Cryoprotective compounds

Finkle BJ, ZavolaME, Ulrich JM. Cryoprotective compounds in the viable freezing of plant tissues, in Cryopreservation of Plant Cells and Organs (Kartha KK, ed.), CRC Press, Boca Raton, FL, 1985, pp. 75-113. 

During hardening, many plant cells produce their own internal supply of cryoprotective compounds. In those instances, nonpenetrating soluble sugars such as raffinose or sucrose are often accumulated, and resistance to freezing increases. 

Cryoprotective Compounds. Cryoprotective effects of sugars, polyalcohols, and compounds of other families have been known since the 1940s. Many works on those effects have been reported in the fields of freezing preservation of blood, microorganisms, etc., but this review will deal with only the works on fish muscle proteins. 

Most proteins are not sufficiently stable in aqueous solution to allow formulation as a sterile solution. Instead, the protein is freeze-dried and reconstituted before use. Development of a freeze-dried protein formulation often requires special attention to the details of the freezing process (potential pH shifts and ionic strength increase with freezing) as well as to potential loss of activity with drying. Formulation additives, such as sugars and polyhydroxy compounds, are often useful as cryoprotectants and lyoprotectants. Residual moisture may also be critical to the stability of the dried preparation [33],... 

For cryostat sectioning, the tissue specimens are cryoprotected in 30% sucrose in 0.1 M phosphate buffer for 12 hr or until they sink to the bottom of the container. They are embedded in O.C.T compound (Miles, Elkhart, IN) and frozen in N-heptane cooled to the temperature of liquid nitrogen. Alternatively, if the antigens are resistant to paraffin embedding, the specimens can be dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. 

Karsten U, Kiick K, Vogt C, Kirst GO (1996) Dimethylsulfoniopropionate production in phototrophic organisms and its physiological function as a cryoprotectant. In Kiene RP, Visscher PT, Keller MD, Kirst GO (eds) Biological and environmental chemistry of DMSP and related sul-fonium compounds. Plenum Press, New York, pp 143-153... 

Cryoprotectants which have so far been found to be effective for fish muscle proteins include such compounds as monosaccharides, oligosaccharides, polysaccharides of relatively small molecular size, di- and polyalcohols, hydroxymonocarboxylic acids, di- and tricarboxylic acids, acidic, basic and some other amino acids, and phosphates and their derivatives (15,16,66,67,72-74, 82,83,93,97-99,112-114,122,140-154). Dimethyl sulfoxide (DMSOT, which is cryoprotective for various biological materials such as red cells, was not effective for fish muscle proteins (147). 

Denaturation during storage at sub-zero temperatures may be minimized by use of suitable cryoprotective agents which include several families of compounds. 

Numerous compounds can provide general cryoprotection to proteins, when used at concentrations of several hundred millimolar. These include sugars, polyols, amino acids, methylamines, and salting-out salts (e.g., ammonium sulfate) [59,61,68-70]. Based on the results of freeze-thawing experiments with LDH and PFK and a review of the literature on protein freezing. Carpenter and Crowe [59] have proposed that this cryoprotection can be explained by the same universal mechanism that Timasheff and Arakawa have defined for solute-induced protein stabilization in nonfrozen, aqueous solution (reviewed in [4,70,78,79]). 

Finally, it is important to consider mechanistically how to explain the much greater potency of PEGs as cryoprotectants relative to other compounds such as sucrose. The data for one case, which are shown in Figure 9 and Table 1, illustrate... 

Organic Compounds. Sucrose, glucose, other sugars, and sorbitol have cryoprotective effects on frozen mince of Alaska pollack (60,118) and carp actomyosin (117,119,120). Furthermore, ethylene glycol, glycerol (121,122), and citrate (123) have cryoprotective effects on the proteins in muscles of Alaska pollack and cod. 

In the authors laboratory, an in vitro test was devised to evaluate the cryoprotective effect of any compound on fish protein. The test system consisted of carp actomyosin either in solution (in 0.6M KC1) or in suspension (in 0.05M KC1). By means of this system about 150 compounds were screened, out of which about 30 compounds were found to be markedly effective and another 20 compounds were found to be moderately effective. Among the former group, monosodium glutamate (MSG) was particularly outstanding. Additives were used at a concentration of 0.1-0.2M in the final mixture and the pH was adjusted to 7 before freezing (51,52,116,117,119,124,125,126). Similar studies have been done by other workers (60,127). 

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