X-Message-Number: 17076 From: Date: Tue, 24 Jul 2001 00:01:18 EDT Subject: Suspended Animation of Cells--Part II Cryonet: To follow is the second section of a reprint of a cryogenics lab manual used for the long-term storage of cells. The first section appeared in yesterday's Cryonet edition. An unfortunate typographical error appeared in yesterday's section where the word "neither" was substituted for the word "rather" in the following sentence regarding the efficacy of cryopreservatives with eukaryotic cells (e.g., mammalian cells): "These agents have little effect on the damage caused by fast freezing (intercellular ice crystal formation), but rather [not "neither" as I inadvertently transposed/mistyped] prevent or lessen the damage caused by slow freezing (dehydration and shrinkage) (8)." I will attempt to better proof the following and subsequent reprint posts for the: GENERAL GUIDE FOR CRYOGENICALLY STORING ANIMAL CELL CULTURES by John A Ryan [Note: Most Cryonet readers might have little interest in the sections titled "STORAGE VESSELS" and "LABELING AND RECORDKEEPING" and may wish to skip these particular sections to follow] QUOTE 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 of 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 off 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 or these cultures are then examined microscopically and tested by direct culture for the presence of bacteria, yeast, fungi, and mycoplasmas. Mycoplasmas present a special problem since they can be found in cultures at very high concentrations (up to 10*8 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 dissociation 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 or 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 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, ethylene glycol and polyvinyl pyrrolidone (7). However, dimethysulfoxide (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 (5 mL). CAUTION: Take special care to avoid contract with solutions containing DMSO. It is a very powerful polar solvent capable of rapidly penetrating intact skin and carrying in with it harmful contaminates 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 4C 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 (5 mL) 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) 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 manufactures' 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 polypropylenes) 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 1). 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 2). 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 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 -1C to -3C 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 umber 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. Insulated cardboard or polystyrene foam boxes are commonly used as freezing chambers in ultracold freezers. These homemade devices work well with many cell lines but do not always give controlled, reproducible or uniform cooling. As a result, their may be serious differences in viability among the vials upon thawing. This homemade approach is not recommended for valuable or irreplaceable cultures. No matter which cooling method is used, transfer from the cooling chamber or device to the final storage location must be done quickly to avoid warming of the vials. Use an insulated container filled with dry ice or liquid nitrogen as a transfer vessel to ensure that the cells remain below -70C. UNQUOTE [More tomorrow. I note that the last sentence above appears to specify -70C as presumably being a "lab tested" maximum temperature limit for keeping cells viable for future rewarming. For this reason, it may be worth considering the addition of some amount of dry ice to liquid nitrogen storage dewars for "insurance" as it might "buys time" in the event of unit failure (e.g., lost vacuum in hard vacuum LN2-based systems). I do not know if this is common practice or not] David C. Johnson, Raleigh, NC Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=17076