阅读说明：本技术 一种防止样品和细胞外冰直接接触的有效低温保存装置 (Effective low-temperature storage device for preventing sample from directly contacting with extracellular ice ) 是由 韩旭 彼得·库伦 约翰·K·克莱泽 于 2019-08-30 设计创作，主要内容包括：一种低温保护装置通过防止生物材料与破坏细胞的大冰晶直接接触,从而保护了含水生物材料免受在低温冷冻和/或低温储存期间由于冰的形成而导致的机械损伤,所述低温保护储存装置具有带有内腔的壳体。壳体配置为在内腔内容纳具有生物材料的可冻结介质。壳体包括半透膜。所述膜对于大于所述膜的平均孔径的冰晶是不可渗透的,以防止这种冰晶从所述壳体的外部进入所述内腔,使得在所述壳体内的介质中形成的冰晶具有与壳体外部介质中形成的冰晶相比较小的晶体尺寸。从而,保护了生物材料免受由直接与大冰晶接触而产生的机械损伤。(A cryoprotective device protects aqueous biological material from mechanical damage due to ice formation during cryogenic freezing and/or cryogenic storage by preventing the biological material from direct contact with large ice crystals that disrupt cells, the cryoprotective storage device having a housing with an internal cavity. The housing is configured to contain a freezable medium having a biological material within the internal cavity. The housing includes a semi-permeable membrane. The membrane is impermeable to ice crystals larger than the average pore size of the membrane to prevent such ice crystals from entering the internal cavity from the exterior of the shell such that ice crystals formed in the medium within the shell have a smaller crystal size than ice crystals formed in the medium exterior to the shell. Thus, the biomaterial is protected from mechanical damage resulting from direct contact with large ice crystals.)

1. A cryoprotective device for protecting a sample from mechanical damage during cryopreservation, the device comprising:

a shell forming an internal cavity, wherein said shell is configured to contain a freezable medium within said internal cavity, wherein said shell comprises a semi-permeable membrane that is impermeable to ice crystals substantially larger than the mean pore size of said membrane to prevent any such ice crystals from entering the internal cavity from a region external to the shell such that ice crystals formed in said medium within said shell have a smaller crystal size than ice crystals formed in a freezable medium external to said shell.

2. The cryogenic protected storage device of claim 1, wherein a first opening is formed at a first longitudinal end of the housing and the membrane is disposed over a second opening formed in the housing.

3. The cryo-protected storage device of claim 2, wherein the housing is configured to receive the sample through the first opening within an internal cavity.

4. The cryogenic protection storage device of claim 1, wherein the enclosure is a buoyant support, the membrane being attached to the buoyant support to define the internal cavity such that the membrane is submersible within the freezable medium outside the enclosure and at least a portion of the buoyant support is not submersible within the freezable medium outside the enclosure.

5. The cryogenic protective storage device of claim 1, wherein the housing is formed substantially entirely of the membrane.

6. The cryo-protected storage device of claim 1, wherein said membrane is a porous solid layer.

7. The cryogenic protective storage device of claim 6, wherein the porous solid layer allows water and liquid material to permeate through, but prevents ice crystals larger than the pore size of the membrane from passing through the membrane, the ice crystals being formed by solidification of cryoconservation outside the housing when the device is exposed to cryogenic temperatures.

8. The cryogenic storage device of claim 6, wherein the porous solid layer comprises pores having a pore size in a range of about 0.1 nanometers (nm) to about 1 millimeter (mm), about 0.5nm to about 0.1mm, about 1nm to about 100nm, or about 2nm to about 10 nm.

9. The cryogenic storage device of claim 1, wherein the membrane comprises a natural or synthetic polymer.

10. The cryo-protected storage device of claim 1, wherein the membrane comprises a synthetic polymer comprising polyacrylonitrile, polymethylmethacrylate, polysulfone, vinyl alcohol copolymer, or any combination or chemical derivative thereof.

11. The cryo-protected storage device of claim 1, wherein said film comprises a cellulosic material.

12. The cryogenic storage device of claim 11, wherein the cellulosic material comprises regenerated cellulose, denatured cellulose, cellulose diacetate, cellulose triacetate, or any combination or chemical derivative thereof.

13. The cryo-protected storage device of claim 1, wherein said membrane is a dialysis membrane made of denatured cellulose.

14. The cryogenic protective storage device of claim 1, wherein the membrane is permeable to water, organic and/or inorganic liquid solvents, or any combination thereof.

15. The cryogenic protective storage device of claim 1, wherein the membrane is a detachable membrane configured to be selectively mounted to cover the second opening.

16. The cryo-protected storage device of claim 1, wherein said medium comprises a cryo-protective medium having a cryo-protective material suspended therein as an additive.

17. The cryogenic protection storage device of claim 1, wherein the housing includes a recess in which the membrane is mounted to the housing to cover the second opening, the device including a support ring configured to be selectively attached to the housing to secure the membrane over the second opening when the support ring is attached to the housing.

18. The cryogenic protected storage device of claim 17, wherein the support ring comprises an outer support member having an aperture formed therethrough such that when the support ring is mounted to a housing, the membrane is exposed to the medium outside the housing through the aperture formed in the support ring.

19. The cryogenic protection storage device of claim 1, wherein the housing comprises at least one locking tab that lockingly engages with the support ring to secure the support ring to the housing.

20. The cryo-protected storage device of claim 1 wherein said first opening is configured such that said sample can pass into said internal cavity.

21. The cryogenically-protected storage device of claim 1 including a cover configured to be secured to the housing to close the first opening.

22. The cryogenic protected storage device of claim 21, wherein the lid comprises at least one vent formed in a perimeter of the lid to allow media in the internal cavity to flow through the at least one vent when the lid is secured to the housing.

23. The cryogenic protected storage device of claim 21, wherein the lid comprises an inner surface having a convex shape such that the lid protrudes deeper into the internal cavity at a center of the lid than at a periphery of the lid.

24. The cryogenic protected storage device of claim 1, wherein the shell is substantially cylindrical in shape with a circular cross section.

25. The cryogenic protective storage device of claim 1, wherein the freezable medium is a liquid at about 25 ℃ and a solid at about-20 ℃.

26. A system for storing a sample at cryogenic temperatures, the system comprising:

at least one cryo-protected storage device according to claim 1;

an outer container configured to house one or more of the cryogenic storage devices; and

a freezable medium is used in an interior cavity of a housing and an outer container outside the housing.

27. The system of claim 23, wherein the freezable medium comprises hydrophilic and non-toxic macromolecules and an aqueous liquid.

28. The system of claim 27, wherein the freezable medium comprises a cryoprotectant.

29. The system of claim 28, wherein the cryoprotectant comprises dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sucrose, glucose, dextran and other polysaccharides, polyvinylpyrrolidone and polyethylene glycol, and other polymers, other non-penetrating cryoprotectants including but not limited to: chondroitin sulfate and lactobionic acid or any combination thereof.

30. The system of claim 28, wherein the hydrophilic and non-toxic macromolecule is a polymer.

31. The system of claim 30, wherein the polymer forms a three-dimensional structure that is substantially spherical when dissolved in an aqueous liquid.

32. The system of claim 27, wherein the aqueous liquid comprises cell culture media, nutrient media, saline, or any combination thereof.

33. The system of claim 27, wherein the aqueous liquid comprises serum, Fetal Bovine Serum (FBS), Dartbox Modified Eagle's Medium (DMEM), 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), rinse maintenance medium (FHM), Phosphate Buffered Serum (PBS), Dartbox Phosphate Buffered Saline (DPBS), rossv pock souvenir institute medium (RPMI), BF5 medium, CaCl-CELL medium, bacteriolysis broth (LB) medium, CaCl-CELL medium, and combinations thereof₂An aqueous solution, an aqueous NaCl solution, an aqueous KCl solution, or any combination thereof.

34. The system of claim 30, wherein the concentration of the polymer in the medium is greater than about 5% (w/v), greater than about 10% (w/v), greater than about 20% (w/v), or greater than about 50% (w/v).

35. The system of claim 30, wherein the concentration of the cryoprotectant within the medium is equal to or greater than about 20%, equal to or greater than about 50%, equal to or greater than about 75%, or equal to or greater than about 100% of the concentration of the polymer in the medium.

36. The system of claim 24, wherein the medium comprises about 10% (w/v) ficoll, about 5% (w/v) DMSO, about 2% (w/v) chondroitin sulfate, and about 1% (w/v) dextran 40.

37. The system of claim 36, wherein the ficoll is a polysaccharide formed by copolymerization of sucrose and epichlorohydrin.

38. The system of claim 30, wherein the polymer is selected from the group consisting of hydrophilic polysaccharides, polymeric cyclodextrins or sugars, globular proteins or globular proteins, globular glycoproteins formed by linking oligosaccharide chains of globular proteins, other derivatives of globular proteins, and combinations.

39. The system of claim 30, wherein the polymer is a hydrophilic polysaccharide.

40. The system of claim 37, wherein the polysaccharide is formed by copolymerization of sucrose and epichlorohydrin.

41. The system of claim 26, wherein the medium fills a portion or all of a space between the outer container and the at least one cryogenic storage device, the medium being provided in a sufficient amount to cover at least a surface of the membrane facing away from the inner cavity.

42. The system of claim 41, wherein a composition of the medium external to the cryogenic storage device is different from or the same as a composition of the medium in the internal cavity of the cryogenic storage device.

43. A method of protecting a sample from damage during freezing, the method comprising:

providing a housing forming an internal cavity, wherein the housing comprises a semi-permeable membrane;

providing a sample and a first freezable medium in an interior cavity of the housing;

placing the shell within a second freezable medium external to the shell such that the shell is partially or fully immersed in the second freezable medium, wherein the second freezable medium is the same as or different from the first freezable medium;

exposing the second freezable medium to a freezing temperature such that ice crystals having a first size form outside the shell in the second freezable medium;

wherein growth of ice crystals of the first size in the second freezable medium is inhibited by the membrane such that only ice crystals of a second size smaller than the pore size of the membrane can pass through the membrane and cause ice to form in the first freezable medium within the internal cavity of the housing such that ice crystals generated within the housing have a size smaller than ice crystals formed in the second freezable medium outside of the housing.

44. The method of claim 43, wherein said freezing comprises cryogenic freezing, and wherein said freezing temperature comprises a cryogenic temperature.

45. The method of claim 43, wherein said freezing comprises non-cryogenic freezing, and wherein said freezing temperature comprises a non-cryogenic temperature.

46. The method of claim 45, wherein the sample and first freezable medium comprise a cell suspension or biological tissue in a natural or conventional medium and/or solution, wherein the first freezable medium is free of cryoprotectants.

47. The method of claim 43, wherein a first opening is formed at a first longitudinal end of the housing and the membrane is disposed over a second opening formed in the housing.

48. The method of claim 47, wherein the housing is configured to receive the sample within the lumen through the first opening.

49. The method of claim 43, wherein the enclosure is a buoyant support, the membrane being attached to the buoyant support to define the internal cavity such that the membrane is submersible within the freezable medium outside the enclosure and at least a portion of the buoyant support is not submersible within the freezable medium outside the enclosure.

50. A method according to claim 73, wherein the first and/or second freezable medium comprises hydrophilic and non-toxic macromolecules and an aqueous liquid.

51. The method of claim 44, wherein:

the first and/or second freezable medium comprises a cryoprotectant; and

as the temperature of the first freezable medium decreases, water in the first freezable medium within the housing permeates through the membrane, thereby increasing the concentration of cryoprotectant within the first freezable medium during cryofreezing.

52. The method according to claim 51, wherein permeating water through said membrane from said first freezable medium to said second freezable medium increases the concentration of solutes in said first freezable medium and/or decreases the freezing temperature of said first freezable medium to prevent supercooling of said first freezable medium.

53. The method of claim 52, wherein:

ice crystals formed in the second freezable medium are larger and form at a higher temperature than ice crystals formed in the first freezable medium;

the membrane comprises a porous material having pore sizes smaller than the diameter of ice crystals formed in the second freezable medium; and/or

Damage to the sample is reduced relative to storing the sample in a housing without a membrane that allows water to permeate through.

54. The method of claim 47, wherein the membrane is positioned over the second opening to allow fluid communication therethrough.

55. The method of claim 47, comprising attaching a support ring to the housing such that the membrane is secured to the housing over the second opening, the support ring having one or more openings formed therein to allow fluid communication between the first and second freezable media through the membrane.

56. The method of claim 47, wherein the housing is part of a cryogenic storage device.

57. The method of claim 47, including providing an outer container containing said second freezable medium prior to exposing the temperature of said second freezable medium to a cryogenic temperature, wherein exposing the temperature of said second freezable medium to said cryogenic temperature includes placing said outer container in an ambient environment having a cryogenic temperature, and wherein placing said housing in an outer container such that said housing is partially or fully immersed in said second freezable medium.

58. The method of claim 57, wherein the outer container comprises a cryovial.

59. The method of claim 43, wherein said sample and said first freezable medium are provided in said lumen through said first opening.

60. The method of claim 43, comprising securing a cover over the first opening.

61. The method of claim 60, wherein any excess first freezable fluid in the internal cavity is displaced from the internal cavity when the cap is secured over the first opening such that the internal cavity is substantially free of air when the cap is secured over the first opening.

62. The method of claim 43, wherein the sample comprises cells or at least one tissue sample suspended in the first freezable medium.

63. The method of claim 62, wherein the tissue sample comprises natural biological tissue, artificial tissue, or a combination thereof.

64. The method of claim 63, wherein the native biological tissue comprises a human multicellular tissue, an animal multicellular tissue, a plant multicellular tissue, or a microbial multicellular tissue, or a combination thereof.

65. The method of claim 63, wherein the artificial tissue comprises an artificial human multicellular tissue, an animal multicellular tissue, a plant multicellular tissue, or a microbial multicellular tissue, or a combination thereof.

66. The method of claim 62, wherein the tissue sample comprises corneal tissue or retinal tissue.

67. The method of claim 62, wherein the cell comprises one or more eukaryotic cells, one or more prokaryotic cells, or a combination thereof.

68. The method of claim 67, wherein the one or more eukaryotic cells comprise at least one mammalian cell.

69. The method of claim 68, wherein the at least one mammalian cell comprises one or more murine cells, one or more porcine cells, one or more human cells, or a combination thereof.

70. The method of claim 68, wherein the at least one mammalian cell comprises one or more stem cells, one or more somatic cells, one or more germ cells, or a combination thereof.

71. The method of claim 67, wherein the one or more prokaryotic cells comprise at least one bacterial cell, at least one archaeal cell, or a combination thereof.

72. The method of claim 43, wherein the freezing temperature is from about-273 ℃ to about 0 ℃, inclusive.

73. The method of claim 43, wherein the freezing temperature is from about-196 ℃ to about-20 ℃, inclusive.

74. The method of claim 43, wherein the freezing temperature is from about-100 ℃ to about-40 ℃, inclusive.

75. The method of claim 43, wherein the freezing temperature is from about-85 ℃ to about-65 ℃, inclusive.

Technical Field

The present invention relates to the field of cryobiology and cryopreservation and ice formation control techniques and the storage of biological and clinical samples.

Background

Cryopreservation is a technique that allows biological materials to be stored at very low temperatures (typically about-80 ℃ to-196 ℃) in, for example, mechanical deep freezers or liquid nitrogen cryofreezers or storage tanks. Cryopreservation is known to potentially store such biological materials indefinitely over a relatively long period of time without or with substantially limited degradation of the biological material. Although critical to cryopreservation efficiency for the promotion of biomedical research and clinical applications, traditional cryopreservation and cryopreservation procedures result in poor viability of many cell and tissue types that are of great value for transplantation, regenerative medicine and personalized medicine.

Among many other cell and tissue types, the cornea is a typical example, which suffers from low viability and impaired function after traditional cryogenic storage. Around the world, there are approximately 100,000 corneal transplant medical procedures performed annually, but 1000 million potential patients. Hypothermia is a common storage method for corneal tissue, where corneal tissue is stored at about 2-8 ℃, but this storage method can only be effectively stored for less than two weeks before the corneal tissue is damaged and improperly transplanted. Thus, at present, for many developing countries, domestic corneal donations or collections are rare due to cultural or policy restrictions and technical restrictions, and patients in these countries are also challenged to obtain a qualified cornea from developed countries. Transplantation of a biosynthetic cornea is a very promising solution and will soon require effective long-term tissue storage, which is not feasible for international distribution. Corneal tissue is also important for medical or basic research applications. There are about 30,000 donor corneas per year for such medical and/or research purposes. Corneal tissue of controlled quality is not available or, in many cases, affordable.

In recent years, the efficacy associated with medical transplantation of human corneas that have been cryo-preserved in liquid nitrogen has only made less significant progress and suffered from endothelial cell loss of about 30% or more. Thus, even after decades of effort, cryopreservation of the cornea has not achieved wide acceptance due to the demanding requirements of its facilities and labor, its cost inefficiency, and the highly variable clinical and experimental results obtained. Similarly, there is no practical method of cryopreservation for other relatively complex tissue types, such as vascular and ovarian tissue, even though such tissue types are important for the treatment of critical diseases or the development of relevant biomedical technologies.

In conventional cryopreservation techniques, two general cooling methods are widely employed to reach storage temperatures; namely non-equilibrium (vitrification freezing) and equilibrium (slow freezing).

For non-equilibrium cooling methods, the biological material (which may be, for example, a cell and/or tissue sample) is typically loaded with a very high concentration (e.g., typically 40-60% by volume) of a cell membrane-permeable cryoprotectant (typically a small organic molecule, for example)Such as dimethyl sulfoxide (DMSO), ethylene glycol, glycerol, and propylene glycol), and then cooled at a high rate (e.g., 10 deg.f) by immersing the biological material directly into liquid nitrogen or other cryogenic liquids or mixtures²To 10⁴K/min), i.e. by vitrification to achieve an amorphous solid state in the biomaterial without formation of ice crystals. The non-equilibrium cooling method not only causes severe cell osmotic damage and toxicity due to high cryoprotectant concentrations, but is also a complex and time-consuming process for loading and removing cryoprotectants into and from cells and tissues. Devitrification (devitrification) of biological materials creates another key technical problem. For example, if the vitrified sample is transferred to an environment without a low temperature (e.g., -80 ℃ or higher) for storage, ice crystallization occurs inside the vitrified sample when the temperature in the sample becomes higher than the devitrification temperature, causing serious mechanical damage to the sample.

For traditional equilibrium or slow freezing methods, the procedures involved typically include adding a relatively low concentration (e.g., typically 10% or less) of cryoprotectant to the biological material (e.g., cells) prior to cooling; inoculating one or more samples of biological material at or below the freezing point of the cell suspension and cooling the cells to a storage temperature at which the cells are stored; heating the cells; the cryoprotectant is removed from the cells. Reducing cell damage caused by the freezing process is critical to improving cell survival after cryopreservation. During freezing, ice forms in the external (e.g., external to the tissue or cells) cryopreservation media, and mechanical stresses associated with extracellular ice crystals have been shown to cause cell damage and lethal tissue mechanical damage. During slow freezing, damage to the isolated cells is due to ice shear or compressive forces on a cellular scale, which is to some extent tolerable for many cell types. However, for tissue, direct contact with tissue and the growing ice surface cause macroscopic damage under both shear and compressive forces, and introduce relatively large ice crystals in the interstitial spaces of tissue, particularly in the microstructure of the tissue, causing not only cell loss but also loss of tissue function due to structural deformation.

Prior to the devices of the present invention, there was no available device or effective physical method to prevent mechanical damage to tissue caused by slow freezing procedures, and vitrification was the general method used for tissue cryopreservation.

In many practical applications of short-term storage (typically less than one week) of biological cells and tissues, samples are treated with a cryogenic medium without any cryoprotectant and refrigerated at a temperature of, for example, 2-8 ℃, below room temperature, but above the freezing point of the cryogenic medium (about 0 ℃). Further lowering of the storage temperature to temperatures below 0 ℃ can prolong the storage time, but in the absence of cryoprotectants, the inevitable formation of ice below the freezing point of the cryogenic medium can cause fatal damage to cells and tissues. Currently, there is no known device or effective method to mitigate cell damage resulting from ice formation without the use of cryoprotectants. This limitation limits the efficiency of the cryogenic process to a short time. Furthermore, the cryogenic temperatures at which such samples are stored are close to the freezing point, and under normal storage conditions, temperature fluctuations caused by normal operation of mechanical refrigerators can lead to accidental damage to the samples due to accidental ice formation. Thus, there is no known device or effective method for minimizing damage resulting from such accidental ice formation during cryopreservation of samples.

Disclosure of Invention

This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely an example of numerous and varied embodiments. Reference to one or more representative features of a given implementation is likewise exemplary. Such embodiments may or may not generally have the mentioned features. Likewise, those features may be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.

According to a first aspect, there is provided a cryoprotective device for protecting a sample from mechanical damage during cryopreservation, the device comprising: a housing forming an internal cavity, wherein the housing is configured to contain a freezable medium within the internal cavity; wherein the shell comprises a semi-permeable membrane that is impermeable to ice crystals that are substantially larger than the mean pore size of the membrane to prevent any such ice crystals from entering the lumen from a region outside the shell such that ice crystals formed in the medium inside the shell have a smaller crystal size than ice crystals formed in the freezable medium outside the shell.

In some embodiments of the device, the first opening is formed at a first longitudinal end of the housing, and the membrane is disposed over a second opening formed in the housing.

In some embodiments of the device, the housing is configured to receive a sample within the lumen through the first opening.

In some embodiments of the device, the housing is a buoyant support, and the membrane is attached to the buoyant support to define the internal cavity such that the membrane is submersible within a freezable medium external to the housing, wherein at least a portion of the buoyant support is not submersible within the freezable medium external to the housing.

In some embodiments, the sample comprises a cell suspension or biological tissue in a natural or conventional medium and/or solution, which may itself be free of any cryoprotectant, as the case may be. It has been found that short term storage at cryogenic temperatures can be effective with minimal or acceptable loss of cells and/or tissue in the absence of such cryoprotectants.

In some embodiments of the device, the membrane is a porous solid layer. Cannot load all results

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In some embodiments of the device, the porous solid layer allows water and liquid materials to permeate therethrough, but prevents ice crystals larger than the pore size of the membrane from passing through the membrane, which are formed by the solidification of cryogenically preserved external to the housing when the device is exposed to cryogenic temperatures.

In some embodiments of the device, the porous solid layer comprises pores having diameters in a range of about 0.1 nanometers (nm) to about 1 millimeter (mm), about 0.5nm to about 0.1mm, about 1nm to about 100nm, or about 2nm to about 10 nm.

In some embodiments of the device, the membrane comprises a natural or synthetic polymer.

In some embodiments of the device, the membrane comprises a synthetic polymer including polyacrylonitrile, polymethylmethacrylate, polysulfone, a vinyl alcohol copolymer, or any combination or chemical derivative thereof.

In some embodiments of the device, the membrane comprises a cellulosic material.

In some embodiments of the device, the cellulosic material comprises regenerated cellulose, denatured cellulose, cellulose diacetate, cellulose triacetate, or any combination or chemical derivative thereof.

In some embodiments of the device, the membrane is a dialysis membrane made of denatured cellulose.

In some embodiments of the device, the membrane is permeable to water, organic and/or inorganic liquid solvents, or any combination thereof.

In some embodiments of the device, the membrane is a detachable membrane configured to be selectively mounted to cover the second opening.

In some embodiments of the device, the medium comprises a cryoprotective medium having a cryoprotective material suspended therein as an additive.

In some embodiments of the device, the housing includes a recess in which the membrane is mounted in the housing to cover the second opening, the device including a support ring configured to be selectively attached to the housing to secure the membrane over the second opening when the support ring is attached to the housing.

In some embodiments of the device, the support ring comprises an outer support member having an aperture formed therethrough such that when the support ring is mounted to the housing, the membrane is exposed to the medium outside the housing through the aperture formed in the support ring.

In some embodiments of the device, the housing comprises at least one locking tab (tab) lockingly engaged with the support ring to secure the support ring to the housing.

In some embodiments of the device, the first opening is configured such that a sample can pass into the lumen.

In some embodiments, the device includes a cover configured to be secured to the housing to close the first opening.

In some embodiments of the device, the cover includes at least one vent formed in a periphery of the cover to allow the medium in the internal cavity to flow through the at least one vent when the cover is secured to the housing.

In some embodiments of the device, the cover includes an inner surface having a convex shape such that the cover protrudes deeper into the internal cavity at a center of the cover than at a periphery of the cover.

In some embodiments of the device, the housing has a substantially cylindrical shape with a circular cross-section.

In some embodiments of the device, the freezable medium is a liquid at 25 ℃ and a solid below 0 ℃.

In another aspect, a system for storing a sample at cryogenic temperatures is provided, the system comprising: at least one cryo-protected storage device as described in any of the embodiments disclosed herein; and an outer container configured to house one or more cryo-protected storage devices; and a freezable medium for use in the interior cavity of the housing and in an external container external to the housing.

In some embodiments of the system, the freezable medium comprises a hydrophilic and non-toxic macromolecule and an aqueous liquid. In some such embodiments of the system, the freezable medium comprises a cryoprotectant.

In some embodiments of the system, the cryoprotectant comprises dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sucrose, glucose, dextran, and other polysaccharides, polyvinylpyrrolidone and polyethylene glycol, and other polymers, other non-penetrating cryoprotectants including, but not limited to, chondroitin sulfate and lactobionic acid, or any combination thereof.

In some embodiments of the system, the hydrophilic and non-toxic macromolecule is a polymer.

In some embodiments of the system, the polymer forms a three-dimensional structure that is substantially spherical in shape when dissolved in an aqueous liquid.

In some embodiments of the system, the aqueous liquid comprises cell culture media, nutrient media, saline, or any combination thereof.

In some embodiments of the system, the aqueous liquid comprises serum, Fetal Bovine Serum (FBS), Dartbuck's Modified Eagle Medium (DMEM), 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), flushing-maintaining Medium (FHM), Phosphate Buffered Serum (PBS), Dartbuck Phosphate Buffered Saline (DPBS), ross parker monument Medium (RPMI), BF5 Medium, EX-CELL Medium, bacteriolysis broth (LB) Medium, CaCl, Modified Eagle Medium (Dulbecco's Modified Eagle Medium) (DMEM), rinsing-maintaining Medium (FHM), Phosphate Buffered Serum (PBS), Dartbuck Phosphate Buffered Saline (DPBS), ross parker monument Medium (RPMI), BF5 Medium, EX-CELL Medium, bacteriolysis broth (LB) Medium, CaCl₂An aqueous solution, an aqueous NaCl solution, an aqueous KCl solution, or any combination thereof.

In some embodiments of the system, the concentration of polymer in the medium is greater than about 5% (w/v), greater than about 10% (w/v), greater than about 20% (w/v), or greater than about 50% (w/v).

In some embodiments of the system, the concentration of cryoprotectant in the medium is equal to or greater than about 20%, equal to or greater than about 50%, equal to or greater than about 75%, or equal to or greater than about 100% of the concentration of polymer in the medium.

In some embodiments of the system, the medium comprises about 10% (w/v) Ficoll (Ficoll), about 5% (w/v) DMSO, about 2% (w/v) chondroitin sulfate, and about 1% (w/v) dextran 40.

In some embodiments of the system, the ficoll is a polysaccharide formed by copolymerization of sucrose and epichlorohydrin.

In some embodiments of the system, the polymer is selected from the group consisting of hydrophilic polysaccharides, polymeric cyclodextrins or sugars, globular proteins or globular proteins, globular glycoproteins formed by linking oligosaccharide chains of globular proteins, other derivatives of globular proteins, and combinations thereof.

In some embodiments of the system, the polymer is a hydrophilic polysaccharide.

In some embodiments of the system, the polysaccharide is formed by copolymerization of sucrose and epichlorohydrin.

In some embodiments of the system, the medium fills a portion or all of the space between the outer container and the at least one cryogenic storage device, providing a sufficient amount of the medium to cover at least the surface of the membrane facing away from the inner cavity.

In some embodiments of the system, the composition of the medium external to the cryo-protected storage device is different from or the same as the composition of the medium in the interior cavity of the cryo-protected storage device.

In another aspect, a method of protecting a sample from damage during cryofreezing is provided, the method comprising: providing a housing having an internal cavity, wherein the housing comprises a semi-permeable membrane; providing a sample and a first freezable medium in the interior cavity of the housing; placing the housing within a second freezable medium external to the housing such that the housing is partially or fully submerged in the second freezable medium, wherein the second freezable medium is the same as or different from the first freezable medium; exposing the second freezable medium to a cryogenic temperature such that ice crystals having a first size form in the second freezable medium outside of the shell; wherein growth of ice crystals of a first size in the second freezable medium is prevented by the membrane such that only ice crystals of a second size having a pore size smaller than the membrane can pass through the membrane and cause ice to form in the first freezable medium within the internal cavity of the housing, such that ice crystals produced within the housing have a size smaller than ice crystals formed in the second freezable medium external to the housing.

In some embodiments of the method, the first opening is formed at a first longitudinal end of the housing and the membrane is disposed over a second opening formed in the housing.

In some embodiments of the method, the housing is configured to receive a sample within the lumen through the first opening.

In some embodiments of the method, the enclosure is a buoyant support, the membrane being attached to the buoyant support to define the internal cavity such that the membrane is submersible within a freezable medium external to the enclosure, wherein at least a portion of the buoyant support is not submersible within the freezable medium external to the enclosure.

In some embodiments of the method, the first and/or second freezable medium comprises a hydrophilic and non-toxic macromolecule and an aqueous liquid.

In some embodiments of the method, the first and/or second freezable medium further comprises a cryoprotectant; and, as the temperature of the first freezable medium decreases, water in the first freezable medium within the chamber permeates through the membrane, thereby increasing the concentration of cryoprotectant within the first freezable medium during cryogenic freezing.

In some embodiments of the method, permeation of the water from the first freezable medium to the second freezable medium through the membrane increases the solute concentration in the first freezable medium and/or decreases the freezing temperature of the first freezable medium to prevent supercooling of the first freezable medium.

In some embodiments of the method, ice crystals formed in the second freezable medium are larger and formed at a higher temperature than ice crystals formed in the first freezable medium; the membrane comprises a porous material having pore sizes smaller than the diameters of ice crystals formed in the second freezable medium; and/or reduced damage to the sample relative to storing the sample in a housing without a membrane that allows water to permeate therethrough.

In some embodiments of the method, the membrane is positioned over the second opening to allow fluid communication therethrough.

In some embodiments, the method includes attaching a support ring to the housing such that the membrane is secured to the housing over the second opening, the support ring having one or more openings formed therein to allow fluid communication between the membrane between the first and second freezable media.

In some embodiments of the method, the housing is part of a cryogenic storage device.

In some embodiments, the method comprises providing an outer container containing the second freezable medium prior to exposing the temperature of the second freezable medium to the cryogenic temperature, wherein exposing the temperature of the second freezable medium to the cryogenic temperature comprises subjecting the outer container to an environment having a cryogenic temperature, and wherein placing the housing in the outer container such that the housing is partially or fully immersed in the second freezable medium.

In some embodiments of the method, the outer container comprises a cryovial.

In some embodiments of the method, the sample and the first freezable medium are provided in the lumen through the first opening.

In some embodiments, the method includes securing a cover over the first opening.

In some embodiments of the method, any excess first cryogenically-condensable fluid in the internal cavity is removed from the internal cavity when the cover is secured over the first opening such that the internal cavity is substantially free of air when the cover is secured over the first opening.

In some embodiments of the method, the sample comprises cells or at least one tissue sample suspended in a first freezable medium.

In some embodiments of the method, the tissue sample comprises natural biological tissue, artificial tissue, or a combination thereof.

In some embodiments of the method, the native biological tissue comprises human, animal, plant, or microbial multicellular tissue or a combination thereof.

In some embodiments of the method, the artificial tissue comprises an artificial human multicellular tissue, an animal multicellular tissue, a plant multicellular tissue, or a microbial multicellular tissue, or a combination thereof.

In some embodiments of the method, the tissue sample comprises corneal tissue or retinal tissue.

In some embodiments of the methods, the cells comprise one or more eukaryotic cells, one or more prokaryotic cells, or a combination thereof.

In some embodiments of the method, the one or more eukaryotic cells comprise at least one mammalian cell.

In some embodiments of the method, the at least one mammalian cell comprises one or more murine cells, one or more porcine cells, one or more human cells, or a combination thereof.

In some embodiments of the method, the at least one mammalian cell comprises one or more stem cells, one or more somatic cells, one or more germ cells, or a combination thereof.

In some embodiments of the method, the one or more prokaryotic cells comprise at least one bacterial cell, at least one archaeal cell, or a combination thereof.

In some embodiments of the method, the cryogenic temperature is from about-273 ℃ to about 0 ℃, inclusive.

In some embodiments of the method, the cryogenic temperature is from about-196 ℃ to about-20 ℃, inclusive.

In some embodiments of the method, the cryogenic temperature is from about-100 ℃ to about-40 ℃, inclusive.

In some embodiments of the method, the cryogenic temperature is from about-85 ℃ to about-65 ℃, inclusive.

The present disclosure relates to devices, systems and methods for improving the efficiency of cryopreservation of cells or tissues by using a semi-permeable membrane to protect the sample from direct contact with extracellular ice. The device also has specific features that facilitate its clinical use for tissue cryopreservation, including alternative structural designs for disposable and single-use designs, and mechanical designs that improve tissue loading and the hermetic seal of the device.

Freezing temperature is defined herein as a temperature in the range of-273 ℃ to 0 ℃.

In some aspects, the sealed housing contains a cell or tissue suspension in a freezable medium, which may be a cryopreservation medium. The dimensions of the housing are selected to accommodate the volume of the freezable medium, wherein one or more walls or sides or covers of the housing are made of or otherwise configured to be secured to a membrane that is semi-permeable at the freezing temperature.

In some aspects, an outer container is provided to contain one or more such shells, wherein the shells are immersed in another freezable medium in the outer container (which may also be the same or a different cryoprotective medium as the freezable medium in the shells).

In some aspects, the membrane separates the freezable medium inside the housing from the freezable medium outside the housing (e.g., in an outer container).

In some aspects, during cryogenic freezing of a sample, ice formation within a freezable medium outside the housing (e.g., within the container) gradually increases the concentration of solutes located outside the chamber.

In some aspects, during cryofreezing of a sample, when the freezable medium outside the housing freezes, the freezable medium inside the housing loses water to the freezable medium outside the housing through the semi-permeable membrane, thereby maintaining equilibrium between the chemical potentials across the membrane.

In some aspects, during cryofreezing of a sample, the freezable medium inside the housing remains free of ice crystals until the temperature of the freezable medium inside the housing is low enough to allow ice crystals located outside the housing (e.g., on the surface of the membrane outside the housing) to pass through the membrane and create ice nuclei and form ice crystals inside the housing, the ice crystals formed inside the housing being of a much smaller size than the ice crystals located outside the chamber. Mechanical damage to samples (e.g., cells or tissue) inside the housing is minimized due to the formation of ice crystals during cryofreezing.

In certain aspects, the temperature at which ice crystals located outside the housing are allowed to pass through the membrane is below the freezing point of the freezable medium, but above the cryogenic storage temperature (e.g., the ambient temperature to which the freezable medium is exposed during cryogenic freezing).

In some aspects, prior to assembly, one of the walls of the cover and/or the housing is separated from the housing to allow the sample to be loaded into the housing. In some such aspects, one of the walls of the lid and/or the housing has a convex surface to repel or push away excess freezable medium within the housing during loading of the sample or during system assembly. In some such aspects, one of the walls of the cover and/or the housing has a vent passage provided and/or formed therein to release excess freezable medium to prevent build up of pressure in the housing that could damage the membrane and cause the housing to leak. In some such aspects, the vent is in the form of a vent channel that is covered by a wall of the housing after assembly to ensure an airtight seal of the housing and prevent the formation of air bubbles.

In certain aspects, below the membrane of the housing, a cross-shaped support ring is attached. In some such aspects, the support ring may be easily removed from the housing with minimal mechanical force. In some such aspects, the support ring provides a single use approach by requiring the end user to remove the support ring after undergoing a thawing process to remove the membrane for retrieval of the sample from the housing interior. In some such aspects, after assembly of the housing with one or more of the cover, the membrane, and the support ring, the housing cannot be opened without damaging the housing in a manner that results in the housing being unsealed.

In some other aspects, the devices and/or systems disclosed herein, or one or more portions thereof, are reusable.

Drawings

Fig. 1 is a cross-sectional view of an exemplary embodiment of an apparatus for cryogenically freezing and storing a specimen (e.g., a biological sample).

Fig. 2 is a perspective view of an apparatus for cryogenically freezing and storing a specimen (e.g., a biological sample).

Fig. 3A is a cross-sectional view of the device shown in fig. 2.

Fig. 3B is an exploded view of the device shown in fig. 2.

FIG. 4 is a graph of temperature measurements inside and outside the device and over a period of time adjacent the membrane of FIG. 2, illustrating the reduction in size of ice crystals formed within the device during cryogenic freezing.

Fig. 5A-5C illustrate the progression of ice crystal formation within the apparatus of fig. 2 at several stages during cryogenic freezing.

Figures 6A-6D show cross-sectional views of the apparatus of figure 2 with a sample placed therein showing the progression of ice crystal formation at several stages during cryogenic freezing.

Fig. 6E-6H show cross-sectional views of the apparatus of fig. 2 with a sample placed therein, but with the membrane omitted, illustrating the progression of ice crystal formation at several stages during cryogenic freezing.

Figure 7 is a graph comparing endothelial cell viability of human corneas thawed in the apparatus shown in figure 2 but cryogenically frozen with membranes having different molecular weight cut-offs.

Figure 8 shows images of human corneal endothelial cells before and after cryofreezing in a device such as that shown in figure 2 having the membranes of different molecular weight cut-offs of figure 7.

Fig. 9 is a top perspective view of a cryogenic storage system including the device of fig. 2 located in an outer vessel.

Fig. 10 is a cross-sectional view of the system of fig. 9.

Fig. 11A and 11B are schematic diagrams from an automated cell counter showing viability of the interior (fig. 11A) and exterior (fig. 11B) liquids of the device of fig. 9 after being thawed after cryo-freezing.

FIG. 12 is a top perspective view of the system of FIG. 9 with a tissue sample placed in the device.

Fig. 13 is a cross-sectional view of the system of fig. 12.

Fig. 14 is an exemplary stereomicroscope image of the organic sample shown in fig. 12 that has been cryogenically frozen and then thawed.

Fig. 15 is an exemplary stereomicroscope image of an organic specimen substantially similar to that shown in fig. 12, but cryogenically frozen according to conventional cryogenic techniques.

Detailed Description

The presently disclosed subject matter, some, but not all embodiments of the presently disclosed subject matter, will now be described more fully hereinafter. Indeed, the disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.

Although the following terms are well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

Unless defined otherwise below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of those techniques or substitutions of equivalent techniques that would be apparent to those skilled in the art. While the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the subject matter of the present disclosure.

In describing the subject matter of the present disclosure, it will be understood that a number of techniques and steps are disclosed. Each of these has its own benefits and each can also be used in conjunction with one or more, or in some cases, all of the other disclosed techniques.

Thus, for the sake of clarity, the description will avoid repeating every possible combination of the various steps in an unnecessary fashion. However, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the disclosure and claims.

All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings related to the sentences and/or paragraphs in which they are cited.

Following long-standing patent law convention, the terms "a", "an" and "the" are used herein, including the claims, to mean "one or more". Thus, for example, reference to "a cell" includes a plurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

As used herein, the term "about" when referring to an index value or amount, mass, weight, temperature, time, volume, concentration, percentage, or the like of a composition is intended to encompass variations from the specified amount, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ± 1%, in some embodiments ± 0.5%, in some embodiments ± 0.1%, as such variations are suitable for performing the disclosed methods or using the disclosed compositions.

The term "comprising" synonymous with "including", "containing" or "characterized by" is inclusive or open-ended and does not exclude additional unrecited elements or method steps. "comprising" is a term of art used in claim language and means that the specified element is essential, but that other elements may be added and still constitute a structural element (construct) within the scope of the claims.

The phrase "consisting of" as used herein does not include any elements, steps or ingredients not specified in the claims. When the phrase "consisting of, rather than immediately following the preamble" appears in the text of the claims, it only limits the elements listed in the clause; other elements are not excluded from the scope of the claims as a whole.

As used herein, the phrase "consisting essentially of … …" limits the scope of the claims to the specified materials or steps, as well as those that do not materially affect the basic and novel characteristics of the claimed subject matter.

With respect to the terms "comprising," "consisting of," and "consisting essentially of," where one of these terms is used herein, the subject matter of this disclosure and claims can include the use of one of the other two terms.

As used herein, the term "and/or," when used in the context of a list of entities, means that the entities exist alone or in combination. Thus, for example, the phrase "A, B, C, and/or D" includes A, B, C, and D, respectively, but also includes any and all combinations and subcombinations of A, B, C, and D.

As used herein, the term "substantially" refers to an amount, mass, weight, temperature, time, volume, concentration, percentage, or the like, of a value, activity, or composition, that is, to encompass variations from the specified amount, in some embodiments, 40%, in some embodiments, 30%, in some embodiments, 20%, in some embodiments, 10%, in some embodiments, 5%, in some embodiments, 1%, in some embodiments, 0.5%, and in some embodiments, 0.1%, as such variations are suitable for performing the disclosed methods or using the disclosed apparatus and devices. For example, a medium or environment is "substantially anoxic" when the medium or environment is at least 60%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, and in some cases, at least 99% anoxic.

As used herein, the term "basal medium" refers to a minimal basic type of medium to which other components may be added, such as dartbox modified eagle's medium, Ham's F12, eagle's medium, RPMI, AR8, and the like. The term does not exclude media that have been prepared or intended for a particular use, but which have been modified for use with other cell types and the like.

As used herein, "compound" refers to any type of substance or agent, and combinations and mixtures thereof, that is generally considered a drug, therapeutic agent, drug, small molecule, or candidate for use therewith.

The use of the word "detecting" and grammatical variations thereof refers to the determination of the species (species) without quantification, whereas the meaning of "determining" or "measuring" and grammatical variations thereof refers to having a quantified measure of the "species". The terms "detecting" and "identifying" are used interchangeably herein.

The term "component" refers to any compound, whether of chemical or biological origin, that can be used in a cell culture medium to maintain or promote proliferation, survival or differentiation of cells. The terms "component," "nutrient," "supplement," and "ingredient" are used interchangeably and all refer to these compounds. Typical non-limiting ingredients used in cell culture media include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, and the like. Other components that promote or maintain ex vivo cell culture may be selected by those skilled in the art according to particular needs.

As used herein, the term "inhibit," based on the context in which the term "inhibits" is used, refers to the ability of a compound, agent, or method to reduce or hinder a described function, level, activity, rate, or the like. Preferably, the inhibition is at least 10%, more preferably at least 25%, even more preferably at least 50%, most preferably the function is inhibited by at least 75%. The terms "inhibit" and "reduce" and "hinder" are used interchangeably.

As used herein, the term "material" refers to synthetic and natural materials, such as matrix components. As used herein, the term "materials and compounds" refers to, inter alia, materials, compounds, cells, peptides, nucleic acids, drugs, matrix components, and imaging agents.

As used herein, the term "modulate" refers to altering the level of activity, function, or process. The term "modulating" encompasses inhibiting and stimulating an activity, function or process. The term "adjust" (modulated) is used herein interchangeably with the term "modulate" (modulated).

As used herein, the term "prevention" refers to preventing something from happening, or taking precautionary measures against what may happen. For example, "prevention" in the context of the presently disclosed subject matter generally refers to measures taken to reduce the chance of ice formation and/or tissue/cell damage during cryopreservation.

The term "modulate" refers to stimulating or inhibiting a function or activity of interest.

As used herein, "sample" refers to a biological sample from a subject, including but not limited to normal tissue samples, diseased tissue samples, biopsy tissue, blood, saliva, stool, semen, tears, and urine. The sample may also be any other source of material obtained from a subject that contains cells, tissues or fluids of interest.

As used herein, the term "stimulating" refers to inducing or increasing a level of activity or function such that it is higher relative to a control value. Stimulation may be by direct or indirect mechanisms. In one aspect, the activity or function is stimulated by at least 10%, more preferably at least 25%, even more preferably at least 50% compared to a control value. As used herein, the term "stimulant" refers to any composition, compound, or agent, the application of which results in stimulation of a process or function of interest, including but not limited to wound healing, angiogenesis, bone healing, osteoblast production and function, and osteoclast production, differentiation, and activity.

"tissue" refers to (1) a group of similar cells that combine to perform a particular function; (2) a portion of an organism consisting of an aggregate of cells with similar structure and function; and/or (3) a group of cells similarly characterized by their structure and function, such as muscle or neural tissue.

As described elsewhere herein, during cryopreservation, when the outer surface of a tissue sample is contacted by ice as the ice front (ice front) grows to wrap the tissue, the sharp ice dendrites on the growing ice front can mechanically damage the cells on the tissue surface. Therefore, tissues having a functional cell layer on their surface (e.g., cornea with an endothelial cell layer) are susceptible to this type of mechanical damage. Thus, in accordance with the subject matter disclosed herein, it is advantageous to provide a semi-permeable membrane in such cryogenic preservation devices and/or systems to "filter" large ice dendrites and allow only ice crystals having a small or fine crystal structure (e.g., without forming large ice dendrites as known in prior art cryogenic preservation devices, systems and methods) to grow through the membrane. By including such a semi-permeable membrane, mechanical damage due to direct contact between the outer cell layers of the tissue sample with the sharp ice dendrites present on the ice surface, as is known in the art, can be prevented.

In accordance with the presently disclosed subject matter, a semi-permeable membrane (e.g., a cytoplasmic membrane) may be used as an ice growth barrier during cryopreservation freezing. For example, the pore size of the channels in the cell membrane may be in the range of 10 a^-10m, which size of pores only allows ice crystals to grow through them under super-cooled conditions of about-20 c. Due to this undercooling of the intracellular solution (e.g., cryopreservation media), the solute concentration of the intracellular solution remains lower than the solute concentration of the extracellular solution, and thus the difference between the chemical potentials of the intracellular and extracellular water drives the intracellular water to permeate through the membrane and mix into the extracellular solution. The process increasesThe concentration of intracellular solutes and further lowers the freezing point of the intracellular solution and, more importantly, prevents direct contact between intracellular organelles and extracellular ice. Thus, when an appropriate cooling rate is selected, the intracellular solution remains ice-free and vitrified, or very small ice crystals (e.g., having a crystal size between 1-100 nanometers (nm)) are formed upon completion of the cryopreservation process. The use of such cell membranes for cryopreservation minimizes mechanical damage to the biological material undergoing cryopreservation by minimizing direct contact between large ice crystals and the biological material. For isolated cells, in addition to the cell membrane, another layer of such membrane will further protect the cell microstructure on the cell surface or cell body, especially for the sperm tail and the neuronal dendrites.

As such, the presently disclosed subject matter relates to a device that separates a sample containing biological material (e.g., tissue or cells in its cryopreservation media) from ice formation during a slow freezing process to minimize the effects of cryoinjury from large ice crystal formation. An exemplary embodiment of such an apparatus, generally indicated at 100, is shown in fig. 2 through 3B. The device uses a semi-permeable membrane 140 to prevent direct contact of the sample with the growing ice front and to prevent the growth of relatively large ice crystals that would otherwise grow through the region occupied by the membrane 140 until the temperature is sufficiently low. During cryogenic freezing, ice formation occurs primarily outside of the housing 110 of the device 100. The formation of ice crystals increases the solute concentration in the cryogenic medium outside the housing 110 of the device 100. The imbalance between the chemical potentials across the membrane 140 (e.g., between the cryogenic media inside and outside the enclosure 100) causes water inside the enclosure 110 to permeate through and/or through the membrane 140 to the outside of the enclosure 110. The permeation of water through the membrane 140 also prevents supercooling of the external compartment of the device containing the sample (see, e.g., fig. 9, 10, 12 and 13), and without such prevention, the supercooling process can result in the instantaneous formation of large ice crystals directly around or within the sample. The penetration of water through the membrane 140 also prevents the formation of sharp ice crystals (e.g., dendrites) that directly contact and destroy functional cells on the outer surface of such a sample of biological material (e.g., the corneal endothelium on the corneal surface).

As described in more detail in various embodiments presented elsewhere herein, the device 100 effectively prevents the growth of large ice crystals through the membrane 140 during the cryogenic freezing process, thereby effectively separating the sample within the device 100 from the growing ice front formed during conventional cryogenic freezing processes. The device 100 significantly improves tissue cryopreservation efficiency by reducing the formation of large ice crystals that grow through the membrane 140 in the sample region. In certain embodiments, membrane 140 is a soft semi-permeable membrane. In some embodiments, the surface area of the membrane 140 is substantially the same as or greater than the surface area of the sample being cryogenically processed. In some embodiments, the surface area of the membrane 140 is substantially equal to or greater than the surface area of a portion of the sample to be cryo-protected, e.g., a valuable region of tissue, which may be, for example, the cornea. The term "semi-permeable" refers to a membrane 140 that is permeable to water but impermeable to salts, ions, any large organic molecules, and the like. In some embodiments, the membrane is permeable or impermeable to small organic molecules (e.g., dimethyl sulfoxide or DMSO and ethylene glycol). In some embodiments, the membrane 140 comprises a synthetic polymer including polyacrylonitrile, polymethylmethacrylate, polysulfone, a vinyl alcohol copolymer, or any combination or chemical derivative thereof. In some embodiments, the membrane 140 is a cellulosic material, which may include regenerated cellulose, denatured cellulose, cellulose diacetate, cellulose triacetate, or any combination or chemical derivative thereof. In some embodiments, the device 100 significantly improves the efficiency of cryopreservation of samples including, for example, corneal tissue.

According to one embodiment shown in fig. 1, the device includes a semi-permeable membrane 140 as described elsewhere herein that acts as an ice barrier between fluid 310 located within membrane 140 and outside membrane 140. The sample 10 is placed within the chamber formed by the membrane 140 and then at least partially immersed in a bath of a freezable medium (e.g., a fluid that freezes at a low or non-low freezing temperature). The freezable medium is frozen and only sufficiently small ice crystals (e.g., having a size or diameter equal to or less than the size or diameter of the pores of the membrane 140) can be frozen near the sample surface by the membrane 140, resulting in the sample being exposed only to ice crystals having a size or diameter substantially similar to (e.g., within 25%, within 10%, within 5%, within 1%) the size or diameter of the pores of the membrane 140. It may be advantageous to attach the membrane 140 to a support assembly, for example, an annular buoyancy assembly, such that the membrane 140 may be in the form shown. The support member may take any shape, including extending in three dimensions, to achieve any desired shape of the membrane 140 during freezing and/or cryopreservation. In some embodiments, the membrane 140 has a substantially flat bottom portion. In some embodiments, the membrane 140 forms a pouch or flexible container for holding the sample 10 during freezing or cryopreservation.

The device 100 includes a housing 110, the housing 110 defining an interior cavity in which a sample is loaded for cryopreservation. The device 100 includes a membrane 140 that is located outside of the internal cavity of the housing 110, but is secured to the housing 110 at a location adjacent to and defining a bottom surface of the internal cavity. The membrane 140 is secured to the housing 110 by a support ring 150, the support ring 150 being attached (e.g., by fastener(s), interference fit, etc.) to the housing 110 on an opposite side of the membrane 140 to secure the membrane 140 to the housing 110. The support ring 150 has a generally annular shape that is substantially similar to the shape of the membrane. The support ring 150 and the membrane 140 may have any suitable shape that will result in the interior cavity of the housing 110 being substantially sealed at its bottom surface. The support shown has two "X" shaped beams (cross-members) to act as vertical supports for the membrane 140. Any number of cross-beams are contemplated for support ring 150, and in some embodiments, support ring 150 may be entirely devoid of cross-beams. The device 100 also includes a cover 120 that may be secured to the upper surface of the housing 110. When the membrane 140 is mounted on the bottom surface of the inner cavity of the housing 110 and the cover 120 is fixed on the top of the inner cavity of the housing 110. The device is assembled such that the housing 110, the cover 120, and the membrane 140 form a substantially sealed chamber. In some embodiments, the housing and membrane 140 may be configured such that the membrane 140 is located in one sidewall of the housing 110 and/or the cover 120 to define a semi-permeable boundary in any portion of the device 100. In some embodiments, the device 100 may have a membrane 140 disposed in two or more or all of the bottom surface of the housing 110, the side walls of the housing 110, and/or the cover 120. As such, portions of the lid 120 and/or the sidewall or bottom of the housing 110 may be made of a semi-permeable membrane material as disclosed herein with respect to the membrane 140. In certain embodiments, the bottom of the device 100 is a structure that secures the denatured cellulose membrane 140 with a frame (e.g., a support ring 150) that is bonded to the device 100 or the housing 110 of the device 100.

In some embodiments, the cover 120 is configured to allow flow of the cryopreservation media within the housing 110 when one or more samples are loaded into the device 100, and to relieve any pressure generated within the device 100 during cryopreservation by sealing the housing 110 filled with the cryopreservation media and containing the samples to prevent the formation of air bubbles within the device 100. In some embodiments, the bottom surface of the cover 120 has a convex shape that faces the internal cavity when placed on and/or secured to the housing 110 of the device 100. In some such embodiments, the cover 120 has at least one vent passage formed therein. This convex shape of the cover 120 pushes the cryopreservation media toward the periphery of the housing without creating bubbles, wherein the vent channels allow the cryopreservation media to be released from within the housing 110 without creating pressure therein.

In some embodiments, support ring 150 protects membrane 140 from mechanical damage or deformation when cover 120 allows cryogenic storage medium to flow into the housing during cryogenic storage. In some embodiments, the support ring 150 comprises a frame coupled to the bottom of the device 100. In some such embodiments, the frame supporting the ring 150 is a cross formed by thin beams. In some embodiments, it is advantageous that the support ring 150 can be removed from the device 100 with minimal mechanical force, but the support ring 150 is still connected to the housing 110, which is strong enough to prevent the membrane from being damaged or deformed by the support ring 150 becoming dislodged from the housing during cryopreservation. When it is desired to use the sample within the device 100, the device 100 is thawed, the support ring 150 is separated from the housing 110, and the membrane is broken, deformed, removed, etc., so that the sample can be used directly. As such, in some embodiments, the device 100 is not reusable for repeated cryopreservation of samples.

Fig. 9, 10, 12 and 13 illustrate an exemplary embodiment of a cryogenic preservation system, indicated generally at 101, including an outer container, indicated generally at 300, to hold a cryogenic preservation device 100, as described above and shown in fig. 2-3A. In certain embodiments, the outer container 300 is a cryovial or cryotube that provides sufficient space to hold the device 100 and a sufficient amount of cryoprotectant solution in a space, generally designated 310, defined as being external to the device 100 but within the outer container 300.

Certain aspects of the invention are illustrated by the following non-limiting examples.

Example 1. the device is designed to prevent the growth of large ice crystals during freezing of the cryopreservation media.

The device 100 has a housing 110, the housing 110 having a generally cylindrical interior cavity formed therein, the device having an outer diameter of 2.5 centimeters (cm) and a height of 1.4 cm. Exemplary embodiments of such devices are shown in fig. 2-3B. The cover 120 is substantially disc-shaped, circular when viewed from the top, has a convex surface formed on the bottom surface thereof to have a height of about 2 millimeters (mm), and forms four small exhaust passages around the periphery of the cover 120. The housing 100 is a generally cylindrical structure having a height of 1cm, an outer diameter of 2.5cm and an inner diameter of 2 cm. The support ring 150 is inserted into a groove formed in the outer surface of the housing 110, and has an outer ring with an outer diameter of 2cm and an inner diameter of 1.8cm, a thin X-shaped frame with a thickness of 1mm, and a beam of the frame has an inner radial surface attached to the ring with a width of 1 mm. The support ring 150 is attached to the housing by being tightly inserted into a complementary shaped recess formed in the bottom outer surface of the housing 110. The term complementary shape means that the recess has an inner diameter substantially the same as the outer diameter of the support ring 150, if not slightly so that in this embodiment the recess of the housing 110 has an inner diameter of 2 cm. The housing has four feet with a height of 3mm so that the housing can be spaced from the contact surfaces of the feet to form a cavity below the housing 110 with a height defined by the height of the feet. The feet of the housing 110 enable the device to remain upright in an outer chamber (e.g., a 15 milliliter (mL) cryobottle) in a cryopreservation system, as shown in fig. 9, 10, 12, and 13. The components of the apparatus 100 may be formed by any suitable manufacturing technique, including, for example, injection molding, additive manufacturing, and the like.

In this example, the device 100 was filled with 2mL of a Dartbox Modified Eagle's Medium (DMEM) solution containing 10% DMSO and 10% polysucrose as a polymeric cryoprotectant to prevent recrystallization during low temperature storage at-80 ℃, and then the lid 120 was closed. The action of closing the lid 120 pushes the excess cryopreservation media out of the housing 110. The apparatus 100 is placed in a 15mL cryovial (e.g., 300, fig. 9, 10, 12 and 13) also containing 10mL of DMEM solution containing 10% DMSO and 10% ficoll. Although not necessary during normal use, to illustrate the advantages provided by the device 100, the first thermocouple is centered in the housing 110 through a hole provided only for the illustrative embodiment. The second thermocouple is located outside the housing 110, in close proximity to the membrane. The second thermocouple is fixed to a cross beam of the support ring 150. The frozen bottle is then exposed to an ambient temperature environment of-80 ℃ (e.g., in a cryogenic freezer) and then slowly cooled to-80 ℃ at a cooling rate of 0.5 ℃/minute

The temperature profiles read from the two thermocouples were recorded and are shown in fig. 4. The temperature change outside the housing 110 shows an exothermic peak when the second thermocouple comes into contact with the ice front during cooling to-80 ℃. Latent heat released from the ice front brings the supercooled thermocouple back to the solution freezing point. In contrast, due to the fact that the film 140 hinders the growth of ice during freezing, the temperature change measured by the first thermocouple located inside the case 110 shows a very small temperature "bump" as an exothermic peak. Thus, the membrane 140 acts as a barrier to prevent large ice crystals from directly contacting the sample contained in the housing 110, resulting in significantly less supercooling, and also prevents mechanical damage to any sample contained in the housing 110. Mechanical damage to the sample during cryopreservation is typically caused by characteristic dendritic ice crystals formed during the supercooling process. These results are in good agreement with the results shown in figures 5A-5C. The results shown in fig. 5A-5C were obtained by directly observing ice formation inside and outside of the shell 110 using neutron Nuclear Magnetic Resonance (NMR) techniques.

Example 2.NMR imaging directly demonstrates the difference in ice formation process of ice separated by the membrane structure of the device of the invention.

NMR imaging of the freezing process of the cryo-preserved medium within the apparatus 100 was performed on a Varian Unity Inova MRI system (Varian Inc., Palo alto, Calif., USA) equipped with a 7T/210mm horizontal bore with a quadrature driven birdcage radio frequency coil of 10cm inside diameter. The device 100 loaded as described in example 1 above was mounted in a small external container (e.g., a polypropylene bucket) 5cm in height and 5cm in diameter with ground dry ice (approximately 10 ml) disposed at its bottom. The bottom of the device 100 (e.g., the bottom surface of the housing 110) directly contacts dry ice (1.01 × 10)⁵Pa lower surface is-78 deg.c) simulating a cooling process in an ambient environment of-80 deg.c. As shown in FIGS. 5A-5C, as the ice front approaches the membrane 140, the progression of the ice front (e.g., the formation of ice crystals) is stopped by the membrane 140, while the ice front continues to grow along the sidewalls of the housing 110. When the temperature of the cryopreservation media at the membrane 140 is sufficiently low (e.g., approximately-40 ℃), small ice crystals can pass through the membrane 140 and form a curved but uniformly shaped ice front within the housing 110, entering the housing 110 from the inner surface of the membrane 140. These small ice crystals located within shell 110 grow at a much slower rate than the larger ice crystals located outside shell 110. The results shown in the NMR images of fig. 5A-5C clearly show that the morphology of the ice located outside of housing 110 has a dendritic structure and is in sharp contrast to the morphology of the ice front located inside of housing 110. Thus, it shows that the membrane 140 acts as an ice screen, which prevents large ice crystals from entering the housing 110, and only allows small ice crystals to pass through the membrane 140 at a sufficiently low temperature.

Fig. 5A shows device 100 at an initial stage of cryopreservation, where a front of ice has moved through membrane 140 and into the interior cavity (generally designated 200) of housing 110. The small ice crystals 202 within the internal cavity 200 are marked as having a finer cross-hatch pattern than the large ice crystals 212 formed in the region 210 outside the shell 110. The majority of the cryogenic preservation medium in the interior chamber is liquid 204 and the majority of the cryogenic preservation medium outside region 210 is liquid 214. Fig. 5B shows device 100 at an intermediate stage of cryopreservation, where the ice front has moved further into interior cavity 200 and region 210. Thus, most of the cryopreservation media within the internal cavity 200 is now in the form of small ice crystals 202, with the remainder of the cryopreservation media being in the form of liquid 204. Similarly, a majority of the cryogenic preservation medium within region 210 is in the form of large ice crystals 212, with the remainder of the cryogenic preservation medium being in the form of liquid 214. Fig. 5C shows the device 100 at a late stage of cryopreservation, where the ice front has moved through the entire inner cavity 200, such that the entire inner cavity 200 is occupied by small ice crystals 202. Similarly, most of the cryogenic preservation medium within region 210 is in the form of large ice crystals 212 as compared to that shown in fig. 5B, with the remainder of the cryogenic preservation medium within region 210 being in a liquid state.

Example 3 NMR imaging shows how the device prevents corneal endothelial cells from coming into direct contact with growing sharp ice dendrites

The same MRI system and cooling procedure as described in example 2 was used in example 3. Ice dendrite details are detected using higher resolution. The results are shown in FIGS. 6A-6H. Two samples 10 (e.g., human corneal tissue) are mounted in two different devices 100, the first such device having a semi-permeable membrane 140 in place as shown in fig. 2-3A, and the second such device 100 having the membrane 140 omitted. The cryopreservation media that fill the devices 100 and hold the respective cryovials (e.g., 300, fig. 9, 10, 12 and 13) of each device 100 are DMEM solutions containing 10% DMSO and 10% polysucrose.

6A-6D, device 100 with membrane 140 effectively slows the formation of ice in shell 110 and also removes sharp ice dendrites from the growing ice front near the corneal endothelial layer (the surface of the tissue facing membrane 140).

In fig. 6A, the ice front is at the membrane 140 and has partially passed through the membrane 140 such that almost all (e.g., 90% or more) of the interior cavity 200 within the housing 110 is occupied by the liquid cryogenic preservation medium 204, while a minority of the interior cavity 200 is occupied by small ice crystals 202. In region 210, the cryopreservation media outside of housing 110 is partially frozen, large ice crystals 212 form adjacent to membrane 140 in the lower region of the bottom of housing 110, and the remainder of the cryopreservation media in region 210 is in a liquid state. As shown by the larger hatching in fig. 6A-6D, the size of the small ice crystals 202 in the internal cavity 200 is smaller (e.g., at least an order of magnitude) than the size of the large ice crystals 212 in the region 210.

In fig. 6B, the ice front has progressed within the lumen 200 such that small ice crystals 202 partially wrap around the sample 10, with most of the cryopreservation media within the lumen 200 still in a liquid state 204. The cryogenic preservation medium outside of housing 110 is frozen to a greater extent in region 210 than in fig. 6A, with large ice crystals 212 formed in the lower bottom region of housing 110 adjacent membrane 140 and progressing around the sides of housing 110, with the remainder of the cryogenic preservation medium in region 210 being in a liquid state. As described elsewhere herein, the small ice crystals 202 formed within the internal cavity 200 remain smaller than the large ice crystals 212 formed outside the shell 110.

In fig. 6C, the ice front has advanced within the lumen 200 such that the small ice crystals 202 almost completely wrap the sample 10, while most of the cryopreservation media within the lumen 200 is now in the form of small ice crystals 202 and a few of the cryopreservation media within the lumen are in liquid form 204. The cryopreservation media outside of the housing 110 in region 210 is further frozen than shown in fig. 6B, with large ice crystals 212 formed in the lower region of the bottom of the housing 110 adjacent the membrane 140 and proceeding further around the sides of the housing 110, with the remainder of the cryopreservation media in the liquid state in region 210. As described elsewhere herein, the small ice crystals 202 formed within the internal cavity 200 remain smaller than the large ice crystals 212 formed outside the shell 110.

In fig. 6D, the ice front has advanced within the lumen 200 such that the small ice crystals 202 completely encapsulate the sample 10, while the entire volume of the lumen 200 now takes the form of the small ice crystals 202 such that no cryopreservation media or substantially no cryopreservation media within the lumen is in liquid form 204. The cryopreservation media outside of the housing 110 in region 210 is further frozen than shown in fig. 6C, with large ice crystals 212 formed in the lower bottom region of the housing 110 adjacent the membrane 140 and around substantially all sides of the housing 110, with the remainder of the cryopreservation media in the liquid state in region 210. As the cryopreservation freezing process progresses, all of the cryopreservation media within region 210 will be frozen to form large ice crystals 212, such that all of the cryopreservation media within housing 110 will be in the form of small ice crystals 202 and all of the cryopreservation media outside of housing 110 will be in the form of large ice crystals 202. As described elsewhere herein, the small ice crystals 202 formed within the internal cavity 200 remain smaller than the large ice crystals 212 formed outside the shell 110.

In fig. 6E-6H, where such a membrane 140 is not present in the device 100, the device 100 does not provide any protective function to the corneal endothelium and results in direct contact of the corneal endothelium with the sharp ice dendrites.

In fig. 6E, the ice front is located in and has partially entered the interior cavity 200 of the housing 110, at or beyond the slit (slot) where the membrane may be installed, such that almost all (e.g., 90% or more) of the interior cavity 200 within the housing 110 is occupied by the liquid cryogenic medium 204, with a small portion of the interior cavity 200 being occupied by large ice crystals 212. The cryogenic preservation medium outside of housing 110 in region 210 is partially frozen, with large ice crystals 212 formed adjacent to membrane 140 in the region below the bottom of housing 110, and the remainder of the cryogenic preservation medium in region 210 is in a liquid state. As shown by the hatched pattern in fig. 6E-6H, for large ice crystals 212, the size of the large ice crystals 212 in the lumen 200 is the same as the size of the large ice crystals 212 in the region 210.

In fig. 6F, the ice front advances within the lumen 200 such that the large ice crystals 212 partially wrap around the sample 10, and most of the cryopreservation media within the lumen 200 remains in the liquid state 204. The cryogenic preservation medium outside of the housing 110 in region 210 is frozen further than shown in fig. 6E, with large ice crystals 212 formed in the lower region of the bottom of the housing 110 adjacent the membrane 140 and progressing around the sides of the housing 110, with the remainder of the cryogenic preservation medium in region 210 being in a liquid state. As described elsewhere herein, the large ice crystals 212 formed within the internal cavity 200 remain substantially the same size as the large ice crystals 212 formed outside the shell 110.

In fig. 6G, the ice front has advanced within the lumen 200 such that the large ice crystals 212 almost completely encase the sample 10, while most of the cryopreservation media within the lumen 200 now exists in the form of large ice crystals 212, and a few of the cryopreservation media within the lumen are in liquid form 204. The cryopreservation media outside of the housing 110 in region 210 is further frozen than shown in fig. 6F, with large ice crystals 212 formed in the lower region of the bottom of the housing 110 adjacent the membrane 140 and further progressing around the sides of the housing 110, with the remainder of the cryopreservation media in the liquid state in region 210. As described elsewhere herein, the large ice crystals 212 formed within the internal cavity 200 remain substantially the same size as the large ice crystals 212 formed outside the shell 110.

In fig. 6H, the ice front has advanced within the lumen 200 such that the large ice crystals 212 completely wrap the sample 10, the entire volume of the lumen 200 now being in the form of the large ice crystals 212 such that no cryopreservation media or substantially no cryopreservation media within the lumen is in liquid form 204. The cryopreservation media outside of the housing 110 in region 210 is further frozen than shown in fig. 6G, with large ice crystals 212 formed in the lower bottom region of the housing 110 adjacent the membrane 140 and around substantially all sides of the housing 110, with the remainder of the cryopreservation media in liquid state in region 210. As the cryopreservation freezing process progresses, all of the cryopreservation media within region 210 will be frozen to form large ice crystals 212, such that all of the cryopreservation media inside and outside of housing 110 are in the form of large ice crystals 212. As described elsewhere herein, the large ice crystals 212 formed within the internal cavity 200 remain substantially the same size as the large ice crystals 212 formed outside the shell 110.

Example 4 improvement of corneal cryopreservation by use of the device for cryopreservation at-80 deg.C

To demonstrate the efficiency of using the device 100 for cryopreserving samples containing biological material via the working mechanisms disclosed herein, thereby preventing direct contact of the sample with large ice crystals during freezing, six human corneas were stored in the device 100 substantially similar to the embodiment shown in fig. 2-3B. For the purpose of this demonstration, the apparatus 100 was constructed using additive manufacturing techniques, but any type of manufacturing may be envisaged without departing from the scope of the subject matter disclosed herein. Two different types of membranes 140 are installed in each device. The first type of membrane installed in the first subset of apparatus 100 is a regenerated cellulose membrane having a molecular weight cut-off (MWCO) of 7500 grams per mole (g/mol). The second type of membrane installed in the second subset of apparatus 100 is a regenerated cellulose membrane having an MWCO of 10,000 g/mol. Such regenerated cellulose membranes are available from manufacturers such as Millipore. Cryopreservation media added inside and around housing 110 included 5% DMSO, 10% ficoll, 1% dextran 40, and 2% chondroitin sulfate. Each cornea is mounted in the housing 110 of one of the devices, which houses the cryopreservation media described above, and then stored in an ambient environment at-80 ℃ (e.g., in a cryogenic refrigerator). After three months of storage, each device 100 was thawed by inserting each device 100 directly into a 37 ℃ water bath. Endothelial cell counts and morphology after thawing are shown in fig. 7 and 8. The efficiency achieved by the first or second type of membrane 140 is significantly improved (e.g., up to about 90% or more) by the device 100 of the present disclosure for cryopreservation of, for example, human corneal tissue, as compared to conventional methods of corneal cryopreservation, which typically results in 50% or more loss of endothelial cells during cryopreservation.

Example 5: the device uses a medium without cryoprotectant additives during a cryofreezing procedure to mitigate damage to mammalian cells

The modification of extracellular ice morphology using device 100 can be used to mitigate cell damage during freezing using media without any cell-permeable cryoprotectant additives. To demonstrate the efficiency of the device 100 even without the use of a specific cryopreservation medium, mouse mesenchymal stem cells (mscs) were used as samples and frozen in the device 100 in the original culture medium at-20 ℃, as described elsewhere herein. Specifically, as disclosed elsewhere herein, a suspension of about 200 microliters (μ L) of mscs from a cell culture sample (generally designated 310) is provided at a cell density of about 10^5-6cells/mL (determined by an automated cell counter), distributed in the device 100 including the membrane 140In the form of a 7500MWCO thin denatured cellulose film. The cell suspension 310 then forms a thin layer of liquid (e.g., about 2cm in diameter, less than 1mm thick) on top of the membrane 140. Another 3mL of cell suspension 310 from the same mscs culture sample is added to a 15mL cryovial with an inner diameter of 2.5cm and a liquid portion of approximately 0.8cm height is formed at the bottom of the cryovial (e.g., 300, fig. 9, 10, 12, and 13) outside of housing 110. The device 100 holding the layer of cell suspension 310 is then mounted into a cryovial, as shown in fig. 9 and 10, with the lower portion of the housing 110 at least partially immersed in the cell suspension 310 contained within the cryovial but outside the housing 110.

Thus, the culture medium is separated from the cell envelope 140 inside and outside the device. The frozen bottles were then placed in a-20 ℃ laboratory freezer. After 90 minutes, all liquid components (e.g., cell suspension 310 inside and outside device 100) are completely frozen (e.g., have completely changed from a liquid phase to a solid phase). The device 100 was then thawed in a 37 ℃ water bath and the thawed viability of the mscs from the thawed cell suspension 310 inside and outside the device 100 was analyzed by standard trypan blue exclusion experiments in a standard automatic cell counter.

The experiment was repeated five times using cell suspensions 310 from five different cell culture samples. The post-thaw viability of the suspension layer within the device 100 was determined to be 45.8 ± 6.0% by an automated cell counter, while the post-thaw viability of the cell suspension outside the device 100 (e.g., within a freezer bottle) was only 16.8 ± 8.1%. FIGS. 11A and 11B show typical results obtained from an automated cell counter for cell suspension 310 protected by device 100 during freezing (FIG. 11A) and cell suspension 310 unprotected by device 100 (e.g., outside the device). In FIG. 11A, the total cell concentration was measured to be 2.46X 10⁵cells/mL, with a viable cell concentration of 1.29X 10⁵cells/mL (52%), whereas the concentration of dead cells was 1.17X 10⁵cells/mL (48%). In contrast, as shown in FIG. 11B, the total cell concentration was measured to be 3.99X 10⁵cells/mL, with a viable cell concentration of 4.69X 10⁴cells/mL (12%) whereas the concentration of dead cells was 3.52X 10⁵cells/mL (88%).

As clearly shown, the post-thaw viability of membrane-protected cells (e.g., within device 100) is almost 200% higher than unprotected cells. One hypothesis as to why half of the cells inside the device become non-viable during freezing is that the freezer temperature of-20 ℃ is very close to the nucleation temperature of intracellular ice of mammalian cells when no osmotic cryoprotectant (e.g., DMSO) is present. This effect may lead to the formation of intracellular ice that is lethal as a process that is random and progressive to the entire cell population. Thus, even without the use of a cell-permeable cryoprotectant, the device of the present invention is capable of mitigating cell damage resulting from extracellular ice formation; however, this function does not fully protect the cells from the damaging effects of random intracellular ice formation.

Example 6: the device reduces damage to plant tissue without the use of any cryoprotectant additives during the freezing procedure

The modification of the extracellular ice morphology using the device 100 can be applied to mitigate damage to samples including plant tissue without the use of any cryoprotective agent during freezing. To demonstrate the efficiency of this application, onion pulp (pulp) tissue, the uppermost layer of which is the epidermal cell layer, was used as sample 10. The samples were cut into 1cm by 1cm dimensions with an average thickness of about 3-4 mm. A 1mL volume sample of PBS (standard saline solution) solution 310 was added to the housing 110 of the device 100, and then one sample 10 was immersed in the PBS solution with the epidermal cell layer facing the denatured cellulose membrane 140 (here with 7500 MWCO). The device 100 is then placed into a 15mL cryovial (e.g., 300) containing 3mL of PBS solution 310, as shown in fig. 12 and 13, such that the housing is at least partially immersed in the PBS solution 310. Thus, the PBS solution 310 located inside and outside the device 100 is separated by the membrane 140. The entire sample is then exposed to an ambient environment at-80 ℃ (e.g., in a cryogenic refrigerator). After 60 minutes, the sample 10, as well as the PBS solution in the device 100 and in the cryovial, was substantially or completely frozen and then thawed by immersion in a 37 ℃ water bath. The thawed sample 10 was stained on its epidermal cell layer with trypan blue and analyzed for cell viability and tissue structure using a standard stereomicroscope.

For comparison, fresh tissue of the same size was loaded directly into 3mL of PBS solution 310 in a 15mL cryovial without using the apparatus 100, and then frozen, thawed and analyzed according to the same procedure described above. Under stereoscopic microscope observation, the typical morphology of the thawed and stained specimen 10 is shown in fig. 14 and 15, either tissue protected by such a device 100 during freezing (fig. 14) or tissue not protected by such a device 100 (fig. 11B). As shown in fig. 14, the tissue structure of the specimen 10 is well preserved for the tissue protected by the device 100, all cell morphology is similar to fresh tissue, about half of the cells of the specimen are non-viable based on their stained nuclear appearance. In contrast, as shown in fig. 15, the tissue structure of the sample 10, which is not protected by such a device 100, is deformed, and all cells are structurally seriously damaged.

Thus, the device 100 disclosed herein can be used to preserve plant tissue structure and improve cell viability thereof without the use of any cryopreservation media with cell-penetrating cryoprotectant additives dissolved therein. Freezing onion tissue at-20 ℃ with or without the protective device 100, no cells lose viability and the tissue structure is not disrupted due to the protective effect of the cell wall, which is similar in structure to the denatured cellulose membrane 140 used with the device 100. It can therefore be concluded that the device of the invention provides better protection against temperatures below-20 ℃ during freezing than that provided by the cell walls of similar plant tissues. Thus, the result is an embodiment that also demonstrates the higher efficiency of the device 100 of the present disclosure in cell protection compared to some similar protection mechanism that naturally occurs from the cell wall of plant tissue (one of its major components is cellulose).

It will be understood that various details of the disclosed subject matter may be changed without departing from the scope of the disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.