EP0921723A1 - Charge et decharge de protecteurs a permeation pour la cryopreservation de cellules, de tissus et d'organes par vitrification - Google Patents

Charge et decharge de protecteurs a permeation pour la cryopreservation de cellules, de tissus et d'organes par vitrification

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Publication number
EP0921723A1
EP0921723A1 EP97928712A EP97928712A EP0921723A1 EP 0921723 A1 EP0921723 A1 EP 0921723A1 EP 97928712 A EP97928712 A EP 97928712A EP 97928712 A EP97928712 A EP 97928712A EP 0921723 A1 EP0921723 A1 EP 0921723A1
Authority
EP
European Patent Office
Prior art keywords
protectant
solute
sample
mol
permeating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97928712A
Other languages
German (de)
English (en)
Inventor
Victor Bronshtein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universal Preservation Technologies Inc
Original Assignee
Universal Preservation Technologies Inc
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Filing date
Publication date
Application filed by Universal Preservation Technologies Inc filed Critical Universal Preservation Technologies Inc
Publication of EP0921723A1 publication Critical patent/EP0921723A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators

Definitions

  • the present invention relates to non-toxic loading and unloading of permeating protectants that is required for the cryopreservation of biological specimens
  • Ice formation at low temperatures can be avoided only if samples are sufficiently dehydrated.
  • Dehydration is also known as a common cell damaging factor. The damaging effect of the dehydration increases with increasing concentration of vitrification solution and depends strongly on whether the vitrification solution contains permeating protectants such as dimethylsulfoxide
  • DMSO ethylene glycol
  • EG ethylene glycol
  • propylene glycol glycerol
  • glycerol glycerol
  • dehydration does not mean decrease in the cell volume, which actually may be very damaging (Marymen, 1967, 1970) .
  • dehydration means removal of water, or increase in the osmotic pressure. Erroneous use of this term has resulted in several misconceptions. For example, as described below, dehydration by itself is not a strong damaging factor. Dehydration may even be a protective factor, if performed according to the present invention.
  • Damage of cells during dehydration in concentrated solutions of non-permeating solutes is believed to be caused by hydration forces occurring between biological macromolecules and membranes when distances between them become small as a result of dehydration Bryant and Wolfe (1992) .
  • permeating protectant protects against cell dehydration because intracellular protectant diminishes these forces. Therefore, some amount of intracellular protectants are required to protect cells during dehydration to high osmotic pressures.
  • vitrification of larger and more complex specimens has not been achieved yet primarily because of the toxic effect of highly concentrated solutions containing permeating protectants. These concentrated solutions are needed to prevent ice formation in complex specimens during cooling and warming. Therefore, one should consider the dependence of cell injury on the time of equilibration in vitrification solution to better understand the mechanism(s) of toxicity. For successful preservation by vitrification, vitrification solution should more effectively diminish both ice formation in the cytosol and extracellular volumes, and toxic effects associated with equilibration (dehydration) of the specimen in concentrated vitrification solution.
  • Fahy et al . (1990) found that biochemical studies of the toxicity to date have not met the basic criteria required for demonstrating mechanisms of toxicity. This actually means that the direct chemical toxicity of typical permeating protectants (ethylene glycol, propylene glycol, glycerol and DMSO) is small. The inventor agrees with the conclusion of Fahy et al . (1990) that present concepts of protectant toxicity are in need of serious revision.
  • This toxic effect is not related to the increase in intracellular osmotic pressure or biochemical toxicity of protectant because after water efflux from loaded cells, the osmotic pressure and concentration of protectant inside cells is approximately equal to that outside the cells.
  • Timasheff (1993) criticized the belief that protectants form some sort of coating (a shell) that protects proteins from denaturation during cryopreservation. His criticism was based on the articles of Gekko and Timasheff (1981) , Lee and Timasheff (1981) , and other publications reporting that protectants excluded from the surface of proteins. The inventor (Bronshtein, 1995) submitted that the above conclusion of Timasheff and his co-workers is questionable for two reasons. First, the thermodynamic equilibrium in the dialysis experiments of Timasheff and his co-workers cannot be obtained if the hydrostatic pressure inside the dialysis bag is equal to the pressure outside the bag.
  • Steponkus et al . (1992) have shown that decreasing osmolarity of the vitrification solution allows one to decrease the damaging effect of dehydration in vitrification solution if the dehydration time is several minutes or less. However, to obtain cell survival after cryopreservation, one should successfully vitrify both the extracellular solution and the cytosol. For this reason, Steponkus et al . (1992) suggested that the better protectant for the loading step is one that allows stable vitrification of cytosol after dehydration in vitrification solution with lower osmolarity. This suggestion was a reflection of a general belief that the presence of protectants inside cells helps to vitrify cytosol.
  • the method should allow for controlled permeation of protectants into the samples during long-term contact of the samples with the vitrification solution.
  • Both loading and unloading methods, vitrification and rehydration solutions should allow for superior survival of the cryopreserved sample.
  • the present invention is directed to a method for minimizing toxic effects of loading and unloading biological specimens with permeating protectants.
  • the method includes the steps of loading the sample by contacting the sample with a solution comprising a permeating protectant and a non-permeating co-solute that limits the amount of the protectant that penetrates into cells of the biological specimen.
  • decreasing the ability of the protectant to enter cells of the biological specimen is achieved most effectively by adding non-penetrating co-solutes that effectively decrease the chemical potential of permeating protectants in the extracellular solution.
  • the more co-solute that is added the less amount of protectant penetrates into the cells; however, some minimum amount of protectant inside the cells is required to protect the cells against dehydration.
  • the concentration of the co-solutes that can be added is limited.
  • the maximum concentration of co-solutes that can be added to the extracellular solution, to limit penetration of protectant inside cells, depends on the minimum amount of protectant required to protect cells against dehydration.
  • the maximum concentration of co-solutes can be found experimentally for every specific combination of permeating protectants, osmotic pressure of the extracellular solution, and type of co-solute.
  • the method also includes gradual or stepwise loading (unloading) of permeating protectants with simultaneous increase (decrease) in concentration of permeating protectants and non-permeating co-solutes.
  • concentration of non- permeating co-solutes should be the maximum possible concentration that still does not damage cells.
  • Co-solutes that decrease the chemical potential of penetrating Protectants or protectants in aqueous solutions include, but are not limited to:
  • Amino acids glycine, alanine, glutamic acid, proline, valine, hydroxy- 1 -proline , betaaminopropionic acid, aminobutyric acid, beta-aminocaproic acid, aminoisobutyric acid, N-methylglycine, norvaline, and others that are soluble in water in concentration greater than 0.1 mol/1, and derivatives of amino acids (sarcosine, iminodiacetic acid, hydroxyethyl glycine, etc.) that are soluble in water in concentration >0.1 mol/1.
  • Betaines betaine and other betaines that are soluble in water in concentration greater than 0.1 mol/1.
  • Carbohydrates a) Monosaccharides (aldoses and ketoses) : glyceraldehyde, lyxose, ribose, xylose, galactose, glucose, hexose, mannose talose, heptose, dihydroxyacetone, pentulose, hexulose, heptulose, octulose, etc., and their derivatives; b) Amino sugars: D-ribose, 3-amino-3 -deoxy- , chitosamine, fucosamine, etc.
  • Alditols and inositols glycerol , erythritol, arabinitol , ribitol, mannitol, iditol, betitol, inositol , etc . ; d) Aidonic, uronic, and aldaric acids that are soluble in water in concentration >0.1 mol/1; and e) disaccharides and polysaccharides (sucrose, trehalose, etc.) .
  • Amino acids most effectively decrease the chemical potential of permeating protectants in aqueous solutions.
  • the invention allows one to significantly increase concentrations of vitrification solution and the times of loading, cell equilibration in the vitrification solution, and unloading, without increasing cell damage. This allows one to solve many problems occurring during loading of organs with protectants and subsequent cooling by decreasing gradients of osmotic pressure within the sample. This is a very important matter, because if a portion of cells in the sample is less dehydrated it may freeze at low temperatures and be damaged.
  • Hydration of the cells after cryopreservation and washing out of protectant (unloading) is achieved by equilibration of the specimens (perfusion with, in the case of organs) in solutions of the same protectant with lower osmotic pressures, but still containing the maximum concentration of the co-solutes (amino acids, betaines or carbohydrates) that do not damage cells.
  • the change of concentration during unloading may be gradual or stepwise. This will speed up efflux of protectant and limit the increase of the cell's volume during rehydration. This is an important issue because the increase in cell volume, more than the initial cell volume, may damage the cells.
  • Fig. 1 shows a plot of the effect of co-solutes on toxicity of 60% ethylene glycol vitrification solution on red blood cells.
  • the present invention is directed toward improving low temperature preservation of cells, multicellular specimens and organs by vitrification.
  • samples should be substantially dehydrated.
  • the dehydration damages cells because of large repulsive forces between macromolecules that occur inside cells.
  • a small amount of protectant should be present inside cells in order to decrease these forces.
  • the amount of protectant inside the cells should be kept as low as possible to decrease the toxic effect of vitrification solution and to increase the stability of the amorphous state inside the cells at low temperatures . This can be achieved by including non-penetrating co-solutes (amino acids, betaines, sugars, etc.) in the vitrification solution in concentrations from 0.1 - 0.7 mol/1.
  • intracellular protectant should be removed from the cells and exchanged for water.
  • the inventor believes that damage during rehydration occurs because of an increase in cell volume to more than the initial cell volume, when cells are transferred from vitrification solution to washing (rehydration) solutions.
  • rehydration solutions amino acids, betaines, carbohydrates, or other non-penetrating, co-solutes that effectively decrease the chemical potential of permeating protectants in aqueous solutions.
  • the co-solutes are used in concentrations from 0.1 - 0.7 mol/1. Higher co-solute concentrations will more effectively limit the mass of intracellular protectant, however, when this mass gets very small the dehydrated cells may be damaged.
  • the method for preserving a biological sample comprising the step of loading the sample by contacting the sample with a solution comprising a permeating protectant and a co-solute that decreases the ability of the protectant to enter cells of the biological specimen.
  • the protectant is one of a group of common permeating protectants including, but not limited to, dimethylsulfoxide, ethylene glycol, propylene glycol and glycerol.
  • the co-solute is one of a number of the following classes of compounds including, but not limited to, amino acids and derivatives thereof soluble in water in concentration greater than 0.1 mol/1, betaines soluble in water in concentration greater than 0.1 mol/1, carbohydrates and sugar alcohols, wherein the carbohydrates are selected from the group consisting of aldose monosaccharides, ketose monosaccharides, amino sugars, alditols, inositols, aidonic, uronic and aldaric acids soluble in water in concentrations of greater than 0.1 mol/1, disaccharides and polysaccharides .
  • the total concentration of non-permeating co-solutes in the vitrification solution is preferably between 0.1 and 0.7 mol/1 and is equal to a maximum possible concentration that does not damage cells.
  • the method of the present invention involves both gradual and/or stepwise loading of permeating protectants with simultaneous increase in concentration of permeating protectants and non-permeating co-solutes.
  • concentration of non-permeating co-solutes should be the maximum possible concentration that still does not damage the biological specimen.
  • the loading step is performed in two or more stages of contacting the sample with increasingly higher concentrations of permeating protectant and co-solute.
  • the loading step is performed by simultaneously increasing concentrations of both the protectant and the co-solute from initial concentrations to final concentrations according to a desired profile.
  • the initial concentration of permeating protectant is zero.
  • the initial concentration of co-solute is preferably zero, but may be greater than zero as long as the co-solute does not damage the sample.
  • the final concentration of co- solute may be determined empirically depending on the nature of the specimen and the choice and concentration of protectant .
  • the unloading of the protectant can be performed in a gradual or step-wise manner.
  • the only limitation in the profile of the simultaneous increase in protectant and co-solute concentration during loading is that the concentrations of the respective elements remain in an optimal proportion to minimize toxic effect of high concentrations of protectants.
  • the increase in concentration of the protectant and co-solute may be performed manually or mechanically and may be accomplished stepwise or according to a desired profile.
  • the shape of the profile curve may be linear or non-linear, depending upon empirical optimization of the profile for a specific cell type.
  • the rehydration or unloading step is directed to the replacement of protectant in the preserved sample with water.
  • the step of unloading the sample is performed by contacting the sample with a rehydration solution which can be an aqueous solution lacking the protectant (having smaller concentration of the protectant) such that the protectant is removed from the cells of the sample.
  • a rehydration solution which can be an aqueous solution lacking the protectant (having smaller concentration of the protectant) such that the protectant is removed from the cells of the sample.
  • the sample is unloaded in a manner opposite that of the loading step.
  • the rehydration solution includes a co-solute and a protectant and the unloading step is performed by simultaneously decreasing concentrations of both the protectant and the co-solute from initial concentrations to final concentrations according to a desired profile.
  • the initial concentrations of both the protectant and the co- solute may be identical to or smaller than the final concentrations thereof, respectively, in the loading process.
  • the protectant and the co-solute used during unloading are the same as those used during loading of the same sample.
  • the protectant and co-solute of the rehydration or unloading solution are selected from the same groups of compounds used in the loading or vitrification solutions.
  • the protectant is one of a group of common permeating protectants including, but not limited to, dimethylsulfoxide, ethylene glycol, propylene glycol and glycerol.
  • the co-solute is one of a number of the following classes of compounds including, but not limited to, amino acids and derivatives thereof soluble in water in concentration greater than 0.1 mol/1, betaines soluble in water in concentration greater than 0.1 mol/1, carbohydrates and sugar alcohols, wherein the carbohydrates are selected from the group consisting of aldose monosaccharides, ketose monosaccharides, amino sugars, alditols, inositols, aidonic, uronic and aldaric acids soluble in water in concentrations of greater than 0.1 mol/1, disaccharides and polysaccharides.
  • the total concentration of non-permeating co-solutes in the unloading solutions is preferably between 0.1 and 0.7 mol/1 and is equal to a maximum possible concentration that does not damage cells.
  • the unloading step involves both gradual and/or stepwise unloading of permeating protectants with a simultaneous decrease in the concentration of the permeating protectants and non- permeating co-solutes.
  • the initial concentration of non- permeating co-solutes should be the maximum possible concentration that still does not damage the biological specimen.
  • the unloading step is also performed in two or more stages of contacting the sample with increasingly lower concentrations of permeating protectant and non-permeating co-solute.
  • the unloading step is performed by simultaneously decreasing concentrations of both the protectant and the co-solute from initial concentrations to final concentrations according to a desired profile.
  • the initial concentrations of protectant and co-solute is preferably the same as the final concentrations in the loading (vitrification) solution.
  • the final concentration of permeating protectant is zero.
  • the final concentration of co-solute may be greater than zero as long as the co-solute does not damage the sample cells.
  • the loading of the protectant can be performed in a gradual or stepwise manner.
  • the only limitation in the profile of the simultaneous decrease in protectant and co- solute concentrations during unloading is that the concentrations of the respective elements remain in an optimal proportion to minimize toxicity and to maximize viability on rehydration.
  • the decrease in concentrations of the protectant and co-solute may be performed manually or mechanically and may be accomplished stepwise or according to a desired profile.
  • the shape of the profile curve may be linear or non-linear, depending on empirical optimization of the profile for a specific cell type.
  • EXAMPLE 1 Gradual Loading and Unloading of Rat Heart with Dimethylsulfoxide (DMSO) .
  • DMSO Dimethylsulfoxide
  • the heart was excised and immediately immersed in ice-cold Krebs- Henseleit buffer (KHB) , which contained (in mM) : 118 NaCl, 11 glucose, 25 NaHC0 3 , 4.7 KC1, 1.2 MgS0 4 , 1.2 KH 2 P0 4 , 0.5 Na-EDTA, and 2.5 CaCl 2 ⁇
  • KHB Krebs- Henseleit buffer
  • the aorta was cannulated and the heart retrograde perfused at 70 mm Hg for 9 min. with 36.5°C KHB equilibrated with 95% 0 2 /% C0 2 .
  • the perfusion was continued for 2 min. at 60 mm Hg with CP-llE saturated with 100% 0 2 .
  • composition of CP-llE (in mM) was: 125 NaCl, 7 glucose, 1.2 KH 2 P0 4 , 10 mannitol, 15 MgS0 4 , 14 KC1, 10 Hepes, 0.02 EDTA, 0.28 CaCl 2 , pH 7.5.
  • Loading and removal of DMSO and manni tol After
  • CP-llE flush the arrested heart was transferred to a perfusion apparatus (Fig. 1) . Both CP-11EB and CP- HE+DMSO+mannitol were bubbled constantly with 100% 0 2 . Perfusate was delivered via an aortic cannula by a peristaltic pump at a flow rate of 1 ml/min. Two experiments were performed at room temperature. Experiment 0: The heart was gradually loaded with 30 wt% DMSO and immediately unloaded. No co-solutes were used. A linear gradient of 0 to 30% DMSO was generated using a gradient maker. DMSO gradient was controlled by the duration of infusion (or the total volume of solution infused) . Loading time was 30 min. During the 30 min.
  • Experiment 1 The heart was gradually loaded with 30 wt% DMSO and immediately unloaded. A linear gradient of both 0 to 30% DMSO and 0 to 3% mannitol was generated using a gradient maker. DMSO gradient was controlled by the duration of infusion (or the total volume of solution infused) . Loading time was 30 min. During the 30 min. loading, the rate of increase in concentration of the solution was 1% DMSO/min. and 0.075% mannitol/min. Unloading began right after the end of loading. Unloading time was 60 min. During the 60 min. unloading, a gradient of decreasing DMSO concentration was 0.5 wt%/min. , the final concentration of mannitol was 1 wt%.
  • Experiment 2 The heart was gradually loaded with 30 wt% DMSO during 30 min, then perfused 30 min. with 30% DMSO + 3% mannitol, and then unloaded during 60 min. A linear gradient of both 0 to 30% DMSO and 0 to 3% mannitol was generated using a gradient maker. DMSO gradient was controlled by the duration of infusion (or the total volume of solution infused) . During the 30 min. loading, the rate of increase in concentration was 1% DMSO/min. and 0.075% mannitol/min. During the 60 min. unloading, a gradient of decreasing DMSO concentration was 0.5 wt%/min, the final concentration of mannitol was 1 wt%.
  • Cardiac function was assessed by working mode reperfusion with KHB at 11 mm Hg preload and 70 mm Hg after load.
  • Heart rate HR, beats/min
  • AF and CF, ml/min. aortic and coronary flow
  • CO AF+CF, ml/min.
  • systolic and diastolic aortic pressure mm Hg
  • Coronary vascular resistance and work were calculated according to Neely et al . , 1967 (Neely et al . "Effect of Pressure Development on Oxygen Consumption by the Isolated Rat Heart” . Am. J. Physiology, 212:804-12, 1967) .
  • Step 1 100 ⁇ l of blood were mixed with 100 ⁇ l of 30 wt% EG + 0.9 wt% NaCl. Equilibration time 10 min.
  • Step 2 1000 ⁇ l of vitrification solution vitrification solution containing mixture of 60 wt% EG + 0.9 wt% NaCl with different amounts of glutamic acid monosodium Salt (GA) per gram of Vitrification solution were slowly (during 3-5 min.) added to the mixture obtained after Step 1.
  • G glutamic acid monosodium Salt
  • Step 1 After 60 min. equilibration in vitrification solution erythrocytes were centrifuged down
  • Step 2 After 10 min. of equilibration, above the erythrocytes were centrifuged down again, then, 0.5 ml of supernatant was removed from each sample, and 0.5 ml of 3 wt% GA + 0.9 wt% NaCl were added to each sample. Then the samples were mixed by vortexing and were equilibrated 10 min.
  • erythrocytes were centrifuged down again and 0.5 ml of supernatant was collected from each sample.
  • Concentration of free hemoglobin in supernatants collected from the samples after equilibration in Vitrification solution, first and second step of unloading was used to characterize the erythrocyte damage (hemolysis) .
  • the concentration of free hemoglobin was measured using 390 Turner spectrophotometer (at 550nm wavelength) after adding 0.5 ml of supernatant to 3 ml of water.
  • the hemolysis was measured as a ratio of hemoglobin concentration in the supernatants to the hemoglobin concentration in the mixture of 41.7 ⁇ l of blood with 3.5 ml water that was taken as 100% hemolysis.
  • 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.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention concerne un procédé pour la cryopréservation d'un échantillon biologique, y compris la charge progressive ou par étape de l'échantillon avec un protecteur à perméation, procédé sans lequel on met en contact l'échantillon avec des solutions renfermant le protecteur et aussi un co-soluté sans perméation qui limite la quantité de protecteur pénétrant dans les cellules du spécimen biologique. Le procédé consiste en outre à décharger (sous forme de réhydratation) progressivement ou par étape l'échantillon par mise au contact d'une ou de plusieurs solutions réhydratantes en abaissant graduellement la concentration du protecteur et du co-soluté, de manière à éliminer le protecteur dans les cellules de l'échantillon. La concentration du co-soluté pendant les opérations de charge et de décharge doit avoir la valeur maximum atteignable sans pour autant endommager l'échantillon aussi bien à température ambiante qu'aux températures inférieures à 0°.
EP97928712A 1996-05-29 1997-05-29 Charge et decharge de protecteurs a permeation pour la cryopreservation de cellules, de tissus et d'organes par vitrification Withdrawn EP0921723A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US1863896P 1996-05-29 1996-05-29
US18638P 1996-05-29
PCT/US1997/009207 WO1997045010A1 (fr) 1996-05-29 1997-05-29 Charge et decharge de protecteurs a permeation pour la cryopreservation de cellules, de tissus et d'organes par vitrification

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EP0921723A1 true EP0921723A1 (fr) 1999-06-16

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EP (1) EP0921723A1 (fr)
JP (1) JP2001517204A (fr)
AU (1) AU3290097A (fr)
CA (1) CA2256714A1 (fr)
WO (1) WO1997045010A1 (fr)

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CA2379111C (fr) * 1999-07-13 2011-07-26 Thomson Licensing S.A. Systeme de gestion des erreurs dans les informations specifiques a des programmes dans un decodeur video
US6869757B2 (en) * 2000-07-31 2005-03-22 21St Century Medicine, Inc. Advantageous carrier solution for vitrifiable concentrations of cryoprotectants, and compatible cryoprotectant mixtures
US7250292B2 (en) * 2000-01-26 2007-07-31 21St Century Medicine Hypertonic reduction of chilling injury
US7094601B2 (en) 2000-05-16 2006-08-22 The General Hospital Corporation Microinjection of cryoprotectants for preservation of cells
US20020045156A1 (en) * 2000-05-16 2002-04-18 Mehmet Toner Microinjection of cryoprotectants for preservation of cells
DE102004030285B4 (de) * 2004-06-23 2007-08-02 Franz Lahnsteiner Gefrierkonservierung von Eiern und Embryonen von Fischen
WO2012067240A1 (fr) 2010-11-19 2012-05-24 セーレン株式会社 Solution de stockage vitrifiée pour des cellules
CN109042628A (zh) * 2018-09-21 2018-12-21 洛阳未羊生物科技有限公司 一种羊膜组织的冻存方法

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US4980277A (en) * 1987-10-16 1990-12-25 Cultor Ltd. Cryoprotectant solution and method
US5071741A (en) * 1988-04-18 1991-12-10 Cryolife, Inc. Cryoprotective agent and its use in cryopreservation of cellular matter
US5595866A (en) * 1994-05-27 1997-01-21 Methodist Hospital Of Indiana, Inc. Step-wise method to remove cryoprotectant from sperm

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Title
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AU3290097A (en) 1998-01-05
JP2001517204A (ja) 2001-10-02
WO1997045010A1 (fr) 1997-12-04
CA2256714A1 (fr) 1997-12-04

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