A combined reagent for animal tissue cryopreservation and its application and method

Abstract

The present application relates to a kind of animal tissue cryoprotective combined reagent and its application and method, specifically, it is related to a kind of combined reagent for the cryoprotection of animal tissue, the application of the combined reagent in animal tissue cryoprotection, and the method for the cryopreservation of animal tissue using the combined reagent.The combined reagent of the present application and its application and method can be combined with ice frozen section technology to carry out section study to animal tissue, and very effectively reduce the formation of ice crystal while maintaining the morphology of tissue.

Description

Technical Field

[0001] This invention relates to the technical field of cryopreservation of animal tissues, and more specifically to a combination reagent for cryopreservation of animal tissues, the application of the combination reagent in cryopreservation of animal tissues, and a method for freezing animal tissues using the combination reagent. Background Technology

[0002] To study the structure of isolated biological tissues in a multi-scale, high-resolution, and systematic manner, tissue sectioning is typically required, followed by molecular labeling and imaging of the sections. Sectioning is a crucial step in tissue processing; the quality of the sections determines the reliability of the tissue sample and whether it is possible to reconstruct it into its original three-dimensional state. This is essential for many biological studies and clinical case examinations.

[0003] Cryosectioning involves cutting frozen biological tissue using a cryostat. Because frozen tissue has a certain degree of rigidity, it can be sliced ​​into thin, uniform sections (typically 20 to 80 micrometers). Cryosections are widely used in rapid pathological examinations. Since the process does not cause irreversible damage to biomolecules, they are commonly used in fluorescence imaging, various immunostaining methods, and other research methods. Their compatibility with fluorescence imaging allows for the imaging of three-dimensional structures. However, preventing ice crystal damage and the destruction of the original structure during freezing remains one of the biggest challenges in cryosectioning technology.

[0004] Existing technologies have proposed using a variety of substances as cryoprotectants for biological samples. For example, CN110250162A proposes using specific proteins, such as RNA-binding proteins, in combination with substances such as human serum albumin, propylene glycol, ethylene glycol, and sucrose.

[0005] CN108112575A uses a combination of dextrin, dextran, isomaltose oligosaccharides, etc., as a cryoprotectant for cells or tissues.

[0006] Current conventional cryopreservation methods use gradient dehydration of animal tissues with 20% wt% sucrose PBS and 30% wt% sucrose PBS. However, animal tissues significantly shrink in volume and change morphology during dehydration. Even after dehydration, ice crystals still form during freezing, irreversibly damaging cell structure. Furthermore, due to the tissue's fragility, sections are easily damaged, making it difficult to cut thin sections (below 20 micrometers). During thawing, significant recrystallization occurs, further amplifying the ice crystal damage. In addition, traditional cryosectioning typically requires drying or even oven-drying the sections attached to the slide to prevent easy detachment. However, this process allows moisture to evaporate from the crystallized pores in the tissue slices, further enlarging the pores and creating a sponge-like structure. This drying process not only damages the tissue's morphology but also the structure of biomolecules, thus affecting the effectiveness of molecular labeling or enhancing background fluorescence during imaging. Then, PBS is used for rinsing. During this process, due to the shrinkage of brain tissue caused by sucrose dehydration, the osmotic pressure returns to its pre-dehydration state. Therefore, the tissue sections will swell on the slide, causing shrinkage or even detachment. Figure 1 (a) It is evident that traditional cryoprotectants, such as sucrose dehydration agents, are imperfect cryoprotection methods. They can only reduce ice crystal formation to a limited extent, but lack protection for the mechanical characteristics of tissues during sectioning, during rewarming, drying, and rehydration. Such shortcomings limit the reliability of frozen sections in revealing the structure of microscopic biological systems. Figure 3 (a) and (b) increase the difficulty for researchers to integrate multi-scale data into the overall organizational structure.

[0007] There is currently no cryoprotectant that can achieve satisfactory results in reducing ice crystal formation during freezing and minimizing damage to tissue morphology and molecular structure during section rewarming and drying, thus maintaining sample integrity at both the macroscopic and microscopic scales. Summary of the Invention

[0008] Technical issues

[0009] The purpose of this invention is to provide a cryoprotection method that fully maintains tissue size and structure on a macroscopic level, fully protects the structure of biomolecules on a microscopic level, forms very few and very small ice crystals during freezing, exhibits good mechanical properties during slicing to avoid breakage and damage, allowing for thinner, smoother sections, effectively inhibits recrystallization during rewarming, protects tissue from drying during drying, and prevents sections from swelling or shrinking after rehydration. This method will enable cryosectioning technology to completely preserve the spatial and molecular structure of tissues, making it possible to fully reconstruct multi-scale three-dimensional tissue structures using frozen sections.

[0010] Technical solution

[0011] To address the problems in the prior art and to achieve the objectives of this invention, this application provides a combined reagent for cryopreservation of animal tissues, the combined reagent comprising:

[0012] Permeation-enhancing molecules,

[0013] Non-permeable sugar cryoprotective molecules,

[0014] Permeable cryoprotective molecules, and

[0015] Recrystallization inhibitory molecules.

[0016] In one embodiment of the combined reagent of the present invention, the combined reagent further comprises:

[0017] Buffering components, preferably those with a pH of 7.2 to 7.4, such as 0.01M phosphate buffer with a pH of 7.2 to 7.4, and

[0018] The fixed component is preferably 4% paraformaldehyde by weight.

[0019] In one embodiment of the combined reagent of the present invention, the permeation-enhancing molecule is selected from any one or more combinations of the following:

[0020] Choline salts, preferably sodium cholate, sodium deoxycholate, sodium lithochondrate, sodium chenodeoxycholate, or other choline salts; the concentration of the choline salt is from 0.5% to 5% by weight relative to the total weight of the combined reagents used for cryoprotection of animal tissues.

[0021] A betaine-based zwitterionic surfactant containing fatty acid chains, preferably lauramidopropyl hydroxysulfonate betaine, lauramidopropyl betaine, dodecyl / tetradecyl dimethyl hydroxypropyl sulfonate betaine, dodecyl dimethyl betaine, or other similar reagents; the concentration of the betaine-based zwitterionic surfactant containing fatty acid chains is from 0.5% to 10% by weight relative to the total weight of the combined reagents used for cryoprotection of animal tissues.

[0022] A betaine-based zwitterionic surfactant containing a cholic acid side chain, preferably CHAPS (3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt); the concentration of the betaine-based zwitterionic surfactant containing a cholic acid side chain is from 0.5% to 5% by weight relative to the total weight of the combined reagents used for cryoprotection of animal tissues.

[0023] Anionic surfactants containing fatty acid chains, preferably sodium dodecyl sulfate or sodium dodecylbenzene sulfonate, are used; the concentration of the anionic surfactant containing fatty acid chains is from 0.5% to 10% by weight relative to the total weight of the combined reagents used for cryoprotection of animal tissues.

[0024] Nonionic surfactants, preferably Triton X-100, etc.; the concentration of nonionic surfactants is from 0.5% to 10% by weight relative to the total weight of the combined reagents used for cryoprotection of animal tissues.

[0025] The total concentration of the permeation-enhancing molecules is 0.5% to 10% by weight, relative to the total weight of the combined reagents used for cryoprotection of animal tissues.

[0026] In one embodiment of the combined reagent of the present invention, the permeation-enhancing molecule is 1% sodium deoxycholate or 3% lauramide propyl hydroxysulfonate betaine.

[0027] In one embodiment of the combined reagent of the present invention, the non-permeable sugar cryoprotectant molecule is selected from any one or more combinations of trehalose, sucrose, raffinose, mannitol, sorbitol, glucose, and galactose.

[0028] The total concentration of the non-permeable sugar cryoprotectant molecules is 3% to 40% by weight relative to the total weight of the combined reagents used for cryoprotection of animal tissues.

[0029] In one embodiment of the combined reagent of the present invention, the non-permeable sugar cryoprotectant molecule is 10% to 35% by weight of trehalose.

[0030] In one embodiment of the combined reagent of the present invention, the permeable cryoprotectant molecule is selected from any one or more combinations of ethylene glycol, dimethyl sulfoxide, 1,2-propanediol, glycerol, and glycine betaine.

[0031] The total concentration of the permeable cryoprotectant molecules is 3% to 15% by weight relative to the total weight of the combined reagents used for cryoprotection of animal tissues.

[0032] In one embodiment of the combined reagent of the present invention, the permeable cryoprotectant molecule is 5% to 10% by weight of ethylene glycol.

[0033] In one embodiment of the combined reagent of the present invention, the recrystallization inhibitor molecule is selected from polymeric recrystallization inhibitors, such as polyvinyl alcohol, preferably 0.05% to 1% by weight of polyvinyl alcohol, more preferably 0.1% by weight of polyvinyl alcohol.

[0034] Another aspect of the present invention provides the application of the combined reagents described in any of the above embodiments in the cryopreservation of animal tissues.

[0035] Another aspect of the present invention provides a method for freezing animal tissue, the method comprising bringing the animal tissue into full contact with a combination reagent as described in any of the above embodiments of the present invention, and then freezing it.

[0036] In one embodiment of the method for freezing animal tissue according to the present invention, the method further includes slicing the frozen tissue.

[0037] Beneficial effects

[0038] The animal tissue cryoprotection combination reagent of the present invention utilizes osmotic-enhancing molecules to effectively deliver high concentrations of non-permeable carbohydrate cryoprotective molecules into animal cells, and works in conjunction with permeable cryoprotective molecules to achieve a balance between intracellular and extracellular cryoprotective molecules, thereby obtaining an effective cryoprotection effect while avoiding tissue and cell shrinkage caused by an osmotic pressure difference where the extracellular molecule concentration is much greater than the intracellular concentration. Due to the sufficient balance between extracellular and intracellular cryoprotective molecules, it is named Balanced Cryopreservation (BCP). Furthermore, the animal tissue cryoprotection combination reagent of the present invention also employs recrystallization inhibitor molecules, which can be combined with the aforementioned non-permeable carbohydrate cryoprotective molecules and permeable cryoprotective molecules to achieve a better recrystallization inhibition effect. Attached Figure Description

[0039] Figure 1 This demonstrates the advantages of the balanced cryoprotection of the present invention compared to the traditional sucrose dehydration cryoprotection. In this diagram, a is a schematic diagram of tissue damage caused during the traditional sucrose dehydration cryoprotection process, and b is a schematic diagram of the balanced cryoprotection process and tissue protection.

[0040] Figure 2 This demonstrates that the balanced cryoprotection of the present invention effectively reduces ice crystal damage and maintains tissue morphology, wherein,

[0041] Image a shows autofluorescence images of mouse brain slices treated with 30% sucrose, dehydrated, unfrozen, balanced cryoprotected, and treated with 2M ethylene glycol, two hours after dehydration and after rehydration. It can be seen that sugars cause the tissue to form numerous small pores, while the permeable small-molecule ethylene glycol causes the tissue to form a few larger pores. The BCP group of this invention is basically similar to the unfrozen group, forming small and few pores. After rehydration, the layered structure of the cerebral cortex, indicated by autofluorescence of the tissue cell nuclei, is clearly visible.

[0042] b shows that gradient sucrose dehydration significantly caused a decrease in tissue volume (using paired t-test, N=6).

[0043] c shows that permeable small-molecule cryoprotectants do not cause significant changes in tissue volume at relatively low concentrations (using paired t-test, N=6).

[0044] d shows that treatment of tissues with surfactant-free BCP-F and BCP resulted in a significant reduction in tissue volume (using paired t-test, N=6);

[0045] e showed that there was no significant change in tissue volume after normal BCP treatment (using paired t-test, N=6);

[0046] The “30% sucrose” group in f shows photographs of mouse brain tissue after fixation, BCP treatment, and sucrose gradient dehydration; the “BCP group” shows mouse brain sections after sucrose gradient dehydration and mouse brain sections after BCP treatment.

[0047] Figure 3 This demonstrates that balanced cryoprotection maintains the cell's micromorphology.

[0048] a shows that the dendritic structures of cortical pyramidal neurons marked by thy1-YFP transgenic mice were cleaved by ice crystals caused by a sucrose gradient dehydration method (as indicated by the triangular arrows);

[0049] b shows the destruction of intracellular structures of cortical pyramidal neurons labeled in thy1-YFP transgenic mice by ice crystals caused by a sucrose gradient dehydration method (as indicated by the triangular arrows);

[0050] c shows that the cell bodies and fiber structures of cortical neurons marked by thy1-YFP transgenic mice were preserved intact after freezing under the protection of the combined reagents of the present invention.

[0051] d shows that the cortical pyramidal neuronal dendritic structures marked by thy1-YFP transgenic mice were preserved intact after freezing under the protection of the combined reagents of the present invention.

[0052] Figure 4 The distribution of cryoprotective molecules both intracellularly and extracellularly is shown.

[0053] The left side shows the situation where the cell membrane is not permeable; only transmembrane-transferable small molecules like EG can enter the cell membrane. Therefore, increasing the concentration of extracellular trehalose will cause cell shrinkage and tissue dehydration.

[0054] The right side shows the cell condition after treatment with the combined reagents of the present invention. High concentrations of trehalose enter the cells, and there is no cell shrinkage caused by osmotic pressure differences.

[0055] Figure 5 This is a schematic diagram of the vitrification and quick-freezing process for tissues.

[0056] Figure 6 The invention demonstrates that the osmotic-enhancing molecules prevent high-concentration sugars from reducing tissue volume. The left figure shows the effect of 30% wt sucrose and the same concentration of sucrose with different osmotic-enhancing molecules on the relative tissue volume; the left figure also shows the effect of 30% wt trehalose and the same concentration of trehalose with different osmotic-enhancing molecules on the relative tissue volume. It can be seen that whether it is sucrose or trehalose, a high concentration of 30% wt will lead to a reduction in the relative tissue volume. However, after the osmotic-enhancing molecules are mixed into the sugar solution, the volume reduction effect disappears.

[0057] Figure 7 The diagram shows the results obtained according to Example 1 of this application, in which the tissue is not deformed after being processed and frozen by the method of the present invention, a small amount of ice crystals are formed, the mesoscopic structure is not destroyed, but a small amount of microstructure may be destroyed.

[0058] Figure 8 The diagram shows the results obtained according to Example 3 of this application, where the tissue showed no deformation and the processing time was less than two days. No ice crystals were formed that would cause damage to the visible mesoscopic and microscopic structures. Detailed Implementation

[0059] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0060] Addressing the problems of existing animal tissue cryoprotective reagents that damage tissue and cell structures during freezing, cause cross-sectional damage during slicing, lead to recrystallization during rewarming, and form voids and sponge-like structures during drying, thus disrupting tissue morphology and molecular structure, the inventors, through extensive research and experimentation, discovered that the combined reagent of this invention for cryoprotecting animal tissues, described in detail below, simultaneously solves the aforementioned problems in the prior art. Macroscopically, it completely maintains tissue size and structure; microscopically, it fully protects the structure of biomolecules; during slicing, it exhibits excellent mechanical properties, avoiding breakage and allowing for thinner, smoother slices; during rewarming, it effectively inhibits recrystallization; during drying, it protects the tissue from drying effects; and after rehydration, it prevents the slices from swelling or shrinking.

[0061] Compared to traditional tissue cryoprotection methods, the balanced cryoprotection technology of this invention has four most significant advantages:

[0062] (1) It does not cause tissue deformation. BCP treatment, by delivering a high concentration of cryoprotectant molecules that are normally non-transmembrane-bound into cells, overcomes the problem of high osmotic pressure caused by high concentrations of cryoprotectants leading to overall tissue shrinkage. After freezing and sectioning BCP-treated tissues, rigorous macroscopic and microscopic morphological measurements can be performed to obtain truly accurate macroscopic, cellular, and subcellular morphological dimensions. Figure 1 b, Figure 2 (bf).

[0063] (2) Make full use of the properties of BCP formula that do not change tissue morphology, and combine fixed components to perform heart perfusion on animals. Due to the high efficiency of the circulatory system in delivering chemical reagents, a ten-minute treatment can have a similar effect to a day of immersion, thereby shortening the entire cryoprotection time to within a few hours.

[0064] (3) Significantly reduces the number and size of ice crystals. BCP combines the properties of permeable small molecules to reduce the number of ice crystals, the properties of impermeable sugar molecules to reduce the size of ice crystals, and the highly efficient recrystallization inhibition effect of PVA macromolecules to achieve optimal intracellular and extracellular cryoprotection effects. Figure 1 b, Figure 2 a). Combined with vitrification and quick-freezing treatment ( Figure 5 This can further reduce ice crystal damage. BCP makes it possible to study microstructures (such as the fiber structure of neurons) using frozen sections, and to reconstruct and restore them in three-dimensional space. Figure 2 (c and d).

[0065] (4) Improves the mechanical structure of frozen tissue and avoids cross-sectional damage. Tissue treated with BCP has fewer and smaller ice crystals (…). Figure 1 b, Figure 2 Due to factors such as a), it has better mechanical properties and very low brittleness. Therefore, tissues treated with BCP can be sectioned into ultrathin sections, as thin as 1 micrometer, to obtain more section samples, ensure better staining effects, or perform super-resolution imaging.

[0066] (5) Effective protection of biomolecules. BCP has the effect of protecting proteins from denaturation during freezing, thawing, and drying, and can better preserve endogenous fluorescence and maintain antigenic determinant structure to obtain better immunofluorescence staining results.

[0067] This invention uses a highly efficient and mild surfactant to make cell membranes permeable to cryoprotective molecules that are otherwise impermeable or have relatively low permeability, thereby avoiding tissue shrinkage caused by high concentrations of cryoprotectants. Tissue cryoprotection methods that use the surfactants mentioned in component a. to alter cell membrane permeability and deliver cryoprotective molecules into cells fall within the scope of this technology.

[0068] This invention combines three types of molecules with different cryoprotective activities: sugars, permeable cryoprotective small molecules, and highly efficient recrystallization inhibitors such as polyvinyl alcohol, with a permeation reagent to obtain the best cryoprotective effect and frozen tissue mechanical properties.

[0069] This invention utilizes the property of balanced cryoprotectants that do not alter tissue morphology, and combines them with fixatives to form a balanced cryoprotective fixative. Simultaneously, it enables rapid and efficient cryoprotection and fixation of animals through careful perfusion. Methods that use morphology-unaltering cryoprotectants combined with fixatives to perfuse animals and accelerate the entire tissue processing procedure fall within the scope of this technology.

[0070] This invention lowers the tissue freezing point to below the freezing point of the embedding medium. It utilizes the temperature difference to allow the embedding medium to solidify slowly first, and then uses liquid nitrogen or dry ice to rapidly freeze the embedded, unsolidified tissue. This avoids compression of the tissue during the embedding medium's solidification process and ensures a rapid cooling effect. Any method that allows the embedding medium to solidify before the tissue and then rapidly freezes the entire tissue falls within the scope of this technology.

[0071] The present invention provides a combined reagent for cryoprotection of animal tissues. This reagent utilizes a permeation-enhancing molecule to effectively deliver a high concentration of non-permeable carbohydrate cryoprotectant molecules into animal cells, and works in conjunction with permeable cryoprotectant molecules to achieve a balance between intracellular and extracellular cryoprotectant molecules. This results in effective cryoprotection while avoiding tissue and cell shrinkage caused by an osmotic pressure difference where the extracellular molecule concentration is much higher than the intracellular concentration. Due to this sufficient balance between extracellular and intracellular cryoprotectant molecules, it is named Balanced Cryopreservation (BCP). Furthermore, the animal tissue cryoprotection combined reagent of the present invention also employs a recrystallization inhibitor molecule, which can be combined with the aforementioned non-permeable carbohydrate cryoprotectant molecules and permeable cryoprotectant molecules to achieve a better recrystallization inhibition effect.

[0072] The combination reagents of this invention for cryoprotection of animal tissues include, but are not limited to: permeation-enhancing molecules, non-permeable sugar cryoprotecting molecules, permeable cryoprotecting molecules, and recrystallization-inhibiting molecules.

[0073] Optionally, in one embodiment, the combination reagent of the present invention for cryoprotection of animal tissues may also contain other components commonly used in biological tissue treatments, such as freezing, fixation, and sectioning methods, for example, buffering components and fixation components.

[0074] The buffer component is preferably a buffer component with a pH of 7.2-7.4, and more preferably a 0.01M phosphate buffer with a pH of 7.2-7.4.

[0075] The preferred fixed component is 4% paraformaldehyde by weight.

[0076] In the combined reagent for cryoprotection of animal tissues of the present invention, the permeation-enhancing molecule is used to increase the permeability to tissues or cells, thereby allowing other components in the combined reagent of the present invention, especially non-permeable sugar cryoprotecting molecules, to permeate into tissues and cells. This achieves a balance of cryoprotecting molecules inside and outside the tissue and cells, effectively cryoprotecting structures, molecules and other biological entities within tissues and cells, and also avoiding tissue and cell shrinkage caused by concentration differences of various reagents inside and outside the tissue and cells.

[0077] The permeation-enhancing molecules of the present invention can efficiently deliver high concentrations of non-permeable cryoprotectants, examples of which include: cholates, betaine-type zwitterionic surfactants containing fatty acid chains, betaine-type zwitterionic surfactants containing cholic acid side chains, anionic surfactants containing fatty acid chains, nonionic surfactants, or combinations of two or more thereof.

[0078] Examples of the cholate salts include sodium cholate, sodium deoxycholate, sodium lithochondrate, sodium chenodeoxycholate, or other cholate salts. Sodium deoxycholate is preferred.

[0079] In the combined reagent of the present invention, the concentration of the choline salt is 0.5% to 5% by weight, preferably 1% to 3% by weight, and more preferably 1.5% to 2% by weight.

[0080] Examples of the betaine-type zwitterionic surfactants containing fatty acid chains include: lauramidopropyl hydroxysulfonate betaine, lauramidopropyl betaine, dodecyl / tetradecyl dimethyl hydroxypropyl sulfonate betaine, dodecyl dimethyl betaine, or other similar reagents. Among these, lauramidopropyl hydroxysulfonate betaine is preferred.

[0081] In the combined reagent of the present invention, the concentration of the betaine-type zwitterionic surfactant containing fatty acid chains is 0.5% to 10% by weight, preferably 1% to 8% by weight, and more preferably 3% to 6% by weight.

[0082] Examples of betaine-based zwitterionic surfactants containing cholic acid side chains include CHAPS (3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt) and similar reagents.

[0083] In the combined reagent of the present invention, the concentration of the betaine-type zwitterionic surfactant containing cholic acid side chains is 0.5% to 5% by weight, preferably 1% to 3% by weight, and more preferably 1.5% to 2% by weight.

[0084] Examples of the anionic surfactants containing fatty acid chains include sodium dodecyl sulfate and sodium dodecylbenzene sulfonate.

[0085] In the combined reagent of the present invention, the concentration of the anionic surfactant containing a fatty acid chain is 0.5% to 10% by weight, preferably 1% to 8% by weight, and more preferably 3% to 6% by weight.

[0086] Examples of such nonionic surfactants include Triton X-100, etc.

[0087] In the combined reagent of the present invention, the concentration of the nonionic surfactant is 0.5% to 10% by weight, preferably 1% to 8% by weight, and more preferably 3% to 6% by weight.

[0088] In a preferred embodiment of the present invention, the combined reagent of the present invention contains 1% by weight sodium deoxycholate or 3% by weight lauramidopropyl hydroxysulfonate betaine as a permeation-enhancing molecule. Compared with other concentrations and / or types of permeation-enhancing molecules, 1% by weight sodium deoxycholate or 3% by weight lauramidopropyl hydroxysulfonate betaine has the least impact on tissues and provides the best effect in cryoprotection and maintenance of tissue cell morphology.

[0089] Examples of the non-permeable sugar cryoprotectant molecules include any one or more combinations of the group consisting of trehalose, sucrose, raffinose, mannitol, sorbitol, glucose, and galactose. Trehalose is preferred, or a combination of trehalose with any one or more of sucrose, raffinose, mannitol, sorbitol, glucose, and galactose.

[0090] In the combined reagent of the present invention, the total concentration of the non-permeable sugar cryoprotectant molecules is 3% to 40% by weight, preferably 5% to 30% by weight, and more preferably 10% to 25% by weight.

[0091] In a preferred embodiment of the present invention, the combined reagent of the present invention contains 10% to 35% by weight of trehalose as a non-permeable sugar cryoprotectant molecule.

[0092] Sugars exceeding 10% by weight have higher intracellular osmotic pressures, directly affecting tissues and causing cell dehydration, leading to tissue shrinkage. For example, 30% by weight of sucrose and trehalose both cause tissue shrinkage. However, after incorporating the osmotic-enhancing molecules of this invention, the tissue size shows no significant difference compared to before the addition of the sugar solution, and exhibits similar cryoprotective effects (e.g., Figure 6 (As shown).

[0093] Examples of the permeable cryoprotectant molecules of the present invention include any one or more combinations of the group consisting of ethylene glycol, dimethyl sulfoxide, 1,2-propanediol, glycerol, and glycine betaine. Ethylene glycol is preferred.

[0094] In the combined reagent of the present invention, the total concentration of the permeable cryoprotectant molecules is 3% to 15% by weight, preferably 5% to 12% by weight, and more preferably 6% to 10% by weight.

[0095] When the total concentration of the permeable cryoprotective molecules exceeds 15% by weight, these molecules significantly lower the tissue's freezing point; excessively high concentrations can prevent the tissue from remaining solid at the temperature of the cryostat, thus making sectioning impossible. When the total concentration of the permeable cryoprotective molecules is below 3% by weight, it does not provide sufficient protection.

[0096] In a preferred embodiment of the present invention, the combined reagent of the present invention contains 5% to 10% ethylene glycol as a permeation cryoprotectant molecule.

[0097] The permeable cryoprotectant molecule can further reduce the number of ice crystals, improve the mechanical strength of frozen tissue sections, and reduce tissue brittleness on the basis of the protection of non-permeable sugar cryoprotectant molecules.

[0098] Examples of recrystallization inhibitors of the present invention include polyvinyl alcohol, or similar polymeric recrystallization inhibitors. Polyvinyl alcohol is preferred, for example, polyvinyl alcohol (PVA, MW = 27000, degree of alcoholysis 89%).

[0099] In the combined reagent of the present invention, the concentration of the recrystallization inhibitor molecule is from 0.05% to 1% by weight, preferably 0.1% by weight.

[0100] In a preferred embodiment of the present invention, the combined reagent of the present invention contains 0.05% to 1% by weight, preferably 0.1% by weight, polyvinyl alcohol as a recrystallization inhibitor molecule.

[0101] The buffer components of this invention may include buffer components commonly used in biological sample processing solutions, such as phosphate buffer, acetate buffer, and citrate buffer, preferably phosphate buffer. The concentration of the buffer is, for example, 0.001 M to 0.1 M, preferably 0.01 M. The pH of the buffer is 7.2 to 7.4.

[0102] The fixative component of this invention can be a fixative commonly used in the fixation of biological tissue specimens, such as paraformaldehyde.

[0103] In the combined reagents of the present invention, the concentration of the fixative is from 1% to 8% by weight, preferably 4% by weight.

[0104] In a preferred embodiment of the present invention, the combined reagent of the present invention contains 4% by weight of paraformaldehyde as a fixing component.

[0105] Example

[0106] Example 1:

[0107] 1. Mice were perfused with pre-cooled heparin-saline solution until the blood was completely drained.

[0108] 2- Mice were perfused with 4% paraformaldehyde (containing 0.01M phosphate buffer) for 2 min via cardiac perfusion.

[0109] 3- Mice were perfused with balanced cryoprotective fixative BCP (1 wt% sodium deoxycholate, 15 wt% trehalose, 10 wt% ethylene glycol, 4 wt% paraformaldehyde, 0.01 M pH 7.2-7.4 phosphate buffer, 0.1 wt% MW = 27000 alcoholysis degree 89% polyvinyl alcohol) for 10 min via cardiac perfusion.

[0110] 4. Remove the brain and place the mouse brain in a balanced cryoprotectant fixative at room temperature for 4 hours.

[0111] 5. The mouse brain was embedded in an embedding agent and then frozen at -80°C for at least 30 minutes.

[0112] 6. Remove the embedded block and transfer it to a cryostat using dry ice.

[0113] 7- Perform frozen sectioning at -30°C and attach the sections to gelatin-coated adhesive slides.

[0114] The process, effects, or tissue sections obtained in Example 1 are as follows: Figure 7 As shown in the figure, after processing and freezing sections using the method of this invention, the tissue shows no deformation, forms a small amount of ice crystals, does not damage the mesoscopic structure, but may damage a small amount of the microstructure. This invention is rapid, easy to operate, and the entire process takes less than 5 hours.

[0115] Example 2:

[0116] 1. Mice were perfused with pre-cooled heparin-saline solution until the blood was completely drained.

[0117] 2- Mice were perfused with 4% paraformaldehyde (containing 0.01M phosphate buffer) for 2 min via cardiac perfusion.

[0118] 3- Mice were perfused with balanced cryoprotective fixative BCP (1 wt% sodium deoxycholate, 15 wt% trehalose, 10 wt% ethylene glycol, 4 wt% paraformaldehyde, 0.01 M pH 7.2 to 7.4 phosphate buffer, 0.1 wt% MW = 27000 alcoholysis 89% polyvinyl alcohol) for 10 min via cardiac perfusion.

[0119] 4. Remove the brain and place the mouse brain in a balanced cryoprotectant fixative at room temperature for 4 hours.

[0120] 5. Transfer mouse brains to balanced cryoprotectant (35 wt% trehalose, 10 wt% ethylene glycol, 0.01 M pH 7.2-7.4 phosphate buffer) at 4°C for 24 h until sedimentation.

[0121] 5. The mouse brain was embedded in an embedding agent and then frozen at -80°C for at least 30 minutes.

[0122] 6. Remove the embedded block and transfer it to a cryostat using dry ice.

[0123] 7- Perform frozen sectioning at -35°C and attach the sections to gelatin-coated adhesive slides.

[0124] Fluorescence images of confocal imaging of frozen sections of Thy1-YFP mouse brain tissue after BCP treatment according to this embodiment are shown below. Figure 3 As shown in c and d; in contrast, Figure 3 Figures a and b show confocal fluorescence images of frozen sections of thy1-YFP mice after dehydration with 30% sucrose. The arrows point to obvious cell structure breakage caused by ice crystal damage.

[0125] Therefore, it can be seen that after treatment using the method of this invention, the tissue shows no deformation, the treatment time is within two days, and the operation is simple. It forms almost no ice crystals, does not damage the mesoscopic structure, and hardly damages the microscopic structure.

[0126] Example 3:

[0127] 1- The mouse brains treated with 5- in Example 2 were bonded to an iron base.

[0128] 2. Immerse the tissue or organ in the embedding agent and remove it until the surface is evenly coated with a thin layer of embedding agent.

[0129] 3. Place the base and organ tissue in a refrigerator at -8°C. The BCP-treated organ will not solidify and form ice crystals at this temperature, while the embedding agent will solidify and form a thin layer on the organ surface.

[0130] 4. Add liquid nitrogen to the outer layer of the double-walled container and isopentane to the inner layer. The temperature of the inner isopentane layer should be approximately -160°C. Use a magnetic cover to hold the iron base in place and immerse the organ in the isopentane for 1 minute.

[0131] 5. Remove the frozen organ and quickly place it in a low-temperature freezer (at -40°C to -80°C) and leave it for at least 2 hours until the isopentane on the surface of the embedding agent has fully evaporated.

[0132] 6. Remove the magnetic cover, iron base, and organ and invert them into a pre-cooled embedding medium at -4°C. Then quickly place them in isopentane with dry ice to allow the embedding medium to solidify.

[0133] 7. Remove the embedded organ (it can be stored long-term in a -80°C freezer).

[0134] 8- Mounted on the base of the cryostat.

[0135] 9. Frozen sectioning was performed at -35°C, and the sections were then mounted on gelatin-coated adhesive slides.

[0136] Example 3: The process, effect, or obtained tissue sections are as follows. Figure 8 As shown in the figure, after processing and freezing sections using the method of this invention, the tissue showed no deformation, and the processing time was within two days. No ice crystals were formed that would cause damage to the visible mesoscopic and microscopic structures.

[0137] Example 4: The balanced cryoprotection of the present invention effectively reduces ice crystal damage and maintains the morphology of tissues.

[0138] This embodiment focuses on describing Figure 2 The experimental procedure for the results shown.

[0139] (a)

[0140] The BCP (balanced cryoprotection combination reagent of the present invention) group was processed according to Example 2 above.

[0141] Mouse brains from the unfrozen group were sectioned using a vibratory microtome, perfused, and fixed with PFA (paraformaldehyde) for 24 hours.

[0142] The sucrose group was treated sequentially with 0.01MPBS+20% sucrose and 0.01MPBS+30% sucrose for 24 h each, followed by PFA fixation for 24 h.

[0143] The ethylene glycol group was treated sequentially with 0.01M PBS + 1.5M ethylene glycol and 0.01M PBS + 2M ethylene glycol for 24 hours each, followed by PFA fixation for 24 hours.

[0144] After exposing the sections of each group to air for 2 hours, the tissue structure was observed using autofluorescence at 488 / 521 nm using a mercury lamp. The results are as follows: Figure 2 As shown on the left side of a. After photographing, the tissue was immersed in PBS for 5 minutes and photographed again to show the rehydrated tissue condition. The results are as follows. Figure 2 As shown on the right side of a.

[0145] (b)

[0146] The volume of brain tissue treated according to (a) sucrose group was determined by water displacement method before each fluid change.

[0147] (c)

[0148] The volume of brain tissue treated with (a) ethylene glycol group was determined by water displacement method before each fluid change.

[0149] (d)

[0150] Following the procedure in Example 2, the volume of brain tissue was measured by water displacement method before step 4 (PFA), after step 4 (BCP-F), and after step 5 (BCP).

[0151] (e) Photographs of PFA, brain tissue dehydrated with 30% sucrose and treated with BCP according to Example 2.

[0152] (f) Photographs of frozen sections of brain tissue after dehydration with 30% sucrose and BCP treatment according to Example 2.

[0153] from Figure 2 As can be seen, the balanced cryoprotection of the present invention effectively reduces ice crystal damage and maintains the morphology of the tissue.

[0154] Example 5: The permeation-enhancing molecule of the present invention prevents high concentrations of sugars from reducing tissue volume.

[0155] Mice were cardiac perfusioned with pre-cooled heparin-saline solution until the blood was completely drained. In the group without osmotic promoters, brains were directly harvested, fixed with PFA for 24 h, and then transferred to 0.01 MPB + 30% sucrose solution for 48 h. Brain volume was determined by the water displacement method.

[0156] Groups treated with osmosis enhancers underwent 10-minute cardiac perfusion with 30% sucrose + 3% LHSB, 30% sucrose + 1% SDC, or 30% sucrose + 2% CHAPS. Brains were harvested after perfusion, and brain volume was measured 8 hours later using the water displacement method. Relative brain volumes from each treatment group were plotted, and the results are shown below. Figure 6 As shown on the left.

[0157] The experiment was repeated by replacing sucrose with trehalose in all the above groups. The relative brain volume of each treatment group was plotted, and the results are as follows: Figure 6 As shown on the right. The permeation-enhancing molecule of this invention prevents high concentrations of sugars from reducing tissue volume.

[0158] Preferred embodiments of the invention have been described above with reference to the accompanying drawings. Many features and advantages of these embodiments are apparent from this detailed description, and therefore the claims are intended to cover all such features and advantages of these embodiments that fall within their true spirit and scope. Furthermore, since many modifications and alterations will readily occur to those skilled in the art, the embodiments of the invention are not intended to be limited to the precise structures and operations illustrated and described, but rather to encompass all suitable modifications and equivalents falling within their scope.

Claims

1. A combination reagent for cryoprotecting animal tissues, the combination reagent comprising: Permeation-enhancing molecules, Non-permeable sugar cryoprotective molecules, Permeable cryoprotective molecules, and Recrystallization inhibitor molecules, in, The permeation-enhancing molecule is one or two of betaine-type zwitterionic surfactants containing fatty acid chains and betaine-type zwitterionic surfactants containing bile acid side chains. Of which, relative to the total weight of the combined reagents used for cryoprotection of animal tissues, the total concentration of the permeation-enhancing molecules is 0.5% to 10% by weight, the total concentration of the non-permeable sugar cryoprotecting molecules is 3% to 40% by weight, the total concentration of the permeable cryoprotecting molecules is 3% to 15% by weight, and the concentration of the recrystallization-inhibiting molecules is 0.05% to 1% by weight. The fatty acid chain-containing betaine-based zwitterionic surfactant is selected from lauramidopropyl hydroxysulfonate betaine, lauramidopropyl betaine, dodecyl / tetradecyl dimethyl hydroxypropyl sulfonate betaine, and dodecyl dimethyl betaine; the concentration of the fatty acid chain-containing betaine-based zwitterionic surfactant is from 0.5% to 10% by weight relative to the total weight of the combined reagents used for cryoprotection of animal tissues. The betaine-type zwitterionic surfactant containing cholic acid side chains is CHAPS (3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt); the concentration of the betaine-type zwitterionic surfactant containing cholic acid side chains is from 0.5% to 5% by weight relative to the total weight of the combined reagents used for cryoprotection of animal tissues.

2. The combined reagent as described in claim 1, wherein, The permeation-enhancing molecule is 3% by weight lauramidopropyl hydroxysulfonate betaine.

3. The combined reagent as described in claim 1, wherein, The non-permeable sugar cryoprotectant molecule is selected from any one or more of the group consisting of trehalose, sucrose, raffinose, mannitol, sorbitol, glucose, and galactose.

4. The combined reagent as described in claim 3, wherein, The non-permeable sugar cryoprotectant molecule is 10% to 35% trehalose.

5. The combined reagent as described in claim 1, wherein, The permeable cryoprotectant molecule is selected from one or more of the group consisting of ethylene glycol, dimethyl sulfoxide, 1,2-propanediol, glycerol, and glycine betaine.

6. The combined reagent as described in claim 1, wherein, The recrystallization inhibitor molecule is selected from polymeric recrystallization inhibitors.

7. The combination reagent as described in claim 6, wherein, The polymer recrystallization inhibitor is polyvinyl alcohol.

8. The combined reagent as described in claim 1, wherein, The recrystallization inhibitor is 0.1% by weight of polyvinyl alcohol.

9. The use of the combined reagent as described in any one of claims 1 to 8 in the cryopreservation of animal tissues.

10. A method for freezing animal tissue, the method comprising bringing the animal tissue into full contact with a combination of reagents as described in any one of claims 1 to 8, and then freezing it.

11. The method for freezing animal tissue as described in claim 10, the method further comprising slicing the frozen tissue.

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