process of cellular dehydration and shrinkage.

When the cooling process is rapid, intracellular ice crystals form before complete cellular dehydration has occurred. These ice crystals disrupt cellular organelles and membranes and lead to cell death during the recovery (thawing) process.

When the cooling process is slow, free intracellular water is osmotically pulled from the cells resulting in complete cellular dehydration and shrinkage. This can also cause cellular death but there is little agreement on the mechanisms involved. The physical stresses of cellular shrinking may cause some damage resulting in irreparable membrane loss and cytoskeletal and organelle disruption. Damage may also be caused by the high concentrations of solutes in the remaining unfrozen extracellular medium (essentially a brine solution). These solutes attack cells both externally and internally, resulting in membrane damage, pH shifts and general protein denaturation.

Figure 1:   Effects of Freezing Rates on Cells

However, when the cooling rate is slow enough to prevent intracellular ice formation, but fast enough to avoid serious dehydration effects, cells may be able to survive the freezing and thawing process. This survival zone or window is readily observed in many bacteria and other prokaryotes, but for most eukaryotic cells it is nonexistent or very difficult to find without using cryoprotective agents. These agents have little effect on the damage caused by fast freezing (intracellular ice crystal formation), but rather prevent or lessen the damage caused by slow freezing (dehydration and shrinkage) (8) .

The final storage temperature is also critical for successful cryopreservation. To completely stop biological time, storage temperatures must be maintained below -130�, the glass transition point below which liquid water does not exist and diffusion is insignificant. While many cell cultures are successfully stored at -70� to -90� for months or even years,- biological time is not stopped, only slowed, and cellular damage or changes will accumulate.

Storage in liquid nitrogen at -196� effectively prevents all thermally driven chemical reactions. Only photo-physical effects caused by background ionizing radiation still operate at this temperature. Thousands of years are estimated to be necessary before background radiation will have a noticeable effect on cryopreserved cultures (2,8).

Practical Aspects of Cell Freezing

Under the best of circumstances the process of freezing remains stressful to all cell cultures. It is important that everything possible be done to minimize these stresses on the cultures in order to maximize their subsequent recovery and survival. The following suggestions and recommendations are designed to augment the protocols referred to earlier.

I. Cell Selection First ensure that the cells are in their best possible condition. Select cultures near the end of log phase growth (approximately 90% confluent) and change their medium 24 hours prior to harvesting. Carefully examine the culture for signs of microbial contamination. Facilitate this by growing cultures in antibiotic-free medium for several passages prior to testing. This allows time for any hidden, resistant contaminants (present in very low numbers) to reach a higher, more easily detected level. Samples of these cultures are then examined microscopically and tested by direct culture for the presence of bacteria, yeasts, fungi, and mycoplasmas.

Mycoplasmas present a special problem since they can be found in cultures at very high concentrations (up to 108 organisms per milliliter of medium) without any visible effects or turbidity. As a result, as many as 20% of all animal cell cultures are contaminated by these ubiquitous but unseen organisms. Although special efforts are required to detect mycoplasmas, the serious consequences of their presence makes testing frozen culture stocks absolutely essential (9,12) .

Check for both the identity of the cultures and the presence of any expected special characteristics. Monitor cell identities by karyology and isoenzyme analysis, ensuring that they are, at the very least, the correct species (10 ).

II. Cell Harvesting Start with the standard harvesting procedure generally recommended for the culture and be as gentle as possible. Remove all dissociating agents by washing or inactivation (especially important when using serum-free medium). Centrifugation, when absolutely necessary, should only be hard enough to obtain a soft pellet; 100 x g for 5 to 6 minutes is usually sufficient. To ensure uniformity of the final frozen stock, pool the contents of all harvested culture vessels. This also makes it much easier to perform essential quality control testing for microbial contamination and culture identity.

Count and then dilute or concentrate the harvested cell suspension to twice the desired final concentration, which is usually 4 to 10 million viable cells per milliliter. An equal volume of medium containing the cryoprotective agent at twice its final concentration will be added later to achieve the desired inoculum. Keep the cells chilled to slow their metabolism and prevent cell clumping. Avoid alkaline pH shifts by gassing with CO2 when necessary.

III. Cryoprotection As mentioned earlier, cryoprotective agents are necessary to minimize or prevent the damage associated with slow freezing. The mechanisms providing this protection, although not completely understood, appear to work primarily by altering the physical conditions of both the ice and solutions immediately surrounding (external to) the cells. Permeation of the cells by cryoprotectants does not appear to be necessary for their proper functions (4) . Remember, protection against fast freezing damage (internal ice formation) is not provided by these agents, but rather by careful control of the freezing rate. A wide variety of chemicals provide adequate cryoprotection, including methyl acetamide, methyl alcohol, ethyleneglycol and polyvinyl pyrrolidone (7 ). However, dimethylsulfoxide (DMSO) and glycerol are the most convenient and widely used. Many of these agents, although providing excellent cryoprotection, have toxic side effects on cultures making their use difficult.

DMSO is most often used at a final concentration of 5-15% (v/v). Always use reagent or other high purity grades that have been tested for suitability. Sterilize by filtration through a 0.2 micron nylon membrane in a polypropylene or stainless steel housing and store in small quantities (5mL). CAUTION: Take special care to avoid contact with solutions containing DMSO. It is a very powerful polar solvent capable of rapidly penetrating intact skin and carrying in with it harmful contaminants such as carcinogens or toxins. Some cell lines are adversely affected by prolonged contact with DMSO. This can be reduced or eliminated by adding the DMSO to the cell suspension at 4� and removing it immediately upon thawing. If this does not help, lower the concentration or try glycerol or another cryoprotectant.

Glycerol is generally used at a final concentration of between 5 and 20% (v/v). Sterilize by autoclaving for 15 minutes in small volumes (5mL) and refrigerate in the dark. Although less toxic to cells than DMSO, glycerol frequently causes osmotic problems, especially after thawing. Always add it at room temperature or above and remove slowly by dilution.

High serum concentrations may also help cells survive freezing. Replacing standard media-cryoprotectant mixtures with 95% serum and 5% DMSO may be superior for some overly sensitive cell lines, especially hybridomas.

Add cryoprotective agents to culture medium (without cells) immediately prior to use to obtain twice the desired final concentration (2X). Mix this 2X solution with an equal volume of the harvested cell suspension (also 2X) to obtain the inoculum for freezing. This method is less stressful for cells, especially when using DMSO as the cryoprotectant.

IV. Storage Vessels After the cryoprotective solution is mixed with the cell suspension, the resulting inoculum is added in small aliquots (usually 1 to 2 milliliters) to each storage vessel. Due to the extremely low temperatures encountered during cryogenic storage, not all vessel materials or designs are suitable or safe. Many materials become very brittle at these temperatures; vessels made from them may shatter or crack during storage or thawing. Carefully check the vessel manufacturers' recommendations on proper selection and use.

Also important is selecting the sealing system or cap design used to maintain the integrity of the vessel, especially for storage in liquid nitrogen. If these vessels leak during storage (as many do) they will slowly fill with liquid nitrogen. When they are eventually returned to room temperature, the liquid nitrogen quickly vaporizes causing a rapid pressure buildup. The vessels may then violently blow off their caps or explode to vent the pressure and release their contents into the atmosphere. This is a very dangerous situation, especially if the vessels contained pathogenic organisms or potentially toxic or harmful substances. Storage above liquid nitrogen to reduce these potential hazards is strongly recommended in such situations.

Two types of vessels are commonly used for cryogenic storage: heat-sealable glass ampules and plastic (usually polypropylene) screw-capped vials. Both are available in a variety of sizes (1 to 5 milliliter capacity) although the smaller sizes are preferred for cryogenic storage (See Figure 2).

Because of sealing and labeling problems, glass ampules are no longer widely used in cell culture laboratories. Invisible pinhole leaks may occur in vials during the sealing process; if these are later stored submerged in liquid nitrogen, they may explode when removed for thawing. Pinholes can usually be detected before freezing by immersing sealed ampules for 30 minutes in a chilled solution of 70% ethanol containing 1% methylene blue. This solution will rapidly penetrate and stain any leaky ampules; after rinsing with water, defective ampules are then easily detected and discarded.

Due to their greater safety and convenience, plastic vials have largely replaced glass ampules for cryogenic storage. The wide variety of styles and special features like printed marking areas and colored caps for easier identification also add to their popularity.

Several cap styles are available, some with the internally-threaded stopper, and others with externally-threaded designs which help minimize contamination (See Figure 3).

V. Labeling and Recordkeeping Providing for long-term location and identification of frozen cultures is the most frequently overlooked area of cryogenic storage. A cryogenic cell repository is expected to outlast the laboratory workers who contribute to it, but poorly maintained or missing inventory records, and improperly or illegibly labeled vials and ampules may prevent this, especially after the people responsible have gone.

Labels must contain enough information to locate the appropriate records; usually the culture's identity, date frozen, and initials of the person responsible are sufficient. Most plastic vials have printed marking spots or areas for easy labeling. On vials and ampules without marking spots use cloth labels with special adhesives formulated for cryogenic conditions.

Special ceramic-based inks are available for labeling glass ampules. These are applied prior to filling and then baked onto the glass, usually during dry heat sterilization. Permanent marking spots can be applied on glass ampules with white nail polish. A laboratory marking pen is then used to write on the spot once it has dried.

No matter which labeling method is chosen, use special care to check its permanency under cryogenic conditions. Some marking spots, inks, and labels may flake off or fade during long-term storage; a trial run of at least several weeks is recommended.

Fully detail in the records the culture's storage conditions, including all of the following information: culture identity, passage or population doubling level, date frozen, freezing medium and method used, number of cells per vial, total number of vials initially frozen and the number remaining, their locations, their expected viability and results of all quality control tests performed (sterility, mycoplasma, species, karyotype, etc.). Additional culture information, especially their origin, history, growth parameters, special characteristics, and applications, is also helpful and should be included whenever possible.

Make special efforts to keep all records up to date and to ensure everyone in the facility is properly using them. Use pre-printed forms to make the information recording process easier and more likely to be completed. Keep updated, duplicate copies of all critical records in a safe place removed from the laboratory area to guard against their accidental loss or destruction. This is especially important if a computer-based recordkeeping system is used; a current back up copy should always be maintained in addition to the information stored in the computer.

VI. Cooling Rate The cooling rate used to freeze cultures must be just slow enough to allow the cells time to dehydrate, but fast enough to prevent excessive dehydration damage. A cooling rate of -1� to -3� per minute is satisfactory for most animal cell cultures. Larger cells, or cells having less permeable membranes may require a slower freezing rate since their dehydration will take longer.

The best way to control cooling rates is using electronic programmable freezing units. Although expensive, they allow precise control of the freezing process, give very uniform and reproducible results, and can freeze large numbers of vials or ampules. Most units are available with chart recorders for a permanent record of the cooling process.

There are a variety of mechanical freezing units that provide adequate control of the cooling rate and are relatively inexpensive. Some units use racks designed to hold vials at predetermined depths in the neck of a liquid nitrogen freezer. The cooling rate is dependent on the total number of vials and the depth at which the rack is placed. Another design uses an alcohol filled metal canister containing a rack with a capacity of 24 vials. The filled canister is placed in an ultracold mechanical freezer where the alcohol acts as a bath to achieve more uniform heat transfer and cooling. After freezing 4 to 5 hours, the vials are removed from the canister and transferred to their final storage locations.