WO2023064216A1 - Composition comprenant un(des) agent(s) de formation de verre pour l'utilisation comme cryoprotecteurs et procédés pour les fabriquer et les utiliser - Google Patents

Composition comprenant un(des) agent(s) de formation de verre pour l'utilisation comme cryoprotecteurs et procédés pour les fabriquer et les utiliser Download PDF

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WO2023064216A1
WO2023064216A1 PCT/US2022/046181 US2022046181W WO2023064216A1 WO 2023064216 A1 WO2023064216 A1 WO 2023064216A1 US 2022046181 W US2022046181 W US 2022046181W WO 2023064216 A1 WO2023064216 A1 WO 2023064216A1
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medium
glass forming
vitrification
concentration
carboxylic acid
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PCT/US2022/046181
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English (en)
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Steven Mullen
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Cook Regentec Llc
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    • 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

  • compositions and methods for vitrification of a biological specimen or material such that the biological specimen remains viable after it is warmed.
  • compositions and a method for vitrification of a biological specimen or material and in particular, a medium for conditioning, vitrification, and long-term storage of the biological specimen or material, including viable cells, such as mature, unfertilized oocytes.
  • Cryo-preservation or cryo-conservation is a process where organelles, cells, tissues, extracellular matrix, organs, or any other biological constructs susceptible to damage caused by unregulated chemical kinetics are preserved by cooling to very low temperatures (typically -80 °C using solid carbon dioxide or an ultra-low freezer, or -196 °C using liquid nitrogen). At low enough temperatures, any enzymatic or chemical activity which might cause damage to the biological material in question is effectively stopped. Cryopreservation methods seek to reach low temperatures without causing damage during the transition to and return from cryogenic temperatures. Traditional cryopreservation has relied on incubating the material to be cryopreserved with a class of molecules termed cryoprotectants.
  • Cryoprotectants include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, sucrose, trehalose, and large macromolecules with a high bonding affinity to water. Cryoprotectants help prevent damage to the materials as a result of the process of cooling and warming.
  • DMSO dimethyl sulfoxide
  • glycerol propylene glycol
  • ethylene glycol ethylene glycol
  • sucrose sucrose
  • trehalose trehalose
  • large macromolecules with a high bonding affinity to water.
  • cryopreservation techniques In conventional cryopreservation techniques, cells are harvested, suspended in a storage solution, then preserved by freezing. Cryopreservation protocols subject the cells to a multitude of stresses and insults throughout the process of cell harvesting, freezing, and thawing. These stresses and insults can cause irreparable damage to the cell.
  • cryopreserve oocytes, embryos, sperm and other similar biological specimens is critical to the widespread application of assisted reproductive technologies.
  • cryopreservation techniques are not well developed in most species.
  • cryoprotectants such as DMSO
  • slow controlled rates of cooling usually in the range of 0.1 - 1.0°C/min slowly dehydrate the cell during freezing to prevent intracellular ice crystallization.
  • cryopreservation of oocytes, embryos and other developmental cells using such procedures often results in a reduced ability to both establish and maintain pregnancy following embryo transfer.
  • Oocytes are particularly susceptible to cryopreservation damage because of disruption of the metaphase spindle microtubule integrity during cooling, and the extensive amount of intracellular lipids present in the oocytes of many species.
  • cryoprotectant concentrations are very toxic to cells, such as oocytes, embryos and other delicate developmental cells. Cryoprotectant toxicity can be reduced by increasing the cooling rate, which allows the use of lower concentrations of cryoprotectants. This has been accomplished by plunging oocytes held on electron microscopy grids, within thinly walled straws (known as open pulled straws) or other devices that facilitate very rapid cooling, directly into liquid nitrogen. However, these procedures are cumbersome and recovery of cells can be problematic. Furthermore, the cryoprotectant concentration necessary to successfully vitrify cells with these systems is still high enough to render the solutions toxic to the cells.
  • One embodiment relates to a method of cryopreservation of a biological material harvested from a donor comprising: (i) suspending the biological material in a first medium, the first medium comprising one or more permeating cryoprotectants to allow cryoprotectants to diffuse into the biological material; (ii) to allow rapid dehydration of the biological material and further cryoprotectant loading: (a) adding a glass forming agent selected from a carboxylic acid or carboxylate salt thereof to the first medium, or (b) moving the biological material from the first medium into a second medium, the second medium comprising an aqueous solution containing traditional cryoprotectants and a glass forming agent selected from a carboxylic acid or carboxylate salt thereof; and (iii) cryopreserving the biological material suspension by vitrifying the suspension, wherein an increase in the concentration of the glass forming agent allows for a relative reduction in the concentration of the cryoprotectant in the first medium or the second medium by at least 2-fold.
  • the carboxylic acid may comprise at least three carbons.
  • the carboxylic acid may be an alpha- hydroxy- and/or beta-hydroxy- carboxylic acids.
  • the carboxylic acid may be an amino acid.
  • the carboxylate salt may be selected from monovalent cations.
  • the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine.
  • the glass forming agent may be selected from the group consisting of sodium propionate, sodium lactate and threonine. In the described method, the glass forming agent may be at a concentration of from about 0.1 to about I molar.
  • the cryoprotectant may be selected from the group consisting of: dimethyl sulfoxide (DMSO), glycerol, a glycol, acetamide, formamide, polyvinylpyrrolidone, a hydroxyethyl starch, a polysaccharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, dextran, or a combination thereof.
  • the relative reduction in the concentration of the cryoprotectant may be by a factor of between 3- and I0-fold, wherein the cryoprotectant is ethylene glycol.
  • the relative reduction in the concentration of the cryoprotectant may be by a factor of up 30-fold, wherein the cryoprotectant is 1 ,2-propanediol.
  • the cooling step may comprise plunging the oocyte suspension in liquid nitrogen.
  • the biological material may be incubated at a temperature of from about 4°C and about 37°C.
  • the method may further comprise the step of de-oxygenating the suspension during the incubation period.
  • the cell vitrification medium may further comprise a cell nutrient matrix comprising a sufficient amount of nutrients to sustain metabolic needs of the biological material during the incubation period, without substantially depleting the nutrients, so as to maintain the viability of the biological material.
  • the cell nutrient matrix comprises at least one amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cystine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, praline, serine, taurine, threonine, tryptophan, tyrosine, and valine.
  • the cell nutrient matrix may comprise at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin, and thiamine.
  • Another embodiment relates to a method of cryopreservation of mature, unfertilized oocytes harvested from a donor comprising: (i) suspending the oocytes harvested from the donor in a first medium, the first medium comprising one or more permeating cryoprotectants to allow cryoprotectants to diffuse into the oocytes; (ii) to allow rapid dehydration of the oocytes and further cryoprotectant loading: (a)adding a glass forming agent selected from a carboxylic acid or carboxylate salt thereof to the first medium, or (b) moving the oocytes from the first medium into a second medium, the second medium comprising an aqueous solution containing traditional cryoprotectants and a glass forming agent selected from a carboxylic acid or carboxylate salt thereof; and (iii)
  • the carboxylic acid may comprise at least three carbons.
  • the carboxylic acid may be an alpha- hydroxy- and/or beta-hydroxy- carboxylic acid.
  • the carboxylic acid may be an amino acid.
  • the carboxylate salt may be selected from monovalent cations.
  • the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine.
  • the glass forming agent may be selected from the group consisting of sodium propionate, sodium lactate and threonine. In the described method, the glass forming agent may be at a concentration of from about 0.1 to about I molar.
  • the cryoprotectant may be selected from the group consisting of: dimethyl sulfoxide (DMSO), glycerol, a glycol, acetamide, formamide, polyvinylpyrrolidone, a hydroxyethyl starch, a polysaccharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, dextran, or a combination thereof.
  • the relative reduction in the concentration of the cryoprotectant may be by a factor of between 3- and I0-fold, wherein the cryoprotectant is ethylene glycol.
  • the relative reduction in the concentration of the cryoprotectant may be by a factor of up 30-fold, wherein the cryoprotectant is 1 ,2-propanediol.
  • the cooling step may comprise plunging the oocyte suspension in liquid nitrogen.
  • the oocytes are incubated at a temperature of from about 4°C and about 37°C.
  • the method may further comprise the step of de-oxygenating the oocyte suspension during the incubation period.
  • the cell vitrification medium may further comprise a cell nutrient matrix comprising a sufficient amount of nutrients to sustain metabolic needs of the oocytes during the incubation period, without substantially depleting the nutrients, so as to maintain the viability of the oocytes.
  • the cell nutrient matrix comprises at least one amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cystine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, praline, serine, taurine, threonine, tryptophan, tyrosine, and valine.
  • the cell nutrient matrix may comprise at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin, and thiamine. In the method, with the proviso that the first medium or the second medium includes less than a cryopreservative amount of one or more molecules that have cryopreservation properties at higher concentrations.
  • Another embodiment relates to the use of at least one glass forming agent selected from a carboxylic acid or carboxylate salt thereof in a method of cryopreservation of mature, unfertilized oocytes harvested from a donor.
  • Cryopreservation may be by vitrification.
  • Yet a further embodiment relates to the use of at least one glass forming agent selected from a carboxylic acid or carboxylate salt thereof in a method of cryopreservation of biological material harvested from a donor.
  • Cryopreservation may be by vitrification.
  • Yet a further embodiment related to a method of reducing the total concentration of solutes by at least 2%, or at least 5%, or at least 10% in an aqueous solution used for cryopreservation of biological material harvested from a donor, comprising adding a glass forming vitrification agent selected from a carboxylic acid or carboxylate salt thereof at a concentration of from about 0.1 to about 0.75 molar to the aqueous solution, wherein the presence of the glass forming vitrification agent in the aqueous solution allows to reduce the total concentration of solutes in the aqueous solution necessary to achieve successful cryopreservation.
  • the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine.
  • Yet another embodiment relates to a method of reducing toxicity of an aqueous solution used for cryopreservation of biological material harvested from a donor comprising adding a glass forming vitrification agent selected from a carboxylic acid or carboxylate salt thereof at a concentration of from about 0.1 to about 0.75 molar to the aqueous solution, wherein the presence of the glass forming vitrification agent in the aqueous solution reduces the toxicity of the solution by at least 2%, or at least 5%, or at least 7.5%, or at least 10%, or at least 15% by allowing a reduction in more toxic compounds.
  • the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine.
  • a further embodiment relates to a cryopreservative composition
  • a cryopreservative composition comprising an aqueous solution containing traditional cryoprotectants, and a glass forming agent selected from a carboxylic acid or carboxylate salt thereof, wherein an increase in the concentration of the glass forming agent in the composition allows for a relative reduction in the concentration of the traditional cryoprotectants in the composition by at least 2-fold.
  • Figure I shows chemical structures of exemplary glass forming agents.
  • Figure 2 depicts a survival plot for vitrification study.
  • Figure 3 depicts a graph showing the relationship between lactate or propionate molality and total solute molality necessary to vitrify in a 1/4 cc CryoStraw (Ethylene Glycol is the additional Solute).
  • Figure 4 depicts a graph showing the relationship between lactate, propionate, or trehalose molality and total solute molality necessary to vitrify in a 1/4 cc CryoStraw (Propylene Glycol is the additional Solute).
  • Described herein are components used to formulate a cell cryopreservation medium and a method that can be used for the effective cryopreservation, e.g. via vitrification, of a biological specimen or material, such as developmental cells (e.g., mature, unfertilized oocytes), such that the biological specimen or material remains viable after it is thawed.
  • a biological specimen or material such as developmental cells (e.g., mature, unfertilized oocytes)
  • developmental cells e.g., mature, unfertilized oocytes
  • the described vitrification media and methods may be applicable to any biological specimen or material, including any viable cells.
  • cryopreservation medium refers to cell medium used during the cryopreservation process of a biological specimen or material.
  • the cryopreservation medium may be a “vitrification medium,” which is specifically used during a vitrification process of a biological specimen or material.
  • viable in the context of the biological specimen or material, refers to a biological specimen or material, which is able to live and function normally (e.g., develop normally) for a period of time.
  • biological specimen or “biological material” can be used interchangeably and refer to a cell or tissue harvested from a suitable human or other animal (e.g., mammalian) donor, and includes developmental cells, or a biological construct derived from such cells (e.g., via 3-D printing or other methods known in the art).
  • developmental cells refers to a reproductive cell of an organism that has the capacity to develop into a new individual organism capable of independent existence (sometimes when combined with another reproductive cell, as when discussing gametes).
  • Developmental cells include, but are not limited to, sperm, oocytes (e.g fashion mature, unfertilized oocytes and immature oocytes), embryos, morulae, blastocysts, and other early embryonic cells.
  • oocytes e.g., sperm
  • oocytes e.g., sperm
  • embryos morulae
  • blastocysts e.g., ocysts
  • embryonic cells e.g., embryonic embryonic cells.
  • the mature, unfertilized oocytes may be harvested from a suitable human or other animal (e.g., mammalian) donor.
  • Other types of biological specimen include a specimen or material obtained from any source including cells obtained from any donor species, organism, organ or tissue. Specific examples of cells include cells commonly used in laboratory experiments, like fibroblasts and CD34+ T-cells; numerous others exist and are available through large local and national repositories
  • cryopreservation refers to the preservation of a biological specimen or material at extremely low temperature.
  • vitrification refers to phenomenon wherein a biological specimen is cooled to very low temperatures such that the water in the specimen forms a glasslike state without undergoing crystallization.
  • vitrification solutions have high concentrations of solutes (i.e., cryoprotectants), e.g., in the range of 5-7 moles per L solution in order to vitrify.
  • cryoprotectant(s) refers to a substance(s) used to protect biological tissue/material from freezing damage (i.e., due to ice formation or solution effects injury).
  • a disadvantage of the vitrification technique with high concentrations of cryoprotectants is that the cryoprotectants are toxic to cells, especially oocytes, embryos and other delicate developmental cells, and particularly at the concentrations used in vitrification procedures.
  • solutes e.g., “glass forming agent(s)”
  • solutes e.g., “glass forming agent(s)”
  • cryoprotectants known to be necessary to achieve and maintain the vitreous state, e.giller 5-7 moles per L, to produce less toxic and more effective vitrification solutions.
  • an effective vitrification media formulation that include at least one glass forming agent selected from a carboxylic acid or carboxylate salt thereof, which has a molecular structure that is predicted to be a more effective glass forming agent that other, commonly used solutes.
  • the glass forming agent used in the described media formulations contributes to an increase in the glass-forming tendency of the vitrification media. It is this superior glass forming ability of the vitrification solution that was demonstrated by the inventors.
  • a less toxic due to lesser amounts of the commonly used cryoprotectant(s), such as ethylene glycol
  • more effective vitrification media is produced.
  • the vitrification medium described herein includes an aqueous cell medium including water, traditional solutes, traditional cryoprotectants, and a glass forming agent selected from a carboxylic acid or carboxylate salt thereof.
  • a glass forming agent refers to an agent used in the described method of vitrification of a biological specimen or material. Exemplary glass forming agents are provided in Figure I .
  • traditional solutes means solutes like salts, energy sources such as glucose, amino acids, and other types of solutes used in cell culture medium for metabolic support of cells and not for cryopreservation purposes.
  • traditional cryoprotectants means cryoprotectants that have been routinely used for biological material cryopreservation, and do not include the novel solutes described in this disclosure.
  • traditional cryoprotectants include dimethyl sulfoxide, ethylene glycol, propylene glycol, and glycerol.
  • a specific embodiment described herein relates to a vitrification medium for oocytes harvested from a donor, which includes an aqueous cell medium including water traditional solutes, traditional cryoprotectants, and a glass forming agent selected from a carboxylic acid or carboxylate salt thereof.
  • the carboxylic acid present in the cryopreservation medium can comprise at least three carbons. In some instances, the carboxylic acid present in the cryopreservation medium can comprise at least four carbons. [0039] In certain other embodiments, the carboxylic acid present in the cryopreservation medium may be an alpha-hydroxy- and/or beta-hydroxy- carboxylic acids.
  • the carboxylic acid may also be an amino acid.
  • the carboxylate salt may be selected from the group consisting of one or more of carboxylate salts with a counter ion such as sodium or potassium, eg., a sodium salt, a potassium salt, etc.
  • the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine.
  • the glass forming agent is selected from the group consisting of sodium propionate, sodium lactate and threonine.
  • Figure I illustrates the chemical structures of the glass forming agents described herein, and as used in the described compositions and methods.
  • the vitrification medium may contain threonine, which is an amino acid that is used in the biosynthesis of proteins.
  • Threonine contains an a-amino group, a carboxyl group, and a side chain containing a hydroxyl group, making it a polar, uncharged amino acid.
  • Threonine is a powder in its purified form.
  • the powder form of threonine would be used in aqueous solutions in combination with other solutes to manufacture vitrification solutions described herein.
  • Threonine has a molecular structure that confers an advantage to the creation of a vitreous aqueous solution compared to other solutes often used in cryopreservation solutions.
  • ethylene glycol Example 2
  • a commonly used solute in cryopreservation solutions and a relatively weak glass forming agent an increase in the concentration of threonine can result in a relative reduction in the concentration of ethylene glycol by a factor of between 3 and 9.67, depending upon the concentration used.
  • the vitrification medium may contain sodium lactate, which is the sodium salt of lactic acid, and has a mild saline taste.
  • sodium lactate is produced by fermentation of a sugar source, such as com or beets, and then, by neutralizing the resulting lactic acid to create a compound having the formula
  • sodium lactate is a white powder in its purified form.
  • the powder form of sodium lactate would be used in aqueous solutions in combination with other solutes to manufacture vitrification solutions described herein.
  • the vitrification medium may contain sodium propionate, which is the sodium salt of propionic acid which has the chemical formula Na(C 2 H 5 COO).
  • This white crystalline solid is deliquescent in moist air. It is produced by the reaction of propionic acid and sodium carbonate or sodium hydroxide.
  • sodium propionate is a crystalline solid in its purified form.
  • the crystalline solid form of sodium propionate would be used in aqueous solutions in combination with other solutes to manufacture vitrification solutions described herein.
  • the glass forming agent(s) described herein is included in the cell vitrification medium described herein, typically in a concentration ranging from about 0.05 to about 1.0 molar; more preferably, in a concentration ranging from about from about 0.1 to about 0.8 molar; more preferably, in a concentration ranging from about from about 0.2 to about 0.75 molar; and most preferably, in a concentration ranging from about from about 0.25 to about 0.5 molar. Other concentration ranges are also contemplated.
  • the glass forming agent(s) is included in the cell vitrification medium described herein, typically in a concentration of about 0.1 molar, or about 0.15 molar, or about 0.2 molar, or about 0.25 molar, or about 0.3 molar, or about 0.35 molar, or about 0.4 molar, or about 0.45 molar, or about 0.5 molar, or about 0.55 molar, or about 0.6 molar, or about 0.65 molar, or about 0.7 molar, or about 0.75 molar. Other concentrations are also contemplated.
  • threonine or lactate at low concentrations (e.g., 0.5 - 2 millimolar) as part of a base solution formulation.
  • those compounds are not present to function as cryoprotectants, but as metabolic support molecules.
  • a cell vitrification medium that contains at least one glass forming agent selected from a carboxylic acid or carboxylate salt thereof at much higher levels, of more than about 10 millimolar; more preferably, more than about 50 millimolar; more preferably, more than about 100 millimolar.
  • the cell vitrification medium includes an aqueous cell medium, including water and traditional solutes, as the base medium to which the glass forming agent(s) are added.
  • base solution or “base medium” means a solid or liquid preparation made specifically for the growth, manipulation, transport or storage of a biological specimen or material present therein.
  • Exemplary cell base media include protein- supplemented Gamete Buffer and Tissue Culture Medium 199 (TCM I99). Some commercially available base media may also be used as a base medium and can be purchased from Invitrogen, Sigma-Aldrich, and other cell culture media manufacturers.
  • the vitrification media described herein may also contain sufficient metabolic substrates to maintain cell integrity, viability, and function throughout the vitrification and recovery processes.
  • the described cell vitrification medium can also include a nutrient-rich matrix that has a sufficient amount of cell metabolites to sustain the metabolic needs of the harvested biological material, including the developmental cells while incubating the cells.
  • Cell metabolites include nutrients that are easily absorbed into the cells to be preserved.
  • nutrients include one or more amino acids selected from alanine, arginine, asparagine, aspartic acid, cystine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, praline, serine, taurine, threonine, tryptophan, tyrosine, and valine.
  • the cell metabolites include one or more vitamins selected from the group comprising pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin, and thiamine.
  • the concentration of the amino acids may be chosen to match the amino acid concentration found in the healthy cytoplasm of the cells. Alternatively, the concentration of amino acids in the medium may be chosen to be proportional to the metabolic needs of the cells during normal cell metabolism.
  • the vitrification medium described herein can also include cell energy sources, such as adenosine, saccharides like glucose, or metabolites of glucose, such as pyruvate. These energy sources may be needed to supply immediate energy to the cells during vitrification process.
  • cell energy sources such as adenosine, saccharides like glucose, or metabolites of glucose, such as pyruvate.
  • the cell vitrification medium may include an inorganic salt.
  • Suitable salt-forming inorganic anions include chloride, phosphate, sulfate, and selenite.
  • Suitable salt-forming inorganic cations include sodium, potassium, magnesium, copper, and zinc cations.
  • the cell medium has a concentration of inorganic salts substantially equal to the concentration of inorganic salts found in the in vivo donor cells.
  • the cell vitrification medium can contain at least one hormone.
  • hormones include insulin; leutropic hormone; transferrin; somatropin; and linoleic acid.
  • the vitrification medium may also include one or more cell-permeating cryoprotectants, one or more non-cell-permeating cryoprotectants, or a combination thereof.
  • the cell vitrification medium can comprise one or more cell-permeating cryoprotectants selected from DMSO, glycerol, a glycol (e.g., a propylene glycol, an ethylene glycol), acetamide, formamide, or a combination thereof.
  • the vitrification medium can comprise one or more non cell-permeating cryoprotectants selected from one or more of various macromolecules (e.g uniform polyvinylpyrrolidone, hydroxyethyl starch, Ficoll), a polysaccharide, a monosaccharide, a dissacharide, a sugar alcohol, trehalose (e.g. trehalose dehydrate), raffinose, a glucan, or a combination thereof.
  • various macromolecules e.g. uniform polyvinylpyrrolidone, hydroxyethyl starch, Ficoll
  • a polysaccharide e.g. uniform polyvinylpyrrolidone, hydroxyethyl starch, Ficoll
  • a polysaccharide e.g. uniform polyvinylpyrrolidone, hydroxyethyl starch, Ficoll
  • a polysaccharide e.g. uniform polyvinylpyrrolidone,
  • the concentration of the cryoprotectants in the described vitrification medium that includes the carboxylic acid or carboxylate salt glass forming agents may be in the range of 15 to 16 molal, which is significantly lower as compared to the typical concentration of 18 to 19 molal when no carboxylic acid or carboxylate salt glass forming agent(s) described herein are present in the medium.
  • the medium may also include a free oxygen radical scavenger to protect the cells from free oxygen radicals produced during cryopreservation.
  • free oxygen radical scavengers include allopurinol and glutathione.
  • the cell medium can comprise glycine, glutamine/glutamic acid, and cystine, amino acids that are rapidly converted by the cell into glutathione.
  • the cell medium may be buffered with a mild buffer solution having a content and concentration such that the cell medium has a first pH that ranges from about 7.3 to about 7.5 at a temperature above 35° C.
  • a suitable buffer includes a sodium carbonate buffer, an N-[Hydroxyethyl] piperazine-N'[2-ethananesulfonic acid] (“HEPES”) buffer, or a (3-(N- morpholino)propanesulfonic acid; (“MOPS”) buffer, or a combination of these.
  • the cell vitrification medium may further include complex biological solutions such as egg yolk or serum. Although these compounds may be difficult to use in a clinical cryopreservation solution, they may be used more readily with animal cells.
  • Certain embodiments relate to a method of vitrification of a biological specimen or material, such as mature, unfertilized oocytes harvested from a donor or infertility patient or embryos used in infertility treatments, using the vitrification medium described herein.
  • the cell vitrification medium is an aqueous solution that contains one or a combination of the glass forming agents selected from a carboxylic acid or carboxylate salt thereof, such as sodium propionate, sodium lactate and threonine.
  • the described method that utilizes the described vitrification medium is useful in preserving a biological specimen or material obtained from any source including cells obtained from any donor species, organism, organ or tissue and especially useful for the preservation of mature, unfertilized oocytes harvested from a donor.
  • the same cell vitrification medium may or may not be used during all phases of the vitrification process, i.e., cell harvesting, preconditioning, dehydrating, cooling, and thawing.
  • the described method of vitrification of mature, unfertilized oocytes harvested from a donor comprises suspending the oocytes harvested from the donor in a solution designed to load cryoprotectants into the cytoplasm of cells.
  • This solution may contain one or more agents selected from a carboxylic acid or carboxylate salt.
  • the cells are transferred into a cell vitrification medium, allowing further loading of cryoprotectants and also rapid dehydration of the oocytes, followed by cooling the oocyte suspension in the cell vitrification medium, wherein the cell vitrification medium comprises an aqueous solution, at least one glass forming agent selected from a carboxylic acid or carboxylate salt thereof, along with other components of the medium (e.g., water and traditional solutes).
  • the cell vitrification medium comprises an aqueous solution, at least one glass forming agent selected from a carboxylic acid or carboxylate salt thereof, along with other components of the medium (e.g., water and traditional solutes).
  • the cells are initially harvested by any process capable of separating the desired cells from a donor source, such as, for example, an organ or tissue sample.
  • a donor source such as, for example, an organ or tissue sample.
  • the process of isolating the cells should minimize damage to the cells being isolated, such that a maximum number of usable cells are obtained for later use.
  • the cells may be harvested at temperatures higher than about 4°C. to maximize the energy state of the cells, so that the cells are maintained in an environment that is similar to their normal environment.
  • the temperature of the donor source is maintained between about 34° C.-38° C., although any temperature which maintains normal cell metabolism and protects the cells against subsequent damage is suitable.
  • the temperature may be maintained at 37°C.
  • cell isolation may be accomplished by any techniques that are well-known to the skilled artisan. Exemplary methods of harvesting biological specimens, including mature, unfertilized oocytes are known in the art and would be available to a person skilled in the art. See, e.g., U.S. Pat. No. 7,384,391 , which is incorporated herein in its entirety.
  • preconditioning may aid the vitrification process in reversing the damage inflicted through harvesting.
  • Preconditioning of the cells may include the steps of washing the cells, forming a suspension of the cells using a suitable cell medium, and then incubating the cells in a suitable cell medium.
  • the cells may be subjected to mild stress to condition them to the stresses associated with the vitrification process.
  • the dehydrating and cooling step of the described method can include suspending the biological material harvested from the donor in a cryoprotectant loading medium, then a cell vitrification medium, and cooling the biological material suspension in the cell vitrification medium to cryogenic temperatures.
  • cryoprotectant loading refers to the process of permeating cryoprotectant diffusion into the cells.
  • vitrification device(s) or “storage device(s)” refer to devices used to vitrify a biological specimen or material.
  • Exemplary vitrification devices include vials, straws, needle assembly/capsule combinations, and other commercially-available devices, including, e.g., Cryotop® Vitrification Device, Cryolock® Vitrification Device, Rapid-1 carrier, and High Security Vitrification devices (e.g., straw).
  • the vitrification devices containing the biological material are cooled fast enough to vitrify the medium containing the biological material by exposing them to cryogenic media.
  • the term “exposing” in the context of “exposing the biological material to a cryogenic media” includes directly or indirectly exposing the material to the freezing material.
  • the term “freezing material” refers to any material, including but not limited to, liquid gases such as liquid nitrogen (including liquid nitrogen slush), liquid propane, liquid helium or ethane slush, which are capable of causing vitrification of a biological material.
  • the term “directly exposing” in the context of the method described herein refers to directly exposing a biological specimen or material, including mature, unfertilized oocytes, to a cryogenic medium if the majority of the surface of the biological specimen, or the medium, solution or material in which the biological specimen resides, is allowed to come into direct contact with the cryogenic medium.
  • the vitrification step of the presently described method can include plunging a biological specimen or a biological specimen suspension (i.e., the biological specimen with any media in which the specimen in suspended) into liquid nitrogen.
  • the term “indirectly exposing” refers to exposing the biological material fully contained and sealed in a storage device to the cryogenic medium.
  • the vitrification step of the presently described method can include plunging a storage device containing the biological specimen or a biological specimen suspension (i.e., the biological specimen with any media in which the specimen in suspended) into liquid nitrogen.
  • the biological specimen Upon exposure to the cryogenic medium, the biological specimen undergoes vitrification.
  • the biological specimen which has undergone vitrification may be stored for a period of time, and then warmed at a later date.
  • the warmed biological specimen remains viable.
  • Preferred biological specimens according to the present invention are developmental cells, such as mature, unfertilized oocytes.
  • the same vitrification medium is used during all phases of cryopreservation process, e.g., preconditioning, vitrifying and thawing.
  • an increase in the concentration of a glass forming agent, such as threonine can result in a relative reduction in the concentration of 1 ,2-propanediol up to 30-fold, depending upon the concentration used (average approximately 8.5 in a range of 0.5 to 3% threonine).
  • the temperature at which cryopreserved preparations are stored affects the length of time after which the material can be recovered successfully.
  • biological material should be stored in liquid nitrogen freezers.
  • Liquid nitrogen units that provide all-vapor storage are ideal as long as the working temperature at the opening of the unit remains below - 130°C.
  • a cryotank e.g. liquid nitrogen LN2 - tank.
  • the method may further include warming the biological material and unloading, by dilution and removal of any cryoprotectants from the cytosol.
  • the vitrified/cryopreserved biological material can be later thawed for use, during which the vitrified cryopreservation medium will convert to a liquid form.
  • the biological material can be removed from the freezer or other cryopreservation storage equipment and allowed to thaw by exposure to room temperature, while in other forms the cryopreserved biological material can be warmed in a liquid bath or a warming medium (e.g., at a heated temperature, such as 37°C), which can for example be set to a constant temperature or progressively warmed to thaw the product.
  • the cells may be post-conditioned, after the cell suspension reaches the desired temperature. Similar to cell preconditioning, the cell post- conditioning is done to reverse stresses caused by freezing and to prevent cell damage caused by rewarming and reoxygenation.
  • the post-conditioning can include the steps of cryoprotectant removal and incubation.
  • the warming process for the biological material includes placing the end of the vitrification device into the warming solution e.g., solution 3 as shown in Table 5; Blastocyst Warming Kit Warming Solution I (0.33 mol/L Trehalose)) to warm the biological material and allow the cryoprotectants to diffuse out of the biological material.
  • the warming solution e.g., solution 3 as shown in Table 5; Blastocyst Warming Kit Warming Solution I (0.33 mol/L Trehalose)
  • the biological material and associated device can be warmed as a whole, with the holding assembly/capsule combination remaining received within the vial or other exterior container (potentially maintained with a sterile seal), or the holding assembly can be separated from the vial or other container prior to warming above cryogenic temperatures.
  • Warming can be performed over any suitable period of time, for example over a period of time from about I second to 30 minutes. However, as noted above, for vitrification, the re- warming processes are rapid enough to avoid ice crystal formation.
  • the biological material can be subsequently exposed to the remaining solutions for the times and temperatures necessary to avoid damage (i.e. as little as 0.1 seconds to several minutes for larger biological materials) and ranging from slightly above the melting temperature of the vitrification solution (e.g., - 15 degrees C) up to 37 degrees C, before using the biological material, e.g., for in vitro fertilization.
  • exemplary solutions to which oocytes were exposed during the cryoprotectant loading (Soln. I &2) and unloading (Soln. 3-5), and the respective times and temperatures of exposure are provided in Table 5 in Example I below.
  • Certain further embodiments relate to a use of at least one glass forming agent selected from a carboxylic acid or carboxylate salt thereof in a method of vitrification of mature, unfertilized oocytes harvested from a donor.
  • Certain additional embodiments relate to a method of reducing the total concentration of solutes by at least 2%, or at least 5%, or at least 10%, or at least 15% in an aqueous solution used for vitrification of mature, unfertilized oocytes harvested from a donor, comprising adding a glass forming vitrification agent selected from a carboxylic acid or carboxylate salt thereof at a concentration of from about 0.1 to about 0.75 molar to the aqueous medium, wherein the vitrification agent reduces the total concentration of solutes in the aqueous solution.
  • the carboxylic acid present in the cryopreservation medium can comprise at least three carbons.
  • the carboxylic acid present in the cryopreservation medium can comprise at least four carbons.
  • the carboxylic acid present in the cryopreservation medium may be an alpha-hydroxy- and/or beta-hydroxy- carboxylic acids.
  • the carboxylic acid may also be an amino acid.
  • the carboxylate salt may be selected from the group consisting of one or more of a sodium salt, a potassium salt, or other monovalent cations.
  • the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine.
  • the glass forming agent is selected from the group consisting of sodium propionate, sodium lactate and threonine.
  • Yet further embodiments relate to a method of reducing toxicity of an aqueous solution used for vitrification of mature, unfertilized oocytes harvested from a donor, comprising adding a glass forming vitrification agent selected from a carboxylic acid or carboxylate salt thereof at a concentration of from about 0.1 to about 0.75 molar to the aqueous solution used for vitrification medium, wherein the vitrification agent reduces the toxicity of the solution by at least 5%, or at least 7.5%, or at least 10%, or at least 15% by substituting for other, more toxic, cryoprotectants.
  • the carboxylic acid present in the cryopreservation medium can comprise at least three carbons.
  • the carboxylic acid present in the cryopreservation medium can comprise at least four carbons.
  • the carboxylic acid present in the cryopreservation medium may be an alpha-hydroxy- and/or beta-hydroxy- carboxylic acids.
  • the carboxylic acid may also be an amino acid.
  • the carboxylate salt may be selected from the group consisting of one or more of a sodium salt, a potassium salt, or other monovalent cations.
  • the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine.
  • the glass forming agent is selected from the group consisting of sodium propionate, sodium lactate and threonine.
  • the CryoBuffer range may be from apx. 40 - 70 % by volume; the Ethylene Glycol range may be from about 0 - 60 % by volume; the DMSO range may be from about 0 - 50 % by volume; the Trehalose Dihydrate range may be from about 0.05 to 1.0 moles per liter of solution.
  • the amount of the more toxic agents listed above may be reduced by between 2 and 20 percent relative to the percent in a cryopreservation solution that did not contain those compounds.
  • the process can simply be described as adding all of the components to water for dissolution, sterilizing said solution in a manner that does not affect its properties (sterile filtration is commonly applied for this type of solution), and then dispensing the solution into appropriate containers such as sterile glass or plastic bottles.
  • Example I Vitrification Study
  • Treatment solution I which is a first newly tested vitrification solution of this current study:
  • Treatment solution 2 which is a second newly tested vitrification solution of this current study:
  • Treatment solution 3 is the standard vitrification solution:
  • Figure 2 shows a survival plot for vitrification study.
  • Table 5 includes solutions to which oocytes were exposed during the cryoprotectant loading (Soln. I &2) and unloading (Soln. 3-5), and the respective times and temperatures of exposure.
  • Mature eggs were collected from an Fl strain of mouse using standard superovulation and collection procedures. After removing the cumulus cells, the eggs were randomly allocated to one of the four treatment conditions (one condition was a control, where the eggs were subjected to in vitro fertilization and embryo culture (IVF/EC) only. Eggs being vitrified were exposed to Solution I as indicated in Table 5, then moved to Solution 2 (the vitrification solution associated with the assigned treatment). After exposure to solution 2, the eggs were pipetted onto a vitrification device and placed in liquid nitrogen for storage. The warming process consisted of placing the end of the vitrification device into the 3rd solution to warm the oocytes and begin to remove the cryoprotectants from them.
  • IVPF/EC in vitro fertilization and embryo culture
  • the oocytes were subsequently pipetted through the remaining solutions for the times and temperatures indicated, before using them for in vitro fertilization.
  • Surviving eggs were co-incubated with sperm from the same mouse strain as the egg donor animals for 4-5 hours in Sydney IVF Fertilization Medium. Subsequent to this incubation, eggs were washed free of sperm and incubated in Sydney IVF Cleavage Medium for apx. 96 hours.
  • Embryos were assessed for cleavage to 2-cell embryos, and also for development to morphologically-normal expanded blastocysts. Those data were used to compare the viability of the embryos developing from oocytes vitrified from the 3 treatments ( Figure 2 and Table 4).
  • Example 2 Reduction in the concentration necessary to vitrify when substituting a glass forming agent selected from a carboxylic acid or carboxylate salt for ethylene glycol.
  • Solutions were made by adding sodium propionate or sodium lactate to ethylene glycol in water so that the minimum concentration of ethylene glycol necessary to vitrify when the sodium lactate or sodium propionate were at concentrations ranging from 0.5 to 5 % by weight, at 0.5% increments, was determined.
  • Figure 3 shows the relationship between lactate or propionate molality and total solute molality necessary to vitrify in a 1/4 cc CryoStraw, where ethylene glycol is the additional solute.
  • ethylene glycol is the additional solute.
  • lactate and propionate have superior glass forming properties over ethylene glycol. Based on the date, lactate and/or propionate are a new class of organic compounds that can be successfully used as glass forming agents for cryopreservation.
  • Example 3 - Reduction in the concentration necessary to vitrify when substituting a glass forming agent selected from a carboxylic acid or carboxylate salt for propylene glycol.
  • Solutions were made by adding sodium propionate or sodium lactate to propylene glycol in water so that the minimum concentration of propylene glycol necessary to vitrify when the sodium lactate or sodium propionate were at concentrations ranging from 0.5 to 5 % by weight, at 0.5% increments, was determined.
  • a control experiment was also assessed, where trehalose was used as the solute substitute for propylene glycol, to determine if a beneficial effect of similar magnitude would be seen with a non-carboxylate compound.
  • Figure 4 shows the relationship between lactate or propionate molality and total solute molality necessary to vitrify in a 1/4 cc CryoStraw, where propylene glycol is the additional solute.
  • lactate and/or propionate have superior glass forming properties over propylene glycol. Based on the data, lactate and/or propionate are a new class or organic compounds that can be successfully used as glass forming agents for cryopreservation solutions.

Abstract

La présente invention concerne un milieu de cryoconservation et un procédé de cryoconservation d'un matériau biologique récolté auprès d'un donneur, tel que des oocytes matures, non fertilisés récoltés auprès d'un donneur. Le milieu de cryoconservation de cellules comprend une solution aqueuse et un agent de formation de verre sélectionné parmi un acide carboxylique ou un sel carboxylate de ce dernier. Une augmentation dans la concentration de l'agent de formation de verre dans le milieu de vitrification permet une réduction relative de la concentration du cryoprotecteur dans le milieu de vitrification.
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