Preservation, Storage And Transportation Of Living Tissues & Cells
Now that very many cell-strains have been isolated, one of the major problems confronting some laboratories is the maintenance of the large number of cultures necessary to carry several strains simultaneously. A further difficulty is the tendency for cell-types to change after they have been kept in a state of rapid proliferation for some years. Similar problems have been encountered in the handling of micro-organisms and the answers which have been suggested for the maintenance and storage of cells are very similar to those, which have been adopted by bacteriologists. All the methods depend on the maintenance of the cells at reduced temperatures but there are some differences in technique depending on the temperature used.
Cells can be maintained at slightly reduced temperatures, refrigerator temperatures or very low temperatures (- 70° to - 190oC.). Room temperature and refrigerator storage are someÂtimes used for short-term maintenance but for long-term storage very low temperatures are necessary. In the deep-frozen state metabolism is, of course, suspended completely.
Maintenance at slightly reduced temperatures
Most mammalian cells and tissue fragments will survive indefinitely at 30°C. provided the medium is renewed when required. By this means the frequency of feeding can be greatly reduced. Thus, compared with 37°C. the intervals between feeding can be about three times as long.
The temperature can be still further reduced and many cells will survive unharmed at about 20°. At this temperature very infrequent renewal of medium is required, although the cells rarely continue to behave quite normally if their normal incubation temperature is of the order of 37°. They tend to round up and may leave the glass and sometimes they become packed with fat droplets. However, if the medium is renewed and the temperature raised to normal once more the cells very rapidly return to normal after a short lag period.
Maintenance at refrigerator temperature
Some reports have appeared concerning the storage of tissue in the refrigerator (2 - 6o). It would appear that sheets of cells and single cells do not survive well for more than a few days in these conditions but that tissue fragments will survive for several weeks in a nutrient medium. This may be due to the high oxygen tension, which can develop in the medium. The main practical use of this method is in the preservation of surviving tissue before explanation, usually only for a few days. The tissue should be cut into pieces of a few cubic millimeters and stored in a nutrient medium.
Preservation by freezing
In several fields advantage has been taken of the observation that deep-frozen tissue may remain viable. This principle was applied to the storage of viruses and bacteria but when it was applied to animal cells they did not survive. At first this was thought to be due to laceration of the cell membranes by ice crystals but more recent evidence suggests that the cause may be osmotic changes which give rise to irreversible changes in lipoprotein complexes resulting in splitting of membranes within the cell. In any event the answer to the preservation of living animal cells proved to be the addition of a substance such as glycerol or ethylene glycol to the medium and slow freezing. The technique was worked out in some detail, by Smith who demonstrated the survival of ovarian granulosa cells after deep freezing. Scherer and Hoogasian demonstrated its effectiveness with some stock lines of cultured cells and since then it has been widely applied.
The following principles summarise generally accepted current practice.
- The cells should be in a healthy state before freezing.
- They should be suspended in growth medium containing glycerol or dimethyl sulphoxide (5-15 per cent.) shortly before freezing, and sealed in a gas-light ampoule.
- Freezing to -70° should be controlled over a period of about an hour or preferably longer. A cooling rate of one degree a minute is recommended.
- Storage temperature should be maintained at - 70° or lower.
- Thawing should be rapid (2 - 3 minutes).
Details of the technique are given below.
Equipment
1. Refrigerator.-Cells may be stored in a low-temperature deepÂfreeze cabinet operating at a temperature in the region of -70° C. (e.g. Revco deep-freeze) or in a dry-ice chest at about the same temperature. However, viability tends to be lost over a period of months at these temperatures. At temperatures of less than - 90oC no progressive loss of viability can be detected over very long periods and these very low temperatures are therefore recommended. Liquid nitrogen is usually used as the refrigerant. It has a temperature of - 196°C. and the overlying vapour in a well-insulated vessel gives temperatures within the range of -150° to - 180°. Suitable containers (often called liquid nitrogen refrigerators) are manufactured by several companies. The Linde division of the Union Carbide Company manufactures a range of suitable vessels, including a small one with a capacity of 972 ampoules, which is suitable for a relatively small laboratory.
2. Devices for recovery of cells.-Cells are usually stored in 1 ml. ampoules in liquid nitrogen refrigerators. Most methods for marking ampoules are unsatisfactory. Hence, it is important that records be kept meticulously and that ampoules be stored systematically. The liquid nitrogen refrigerator amounts to a Dewar flask, similar to a large Thermos flask. The contents are distributed to a series of containers, usually deep cans, like pipette canisters, which can be recovered individually by some device or another. Within these containers the ampoules are held either in tubes or clipped to 'canes'. A cane is a metal rod bearing a number of clips to which individual ampoules can be attached. Each cane or tube carries a reference number.
3. Cooling devices.-In order to ensure a steady slow rate of cooling several devices have been developed. The simplest can be made from a block of expanded polystyrene insulating material about four inches thick. This is sliced down the middle to give a top and bottom half and matching cavities are then made in each half with a large cork borer to hold ampoules. Ampoules are placed in the cavities, the two halves held together with an elastic band, and the whole device placed in a Revco deep-freeze cabinet for two hours. The cells are transferred direct to the liquid nitrogen refrigerator.
A similar device, invented by Greaves and his colleagues, consists of a modified stopper for a liquid nitrogen refrigerator and operates on exactly the same principle. It is available from the Linde company.
For meticulous large-scale work programmed cooling devices have been developed but these are not necessary for the ordinary laboratory. Simpler methods than the ones mentioned above are also available. For instance, reasonably good results can be obtained by packing the ampoules in cotton-wool in an ice-cream carton and putting them first in the 4° refrigerator for one hour then in the -20° freezer for one hour before transferring them to the coldest temperature available. Results are not so good by this method but in a laboratory not equipped to handle cell-freezing routinely it provides a satisfactory makeshift.
4. Other equipment.-A large Dewar flask will be required to store or transport liquid nitrogen if a liquid nitrogen refrigerator is used. (It may be remarked that it is best to have a well-established routine of topping-up every week, although the reservoir of liquid nitrogen may be sufficient for many weeks.)
General Procedure
The procedure for preparing twelve ampoules of a cell strain is described.
- Harvest 1-2x108 cells, which must be growing exponentially) by a standard method, such as trypsinisation. Resuspend in 21.5 ml. of fresh growth medium. Open ampoules.
- Add 2.5 ml. dimethyl sulphoxide (to give a final concentration of 10 per cent.). Mix quickly and dispense approximately 2 ml rapidly to each ampoule. Store ampoules on ice,
- Seal ampoules (practice may be required). Return to ice. (3A. Test ampoules by submerging in a solution of crystal violet in methyl alcohol. If the ampoules arc leaky some dye will be sucked in. This step is unnecessary if the operator is proficient.)
- Place ampoules in cooling device and leave 1-2 hours to cool to below -30°.
- Rapidly transfer ampoules to liquid nitrogen refrigerator. (Thawing at this stage will kill the cells.)
- Complete records.
- Within the next few days remove two ampoules, thaw as described below, and lest for viability by dye exclusion or plating efficiency. If viability is good confirm the record. If it is bad prepare a new batch of cells to replace the bad batch.
- To recover cells from the deep-freeze proceed as follows. Prepare a beaker by filling it with water at 37oC. and provide it with a cover. Put on protective goggles. (Occasionally a defective ampoule may fill with liquid nitrogen and explode on thawing.) Remove an ampoule with forceps and slip it under the cover into the beaker. When it has thawed open the ampoule and transfer the contents to a suitable vessel. Adding medium slowly at first, make the volume up to at least 25 ml. Transfer to culture vessels and incubate.
In the procedure described above dimethyl sulphoxide was recommended as the protecting agent. In our experience better results are obtained with it than with glycerol and this is probably the general experience. The reason may be that it can diffuse in and out of cells more readily with the result that there is less osmotic damage when it is added to medium or diluted out.
Transportation of cells
It is now common practice to transport samples of cell strains over long distances. The availability of airfreight and reliable mail services reduces the problem to the relatively simple one of maintaining the cells alive for, at the most, two days in transit.
It has to be accepted that the culture vessel will be agitated during handling so that there is no point in attempting to maintain the cells as a monolayer on the wall of a vessel partially filled with medium which may slop about. If it is desirable to despatch them as a monolayer practically all the medium should be removed from the vessel or else it should be completely filled. Alternatively the cells may be transported as a suspension.
The main hazard in transporting cells is exposure to extremes of temperature. Thus, in winter if cells are sent by ordinary mail it is possible that they will be frozen. If they are sent by airfreight at any time of the year the same may happen due to low temperatures in the uninsulated freight compartment of a high-flying aircraft. In summer in many parts of the world the air temperature alone may rise to levels lethal to cells, while they may be killed rapidly even in temperate climates if the sun shines directly on the package containing them. However, awareness of these factors is usually all that is necessary to ensure that adequate steps are taken to deal with them.
At most times of the year in Great Britain it has been found adequate to send the cells, either as a suspension or as a monolayer with about 2 ml, of medium in the bottle, by Express letter post. An ordinary prescription bottle or a large test-tube is used. It is protected, with some wadding and wrapped up securely in a parcel which is despatched in the late afternoon. From most parts of the country this ensures delivery first thing the following morning. In cold or hot weather, it is necessary to insulate the package much more thoroughly. If it is to be sent over a distance in the winter it is advisable to send it by passenger train with an arrangement to have it collected at the other end.
If cells have to be carried over a longer distance, it is best to send them by air. In making arrangements for international shipments it is advisable to consult a government organisation to avoid unnecessary customs delay. Transatlantic shipments to Great Britain from the United States have been arranged by the United Kingdom Treasury and Supply Delegation by arrangement with the Medical Research Council. It should be emphasised to those responsible that the package must not be exposed to extremes of temperature.
In countries such as the United States, in which extremes of temperature are encountered, it is necessary to send cells in insulated packages most of the year. Foam plastics, powdered cork and cotton are effective insulators.
If for any reason cells have to be transported over a long distance by slower methods this can be achieved satisfactorily by filling the culture vessel completely with fresh medium immediately before transportation and then keeping them at a temperature of about 20°C. By this means cells have been known to survive in a suitcase for a period of at least three weeks. Where possible the other methods described are to be preferred.
