the present invention is directed to a method for preserving a biological specimen and compositions for achieving the same . suitable specimens can be single cells ( erythocyte , stem cells , sperm , e . coli , yeasts and other cellular microorganisms , etc .) or multicellular tissues ( skin , blood vessels , organs , embryos , etc .). the method , vitrification solutions and rehydration solutions described herein minimize toxicity of the vitrification and rehydration solutions and increase intracellular and extracellular vitrification temperatures . the method includes the step of contacting a specimen or sample with a cryopreservation or vitrification solution . the cryopreservation solution includes a permeating ( i . e ., low molecular weight ) cryoprotectant , a non - permeating ( i . e ., high molecular weight ) cryoprotectant and a non - permeating co - solute that effectively decrease the chemical potential of penetrating cryoprotectants in the vitrification solution . addition of high molecular weight non - permeating cryoprotectants will increase the vitrification temperature of the cryopreservation solution outside cells . the co - solutes will limit the amount of permeating cryoprotectants that move inside cells and therefore increase the mass / mass ratio of intracellular protein to permeating cryoprotectant in a dehydrated specimen in cryopreservation solution . this will increase the intracellular vitrification temperature for a given osmotic pressure of cryopreservation solution . the more co - solutes added , the less cryoprotectant penetrates inside the specimen . the more protein / cryoprotectant ratio inside cells , the higher the intracellular vitrification temperature . however , some minimum amount of cryoprotectant is required inside the cells of the specimen in order to protect the cells against dehydration . for this reason , the concentration of the co - solutes that can be added is limited . the maximum concentration of co - solutes that can be added to cryopreservation solution , to limit penetration of cryoprotectant inside cells , depends upon the minimum amount of cryoprotectant required to protect cells against dehydration in cryopreservation solution . the maximum concentration of co - solutes can be found experimentally for every specific type of permeating cryoprotectants , osmotic pressure of cryopreservation solution , type of co - solute and type of specimen . as noted above , the invention provides a method for shelf preservation of cells and multicellular specimens at refrigeration or higher temperatures . to increase vitrification temperature outside the cells , cryopreservation solution should contain high molecular weight cryoprotectants , such as dextrans , starches , polyethylene glycol , polyvinylpyrrolidone , ficol , peptides , etc . co - solutes that decrease the chemical potential of penetrating cryoprotectants in aqueous solutions include , but are not limited to : 1 . amino acids : glycine , alanine , glutamic acid , proline , valine , hydroxy - 1 - proline , beta - aminopropionic acid , aminobutyric acid , beta - ainocaproic acid , aminoisobutyric acid , n - methylglycine , norvaline , and others that are soluble in water in concentration & gt ; 0 . 1 mol / l , and derivatives of amino acids ( sarcosine , iminodiacetic acid , hydroxyethyl glycine , etc .) that are soluble in water in concentration & gt ; 0 . 1 mol / l . 2 . betaines : betaine and other betaines that are soluble in water in concentration & gt ; 0 . 1 mol / l . 3 . carbohydrates : monosaccharide ( aldose and ketoses ) glyceraldehyde , lyxose , ribose , xylose , galactose , glucose , hexose , mannose , talose , heptose , dihydroxyacetone , pentulose , hexulose , heptulose , octulose , etc ., and their derivatives a . amino sugars : d - ribose , 3 - amino - 3 - deoxy -, chitosamine , fucosamine , etc . ; b . alditols and inositols : glycerol , erythritol , arabinitol , ribitol , mannitol , iditol , betitol , inositol , etc . ; c . aldonic , uronic , and aldaric acids that are soluble in water in concentration & gt ; 0 . 1 mol / l . ; and d . disaccharides ( sucrose , trehalose , etc .). 4 . sugar alcohols ( sorbitol , etc .). to obtain a high intracellular vitrification temperature , the cells should be substantially dehydrated . the dehydration damages the cells due to large repulsive forces between macromolecules inside cells . a small amount of cryoprotectant should be present inside cells in order to decrease these forces . however , the amount of cryoprotectant inside the cells should be kept as low as possible to decrease the toxic effect of the vitrification solution and to increase the intracellular vitrification temperature . all these requirements can be achieved by using cryopreservation solution that contain mixtures of permeating ( i . e ., low molecular weight ) and non - permeating ( i . e ., high molecular weight ) cryoprotectants along with non - permeating co - solutes ( amino acids , betaines , sugars , etc . in concentrations from 0 . 1 - 0 . 6 mol / l ) that effectively decrease the chemical potential of penetrating cryoprotectants in cryopreservation solution . after dehydration in cryopreservation ( vitrification ) solution , cells can be stored at a temperature that is lower than the vitrification temperatures both inside and outside the cells of the specimen . prior to dehydration , cells may be loaded in a low concentration ( 5 - 40 wt %), non - damaging solution of permeating cryoprotectant to protect cells from damage during dehydration in cryopreservation solution . after storage , the samples should be rehydrated and returned to normal physiological medium . in other words , intracellular cryoprotectant should be removed from the cells and exchanged for water . it is believed that damage during rehydration , when cells are transferred from cryopreservation ( vitrification ) solution to a rehydration ( washing ) solution , occurs because of an increase in cellular volume beyond initial cellular volumes . to avoid this possibility of damage , one has to include in rehydration solutions , co - solutes , as described above , such as : amino acids , betaines , carbohydrates , or other non - permeating co - solutes that effectively decrease the chemical potential of permeating cryoprotectants in aqueous solutions . the co - solutes are used in concentrations from 0 . 1 - 0 . 6 mol / l . higher co - solute concentrations will more effectively limit the mass of intracellular cryoprotectants , however , when this mass gets very small , the dehydrated cells may be damaged . the invention allows one to significantly decrease the osmotic pressure of vitrification solution required to obtain a stable vitrification of cells during cooling , to significantly increase extracellular and intracellular vitrification temperatures and the time of cell equilibration ( dehydration ) in the vitrification solution , without increasing cell damage . this allows one to solve many related problems occurring during equilibration in vitrification solution , storage and rehydration and washing out of intracellular cryoprotectant . to improve the ability of cells to survive the cryopreservation process described herein , the amounts of permeating cryoprotectant and other components of the cryopreservation solution may be increased in the cryopreservation solution in a stepwise fashion , a linear fashion or according to a desired profile from an initial concentration (. gtoreq . 0 %) to an optimal final concentration . the cryopreservation solution and the relative amounts of components thereof may be controlled mechanically or manually . similarly , to optimize the rehydration process , the contents of the rehydration solution and timing of the rehydration process can be similarly controlled . the optimal initial and final concentrations , as well as the optimum method for increasing the relative concentrations of the components of the cryopreservation and rehydration solutions is determined empirically . by increasing the intracellular and extracellular vitrification temperatures , one will be able to increase storage temperature up to refrigeration or even room temperature and , therefore , develop method of long - term shelf preservation of cells . by increasing the equilibration time in vitrification solution , osmotic pressure gradients arising during dehydration of multicellular specimens can be decreased . this is a very important matter because if a portion of cells in the sample is less dehydrated than other portions , it may freeze during subsequent cooling and be damaged . limiting the amount of cryoprotectant inside cells simplifies the washing out procedure or completely avoids washing of the intracellular cryoprotectant from cells prior to transfusion or transplantation . this is a very important achievement for blood transfusion , transplantation of embryos and artificial insemination services . the method of the present invention encompasses dehydration of specimens , cooling samples to a storage temperature , warming of the samples to ambient temperature , rehydration and washing out of cryoprotectants in rehydration solution , and returning to normal physiological conditions for various medical procedures ( transfusions , transplantation , etc .). the above invention has been described with reference to the preferred embodiment . obvious modifications and alterations will occur to others upon reading and understanding the preceding detailed description . it is intended that the invention be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof . bronshtein , v . l . 1995 . a heresy about an organ cryopreservation by vitrification . in : “ cryo &# 39 ; 95 program ,” abstract p2 - 66 of a paper presented at the 32nd annual meeting of the society for cryobiology , madison , wis . bronshtein , v . l . 1995 . binding , of protectants to the biological macromolecules . in : “ cryo &# 39 ; 95 program ”, abstract p2 - 65 of a paper presented at the 32nd annual meeting of the society for cryobiology , madison , wis . bronshteyn , v . l . and steponkus , p . l . 1994 . amino acids and carbohydrates limit permeation of ethylene glycol in drosophila melanogaster embryos . in : “ abstract of papers presented at the 31st annual meeting of the society for cryobiology ,” abstract 54 , kyoto , japan . bronshteyn , v . l . and steponkus , p . l . 1993 . differential scanning calorimeter studies of heterogeneous ice nucleation in drosophila melanogaster embryos . in : “ abstract of papers presented at the 30th annual meeting of the society for cryobiology ”, abstract 22 , atlanta , ga . bronshteyn , v . l . and steponkus , p . l . 1992 . differential scanning calorimeter studies of ice formation in drosophila melanogaster embryos . cryobiology 29 : 764 - 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( abstract ) bryant , g . and wolfe , j . interfacial forces in cryobiology and anhydrobiology . cryo - letters 13 , 23 - 36 , 1992 . crowe , j . h ., carpenter , j . f ., crowe , l . m . and anchorodoguy , t . j . ( 1990 ). are freezing and dehydration similar stress vectors ? a comparison of modes of interaction of stabilizing solutes with biomolecules , cryobiology 27 : 219 - 231 . fahy , g . m ., lilley , t . h ., linsdell , h ., st . john douglas m . and meryman , h . t . 1990 . protectant toxicity and protectant toxicity reduction : in search of molecular mechanisms . cryobiology 27 : 247 - 268 . fahy , g . m ., macfarlane , d . r ., angell , c . a . and meryman , h . t . 1984 . vitrification as an approach to cryopreservation . cryobiology 21 : 407 - 426 . gekko , k . and timasheff , s . n . 1981 . mechanism of stabilization by glycerol : preferential hydration in glycerol - water mixtures . biochemistry 20 : 4667 - 76 . langis , r . and steponkus , p . l . 1990 . cryopreservation of rye protoplasts by vitrification . plant physiol . 92 : 666 - 671 . lee , j . c . and timasheff , s . n . ( 1981 ) the stabilization of proteins by sucrose . j . biol . chem . 256 : 7193 - 7201 kasai , m ., komi , j . h ., takakamo , a ., tsudera , h ., sakurai , t . and machida , t . 1990 . a simple method for mouse embryo cryopreservation in a low toxicity vitrification solution , without appreciable loss of viability . j . reprod . fertil . 89 : 91 - 97 . kedem , o . and katchalsky , a . ( 1958 ) thermodynamic analysis of the permeability of biological membranes to non - electrolytes . biochem . biophys . acta 27 : 229 - 246 . meryman , h . t . 1967 . the relationship between dehydration and freezing injury in the human , erythrocyte . in : cellular injury and resistance in freezing organisms . vol . 11 , edited by e . asahina . pp . 231 - 244 . institute of low temperature science , hokkaido university , sapporo , japan . meryman , h . t . 1970 . the exceeding of a minimum tolerable cell volume in hypertonic suspension as a cause of freezing injury . in : the frozen cell , edited by g . e . w . wolstenholme and m . o &# 39 ; conner . pp . 51 - 67 . j . & amp ; a . churchill , london . rall , w . f . and fahy , g . m . 1985a . vitrification : a new approach to embryo cryopreservation . theriogenology 23 : 220 . rall , w . f . and fahy , g . m . 1985b . ice - free cryopreservation of mouse embryos at − 196 . degree . c . by vitrification . nature 313 : 573 - 575 . steponkus , p . l ., bronshteyn , v . l . and caldwell , sh . 1994 . cryopreservation of drosophila melanogaster embryos : formulation of improved vitrification solutions . in : “ abstract of papers presented at the 31st annual meeting tcdr of the society for cryobiology ”, abstract 51 , kyoto , japan . steponkus , p . l ., langis , r . and fujikawa , s . 1992 . cryopreservation of plant tissues by vitrification . in : advances in low - temperature biology , vol . 1 , edited by p . l . steponkus . pp . 1 - 61 . jai press , ltd ., london . tachikawa , s ., otoi , t ., kondo , s ., machida , t . and kasai , m . 1993 . successful vitrification of bovine blastocysts , derived by in vitro maturation and fertilization . mol . reprod . dev . 34 : 266 - 271 . timasheff , s . n . 1993 . the control of protein stability and association by weak interaction with water : how do solvents affect these processes ? annu . rev . biophys . biolmol . strict . 22 : 67 - 97 . zhu , s . e ., kasai , m ., otoge , h ., sakurai , t . and machida , t . 1993 . cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycol - based solutions . j . reprod . fertil . 98 : 139 - 145 .
the method of preservation by vitrification , described in the present application , provides for storage of samples at higher temperatures than in conventional methods and can be applied to cells , multicellular tissues , organs and organisms . the method of the present invention includes preparing a solution of vitrification non - permeating co - solutes , a permeating cryoprotectant and a non - permeating cryoprotectant , contacting a sample with the vitrification solution and storing the sample at a storage temperature . the method also includes the step of rehydrating the preserved sample in a rehydration solution prepared in the manner of the vitrification storage solution . the present invention is also directed to a vitrification solution and a rehydration solution as described in connection with the method .