An efficient, biocompatible cryopreservation medium that eliminates the need for cell-permeant cryoprotectants

Abstract

A cryopreservation medium comprising: a first cryoprotected particle or macromolecule; a second cryoprotected particle or macromolecule; and an aqueous liquid, wherein the first cryoprotected particle or macromolecule is hydrophilic and has a spherical shape when dissolved or suspended in the aqueous liquid, and the second cryoprotected particle or macromolecule has an affinity for the first cryoprotected particle or macromolecule and an affinity for the plasma membrane of a cell.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is based on and claims priority to U.S. Provisional Application No. 63 / 170,673, filed April 5, 2021, the disclosure of which is incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with United States Government support under NIH 2R44OD020163-02A1 awarded by the National Institutes of Health (NIH) Small Business Innovation Research (SBIR). The Government has certain rights in the invention. Technical Field The present disclosure is directed to the fields of cryobiology, cryopreservation, and controlled ice formation techniques, as well as storage of biological and clinical samples. [Background technology]

[0002] Cryopreservation is a technique that allows biological materials to be preserved at temperatures below the freezing point of water (i.e., 0 °C). Cryopreservation is a field in which progress has been slow, due in large part to a limited understanding of ice formation mechanisms at nanoscale dimensions, as well as a lack of efficient means to control ice formation at the cellular scale. Practical cryopreservation began in 1949, when it was surprisingly discovered that animal sperm could be cryopreserved using a glycerol-rich medium (Polge C, Smith Au, Parkes As. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature. 1949 Oct 15;164(4172):666). Despite decades of cryopreservation innovation efforts since then, all existing cryopreservation techniques (except for a few that can be adopted only for a few cell types with unique biophysical characteristics) and all products on the market still rely on the use of a variety of biologically incompatible (i.e., cell-permeable and reactive) small molecule cryoprotectants. These cell-permeable cryoprotectants include, but are not limited to, glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, and propanediol. Unfortunately, the inclusion of these small molecule reagents in cryopreservation media causes many technical, practical, and regulatory challenges. More importantly, a myriad of cell and tissue types respond poorly to existing cryopreservation protocols, exhibiting poor post-thaw survival and impaired function. Summary of the Invention [Problem to be solved by the invention]

[0003] In the face of recent rapid advances in cell-based tissue engineering and regenerative medicine, as well as the continuing development of transplantation techniques using human donor or xenogeneic tissues, there is an urgent need for improved cryopreservation media and methods that are clinically practical and overcome long-standing challenges. In particular, there is a need for efficient cryopreservation media that eliminate the need to include cell-permeable cryoprotectants, and practical methods of use that reduce the complications and inefficiencies associated with cell-permeable cryoprotectants. [Means for solving the problem]

[0004] overview Disclosed herein is an efficient cryopreservation medium that eliminates the need to include a cell-permeable cryoprotectant. Methods of using the cryopreservation medium are also disclosed.

[0005] The present specification discloses a cryopreservation medium comprising: a first cryoprotected particle or macromolecule; a second cryoprotected particle or macromolecule; and an aqueous liquid, wherein the first cryoprotected particle or macromolecule is hydrophilic and has a spherical shape when dissolved or suspended in the aqueous liquid, and the second cryoprotected particle or macromolecule has an affinity for the first cryoprotected particle or macromolecule and an affinity for the plasma membrane of a cell or a lipid membrane of a lipid membrane-bound biological structure.

[0006] The present specification also discloses a method for protecting a lipid membrane of a lipid membrane-bound biostructure, the method comprising contacting the lipid membrane-bound biostructure with a cryopreservation medium before cooling the lipid membrane-bound biostructure to a temperature of about -70°C to about -273°C, wherein nanoscale cubic ice is formed around the lipid membrane at a temperature of about -70°C to about -273°C.

[0007] These and other features are illustrated by the following drawings and detailed description.

[0008] The following drawings are exemplary embodiments in which like elements are numbered likewise: [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram showing the mechanism of action of the cryopreservation medium of the present invention. [Diagram 2] Figure 2 shows experimental results revealing the formation of nanoscale cubic ice in media containing Ficoll 70 through cryogenic X-ray diffraction and transmission electron microscopy of replicas of freeze-fractured samples. [Diagram 3] FIG. 3 shows the results of an experiment using a fluorescent microscope, which shows that chondroitin sulfate A sodium salt molecules significantly promote the affinity between Ficoll 70 molecules and cell membranes. [Figure 4] FIG. 4 shows the results of experiments using cryomicroscopy to demonstrate that the medium of the present invention prevents the formation of intracellular ice during freezing. [Diagram 5] FIG. 5 shows the efficiency of the medium of the present invention in cryopreserving Sf9 cells at both −80° C. and liquid nitrogen temperatures. [Figure 6] FIG. 6 shows the efficiency of the cryopreservation medium of the present invention in the cryopreservation of human adipose stem cells at -80°C. [Figure 7] FIG. 7 shows the efficiency of the cryopreservation medium of the present invention in the cryopreservation of bovine chromaffin cells at -80°C. [Figure 8] FIG. 8 shows the efficacy of the cryopreservation medium of the present invention in the cryopreservation of human skin grafts at -80°C. [Figure 9] FIG. 9 shows the effectiveness of the cryopreservation medium of the present invention in cryopreserving human limbal tissue at -80°C. [Figure 10] FIG. 10 shows the effectiveness of the cryopreservation medium of the present invention in cryopreserving bovine adrenal tissue at -80°C. [Figure 11] FIG. 11 shows the effectiveness of the cryopreservation medium of the present invention in the cryopreservation of 2D iPSC-derived RPE tissue at -80° C. [Figure 12] FIG. 12 shows the effectiveness of the cryopreservation medium of the present invention in the cryopreservation of 3D differentiated neural tissue at -80° C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description Cryopreservation is a technique that allows biological materials to be stored at very low temperatures, usually around -80 °C to -196 °C, such as in mechanical freezers or liquid nitrogen cryogenic freezers or tanks. Cryopreservation or cryopreservation is known to preserve such biological materials for relatively long periods, sometimes indefinitely, without functional deterioration of the biological material or with substantially limited decomposition. Practical cryopreservation began in 1949, when it was accidentally discovered that animal semen could be cryopreserved using a glycerol-rich medium. Since then, with regard to the cryopreservation of cells, almost all cryopreservation techniques in practical use and all existing products on the market continue to rely on the use of biologically reactive small molecule cryoprotectants of various types and concentrations that penetrate the cell membrane and enter the cells (i.e., cell permeation). Cell-permeant cryoprotectants will always be necessary for the cryopreservation of any kind of tissue, as long as the purpose of the cryopreservation practice is to maintain the viability and function of the majority of cells in the tissue. If the cryoprotectant does not penetrate, then in the tissue, very little, only genetic material or pathological features will be preserved. The use of cell-permeant small molecule cryoprotectants provides three main cryoprotective functions:

[0011] First, cell-permeant small molecule cryoprotectants increase the viscosity of the cryoprotectant solution: at relatively low concentrations, viscous, cell-permeant small molecule cryoprotectants reduce the size of extracellular ice crystals that form during freezing, and at very high concentrations, they prevent ice formation, i.e., the so-called vitrification approach.

[0012] Second, cell-permeant small molecule cryoprotectants prevent intracellular ice formation. The viscous liquid enters the cell by osmosis, thereby either reducing the size of intracellular ice to an extent that organelles are not damaged, or preventing intracellular ice formation altogether. Intracellular ice formation is generally thought to result from large extracellular ice crystals disrupting damaged cell membranes.

[0013] Third, cell-permeant small molecule cryoprotectants slow the cell-damaging recrystallization process by increasing the viscosity of the solution. Storage at temperatures above the ice recrystallization range, e.g., about -80°C or higher, slows the cell-damaging recrystallization process due to the aforementioned viscosity increase mechanism. The damage mechanism is believed to be due to the physical properties of hexagonal ice crystals (i.e., Ice Ih, the hexagonal crystal form of ordinary ice, or frozen water) at temperatures above about -100°C. The small hexagonal ice crystals formed during the freezing process are thermally unstable and tend to spontaneously combine to form larger ice crystals or simply continue to grow, thereby forming larger crystals, resulting in damage to cells or tissues during storage or thawing processes at about -80°C (the typical operating temperature of a regular laboratory freezer).

[0014] To further improve the efficiency of these protective mechanisms, various types of non-permeant cryoprotectants have been utilized and developed for use in various cryopreservation methods. Additives include, but are not limited to, oligosaccharides such as sucrose, raffinose, and trehalose; polymers such as hydroxyethyl starch (HES), polysaccharides, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), chondroitin sulfate, albumin, some of the antifreeze proteins and their analogs; and natural or newly formulated biological compounds such as human and animal serum and serum substitutes. However, existing cryoprotectants containing these components cannot completely eliminate the need for cell-permeant cryoprotectants in the media mixture to ensure post-thaw viability on a scale sufficient for practical cryopreservation applications for most cell types and all tissue types. Uchida et al. (Uchida T, Takeya S. Powder X-ray diffraction observations of ice crystals formed from disaccharide solutions. Phys Chem Phys. 2010 Dec 7;12(45):15034-9) reported that when a solution containing extremely high concentrations (approximately 50% w / v) of disaccharides (e.g., sucrose and trehalose) is frozen at a relatively high cooling rate (e.g., several hundred degrees per minute), the sugar molecules spontaneously precipitate due to their solubility limit in cold water, spontaneously forming nanoscale spherical particles (approximately 10-20 nm in size), minimizing the energy of the system. Around these sugar particles, stable cubic ice (i.e., ice I) with a size of less than 10 nm is formed. c ) crystals, i.e. nanoscale cubic ice, have been identified. Unlike hexagonal ice, cubic ice is a metastable cubic crystalline variant of ice that forms very small crystals, which may enhance cell viability during cryopreservation procedures by preventing mechanical damage caused by the formation of relatively large (usually over 10 μm) hexagonal ice crystals. However, the formation of nanoscale cubic ice using the above method requires very high concentrations of disaccharides and fast cooling rates, making such procedures impractical for cryopreservation.

[0015] Few cell types can be cryopreserved without cell-permeable cryoprotectants. For example, red blood cells (lacking the nucleus and some organelles) can be cryopreserved using analogues of HES or antifreeze proteins; certain liver cell types can accumulate high concentrations of glucose intracellularly through active transport and achieve relatively high post-thaw survival rates. Obviously, these specific functions do not exist in normal cell types, so the application of the related techniques is highly limited. Trehalose has been transported into various cell types by electroporation or acoustic methods to achieve cell cryopreservation without permeating cryoprotectants. However, these techniques are not suitable for cryopreservation of any kind of tissues due to the limited transport distance of the electric or acoustic fields. Meanwhile, in the above methods, both trehalose and glucose molecules actually permeate the cell membrane, so these methods still employ the so-called cell-permeable approach. All of these methods require complicated operations and expensive equipment to prevent extensive damage to cells, and more importantly, liquid nitrogen facilities are required for long-term storage.

[0016] On the other hand, the use of non-permeating cryoprotectants as listed and described above does not effectively mitigate the well-known adverse effects resulting from the use of permeating cryoprotectants, including: Introduction of varying degrees of biochemical damage or complications, including but not limited to toxicity, apoptosis, and unwanted differentiation of stem cells. Some of these effects may raise regulatory concerns, especially in the development of cell-based therapies or regenerative medicines; and · Physical osmotic damage to cells and tissues during the loading procedure (i.e., before freezing) and thawing procedure (i.e., after thawing), resulting in damage to cell and tissue structure and ultrastructure.

[0017] As a result, the cryopreservation methods described above that utilize cell-permeable cryoprotectants face insurmountable challenges for further improvement, examples of which are discussed in more detail below.

[0018] Vitrification approaches using high concentrations, typically 40%-50% per volume (v / v), of cell-permeable cryoprotectants result in significant chemical and osmotic effects that are detrimental to therapeutic and tissue engineering applications, which need to be mitigated or completely eliminated. Vitrification approaches also require the determination of so-called critical cooling rates (e.g., 10 4 A cooling rate higher than 10 Kelvin (K) / min and an even higher heating rate (e.g., 10 K / min) to prevent both the devitrification (crystallization of the vitrified solution during warming) and the subsequent recrystallization process of the devitrified solution. 5 K / min) is necessary. Both requirements limit the sample size utilized in the vitrification approach due to the limited thermal conduction of biological samples. Furthermore, due to the fact that the vitrification and devitrification temperatures of the osmotic cryoprotectant solutions are almost always lower than -100 °C, the vitrification procedure requires the use of cryogenic fluids (e.g., liquid nitrogen, between -196 °C in the liquid phase and 120 °C to -196 °C in the gas phase in a sealed container) and related equipment, or very expensive ultra-low temperature freezers instead of regular freezers operating at about -80 °C. However, the liquid nitrogen equipment or devices are expensive and large-scale, which significantly increases the costs of storage, transportation, and maintenance. Therefore, even if the vitrification method can achieve a high survival rate after thawing, industrial users prefer to avoid this approach. Taking human skin allografts as an example, the vitrification of small (e.g., 5 cm 2 It has been demonstrated that human skin samples can be efficiently cryopreserved using vitrification media and procedures, however, a large number of conventional donor tissues (each of which varies in size, typically over 100 cm) are required. 2 For preservation of these tissues (above 100%), skin banks use the traditional approach of freezing in 15%–30% v / v glycerol and storing in a conventional freezer, even though the post-thaw viability is only about half that achieved with vitrification.

[0019] Slow-cooling approaches using low concentrations (typically 5–15% v / v) of cell-permeable cryoprotectants result in less chemical and osmotic damage than vitrification approaches. Some mechanically tough cells (e.g., bacterial cells, certain insect cells, mammalian red blood cells, etc.) that can withstand damage from recrystallization can be stored long-term in -80 °C freezers. However, for most mammalian cell types, liquid nitrogen facilities are required for long-term storage unless recrystallization can be prevented. For tissues, slow-cooling methods generally result in poor post-thaw survival and significant structural tissue damage associated with ice formation during freezing and recrystallization during warming. Again, using human skin allografts as an example, traditional glycerol-derived approaches result in more than 50% cell loss in both laboratory-based and tissue bank operations. Results are similar for other human tissue types.

[0020] To prevent ice recrystallization at storage temperatures around -80℃, a cryopreservation medium containing a relatively high concentration (approximately 10% w / v after mixing with the cell suspension) of very dense spherical polysaccharide molecules such as Ficoll 70 (a spherical dense polysucrose molecule with a molecular weight (MW) of approximately 70 kDa) and a low concentration of DMSO (5-10% v / v) was disclosed by Han et al., 2017 (Han X, Yuan Y, and Roberts RM 2017. Cryopreservation Medium and Method to Prevent Recrystallization, PCT / US2017 / 032606) and Yuan et al., 2016 (Yuan Y, Yang Y, Tian Y, Park J, Dai A, Roberts RM, Liu Y, Han X. Efficient long-term cryopreservation of pluripotent stem cells at -80℃. Nature, Scientific Reports. 2016). 6:34476). This medium allows short-term storage of mammalian cells in regular freezers, thus eliminating the need for liquid nitrogen facilities for long-term storage of mammalian and insect cells, as well as tissue cryopreservation. As demonstrated in previous thermal studies (Yuan Y, Yang Y, Tian Y, Park J, Dai A, Roberts RM, Liu Y, Han X. Efficient long-term cryopreservation of pluripotent stem cells at -80°C. Nature, Scientific Reports. 2016 6:34476), medium containing 10% w / v to 20% w / v Ficoll 70 prevents ice recrystallization at temperatures up to about -65°C, making this method suitable for long-term storage at temperatures below about -70°C, including the typical operating temperatures of regular laboratory mechanical freezers. A commercial product (C80EZ® medium) has been used successfully in numerous industrial applications and continues to be used today.However, as one of our examples shows, the use of Ficoll 70 alone, even at concentrations above 20%, cannot completely remove DMSO or other cell-permeable cryoprotectants for efficient cell and tissue cryopreservation.

[0021] The present disclosure relates to a cryopreservation medium that combines the use of two types or classes of cryoprotective particles or macromolecules in an aqueous liquid, eliminating the need for the use of small molecule cell-permeant cryoprotectants to achieve long-term preservation of biological samples while maintaining cellular viability and function. A "cryopreservation medium" is a solution that allows living cells (or cellular components, or artificially created structures that resemble cells or cellular components) to be preserved in a frozen state and to retain all or substantially all cellular properties and function (or in the case of cellular components, the respective properties) after thawing.

[0022] The present specification discloses a cryopreservation medium comprising: a first cryoprotected particle or macromolecule; a second cryoprotected particle or macromolecule; and an aqueous liquid, wherein the first cryoprotected particle or macromolecule is hydrophilic and has a spherical shape when dissolved or suspended in the aqueous liquid, and the second cryoprotected particle or macromolecule has an affinity for the first cryoprotected particle or macromolecule and an affinity for the plasma membrane of a cell.

[0023] The first cryoprotected particles or macromolecules are hydrophilic and have nanoscale characteristics that are very dense and spherical or nearly spherical when dissolved or suspended in water, with a highly hydrophilic surface. A representative cryoprotected particle or macromolecule of the first type 10 is identified in Figure 1. In solution, the first cryoprotected particle or macromolecule promotes the formation of nanoscale cubic ice crystals 30 near its surface while also preventing the formation of hexagonal ice crystals 40 near its surface.

[0024] The second cryoprotected particle or macromolecule has a high affinity for the first particle or macromolecule. In Figure 1, a representative bond 50 between a first and a second cryoprotected particle or macromolecule is shown.

[0025] Specific examples of the first cryoprotected particle or macromolecule include spherical hydrophilic polysaccharides, polymeric cyclodextrins, polymeric saccharides, globular proteins, globular glycoproteins containing oligosaccharide chains attached to the outer surface of globular proteins, globular protein derivatives, globular polypeptides, and globular nucleic acids.

[0026] The second cryoprotective particle or macromolecule also has a high affinity for structures / materials within the cell membrane or plasma membrane of the cell-like structure that associates with the cell or tissue to be cryopreserved. Such structures / materials of the plasma membrane include, for example, a phospholipid layer, proteins, or other macromolecules located on the outer surface of the cell plasma membrane. Figure 1 shows a representative bond 60 between the second type of cryoprotective particle or macromolecule and a cell membrane that associates with the cell or tissue to be cryopreserved.

[0027] The above unique combination of bonds generated by the use of two types of cryoprotective particles or macromolecules acting together greatly increases the probability that the cell membrane will come into contact only with nanoscale cubic ice crystals formed near the surface of the first cryoprotective particle or macromolecule throughout the duration of the cryopreservation process. As demonstrated by the transmission electron microscopy results shown in Figure 2B, each of the first particles or macromolecules can generate a layer of cubic ice with a thickness of about 10-50 nm around it after being cooled to about -80 °C. Therefore, the cell plasma membrane is less susceptible to damage by the large hexagonal ice crystals because the hexagonal ice crystals are located far away from the first cryoprotective particle or macromolecule due to the presence of the second cryoprotective particle or macromolecule in between. As a result, the plasma membrane is well protected by this layer during freezing, and the formation of nanoscale ice on the outside of the membrane does not introduce intracellular ice formation, or the size and number of intracellular ice crystals induced in this scenario are much smaller than the nanoscale cubic ice crystals on the outside of the membrane. Therefore, intracellular components are also efficiently protected even in the absence of a cell-permeable cryoprotectant in the cryopreservation medium.

[0028] The presence of nanoscale cubic ice structures and primary cryoprotective particles or macromolecules also acts as a mechanism to prevent the recrystallization of hexagonal ice by limiting the direct contact of hexagonal ice crystals with each other by separating them and preventing them from combining or fusing to form larger crystals. Such a mechanism to prevent the recrystallization of hexagonal ice cannot be achieved by conventional cryopreservation media containing small molecule cell-permeable cryoprotectants or other types of non-permeable molecules. Nanoscale cubic ice is a type of Ice I on the scale of 0.1 nm to 10 nm in size. c The formation of.

[0029] As demonstrated in a thermal study by Yuan et al., 2016 (Yuan Y, Yang Y, Tian Y, Park J, Dai A, Roberts RM, Liu Y, Han X. Efficient long-term cryopreservation of pluripotent stem cells at -80°C. Nature, Scientific Reports. 2016 6:34476), the first cryoprotectant particles or macromolecules prevent ice recrystallization at temperatures up to about -65°C. Thus, the method of the present invention is suitable for long-term storage at any temperature below about -70°C, including the typical operating temperatures of conventional laboratory mechanical freezers, the storage temperatures of liquid nitrogen facilities (about -120°C to -196°C), and the low temperatures provided by other cryogenic fluids or physical processes (-196°C to -273°C).

[0030] In one embodiment, the first cryoprotected particle or macromolecule is a polymer.The polymer may comprise a molecule that forms a roughly spherical, compact, three-dimensional structure when dissolved in aqueous liquid.In one embodiment, the first cryoprotected particle or macromolecule comprises a spherical hydrophilic polysaccharide, a polymerized cyclodextrin, a polymerized sugar, a spherical protein, a spherical glycoprotein that comprises an oligosaccharide chain attached to the outer surface of a globular protein, a globular protein derivative, or a combination thereof.

[0031] The first cryoprotected particle or macromolecule has a nanometer-sized particle size.In one embodiment, the first cryoprotected particle or macromolecule has a particle size of about 50 nm or less, or about 25 nm or less, or about 10 nm or less.In one embodiment, the first cryoprotected particle or macromolecule has a particle size of about 10 nm.

[0032] In one embodiment, the first cryoprotected particle or macromolecule comprises a spherical hydrophilic polysaccharide comprising a copolymer of sucrose and epichlorohydrin. Examples of copolymers of sucrose and epichlorohydrin include FICOLL TM The spherical hydrophilic polysaccharides include molecules. The spherical hydrophilic polysaccharides have an average molecular weight of about 50,000 Da to about 100,000 Da, or about 60,000 Da to about 80,000 Da, or about 68,000 Da to about 72,000 Da, or about 69,000 Da to about 71,000 Da. In one embodiment, the spherical hydrophilic polysaccharides have an average molecular weight of 70,000 Da. In another embodiment, the spherical hydrophilic polysaccharides have an average molecular weight of about 5,000 Da to about 1,000,000 Da.

[0033] In one embodiment, the first cryoprotected particle or macromolecule is a high molecular weight sucrose polymer formed by copolymerization of sucrose and epichlorohydrin, also commonly referred to herein as "Ficoll 70." TM Ficoll 70. The Ficoll 70 molecule is highly branched and has a high content of hydroxyl groups, which gives the material excellent solubility in aqueous media. Ficoll 70 has an average molecular weight of approximately 70,000 Da.

[0034] In one embodiment, the second cryoprotected particle or macromolecule is also a polymer. The polymer may comprise a molecule that is a polysaccharide, particularly a polysaccharide formed of chains of different types of sugars or a single type of sugar. In one embodiment, the polysaccharide comprises a glycosaminoglycan (GAG), a modified GAG, a salt thereof, or a combination thereof. In one embodiment, the GAG ​​comprises chondroitin sulfate, dermatan sulfate, or a combination thereof. In one embodiment, the chondroitin sulfate comprises chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, chondroitin sulfate E, a salt thereof, or a combination thereof. An example of a modified GAG includes, for example, sulfated GAG.

[0035] The salts disclosed herein retain the biological effectiveness and properties of the given compounds and are not biologically or otherwise undesirable. Acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include, by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di(substituted alkyl)amines, tri(substituted alkyl)amines, alkenylamines, dialkenylamines, trialkenylamines, substituted alkenylamines, di(substituted alkenyl)amines, tri(substituted alkenyl)amines, cycloalkylamines, di(cycloalkyl)amines, tri(cycloalkyl)amines, substituted cycloalkylamines, disubstituted cycloalkylamines, trisubstituted cycloalkylamines, cycloalkenylamines, di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substituted cycloalkenyl ...alkenylamines, disubstituted cycloalkylamines, trisubstituted cycloalkylamines, cycloalkenylamines, di(cycloalkenyl)amines, tri(cycloalkenyl)amines, substituted cycloalkenylamines, disubstituted cycloalkenylamines, disubstituted cycloalkenylamines, disubstituted cycloalkenylamines, disubstituted cycloalken Included are salts of primary, secondary, and tertiary amines such as substituted cycloalkenylamines, trisubstituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed di- and triamines, where at least two of the substituents on the amine are different and are selected from the group consisting of, but not limited to, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic, etc. Also included are amines where two or three substituents together with the amino nitrogen form a heterocyclic or heteroaryl group.

[0036] Examples of pharma- ceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, etc. Pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, mesylic acid, esylic acid, besylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC-(CH2)n-COOH (wherein n=0-4), and the like.

[0037] In one embodiment, the second cryoprotected particle or macromolecule comprises chondroitin sulfate A sodium salt.

[0038] The cryoprotective medium disclosed herein includes an aqueous liquid. An "aqueous liquid" is a liquid that contains mainly water and is in a liquid state over the temperature range in which water is in a liquid state. In one embodiment, the aqueous liquid includes a saline solution, a cell or tissue culture medium, a buffer solution, or a combination thereof.

[0039] Examples of saline solutions include Alsever's solution, Earle's balanced salt solution (EBSS), Gey's balanced salt solution (GBSS), Hank's balanced salt solution (HBSS), (Dulbecco's) phosphate buffered saline (DPBS or PBS), Ringer's balanced salt solution (RBSS), Shim's balanced salt solution (SBSS), TRIS buffered saline (TBS), Tyrode's balanced salt solution (TBSS), HEPES (4-(2-hydroxyethyl)-piperazin-1-yl]ethane-1-sulfonic acid), CaCl2 aqueous solution, NaCl aqueous solution, KCl aqueous solution, or combinations thereof.

[0040] In one embodiment, the aqueous liquid may include a cell or tissue culture medium. The cell or tissue culture medium includes components that promote the growth and / or maintenance of cells and / or tissues. The specific composition of the cell or tissue culture medium varies depending on the type of cells and / or tissues used. Non-limiting examples of components in cell or tissue culture medium include, for example, serum (e.g., fetal bovine serum: FBS), carbohydrates (e.g., sucrose, galactose, fructose, maltose), amino acids, vitamins, minerals, inorganic salts, pH buffer systems, hormones, basic and trace elements (iron, zinc, copper, selenium, magnesium), supplements, and antibiotics. Specific examples of cell or tissue culture media, which may be used alone or in combination with additional components (serum, antibiotics, etc.), include Dulbecco's Modified Eagle's Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), Flushing's Maintenance Medium (FHM), DPBS (Dulbecco's Phosphate Buffered Saline), RPMI (Roswell Park Memorial Institute) medium, BF5 medium, EX-CELL® medium, Lysogeny Broth (LB), or combinations thereof.

[0041] The amount of the first cryoprotected particle or macromolecule in the cryopreservation medium is from about 10% (w / v) to about 50% (w / v), or from about 15% (w / v) to about 30% (w / v), or from about 15% (w / v) to about 25% (w / v), or from about 18% (w / v) to about 22% (w / v).

[0042] The amount of the second cryoprotected particle or macromolecule in the cryopreservation medium is from about 1% (w / v) to about 15% (w / v), or from about 2.5% (w / v) to about 10% (w / v), or from about 4.5% (w / v) to about 10% (w / v), or from about 4.5% (w / v) to about 7.5% (w / v).

[0043] The present disclosure provides a method for cryopreserving cells and tissues using the cryopreservation medium disclosed herein. In one embodiment, the cryopreservation medium is substantially free of cell-permeable cryoprotectants. In other words, the cryopreservation medium is substantially free of cell-permeable cryoprotectants. As used herein, "substantially free of cell-permeable cryoprotectants" and / or "substantially no cell-permeable cryoprotectants" means that the cryopreservation medium contains less than 5%, less than 2.5%, less than 1%, less than 0.5% cell-permeable cryoprotectants. In one embodiment, the cryopreservation medium is free of cell-permeable cryoprotectants, i.e. does not include cell-permeable cryoprotectants.

[0044] The cryopreservation medium can be used for the protection of the lipid membrane of lipid membrane-bound biostructure. As used herein, "lipid membrane-bound biostructure" refers to a biostructure that has a lipid membrane that defines the outer surface of the biostructure. Such lipid membrane-bound biostructures include cells, extracellular vesicles, and / or lipid-bound vesicles. This term also includes "tissues", which are structures that are composed of at least one cell that has an outer surface that is defined by a lipid membrane. Thus, in one embodiment, lipid membrane-bound biostructures include cells, tissues, extracellular vesicles, lipid-bound vesicles, organs, organisms, or combinations thereof.

[0045] In one embodiment, the lipid membrane-bound biostructure comprises a cell, an extracellular vesicle, a lipid-bound vesicle, an organ, an organism, or a combination thereof.

[0046] In one embodiment, the lipid membrane bound biostructure is a tissue comprising a plurality of cells, an organ comprising a plurality of cells, or an organism comprising a plurality of cells.

[0047] In one embodiment, disclosed herein is a method of protecting a lipid membrane of a lipid membrane-bound biostructure, comprising contacting the lipid membrane-bound biostructure with a cryopreservation medium prior to cooling the lipid membrane-bound biostructure to a temperature between about -70°C and about -273°C, whereby ice cubes form around the lipid membrane at temperatures between about -70°C and about -273°C.

[0048] Also disclosed is a method for cryopreserving a lipid membrane-bound biostructure, the method comprising: contacting the lipid membrane-bound biostructure with a cryopreservation medium disclosed herein to treat the lipid membrane-bound biostructure; cooling the treated lipid membrane-bound biostructure to a temperature of about -70°C to about -273°C to freeze the lipid membrane-bound biostructure; and maintaining the frozen lipid membrane-bound biostructure at a temperature of about -70°C to about -273°C.

[0049] In one embodiment, the contacting comprises adding a certain amount of cryopreservation medium to the two-dimensional or three-dimensional culture comprising the lipid membrane-bound biostructure and culture medium.The cryopreservation medium can be added directly to the lipid membrane-bound biostructure in the two-dimensional or three-dimensional culture without removing the culture medium.In one embodiment, the medium can be removed before adding the cryopreservation medium.

[0050] In one embodiment, the concentrated preparation of cryopreservation medium is added directly to the culture or suspension containing the lipid membrane-bound biostructures without first removing the culture medium (or the washing medium if the lipid membrane-bound biostructures are washed). The concentrated cryopreservation medium contains an increased amount of the first and second particles or macromolecules such that the concentration of the first and second particles or macromolecules is about 1.5 times, or about 2 times, or about 2.5 times, or about 3 times, or about 3.5 times, or about 4 times, or about 4.5 times, or about 5.5 times, or about 6 times, or about 6.5 times, or about 7 times, or about 7.5 times, or about 8 times, or about 8.5 times, or about 9 times, or about 9.5 times, or about 10 times higher than the respective concentrations in the non-concentrated cryopreservation medium. The concentrated cryopreservation medium is added in a specific volume ratio to dilute the concentration of the first and second particles or macromolecules in the medium to the above amount.

[0051] In one embodiment, the lipid membrane-bound biostructures are in suspension and are pelleted (e.g., by centrifugation) prior to contacting the lipid membrane-bound biostructures with a storage medium. Once pelleted, any culture or wash medium present is removed from the lipid membrane-bound biostructures, cryopreservation medium is added directly to the pellet to resuspend the lipid membrane-bound biostructures, and the new suspension is cooled to cryogenic temperatures for storage. In one embodiment, the volume ratio of cryopreservation medium to lipid membrane-bound biostructure pellet is between 10:1 and about 10,000:1.

[0052] In one embodiment, the volume ratio of cryopreservation medium to the relatively large volume of tissue or other membrane-bound biological structure is about 1:1 to about 10:1.

[0053] In one embodiment, the lipid membrane-bound biostructure to be cryopreserved is contacted with a cryopreservation medium for a time sufficient for the first and second particles or macromolecules present in the medium to completely diffuse through the lipid membrane-bound biostructure. The lipid membrane-bound biostructure thus treated is then cooled to cryogenic temperatures for storage.

[0054] In one embodiment, the treated lipid membrane-bound biostructure is frozen by cooling to a temperature between about -70°C and about -273°C. In one embodiment, the treated lipid membrane-bound biostructure is frozen by cooling to a temperature between about -196°C and about -70°C, or about -120°C and about -80°C. In one embodiment, the lipid membrane-bound biostructure is contacted with a cryopreservation medium at room temperature for about 30 minutes to about 120 minutes, and then cooled.

[0055] In some embodiments, cooling occurs at a rate of about 0.01° C. / min to about 1000° C. / min, or at a rate of about 0.1° C. / min to about 100° C. / min, or at a rate of about 0.5° C. / min to about 1° C. / min, or at a rate of about 1° C. / min to about 5° C. / min. In some aspects, cooling occurs immediately after contacting the lipid-bound biostructure with the cryopreservation medium.

[0056] Optionally, prior to the cooling step, the lipid membrane-bound biostructure can first be frozen at a temperature of about -18°C to about -25°C for a period of time. This period is long enough to ensure complete freezing of the lipid membrane-bound biostructure or its relatively large size, but not so long as to adversely affect its structure and / or viability. The period can be, but is not necessarily limited to, from about 6 hours or about 12 hours to a week or more.

[0057] In the methods disclosed herein, after cooling, the lipid membrane-bound biostructure is maintained (preserved) at a temperature between about -70°C and about -273°C.

[0058] As discussed above, during cooling, the water in the cryopreservation medium forms cubic ice on the outer surface of the lipid membrane of the lipid membrane-bound biostructure. Thus, the frozen lipid membrane-bound biostructure remains substantially intact when maintained at a temperature of about -70°C to about -85°C for a period of at least 3 weeks. In one embodiment, the period is at least 1 year.

[0059] In one embodiment, the lipid membrane-bound biostructure comprises a plurality of cells, and the post-thaw viability of the frozen plurality of cells is greater than or equal to about 60%, or about 75%, or about 80%, or about 90% of the total number of viable cells prior to cooling.

[0060] In one embodiment, the lipid membrane-bound biostructure comprises a eukaryotic cell or a prokaryotic cell. The eukaryotic cell may be a mammalian cell, a plant cell, an insect cell, or a combination thereof. The mammalian cell is not necessarily limited to, and may include, for example, a human cell, a mouse cell, a pig cell, a cow cell, a dog cell, a cat cell, or a combination thereof. The mammalian cell may include a stem cell, an adipocyte, a somatic cell, a germ cell, a chromaffin cell, a dermal cell, an epithelial cell, a neural progenitor cell, an embryonic stem cell, a pluripotent stem cell, an erythrocyte, a leukocyte, or a combination thereof.

[0061] In one embodiment, the lipid membrane-bound biostructure comprises tissue. The tissue is a living or bioartificial eukaryotic tissue. The eukaryotic tissue can be mammalian tissue. Mammalian tissue includes, but is not limited to, for example, human tissue, mouse tissue, porcine tissue, bovine tissue, canine tissue, feline tissue, or combinations thereof.

[0062] In one embodiment, the viability of a frozen tissue after thawing is greater than the viability of the same frozen tissue contacted with a cryopreservation medium comprising a cell-permeable cryoprotectant.

[0063] The present disclosure combines the use of the highly dense, spherical, and hydrophilic Ficoll 70 and chondroitin sulfate, which, when used in combination, fundamentally alters ice formation mechanisms at both the nanoscale and cellular levels, along with the hypothesized essential physical and biophysical mechanisms of action, as shown in Figure 1.

[0064] As shown in FIG. 1, the first cryoprotective particles or macromolecules 10 (e.g., Ficoll 70) promote the formation of nanoscale cubic ice 30 near their surface. The first cryoprotective particles or macromolecules 10 have a unique combination of characteristics, such as having a nearly perfect sphere and nanometer-sized particle size (e.g., particle size (diameter) of about 10 nm), being very dense, and having high hydrophilicity, resulting in nanoscale cubic ice structures. To our knowledge, such nanoscale cubic ice structures do not exist in all other existing cryoprotective media that use other types of polymers.

[0065] The second cryoprotective particle or macromolecule 20 (e.g., chondroitin sulfate) acts as a "glue" or "connector" that improves the connection between the cell's plasma membrane and the first cryoprotective particle or macromolecule 10, but also between the first cryoprotective particle itself, thereby forming a special network composed of the cell plasma membrane and the first cryoprotective particle or macromolecule near the surface of the cell. This unique combination and use of the first and second cryoprotective particles or macromolecule significantly increases the likelihood that the cell membrane will come into contact only with the nanoscale cubic ice crystals that form near the surface of the first cryoprotective particle or macromolecule. Thus, the cell plasma membrane is less prone to be damaged by the large hexagonal ice crystals 40 that are located far away from the surface of the first cryoprotective particle or macromolecule 10. As a result, the cell membrane is well protected during freezing, and either the formation of nanoscale ice on the outside of the membrane does not introduce ice formation inside the cell, or the size and number of the induced intracellular ice crystals are much smaller than the size and number of the nanoscale cubic ice crystals on the outside of the membrane. Therefore, intracellular components are also efficiently protected.

[0066] The mechanism of action of the combination of the first and second cryoprotected particles or macromolecules is described in further detail below, using Ficoll 70 and chondroitin sulfate molecules as illustrative examples.

[0067] In stark contrast to other macromolecules used in cryopreservation, Ficoll 70 forms nearly perfectly spherical and highly compact structures in aqueous solutions. Advantageously, it has been discovered that the special structure and highly hydrophilic surface of Ficoll 70 (i.e., highly branched sucrose network) promotes the formation of nanoscale cubic ice during freezing.

[0068] According to the phase diagram of pure water, at 1 atmosphere, cubic ice forms only at temperatures below -100°C. At pressures significantly lower than 1 atmosphere, water can form cubic ice at temperatures above -100°C. When Ficoll 70 macromolecules are present in water, the formation of nanoscale cubic ice can be achieved at relatively high temperatures (e.g., above -80°C) near the surface of the Ficoll 70 molecules. This effect is evidenced by results obtained from experiments using cryogenic X-ray diffraction and transmission electron microscopy of replicas of freeze-fractured samples of medium containing 10% Ficoll 70, as shown in Figures 2A and 2B.

[0069] Uchida et al., 2010 (Uchida T, Takeya S. Powder X-ray diffraction observations of ice crystals formed from disaccharide solutions. Phys Chem Phys. 2010 Dec 7;12(45):15034-9) reported that when solutions containing extremely high concentrations (approximately 50% w / v) of disaccharides (e.g., sucrose and trehalose) were frozen at relatively high cooling rates (e.g., several hundred degrees per minute), the sugar molecules spontaneously precipitated due to their solubility limit in cold water, and spontaneously formed nanoscale spherical particles to minimize the energy of the system. As shown in Figure 2A, X-ray diffraction patterns similar to those of Ficoll-rich medium were found for frozen solutions containing nanoscale granular sugar particles. However, when these same solutions were cooled at a slower cooling rate (e.g., 1 degree per minute, as used in slow freezing procedures for cells and tissues), those nanoscale spherical structures did not form, and instead, only the formation of regular hexagonal ice was detected in the same solutions. The use of such high concentrations of small molecule sugars causes lethal osmotic damage to nearly all cell types, making this an impractical cryopreservation approach.

[0070] On the other hand, cubic ice formation has been predicted for certain nanostructures (Davies MB, Fitzner M, Michaelides A. Routes to cubic ice through heterogeneous nucleation. Proc Natl Acad Sci US A. 2021 Mar 30;118(13):e2025245118). However, due to multiple influencing factors such as the surface of normal substances (such as hemispherical proteins) being uneven at the nanoscale, and / or their molecules being relatively loose and not as dense as Ficoll 70 molecules, and / or their surfaces being not as hydrophilic as Ficoll 70, and / or their solubility being very low, the resulting ice structures are generally hybrid or the cubic ice portion is too minimal due to the low solubility of the particles or macromolecules involved. Thus, what has been advantageously discovered by the composition and method of the medium of the present invention is the unique ice formation mechanism generated by Ficoll 70 molecules.

[0071] Without being limited by theory, other artificial spherical nanoparticles (e.g., highly spherical organic or inorganic nanoparticles), with or without specific surface modifications (e.g., conjugation with sugar molecules), may also achieve similar effects as Ficoll 70 if their hydrophilicity and solubility are sufficiently high. Very dense spherical polysaccharide molecules (e.g., polytrehalose and polymannitol) may also achieve similar effects. As for existing polysaccharides, almost all types form loose structures or are irregularly shaped when dissolved in water, with the exception of dextrans of various molecular weights, which form long rod-like structures, which are not suitable for promoting the formation of cubic ice. On the other hand, Ficoll 400 (polysucrose with a molecular weight of about 400 kDa) is another example of a very dense and spherical polysucrose type, but its diameter is much larger than that of Ficoll 70, which reduces the surface tension and makes it less efficient.

[0072] However, the above nanoscale cubic ice formation phenomenon is localized near the surface of Ficoll 70, while hexagonal ice, which forms at a relatively far distance from the surface of Ficoll 70 molecules, still dominates in frozen Ficoll 70 solutions. As shown in Figure 2A, the major TE111 peak (typical of cubic ice) observed by X-ray diffraction of frozen Ficoll 70 solutions is mainly due to the fact that cubic ice produces strong diffraction intensity comparable to X-rays due to the nanoscale crystal size. Therefore, the use of Ficoll 70 alone is insufficient, and cells suspended in Ficoll 70 solutions are more likely to be damaged by hexagonal ice.

[0073] Chondroitin sulfate has a high affinity for cell membranes and significantly increases the adhesion of cell membranes to other organic substances. Chondroitin sulfate is also frequently used in tissue engineering to promote cell adhesion to tissue scaffolds. Chondroitin sulfate also has a natural affinity for Ficoll 70, which has a repeating disaccharide unit and a surface formed by a highly branched sucrose network. Thus, the addition of a sufficient concentration of chondroitin sulfate to an aqueous solution of Ficoll 70 significantly increases the opportunity and probability for cell membranes to bind to Ficoll 70 molecules, as shown in Figure 1, and at the same time, the Ficoll molecules bind to each other to form a network. The results shown in Figure 3 using a fluorescence microscopy vocabulary book support this novel mechanism of action.

[0074] As a result, during freezing, this newly discovered mechanism of action efficiently prevents the formation of hexagonal ice near the cell membrane in close proximity to Ficoll 70 macromolecules. On the other hand, intracellular ice is generally introduced by extracellular ice, and the size of intracellular ice crystals is always much smaller than that of extracellular ice crystals, so the presence of high concentrations of intracellular macromolecules (approximately 30%-50% v / v in mammalian cells before freezing, which increases significantly due to the loss of intracellular water during freezing) may play a role in preventing the formation of nanoscale cubic ice near the cell membrane. Even if intracellular ice induced in this scenario is formed, the size of such crystalline ice is much smaller than the nanoscale ice structures outside the cell membrane, causing no or negligible damage to intracellular organelles and cell ultrastructures. Therefore, the combined use of both Ficoll 70 and chondroitin sulfate at sufficient concentrations in aqueous media provides adequate protection to both cell membranes and intracellular structures, thereby improving survival after thawing.

[0075] The efficiency of such cryopreservation media may also be due to several other beneficial factors, especially those contributed by the presence of chondroitin sulfate: these factors include, but are not limited to, the role of chondroitin sulfate as an anti-apoptotic agent, reducing cell death due to certain biophysical effects (e.g., loss of intracellular water during freezing), the role of chondroitin sulfate in stimulating cellular synthesis of proteoglycans and hyaluronic acid, thus stimulating their proper structure and function and reducing freeze damage, and / or the role of chondroitin in slowing down cell damage processes through various mechanisms.

[0076] The present disclosure is further illustrated by the following non-limiting examples. EXAMPLES

[0077] Detection of nanoscale cubic ice in Ficoll 70-containing media by cryogenic X-ray diffraction and transmission electron microscopy of replicas of freeze-fractured samples The formation of nanoscale cubic ice in media containing relatively high concentrations of Ficoll 70 is demonstrated by both cryogenic X-ray diffraction and transmission electron microscopy results as a unique cryoprotective mechanism.

[0078] A standard sample holder containing 10% Ficoll 70 in water was first slowly frozen to -80°C and then examined in a cryogenic X-ray diffraction chamber using the same instrument system, following the protocol described by Holm et al., 2004 (Holm AP, Pecharsky VK, Gschneidner KA, Rink R, and Jirmanus MN, X-ray powder diffractometer for in situ structural studies in magnetic fields from 0 to 35 kOe between 2.2 and 315 K, Rev. Sci. Instrum. 2004 75:1081). As shown in Figure 2A, detection of X-ray diffraction (line A) at -80°C demonstrated a major TE111 peak characteristic of cubic ice formation, and the size of the cubic ice crystals produces strong diffraction intensities comparable to the wavelength of X-rays due to the nanoscale crystal size. In contrast, for 10% DMSO in water, or 10% DMSO and 10% PVP or PEG in water, the diffraction patterns (line B) obtained following the same procedure were nearly identical to those from regular hexagonal ice and lacked the dominant TE111 peak.

[0079] A standard sample holder for freeze-fracturing containing 10% Ficoll 70 aqueous solution was first slowly frozen to -80 °C and then transferred to a standard freeze-fracture replica sample preparation system (Leica EM ACE900). Replicas of the fractured surfaces were made with gold and nickel nanoparticles. The replicas were then analyzed using a conventional transmission electron microscope. A 10,000-fold amplification clearly revealed the structure of hexagonal ice crystals (12 and 12' in Figure 2B) separated by a mixture of Ficoll molecules and nanoscale ice (11 and 11' in Figure 2B). Further amplification at 11 or 11' then demonstrated that the Ficoll molecules are surrounded by finer ice structures, which are cubic ice as determined from the results of the X-ray diffraction experiment shown in Figure 2A.

[0080] These studies explain the unique mechanism of action of Ficoll 70 molecules to form cubic ice near the surface at temperatures above -100°C under 1 atm pressure, which is physically impossible in normal aqueous solutions, and how Ficoll 70 medium prevents the recrystallization of hexagonal ice by separating them from each other, explaining the mechanism underlying the thermal studies demonstrated by Han et al., 2017 (Han X, Yuan Y, and Roberts RM 2017. Cryopreservation Medium and Method to Prevent Recrystallization, PCT / US2017 / 032606) and Yuan et al., 2016 (Yuan Y, Yang Y, Tian Y, Park J, Dai A, Roberts RM, Liu Y, Han X. Efficient long-term cryopreservation of pluripotent stem cells at -80°C. Nature, Scientific Reports. 2016 6:34476). EXAMPLES

[0081] Fluorescence microscopy studies showing that chondroitin sulfate A sodium salt molecules significantly enhance the affinity between Ficoll 70 molecules and cell membranes Adding a sufficient concentration of chondroitin sulfate to a Ficoll 70 solution (DMEM) increases the chance and probability that cell membranes will bind to Ficoll 70 molecules, and at the same time increases the chance that Ficoll molecules will bind to each other to form a network. Fluorescence microscopy experiments were performed to demonstrate this unique mechanism.

[0082] Ficoll 70 fluorescein isothiocyanate form (FITC-Ficoll 70) was purchased. Retinal pigment epithelial (RPE) cell sheets were combined with four different solutions based on DMEM medium, including: (A) 20% w / v regular Ficoll 70, (B) 20% Ficoll + 0.01% FITC-Ficoll, (C) 20% Ficoll + 0.01% FITC-Ficoll + 2.5% chondroitin sulfate A sodium salt, and (D) 20% Ficoll + 0.01% FITC-Ficoll + 5% chondroitin sulfate A thorium salt. The FITC fluorescence intensity near the cell sheet surface was measured, and the results are shown in Figure 3.

[0083] As seen in Figure 3, after only a short time (15 min), the solution containing 5% chondroitin sulfate significantly promoted the attachment of FITC-Ficoll to the cell surface. Thus, this experiment elucidated the mechanism of action of the cryopreservation medium and verified the hypothesis shown in Figure 1. As a result, during freezing, this newly discovered mechanism of action efficiently prevents the formation of regular hexagonal ice near the cell membrane in close proximity to Ficoll 70 macromolecules. On the other hand, since intracellular ice is generally introduced by extracellular ice and the size of intracellular ice crystals is always much smaller than that of extracellular ice crystals, the formation of nanoscale cubic ice near the cell membrane due to the presence of a high concentration of intracellular macromolecules (which is about 30%-50% v / v in mammalian cells before freezing and increases significantly due to the loss of intracellular water during freezing) should play a role in preventing the formation of intracellular ice. Therefore, the combined use of both Ficoll 70 and chondroitin sulfate in an aqueous medium can provide sufficient protection for both cell membranes and intracellular structures, thereby improving survival after thawing. EXAMPLES

[0084] Cryomicroscopy demonstrating that the medium of the present invention prevents intracellular ice formation during freezing cell density 10 8 Sf9 cells (a standard insect cell line) at 100 / ml (total volume of cells to total volume of medium is approximately 1:2) were suspended in regular medium EX-CELL medium containing 20% ​​w / v Ficoll 70 and 5% chondroitin sulfate A sodium salt (A), EX-CELL medium containing 10% v / v DMSO and 10% v / v FBS (B), and EX-CELL alone (C), respectively. The cell suspension samples were loaded into the freezing chamber of a standard cryomicroscope (Linkam, UK) and cooled from 0°C to -196°C at a cooling rate of 1 K / min. Both the medium of the present invention (A) and the regular cryopreservation medium (B) prevented the formation of intracellular ice, which is usually much darker than the extracellular ice area when viewed under a cryomicroscope, while severe intracellular ice occurred during this procedure when the medium did not contain a cryoprotectant (C). The ice crystals in A were also much smaller than those in B, whereas the size of the hexagonal ice crystals was significantly reduced in the medium of the present invention by the mechanism shown in Example 2. EXAMPLES

[0085] Efficacy of the medium of the present invention in cryopreservation of Sf9 cells at both -80°C and liquid nitrogen temperatures The cell suspensions from Example 3 were also transferred into cryovials and frozen at -80°C in a regular laboratory freezer or liquid nitrogen tank for storage for 2, 4 and 8 weeks. The treatments were EX-CELL medium with 20% w / v Ficoll 70 and 5% Chondroitin Sulfate A Sodium Salt (A), EX-CELL medium with 10% v / v DMSO and 10% v / v FBS (B) and EX-CELL alone (C). The post-thaw viability results measured by a standard automated cell counter (Countess II) and trypan blue assay are shown in Figure 5 for storage at -80°C (Figure 5A) and liquid nitrogen temperature (Figure 5B). Clearly, for Sf9 cells, the medium of the present invention achieved a similar efficiency as using regular cryopreservation medium in both storage conditions, whereas the observed cell survival was negligible when using medium without cryoprotectant. As demonstrated in the Examples below, using conventional methods, Sf9 cells can be cryopreserved long term at -80°C, whereas most other cells and all tissues cannot. EXAMPLES

[0086] Efficacy of the cryopreservation medium of the present invention in cryopreservation of human adipose stem cells at -80°C Long-term storage of human adipose-derived mesenchymal stem cells at -80°C in cryoprotective medium containing 20% ​​w / v Ficoll 70 and 5% w / v chondroitin sulfate A sodium salt was compared with conventional medium containing 10% DMSO and 10% fetal bovine serum.

[0087] Human adipose-derived mesenchymal stem cells (hASCs) from donors were passaged to generate a sufficient number of seeded cell culture flasks following standard culture protocols for hASCs. Cells were transferred to centrifuge tubes, formed a cell pellet by centrifugation, and the supernatant was removed. Cryopreservation medium containing 20% ​​w / v Ficoll 70 and 5% w / v sodium chondroitin sulfate A in Dulbecco's Modified Eagle's (DMEM) medium without cell-permeable cryoprotectants was added directly to the cell pellet for approximately 10 min. 6A new suspension was formed with a cell density of 1000 cells / ml (total volume of cells to total volume of medium is approximately 1:200). The new suspension was aliquoted into standard cryovials. The cryovials were then cooled to -80°C in a regular laboratory freezer using a standard cooling box at a cooling rate of approximately 1°C / min and stored in the -80°C freezer for 2 months. The cryovials were thawed in a 37°C water bath and post-thaw viability was measured using a standard automated cell counter based on trypan blue exclusion. As a comparison, cells from the same donor were prepared using conventional cryopreservation medium, a standard medium containing 10% v / v DMSO and 10% v / v fetal bovine serum, or DMEM containing only 20% w / v Ficoll 70. The significantly improved post-thaw viability obtained using the cryopreservation medium of the present invention is shown in Figure 6A (black bars).

[0088] As shown in Figure 6A, greater than 80% viability was observed when cryoprotective medium containing 20% ​​w / v Ficoll 70 and 5% w / v chondroitin sulfate A sodium salt was used. In comparison, only about 20% viability was observed when conventional medium containing 10% DMSO and 10% fetal bovine serum was used, and also only about 20% viability was observed when 20% w / v Ficoll 70 alone was used. See Figure 6A.

[0089] Thawed cells from treatment with cryoprotective medium also proliferated efficiently and expressed adipogenesis as shown in Figure 6B. Thus, cryoprotective medium containing 20% ​​w / v Ficoll 70 and 5% w / v chondroitin sulfate A sodium salt efficiently enabled long-term storage of hASCs at -80 °C, preserving cell viability and pluripotency. Although conventional medium containing 10% DMSO and serum enabled similar efficiency in long-term storage in liquid nitrogen facilities, such an approach, as described here, is not suitable for storage at -80 °C due to recrystallization that is damaging to the cells. EXAMPLES

[0090] Efficacy of the cryopreservation medium of the present invention in cryopreservation of bovine chromaffin cells at -80°C Efficient long-term storage of primary bovine chromaffin cells at -80°C using a cryopreservation medium containing 20% ​​w / v Ficoll 70 and either 5% or 10% w / v chondroitin sulfate A sodium salt was compared with conventional medium containing 10% DMSO and 10% fetal bovine serum.

[0091] Bovine primary chromaffin cells were isolated from bovine adrenal glands. Using the same procedure as described in Example 1, the cells were preserved as follows: (A) DMEM containing 20% ​​w / v Ficoll 70 and 5% chondroitin sulfate A sodium salt; (B) DMEM containing 20% ​​w / v Ficoll 70 and 10% w / v chondroitin sulfate A sodium salt; (C) conventional medium (DMEM containing 10% DMSO and 10% serum); (D) control medium (DMEM and 20% w / v Ficoll 70); and (E) DMEM containing 10% w / v Ficoll 70 and 5% w / v chondroitin sulfate A sodium salt. The storage temperature was -80°C, and the storage period was 4 months. It is shown in Figure 7 (two black bars) that the survival rate after thawing was significantly improved by using the medium of the present invention. The results were similar to those of Example 5.

[0092] Primary bovine chromaffin cells stored in cryopreservation medium containing 20% ​​w / v Ficoll 70 and either 5% or 10% w / v chondroitin sulfate A sodium salt showed a viability of over 70%, while cells stored in medium containing 10% w / v Ficoll 70 and 5% w / v chondroitin sulfate A sodium salt showed a viability of approximately 30%, whereas cells stored in conventional medium containing 10% DMSO and 10% fetal bovine serum showed a viability of approximately 20%, and cells stored in medium containing only 20% w / v Ficoll 70 showed a viability of approximately 10%. EXAMPLES

[0093] Efficacy of the cryopreservation solution of the present invention in cryopreservation of human skin grafts, human corneal limbal tissue, and bovine adrenal tissue at -80°C The efficiency of using the medium of the present invention (DMEM containing 20% ​​w / v Ficoll 70 and 5% w / v chondroitin sulfate A sodium salt) for cryopreservation of donor tissue was evaluated.

[0094] In particular, the efficiency of the medium of the present invention for cryopreservation of human skin grafts from seven different donors was tested. Each skin graft had a median thickness of 0.5 mm. Each tissue (approximately 10 cm x 10 cm in size) was mixed with two volumes of the medium of the present invention in a sterile freezing bag. The filled freezing bag was first cooled overnight in a -20°C freezer and then transferred to a -80°C freezer for storage. After one month of storage at -80°C, the post-thaw functionality of the skin grafts from seven different donors was analyzed by standard PrestoBlue assay, the tissue quality was evaluated by standard TUNEL staining, and the tissue ultrastructure was observed by transmission electron microscopy (TEM). Similar tissues from the same donors were also frozen in standard medium with 15% glycerol in Ringer's solution as a conventional approach for comparison. The results are shown in Figures 8A-8C. The test results show that the cryoprotection provided by the medium of the present invention is significantly better (reduced cell apoptosis and ultrastructural damage) or equivalent (cell number) to conventional media containing high concentrations of glycerol.

[0095] The medium of the present invention (DMEM containing 20% ​​w / v Ficoll 70 and 5% w / v chondroitin sulfate A sodium salt) was evaluated for its effect on cryopreservation of limbal tissue at -80°C. Two pairs of eligible human corneas were sent to a surgical team at Washington University with expertise in limbal stem cell (LSC) transplantation. For each pair of corneas, one cornea was cut radially into four sections (quadrants) and each quadrant was cryopreserved individually in a standard sterile 15 ml cryovial (Nalgene™) containing 10 ml of the medium of the present invention. For the other cornea of ​​the same pair, a quadrant was cryopreserved individually in standard DMEM medium with 5% DMSO and 10% FBS as a control. The tissue was also initially frozen overnight at -20°C and then thawed after storage at -80°C for one week or one month. After thawing, for each corneal quadrant, one of the quadrants was fixed in standard fixative for TUNEL staining, another quadrant was fixed for transmission electron microscopy (TEM), and the remaining two were radially cut into thirds each (total of 2x3=6 pieces) for in vitro culture to determine proliferation of LSCs from the thawed tissue.

[0096] After one week of storage at -80°C, LSC proliferation was similar in the two groups, but after one month, LSC proliferation was more prominent in the group cryopreserved with the medium of the present invention. Notably, evaluation of six tissue pieces from each group showed that after seven days of culture, 6 / 6 in the group using the medium of the present invention achieved proliferation of LSCs, whereas 3 / 6 in the DMSO+FBS group achieved proliferation of LSCs. Without being limited by theory, it is believed that this difference may be due to the fact that the invented protocol does not involve the use of toxic DMSO. Figure 9 shows representative results for cryopreservation of human limbal tissue for one month at -80°C using the cryopreservation medium of the present invention. Figure 9A: Representative image showing proliferation of limbal stem cells from tissue after thawing. Figure 9B: Representative image of cell staining (AE5 antibody against CK3) showing well-differentiated cells. Figure 9C: Representative image showing TUNEL staining of thawed tissue, showing normal limbal structure and cell health (few apoptotic cells). FIG. 9D: Representative transmission electron microscopy showing normal LSC ultrastructure after cryopreservation.

[0097] Considering the clinical value of cryopreservation of neuroendocrine tissues (e.g., pancreatic islets), we evaluated the efficiency of the medium of the present invention in the long-term preservation of adrenal medulla tissue, paving the way for the future adoption of the medium of the present invention in transplantation of pancreatic islets. Adrenal medulla was processed into small pieces of approximately 2 mm on each side. Approximately 30-40 samples were transferred into one 15 ml cryovial containing 10 ml of the medium of the present invention as the preservation medium, or into one 15 ml cryovial containing 10 ml of tissue culture medium with 10% DMSO and 10% FBS as a control. The cryovials were first frozen overnight at -20°C and then transferred to a -80°C freezer for storage. After storage at -80°C for one year, the quality of the tissue was analyzed by TUNEL staining, and the function of chromaffin cells was evaluated by detection of single vesicle catecholamine release using a microelectrochemical microelectrode. Typical results are shown in Figure 10A-B.

[0098] As shown in the figure, the control group (Figure 10B, using conventional medium) had low cell viability after thawing, the tissue structure was significantly damaged, and there was no detectable signal of catecholamine release. In contrast, treatment with the medium of the present invention (Figure 10A) well preserved the tissue structure, cell viability, and functionality. This study demonstrated the effectiveness of the cryopreservation medium of the present invention in achieving high throughput in long-term tissue preservation. EXAMPLES

[0099] Efficacy of the cryopreservation medium of the present invention in cryopreservation of bioartificial tissues by example of 2D RPE tissue and 3D neural tissue derived from differentiated iPSCs at -80°C The efficiency of the medium of the present invention (DMEM containing 20% ​​w / v Ficoll 70 and 5% w / v chondroitin sulfate A sodium salt) in cryopreservation of iPSC-derived bioartificial tissues was evaluated. Considering the success and simplicity of using standard 15 ml cryovials (2.5 cm in diameter and 5 cm in height) and the medium of the present invention for freezing small tissues, as shown above, it was decided to use the same cryovials, which are usually round and 1 cm in diameter, for iPSC-derived tissues as well. The cooling procedure involved mounting the cryovial containing 10 ml of the medium of the present invention and one tissue directly in a -80 °C freezer. The cooling rate of the tissue was estimated by inserting a thermocouple into the medium of the present invention at the bottom of the cryovial (as the density of the tissue is always greater than that of the medium of the present invention). Within the typical temperature range of the crystallization process, i.e., between -1 °C and -40 °C, the average cooling rate measured by this method was in the range of 1-2 °C / min, which is also close to the optimal cooling rate for relatively small tissues. The warming / thawing process involves placing the frozen cryovials into a 37 °C water bath with a warming rate of approximately 10 °C / min. TM Differentiated neural structures (e.g., 10x10 cells) were generated by following standard protocols.

[0100] Tissues were directly chilled as above with 10 ml of the medium of the present invention or conventional medium containing 10% DMSO and 10% FBS in 15 ml cryovials and stored in a -80°C freezer. After 2 months of storage, post-thaw viability was assessed by standard staining and confocal microscopy of RPE and ReN cells, and representative results are shown in Figures 11A-11B and 12A-12B. The group using the medium of the present invention (Figures 11A and 12A) yielded much higher viability and tissue quality than the conventional medium group (Figures 11B and 12B) and was comparable to the non-frozen control.

[0101] The efficiency of long-term storage (eg, 6 and 12 months) of the media of the present invention is also investigated.

[0102] Alternatively, the compositions, methods, and articles can comprise, consist of, or consist essentially of any suitable materials, steps, or components disclosed herein. The compositions, methods, and articles can additionally or alternatively be formulated so as to be free or substantially free of materials (or species), steps, or components that are not necessary for the function or achievement of the function of the compositions, methods, and articles.

[0103] All ranges disclosed herein are inclusive of the endpoints, and the endpoints can be combined independently of each other (e.g., "up to 25 wt.%, more specifically 5 wt.% to 20 wt.%, includes the endpoint value and all intermediate values ​​in the range "5 wt.% to 25 wt.%, etc."). "Combination" includes blends, mixtures, alloys, reaction products, and the like. Terms such as "first", "second", and the like do not denote any order, quantity, or importance, but are used to distinguish one element from another. The terms "a", "an", and "the" do not imply limitations of quantity, and should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. "Or" means "and / or" unless otherwise indicated. As used herein, terms such as "comprising", "including", "having", "containing", "involving", and the like should be understood to be open-ended. Unless otherwise specified, "including" means, but is not limited to, "about" or "approximately" as used herein means that the stated value is inclusive and within an acceptable range of deviation of the particular value as determined by one of ordinary skill in the art taking into account the measurement in question and the error associated with the particular quantity measurement (i.e., limitations of the measurement system). For example, "about" means within one or more standard deviations, or within ±10% or ±5% of the stated value. Any examples, or use of exemplary language (e.g., "such as"), are intended merely to better illustrate the invention and do not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention as used herein.

[0104] References throughout this specification to "aspects," "embodiments," and the like mean that a particular element described in connection with that embodiment is included in at least one embodiment described herein and may or may not be present in other embodiments. Moreover, it should be understood that the described elements can be combined in any suitable manner in the various embodiments. "Combinations thereof" are open-ended and include any combination that includes at least one of the recited components or features and, where appropriate, similar or equivalent components or features.

[0105] Although the present invention has been described with reference to exemplary embodiments, those skilled in the art will recognize that various changes can be made and equivalents can be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications can be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope of the invention. Therefore, it is not intended that the invention be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but rather, it is intended to include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. A first cryoprotected particle or macromolecule; A second cryoprotected particle or macromolecule; and 1. A cryopreservation medium comprising an aqueous liquid, the first cryoprotected particle or macromolecule is hydrophilic and has a spherical shape when dissolved or suspended in an aqueous liquid; and A cryopreservation medium, wherein the second cryoprotected particle or macromolecule has an affinity for the first cryoprotected particle or macromolecule and an affinity for the plasma membrane of a cell or a lipid membrane of a lipid membrane-bound biostructure.

2. 2. The cryopreservation medium of claim 1, wherein the amount of the first cryoprotected particle or macromolecule is from about 10% (w / v) to about 50% (w / v).

3. 2. The cryopreservation medium of claim 1, wherein the amount of the first cryoprotected particle or macromolecule in the medium is from about 15% (w / v) to about 30% (w / v).

4. 2. The cryopreservation medium of claim 1, wherein the amount of the second cryoprotectant particle or macromolecule in the medium is from about 1% (w / v) to about 15% (w / v).

5. 2. The cryopreservation medium of claim 1, wherein the amount of the second cryoprotectant particle or macromolecule in the medium is from about 2.5% (w / v) to about 10% (w / v).

6. 2. The cryopreservation medium of claim 1, wherein the cryopreservation medium is substantially free of cell-permeable cryoprotectants.

7. 2. The cryopreservation medium of claim 1, wherein the cryopreservation medium comprises less than 5% w / v of a cell-permeable cryoprotectant.

8. 7. The cryopreservation medium of claim 6, wherein the cell-permeable cryoprotectant comprises dimethylsulfoxide, glycerol, ethylene glycol, or a combination thereof.

9. The cryopreservation medium of claim 7, wherein the cell-permeable cryoprotectant comprises dimethyl sulfoxide, glycerol, ethylene glycol or a combination thereof.

10. 2. The cryopreservation medium of claim 1, wherein the first cryoprotective particle or macromolecule comprises a polymer that forms a compact three-dimensional structure.

11. The cryopreservation medium of claim 1, wherein the first cryoprotected particle or macromolecule comprises a globular hydrophilic polysaccharide, a polymerized cyclodextrin, a polymerized saccharide, a globular protein, a globular glycoprotein comprising an oligosaccharide chain attached to the outer surface of a globular protein, a globular protein derivative, a globular polypeptide, a globular nucleic acid, or a combination thereof.

12. 2. The cryopreservation medium of claim 1, wherein the first cryoprotected particle or macromolecule comprises a globular glycoprotein comprising oligosaccharide chains attached to the outer surface of the globular protein.

13. 2. The cryopreservation medium of claim 1, wherein the first cryoprotective particle or macromolecule comprises a spherical hydrophilic polysaccharide comprising a copolymer of sucrose and epichlorohydrin.

14. The cryopreservation medium of claim 13, wherein the globular hydrophilic polysaccharide has an average molecular weight of about 5,000 Da to about 1,000,000 Da.

15. 2. The cryopreservation medium of claim 1, wherein the globular hydrophilic polysaccharide has an average molecular weight of about 68,000 Da to about 72,000 Da.

16. The cryopreservation medium of claim 1 , wherein the second cryoprotective particle or macromolecule comprises a glycosaminoglycan, a modified glycosaminoglycan, a salt thereof, or a combination thereof.

17. 2. The cryopreservation medium of claim 1, wherein the second cryoprotective particle or macromolecule comprises chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, dermatan sulfate, a salt thereof, or a combination thereof.

18. 2. The cryopreservation medium of claim 1, wherein the second cryoprotective particle or macromolecule comprises chondroitin sulfate A sodium salt.

19. 1. A method for protecting a lipid membrane of a lipid membrane-bound biostructure, comprising: contacting the lipid membrane-bound biostructure with a cryopreservation medium according to any one of claims 1 to 18 before cooling the lipid membrane-bound biostructure to a temperature of about -70°C to about -273°C, The method comprises forming nanoscale cubic ice around the lipid membrane at temperatures between about -70°C and about -273°C.

20. A method for cryopreserving lipid membrane-bound biostructures, comprising: treating a lipid membrane-bound biostructure by contacting the lipid membrane-bound biostructure with a cryopreservation medium according to any one of claims 1 to 18; Cooling the treated lipid membrane-bound biostructure to a temperature of about -70°C to about -273°C to freeze the lipid membrane-bound biostructure; and maintaining the frozen lipid membrane-bound biostructure at a temperature of about -70°C to about -273°C.

21. 21. The method of claim 20, wherein the lipid membrane-bound biological structure comprises a cell, a tissue, an extracellular vesicle, a lipid-bound vesicle, an organ, an organism, or a combination thereof.

22. 21. The method of claim 20, further comprising freezing the lipid membrane-bound biostructure at a temperature of about -18°C to about -25°C for a period of time prior to cooling to a temperature of about -70°C to about -273°C.

23. 23. The method of claim 22, wherein the period is from about 6 to about 12 hours.

24. 21. The method of claim 20, wherein the cryopreservation medium is contacted with the lipid membrane-bound biostructure at room temperature for about 30 minutes to about 120 minutes, and then cooled.

25. 21. The method of claim 20, wherein the frozen lipid membrane-bound biostructure remains substantially intact when maintained at a temperature of about -70°C to about -85°C for a period of at least 3 weeks.

26. 26. The method of claim 25, wherein the period is at least one year.

27. 21. The method of claim 20, wherein the volume ratio of cryopreservation medium to lipid membrane-bound biostructure is about 1:1 to about 10:

1.

28. 21. The method of claim 20, wherein the volume ratio of cryopreservation medium to lipid membrane-bound biostructure is about 10:1 to about 10,000:

1.

29. The method of claim 20, wherein the contacting comprises adding a volume of a cryopreservation medium to a two-dimensional or three-dimensional culture comprising the lipid membrane-bound biostructure and a culture medium, and optionally removing the culture medium from the two-dimensional or three-dimensional culture prior to the contacting.

30. 21. The method of claim 20, wherein the cooling is at a rate of about 0.01° C. / min to about 100° C. / min.

31. 21. The method of claim 20, wherein the cooling is at a rate of about 1° C. / min to about 5° C. / min.

32. 21. The method of claim 20, wherein the lipid membrane-bound biostructure comprises a plurality of cells, and the post-thaw viability of the frozen plurality of cells is about 60% or more of the total number of viable cells prior to cooling.

33. 21. The method of claim 20, wherein the post-thaw viability of the frozen plurality of viable cells is greater than or equal to about 80% of the total number of viable cells prior to cooling.

34. 21. The method of claim 20, wherein the lipid membrane-bound biological structure is a tissue comprising a plurality of cells, an organ comprising a plurality of cells, or an organism comprising a plurality of cells.

35. 21. The method of claim 20, wherein the plurality of cells is mammalian cells, insect cells, plant cells, or a combination thereof.

36. 36. The method of claim 35, wherein the viability of the frozen tissue after thawing is greater than the viability of the same frozen tissue contacted with a cryopreservation medium comprising a cell-permeable cryoprotectant.

37. 21. The method of claim 20, wherein during cooling, the water in the cryopreservation medium forms ice cubes on the outer surface of the lipid membrane of the lipid membrane-bound biostructure.

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