cryopreservation and freeze-drying protocols

CHAPTER 1 Overview Mark R. McLellanand John G. Day 1. Introduction Nature dictates that biological material will decay and die. The struc- ture and function of organisms will change and be lost with time, as surely in laboratory cultures as in the biologists who study and manipu- late them. Attempts to stop the biological clock have been conjured by minds ancient and modern; at the heart of many such schemes have been experiments with temperature and water content. Whereas refrigeration technology provides a means of slowing the rate of deterioration of perishable goods, the use of much lower temperatures has proved a means of storing living organisms in a state of suspended animation for extended periods. The removal of water from viable biolog- ical material in the frozen state (freeze-drying) provides another means of arresting the biological clock by withholding water, and commencing again by its addition. Over 40 years have passed since the first demonstration of the effective cryopreservation of spermatozoa was made (I). The potential of storing live cells for extended, even indefinite, periods quickly caught the imagi- nation of biologists and medics working in diverse fields, and experi- ments to cryopreserve many thousands of organelle, cell, tissue, organ, and body types have been, and continue to be, performed. Key mile- stones have been the successful cryopreservation of bull spermatozoa (2); the first successfully frozen and thawed erythrocytes (3); the first live birth of calves after insemination using frozen spermatozoa (4); suc- cessful cryopreservation of plant cell cultures (5); cryopreservation of a From: Methods in Molecular Biology, Vo/. 38: Cryopreservatlon and Freeze-Drymg Protocols Edited by: J. G. Day and M R McLellan Copyright Q 1995 Humana Press Inc., Totowa, NJ 2 McLellan and Day plant callus (6); the successful recovery of frozen mouse embryos (7,8); and the use of cryopreservation to store embryos for use in human in vitro fertilization programs (9). Furthermore, cryopreservation has become widely accepted as the optimal method for the preservation of microor- ganisms (10-13). Cryopreservation and freeze-drying are widely employed to conserve microbial biodiversity (11-13) (see Chapters 2-7, and 9). This is one of the key roles performed by microbial service culture collections. More recently, cryopreservation has been accepted as an appropriate technique to preserve endangered plant (14) and animal (15) species (see Chapters 14 and 20). However, many cells and tissues for which there is a need for long-term biostorage await suitable methodologies. It is to be hoped that we are on the verge of cryopreserved transplant organs, frozen by vitrifi- cation; reproducible freezing of teleost eggs or embryonic stages; as well as the successful cryopreservation of human oocytes; a greater range of plant tissues; and a broader range of microalgae and protozoa. A common misconception among noncryobiologists is that successful cryopreservation methods for one strain or species are transferable to similar cells or organisms. Although this is sometimes true, it is far from the rule. With different biology comes a different response to cryopro- tectants and freezing; a preservation protocol may need adjustments, or to be constructed afresh for the material under study. It is worth a brief word on how such methods are determined. It is usually the case that cryoprotectants must be added to protect cells during cooling, and careful manipulation of temperature excur- sion is required to control the size, configuration, and location of ice crystals. Therefore, choice and concentration of cryoprotectants, and rate of cooling must be optimized as the basis for any protocol. An accidental discovery was the spur for modern cryobiology (1); Polge’s discovery of glycerol as an effective protectant allowed rapid advances in mammalian spermatozoa freezing. Dimethyl sulfoxide (DMSO), methanol, ethylene glycol, and hydroxyethyl starch (HES) have been added to the list of effective cryoprotectants. Many successful proto- cols have been developed empirically, by optimizing choice, concen- tration, time, and temperature of addition of cryoprotectant; along with the rate of cooling. Much is known of the response of cells to low temperatures, and the effects of cryoprotectants, as a result of the efforts of scientists from a Overview 3 range of disciplines over the past 50 yr. The subject has its own consider- able and complex literature to which the reader is referred for further information (16-21). An outline of the major principles is given in the Introduction to Chapter 10. Such understanding has aided formulation of cryopreservation protocols by predicting optimum cooling rates from measured biophysical characters (22) and by direct visualization of cells and organisms during cooling (23). The formulation of freeze-drying protocols are as yet firmly empiri- cally based; it has until recently been the case that the freeze-drying community have not accessed relevant information available from cryo- biological studies. Further understanding of the effects of the sublima- tion phase of freeze-drying on cell biology is required, if techniques employed by microbiologists are to be extended to a range of eukaryotic cells, including erythrocytes and mammalian spermatozoa. In order for a biostorage method to be acceptable as a routine labora- tory practice, several criteria need to be fulfilled. Ideally, it should be relatively simple; complex procedures prior to freezing or freeze-drying may make the method more cumbersome or expensive than the culture methods it replaces. In addition, postthaw viability should be high, in order that cultures can regenerate rapidly, and preexisting freeze-resis- tant mutants are not selected. Many culture collections and gene banks insist on high recovery values prior to a protocol being adopted for reg- ular use; 50% viability postthaw has been accepted in some culture collections as a nominal cutoff for adopting maintenance by cryopreser- vation alone (2#,25). Additionally, the storage method adopted should give level recovery rates with time; there is good evidence that a cryo- preservation method yielding high initial recovery values, maintains viability at that level on prolonged storage (26,27). The same may not be true of freeze-dried cultures or macromolecules, which are recommended to be stored at refrigerator or freezer temperatures. As evidenced by the list of contributors to this volume, the cryobio- logical community embraces a wide range of specialists; medical scien- tists, plant-, animal, and microbiologists. Since much of the information on cryopreservation and freeze-drying is scattered, or bound in with theo- retical literature, it is sometimes difficult to supply a recipe methodology for a particular purpose. We hope this handbook will be useful in provid- ing clear and concise instructions for the long-term storage of a wide range of materials across the biological kingdoms. McLellan and Day References 1. Polge, C , Smith, A. U., and Parkes, A. S. (1949) Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164,666. 2. Smith, A. U. and Polge, C. (1950) Storage of bull spermatozoa at low tempera- tures. Vet. Rec. 62,115-l 17. 3. Smith, A. U. (1950) Prevention of haemolysis during freezing and thawing of red blood cells. Lancet ii, 910,911. 4. Stewart, D. L. (1951) Storage of bull spermatozoa at low temperatures. Vet. Ret 63,65,66. 5. Latta, R. (1971) Preservation of suspension cultures of plant cells by freezing. Can. J. Bot. 49, 1253,1254. 6. Bannier, L. J. and Steponkus, P. L. (1972) Freeze preservation of callus cultures of Chrysanthemum morifolium Ramat. HortScience 7, 194. 7. Whittmgham, D. G., Leibo, S. P., and Mazur, P. (1972) Survival of mouse embryos frozen to -196 and -296°C. Sctence 178,411-414. 8. Wilmut, I. (1972) The effects of cooling rate, cryoprotectant agent and stage of development on survival of mouse embryos during freezing and thawing Life Sci 11,1071-1079. 9 Cohen, J., Simons, R., Fehilly, C. B , Fishel, S. B., Edwards, R. G., Hewitt, J., Rowland, G. F., Steptoe, P. C., and Webster J M. (1985) Birth after replacement of hatching blastocyst cryopreserved at expanded blastocyst stage. Lancet i, 647. 10. Heckley, R. J. (1978) Preservation of microorgamsms. Adv. Appl. Microbial. 24, l-54. 11. Hatt, H. (ed.) (1980) American Type Culture Collection Methods. I. Laboratory Manual on Preservation Freezing and Freeze-Drying. ATCC, Rockville, MD. 12. Kirsop, B. E. and Snell, J. S. S. (eds.) (1984) Maintenance of Microorganisms. Academic, London 13. Kirsop, B. E. and Doyle, A. (eds.) (1991) Mumtenance of Microorganisms and Cultured Cells. Academic, London. 14. Withers, L. A. (1987) The low temperature preservation of plant cell, tissue and organ cultures and seed for genetic conservation and improved agriculture, in The Erects of Low Temperatures on Biological Systems (Grout, B. W. W. and Morris G. J., eds.), Edward Arnold, London, pp. 389-409. 15. Seymour, J. (1994) Freezing trme at the zoo. New Scientist No. 1910,21-23. 16. Grout, B. W. W. and Morris G. J. (eds.) (1987) The Effects ofLow Temperatures on Biological Systems Edward Arnold, London 17. Morris, G. J. and Clarke, A. (eds.) (1981) The Esfects of Low Temperatures on Biological Systems Academic, London. 18. Ashwood-Smith, M. J. and Farrant, J. (eds.) (1980) Low Temperature Preserva- tion in Biology and Medicine, Pitman Medical, Tunbridge Wells, Kent. 19. Franks, F. (1985) Biophysics and Biochemistry at Low Temperatures. Cambridge University Press, London. 20. Steponkus, P. L (ed.) (1992) Advances tn Low Temperature Biology vol. 1. JAI, London. Overview 5 21. Steponkus, P. L. (ed.) (1993) Advances in Low Temperature Biology vol. 2. JAI, London. 22. Pitt, R. E. and Steponkus, P L. (1989) Quantitative analysis of the probability of intracellular ice formation during freezing of isolated protoplasts. Cryobiology 26, 44-63. 23. McGrath, J J. (1987) Temperature controlled cryogenic light microscopy-an introduction to cryomicroscopy, in The Effects of Low Temperatures on Biological Systems (Grout, B. W. W. and Morris G. J., eds.), Edward Arnold, London, pp. 234-268. 24. Leeson, E. A., Cann, J. P., and Morris, G. J. (1984) Maintenance of algae and protozoa, in Maintenance of Microorganisms (Kirsop B. E. and Snell, J. S. S., eds.), Academic, London, pp. 131-160. 25. McLellan, M. R., Cowling, A. J., Turner, M., and Day, J. G. (1991) Maintenance of algae and protozoa, in Maintenance of Microorganisms and Cultured Cells (Kirsop B. E. and Doyle A., eds.), Academic, London, pp. 183-208. 26. McLellan, M. R. (1989) Cryopreservation of diatoms. Diatom. Res. 4,301-318. 27 Brown, S. and Day, J. G. (1993) An improved method for the long-term preserva- tion of Naegleria gruberi. Cryo-Lett. 7,347-352. [...]... preservation and storageof plant viruses, in Methods in Virology, vol IV (Maramarosch,K and Koprowski, H., eds.),Academic,London, pp 491-501 4 Ward, T G (1968) Methodsof storageandpreservationof animal viruses,in Methods in Virology, vol IV (Maramarosch, andKoprowski,H., eds.),Academic,LenK don, pp 481-489 2 CHAPTER3 Freeze-Drying and Cryopreservation Stephen of Bacteria E Perry 1 Introduction Freezing and freeze-drying. .. parallel 1.1 Freeze-Drying Simple freezing and freeze-drying regimes are often established empirically However, it is possible to apply scientific principles to the control of parameters allowing the optimization of processes for the freezing and drying of organisms (2) Thus, heat and vapor transfer can be manipulated to maintain sublimation under optimal conditions of temperature and time, Freeze-drying. .. status of culture collections and theu contribution to biotechnology Crit Rev Biotechnol 2,287-3 14 2 Franks, F (1990) Freeze-drying: from empiricism to predictability Cryo-Lett 11, 93-l 10 3 Meryman, H T (1966) Freeze-drying, in Cryobiology (Meryman, H T., ed.), Academic, London, pp 609-663 4 Mackey, B M (1984) Lethal and sublethal effects of refrigeration, freezing and freeze-drying on micro-organisms,... W W and Morris, G J (1987) Freezing and cellular organization, in The Effects of Low Temperatures on Biological Systems (Grout, B W W and Morris, G J., eds.), Edward Arnold, London, pp 147-174 11 Feltham, R K A., Power, A K., Pell, P A., and Sneath, P H A (1978) A simple method for storage of bacteria at -76°C J Appl Bact 44,3 13-3 16 12 Redway, K F and Lapage, S P (1974) Effect of carbohydrates and. .. Bioscience and Human-Technology, Japan (formerly, The Fermentation Research Institute) From Methods m Molecular B!ology, Vol 38: Cryopresmatron and Freeze-Drymg Protocols Ed&d by J G Day and M f? Mclellan Copyright Q 1995 Humana Press Inc., Totowa, NJ 31 32 Kawamura et al 2 Materials 1 Growth medium: YM agar medium (see Notes 1 and 2) Dissolve 3 g yeast extract, 3 g malt extract, 5 g peptone, 10 g glucose, and. .. the cap tightly, and repeat the vortexing procedure (see Note 8) 5 Centrifuge the suspension 2000g for 10 mm at 6 Dispense the clarified supernatant medium in properly labeled cryotubes, replace the cap securely, and freeze at -70°C as described in Section 3.2., step 4 7 For recovery of viruses use method detailed in Section 3.2., step 5 Virus Cryopreservation and Storage 15 3.5 Cryopreservation in... increase the surface area and prevent frothing For large culture collections, the centrifugal method has advantages in minimizing the likelihood of cross-contamination as ampules may be plugged after filling and sealed under vacuum on a manifold at the end of the secondary drying stage However, for the inexperienced and infrequent user, centrifugal freeze-drying is more technically demanding The method described... -30°C freezer and incubate for 2 h The stoppering unit is also precooled to -30°C thus ensuring that when the trays of vials are transferred to the unit, the contents of the vials will not thaw 8 Switch on the condenser unit of the freeze-drying machine and close the condenser drain valve When the condensertemperature falls to below -5O”C, quickly transfer the stoppermg unit and vials to the freeze-drying. .. factors affecting the outcome of freeze-drying have been discussed by Meryman (3) Many of these factors, e.g., incubation temperature of the culture and phase of growth, can be controlled and adjusted to give optimum survival rates A standard method will work for most species of bacteria, but more fastidious organisms, for instance, anaerobic Bacteria 10 11 12 13, 14 15 16 17 18 Cryopreservation 29 photobacteria... thick and, therefore, strong seal Virus Cryopreservation and Storage 17 12 If available, a high voltage spark tester can be used to test the integrity of the vacuum, but this is not absolutely essential (see Notes 19 and 20) 13 Label the ampules in such a way that they can be identified many years later White cloth tape is ideal for this purpose 14 Store the ampules at 4°C or lower if possible and avoid . fields, and experi- ments to cryopreserve many thousands of organelle, cell, tissue, organ, and body types have been, and continue to be, performed. Key mile- stones have been the successful cryopreservation. virus stocks and these depend to some extent on the peculiar properties of the particular viruses. Although the protocols in this chapter are devoted to cryopreservation and freeze-drying. Biology, Vo/. 38: Cryopreservatlon and Freeze-Drymg Protocols Edited by: J. G. Day and M R McLellan Copyright Q 1995 Humana Press Inc., Totowa, NJ 2 McLellan and Day plant callus (6); the successful