EP0932337A1 - Solutions de vitrification servant a conserver a long terme des cellules, des tissus, des organes et des organismes - Google Patents

Solutions de vitrification servant a conserver a long terme des cellules, des tissus, des organes et des organismes

Info

Publication number
EP0932337A1
EP0932337A1 EP97940831A EP97940831A EP0932337A1 EP 0932337 A1 EP0932337 A1 EP 0932337A1 EP 97940831 A EP97940831 A EP 97940831A EP 97940831 A EP97940831 A EP 97940831A EP 0932337 A1 EP0932337 A1 EP 0932337A1
Authority
EP
European Patent Office
Prior art keywords
permeating
cryoprotectant
cell
rehydration
solution
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Withdrawn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
EP97940831A
Other languages
German (de)
English (en)
Other versions
EP0932337A4 (fr
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
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Universal Preservation Technologies Inc filed Critical Universal Preservation Technologies Inc
Publication of EP0932337A1 publication Critical patent/EP0932337A1/fr
Publication of EP0932337A4 publication Critical patent/EP0932337A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • 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
    • A01N1/02—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
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents

Definitions

  • This invention relates to the long-term shelf preservation of cells and multicellular specimens by vitrification.
  • the invention is directed to the optimization of vitrification and rehydration solutions, as well as vitrification, and rehydration procedures.
  • Vitrification is an alternative approach to cryopreservation that utilizes solidification of samples during cooling, without formation of ice crystals (Fahy, G.M. et al . , 1984). Conventionally, cryopreservation by vitrification of single cell
  • Ice formation at low temperatures can be avoided only if samples are sufficiently dehydrated.
  • Dehydration is known to damage cells.
  • the damaging effect of dehydration increases with increasing osmotic pressure (concentration) and depends strongly upon whether the vitrification solution contains permeating cryoprotectants.
  • concentration osmotic pressure
  • cells normally cannot survive equilibration in solutions containing only non-permeating solutes in concentration >1 mol/1.
  • many types of cells can easily tolerate equilibration in solutions containing permeating cryoprotectants in much higher concentrations. This is because penetration of cryoprotectants protects cells against dehydration damage.
  • dehydration does not mean a decrease in the cell volume which actually may be very damaging (Meryman, H.T., 1967, Meryman, H.T., 1970) .
  • the term "dehydration” means removal of water, or increase in the osmotic pressure. Erroneous use of this term 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, as performed according to the present invention.
  • cryoprotectants help to vitrify cytosol, and the fact that some intracellular cryoprotectant is required to protect cells during dehydration, penetration of cryoprotectant inside cells may be considered as a beneficial phenomena.
  • a negative aspect of this penetration, considered in the literature, is associated with direct chemical toxicity of cryoprotectants (Fahy et al . , 1990). Because the toxicity is believed to be proportional to the concentration of cryoprotectants (not to the amount of cryoprotectants inside a cell) three basic approaches have been proposed to minimize the toxicity (for details see review of Steponkus, P.L. et al . , 1992): 1. to use a mixture of different cryoprotectants ;
  • thermodynamic force responsible for cryoprotectant permeation inside cells is proportional to the cryoprotectant concentration gradient across the cell membrane independent of the composition of the vitrification solution.
  • Bronshteyn, V.L. et al . (1994) found that amino acids (glycine and glutamic acid) and carbohydrates (sucrose and sorbitol) significantly diminished ethylene glycol permeation inside Drosophila melanogaster embryos.
  • the preventive effect of amino acids was impressive because 1 wt% of glutamic acid + 0.5 wt% glycine practically prevented ethylene glycol permeation inside embryos for up to three hours of equilibration in vitrification solution containing 42 wt% ethylene glycol.
  • Steponkus, P.L. et al . (1992) have shown that decreasing osmolarity of the vitrification solution decreases the damaging effect of dehydration in vitrification solution if the dehydration time is several minutes or less.
  • Steponkus et al . (1992) suggested that the better cryoprotectant 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 cryoprotectants inside cells helps to vitrify cytosol .
  • an object of the present invention to provide a preservation method and a cryoprotectant for cryopreserving cells and multicellular specimens that accounts for the newfound facts that use of low molecular weight cryoprotectants can be detrimental to the cryopreserva ion process. It is a further object of the present invention to provide a preservation method and a vitrification solution for preserving by vitrification extracellular spaces in the specimen.
  • the present invention is directed to a method of preserving cells or multicellular specimens including the step of contacting the specimen with a vitrification solution comprising a permeating cryoprotectant, a non- permeating cryoprotectant and a non-permeating co-solute that limits the amount of the permeating cryoprotectant that permeates the specimen.
  • the method further includes the step of unloading the specimen by contacting the loaded specimen with a rehydration solution comprising a non- permeating co-solute and, optionally, a permeating cryoprotectant and a non-permeating rehydration cryoprotectant, such that cryoprotectant is removed from the cells of the specimen.
  • the cryoprotectants can be loaded or unloaded in a stepwise manner, in a linear manner, or according to a desired profile .
  • the present invention is also directed to the vitrification and rehydration solutions for use in connection with the method described above.
  • 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 erythocyte, stem cells, sperm, E. Coli , yeasts and other cellular microorganisms, etc.
  • multicellular tissues erythocyte, stem cells, sperm, E. Coli , yeasts and other cellular microorganisms, etc.
  • 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.
  • some minimum amount of cryoprotectant is required inside the cells of the specimen in order to protect the cells against dehydration.
  • 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.
  • 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:
  • Amino acids glycine, alanine, glutamic acid, proline, valine, hydroxy-1-proline, beta- aminopropionic acid, a inobutyric acid, beta-ainocaproic acid, aminoisobutyric acid, N-methylglycine, norvaline, and others that are soluble in water in concentration >0.1 mol/1, and derivatives of amino acids (sarcosine, i inodiacetic 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 >0.1 mol/1.
  • 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 >0.1 mol/1.; and d. disaccharides (sucrose, trehalose, etc.). 4. Sugar alcohols (sorbitol, etc.) .
  • 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.
  • 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
  • cryoprotectants along with non-permeating co-solutes (amino acids, betaines, sugars, etc. in concentrations from 0.1 - 0.6 mol/1) that effectively decrease the chemical potential of penetrating cryoprotectants in cryopreservation solution.
  • cells can be stored at a temperature that is lower than the vitrification temperatures both inside and outside the cells of the specimen.
  • cells 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.
  • 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/1. 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 .
  • 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.
  • 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 .
  • 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.).
  • Steponkus P.L., Langis, R. and Fujikawa, S. 1992. Cryopreservation of plant tissues by vitrification. In: Advances in Low-Tempera ture Biology, Vol. 1, edited by P.L. Steponkus. pp. 1-61. JAI Press, Ltd., London.

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Environmental Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Dentistry (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Le procédé de conservation par vitrification décrit dans cette demande permet de stocker des échantillons à des températures supérieures à celles utilisées dans les procédés classiques et peut être appliqué à des cellules, des organes, des organismes et des tissus multicellulaires. Le procédé de cette invention consiste à préparer une solution de co-solutés non perméables de vitrification (acides aminés, betaïnes, glucides ou autres co-solutés non perméables qui réduisent efficacement le potentiel chimique de cryoprotecteurs à perméation dans des solutions aqueuses), un cryoprotecteur à perméation et un cryoprotecteur non perméable (polyvinyl-pyrrolidone, polyéthylène glycol, dextrane, amidon d'hydroxy éthyle, Ficol, etc...); à mettre un échantillon en contact avec la solution de vitrification et à stocker l'échantillon à une température de stockage. Le procédé comprend également une étape de réhydratation de l'échantillon conservé dans une solution de réhydratation préparée de la même manière que la solution de stockage par vitrification. Cette invention concerne également une solution de vitrification et une solution de réhydratation telles que cells décrites dans le procédé.
EP97940831A 1996-09-06 1997-09-05 Solutions de vitrification servant a conserver a long terme des cellules, des tissus, des organes et des organismes Withdrawn EP0932337A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US2557096P 1996-09-06 1996-09-06
US25570P 1996-09-06
PCT/US1997/015611 WO1998009514A1 (fr) 1996-09-06 1997-09-05 Solutions de vitrification servant a conserver a long terme des cellules, des tissus, des organes et des organismes

Publications (2)

Publication Number Publication Date
EP0932337A1 true EP0932337A1 (fr) 1999-08-04
EP0932337A4 EP0932337A4 (fr) 1999-12-29

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Application Number Title Priority Date Filing Date
EP97940831A Withdrawn EP0932337A4 (fr) 1996-09-06 1997-09-05 Solutions de vitrification servant a conserver a long terme des cellules, des tissus, des organes et des organismes

Country Status (5)

Country Link
EP (1) EP0932337A4 (fr)
JP (1) JP2001502664A (fr)
AU (1) AU4252097A (fr)
IL (1) IL128855A0 (fr)
WO (1) WO1998009514A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US20130236960A1 (en) * 2010-11-19 2013-09-12 Yoshihiro Kunitomi Vitrificated storage solution for cells
WO2013096659A1 (fr) * 2011-12-20 2013-06-27 Cook General Biotechnology Llc Procédés et compositions pour le stockage de cellules animales

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4865871A (en) 1983-08-23 1989-09-12 Board Of Regents The University Of Texas System Method for cryopreparing biological tissue

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4559298A (en) * 1982-11-23 1985-12-17 American National Red Cross Cryopreservation of biological materials in a non-frozen or vitreous state
US4980277A (en) * 1987-10-16 1990-12-25 Cultor Ltd. Cryoprotectant solution and method
US5145770A (en) * 1990-06-04 1992-09-08 Biosurface Technology, Inc. Cryopreservation of cultured epithelial sheets
CA2051092C (fr) * 1990-09-12 2002-07-23 Stephen A. Livesey Methode et appareillage pour la cryopreservation, la stabilisation a sec et la rehydratation de suspensions biologiques
US5160313A (en) * 1991-05-14 1992-11-03 Cryolife, Inc. Process for preparing tissue for transplantation
US5217860A (en) * 1991-07-08 1993-06-08 The American National Red Cross Method for preserving organs for transplantation by vitrification
IT1256621B (it) * 1992-12-04 1995-12-12 Soluzioni crioprotettrici

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4865871A (en) 1983-08-23 1989-09-12 Board Of Regents The University Of Texas System Method for cryopreparing biological tissue

Also Published As

Publication number Publication date
WO1998009514A1 (fr) 1998-03-12
AU4252097A (en) 1998-03-26
JP2001502664A (ja) 2001-02-27
EP0932337A4 (fr) 1999-12-29
IL128855A0 (en) 2000-01-31

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