A cryopreservation solution for kidney organoids, preparation method and use method

Abstract

The present invention provides a cryopreservation solution, a preparation method and a use method for kidney organoids, and relates to the field of cell and tissue engineering technology. The cryopreservation solution includes the following components by weight: 4.7 to 5.3 parts of cell cryopreservation solution; 3.5 to 4.5 parts of KOSR serum substitute; 0.8 to 1.2 parts of improved culture medium; wherein the improved culture medium includes RPMI 1640 culture medium and L-alanyl-L-glutamine, and the weight of RPMI 1640 culture medium is 3.5 to 4.5 times that of L-alanyl-L-glutamine. KOSR serum substitute and improved culture medium are added to CS10 cell cryopreservation solution to dilute the concentration of dimethyl sulfoxide, so that the dimethyl sulfoxide in the cryopreservation solution reaches the required minimum concentration, which not only reduces the formation of ice crystals, but also avoids the cytotoxicity caused by the excessively high concentration of dimethyl sulfoxide, thereby reducing the damage of kidney organoids.

Description

Technical Field

[0001] The present invention relates to the field of cell and tissue engineering technology, and in particular to a cryopreservation solution for kidney organoids, a preparation method and a use method thereof. Background Art

[0002] Kidney organoids are a three-dimensional cell complex cultivated in vitro that can simulate the complex structure and function of the kidney and have become an important tool for disease model construction, drug screening and regenerative medicine research. The cryopreservation and thawing of kidney organoids are important links in the entire technology application. Currently, cryopreservation mostly adopts slow freezing and vitrification methods. Among them, the slow freezing method uses low concentrations of cryoprotective agents (Cryoprotective Agents, CPAs), and the vitrification method uses high concentrations of cryoprotective agents. If ice crystals form and dissolve during the freezing and thawing process, the connection between cells will be destroyed, resulting in the collapse of the three-dimensional structure or loss of function of kidney organoids. Cryoprotectants include dimethyl sulfoxide and serum. Although dimethyl sulfoxide can prevent cell frostbite, it is potentially toxic to cells and tissues, and may cause cell membrane damage, protein denaturation and metabolic disorders, thereby affecting the survival rate and function of kidney organoids. Although serum can provide nutrition for cells, it has uncontrollable potential differentiation effects on kidney organoids derived from stem cells, affecting the success rate of thawing after freezing. Therefore, it is necessary to develop cryopreservation fluids and methods suitable for kidney organoids to improve the preservation effect of kidney organoid cryopreservation and the success rate of cryopreservation and thawing. Summary of the invention

[0003] The purpose of the present invention is to provide a cryopreservation solution, a preparation method and a use method for kidney organoids. The technical problem to be solved is how to reduce the damage to kidney organoids after cryopreservation.

[0004] The present invention is achieved through the following technical solutions:

[0005] A first aspect provides a cryopreservation solution for kidney organoids, comprising the following components by weight:

[0006] 4.7-5.3 portions of CS10 cell cryopreservation solution;

[0007] 3.5-4.5 parts KOSR serum replacement;

[0008] 0.8-1.2 parts of modified culture medium;

[0009] The improved culture medium comprises RPMI 1640 culture medium and L-alanyl-L-glutamine, and the weight of the RPMI 1640 culture medium is 3.5 to 4.5 times that of L-alanyl-L-glutamine.

[0010] Because kidney organoid cryopreservation is different from ordinary cell or stem cell cryopreservation, the specific differences are as follows:

[0011] (1) Tissue complexity of kidney organoids: Kidney organoids have complex three-dimensional structures and multiple cell types (such as podocytes, renal tubular epithelial cells in different regions, etc.); while cryopreservation of ordinary cells or stem cells usually targets a single cell type;

[0012] (2) Cryopreservation medium: cryopreservation of kidney organoids requires optimized cryopreservation medium to protect multiple cell types and tissue structures, and specific protective agents need to be added to prevent damage to the organoid structure; while cryopreservation of ordinary cells or stem cells usually uses standard cryopreservation medium (such as a solution containing dimethyl sulfoxide and serum);

[0013] (3) Preservation time: The expected cryopreservation time of kidney organoids can be from several hours to several days, but current technology is still limited; whereas the cryopreservation time of ordinary cells or stem cells can be several years or even longer.

[0014] Based on the above cryopreservation differences, KOSR serum substitute and modified culture medium were added to the above CS10 cell cryopreservation solution to dilute the concentration of dimethyl sulfoxide so that the dimethyl sulfoxide in the cryopreservation solution reached the required minimum concentration, which not only reduced the formation of ice crystals but also avoided the cytotoxicity caused by excessive dimethyl sulfoxide concentration. The uncontrollable effect of serum on the differentiation of renal organoids was solved by the KOSR serum substitute.

[0015] In addition, since CS10 cell cryopreservation medium contains balanced salt solution, antioxidants, carbohydrates and protein stabilizers, these ingredients work together on kidney organoids to provide an optimized freezing environment and reduce cell damage during freezing and thawing; balanced salt solution helps maintain the balance of osmotic pressure inside and outside cells, preventing cells from being damaged by osmotic pressure imbalance; antioxidants can remove free radicals produced during cryopreservation and reduce oxidative stress damage to cells; carbohydrates (such as glucose) serve as energy sources to support the metabolic needs of cells during cryopreservation; protein stabilizers can protect the protein structure in cells and prevent them from denaturing or degrading due to freezing. The modified culture medium contains rich amino acids, vitamins and inorganic salts, which provide necessary nutrition and environmental support for kidney organoids, help cells maintain metabolic activity and functional integrity, and help them recover and survive after cryopreservation; the addition of L-alanyl-L-glutamine helps maintain the metabolic activity of kidney organoids during cryopreservation and reduce cell damage caused by slowed metabolism. KOSR serum replacement serves as a culture medium supplement rich in growth factors and nutrients, providing nutritional support for kidney organoids and helping to maintain the structural and functional integrity of kidney organoids.

[0016] Furthermore, the following components are included by weight:

[0017] 5 cell cryopreservation solutions;

[0018] 4 KOSR serum replacements;

[0019] 1 modified medium;

[0020] The weight of the RPMI 1640 medium in the above-mentioned improved medium is 4 times that of L-alanyl-L-glutamine.

[0021] The dimethyl sulfoxide in the above-mentioned cell freezing solution and the balanced salt solution in the matrix solution work synergistically to help maintain the balance of osmotic pressure inside and outside the cells and prevent cells from rupturing due to osmotic pressure imbalance; dimethyl sulfoxide and the growth factors and nutrients in the KOSR serum substitute work together on kidney organoids to provide necessary protection and support for cells and promote cell recovery and growth after cryopreservation. The antioxidants in the matrix solution can assist the growth factors in the KOSR serum substitute to jointly resist the oxidative stress damage generated during the cryopreservation process; the carbohydrates and protein stabilizers in the matrix solution work together with the nutrients such as amino acids in the modified culture medium to provide comprehensive nutritional support for kidney organoids and help maintain the structural and functional integrity of kidney organoids. The nutrients in the KOSR serum substitute and the modified culture medium complement each other to provide comprehensive nutritional support for kidney organoids, which helps the recovery and growth of kidney organoids after cryopreservation and maintains their structural and functional integrity. Compared with the 10% concentration of dimethyl sulfoxide in the prior art, the present invention reduces the concentration of dimethyl sulfoxide in the cryopreservation solution to only 5%, thereby weakening the potential cytotoxicity of the cryopreservation solution to kidney organoids; since the cryopreservation solution does not contain serum components, the effect of the cryopreservation solution on the differentiation of kidney organoids is solved, thereby improving the success rate of resuscitation of kidney organoids after cryopreservation.

[0022] Furthermore, the concentration of L-alanyl-L-glutamine in the above improved culture medium is 2 mM.

[0023] A second aspect provides a method for preparing a cryopreservation solution, the method being used to prepare the above-mentioned cryopreservation solution;

[0024] The preparation method comprises the following steps:

[0025] The RPMI 1640 medium and L-alanyl-L-glutamine are taken in a ratio of 4:1, put into a container and mix well to obtain a modified medium;

[0026] The modified culture medium was stored at 4°C.

[0027] At room temperature, CS10 cell freezing solution, KOSR serum replacement and improved culture medium with a storage temperature of 4°C were taken out in a ratio of 5:4:1, put into a container and mixed evenly to obtain a freezing solution.

[0028] First, RPMI 1640 medium and L-alanyl-L-glutamine were mixed in a ratio of 4:1 to prepare the modified medium for use; since the concentration of dimethyl sulfoxide in the CS10 cell cryopreservation solution was 10%, the concentration of dimethyl sulfoxide was reduced by adding KOSR serum replacement and the modified medium to avoid the cytotoxicity caused by excessive dimethyl sulfoxide concentration. The CS10 cell cryopreservation solution, KOSR serum replacement and modified medium were stored at 4°C to ensure their stability before mixing with other components; mixing the CS10 cell cryopreservation solution, KOSR serum replacement and modified medium at room temperature helped to quickly and evenly disperse the components and avoid the influence of low temperature on the mixing process.

[0029] Further, at room temperature, the CS10 cell freezing solution, KOSR serum replacement and improved culture medium are mixed to obtain the freezing solution in the following specific steps:

[0030] The CS10 cell freezing solution and the improved culture medium are mixed in a ratio of 1:1, and the CS10 cell freezing solution and the improved culture medium stored at 4° C. are placed in a container, and the CS10 cell freezing solution and the improved culture medium are mixed in the container to obtain a second mixed solution;

[0031] The second mixed solution and the KOSR serum substitute are in a ratio of 1:1, the KOSR serum substitute stored at 4° C. is placed in a container, and the KOSR serum substitute and the second mixed solution are mixed to obtain a third mixed solution;

[0032] The third mixed solution and the CS10 cell freezing solution are in a ratio of 4:3, the CS10 cell freezing solution with a storage temperature of 4° C. is taken and placed in a container, and the third mixed solution and the CS10 cell freezing solution are mixed to obtain a fourth mixed solution;

[0033] The fourth mixed solution and the KOSR serum substitute are in a ratio of 7:2, the KOSR serum substitute stored at 4° C. is taken and placed in a container, and the fourth mixed solution and the KOSR serum substitute are mixed to obtain a fifth mixed solution;

[0034] The fifth mixed solution and the CS10 cell freezing solution are in a ratio of 9:1. The CS10 cell freezing solution with a storage temperature of 4° C. is taken and placed in a container. The fifth mixed solution and the CS10 cell freezing solution are mixed to obtain a freezing solution.

[0035] Adding improved culture medium to the above-mentioned CS10 cell cryopreservation solution not only adds nutrients to the cryopreservation solution, but also dilutes the CS10 cell cryopreservation solution, reduces the concentration of dimethyl sulfoxide, and avoids the cytotoxicity caused by excessive dimethyl sulfoxide concentration; adding KOSR serum substitute to the second mixed solution not only supplements nutrients to the cryopreservation solution, but also dilutes the concentration of dimethyl sulfoxide and reduces cytotoxicity; adding CS10 cell cryopreservation solution and KOSR serum substitute in multiple steps is intended to further evenly disperse the components and ensure their balanced distribution in the cryopreservation solution.

[0036] The third aspect provides a method for using a cryopreservation solution, wherein the cryopreservation solution prepared by the above preparation method adopts the method for using;

[0037] The method of use includes the following steps:

[0038] The prepared freezing solution is injected into a 2 ml cryotube, and each cryotube is injected with 800 ul of freezing solution to obtain a cryotube to be used;

[0039] placing the kidney organoid into the cryopreservation tube to be used, contacting the kidney organoid with the cryopreservation solution in the cryopreservation tube to be used, and obtaining the cryopreservation tube to be frozen;

[0040] The cryopreservation tubes to be frozen are placed in a gradient freezing box, and then the gradient freezing box is placed in a -80°C ultra-low temperature refrigerator; after the temperature of the kidney organoids drops to -80°C, the cryopreservation tubes to be frozen are transferred to a liquid nitrogen tank filled with liquid nitrogen.

[0041] The above-mentioned cryopreservation solution is separated into liquids. 800uL of cryopreservation solution can usually provide an appropriate cell density, which helps to improve the survival rate of cells during freezing and thawing. In addition, during the freezing process, the liquid will freeze and expand. Adding only 800ul of liquid to a 2ml cryopreservation tube can reserve a certain amount of space to prevent the cryopreservation tube from rupturing due to excessive internal pressure. Place the kidney organoids in the cryopreservation tube to be used, so that the kidney organoids are fully immersed in the cryopreservation solution to ensure that the kidney organoids receive sufficient and comprehensive nutritional support and protection during the freezing process to reduce damage. Through the gradient cooling method, the kidney organoids gradually adapt to the low temperature environment, reduce cell damage caused by sudden temperature drops, improve the survival rate of kidney organoids during the freezing process, and reduce the formation of ice crystals and damage to cell structures; use the ultra-low temperature environment of liquid nitrogen to preserve kidney organoids for a long time, ensure that the kidney organoids remain stable at ultra-low temperatures, extend the storage time, and reduce cell damage caused by temperature changes.

[0042] Furthermore, the time taken to transfer the cryotubes to be frozen to the liquid nitrogen tank is less than 2 minutes.

[0043] Avoid exposing the cryopreservation tubes to room temperature for a long time, as this may affect the cryopreservation effect of the kidney organoids.

[0044] The above kidney organoids have reached a stable low temperature state in the -80℃ ultra-low temperature freezer. If the transfer time is too long, it may cause temperature fluctuations, thus affecting the cryopreservation effect of the kidney organoids. Controlling the transfer within 2 minutes can minimize temperature fluctuations and keep the kidney organoids in a stable low temperature environment. At -80℃, the water in the kidney organoids has formed a stable ice crystal structure. If the transfer time is too long, the ice crystals may rearrange or form new ice crystals due to temperature fluctuations, thereby causing damage to the kidney organoids. Rapid transfer to a liquid nitrogen tank can maintain the stability of the ice crystal structure and prevent cell damage caused by the re-formation of ice crystals. The ability of cells to remain active at low temperatures is limited. Long-term temperature fluctuations or exposure to non-ideal environments may cause cell activity to decrease. Rapid transfer to a liquid nitrogen tank can maximize the activity of cells and provide a better material basis for subsequent experiments and applications. In addition, rapid transfer to a liquid nitrogen tank can also reduce other potential risks caused by long-term exposure to non-liquid nitrogen environments, such as contamination, oxidation, etc. These factors may affect the preservation quality of kidney organoids, so rapid transfer helps to improve the overall preservation effect.

[0045] Further, after obtaining the above-mentioned cryopreservation tube to be used, the storage temperature of the cryopreservation tube to be used is determined according to the waiting time of the cryopreservation solution, and the specific steps include:

[0046] The time when the preparation of the above-mentioned freezing solution is completed is preset as the initial time, the time when the kidney organoid is placed in the cryopreservation tube to be used is the use time, and the difference between the use time and the initial time is the waiting use time;

[0047] If the above waiting time is less than 30 minutes, place the cryotube to be used at room temperature;

[0048] If the above waiting time for use is greater than 30 minutes and less than 24 hours, place the cryovials to be used at 4°C;

[0049] If the waiting time is longer than 24 hours, place the cryovials at -20°C.

[0050] The waiting time for the cryopreservation solution is short, and the composition and stability of the cryopreservation solution will not be significantly affected. Therefore, placing the cryopreservation tubes to be used in a room temperature environment can facilitate subsequent operations without adversely affecting the cryopreservation of kidney organoids. As the waiting time increases, some components in the cryopreservation solution may begin to undergo subtle changes, such as protein denaturation or degradation of certain chemicals. Placing the cryopreservation tubes to be used in a 4°C environment can slow down the rate of these changes, thereby maintaining the stability and effectiveness of the cryopreservation solution. In addition, the temperature of 4°C can also prevent the growth of bacteria and other microorganisms, ensuring the hygiene and safety of the cryopreservation solution during the long waiting process. When the waiting time exceeds 24 hours, the composition of the cryopreservation solution at room temperature will change significantly, and even lose its protective effect on kidney organoids. Placing the cryopreservation tubes to be used at -20°C can further slow down the rate of composition changes and extend the storage time of the cryopreservation solution.

[0051] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0052] Based on the above cryopreservation differences, KOSR serum substitute and modified culture medium were added to the above CS10 cell cryopreservation solution to dilute the concentration of dimethyl sulfoxide, so that the dimethyl sulfoxide in the cryopreservation solution reached the required minimum concentration (only 5%), which not only reduced the formation of ice crystals, but also avoided the cytotoxicity caused by excessive dimethyl sulfoxide concentration; the uncontrollable effect of serum on the differentiation of renal organoids was solved by KOSR serum substitute.

[0053] In addition, since CS10 cell cryopreservation medium contains balanced salt solution, antioxidants, carbohydrates and protein stabilizers, these ingredients work together on kidney organoids to provide an optimized freezing environment and reduce cell damage during freezing and thawing; balanced salt solution helps maintain the balance of osmotic pressure inside and outside cells, preventing cells from being damaged by osmotic pressure imbalance; antioxidants can remove free radicals produced during cryopreservation and reduce oxidative stress damage to cells; carbohydrates (such as glucose) serve as energy sources to support the metabolic needs of cells during cryopreservation; protein stabilizers can protect the protein structure in cells and prevent them from denaturing or degrading due to freezing. The modified culture medium contains rich amino acids, vitamins and inorganic salts, which provide necessary nutrition and environmental support for kidney organoids, help cells maintain metabolic activity and functional integrity, and help them recover and survive after cryopreservation; the addition of L-alanyl-L-glutamine helps maintain the metabolic activity of kidney organoids during cryopreservation and reduce cell damage caused by slowed metabolism. KOSR serum replacement serves as a culture medium supplement rich in growth factors and nutrients, providing nutritional support for kidney organoids and helping to maintain the structural and functional integrity of kidney organoids.

[0054] Since the cryopreservation fluid does not contain serum components, the effect of the cryopreservation fluid on the differentiation of kidney organoids is solved, and the success rate of resuscitation of kidney organoids after cryopreservation is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:

[0056] Figure 1 This is a kidney organoid before cryopreservation;

[0057] Figure 2 The present invention is a kidney organoid cryopreserved by using the cryopreservation solution of the present invention. DETAILED DESCRIPTION

[0058] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0059] First embodiment:

[0060] A cryopreservation solution for kidney organoids, comprising the following components by weight:

[0061] 5 g CS10 cell freezing solution, wherein the CS10 cell freezing solution contains 0.5 g dimethyl sulfoxide and 4.5 g matrix solution;

[0062] 4 g KOSR serum replacement;

[0063] 1g modified culture medium;

[0064] Wherein, the RPMI 1640 medium itself does not contain glutamine, and 0.2 g of L-alanyl-L-glutamine is added to 0.8 g of RPMI 1640 medium to obtain 1 g of improved medium, and the concentration of L-alanyl-L-glutamine in the improved medium is 2 mM;

[0065] Among them, the full name of CS10 cell freezing medium is CryoStor CS10, the full name of KOSR serum substitute is KnockOut™ Serum Replacement, and the full name of RPMI 1640 medium is Advanced RPMI 1640 medium.

[0066] KOSR serum replacement and modified culture medium were added to the above-mentioned CS10 cell cryopreservation solution to dilute the concentration of dimethyl sulfoxide (DMSO), reducing the concentration of DMSO in the cryopreservation solution to 5% (this concentration is the minimum concentration of DMSO required for the cryopreservation solution). This can not only reduce the formation of ice crystals, but also avoid the cytotoxicity caused by excessive DMSO concentration. Since KOSR serum replacement is a serum-free culture medium supplement, the uncontrollable effect of serum on the differentiation of renal organoids is solved by KOSR serum replacement.

[0067] Since the above-mentioned matrix solution includes balanced salt solution, antioxidants, carbohydrates and protein stabilizers, etc., these components work together on kidney organoids to provide an optimized freezing environment and reduce cell damage during freezing and thawing. The modified culture medium is rich in amino acids, vitamins and inorganic salts, which provides necessary nutrition and environmental support for kidney organoids, helps cells maintain metabolic activity and functional integrity, and helps them recover and survive after freezing; the addition of L-alanyl-L-glutamine helps maintain the metabolic activity of kidney organoids during freezing and reduces cell damage caused by metabolic slowdown. KOSR serum replacement is rich in growth factors and nutrients, provides nutritional support for kidney organoids, reduces cell damage caused by metabolic slowdown, helps maintain the structural and functional integrity of kidney organoids, and promotes growth and survival after recovery.

[0068] The above-mentioned dimethyl sulfoxide and the balanced salt solution in the matrix solution work synergistically to help maintain the balance of osmotic pressure inside and outside the cells and prevent cells from rupturing due to osmotic pressure imbalance; dimethyl sulfoxide and the growth factors and nutrients in the KOSR serum substitute work together on kidney organoids to provide necessary protection and support for cells and promote cell recovery and growth after cryopreservation. The antioxidants in the matrix solution can assist the growth factors in the KOSR serum substitute to jointly resist the oxidative stress damage generated during the cryopreservation process; the carbohydrates and protein stabilizers in the matrix solution work together with the nutrients such as amino acids in the modified culture medium to provide comprehensive nutritional support for kidney organoids and help maintain the structural and functional integrity of kidney organoids. The nutrients in the KOSR serum substitute and the modified culture medium complement each other to provide comprehensive nutritional support for kidney organoids, which helps the recovery and growth of kidney organoids after cryopreservation and maintains their structural and functional integrity.

[0069] Compared with the 10% concentration of dimethyl sulfoxide in the prior art, the present invention reduces the concentration of dimethyl sulfoxide in the cryopreservation solution to only 5%, which weakens the potential cytotoxicity of the cryopreservation solution to kidney organoids; since the cryopreservation solution does not contain serum components, the effect of the cryopreservation solution on the differentiation of kidney organoids is solved, and the success rate of resuscitation of kidney organoids after cryopreservation is improved. In summary, the synergistic effect of the above components helps to reduce the damage of kidney organoids after cryopreservation and improve their survival rate and functional recovery after resuscitation.

[0070] In addition, although the present invention does not explicitly list buffers, electrolytes, nutrients, and osmotic protectants, etc., the components contained in this cryopreservation solution are: RPMI 1640 culture medium contains a buffer system, such as a variety of essential electrolytes, such as sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺), magnesium (Mg²⁺), chloride (Cl⁻), phosphate (PO4³⁻) and sulfate (SO4²⁻), etc., which are important components for maintaining electrolyte balance in cells and tissues; CS10 cell cryopreservation solution contains buffer components to maintain the acid-base balance of the solution to ensure the stability of the physiological environment of organoids under low temperature conditions; KOSR serum substitute provides essential proteins, amino acids, vitamins and growth factors to meet the metabolic needs of organoids during freezing and thawing. Through reasonable proportion design and ingredient selection, this cryopreservation solution has covered and synergistically achieved the ability to maintain osmotic pressure, provide a buffer environment, electrolyte balance and rich nutritional support after mixing. The addition of KOSR serum replacement and RPMI 1640 culture medium dilutes the concentration of dimethyl sulfoxide, thereby affecting its effect in preventing water crystallization. In order for the mixed solution to have a better preservation effect, other cryoprotectants (such as glycerol, ethylene glycol, etc.) can be added to ensure that "the water in the cells will not crystallize when the cells are close to the freezing point". The freezing conditions can also be optimized (by adjusting parameters such as the freezing rate and freezing temperature) to reduce damage to cells during the freezing process, thereby compensating for the adverse effects of the reduced DMSO concentration to a certain extent.

[0071] The first comparison:

[0072] The freezing solution includes the following components by weight: 2 g CS10 cell freezing solution; 7 g KOSR serum replacement; 1 g improved culture medium.

[0073] When this cryopreservation fluid is used for the cryopreservation of kidney organoids, ice crystal formation and cell structure disorder will occur in the kidney organoids during the cryopreservation period, and the cell survival rate of the kidney organoids will decrease after thawing.

[0074] Second comparison:

[0075] The freezing solution includes the following components by weight: 5 g CS10 cell freezing solution; 4 g KOSR serum replacement; 1 g RPMI 1640 culture medium.

[0076] When this cryopreservation fluid is used for the cryopreservation of kidney organoids, the cell volume in the kidney organoids will shrink and the intercellular spaces will increase during the cryopreservation period. The overall function of the kidney organoids will be difficult to recover after thawing (e.g., the glomerular filtration rate and tubular reabsorption function may be seriously affected), and the cell survival rate will decrease.

[0077] Second embodiment:

[0078] A method for preparing a freezing solution, the method being used to prepare the above-mentioned freezing solution;

[0079] The preparation method comprises the following steps:

[0080] Take the above RPMI 1640 medium and L-alanyl-L-glutamine in a ratio of 4:1, put them into a container (a test tube or a centrifuge tube can be used) and mix them evenly to obtain a modified medium;

[0081] The modified culture medium was stored at 4°C.

[0082] At room temperature, CS10 cell freezing solution, KOSR serum replacement and improved culture medium with a storage temperature of 4°C were taken out in a ratio of 5:4:1, put into a container and mixed evenly to obtain a freezing solution.

[0083] First, RPMI 1640 medium and L-alanyl-L-glutamine were mixed in a ratio of 4:1 to prepare the modified medium for use; since the concentration of dimethyl sulfoxide in the CS10 cell cryopreservation solution was 10%, the concentration of dimethyl sulfoxide was reduced by adding KOSR serum replacement and the modified medium to avoid the cytotoxicity caused by excessive dimethyl sulfoxide concentration. The CS10 cell cryopreservation solution, KOSR serum replacement and modified medium were stored at 4°C to ensure their stability before mixing with other components; mixing the CS10 cell cryopreservation solution, KOSR serum replacement and modified medium at room temperature helped to quickly and evenly disperse the components and avoid the influence of low temperature on the mixing process.

[0084] In a specific embodiment, at room temperature, the CS10 cell freezing solution, KOSR serum replacement and improved culture medium are mixed to obtain the freezing solution, comprising:

[0085] The CS10 cell freezing solution and the improved culture medium are mixed in a ratio of 1:1, and the CS10 cell freezing solution and the improved culture medium stored at 4° C. are placed in a container, and the CS10 cell freezing solution and the improved culture medium are mixed in the container to obtain a second mixed solution;

[0086] The second mixed solution and the KOSR serum substitute are in a ratio of 1:1, the KOSR serum substitute stored at 4° C. is placed in a container, and the KOSR serum substitute and the second mixed solution are mixed to obtain a third mixed solution;

[0087] The third mixed solution and the CS10 cell freezing solution are in a ratio of 4:3, the CS10 cell freezing solution with a storage temperature of 4° C. is taken and placed in a container, and the third mixed solution and the CS10 cell freezing solution are mixed to obtain a fourth mixed solution;

[0088] The fourth mixed solution and the KOSR serum substitute are in a ratio of 7:2, the KOSR serum substitute stored at 4° C. is taken and placed in a container, and the fourth mixed solution and the KOSR serum substitute are mixed to obtain a fifth mixed solution;

[0089] The fifth mixed solution and the CS10 cell freezing solution are in a ratio of 9:1. The CS10 cell freezing solution with a storage temperature of 4° C. is taken and placed in a container. The fifth mixed solution and the CS10 cell freezing solution are mixed to obtain a freezing solution.

[0090] Adding improved culture medium to the above-mentioned CS10 cell cryopreservation solution not only adds nutrients to the cryopreservation solution, but also dilutes the CS10 cell cryopreservation solution, reduces the concentration of dimethyl sulfoxide, and avoids the cytotoxicity caused by excessive dimethyl sulfoxide concentration; adding KOSR serum substitute to the second mixed solution not only supplements nutrients to the cryopreservation solution, but also dilutes the concentration of dimethyl sulfoxide and reduces cytotoxicity; adding CS10 cell cryopreservation solution and KOSR serum substitute in multiple steps is intended to further evenly disperse the components and ensure their balanced distribution in the cryopreservation solution.

[0091] Third embodiment:

[0092] A method for using a cryopreservation solution, wherein the cryopreservation solution prepared by the above preparation method is used in the method;

[0093] The method of use includes the following steps:

[0094] The prepared freezing solution is injected into a 2 ml cryotube, and each cryotube is injected with 800 ul of freezing solution to obtain a cryotube to be used;

[0095] Place 4 kidney organoids into the above-mentioned cryopreservation tubes to be used (a sterilized 20ul range pipette can be used, equipped with a corresponding "gun tip", and the tip area of ​​about 2mm at the front of the gun tip can be cut off, and then the kidney organoids are sucked up and moved to the pipette tip. Generally, the kidney organoids are located at the top of the liquid in the gun tip, and then the liquid at the bottom of the gun tip is slowly pumped out until the kidney organoids reach the bottom of the gun tip, and then the kidney organoids are pumped from the bottom of the gun tip into the cryopreservation tubes containing the cryopreservation solution), and the kidney organoids are in contact with the cryopreservation solution in the cryopreservation tubes to be used to obtain cryopreservation tubes to be frozen;

[0096] The cryopreservation tubes to be frozen are placed in a gradient freezing box, and then the gradient freezing box is placed in a -80°C ultra-low temperature refrigerator; after the temperature of the kidney organoids drops to -80°C, the cryopreservation tubes to be frozen are transferred to a liquid nitrogen tank filled with liquid nitrogen.

[0097] The above-mentioned cryopreservation solution is separated into liquids. 800uL of cryopreservation solution can usually provide an appropriate cell density, which helps to improve the survival rate of cells during freezing and thawing. In addition, during the freezing process, the liquid will freeze and expand. Adding only 800ul of liquid to a 2ml cryopreservation tube can reserve a certain amount of space to prevent the cryopreservation tube from rupturing due to excessive internal pressure. Place the kidney organoids in the cryopreservation tube to be used, so that the kidney organoids are fully immersed in the cryopreservation solution to ensure that the kidney organoids receive sufficient and comprehensive nutritional support and protection during the freezing process to reduce damage. Through the gradient cooling method, the kidney organoids gradually adapt to the low temperature environment, reduce cell damage caused by sudden temperature drops, improve the survival rate of kidney organoids during the freezing process, and reduce the formation of ice crystals and damage to cell structures; use the ultra-low temperature environment of liquid nitrogen to preserve kidney organoids for a long time, ensure that the kidney organoids remain stable at ultra-low temperatures, extend the storage time, and reduce cell damage caused by temperature changes.

[0098] Combination Figure 1 and Figure 2 It can be seen that complex tubular structures can still be seen on the edges of kidney organoids that were revived after freezing, and that successfully revived kidney organoids have less structural loss.

[0099] In a specific embodiment, the time taken to transfer the cryotubes to be frozen to the liquid nitrogen tank is less than 2 minutes.

[0100] Avoid exposing the cryopreservation tubes to room temperature for a long time, as this may affect the cryopreservation effect of the kidney organoids.

[0101] The above kidney organoids have reached a stable low temperature state in the -80℃ ultra-low temperature freezer. If the transfer time is too long, it may cause temperature fluctuations, thus affecting the cryopreservation effect of the kidney organoids. Controlling the transfer within 2 minutes can minimize temperature fluctuations and keep the kidney organoids in a stable low temperature environment. At -80℃, the water in the kidney organoids has formed a stable ice crystal structure. If the transfer time is too long, the ice crystals may rearrange or form new ice crystals due to temperature fluctuations, thereby causing damage to the kidney organoids. Rapid transfer to a liquid nitrogen tank can maintain the stability of the ice crystal structure and prevent cell damage caused by the re-formation of ice crystals. The ability of cells to remain active at low temperatures is limited. Long-term temperature fluctuations or exposure to non-ideal environments may cause cell activity to decrease. Rapid transfer to a liquid nitrogen tank can maximize the activity of cells and provide a better material basis for subsequent experiments and applications. In addition, rapid transfer to a liquid nitrogen tank can also reduce other potential risks caused by long-term exposure to non-liquid nitrogen environments, such as contamination, oxidation, etc. These factors may affect the preservation quality of kidney organoids, so rapid transfer helps to improve the overall preservation effect.

[0102] In a specific embodiment, after obtaining the cryopreservation tube to be used, the storage temperature of the cryopreservation tube to be used is determined according to the waiting time of the cryopreservation solution, and the specific steps include:

[0103] The time when the preparation of the above-mentioned freezing solution is completed is preset as the initial time, the time when the kidney organoid is placed in the cryopreservation tube to be used is the use time, and the difference between the use time and the initial time is the waiting use time;

[0104] If the above waiting time is less than 30 minutes, place the cryotube to be used at room temperature;

[0105] If the above waiting time for use is greater than 30 minutes and less than 24 hours, place the cryovials to be used at 4°C;

[0106] If the waiting time is longer than 24 hours, place the cryovials at -20°C.

[0107] The waiting time for the cryopreservation solution is short, and the composition and stability of the cryopreservation solution will not be significantly affected. Therefore, placing the cryopreservation tubes to be used in a room temperature environment can facilitate subsequent operations without adversely affecting the cryopreservation of kidney organoids. As the waiting time increases, some components in the cryopreservation solution may begin to undergo subtle changes, such as protein denaturation or degradation of certain chemicals. Placing the cryopreservation tubes to be used in a 4°C environment can slow down the rate of these changes, thereby maintaining the stability and effectiveness of the cryopreservation solution. In addition, the temperature of 4°C can also prevent the growth of bacteria and other microorganisms, ensuring the hygiene and safety of the cryopreservation solution during the long waiting process. When the waiting time exceeds 24 hours, the composition of the cryopreservation solution at room temperature will change significantly, and even lose its protective effect on kidney organoids. Placing the cryopreservation tubes to be used at -20°C can further slow down the rate of composition changes and extend the storage time of the cryopreservation solution.

[0108] The above specific implementation methods further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific implementation methods of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cryopreservation solution for kidney organoids, characterized in that: By weight Includes the following ingredients: 4.7 to 5.3 portions of cell freezing solution; wherein the cell freezing solution is CryoStor CS10; 3.5-4.5 parts KOSR serum replacement; 0.8-1.2 parts of modified culture medium; The improved culture medium comprises RPMI 1640 culture medium and L-alanyl-L-glutamine, and the weight of the RPMI 1640 culture medium is 3.5 to 4.5 times that of L-alanyl-L-glutamine.

2. The cryopreservation solution for kidney organoids according to claim 1, characterized in that: By weight Includes the following ingredients: 5 aliquots of cell cryopreservation solution; 4 KOSR serum replacements; 1 modified medium; Wherein, the weight of RPMI 1640 culture medium in the improved culture medium is 4 times that of L-alanyl-L-glutamine.

3. The cryopreservation solution for kidney organoids according to claim 2, characterized in that: The concentration of L-alanyl-L-glutamine in the modified medium is 2 mM.

4. A method for preparing a freezing solution, characterized in that: The method is used to prepare the freezing solution according to any one of claims 1 to 3; The preparation method comprises the following steps: The RPMI 1640 medium and L-alanyl-L-glutamine are taken in a ratio of 4:1, put into a container and mix well to obtain a modified medium; The modified culture medium is stored at a temperature of 4°C; At room temperature, CS10 cell freezing solution, KOSR serum replacement and improved culture medium with a storage temperature of 4°C were taken out in a ratio of 5:4:1, put into a container and mixed evenly to obtain a freezing solution.

5. The method for preparing a cryopreservation solution according to claim 4, characterized in that: The specific steps of mixing the CS10 cell freezing solution, KOSR serum replacement and improved culture medium at room temperature to obtain the freezing solution include: The CS10 cell freezing solution and the improved culture medium are in a ratio of 1:1, and the CS10 cell freezing solution and the improved culture medium with a storage temperature of 4° C. are taken and placed in a container, and the CS10 cell freezing solution and the improved culture medium are mixed in the container to obtain a second mixed solution; The second mixed solution and the KOSR serum substitute are in a ratio of 1:1, a KOSR serum substitute stored at 4° C. is taken and placed in a container, and the KOSR serum substitute and the second mixed solution are mixed to obtain a third mixed solution; The third mixed solution and the CS10 cell freezing solution are in a ratio of 4:3, the CS10 cell freezing solution with a storage temperature of 4° C. is taken and placed in a container, and the third mixed solution and the CS10 cell freezing solution are mixed to obtain a fourth mixed solution; The fourth mixed solution and the KOSR serum substitute are in a ratio of 7:2, a KOSR serum substitute stored at 4° C. is taken and placed in a container, and the fourth mixed solution and the KOSR serum substitute are mixed to obtain a fifth mixed solution; The fifth mixed solution and the CS10 cell freezing solution are in a ratio of 9:

1. The CS10 cell freezing solution with a storage temperature of 4° C. is taken and placed in a container. The fifth mixed solution and the CS10 cell freezing solution are mixed to obtain the freezing solution.

6. A method for using a freezing solution, characterized in that: The cryopreservation solution prepared by the preparation method according to any one of claims 4 to 5 adopts the use method; The method of use includes the following steps: The prepared freezing solution is injected into a 2 ml freezing tube, and each freezing tube is injected with 800 ul of freezing solution to obtain a freezing tube to be used; placing the kidney organoid into the cryopreservation tube to be used, so that the kidney organoid contacts the cryopreservation solution in the cryopreservation tube to be used, thereby obtaining the cryopreservation tube to be frozen; The cryotubes to be frozen are placed in a gradient freezing box, and then the gradient freezing box is placed in a -80°C ultra-low temperature refrigerator; after the temperature of the kidney organoid drops to -80°C, the cryotubes to be frozen are transferred to a liquid nitrogen tank filled with liquid nitrogen.

7. The method for using the freezing solution according to claim 6, characterized in that: The time taken to transfer the cryotube to be frozen to the liquid nitrogen tank is less than 2 minutes.

8. The method for using the freezing solution according to claim 6, characterized in that: After obtaining the cryopreservation tube to be used, determining the storage temperature of the cryopreservation tube to be used according to the waiting time of the cryopreservation solution, the specific steps include: The time when the preparation of the freezing solution is completed is preset as the initial time, the time when the kidney organoid is placed in the cryopreservation tube to be used is the use time, and the difference between the use time and the initial time is the waiting use time; If the waiting time for use is less than 30 minutes, place the cryotube to be used at room temperature; If the waiting time is greater than 30 minutes and less than 24 hours, place the cryovials to be used at 4°C; If the waiting time for use is greater than 24 hours, the cryovials to be used are placed at -20°C.

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