X-Message-Number: 17088 From: Date: Tue, 24 Jul 2001 21:57:49 EDT Subject: Suspended Animation of Cells--Part III Cryonet: To follow is the third and final section of a reprint of a cryogenics lab manual used for the long-term storage of cells. The first and second parts appeared in yesterday's and the day before's Cryonet edition. References cited in the guide are included at the end of the post for Part I. QUOTE: GENERAL GUIDE FOR CRYOGENICALLY STORING ANIMAL CELL CULTURES by John A. Ryan VII. CRYOGENIC STORAGE Only freezers capable of continually maintaining temperature below -130C should be considered for long-term cryogenic storage. Although most liquid nitrogen-cooled freezers and some special designed mechanical freezers meet this requirement, most cell culture laboratories prefer liquid nitrogen freezers (See Figure 3). The final choice is often based on the availability of a reliable supply of liquid nitrogen, the storage capacity required, and the size of the budget. Liquid nitrogen freezers permit storage either in the vapor phase above the liquid at temperature between -140C and -180C, or submerged in the liquid at a temperature below -196C. Using vapor phase storage greatly reduces the possibility of leaky vials or ampules exploding during removal. However, since the amount of liquid nitrogen is the freezer is reduced to provide space for vapor phase storage, the freezer's holding time (the period it can maintain its storage temperature without adding more liquid nitrogen) is also reduced. This lowers the freezer's margin of safety and requires more frequent monitoring and filling. Give careful consideration to these safety issues when deciding upon a storage method. Frequently check nitrogen levels in freezers; a schedule should be established and strictly adhered to. Nitrogen evaporation is dependent of both the degree of use and the static holding time of the freezer. Sudden, unexplained increases in the evaporation rate may signal damage to the insulation or other problems with the freezer and must be carefully investigated. Avoid frost or ice buildup around freezer openings; this increases the nitrogen evaporation rate and can cause elevated temperatures in the upper portion of vapor phase freezers. Audible alarm systems for detecting low liquid nitrogen levels are available to provide additional safeguards; however, they provide a false sense of security if not monitored 24 hours a day. VIII. THAWING CAUTION: Always use appropriate safety equipment when removing vials and ampules from liquid or vapor phase nitrogen freezers. A full face shield, heavy gloves and lab coat are strongly recommended for protection against exploding vials or ampules. Remove the vial or ampule from its storage location and carefully check both the label and storage record to ensure that it is the correct culture. Place the vessel in warm water, agitating gently until completely thawed. Rapid thawing (60 to 90 seconds at 37 C) provides the best recovery for most cell cultures; it reduces or prevents the formation of damaging ice crystals within cells during rehydration. IX. RECOVERY Since some cryoprotective agents may damage cells upon prolonged exposure, remove the agents as quickly and gently as possible. Several approaches are used depending on both the cryoprotective agents and characteristics of the cells. Most cells recover normally if they have the cryoprotective agent removed by a medium change within 6 to 8 hours of thawing. Transfer the contents of the ampule or vial to a T-75 flask or other suitable vessel containing 15 to 20 milliliters of culture medium and incubate normally. As soon as a majority of the cells have attached, remove the medium containing the how diluted cryoprotective agent and replace with fresh medium. For cells that are sensitive to cryoprotective agents, removing the old medium is easily accomplished by gentle centrifugation. Transfer the contents of the vial or ampule to a 15 mL centrifuge tube containing 10 mL of fresh medium and spin for 5 minutes at 100 x g, discard the supernatant containing the cryoprotectant and resuspend the cell pellet in fresh medium. Then transfer the cell suspension to a suitable culture vessel and incubate normally. When glycerol is used as the cryoprotectant, the sudden addition of a large volume of fresh medium to the thawed cell suspension can cause osmotic shock, damaging or destroying the cells. Use several stepwise dilutions with an equal volume of warm medium every 10 minutes before further processing to give the cells time to readjust their osmotic equilibrium. X. PROBLEM SOLVING SUGGESTIONS Viability problems associated with cryogenic storage are usually noticed soon after cultures are thawed and plated. There are four major areas where problems occur: 1) During harvesting and processing of the cells. Problems may be caused by excessive exposure of the cells to dissociating agents; using a cryoprotective agent that is toxic; or allowing high density cell suspensions to remain too long at room temperature or at a pH that is too basic. 2) During the cooling (freezing) process. Excessive cell damage and reduced culture viability often result from using a cooling rate that is too fast or too slow, or when the cooling process is temporarily interrupted. Not using a suitable cryoprotective agent at an appropriate concentration will also result in viability problems. 3) During cryogenic storage. Culture viability is often reduced when vials are allowed to warm up during transfer to the freezer, or if the repository temperature is not consistently maintained at appropriate cryogenic temperatures. 4) During thawing and recovery. Problems arise when the thawing process is two slow or the cryoprotectants are improperly removed (see above). These viability problems can often be corrected by using the following technique to identify the stage in the freezing process where the problem originates. Harvest enough cells to prepare at least four vials. Then remove a sample of cell suspension, equivalent in cell number to that which will be placed into the vials, and immediately place it into a culture vessel with an appropriate amount of medium and incubate. This culture will be used as a control to compare with the cultures set up in the remaining steps. Next add the cryoprotective agent to the remaining cells and divide among three vials. Place one vial at 4 C for one hour. Then remove the cells from the vial, process as though they had just been thawed from the freezer, and plate in medium as above. This culture will be compared with the control culture to determine if there are any problems associated with the slow cooling process. The remaining vial is then transferred to the cryogenic freezer and stored overnight before being thawed and processed as above. This culture will be compared with the control culture to determine if there are any problems associated with the cryogenic storage conditions. If additional vials of cells are available, several different recovery techniques should be used to determine if the recovery technique is the source of the problem. By comparing all of the cultures to the original culture, it should then be possible to determine at which state of the freezing process the problem occurred. Once this is known, the information presented in this guide and its references should be enough to eliminate the problem. XI. MANAGING A CELL REPOSITORY The effort and expenses of managing a repository should be kept in line with the value of the cultures stored within it. This value is determined by answering two questions: How much time, money and effort is already invested in these stored cell cultures? And, what are the consequences of losing them? Cultures that are easily replaced through other labs or commercial sources may not require special efforts, but unique cultures, such as hybridomas and other genetically engineered cells, are irreplaceable and require that special efforts be made to ensure their safety. The answers to these questions will help determine just how extensive and thorough your efforts should be. Next, identify the potential problem areas that can cause the loss of these cultures. Some of these areas, such as vessel selection, recordkeeping, labeling, freezer monitoring, storage conditions, and quality issues (contamination and species identity), have already been discussed in this guide. Decide what steps are necessary to eliminate or minimize these problems. Split irreplaceable or extremely valuable cultures among several freezers, with at least one freezer in a separate location to protect against fire or other natural disasters. Colleagues in other labs or buildings may be able to provide good backup storage, especially if a reciprocal arrangement is made for them. One final step remains; plan ahead for emergencies! One of the most serious and unexpected emergencies is the failure of a cryogenic freezer. Careful monitoring of the liquid nitrogen level or charting the temperature may give an early warning that failure is occurring, but middle of the night failures can and do happen. Have plans prepared in advance to deal with freezer failure and other problems. If these involve a colleague's equipment, get permission and make all necessary arrangements in advance--last night phone calls are usually not appreciated. This information has been compiled to provide a guide for better understanding of the cryogenic preservation process. UNQUOTE David C. Johnson, Raleigh, NC Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=17088