Efficient cryopreservation medium that preserves tissue ultra-structures and eliminates tissue cells

Abstract

A cryopreservation medium including: a cryoprotective particle or macromolecule; and an aqueous liquid, wherein the cryoprotective particle or macromolecule is hydrophilic and has a spherical shape when dissolved or suspended in the aqueous liquid. A cryopreservation method that uses the medium of the present invention to preserve the ultrastructures of the tissues and simultaneously decellularize the tissues in a simple freezing procedure.

Description

EFFICIENT CRYOPRESERVATION MEDIUM THAT PRESERVES TISSUE ULTRA¬STRUCTURES AND ELIMINATES TISSUE CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on and claims priority to U.S Provisional Application Serial No. 63 / 513,624 filed July 14, 2023, which is incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The present disclosure is directed to the fields of cryobiology, cry opreservation, and ice formation control technologies, as well as storage of biological and clinical samples.BACKGROUND

[0003] Cryopreservation is a technique that enables long-term storage of biological materials at temperatures below the freezing point of water (i.e., 0°C). Practical cryostorage began from the discovery that animal semen could be cryopreserved using glycerol rich media in 1949 (Polge C, Smith Au, Parkes As. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature. 1949 Oct 15; 164(4172) :666). To provide protection for cells in tissues against ice formation, existing cryopreservation technologies (except a few that can be adopted only for few cell types with unique biophysical features) and all products in the marketplace, remain dependent on the use of various biologically incompatible (i.e., cell permeating and reactive) small molecule cryoprotectants. These cell-permeating cryoprotectants include but are not limited to, glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, and propanediol.

[0004] With recent advancements in tissue engineering, tissue transplantation, and xenotransplantation, there is a growing need for preserving tissue ultrastructures without necessarily preserving cell viability or even with the intention to completely eliminate cells from the donor tissue, whether it is sourced from humans or animals. These ultrastructures include but are not limited to, the structures formed by various collagen and elastin fibers, by fibrillin and other structural proteins, or by glycosaminoglycan (e.g., chondroitin sulfate and hyaluronic acid) network. The decellularized tissues with said ultrastructure play a critical role in tissue engineering as they serve as the base or scaffold materials for regrowing patient- derived cells. One of the key applications of decellularized tissues is in personalized therapeutics, where patient- specific cells can be regenerated within the processed donor tissues, such as skins, cartilages and blood vessels, to be transplanted back to the patients. Thisapproach enables the creation of tailored treatments that match the recipient’s unique needs for modification. Moreover, decellularization is particularly desirable for certain transplantations using allogeneic or xenogeneic tissues. For instance, in corneal stromal transplantation for the treatment of keratoconus, the removal of donor (either human or genetically modified animals) cells from the tissues is highly beneficial. By eliminating the donor cells, the risk of immune rejection from the recipients is significantly reduced, thereby increasing the success rate of the surgery. Additionally, the removal of donor cells eliminates or minimize the need for recipients to undergo immunosuppression, which can have its own set of complications and side effects.

[0005] However, the conventional methods using permeating cryoprotectants either directly damage tissue structures due to osmotic damages or fail in killing donor cells in the tissues. When low concentrations of permeating cryoprotectants, such as 10%-20% DMSO, are employed, the cryoprotective effects enable approximately 50% of donor cells to survive the procedure. However, the formation of relatively large ice crystals significantly disrupts tissue ultrastructures. For instance, cryopreservation of corneas with 10% DMSO leads to increased tissue thickness and disruption of collagen fiber arrangement in the corneal stroma. In contrast, the use of very high concentrations of permeating cryoprotectants causes significant osmotic stress during the permeation process of these small molecules. While this is advantageous for cell elimination due to both the osmotic stress and the toxicity of those small molecules, it also results in mechanical damage to tissue ultrastructures.

[0006] For example, approximately 10% of corneas donated in the US were frozen in 90% or pure glycerol for decellularization and storage in freezers (typically -80°C), but the thawed tissues lack the mechanical properties of fresh tissues. As a consequence, these tissues are currently limited to serving as surgical tools for glaucoma shunts, rather than being suitable for transplantation to treat conditions like keratoconus and other comeal stromal diseases. Consequently, the treatment of keratoconus still heavily relies on procuring fresh donor tissues, which poses significant challenges in countries other than the US.

[0007] Other methods for eliminating donor cells, such as those involving radioactive, chemical, or thermal effects, also have an impact on the quality of tissue ultrastructures. Additionally, these methods often require expensive instruments, complex operations, and may need to be combined with tissue cryopreservation or freezing protocols for practical implementation in storage of the processed tissues. For example, the use of high hydrostatic pressure generating equipment can efficiently achieve decellularization of human stroma. However, this method alone cannot achieve long-term storage of processed tissues. Based on the use of complicated freezing followed by gamma radiation protocols, the Lions Eye Institutefor Transplant & Research commercialized the sterilized and decellularized corneal stromal product, trademarked as OptiGraft®. However, it was known that the sterilization radiation procedure performed on human corneas results in considerably thinner (approximately 50% reduction in diameter) collagen fibrils that those in the fresh corneas. Additionally, the density of collagen fibrils in gamma-irradiated corneal stromal samples is only half or one third of that in fresh corneas.

[0008] Therefore, the current approaches for preserving donor tissue ultrastructures have limitations that compromise either the structural integrity or the decellularization efficiency. Addressing these challenges is crucial to enhance the success and accessibility of tissue preservation techniques in various medical applications.

[0009] Other various types of decellularized tissues also play a crucial role in clinical applications due to their ability to provide a scaffold that promotes tissue regeneration while minimizing the risk of immune rejection. For instance, decellularized skin is widely used in skin transplantation to treat severe bums and chronic wounds, offering a natural extracellular matrix (ECM) that supports cell attachment, proliferation, and differentiation. Similarly, decellularized amniotic membranes are employed in ocular surgeries and wound healing due to their anti-inflammatory, anti-scarring, and antimicrobial properties. Small intestinal submucosa (SIS), another decellularized tissue, is utilized in various surgical repairs, including hernia and urinary incontinence treatments, because of its robust ECM that fosters tissue integration and regeneration. These applications highlight the significance of decellularized tissues in enhancing the efficacy and safety of transplants and regenerative therapies, ultimately improving patient outcomes.

[0010] Meanwhile, tissue engineering technologies, especially those involving induced pluripotent stem cell (iPSC)-derived techniques for generating comeal endothelial and epithelial cells on tissue scaffolds, have witnessed rapid advancements. While decellularized donor stromal tissues represent a naturally optimal choice for scaffold materials, it is highly likely that the aforementioned decellularization process might hinder the efficiency of meeting the growing demand for qualified stromal tissues in the research and development of corneal tissue engineering technologies. Simultaneously, the field has seen remarkable progress in the development of bioengineered stromal tissues through various bioartificial methods. These methods encompass a wide range of innovative approaches, including but not limited to corneal substitutes crafted from recombinant human collagens, fibrin-agarose scaffolds, tissue adhesive gels, 3D-printed tissues, and materials sourced from xenogeneic origins such as porcine skin, corneas, or fish scales. However, a significant obstacle in the mass production,distribution, and transportation of these bioengineered tissues lies in the absence of suitable long-term storage solutions. This challenge needs to be addressed to fully harness the potential of these cutting-edge technologies.SUMMARY OF THE INVENTION

[0011] Disclosed herein is an efficient polymer-based cry opreservation medium without using any cell permeating cryoprotectant for both tissue structural preservation and decellularization. Methods of using the cryopreservation medium are also disclosed.

[0012] Disclosed herein is a cryopreservation medium comprising: a cryoprotective particle or macromolecule and an aqueous liquid, wherein the cryoprotective particle or macromolecule is hydrophilic and has a highly compact semi-spherical shape when dissolved or suspended in the aqueous liquid.

[0013] Disclosed herein also is a method of tissue decellularization and cryopreservation, contacting the tissue with a cryopreservation medium prior to cooling to a temperature of about -70°C to about -273°C, wherein both nano scale cubic ice and hexagonal ice of significantly reduced size is formed inside tissues a temperature of about -70°C to about -273 °C. After thawing, cells in the tissues are damaged and lost viability or functionality, and the tissue ultrastructures are well preserved and suitable for transplantation, or regrowth of a recipient’s’ cells, or other applications with further processing.

[0014] The above described and other features are exemplified by the following figures and detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following figures are exemplary embodiments wherein the like elements are numbered alike.

[0016] Figure 1 shows an illustration of the cryopreservation and decellularization mechanisms of the medium of the present invention. In a frozen solution, the Ficoll particles (10) promote nano scale cubic ice crystals (20) formation near its surface, while hexagonal ice crystals (30) form randomly elsewhere and damages cells (40). It is noteworthy that the ultrastructures of the tissues remain unharmed by the hexagonal ice crystals. This can be attributed to the significant reduction in size of the hexagonal ice crystals, achieved through the combined effect of Ficoll and cubic ice formation.

[0017] Figure 2 shows a comparison of collagen fiber density and size in corneal stroma tissues cryopreserved using two different methods after six months of storage: the traditional90% glycerol medium and the medium of the present invention. The assessment was conducted using transmission electron microscopy (TEM). Left Panel: TEM images at 30,000X magnification show a reduced fiber density and increased cross-sectional area in tissues treated with the glycerolization method. In contrast, tissues treated with the medium of the present invention exhibit collagen fiber density and size similar to those of fresh tissues. Right Panel: Quantitative analyses of the same comparison confirm the observations from the TEM images, demonstrating the superior preservation of collagen fibers with the new medium.

[0018] Figure 3 shows transmission electron microscopy that demonstrates the decellularization efficiency of the medium of the present invention for corneal stroma tissues. Fresh tissues contain intact keratocytes, whereas both the invented medium and the glycerol- treated samples show keratocytes with nuclear condensation, indicating cell destruction.

[0019] Figure 4 shows experimental results from Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) staining that demonstrate the efficiency of invented medium in killing all cells inside or on the surface the corneal stroma tissues.

[0020] Figure 5 shows the experimental results from Haematoxylin-eosin (H / E) staining that demonstrates the efficiency of invented medium in killing all cells inside the human skin tissues.

[0021] Figure 6 shows the experimental results from transmission electron microscopy that demonstrate the efficiency of invented medium in maintaining the tight junctions between the destroyed cells.DETAILED DESCRIPTION

[0022] A cry opreservation medium including highly compact spherical polysaccharide molecules such as Ficoll 70 (a spherical compact polysucrose molecule having a molecular weight (MW) approximating 70k Da) at a relatively high concentration (about 10% w / v after mixing with cell suspensions) together with small concentration of DMSO (5-10% v / v) to prevent ice recrystallization at the storage temperature near -80°C, has been disclosed by Han et al., 2017 (Han X, Yuan Y, and Roberts R.M. 2017. Cryopreservation Medium and Method to Prevent Recrystallization, PCT / US2017 / 032606) and described 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). This medium enabled low-term storage of mammalian cells in a regular deep freezer and thus removed the need for a liquid nitrogen facility for long-term storage of mammalian and insect cells, and also for tissue cry opreservation. As demonstrated by former thermal studies (YuanY, 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 medium containing 10% w / v to 20% w / v Ficoll 70 prevents ice recrystallization at the temperature up to about -65°C, so the method is suitable in long-term storage at any temperature below about -70°C, including the typical working temperatures of regular laboratory mechanical deep freezers. The commercialized product (C80EZ® medium) has been successfully used in numerous industrial applications and continues to be used.

[0023] Ficoll 70 or other Ficoll molecules have been used together with very high concentration of ethylene glycol (approx. 20%) or DMSO together with high concentration of sucrose (approx. 10%) for vitrification of oocytes or embryos, but those methods are only suitable for liquid nitrogen storage.

[0024] Ficoll 70 has also been used together with high concentration of glycosaminoglycan molecules to preserve the viability of cells without any permeating cryopreservation (Han et al. Efficient Biocompatible Cryopreservation Medium That Eliminates the Need for Cell Permeating Cryoprotectants. PCT / USA63 / 170673)|0025| None of the above media or methods are designed to kill or eliminate cells in the tissues.

[0026] Different from all above inventions or methods, the present invention involves the use of high concentration of Ficoll or similar molecules alone to achieve the preservation of the ultra-structures of the tissues and decellularization in a single freezing step and enable the decellularized tissues to be store in regular deep freezers, typically at -80°C for definitely long.

[0027] Disclosed herein is a cryopreservation medium comprising: a cryoprotective particle or macromolecule is hydrophilic and has the nano-scale features of being highly compact and spherical in shape, or nearly spherical, when dissolved or suspended in water, and also has a highly hydrophilic surface. In Figure 1 , a representative cryoprotective particle or macromolecule as 10 is identified. In solution, the cryoprotective particle or macromolecule promotes nano scale cubic ice crystal 20 formation near its surface, while hexagonal ice crystal 30 forms randomly elsewhere and damages cells 40.

[0028] Specific examples of the cryoprotective particle or macromolecule include a spherical hydrophilic polysaccharide, a polymerized cyclodextrin, a polymerized saccharide, a globular protein, a spherical glycoprotein comprising oligosaccharide chains attached to an outer surface of a globular protein, a globular protein derivative, a globular polypeptide, a spherical nucleic acid, or a combination thereof.

[0029] The cryoprotective particle or macromolecule has a nanometer-sized particle diameter. In an aspect, the cryoprotective 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 an aspect, the cryoprotective particle or macromolecule has a particle size of about 10 nm.

[0030] In an aspect, the cryoprotective particle or macromolecule comprises a spherical hydrophilic polysaccharide comprising a copolymer of sucrose and epichlorohydrin. Examples of the copolymer of sucrose and epichlorohydrin include FICOLL™ molecules. The spherical hydrophilic polysaccharide can 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 DI to about 71,000 Da. In an aspect, the spherical hydrophilic polysaccharide has an average molecular weight of 70,000 Da. In another aspect, the spherical hydrophilic polysaccharide has an average molecular weight of about 5,000 Da to about 1,000,000 Da.

[0031] In an aspect, the cryoprotective particle or macromolecule comprises FICOLL™ 70, also referred to herein generally as “Ficoll 70”, which is a high molecular weight sucrose polymer formed by copolymerization of sucrose and epichlorohydrin. Ficoll 70 molecules are highly branched and have a high content of hydroxyl groups, which leads to very good solubility of the material in aqueous media. Ficoll 70 has an average molecular weight of about 70,000 Da.

[0032] In an aspect, the aqueous liquid may comprise a cell or tissue culture medium. The cell or tissue culture medium includes components which facilitate growth and / or maintenance of cells and / or tissues. The specific composition of the cell or tissue culture medium varies depending upon the type of cell and / tissue with which it is used. Non-limiting examples of components in the 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 system, hormones, basic and trace elements (iron, zinc, copper, selenium, magnesium), supplements, antibiotics. Specific examples of cell or tissue culture medium, which can be used alone or in combination with additional components (e.g., serum, antibiotics, etc.), include Dulbecco's Modified Eagle Medium (DMEM), Iscove’s Modified Dulbecco’s Medium (IMDM), flushing / holding medium (FHM), DPBS (Dulbecco's phosphate-buffered saline), RPMI (Roswell Park Memorial Institute) medium, BF5 medium, EX-CELL® medium, Lysogeny broth (LB), or a combination thereof.

[0033] An amount of the cryoprotective particle or macromolecule in the cryopreservation medium is about 10 % (w / v) to about 50% (w / v), or about 15 % (w / v) to about 30% (w / v), or about 15% (w / v) to about 25% (w / v), or about 18% (w / v) to about 22% (w / v).

[0034] The present disclosure provides a method for cryopreserving tissues using the cryopreservation medium disclosed herein. In an aspect, the cryopreservation medium substantially no cell permeating cryoprotectant. Put another way, the cryopreservation medium is substantially free of cell permeating cryoprotectant. As used herein “substantially free of cell permeating cryoprotectant” and / or “substantially no cell permeating cryoprotectant” means that the cryopreservation medium contains less than 5%, or less than 2.5%, or less than 1%, or less than 0.5% of a cell permeating cryoprotectant. In an aspect, the cryopreservation medium is free of cell permeating cryoprotectant i.e., does not include any cell permeating cryoprotectant.

[0035] The cry opreservation medium can be used for the protection of tissue ultrastructures. As used herein “tissue ultrastructure” refers to a biological structure constructed by large organic or inorganic molecules. Representative tissue ultrastructures include but are not limited to, the structures formed by various collagen and elastin fibers, by fibrillin and other structural proteins, or by glycosaminoglycan (e.g., chondroitin sulfate and hyaluronic acid) network.

[0036] In an aspect, disclosed herein is a method of protecting tissue ultrastructures, contacting the tissues with a cryopreservation medium prior to cooling the tissues directly to a temperature between about -70°C and about -273°C, wherein cubic ice is formed inside tissues at the temperature between about -70°C and about -273°C.

[0037] In some embodiments, the cooled 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 l°C / min to about 10°C / min. In some respects, the cooling is performed directly after contacting tissues with the cry opreservation medium.

[0038] The tissues maintained at the temperature of about -70°C to about -85 °C for a time period of at least three weeks. In an aspect, the time period is at least one year.

[0039] In an aspect, after thawing, a post-thaw survival rate of cells of the tissues is less than or equal to about 50%, or about 30%, or about 20%, or about 10%, or about 5%, or about 1%, of the total number of viable cells prior to the cooling.EXAMPLESExample 1: The efficacy of the cryopreservation medium of the present invention in decellularization and cry opreservation of human comeal stromal tissues at -80°C

[0040] The efficiency of using the medium of the present invention (DMEM containing 20% w / v Ficoll 70) for the cryopreservation of human corneal stromal tissues was evaluated. Human corneas with both viable cell types (including but not limited to endothelial cells in the inner layer of corneas, epithelial cells in the outer layer of corneas, keratocytes and fibroblasts inside stroma) were sent to a Washington University surgical team. For each pair of corneas, one cornea was radially cut into half, with each cryopreserved in a standard 90% (v / v) glycerol solutions and the medium of the present invention, respectively. The tissues were merged in sterile 15 ml cryovial (Nalgene™) containing 10 ml of the either of two media. After one hour of incubation, the tissues were directly frozen at -80°C for six months, and then thawed. After thawing, each tissue was washed by balanced salt solutions and then either fixed for transmission electron microscopy (TEM) to study both tissue ultrastructures and cellular structures or fixed for standard Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) staining to study cell viability.

[0041] As shown in Fig.2, the efficiency of the medium of the present invention in preserving the tissue ultra-structures is demonstrated in comparison of the density (i.e., the number of fibers per unit area) and the average size (measured by the average pixel number per fiber) of collagen fibers inside the thawed tissues. The tissues cryopreserved using glycerol exhibited an average intersectional diameter that was 20% larger than those treated with the present invention. The increased size of collagen fibers can be attributed to the inclusion of high concentrations of glycerol during perfusion. This occurs due to the significant variation in osmolarity within the liquid inside the tissues. As a result, the fiber structure thickens, leading to the loss of their original mechanical properties. The use of glycerol also reduces the fiber density of the tissues, consistent with previous studies that show an increase in the overall thickness of stromal tissues due to decreased fiber density. In other words, conventional glycerolization methods result in looser tissue structure. In contrast, the medium of the present invention effectively prevented such thickening, suggesting the preservation of the tissue's mechanical properties. The working mechanism may rely on the facts that the impact on the osmolarity of solutions by using Ficoll polymers is negligible. Meanwhile, the formation of nano scale cubic ice and the hexagonal ice crystals with reduced sizes efficiently minimize the disruption of the collagen fibers during the freezing process.

[0042] As shown in Figs. 3 and 4, using the medium of the present invention, various cell types inside the thawed tissues are all severely damaged. The degree of the damage is comparable to that on the cells in the tissues treated by the traditional medium.

[0043] Therefore, by maintaining the original size and density of collagen fibers, the present invention is expected to retain the tissue’s mechanical properties, which is a significant advantage over the traditional glycerol-based preservation methods. The present invention is also efficient in decellularization for the tissues.Example 2: Efficacy of the Present Invention’s Cry opreservation Medium in Decellularization and Cryopreservation of Human Skin Tissues at -80°C

[0044] Similar results were achieved using the medium of the present invention (i.e., 20% Ficoll 70 in DMEM) for the decellularization of human skin tissues. Split-thickness human donor skin tissues were immersed in sterile 15 ml cryovials containing 10 ml of the medium. After one hour of incubation, the tissues were directly frozen at -80°C for two months. Glycerolization is not a standard method for skin decellularization, so it was not used as a control in this study.

[0045] As shown in Fig. 5, H / E staining results indicated that all cell types in the tissues treated with the medium of the present invention destroys. Furthermore, as shown in Fig. 6, TEM results demonstrated the maintenance of tight junctions between destroyed cells, indicating well-preserved ultrastructures of the tissues.

[0046] The compositions, methods, and articles can alternatively comprise, consist of, or consist essentially of, any appropriate materials, steps, or components herein disclosed. The compositions, methods, and articles can additionally, or alternatively, be formulated so as to be devoid, or substantially free, of any materials (or species), steps, or components, which are otherwise not necessary to the achievement of the function or objectives of the compositions, methods, and articles.

[0047] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other (e.g., ranges of “up to 25 wt.%, or, more specifically, 5 wt.% to 20 wt.%”, is inclusive of the endpoints and all intermediate values of the ranges of “5 wt.% to 25 wt.%,” etc.). “Combinations” is inclusive of blends, mixtures, alloys, reaction products, and the like. The terms “a” and “an” and “the” do not denote a limitation of quantity and are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. “Or” means “and / or” unless clearly stated otherwise. As used herein, the terms “comprising” “including,” “having,” “containing,” “involving,” andthe like are to be understood to be open-ended, i.e., to mean “including” but not limited to, unless otherwise noted. “About” or “approximately” as used herein is inclusive of the stated value and means within an acceptable range of deviation for the particular value as determined by one of ordinary skill in the art, considering the measurement in question and the error associated with measurement of the particular quantity (i.e., the limitations of the measurement system). For example, “about” can mean within one or more standard deviations, or within ± 10% or ± 5% of the stated value. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

[0048] Reference throughout the specification to “an aspect”, “an embodiment”, and so forth, means that a particular element described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments. A “combination thereof’ is open and includes any combination comprising at least one of the listed components or properties optionally together with a like or equivalent component or property.

[0049] While the invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention is not limited to the particular embodiment disclosed as the best mode contemplated for carrying out the present invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMSWhat is claimed:

1. A cryopreservation medium comprising: a cryoprotective particle or macromolecule; and an aqueous liquid, wherein the cryoprotective particle or macromolecule is hydrophilic and has a spherical shape when dissolved or suspended in the aqueous liquid, and wherein the cryopreservation medium comprises less than 5% w / v of a cell permeating cryoprotectant.

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

3. The cryopreservation medium of any of claims 1-2, wherein an amount of the cryoprotective particle or macromolecule in the medium is about 15 % (w / v) to about 30% (w / v).

4. The cryopreservation medium of any of claims 1-3, wherein the cryopreservation medium comprises substantially no cell permeating cryoprotectant.

5. The cryopreservation medium of any of claims 1-4, wherein the cell permeating cryoprotectant comprises dimethyl sulfoxide, glycerol, ethylene glycol, propanediol, or a combination thereof.

6. The cryopreservation medium of any of claims 1-5, wherein the cryoprotective particle or macromolecule comprises a polymer that forms a compact, three-dimensional structure.

7. The cryopreservation medium of any of claims 1-6, wherein the cryoprotective particle or macromolecule comprises a spherical hydrophilic polysaccharide, a polymerized cyclodextrin, a polymerized saccharide, a globular protein, a spherical glycoprotein comprising oligosaccharide chains attached to an outer surface of a globular protein, a globular protein derivative, a globular polypeptide, a spherical nucleic acid, or a combination thereof.

8. The cry opreservation medium any of claims 1-7, wherein the cryoprotective particle or macromolecule comprises a globular protein having oligosaccharide chains attached to the outer surface of the globular protein.

9. The cry opreservation medium of any of claims 1-8, wherein the cryoprotective particle or macromolecule comprises a spherical hydrophilic polysaccharide comprising a copolymer of sucrose and epichlorohydrin.

10. The cryopreservation medium of claim 9, wherein the spherical hydrophilic polysaccharide has an average molecular weight of about 5,000 Da to about 1,000,000 Da.

11. The cry opreservation medium of any of claims 9-10, wherein the spherical hydrophilic polysaccharide has an average molecular weight of about 68,000 Da to about 72,000 Da.

12. The cryopreservation medium of any of claims 10-11, wherein the spherical hydrophilic polysaccharide has an average molecular weight of about 68,000 Da to about 72,000 Da.

13. A method of protecting ultrastructures of biological or bioartificial tissues while decellularizing the tissues, comprising: contacting the tissue with the cryopreservation medium of claims 1-12 prior to cooling; and cooling the treated tissue to a temperature of about -70°C to about -273 °C; wherein cubic ice is formed inside tissues at the temperature of about -70°C to about -273°C.

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