Patent Application: US-25456399-A

Abstract:
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 organismes . 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 .

Description:
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 - l - 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 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 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 (≧ 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 - 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