RELATED APPLICATIONS

[0001] The present patent document claims the benefit of the filing date under 35 U.S.C. § 1 19(e) of Provisional U.S. Patent Application Serial No. 63/255, 176, filed October 13, 2021 , which is hereby incorporated by reference.

BACKGROUND

[0002] Described herein is a composition and a method for vitrification of a biological specimen or material, such that the biological specimen remains viable after it is warmed.

[0003] Specifically, described here is a composition 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.

[0004] Preserving cells for use in future applications has become critical in many areas of biomedicine and agriculture.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] Traditionally, embryos have been cryopreserved using “slow freezing techniques” that utilize relatively low concentrations of cryoprotectants.

[0009] The ability to cryopreserve oocytes, embryos, sperm and other similar biological specimens is critical to the widespread application of assisted reproductive technologies. However, due to several factors such as the large volume of the cells and the high chilling sensitivity of oocytes and early embryos, cryopreservation techniques are not well developed in most species.

[0010] Low concentrations of cryoprotectants, such as DMSO, and 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. However, 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.

[0011] Alternative cryopreservation methods have relied on vitrification with high concentrations of cryoprotectants, which when cooled result in a glass-like state. However, a disadvantage of vitrification techniques is that the necessary 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.

[0012] Therefore a need remains for an improved media composition and new methods for the vitrification of biological specimens, which will be less toxic and able to maximize the viability of the specimen during vitrification and subsequent thawing. Ideal methods will prevent thermomechanical stress to the specimen, have limited toxicity, and provide ease of manipulations during cryopreservation and recovery.

SUMMARY

[0013] 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. In the method, the carboxylic acid may comprise at least three carbons. In the method, the carboxylic acid may be an alpha- hydroxy- and/or beta-hydroxy- carboxylic acids. In the method, 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. In the method, 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. In the method, 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. In the method, 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. In the method, the cooling step may comprise plunging the oocyte suspension in liquid nitrogen. In the method, 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. In the method, 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. 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. [0014] 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) cropreserving the oocyte suspension by vitrifying the oocyte 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. In the method, the carboxylic acid may comprise at least three carbons. In the method, the carboxylic acid may be an alpha- hydroxy- and/or beta-hydroxy- carboxylic acid. In the method, 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. In the method, 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. In the method, 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. In the method, 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. In the method, the cooling step may comprise plunging the oocyte suspension in liquid nitrogen. In the method, 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. In the method, 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.

[0015] 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.

[0016] 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.

[0017] 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. In the method, the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine.

[0018] 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. In the method, the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine.

[0019] Yet a further embodiment relates to 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure I shows chemical structures of exemplary glass forming agents.

[0021] Figure 2 depicts a survival plot for vitrification study.

[0022] 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).

[0023] 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).

DETAILED DESCRIPTION OF THE DRAWINGS AND THE PRESENTLY PREFERRED EMBODIMENTS

[0024] While the present invention may be embodied in many different forms, for the purpose of promoting an understanding of the principles of the present invention, reference will now be made to embodiments, some of which are illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications in the described embodiments and any further applications of the principles of the present invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates. Additionally, in the detailed description below, numerous alternatives are given for various features related to the structure or composition of materials, or to modes of carrying out methods. It will be understood that each such disclosed alternative, or combinations of such disclosed alternatives, can be combined with the more generalized features discussed in the Summary above, or set forth in the Listing of Certain Embodiments below, to provide additional disclosed embodiments herein. [0025] 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. Although throughout this specification, specific embodiments refer to oocytes, the described vitrification media and methods may be applicable to any biological specimen or material, including any viable cells.

[0026] The terms “cryopreservation medium” or “cell cryopreservation medium” refer 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. [0027] The term “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.

[0028] Specifically, described herein are components used to formulate a cell vitrification medium and a method of vitrification of a biological specimen or material. The terms “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). The term “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„ mature, unfertilized oocytes and immature oocytes), embryos, morulae, blastocysts, and other early embryonic cells. For example, 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.

[0029] Vitrification, or the transition of an aqueous solution from a liquid state to a glassy state, is one cryopreservation method. The term “cryopreservation" refers to the preservation of a biological specimen or material at extremely low temperature.

[0030] The term “vitrification” (vitrify) 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.

[0031] There are several ways to maximize survival using vitrification procedures. Osmotic damage can be mitigated. Separate solutions for the cryoprotectant loading and the dehydration/vitrification steps can be used. Loading solutions that will not only increase the glass-forming tendency of the cytoplasm but will also reduce injury during the dehydration step can be formulated. Less toxic and more effective vitrification solutes can be combined. Cold acclimation to increase both the tolerance of cellular membranes to dehydration and the glass-forming tendency of the cytosol can be applied. [0032] For practical application, aqueous solutions must be formulated that are able to undergo vitrification at easily attainable cooling and warming rates. Typically, 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. The term “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). However, as noted above, 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.

[0033] Described herein is the use of solutes (e.g., “glass forming agent(s)”) that reduce the total concentration or cryoprotectants known to be necessary to achieve and maintain the vitreous state, e.g„ 5-7 moles per L, to produce less toxic and more effective vitrification solutions.

[0034] Also, described herein are effective vitrification media formulations 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. Also, surprisingly, by using the described glass forming agent(s) in the described media formulations, a less toxic (due to lesser amounts of the commonly used cryoprotectant(s), such as ethylene glycol) and more effective vitrification media is produced.

[0035] Cell Cryopreservation/Vitrification Medium

[0036] 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. The term 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 . The term “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. The term “traditional cryoprotectants” means cryoprotectants that have been routinely used for biological material cryopreservation, and do not include the novel solutes described in this disclosure. Examples of traditional cryoprotectants include dimethyl sulfoxide, ethylene glycol, propylene glycol, and glycerol.

[0037] 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.

[0038] In certain embodiments, 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.

[0040] In certain further embodiments, the carboxylic acid may also be an amino acid.

[0041] In certain embodiments, 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.

[0042] In certain embodiments, the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine.

[0043] In specific, preferable embodiments, 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.

[0044] For example, in certain embodiments, 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. In certain embodiments, the powder form of threonine would be used in aqueous solutions in combination with other solutes to manufacture vitrification solutions described herein.

[0045] 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. As demonstrated in the Examples, unexpectedly and surprisingly, in the case of 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. In the case of 1 ,2-propanediol, a relatively strong glass forming agent, an increase in the concentration of 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).

[0046] In certain other embodiments, 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

NaC ₃ H ₅ O ₃ . Similarly to threonine, sodium lactate is a white powder in its purified form. In certain embodiments, the powder form of sodium lactate would be used in aqueous solutions in combination with other solutes to manufacture vitrification solutions described herein.

[0047] In certain further embodiments, 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. As noted above, sodium propionate is a crystalline solid in its purified form. In certain embodiments, the crystalline solid form of sodium propionate would be used in aqueous solutions in combination with other solutes to manufacture vitrification solutions described herein.

[0048] In certain embodiment, 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.

[0049] In certain embodiments, 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. [0050] It needs to be noted that some commercially available media solutions may already contain threonine or lactate at low concentrations (e.g., 0.5 - 2 millimolar) as part of a base solution formulation. However, those compounds are not present to function as cryoprotectants, but as metabolic support molecules. In contrast, described herein is 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.

[0051] 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. The term “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. [0052] In certain embodiments, the vitrification media described herein may also contain sufficient metabolic substrates to maintain cell integrity, viability, and function throughout the vitrification and recovery processes.

[0053] For example, in certain embodiments, 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.

[0054] Cell metabolites include nutrients that are easily absorbed into the cells to be preserved. Examples of 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. Additionally, 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.

[0055] In certain embodiments, 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.

[0056] In some embodiments, 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. In additional embodiments, the cell medium has a concentration of inorganic salts substantially equal to the concentration of inorganic salts found in the in vivo donor cells.

[0057] In certain embodiments, the cell vitrification medium can contain at least one hormone. Examples of hormones include insulin; leutropic hormone; transferrin; somatropin; and linoleic acid.

[0058] In certain embodiments, the vitrification medium may also include one or more cell-permeating cryoprotectants, one or more non-cell-permeating cryoprotectants, or a combination thereof. In these regards, 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. In addition or alternatively, the vitrification medium can comprise one or more non cell-permeating cryoprotectants selected from one or more of various macromolecules (e.g„ 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. However, as shown in the Examples below, surprisingly, the presence of the carboxylic acid or carboxylate salt glass forming agents in the cell vitrification medium described herein allows for significantly lower total concentrations of the cryoprotectants.

[0059] For example, 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.

[0060] In certain embodiments, the medium may also include a free oxygen radical scavenger to protect the cells from free oxygen radicals produced during cryopreservation. Examples of the free oxygen radical scavengers include allopurinol and glutathione. Alternatively, the cell medium can comprise glycine, glutamine/glutamic acid, and cystine, amino acids that are rapidly converted by the cell into glutathione.

[0061] In certain embodiments, 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. Preferably, the solution allows pH buffering to prevent extremes in pH during the process of vitrification. 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.

[0062] In certain embodiments, 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.

[0063] Preferred embodiments of the vitrification medium described herein are provided in the Examples section below.

[0064] Methods and Uses

[0065] 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.

[0066] 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.

[0067] In certain embodiments, 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. Then 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).

[0068] I) Cell Harvesting

[0069] 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. 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.

[0070] In certain embodiments, 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. In certain embodiments, 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. In one embodiment, the temperature may be maintained at 37°C. Also, 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. [0071] 2) Preconditioning

[0072] Once the cells have been harvested, they may or may not be preconditioned. In certain embodiments, 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. In other embodiments, the cells may be subjected to mild stress to condition them to the stresses associated with the vitrification process.

[0073] 3) Dehydrating and Cooling

[0074] 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.

[0075] The critical components of the described process of vitrification are described in detail below.

[0076] First, a biological material, such as eggs or embryos, is exposed to the cryoprotectant loading medium that includes at least one permeating cryoprotectant, and subsequently is exposed to the vitrification medium described herein that contains at least one carboxylic acid or carboxylate salt to achieve rapid dehydration of cells and final intracellular cryoprotectant concentration. The term “cryoprotectant loading” refers to the process of permeating cryoprotectant diffusion into the cells.

[0077] Second, the biological material is loaded into vitrification or storage devices that will facilitate cooling at a rate to achieve vitrification. The term “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).

[0078] Third, 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. [0079] 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.

[0080] 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. For example, 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. [0081] The term “indirectly exposing” refers to exposing the biological material fully contained and sealed in a storage device to the cryogenic medium. For example, 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.

[0082] 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.

[0083] Advantageously, in some embodiments, the same vitrification medium is used during all phases of cryopreservation process, e.g., preconditioning, vitrifying and thawing.

[0084] As demonstrated in the Examples, unexpectedly and surprisingly, in the case of ethylene glycol (Example 2), a commonly used solute in cryopreservation solutions and a relatively weak glass forming agent, an increase in the concentration of a glass forming agent, such as 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. In the case of 1 ,2-propanediol, a relatively strong glass forming agent, 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).

[0085] 4) Storage

[0086] The temperature at which cryopreserved preparations are stored affects the length of time after which the material can be recovered successfully. In certain embodiments, for ultimate security and maximum stability, 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. One example of a suitable storage unit is a cryotank (e.g. liquid nitrogen LN2 - tank).

[0087] 5) Thawing/Re-warming

[0088] The method may further include warming the biological material and unloading, by dilution and removal of any cryoprotectants from the cytosol.

[0089] For vitrification, the cooling and re-warming processes are rapid enough to avoid ice crystal formation.

[0090] Following storage, the vitrified/cryopreserved biological material can be later thawed for use, during which the vitrified cryopreservation medium will convert to a liquid form. This can be accomplished in any suitable manner. In some forms, 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.

[0091] Similar to freezing, damage can occur as the result of thawing. T o minimize or avoid potential damage, 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.

[0092] In one embodiment, 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.

[0093] In these or other warming modes, during the warming, 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.

[0094] 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.

[0095] 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.

[0096] 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. In certain embodiments, 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. 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. In certain embodiments, the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine. In specific, preferable embodiments, the glass forming agent is selected from the group consisting of sodium propionate, sodium lactate and threonine.

[0097] 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. In certain embodiments, 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. 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. In certain embodiments, the carboxylic acid may be one or more of propionic acid, lactic acid, and threonine. In specific, preferable embodiments, the glass forming agent is selected from the group consisting of sodium propionate, sodium lactate and threonine.

[0098] Manufacturing of the compositions described herein [0099] Manufacturing a solution containing a glass forming agent comprised of a carboxylic acid or carboxylate salt would be similar to the manufacture of similar solutions without the glass forming agents described herein. The various components of the solution were described above, and can include, e.g„ Cryo Buffer (Cook Medical), Ethylene Glycol (Sigma Alrich), DMSO (Avantor), and Trehalose Dihydrate (Acros Organics).

[00100] 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.

[00101] In certain embodiments, by using the glass forming agents described herein, 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.

[00102] 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.

[00103] EXAMPLES

[00104] Example I : Vitrification Study

[00105] I) Treatment formulations

[00106] The vitrification solution formulations used in this study were as follows:

[00107] Treatment solution I, which is a first newly tested vitrification solution of this current study:

[00108] Table I.

[00109] Treatment solution 2, which is a second newly tested vitrification solution of this current study:

[001 10] Table 2.

[00111] Treatment solution 3 is the standard vitrification solution:

[00112] Table 3.

[00113] Figure 2 shows a survival plot for vitrification study.

[00114] Table 4.

[00115] 2) Solutions Associated with the Steps of the Vitrification Procedure

[00116] 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.

[00117] Table 5.

[00118] Mature eggs (oocytes) 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. 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).

[00119] 3) Comparison of Three T reatment Solutions [00120] Table 6.

[00121] It was found that solutions containing threonine and/or lactate at concentrations of 0.25 molal had advantageous and surprising effect of allowing for significantly lower concentration of standard cryoprotectants as compared to the commercially available vitrification or cryopreservation solutions.

[00122] It needs to be noted that some solutions from some companies may already contain Threonine or Lactate at low concentrations (e.g., I - 2 millimolar) as part of a base solution formulation. However, at those concentrations, the compounds function as metabolic support molecules rather than as cryoprotectants. Furthermore, these results were not derived from an attempt to optimize the new vitrification solution formulation, meaning that even better results may be obtained after such an optimization.

[00123] 4) Conclusion

[00124] The data suggests a meaningful improvement was seen in the outcomes using the new cryopreservation solutions (treatment I and 2) with the compounds under consideration in this disclosure compared to the standard cryopreservation solution [00125] Survival for one of the two treatments using new formulations was superior to the standard treatment by 4 % (89% survival vs. 85%) and the other new formulation had 7 % better survival (92% vs 85%).

[00126] Development of embryos in vitro relative to the untreated controls resulted in one of the two new formulations being superior to the standard treatment by 10% (68% vs. 58%), which is a clinically-relevant improvement. The other formulation had similar, although slightly better, results compared to the standard treatment [00127] Based on the above, it may be possible to develop a formulation that provides results even better than those that we have seen to date, upon further optimization of the chemistry.

[00128] 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.

[00129] Formulation

[00130] The concentration of ethylene glycol dissolved in water necessary for the solution to maintain the vitrified state during cooling and warming in a 1/4 cc CryoStraw was initially determined to the precision of ± I wt %.

[00131] 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.

[00132] The corresponding concentrations were graphed to assess the relationship between the concentration of sodium propionate and sodium lactate, and the total solute concentration in the solution, could be assessed.

[00133] If the sodium lactate or sodium propionate solute had an equal effect on the vitrifiability of the solution as ethylene glycol, then the lines on the graph should be approximately horizontal.

[00134] 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. Surprisingly, as the concentration of lactate or propionate increases, the total solute concentration necessary to vitrify decreases, suggesting that lactate and propionate are stronger glass forming agents as compared to ethylene glycol. [00135] Clearly, lactate and/or 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. [00136] 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.

[00137] Formulation

[00138] The concentration of propylene glycol dissolved in water necessary for the solution to maintain the vitrified state during cooling and warming in a 1/4 cc CryoStraw was initially determined to the precision of ± I wt %.

[00139] 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. [00140] 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.

[00141] The corresponding concentrations were graphed to assess the relationship between the concentration of sodium propionate and sodium lactate, and the total solute concentration in the solution, could be assessed.

[00142] If the sodium lactate or sodium propionate solute, or trehalose solute, had an equal effect on the vitrifiability of the solution as propylene glycol, then the lines on the graph should be approximately horizontal.

[00143] 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. Surprisingly, as the concentration of lactate or propionate increases, the total solute concentration necessary to vitrify decreases, suggesting that lactate and propionate are stronger glass forming agents compared to propylene glycol. The results with the trehalose solution suggest that the effect is specific for the carboxylate compounds, as the graph for the trehalose data is approximately horizontal.

[00144] Clearly, 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.