Patent Description:
The present disclosure relates to the field of cell, tissue and organ preservation, particularly new ice-free formulations (e.g., for vitrification) incorporating sugars, such as disaccharides (e.g., trehalose and sucrose), and protocols that improve sample material properties and biological viability, even as sample volumes are increased to greater than <NUM>. More specifically, the invention relates to a method for preserving large volume cellular materials having a volume greater than <NUM>, such as greater than <NUM>, with at least one of the disaccharides trehalose and sucrose, in an effort to enhance cell survival and tissue functions post-preservation.

Conventional approaches to ice-free cryopreservation have been successful for storage of relatively small sample sizes. For example, human oocyte storage where it has revolutionized clinical in vitro fertilization practice.

In order for the cells or tissues to be preserved, cryoprotectant solutions are typically used to prevent damage due to freezing during the cooling or thawing/warming process. For cryopreservation to be useful, the preserved sample should retain the integrity and/or viability thereof to a reasonable level post-preservation. Thus, the process of preserving the sample should avoid and/or limit the damage or destruction of the cells and/or tissue architecture.

Vitrification, cryopreserved storage in a "glassy" rather than crystalline phase, is an important enabling approach for tissue banking and regenerative medicine, offering the ability to store and transport cells, tissues and organs for a variety of biomedical uses. In ice-free cryopreservation by vitrification the formation of ice is prevented by the presence of high concentrations of chemicals known as cryoprotectants that both interact with and replace water and, therefore, prevent water molecules from forming ice. This approach essentially stops biological time during storage below the cryoprotectant formulations glass transition temperature (Tg), and has been used successfully to maintain the viability and function of small-scale cell and thin tissue samples due to diffusive (heat and mass transfer) and phase change limitations that preclude use in bulk systems such as organs and larger tissues. While previous vitrification techniques (and cryoprotecting agents used therewith, such as DSMO) employing conventional boundary convection warming techniques can sometimes be successful for samples up to <NUM>-<NUM>, ice-formation still occurs as the sample volume approaches <NUM> because conventional boundary convection warming in a bath does not provide fast enough warming rates. Ice formation results in cell and tissue destruction. The major limitations of vitrification for large tissue samples are potential cytotoxicity due to prolonged exposure to the cryoprotectants employed and ice-formation during rewarming.

It is known from document <CIT> (<NUM>-<NUM>-<NUM>) a vitrification solution for cryopreservation of organs, comprising dimethyl sulfoxide, acetamide, glycerine, glycols, propylene glycol, sucrose and polyglycol.

It was found that supplementation of ice-free vitrification formulations employed for large volume cellular materials (e.g., having a volume greater than <NUM>, such as greater than <NUM>) with <NUM> to <NUM> of at least one sugar selected from trehalose and/or sucrose, wherein the formulation contains the at least one sugar and a further cryoprotectant at a concentration of from <NUM> to <NUM>, resulted in increased cell survival post-preservation and tissue functions.

The present application thus provides new methodology for treatment of large volume cellular materials in which at least one sugar selected from trehalose and/or sucrose is added to ice-free vitrification cryoprotectant formulations Supplementation with these sugars reduces both cryoprotectant-induced cytotoxicity and the risk of ice formation during cooling and most importantly during rewarming.

The invention is directed to a method for preserving living large volume cellular material as set out in the claims.

In the following description, numerous details are set forth to provide an understanding of the present disclosure. However, it may be understood by those skilled in the art that the methods of the present disclosure may be practiced without these details.

At the outset, it should be noted that in the development of any such actual embodiment, numerous implementation-specific decisions may be made to achieve the developer's specific goals, such as compliance with system related and business related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort might be complex and time consuming but would nevertheless be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure. In addition, the composition used/disclosed herein can also comprise some components other than those cited. In the summary and this detailed description, each numerical value should be read once as modified by the term "about" (unless already expressly so modified), and then read again as not so modified unless otherwise indicated in context.

As used herein, the term "about" used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context. For example, it includes at least the degree of error associated with the measurement of the particular quantity. When used in the context of a range, the modifier "about" should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the range "from about <NUM> to about <NUM>" also discloses the range "from <NUM> to <NUM>.

Unless otherwise expressly stated herein, the modifier "about" with respect temperatures (°C) refers to the stated temperature or range of temperatures, as well as the stated temperature or range of temperatures +/- <NUM>-<NUM>% (of the stated temperature or endpoints of a range of temperatures) of the stated. Regarding cell viability and cell retention (%), unless otherwise expressly stated herein, the modifier "about" with respect to cell viability and cell retention (%) refers to the stated value or range of values as well as the stated value or range of values +/- <NUM>-<NUM>%. Regarding expression contents, such as, for example, with the units in either parts per million (ppm) or parts per billion (ppb), unless otherwise expressly stated herein, the modifier "about" with respect to cell viability and cell retention (%) refers to the stated value or range of values as well as the stated value or range of values +/- <NUM>-<NUM>%. Regarding expressing contents with the units µg/mL, unless otherwise expressly stated herein, the modifier "about" with respect to value in µg/mL refers to the stated value or range of values as well as the stated value or range of values +/- <NUM>-<NUM>%. Regarding molarity (M), unless otherwise expressly stated herein, the modifier "about" with respect to molarity (M) refers to the stated value or range of values as well as the stated value or range of values +/-<NUM>-<NUM>%. Regarding, cooling rates (°C/min), unless otherwise expressly stated herein, the modifier "about" with respect to cooling rates (°C/min) refers to the stated value or range of values as well as the stated value or range of values +/- <NUM>-<NUM>%.

Also, in the summary and this detailed description, it should be understood that a range listed or described as being useful, suitable, or the like, is intended to include support for any conceivable sub-range within the range at least because every point within the range, including the end points, is to be considered as having been stated. For example, "a range of from <NUM> to <NUM>" is to be read as indicating each possible number along the continuum between about <NUM> and about <NUM>. Additionally, for example, +/- <NUM>-<NUM>% is to be read as indicating each possible number along the continuum between <NUM> and <NUM>. Furthermore, one or more of the data points in the present examples may be combined together, or may be combined with one of the data points in the specification to create a range, and thus include each possible value or number within this range. Thus, (<NUM>) even if numerous specific data points within the range are explicitly identified, (<NUM>) even if reference is made to a few specific data points within the range, or (<NUM>) even when no data points within the range are explicitly identified, it is to be understood (i) that the inventors appreciate and understand that any conceivable data point within the range is to be considered to have been specified, and (ii) that the inventors possessed knowledge of the entire range, each conceivable sub-range within the range, and each conceivable point within the range. Furthermore, the subject matter of this application illustratively disclosed herein suitably may be practiced in the absence of any element(s) that are not specifically disclosed herein.

In addition, use of the "a" or "an" are employed to describe elements and components of the embodiments herein. This is done merely for convenience and to give a general sense of concepts according to the disclosure. This description should be read to include one or at least one and the singular also includes the plural unless otherwise stated.

The terminology and phraseology used herein is for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited.

Also, as used herein any references to "one embodiment" or "an embodiment" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily referring to the same embodiment.

As used herein, the term "room temperature" refers to a temperature of about <NUM> to about <NUM> at standard pressure. In various examples, room temperature may be about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM>.

As used herein, "cellular material" or "cellular sample" refers to living biological material containing cellular components, whether the material is natural or man-made and includes cells, tissues and organs, whether natural or man-made. Such terms also mean any kind of living material to be cryopreserved, such as cells, tissues and organs. In some embodiments, the cells, tissues and organs may be mammalian organs (such as human organs), mammalian cells (such as human cells) and mammalian tissues (such as human tissues).

As used herein, "large volume" as used in the phrase large volume cellular material" or " large volume sample" or "large volume cellular sample" refers to living biological material containing cellular components, whether the material is natural or man-made and includes cellular materials, tissues and organs, whether natural or man-made, where such living biological material containing cellular components has a volume greater than <NUM>, such as a volume greater than about <NUM>, or a volume greater than about <NUM>, or a volume greater than about <NUM>, or a volume greater than about <NUM>, or a volume greater than about <NUM>, or a volume greater than about <NUM>, or a volume in a range of from <NUM> to about <NUM>, such as a volume in a range of from <NUM> to about <NUM>, a volume in a range of from <NUM> to about <NUM>, or a volume in a range of from about <NUM> to about <NUM>, such as a volume in a range of from about <NUM> to about <NUM>, or a volume in a range of from about <NUM> to about <NUM>, or a volume in a range of from about <NUM> to about <NUM>, or a volume in a range of from about <NUM> to about <NUM>, or a volume in a range of from about <NUM> to about <NUM>, or a volume in a range of from about <NUM> to about <NUM>, or a volume in a range of from about <NUM> to about <NUM>, or a volume in a range of from about <NUM> to about <NUM>, or a volume in a range of from about <NUM> to about <NUM>. Such terms also include any kind of living material having such a volume to be cryopreserved, such as cellular materials, tissues and organs. In some embodiments, the tissues and organs having such a volume may be mammalian organs (such as human organs), mammalian cells and mammalian tissues (such as human tissues).

As used in the claimed method, the term "large volume cellular material" refers to cellular material having a volume greater than <NUM> that is obtained from liver, lung, intestine, heart, pancreas, testes, placenta, thymus, adrenal gland, arteries, veins, lymph nodes, bone or skeletal muscle. In one embodiment according to the claims, the cellular material can be selected from human organs and human tissues, selected from intestine, pancreas, testes, placenta, thymus, adrenal gland, arteries, veins, lymph nodes, bone or skeletal muscle. In some embodiments, the tissue or organ is obtained from a human such as a human liver, human lung, human intestine, human heart, human pancreas, human testes, human placenta, human thymus, human adrenal gland, human arteries, human veins, human lymph nodes, human bone or human skeletal muscle.

As used herein, the term "cell(s)" comprises any type of cell, such as, for example, somatic cells (including all kind of cells in tissue or organs), fibroblasts, keratinocytes, hepatocytes, cardiac myocytes, smooth muscle cells, stem cells, progenitor cells, oocytes, and germ cells. Such cells may be in the form of a tissue or organ. In some embodiments, the cells are from a mammal tissue or organ, such as a human tissue or organ described above.

As used herein, "preservation protocol" refers to a process for provision of shelf life to a cell containing, living biological material. Preservation protocols may include cryopreservation by vitrification and/or anhydrobiotic preservation by either freeze-drying or desiccation.

As used herein, the term "vitrification" refers to solidification either without ice crystal formation or without substantial ice crystal formation. In some embodiments, a sample to be preserved (e.g., such as a tissue or cellular material) may be vitrified such that vitrification and/or vitreous cryopreservation (in its entirety-from initial cooling to the completion of rewarming) may be achieved without any ice crystal formation. In some embodiments, a sample to be preserved (e.g., such as a tissue or cellular material) may be vitrified such that vitrification and/or vitreous cryopreservation may be achieved where the solidification of the sample to be preserved (e.g., such as a tissue or cellular material) may occur without substantial ice crystal formation (i.e., the vitrification and/or vitreous cryopreservation (in its entirety-from initial cooling to the completion of rewarming) may be achieved even in the presence of a small, or restricted amount of ice, which is less than an amount that causes injury to the tissue).

As used herein, a sample to be preserved (e.g., such as a tissue or cellular material) is vitrified when it reaches the glass transition temperature (Tg). The process of vitrification involves a marked increase in viscosity of the cryoprotectant solution as the temperature is lowered such that ice nucleation and growth are inhibited. Generally, the lowest temperature a solution can possibly supercool to without freezing is the homogeneous nucleation temperature Th, at which temperature ice crystals nucleate and grow, and a crystalline solid is formed from the solution. Vitrification solutions have a glass transition temperature Tg, at which temperature the solute vitrifies, or becomes a non-crystalline solid.

As used herein, the "glass transition temperature" refers to the glass transition temperature of a solution or formulation under the conditions at which the process is being conducted. In general, the methodology of the present disclosure is conducted at physiological pressures. However, higher pressures can be used as long as the sample to be preserved (e.g., such as a tissue or cellular material) is not significantly damaged thereby.

As used herein, "physiological pressures" refer to pressures that tissues undergo during normal function. The term "physiological pressures" thus includes normal atmospheric conditions, as well as the higher pressures that various tissues, such as vascularized tissues, undergo under diastolic and systolic conditions.

As used herein, the term "cryoprotectant" means a chemical that minimizes ice crystal formation in and around a tissue/organ when the tissue is cooled to subzero temperatures and results in substantially no damage to the tissue/organ after warming, in comparison to the effect of cooling without cryoprotectant.

As used herein, the term "sugar" may refer to any sugar. In some embodiments, the sugar is a polysaccharide. As used herein, the term "polysaccharide" refers to a sugar containing more than one monosaccharide unit. That is, the term polysaccharide includes oligosaccharides such as disaccharides and trisaccharides, but does not include monosaccharides. The sugar may also be a mixture of sugars, such as where at least one of the sugars is a polysaccharide. In some embodiments, the sugar is at least one member selected from the group consisting of a disaccharide and a trisaccharide. In some embodiments, the sugar is a disaccharide, such as, for example, where the disaccharide is at least one member selected from the group consisting of trehalose and sucrose. In some embodiments, the sugar is a trisaccharide, such as raffinose. The sugar may also be a combination of trehalose and/or sucrose and/or raffinose and/or other disaccharides or trisaccharides. In some embodiments, the sugar comprises trehalose. According to the claimed method the sugar is at least one of trehalose and/or sucrose.

As used herein, the term "functional after cryopreservation" in relation to a cryopreserved material means that the cryopreserved material, such as organs or tissues, after cryopreservation retains an acceptable and/or intended function after cryopreservation. In some embodiments, the cellular material after cryopreservation retains all its indented function. In some embodiments, the cellular cryopreserved material preserved by the methods of the present disclosure retains at least <NUM>% of the intended function, such as at least <NUM>% of the intended function, such as at least <NUM>% of the intended function, such as at least <NUM>% of the intended function, such as at least <NUM>% of the intended function, such as at least <NUM>% of the intended function, such as <NUM>% of the intended function. For example, along with preserving the viability of the cells, it may be important to also maintain/preserve the physiological function of the tissue/organ, e.g. for a heart the pumping function, and/or the ability of a tissue (e.g., those to be transplanted) to integrate with surrounding tissue.

As used herein, the term "sterile" means free from living germs, microorganisms and other organisms capable of proliferation.

As used herein, the term "substantially free of cryoprotectant" means a cryoprotectant in an amount less than <NUM> w/w %. In some embodiments, the methods of the present disclosure may use and/or achieve a medium/solution and/or cellular material that is substantially free of cryoprotectant, such as a cellular material that is substantially free of DMSO (i.e., the DMSO is in an amount less than <NUM> w/w %). In some embodiments, the methods of the present disclosure may use and/or achieve a medium/solution and/or cellular material that is substantially free of any cryoprotectant other than the sugar, such as sucrose and/or trehalose).

The present disclosure is directed to methods for preserving large volume living materials/samples/organ(s)/tissue(s) (The terms "materials," "samples,", "organ(s)", and "tissue(s)" are used interchangeably and encompass any living biological material containing cellular components).

The methods of the present disclosure comprise exposing the cellular material having a volume greater than <NUM> to a cryoprotectant formulation containing from <NUM> to <NUM> of at least one sugar selected from trehalose and/or sucrose, wherein the cellular material is obtained from liver, lung, intestine, heart, pancreas, testes, placenta, thymus, adrenal gland, arteries, veins, lymph nodes, bone or skeletal muscle,.

For example, in some embodiments, the cellular cryopreserved material preserved by the methods of the present disclosure retains at least <NUM>% of the intended function, such as at least <NUM>% of the intended function, such as at least <NUM>% of the intended function, such as at least <NUM>% of the intended function, such as at least <NUM>% of the intended function, such as at least <NUM>% of the intended function, such as <NUM>% of the intended function. For example, along with preserving the viability of the cells in tissues and organs, it may be important to also maintain/preserve the physiological function of the cell/tissue/organ, e.g. for a heart the pumping function, and/or the ability of a tissue/cell(s) (e.g., those to be transplanted) to integrate with surrounding tissue/cell(s).

In the methods of the present disclosure, the cells of the large volume cellular material (hereinafter referred to as "cells") are protected during cryopreservation after being brought into contact with the at least one sugar selected from trehalose and/or sucrose in combination with other cryoprotectants during cooling to the cryopreservation temperature and rewarming. In embodiments, being brought into contact with the at least one sugar selected from trehalose and/or sucrose in combination with other cryoprotectants during cooling and rewarming means that the risks of ice formation is minimized such that the viability of the cells does not significantly deteriorate because the cryoprotectant solution has been stabilized/protected by the at least one sugar selected from trehalose and/or sucrose in the cryopreservation formulation/solution/composition.

In embodiments, the solution, such as a known solution well suited for organ storage of cells, tissues and organs, may contain any effective amount of sugar that is effective to provide an environment more conducive to survival of the cells of the large volume cellular material during the preservation protocol.

In some embodiments, in the methods of the present disclosure a medium (the terms "medium" and "solution" are used interchangeably) containing the at least one sugar selected from trehalose and/or sucrose in combination with other cryoprotectants may be combined with cellular materials, such as tissues and organs to prepare a cryopreservation composition. The medium (which may be an aqueous medium) can contain any suitable concentration of the at least one sugar selected from trehalose and/ or sucrose in combination with other cryoprotectants for these purposes.

In some embodiments, at least one sugar, such as a disaccharide (e.g., trehalose and/or sucrose) in combination with other cryoprotectants, is used in an amount in the methods of the present disclosure such that it results in an improved viability (post-cryopreservation) of the living cellular material/sample selected from the group consisting of organs, cells and tissues, such as mammalian organs, mammalian cells, and mammalian tissues (including those which may be subsequently transplanted). The phrases, "improved cell viability" or "improved viability," refer, for example, to a cell viability (%) of at least <NUM>%, such as <NUM>% or more. The improved cell viability (%) may be <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, or <NUM>% or more.

In claimed embodiments, at least one sugar selected from trehalose and/or sucrose (and a further cryoprotectant) is used in an amount in the methods of the present disclosure such that it is effective to accomplish one or more of the following: inhibit ice nucleation, modify ice structure, decrease ice formation, prevent ice formation, decrease/prevent ice formation to an extent that would allow the use of more rapid cooling rates, decrease/prevent ice formation to an extent that would allow a reduction in the amount of cryoprotectant required providing an environment more conducive to cell survival.

In some embodiments, the at least one sugar selected from trehalose and/or sucrose represents from about <NUM> to about <NUM>% of the total weight of the medium comprising the cells to be preserved, such as from about <NUM> to about <NUM>%, or from about <NUM> to about <NUM>%, or from about <NUM> to <NUM>%, or from about <NUM> to about <NUM>%, or from about <NUM> to <NUM>%, or from about <NUM> to about <NUM>%, or from about <NUM> to about <NUM>% of the total weight of the formulation/solution/medium being used with the cells to be preserved.

In claimed embodiments, the formulation/solution/medium contains the at least one sugar selected from trehalose and/or sucrose at a concentration ranging from <NUM> to <NUM>, wherein any concentration occurring within the above ranges can also serve as an endpoint for a range.

In embodiments, the formulation/solution/medium comprising the at least one sugar selected from trehalose and/or sucrose may be contacted with the sample to be preserved for any desired duration, such as until a desired dosage (such as an effective dosage) of the at least one sugar selected from trehalose and/or sucrose is present on/in the cells or tissues to afford an improved viability (post-cryopreservation), and/or to prevent/protect against tissue damage and/or to accomplish one or more of the following: inhibit ice nucleation, modify ice structure, decrease ice formation, prevent ice formation, decrease/prevent ice formation to an extent that would allow the use of slower cooling and warming rates, decrease/prevent ice formation to an extent that would allow a reduction in the amount of cryoprotectant required to provide an environment more conducive to cell survival to preserve tissues.

In some embodiments, the cells to be cryopreserved may also be in contact with a freezing-compatible pH buffer comprised of, for example, at least a basic salt solution, an energy source (for example, glucose), and a buffer capable of maintaining a neutral pH at cooled temperatures. Well known such materials include, for example, Dulbecco's Modified Eagle Medium (DMEM). This material may also be included as part of the cryopreservation composition. See, e.g., <NPL>); and <NPL>).

In claimed embodiments, the cryoprotectant compounds (in total, including the sugars and any other cryoprotectant) are present in the cryopreservation composition in an amount of from about <NUM> to about <NUM>.

In unclaimed embodiments, the cellular material to be preserved may be brought into contact with a cryoprotectant-containing solution/medium/formulation/composition before, during or after incubating the cellular material to be preserved in a solution/medium/formulation/composition containing at least one sugar, such as a disaccharide (e.g., trehalose and/or sucrose). The duration that the tissue may be contacted by immersion and or perfusion in such solution/medium/formulation/composition will be a function of the mass of the tissue. In embodiments, the cooling rates of such solutions/mediums/formulations/compositions may be adjusted to provide adequate tissue permeation (function of concentration and time) to prevent ice formation.

Suitable further cryoprotectants may include, for example, acetamide, agarose, alginate, alanine, albumin, ammonium acetate, anti-freeze proteins, butanediols (such as <NUM>,<NUM>-butanediol), chondroitin sulfate, chloroform, choline, cyclohexanediols, cyclohexanediones, cyclohexanetriols, dextrans, diethylene glycol, dimethyl acetamide, dimethyl formamide (such as n-dimethyl formamide), dimethyl sulfoxide, erythritol, ethanol, ethylene glycol, ethylene glycol monomethyl ether, formamide, glucose, glycerol, glycerophosphate, glyceryl monoacetate, glycine, glycoproteins, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methoxy propanediol, methyl acetamide, methyl formamide, methyl ureas, methyl glucose, methyl glycerol, phenol, pluronic polyols, polyethylene glycol, polyvinylpyrrolidone, proline, propanediols (such as <NUM>,<NUM>-propanediol and <NUM>,<NUM>-propanediol), pyridine N-oxide, raffinose, ribose, serine, sodium bromide, sodium chloride, sodium iodide, sodium nitrate, sodium nitrite, sodium sulfate, sorbitol, triethylene glycol, trimethylamine acetate, urea, valine and xylose. Other cryoprotectants that may be used in the present disclosure are described in <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; and/or <CIT>, which corresponds to <CIT>.

The cryoprotectant composition may also include at least one cyclohexanediol (CHD) compound, for example the cis or trans forms of <NUM>,<NUM>-cyclohexanediol (<NUM>,3CHD) or <NUM>,<NUM>-cyclohexanediol (<NUM>,4CHD), or racemic mixtures thereof, as a cryoprotectant compound.

The CHD compound may be present in the cryopreservation composition in an amount of from, for example, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, or about <NUM> to about <NUM>.

The cryopreservation composition also may include (or be based on) a solution well suited for storage of cells, tissues and organs. The solution may include well known pH buffers. In some embodiments, the solution may be, for example, the EuroCollins Solution, which is composed of dextrose, potassium phosphate monobasic and dibasic, sodium bicarbonate, and potassium chloride, described in <NPL>), or a "VS55" solution, which is an optimized cryoprotectant cocktail that has demonstrated successful vitrification of many biological systems. VS55 solution is composed of <NUM> dimethyl sulfoxide (DMSO), <NUM> propylene glycol, and <NUM> formamide in a base Euro-Collins solution, for a total of <NUM>. Alternatively the cryoprotectant solution may be formulated in an alternative solution, such as Unisol.

Still further, the cryopreservation composition for use in the methods of the present disclosure may also include an anti-freeze glycolipid (AFGL), anti-freeze protein/peptide (AFP), "thermal hysteresis" proteins, (THPs) or ice recrystallization inhibitors (IRIs). Such materials may be present in the cryopreservation composition in an amount of from, for example, about <NUM> to about <NUM>/mL, about <NUM> to about <NUM>/mL, or about <NUM> to about <NUM>/mL of composition.

In some embodiments, the at least one sugar, selected from trehalose and/or sucrose, may act as a replacement for a cryoprotectant, such as, for example, DMSO, or as a supplement to such other cryoprotectants to reduce the concentration thereof, such as to non-toxic concentrations, at which the cryoprotectant achieves the same or better protective effects with regard to preserving as much functionality of the cryopreserved material/sample during the cryopreservation procedure. For example, in some embodiments, the at least one sugar, selected from trehalose and/or sucrose, may act as a replacement for a cryoprotectant, such as, for example, DMSO, in a solution known as "VS55", which is an optimized cryoprotectant cocktail that has demonstrated successful vitrification of many biological systems (VS55 solution is composed of <NUM> dimethyl sulfoxide (DMSO), <NUM> propylene glycol, and <NUM> formamide in a base Euro-Collins solution, for a total of <NUM>). In this regard, the at least one sugar selected from trehalose and/or sucrose may act as a replacement for the cryoprotectant in the VS55 solution, to reduce the concentration thereof, such as to non-toxic concentrations, or as a supplement to the other cryoprotectants in VS55 at which the cryoprotectant achieves the same or better protective effects with regard to preserving as much functionality of the cryopreserved material/sample during the cryopreservation procedure.

In some embodiments, at least one sugar, selected from trehalose and/or sucrose, is used in an amount in the methods of the present disclosure such that it is effective to act as a cryoprotectant for a living material/sample selected from the group consisting of organs, cells and tissues, such as mammalian organs, mammalian cells, and mammalian tissues (including those which may be subsequently transplanted).

The cells in the cellular materials that may be used in the methods of the present disclosure can be any suitable cell composition. In some embodiments, the cells can be skin cells, keratinocytes, skeletal muscle cells, cardiac muscle cells, lung cells, mesentery cells, adipose cells, stem cells, hepatocytes, epithelial cells, Kupffer cells, fibroblasts, neurons, cardio myocytes, myocytes, chondrocytes, pancreatic acinar cells, islets of Langerhans, osteocytes, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, biliary epithelial cells, and progenitor cells or combinations of any of these cell types. According to the claimed invention, the cellular material has to be obtained from liver, lung, intestine, heart, pancreas, testes, placenta, thymus, adrenal gland, arteries, veins, lymph nodes, bone or skeletal muscle.

In some embodiments, the cells used in the methods of the present disclosure may be from any suitable species of animal, for example a mammal, such as a human, canine (e.g. dog), feline (e.g. cat), equine (e.g. horse), porcine, ovine, caprine, or bovine mammal.

The formulation/composition used to prepare the cryopreservation solution can be combined with the at least one sugar, selected from trehalose and/or sucrose, in a variety of ways. In some embodiments, a cellular material can be combined with an aqueous liquid medium, such as an aqueous solution, containing the at least one sugar, selected from trehalose and/or sucrose. For example, a gradual combination, optionally with gentle agitation, can be conducted.

Once the cryopreservation composition has been prepared (and the at least one sugar, selected from trehalose and/or sucrose and associated with the cellular material to be preserved), the cooling for ice-free vitrified cryopreservation may be conducted in any manner, and may use any additional materials to those described above. Protocols for preserving cellular material are described in the following patents and publications: <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; and <CIT>, which corresponds to <CIT>.

The cryopreservation portion of the preservation protocol typically involves cooling cells to temperatures well below the freezing point of water, e.g., to about -<NUM> or lower, more typically to about -<NUM> or lower. Any method of cryopreservation known to practitioners in the art may be used. For example, the cooling protocol for cryopreservation may be any suitable type in which the cryopreservation temperature may be lower (i.e., colder) than -<NUM> or lower (i.e., colder).

In some embodiments, the preservation protocol may include continuous controlled rate cooling from the point of initiation temperature (+<NUM> to -<NUM>) to -<NUM> or any of the above disclosed cooling temperatures, with the rate of cooling depending on the characteristics of the cells/tissues being cryopreserved. According to the claimed invention, the cooling rate is about -<NUM> to about -<NUM> per minute.

Once the samples to be preserved (e.g., cellular materials and/or tissues) are cooled to -<NUM> or lower by this continuous rate cooling, they may be transferred to liquid nitrogen or the vapor phase of liquid nitrogen for further cooling to the cryopreservation temperature, which is typically below the glass transition temperature of the freezing solution. The samples to be preserved (e.g., cellular materials and/or tissues) are cooled to -<NUM> before further cooling to the cryopreservation temperature. However, it is anticipated that the outcome is independent of cooling rate because ice formation will not occur. The limiting factor for retention of cell viability will be the duration of cryoprotectant exposure at temperatures close to zero centigrade, the lower the temperature the less the risk of cytotoxic effects until storage temperatures are achieved at which no deterioration of viability is anticipated.

The cryoprotectant formulations supplemented with at least one sugar selected from trehalose and/or sucrose have a reduced propensity for ice nucleation during exposure to temperatures above the glass transition temperature. Thus, cellular materials in these formulation will tolerate short term exposure to temperatures such as -<NUM>, for minutes or hours. The precise duration depending upon the cryoprotectant/sugar formulation. The duration tolerated at each temperature will depend upon the relative cytotoxicity of the cryoprotectant formulation employed at that temperature. Furthermore, it is anticipated that these cryoprotectant formulations can be used for storage of tissues, where cell viability is not desired (some heart valves, skin, tendons and peripheral nerve grafts for example), at temperatures ranging from liquid nitrogen to physiological temperatures below the denaturation temperature range of collagen (approximately <NUM>).

Some embodiments (not according to the invention) may comprise a stepwise cooling process, in which the temperature of the tissue is decreased to a first temperature in a first solution containing cryoprotectant at a first temperature between the glass transition temperature of the first solution and -<NUM>, then is further decreased to a second temperature in a second solution containing cryoprotectant at temperature between the glass transition temperature of the first solution and -<NUM>, and this process may be repeated with a third, fourth, fifth, sixth, seventh, etc., solution until the desired temperature is achieved.

In embodiments, the glass transition temperature of the first solution (such as a cryoprotectant formulation) may be in set at any desired level, such as, for example, in a range of from about -<NUM> to about -<NUM>, such as about -<NUM> to about -<NUM>, or - <NUM> to about -<NUM>. In embodiments, the tissue may be cooled and subsequently stored at temperatures between the glass transition temperature and about -<NUM>, such as about -<NUM> to about -<NUM>, such as between about -<NUM> to about -<NUM>, or between about -<NUM> and about -<NUM>.

After being immersed in an initial solution, the sample to be preserved (such as a cellular material or tissue) may be immersed in a solution containing cryoprotectant. The final cryoprotectant concentration may be reached in a stepwise cooling process in which the sample to be preserved (such as a cellular material or tissue) may be immersed in a first solution containing a first cryoprotectant concentration, then the tissue may be immersed in a second solution containing a second cryoprotectant concentration (which is higher than the first cryoprotectant concentration), and this process may be repeated with a third, fourth, fifth, sixth, seventh, etc., solution until the desired concentration is achieved. The cryoprotectant solution may contain any combination of cryoprotectants. In some embodiments, the final desired cryoprotectant concentration may be achieved at any suitable temperature that limits the growth of ice during cooling such that ice-induced damage does not occur, for example the final desired cryoprotectant concentration may be achieved at a temperature in the range of from <NUM> to about -<NUM>, such as about -<NUM> to about -<NUM>, or about -<NUM> to about -<NUM>, or -<NUM> to about -<NUM>, or a temperature of about -<NUM>.

In embodiments, the sample to be preserved (such as a cellular material or tissue) may remain free from ice and/or free from ice-induced damage during the preservation protocol (e.g., the cooling protocol, storage, and warming protocol). For example, after completion of the cooling process, the sample to be preserved (such as a cellular material or tissue) may remain free from ice and/or free from ice-induced damage during the storage step/phase for a long period of time, such as a period of at least <NUM> days, or a period of at least <NUM> days, or a period of at least <NUM> days, or a period of at least <NUM> days.

In some embodiments, upon initiation of the cooling process, the sample to be preserved (such as a cellular material or tissue) may remain free from ice and/or free from ice-induced damage during the entire preservation protocol (i.e., during the cooling protocol, storage, and warming protocol), where the entire preservation protocol (e.g.. , the cooling protocol, storage step/phase, and warming protocol) has a duration in a range of from at least <NUM> days to up to about <NUM> months, or a duration in a range in a range of from at least <NUM> days up to about <NUM> months, or a duration in a range in a range of from at least <NUM> days up to about <NUM> month, or a duration in a range in a range of from at least <NUM> days up to about <NUM> days, or a duration in a range in a range of from at least <NUM> days up to about <NUM> days. Additionally, in embodiments, during such preservation protocols the sample to be preserved (such as a cellular material or tissue) will experience minimal cytotoxicity during the duration of the preservation protocol.

The warming protocol may involve a two-step warming procedure (such as that described by Campbell et al. , Two stage method for thawing cryopreserved cells; see, for example, <CIT>. In the two-step warming protocol, the cryopreserved cellular materials (cryopreserved at the cryopreservation temperature) may be removed from the storage freezer. The cryopreserved cellular materials are allowed to first slowly warm in a first environment in the first step of the two-step protocol. The environment is not required to undergo any special treatment or have any particular make-up, and any environment may be used. The environment may be a gaseous atmosphere, for example, air. To effect the slow warming of the first stage, the environment may be at a first warming temperature greater than the cryopreservation temperature. The first warming temperature may be below freezing temperature. For example, temperatures of -<NUM> or, such as about -<NUM> to about - <NUM>, about -<NUM> to about -<NUM>, or about -<NUM>° to about -<NUM> may be used. The advantage of warming in the first step to a sub-zero centigrade temperature is that the potential cytotoxic effects of the cryoprotectant formulation at warmer temperatures will be minimized.

The second step of the two-step warming procedure involves rewarming the cellular material rapidly in a second environment at a second warming temperature that is greater than the warming temperature used in the first warming step. The second warming temperature may be <NUM> or more, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, or about <NUM>. Again, any suitable environment such as gas (air), liquid, or fluid bed may be used as the second environment. For example, a water or dimethylsulfoxide bath at the warm temperature may be used to effect this rapid rewarming. During this step dilution of the cryoprotectants can be initiated using warm diluent solutions that will also contribute to the warming step.

In some embodiments, the conventional heating methods that may be used to warm the samples include, for example, convection and microwave heating. Prior to the methodology of the present disclosure, conventional methodology including convection heating, which heats from the outer boundary, is effective for small vitrified samples but ineffective for large samples (e.g., having a volume greater than <NUM>) due to cytotoxicity and ice formation. In some embodiments, low radiofrequencies and inductive heating may be used to heat when combined with distributed biocompatible magnetic nanoparticles (mNPs). See, for example, <CIT>.

In embodiments, a majority or all of the cells of the sample (e.g., tissue or cellular material) to be preserved may remain viable after the preservation protocol as the majority or all of the cells of the sample will be exposed to minimal cytotoxicity. In other words, the methods of the present disclosure avoid the cytotoxicity of some conventional cryoprotectant solutions by avoiding exposure of the sample to be preserved to the increase of cytotoxicity of the cryoprotectant solution that occurs as the tissue (and solution) temperatures approaches <NUM>. In embodiments, the methods of the present disclosure avoid exposing the sample to be preserved to any conditions and/or cryoprotectants (e.g., by exposure to the extreme conditions, such as severe osmotic stresses and/or chemical cytotoxicity) that may kill a majority or all of the cells (e.g., because of the increased level of cytotoxicity of the cryoprotectant solution at temperatures approaching <NUM>) of the tissue to be preserved. However, it should be noted that in embodiments where cell viability is not desired chemical toxicity or severe osmotic stresses may be employed to render the cellular materials essentially free of living cells.

In unclaimed embodiments, the cryopreserved cellular materials preserved by the methods of the present disclosure may be put to any suitable use, including, for example, research or therapeutic uses. For example, regarding therapeutic uses, the cryopreserved cellular materials may be administered to a human or animal patient to treat or prevent a disease or condition such as aortic heart disease, degenerative joint disease, degenerative bone disease, colon or intestinal diseases, degenerative myelopathy, chronic renal failure disease, heart disease, intervertebral disc disease, corneal disease, spinal trauma and replacement of parts lost due to trauma, such as fingers, limb extremities, and faces.

The cryopreserved cellular materials can be administered to a patient in any suitable manner. In unclaimed embodiments, the cryopreserved cellular materials may be delivered topically to the patient (e.g. in the treatment of burns, wounds, or skin disorders). In some embodiments, the cryopreserved cellular materials may be delivered to a local implant site within a patient. Any of these or any combination of these modes of administration may be used in the treatment of a patient.

In a first aspect, the present disclosure relates to a method for preserving living large volume cellular material, wherein the cellular material has a volume greater than <NUM>, comprising:.

In a second aspect the method of the first aspect may be a method in which the cellular material has a volume greater than <NUM>. In a third aspect, the method of any of the above aspects may be a method in which the preservation protocol includes a vitrification strategy that limits the growth of ice during cooling and warming such that ice-induced damage does not occur during the preservation protocol. In a fourth aspect, the method of any of the above aspects may be a method in which the at least one sugar is trehalose. In a fifth aspect, the method of any of the above aspects may be a method in which the further cryoprotectant is selected from the group consisting of acetamide, agarose, alginate, alanine, albumin, ammonium acetate, anti-freeze proteins, butanediol, chondroitin sulfate, chloroform, choline, cyclohexanediols, cyclohexanediones, cyclohexanetriols, dextrans, diethylene glycol, dimethyl acetamide, dimethyl formamide, dimethyl sulfoxide, erythritol, ethanol, ethylene glycol, ethylene glycol monomethyl ether, formamide, glucose, glycerol, glycerophosphate, glyceryl monoacetate, glycine, glycoproteins, hydroxyethyl starch, ice recrystalization inhibitors, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methoxy propanediol, methyl acetamide, methyl formamide, methyl ureas, methyl glucose, methyl glycerol, phenol, pluronic polyols, polyethylene glycol, polyvinylpyrrolidone, proline, propanediol, pyridine N-oxide, raffinose, ribose, serine, sodium bromide, sodium chloride, sodium iodide, sodium nitrate, sodium nitrite, sodium sulfate, sorbitol, triethylene glycol, trimethylamine acetate, urea, valine and xylose. In a sixth aspect, the method of any of the above aspects may be a method in which the cellular material is selected from human organs and human tissues, selected from intestine, pancreas, testes, placenta, thymus, adrenal gland, arteries, veins, lymph nodes, bone or skeletal muscle. In a seventh aspect, the method of any of the above aspects may be a method in which the medium does not contain DMSO, formamide and/or propylene glycol.

The foregoing is further illustrated by reference to the following examples, which are presented for purposes of illustration and are not intended to limit the scope of the present disclosure.

In the following ice-free cryopreservation formulation supplementation experiments, disaccharides (trehalose and sucrose) are added to various vitrification formulations (VS49, DP6, and VS55) in the amounts shown in <FIG> and compared to a solution (without trehalose and/or sucrose) to access the effectiveness at inhibiting ice formation in various tissues.

In particular, the <FIG> shows that there are significant increases in tissue viability induced by trehalose and sucrose supplementation of vitrification formulations. Data shown in % of control VS55 results performed with the final cryoprotectant concentration added at <NUM>, N= <NUM>, *p<<NUM> by ANOVA or #T-test compared with no additive controls.

In this experiment, addition of either <NUM> trehalose or sucrose to VS49, DP6 and VS55 formulations was compared. The tissues were vitrified in <NUM> of solution. Trehalose supplementation increased leaflet viability in VS49 and VS55. Sucrose supplementation increased leaflet viability in all three formulations. These differences were significantly different, p<<NUM> by ANOVA or T-test. DP6 consistently had the best muscle preservation in all treatment groups but did not achieve statistical significance in this experiment, however, this outcome suggested that cardiac muscle is more tolerant of ice formation than the other tissue types (leaflet and artery) and that formamide might be causing some cytotoxicity. This led to plans to test DP6 with sugars more thoroughly in later experiments. Repetitive experiments were performed using sucrose supplemented VS55 and obtained outcomes similar to those for supplemented VS55 shown here.

Heart Valve and Blood Vessel Procurement: Bonafide excess heart valves or blood vessels were obtained from animals employed in other IACUC approved research studies or from a food processing plant post-mortem. The tissues were dissected, rinsed and placed in sterile cups with ice-cold tissue culture medium containing antibiotics overnight and then allocated for in vitro studies. The heart valve tissues in <FIG> each consisted of a piece of tissue consisting of pulmonary artery, leaflet and cardiac muscle. They were separated and individually assayed for viability using an assay that assesses metabolic activity that is described below.

Vitrification Method: Tissues were gradually infiltrated with VS55 consisting of <NUM> DMSO, <NUM> formamide, and <NUM> propylene glycol in Euro- Collins solution at <NUM> using methods previously described for blood vessel vitrification, a dilution of VS55 to VS49 or DP6 consisting of <NUM> DMSO, <NUM> propylene glycol. Precooled dilute vitrification solutions (<NUM>) are added in five sequential <NUM>-min steps of increasing concentration on ice. The last cryoprotectant concentration with mNPs was added in a final sixth addition step in either precooled -<NUM> or <NUM> full strength vitrification solution and kept in a -<NUM> bath for <NUM> minutes or at <NUM> on ice in plastic tubes. The samples were then cooled in two steps, first rapid cooling to -<NUM> by placing in a precooled <NUM>-methylbutane bath at -<NUM> and then by transfer to vapor phase nitrogen storage for slower cooling to below -<NUM>. Finally, the samples were stored below -<NUM> in vapor phase nitrogen for at least <NUM> before testing.

Warming: Warming was performed by either convection warming or nanowarming. Convection warming is a two-stage process including slow warming to -<NUM> and then as rapid as possible warming to melting. The slow warming rate is created by taking the sample to the top of the -<NUM> freezer and the control warming rate is generated by placing the plastic container in the mixture of <NUM>% DMSO/H2O at room temperature. After rewarming, the vitrification solution was removed in a stepwise manner on ice to keep the tissues cold and minimize cytotoxicity due to the presence of residual cryoprotectants.

Viability Assessment: Metabolic Activity - An alamarBlue™ assay will be used to evaluate the metabolic activity of control and treated tissue samples. Tissues were incubated in <NUM> of DMEM+<NUM>%FBS culture medium for one hour to equilibrate followed by the addition of <NUM>% AlamarBlue under standard cell culture conditions for <NUM> hours. AlamarBlue is a non-toxic fluorometric indicator based on detection of metabolic activity. Fluorescence was measured at an excitation wavelength of <NUM> and an emission wavelength of <NUM>. This evaluation was performed after rewarming (day <NUM>) to demonstrate cell viability. Control and experimental tissues were dried at the end of each experiment, weighed, and results expressed as relative fluorescence units (RFU)/mg tissue dry weight.

The results of these experiments indicate that <NUM> of trehalose or sucrose prevents visible ice formation in both DP6 and VS49 formulations and increases post-vitrification viability in these solutions (DP6, VS49 and VS55) with leaflet viability demonstrating <NUM>-<NUM> fold increases. Notably, the VS49 or DP6 formulations are not effective at controlling ice formation using conventional convection warming, described in <CIT>. However, no ice formation was observed in the presence of either trehalose or sucrose during cooling and rewarming in these experiments with nanowarming.

A further series of experiments was conducted with a <NUM> sucrose DP6 formulation with blood vessels using a device called a Cryomacroscope (used to visualize events during cryopreservation). The results are shown in <FIG> (Contractile responses of fresh and vitrified rabbit carotid arteries, (top) Norepinephrine and Phenylephrine (lower) dose response curves).

The above study was performed on <NUM> samples using convection warming without nanowarming. Preparation, vitrification and rewarming was performed as previously indicated. The blood vessels were cut into <NUM> rings for testing. The artery samples preserved with DP6 supplemented with <NUM> sucrose yielded greater than <NUM>% viability by alamarBlue using methods described above in the first set of experiments(data not shown). The samples were also checked for smooth muscle function and excellent responses to two contractile drugs were observed, as seen in <FIG>.

Blood vessel physiology: Two-mm rings of fresh and ice free cryopreserved blood vessels were prepared for vascular physiology studies to document the function of rabbit femoral artery rings in a Radnoti organ bath system using a panel of constrictive drugs. Isometric contractile tensions were measured after addition of increasing concentrations of each drug using methods previously employed. The physiology results were expressed as grams tension/mg tissue dry weight.

Pig femoral artery cryopreservation was performed in VS55 ± <NUM> sucrose at <NUM> and <NUM> cellular material volumes. Significant preservation was demonstrated in all formulations at <NUM>, whereas at <NUM> the VS55 without sucrose demonstrated low viability. In contrast, the sucrose supplemented formulations demonstrated preservation of viability by both the alamarBlue (<FIG>; porcine femoral artery comparison of <NUM> and <NUM> samples of VS55 ± <NUM> sucrose, where tissue rings were assessed using the alamarBlue metabolic assay) and physiology assays (<FIG>; porcine smooth muscle relaxation induced by sodium nitroprusside after pre-contraction, where VS55 alone works well for a <NUM> sample, but fails at <NUM> sample, and sucrose supplementation preserves the functionality (relaxation) at <NUM> volume sample) employed. These results demonstrate that disaccharide supplementation results in tissue survival at large volumes (e.g., <NUM> volume) where unsupplemented VS55 solution (i.e., no disaccharide added) fails (due to ice formation with loss of tissue viability). Similar results were obtained for two contractile drugs.

The methods used here were the same as in the earlier examples using convection warming.

Rabbit thoracic aorta samples were vitrified in <NUM> volumes of VS55 with or without <NUM> sucrose with or without nanoparticles for comparison of convection versus nanowarming. The results are set forth in <FIG> (<NUM> rabbit thoracic aorta results demonstrating improved outcomes with sucrose supplementation using either convection (middle bar) or nanowarming (far right bar)).

The methods were as described for earlier examples regarding addition of cryoprotectants and cooling. However, in this experiment we compared convection warming and nanowarming. Nanowarming was performed after at least <NUM> at -<NUM>. The samples were vitrified with <NUM>/ml Fe nanoparticles (EMG-<NUM>, Ferrotec) and the samples were inserted into the coil of an inductive heater for rewarming. More specifically we employed a <NUM> kW terminal, <NUM>-<NUM>, EASYHEAT 5060LI solid state induction power supply with a remote work head and custom multi-turn helical coil with <NUM>-<NUM> turns to create a <NUM> kA/m magnetic field in the center. Good results (just under <NUM>% viability) were achieved with either convection heating or nanowarming (conducted as described in <CIT>) as long as sucrose was present in the vitrification solution. VS55 without sucrose performed poorly due to ice formation that was observed in the rewarming process.

In this series of experiments (the results of which are illustrated in <FIG> and <FIG>), the viability of heart valve tissues in which intact heart valves were preserved in <NUM> cryoprotectant volumes and rewarmed by either convection or nanowarming methods are compared.

Three tissue samples were taken from each leaflet and associated pulmonary artery and cardiac muscle for a total of <NUM> samples of each tissue type from each valve. The ice-free vitrification and rewarming procedures were performed as previously described except that a stepwise addition of DP6 was employed with sucrose or trehalose in the last step. In some cases the DP6+sugar step was followed by immersion in VS55+sugar at -10C followed by immediate vitrification. It should be noted that in this experiment, as also observed in <FIG>, DP6 alone results in good cardiac muscle cell viability in spite of the freezing that occurs with DP6, however in this experiment two cryopreservation groups achieved control values (<FIG>, bottom) after addition of sucrose.

With respect to the data shown in <FIG> and <FIG>, individual porcine heart valves were loaded with DP6 and then cryopreserved in either DP6 alone, DP6 with either <NUM> trehalose, <NUM> sucrose or a mixture of <NUM> sucrose and <NUM> trehalose, or transferred at the last moment from DP6 with sugar to VS55 with the same sugar. The total volume of tissue and solution was at least <NUM> mLs. The results from top to bottom are for leaflet, then conduit and finally cardiac muscle. The results in <FIG> are for convection warming and the results in <FIG> are after nanowarming (n=<NUM>). The results are expressed as the mean ± <NUM> standard error in percent of untreated control heart valve tissues.

These results demonstrate that DP6 (<NUM> dimethylsulfoxide plus <NUM> in EuroCollins Solution) with <NUM> sucrose results in excellent preservation of all three tissue components in heart valves, particularly after nanowarming. Several other treatment groups including loading with DP6 and then transferring to VS55 with either sucrose or trehalose also improved viability after either convection warming or nanowarming. There was a tendency for treatment groups with sucrose to be better than with trehalose.

The above results (<FIG>) combine to demonstrate that the use of sugars, such as disaccharides, e.g., sucrose and trehalose, with VS55 and more dilute cryoprotectant formulations (DP6, <FIG>) result in unexpected improved outcomes of ice-free vitrification. These sugars help during both convection and rapid warming with inductive heating of magnetic nanoparticles but it appears that high sucrose and trehalose formulations do not need rapid warming (<FIG>).

The use of sugars, such as disaccharides, e.g., sucrose and trehalose, permits preservation of large volume samples and slow warming with less risk of ice formation and increased post-rewarming viability.

Storage of VS55 with a range of trehalose and sucrose concentrations demonstrates freedom from ice formation at -80C for <NUM> week. In this experiment <NUM> samples of VS55 with increasing concentrations of sugars were placed in <NUM> plastic tubes and stored in a -80C mechanical storage freezer for <NUM> days. The tubes were checked daily for ice formation. The tubes demonstrated ice in a sugar concentration dependent manner with the highest concentrations (such as in a range of from <NUM>-<NUM>) not showing ice formation after <NUM> days, <FIG> shows the tubes after <NUM> and <NUM> days of storage. After one day <NUM> to <NUM> trehalose and <NUM>-<NUM> sucrose were free of ice, while after <NUM> days <NUM>-<NUM> trehalose and <NUM> sucrose groups were free of ice. Lower concentrations of these sugars may still be used for ice-free vitrification but more rapid cooling and warming will be needed because the risk of ice formation will be greater and nanowarming rather than convection warming may be needed.

Specifically, <FIG> is photograph illustrating the ice formation in VS55 supplemented with sugars at -<NUM>: Top left: VS55 with <NUM>-<NUM> sucrose at <NUM> hours; Top right: VS55 with <NUM>-<NUM> trehalose at <NUM> hours. At this time point the VS55 supplemented with either <NUM>-<NUM> sucrose or <NUM>-<NUM> trehalose demonstrated ice (white) formation. Higher concentrations were clear without ice. Bottom left: VS55 with <NUM>-<NUM> sucrose at <NUM> days; and Bottom right: VS55 with <NUM>-<NUM> trehalose at <NUM> days. After a week at -<NUM> the highest concentrations of sugars are still free of ice.

Incorporation of sugars, such as disaccharides, e.g., sucrose and trehalose, in to such ice-free vitrification formulations permits relatively slow cooling and warming rates (such as on the order of hours or days) to be used without ice formation and loss of cell/tissue viability. Additionally, both convection warming and nanowarming methods may be used in the methods of the present disclosure with the formulations described herein. Rapid warming methods, such as nanowarming methods may still be desired because at rapid warming rates there is less opportunity for cryoprotectant exposure induced cytotoxicity. These observations also suggest that other cryoprotectant formulations with sugars can be developed that have even less risk of cryoprotectant cytotoxicity.

Claim 1:
A method for preserving living large volume cellular material, wherein the cellular material has a volume greater than <NUM>, comprising:
exposing the cellular material to a cryoprotectant formulation containing from <NUM> to <NUM> of at least one sugar selected from trehalose and/or sucrose, wherein the cellular material is obtained from liver, lung, intestine, heart, pancreas, testes, placenta, thymus, adrenal gland, arteries, veins, lymph nodes, bone or skeletal muscle,
subjecting the cellular material to a preservation protocol in which ice-induced damage to the cellular material does not occur, wherein the preservation protocol includes a cooling protocol, a storage phase, and a warming protocol, where
a further cryoprotectant is added to the cryoprotectant formulation prior to or during the cooling protocol, the further cryoprotectant being different from the at least one sugar, and the cryoprotectant formulation contains the at least one sugar and the further cryoprotectant at a concentration of from <NUM> to <NUM>, and
during the cooling protocol a continuous controlled rate of about -<NUM> to about -<NUM> per minute is used to cool the cellular material from a point of initiation temperature to -<NUM> or lower, the point of initiation temperature being in a range of from about +<NUM> to -<NUM>, and after completion of the preservation protocol
obtaining a cryopreserved cellular material.