Primary tissue or organoid cryopreservation liquid and tissue and organoid cryopreservation and recovery method

Abstract

The invention discloses a primary tissue or organ-like cryopreservation solution which is prepared from dimethyl sulfoxide, dextran-20, primary cell antibiotics, Penicillin-streptomysin, bovine serum albumin, insulin, transferrin, growth hormone, fibroblast growth factors, human platelet growth factors, bone morphogenetic protein 4, human epidermal growth factors, insulin-like growth factors and the balance of distilled water. The culture medium is prepared from a hepatocyte growth factor, L-glutamic acid, alanine, glycine, L-asparaginic acid, L-proline, serine, sodium pyruvate, glutamine, nicotinamide, N-acetylcysteine, beta-mercaptoethanol, sodium selenite, beta glycerophosphate, cycloastragenol, Y-27632, a DMEM (Dulbecco Modified Eagle Medium) basal culture medium and the like. After the cryopreserved tissue is recovered, the isolated culture of the primary cells can be normally carried out, the viability of the primary cells reaches 93%, and the survival rate of the recovered organoid reaches 92%.

Description

Technical Field

[0001] This invention belongs to the field of cryopreservation technology for primary tissues and organoids, specifically relating to a cryopreservation solution for primary tissues or organoids and a method for cryopreservation and thawing of tissues and organoids. Background Technology

[0002] Primary tissues are a crucial component of biological resources. With the advent of the omics era and the development of bioinformatics and molecular imaging technologies, the demand for biological samples is increasing. However, current methods of primary tissue preservation are limited to simple cryopreservation, which largely fails to preserve the viability of primary tissues, posing significant challenges to subsequent research and applications. Achieving viable preservation of primary tissues has long been a difficult problem in the biomedical field. Currently, there are no commercially available reagents for tissue cryopreservation, and existing cell cryopreservation methods still struggle to preserve tissue viability.

[0003] Tissue organoids are tissue analogs derived from stem cells in organisms (humans and animals). They originate from patient tissues of specific disease types (most commonly tumor tissues) and are cultured in the laboratory to form organoids that retain the pathological tissue structure, genetic characteristics, and cell type heterogeneity of the source tissue. Organoids can be cultured, passaged, cryopreserved, and thawed in vitro for extended periods. Therefore, organoids provide a highly flexible tool for basic research and clinical translational medicine, and are of great significance in disease research, regenerative medicine, new drug development, and precision medicine.

[0004] Achieving viable cryopreservation of primary tissues and organoids, and protecting these valuable specimens, is a crucial step and method for clinical translational research. It allows cells to be removed from their vegetative state for a certain period while preserving their cellular characteristics, enabling them to be thawed and used in scientific experiments or clinical applications when needed. Highly efficient tissue cryopreservation techniques are essential for maintaining the viability of cryopreserved cells and minimizing cell damage.

[0005] Current tissue and organoid cryopreservation technologies still have significant limitations. Traditional cell cryopreservation solutions use culture medium, serum, and DMSO mixed in specific ratios. Fetal bovine serum (FBS), the most commonly used, has a complex composition and significant batch-to-batch variations, increasing the instability of cryopreservation results. Furthermore, the introduction of animal-derived components increases the risk of fungal and mycoplasma contamination, as well as the possibility of allergic reactions. Additionally, the introduction of animal-derived components limits its application in clinical medicine and translational medicine. Moreover, the cryopreservation process typically employs gradient cooling, which is time-consuming and labor-intensive.

[0006] Based on the above-mentioned demand for new cryopreservation technologies for primary tissues and organoids, and the shortcomings of existing cryopreservation technologies, there is an urgent need in the market for a cryopreservation reagent that can be widely used for cryopreservation of various primary tissues and organoids with good results and is simple and practical to operate. Summary of the Invention

[0007] This invention addresses the shortcomings of existing cryopreservation technologies for primary tissues and organoids by providing a cryopreservation solution for primary tissues or organoids, as well as a method for cryopreservation and thawing of tissue organoids.

[0008] This invention provides a cryopreservation solution for primary tissues or organoids, formulated from the following components: dimethyl sulfoxide, dextran-20, primary cell antibiotics, penicillin-streptomysin, bovine serum albumin, insulin, transferrin, growth hormone, fibroblast growth factor, human platelet-like growth factor, bone morphogenetic protein 4, human epidermal growth factor, insulin-like growth factor, hepatocyte growth factor, L-glutamate, alanine, glycine, L-aspartic acid, L-proline, serine, sodium pyruvate, glutamine, nicotinamide, N-acetylcysteine, β-mercaptoethanol, sodium selenite, β-glycerophosphate, cycloastragaloyl alcohol, Y-27632, hydrocortisone, and anti-inflammatory drugs. Ascorbic acid, vitamin B12, vitamin B6, vitamin B2, DMEM basal medium, with dimethyl sulfoxide at a concentration of 5–8 g / L, dextran-20 at a concentration of 5–8 g / L, primary cell antibiotic at a concentration of 0.05–0.15 g / L, penicillin-streptomysin at a concentration of 0.5–1.5 g / L, bovine serum albumin at a concentration of 2–3 mg / L, insulin at a concentration of 5–20 mg / L, transferrin at a concentration of 0.02–0.2 mg / L, growth hormone at a concentration of 1–5 μg / L, fibroblast growth factor at a concentration of 5–30 μg / L, human platelet-derived growth factor at a concentration of 5–20 μg / L, bone morphology... The dosage of morphogenetic protein 4 is 0.1–0.2 μg / L, human epidermal growth factor is 10–20 μg / L, insulin-like growth factor is 50–200 μg / L, hepatocyte growth factor is 10–30 μg / L, L-glutamate is 5–20 mg / L, alanine is 5–30 mg / L, glycine is 2–10 mg / L, L-aspartic acid is 5–20 mg / L, L-proline is 5–20 mg / L, serine is 5–20 mg / L, sodium pyruvate is 30–70 mg / L, glutamine is 100–500 mg / L, and nicotinamide is 0.6–2.4 g. The concentrations of N-acetylcysteine ​​(70–700 mg / L), β-mercaptoethanol (1.5–4.5 mg / L), sodium selenite (1.7–8.5 mg / L), β-glycerophosphate (0.3–3 g / L), cycloastragalool (0.05–5 mg / L), Y-27632 (1.2–2.4 mg / L), hydrocortisone (0.2–1.0 mg / L), ascorbic acid (0.2–0.5 mg / L), vitamin B12 (0.2–1.0 mg / L), vitamin B6 (10–100 μg / L), and vitamin B2 (0.1–0.8 mg / L) are all in DMEM basal medium.

[0009] The preferred dosages are: dimethyl sulfoxide (DMSO) 6 g / L, dextran-20 6 g / L, primary cell antibiotic 0.1 g / L, penicillin-streptomysin 1 g / L, bovine serum albumin 2.5 mg / L, insulin 12 mg / L, transferrin 0.11 mg / L, growth hormone 3 μg / L, and fibroblast growth factor. The dosages were as follows: 17.5 μg / L for [specific drug], 12.5 mg / L for human platelet-like growth factor, 0.15 μg / L for bone morphogenetic protein 4, 15 μg / L for human epidermal growth factor, 125 μg / L for insulin-like growth factor, 20 μg / L for hepatocyte growth factor, 12.5 mg / L for L-glutamate, 17.5 mg / L for alanine, and [specific dosages for glycine]. The dosages are as follows: L-aspartic acid 12.5 mg / L, L-proline 12.5 mg / L, serine 12.5 mg / L, sodium pyruvate 50 mg / L, glutamine 300 mg / L, nicotinamide 1.5 g / L, N-acetylcysteine ​​448 mg / L, β-mercaptoethanol 3.1 mg / L, and sodium selenite 5 mg / L. The concentrations of β-glycerophosphate (1.8 g / L), cycloastragalool (2.4 mg / L), Y-27632 (1.8 mg / L), hydrocortisone (0.6 mg / L), ascorbic acid (0.35 mg / L), vitamin B12 (0.6 mg / L), vitamin B6 (55 μg / L), vitamin B2 (0.45 mg / L), and the remainder is DMEM basal medium.

[0010] The preferred dosages are: dimethyl sulfoxide (DMSO) 8 g / L, dextran-20 8 g / L, primary cell antibiotic 0.15 g / L, penicillin-streptomysin 1.5 g / L, bovine serum albumin 2 mg / L, insulin 5 mg / L, transferrin 0.02 mg / L, growth hormone 5 μg / L, and fibroblast... The dosages of the following medications were used: human cytokinesin-1 (CGF) 5 μg / L, human platelet-derived growth factor (GF) 5 mg / L, bone morphogenetic protein 4 (BGF) 0.1 μg / L, human epidermal growth factor (GF) 20 μg / L, insulin-like growth factor (IGF) 200 μg / L, hepatocyte growth factor (HGF) 10 μg / L, L-glutamate 5 mg / L, alanine 30 mg / L, and glycine 2 mg / L. The dosages are as follows: L-aspartic acid 20 mg / L, L-proline 5 mg / L, serine 20 mg / L, sodium pyruvate 70 mg / L, glutamine 100 mg / L, nicotinamide 0.6 g / L, N-acetylcysteine ​​80 mg / L, β-mercaptoethanol 1.5 mg / L, and sodium selenite 8.5 mg / L. The dosage of β-glycerophosphate was 0.3 g / L, the dosage of cycloastragalool was 5 mg / L, the dosage of Y-27632 was 2.47 mg / L, the dosage of hydrocortisone was 0.2 mg / L, the dosage of ascorbic acid was 0.5 mg / L, the dosage of vitamin B12 was 0.2 mg / L, the dosage of vitamin B6 was 10 μg / L, the dosage of vitamin B2 was 0.8 mg / L, and the balance was DMEM basal medium.

[0011] The preferred dosages are: dimethyl sulfoxide 5 g / L, dextran-20 5 g / L, primary cell antibiotic 0.05 g / L, penicillin-streptomysin 0.5 g / L, bovine serum albumin 3 mg / L, insulin 20 mg / L, transferrin 0.2 mg / L, growth hormone 1 μg / L, and fibroblast... The dosages of the following drugs were as follows: human cytokine growth factor (CGF) 30 μg / L, human platelet-derived growth factor (PGF) 20 mg / L, bone morphogenetic protein 4 (BGF) 0.2 μg / L, human epidermal growth factor (HGF) 10 μg / L, insulin-like growth factor (IGF) 50 μg / L, hepatocyte growth factor (HGF) 30 μg / L, L-glutamate 20 mg / L, alanine 5 mg / L, and glycine 1 mg / L. The dosages are as follows: 0 mg / L for L-aspartic acid, 5 mg / L for L-proline, 20 mg / L for serine, 5 mg / L for sodium pyruvate, 30 mg / L for glutamine, 500 mg / L for nicotinamide, 2.4 g / L for N-acetylcysteine, 0.8 g / L for β-mercaptoethanol, and 1.7 mg / L for sodium selenite. The dosage of β-glycerophosphate was 3.4 g / L, the dosage of cycloastragalool was 50 μg / L, the dosage of Y-27632 was 1.2 mg / L, the dosage of hydrocortisone was 1.0 mg / L, the dosage of ascorbic acid was 0.2 mg / L, the dosage of vitamin B12 was 1.0 mg / L, the dosage of vitamin B6 was 100 μg / L, the dosage of vitamin B2 was 0.1 mg / L, and the balance was DMEM basal medium.

[0012] This invention also provides a method for cryopreservation and thawing of primary tissues, the method comprising the following steps:

[0013] Primary tissue collection: Collect fresh tissue removed by surgery, cut it into 5mm×5mm×5mm pieces, avoiding necrotic and ulcerated areas, and place the tissue into a sample tube containing primary tissue preservation solution as soon as possible after removal from the body, and store it at 4℃ or transport it via cold chain.

[0014] Primary tissue cryopreservation: The sample is transported to the ultra-clean cell room and operated in a biosafety cabinet. The tissue is transferred to a culture dish, and any one of the primary tissue or organoid cryopreservation solutions of claims 1 to 4 is added to ensure that the tissue surface is moist. The tissue is divided using a clean scalpel blade and placed into cryovials containing cryopreservation solution. The tissue is gently shaken to equilibrate and placed directly into the freezer. After overnight storage, the cryovials are placed in liquid nitrogen for long-term preservation.

[0015] Primary tissue thawing: Remove the cryovial containing the tissue to be thawed from liquid nitrogen and quickly place it in a constant temperature water bath to gently thaw until only a single ice crystal remains after the cryopreservation solution has melted. Remove the cryovial and extract the tissue in a clean bench. Place the tissue in a sterile culture dish and rinse with PBS containing antibiotics. Then transfer the tissue block to a centrifuge tube containing tissue basal culture medium. After thawing, the primary tissue is processed using the same methods as fresh tissue.

[0016] In the primary tissue cryopreservation step, the tissue is divided into tissue fragments with a diameter of 1±0.1mm using a clean surgical blade. The fragments are then placed in cryovials containing 1ml of cryopreservation solution in groups of 3-5 tissue fragments. The tissues are gently shaken at 4℃ for 20 minutes to equilibrate, and then placed directly into a -80℃ freezer.

[0017] In the primary tissue resuscitation procedure: the cryovial containing the tissue to be resuscitated is removed from liquid nitrogen and quickly placed in a 37°C constant temperature water bath to gently thaw until only a single ice crystal remains after the cryopreservation solution has melted. The cryovial is then removed, and the tissue is taken out in a laminar flow hood and placed in a 10cm diameter sterile culture dish. The tissue is rinsed three times with PBS containing double antibodies. The tissue block is then transferred to a centrifuge tube containing Advanced DMEM / F12 or DMEM / F12. The resuscitated primary tissue is then processed using the same methods as fresh tissue.

[0018] The present invention also provides a method for cryopreservation, thawing, and culture of organoids, wherein the cryopreservation, thawing, and culture include the following steps:

[0019] Organoid collection: Remove the culture medium from the well plate containing organoids, add cold PBS, place on ice to dissolve the gel, use a pre-chilled pipette tip to collect all the organoids into a centrifuge tube, centrifuge, remove the supernatant, add cold PBS again, tighten the centrifuge tube cap, gently invert to mix, centrifuge, and collect the organoids.

[0020] Organoid cryopreservation: Add primary tissue / organoid cryopreservation solution to the centrifuge tubes containing collected organoids, gently resuspend the organoid precipitate with a low-absorption pipette tip, then aliquot the suspension into cell cryopreservation tubes, shake to equilibrate, place in a freezer, and after overnight storage, place the cryopreservation tubes in liquid nitrogen for long-term preservation.

[0021] Organoid resuscitation: Remove the cryopreservation tubes from liquid nitrogen and quickly place them in a water bath to thaw gently until only one ice crystal remains. Collect all the solution into centrifuge tubes, add organoid culture medium to rinse the cryopreservation tubes, collect the liquid into the centrifuge tubes, centrifuge, and remove the supernatant.

[0022] Organoid culture: Add organoid culture medium to centrifuge tubes, slowly resuspend the organoids, centrifuge, discard part of the supernatant, add matrix gel on ice, and slowly mix with a pre-cooled pipette tip, being careful not to generate air bubbles. Then, drop the cell-containing matrix gel onto a preheated low-adsorption cell culture plate. Place the culture plate in a CO2 incubator and heat until the gel solidifies. Remove the culture plate, add preheated organoid culture medium to each well, and place the culture plate in a CO2 incubator for incubation. Change the culture medium every 2-3 days. Observe and photograph the organoids daily to understand the initial cell number, cell proliferation rate, organoid morphology, and microbial contamination.

[0023] Preferred cryopreservation, thawing, and culture include the following steps:

[0024] Before starting the experiment, pre-cool the PBS on ice and freeze the pipette tip that will be in contact with the matrix gel at -20°C for 1 hour. Organoids can be cryopreserved when their diameter reaches 100-200 μm, at which point the cryopreservation effect is optimal.

[0025] Organoid collection: Remove the culture medium from the well plate containing the organoids, add cold PBS, and place on ice for at least half an hour to allow the gel to thaw. Use a pre-chilled pipette tip to collect all the organoids into a 15 mL centrifuge tube, then centrifuge at 4°C and 1300 rpm for 5 min. Discard the supernatant (Note: The melting range of the matrix gel is 0°C-8°C. To ensure smooth gel thawing, it must be thawed on ice. During this process, you can shake it on a horizontal shaker. Be sure to centrifuge at 4°C to prevent the matrix gel from solidifying again and affecting the cryopreservation effect). Add cold PBS again, tighten the centrifuge tube cap, gently invert to mix, and centrifuge at 4°C and 1300 rpm for 5 min to collect the organoids.

[0026] Organoid cryopreservation: Add an appropriate volume of tissue / organoid cryopreservation solution to a 15ml centrifuge tube containing collected organoids, gently resuspend the organoid precipitate with a low-absorption pipette tip, then aliquot the suspension into cell cryopreservation tubes, gently shake to equilibrate at 4℃ for 10min, and place directly into a -80℃ freezer. After overnight storage, place the cryopreservation tubes in liquid nitrogen for long-term preservation.

[0027] Organoid resuscitation: Before the experiment, PBS was pre-cooled on ice. The pipette tip in contact with the matrix gel was frozen at -20°C for 1 hour. The organoid cryopreservation tubes were taken out from liquid nitrogen or -80°C and quickly placed in a 37°C water bath to thaw gently until the cryopreservation solution thawed down to the point where only one ice crystal remained. All the solution was collected into a 15 mL centrifuge tube. 2 mL of routine organoid culture medium was added to rinse the cryopreservation tube once. The liquid was collected into the centrifuge tube and centrifuged at 4°C, 1300 rpm for 5 min. The supernatant was then removed.

[0028] Organoid culture: Add 2 ml of organoid culture medium to a centrifuge tube, slowly resuspend the organoids, centrifuge at 1300 rpm for 5 min at 4°C, discard part of the supernatant, add 50 μl-200 μl of matrix gel on ice, and slowly mix with a pre-chilled 200 μl pipette tip, being careful not to generate air bubbles. Then, drop approximately 70 μl of the cell-containing matrix gel into a preheated 24-well low-adsorption cell culture plate. Place the culture plate in a 37°C, 5% CO2 incubator and heat for 30 min. After the gel solidifies, remove the culture plate and add 700 μl of preheated organoid culture medium to each well. Place the culture plate in a 37°C, 5% CO2 incubator for incubation. Change the culture medium every 2-3 days. When adding the culture medium, point the pipette tip towards the side wall and add slowly to avoid damaging the matrix gel. Observe and photograph the organoids daily to understand the initial cell number, cell proliferation rate, organoid morphology, and microbial contamination.

[0029] Cell counting was performed using a cell counter, and cell viability was determined using trypan blue staining.

[0030] The cryopreservation solution of this invention incorporates the small molecule cycloastragenol (CAG). CAG is a triterpenoid saponin compound obtained by hydrolysis of astragaloside A, with a molecular weight of 490.71. CAG is a telomerase activator with anti-apoptotic, anti-fibrotic, and anti-oxidative stress functions. In the cryopreservation solution, it can effectively protect tissue cells from death caused by rapid temperature changes, reduce oxidative stress and apoptosis during cell resuscitation, and improve the resuscitation efficiency of tissues and organoids.

[0031]

[0032] Cycloastragenol (CAG)

[0033] The cryopreservation solution of this invention incorporates the small molecule Y-27632. Y-27632 is an ATP-competitive small molecule inhibitor of ROCK-I and ROCK-II. By targeting and binding to the catalytic sites of ROCK-1 / 2, Y-27632 inhibits their kinase activity. Therefore, Y-27632 can act on Rho-mediated stress fiber formation, inhibiting G1-S phase cell cycle progression and cytokinesis. Y-27632 can attenuate doxorubicin-induced apoptosis in human cardiac stem cells and inhibit isolation-induced apoptosis in mouse prostate stem / progenitor cells. Y-27632 is not only indispensable in stem cell culture but also in organoid construction, where it can prevent stem cell apoptosis, improve clonogenic efficiency, and increase the viability of revived cells in tissues and organoids.

[0034]

[0035] This invention incorporates two freezing point protectants into the cryopreservation solution. DMSO is a small molecule compound with strong cell permeability, allowing it to enter cells, alter intracellular osmotic pressure, lower the freezing point of the intracellular solution, and prevent ice crystals from damaging cells. Dextran-20 is a low molecular weight (approximately 20,000–40,000) dextran, an extracellular protectant. Clinically, it is commonly used as a plasma substitute, reducing intercellular adhesion, lowering the freezing point of extracellular solutions, and reducing damage to cells from both extracellular and intracellular ice crystals. DMSO and dextran-20 can respectively lower the freezing point of intracellular and extracellular tissue fluids, playing a crucial role in the preservation of tissue cells and multicellular aggregates, preventing ice crystals in tissue fluid from damaging cells, and are essential for the preservation of tissues and organoids.

[0036] The tissue cryopreservation solution of the present invention can be used for the cryopreservation of solid tumor tissues of humans and animals, organoids derived from lung, liver, digestive tract, testis, pancreas, and nerve tissue, as well as organoids such as lung, liver, digestive tract, and pancreas formed by iPSC cell directed differentiation, and cryopreservation of various serum-free cultured cells.

[0037] This invention provides a complete set of methods for the cryopreservation, thawing, and digestion of tissues and organoids. After cryopreservation, the thawed and digested cells can be used for routine organoid culture, truly solving the problem of low success rates in organoid culture from long-distance tissue transport and long-term cryopreservation.

[0038] The small tissue fragments frozen in this invention can be revived and then used for primary cell isolation and culture. The survival rate of the primary cells after revival can reach 93%, and the survival rate of the organoids after revival can reach 92%.

[0039] The serum-free tissue and organoid cryopreservation solutions of the present invention do not contain animal-derived serum, thus avoiding the risks of mycoplasma, fungal contamination, and allergens; the chemical composition is clearly defined, the cryopreservation effect is stable, and the safety of organoid clinical applications is guaranteed.

[0040] The small molecule cycloastragaloyl alcohol added in this invention can enhance the antioxidant and anti-stress capabilities of cells, prolonging telomeres, reducing cell senescence, and improving cell membrane stability. This study tested the stability of cycloastragaloyl alcohol on tissues under rapid temperature changes, showing that it can significantly reduce cellular oxidative stress damage caused by rapid temperature changes. Furthermore, the reduced concentration of DMSO used minimizes its cytotoxic effects and improves tissue recovery efficiency.

[0041] The small molecule Y-27632 added in this invention can inhibit the kinase activity of ROCK-1 / 2 by targeting and binding to its catalytic site, thereby inhibiting apoptosis of tissue and organoid cells.

[0042] This invention reveals that cycloastragaloyl alcohol (CAG) and Y-27632 at a concentration ratio of 1:0.5 to 1:2 can exert a synergistic protective effect on tissues and organoids. When CAG:Y-27632 = 1:0.75 (2.4 mg / L: 1.8 mg / L), the survival rate of liver tissue after resuscitation can reach 93%, which is much higher than that of the control group.

[0043] This invention is designed for immediate use and does not require gradient cooling. Tissues and organoids can be placed at -80°C overnight and then transferred to liquid nitrogen for long-term preservation. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0045] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following examples that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the reagents and raw materials used in this invention are commercially available.

[0046] Example 1

[0047] Prepare cryopreservation solutions according to the following components and concentrations, for later use:

[0048] A cryopreservation solution for primary tissues or organoids is prepared from the following components: dimethyl sulfoxide, dextran-20, primary cell antibiotics, penicillin-streptomysin, bovine serum albumin, insulin, transferrin, growth hormone, fibroblast growth factor, human platelet-derived growth factor, bone morphogenetic protein 4, human epidermal growth factor, insulin-like growth factor, hepatocyte growth factor, L-glutamate, alanine, glycine, L-aspartic acid, L-proline, serine, sodium pyruvate, glutamine, nicotinamide, N-acetylcysteine, β-mercaptoethanol, sodium selenite, β-glycerophosphate, cycloastragaloyl alcohol, Y-27632, hydrocortisone, ascorbic acid, vitamin B12, vitamin B6, vitamin B2, and DMEM basal medium, wherein dimethyl sulfoxide is 6 g / L and dextran-20 is... 6 g / L, primary cell antibiotic 0.1 g / L, Penicillin-streptomysin 1 g / L, bovine serum albumin 2.5 mg / L, insulin 12 mg / L, transferrin 0.11 mg / L, growth hormone 3 μg / L, fibroblast growth factor 17.5 μg / L, human platelet-12.5 mg / L, bone morphogenetic protein-40.15 μg / L, human epidermal growth factor 15 μg / L, insulin-like growth factor 125 μg / L, hepatocyte growth factor 20 μg / L, L-glutamate 12.5 mg / L, alanine 17.5 mg / L, glycine 6 mg / L L-Aspartic acid 12.5 mg / L, L-proline 12.5 mg / L, serine 12.5 mg / L, sodium pyruvate 50 mg / L, glutamine 300 mg / L, nicotinamide 1.5 g / L, N-acetylcysteine ​​448 mg / L, β-mercaptoethanol 3.1 mg / L, sodium selenite 5 mg / L, β-glycerophosphate 1.8 g / L, cycloastragalol 2.4 mg / L, Y-27632 1.8 mg / L, hydrocortisone 0.6 mg / L, ascorbic acid 0.35 mg / L, vitamin B12 0.6 mg / L, vitamin B6 55 μg / L, vitamin B2 0.45 mg / L, with the remainder being DMEM basal medium.

[0049] Example 2

[0050] Prepare cryopreservation solutions according to the following components and concentrations, for later use:

[0051] A cryopreservation solution for primary tissues or organoids is prepared from the following components: dimethyl sulfoxide, dextran-20, primary cell antibiotics, penicillin-streptomysin, bovine serum albumin, insulin, transferrin, growth hormone, fibroblast growth factor, human platelet-derived growth factor, bone morphogenetic protein 4, human epidermal growth factor, insulin-like growth factor, hepatocyte growth factor, L-glutamate, alanine, glycine, L-aspartic acid, L-proline, serine, sodium pyruvate, glutamine, nicotinamide, N-acetylcysteine, β-mercaptoethanol, sodium selenite, β-glycerophosphate, cycloastragaloyl alcohol, Y-27632, hydrocortisone, ascorbic acid, vitamin B12, vitamin B6, vitamin B2, and DMEM basal medium, wherein dimethyl sulfoxide is 8 g / L and dextran-20 is... 8 g / L, primary cell antibiotic 0.15 g / L, Penicillin-streptomysin 1.5 g / L, bovine serum albumin 2 mg / L, insulin 5 mg / L, transferrin 0.02 mg / L, growth hormone 5 μg / L, fibroblast growth factor 5 μg / L, human platelet-1 growth factor 5 mg / L, bone morphogenetic protein-4 0.1 μg / L, Human Epidermal Growth Factor 20 μg / L, Insulin-like Growth Factor 200 μg / L, Hepatocyte Growth Factor 10 μg / L, L-Glutamate 5 mg / L, Alanine 30 mg / L, Glycine 2 mg / L, L-Aspartic Acid 20 mg / L, L-Proline 5 mg / L, Serine 20 mg / L, Sodium Pyruvate 70 mg / L, Glutamine 100 mg / L, Nicotinamide 0.6 g / L, N-Acetylcysteine ​​80 mg / L, β-Mercaptoethanol 1.5 mg / L, Sodium Selenite 8.5 mg / L, β-Cycloastragaloside 0.3 g / L, Cycloastragalool 5 mg / L, Y-27632 2.47 mg / L, Hydrocortisone 0.2 mg / L, Ascorbic Acid 0.5 mg / L, Vitamin B12 0.2 mg / L, Vitamin B6 10 μg / L, vitamin B2 0.8 mg / L, the remainder was DMEM basal medium.

[0052] Example 3

[0053] Prepare cryopreservation solutions according to the following components and concentrations, for later use:

[0054] A cryopreservation solution for primary tissues or organoids is prepared from the following components: dimethyl sulfoxide, dextran-20, primary cell antibiotics, penicillin-streptomysin, bovine serum albumin, insulin, transferrin, growth hormone, fibroblast growth factor, human platelet-derived growth factor, bone morphogenetic protein 4, human epidermal growth factor, insulin-like growth factor, hepatocyte growth factor, L-glutamate, alanine, glycine, L-aspartic acid, and L-proline. Serine, sodium pyruvate, glutamine, nicotinamide, N-acetylcysteine, β-mercaptoethanol, sodium selenite, β-glycerophosphate, cycloastragalool, Y-27632, hydrocortisone, ascorbic acid, vitamin B12, vitamin B6, vitamin B2, DMEM basal medium containing 5 g / L dimethyl sulfoxide, 5 g / L dextran-20, 0.05 g / L primary cell antibiotic, and penicillin-streptomysin. 0.5 g / L, bovine serum albumin 3 mg / L, insulin 20 mg / L, transferrin 0.2 mg / L, growth hormone 1 μg / L, fibroblast growth factor 30 μg / L, human platelet-derived growth factor 20 mg / L, bone morphogenetic protein 4 0.2 μg / L, human epidermal growth factor 10 μg / L, insulin-like growth factor 50 μg / L, hepatocyte growth factor 30 μg / L, L-glutamate 20 mg / L, alanine 5 mg / L, glycine 10 mg / L, L-aspartic acid 5 mg / L, L-proline 20 mg / L, serine The following concentrations of acetic acid were used: 5 mg / L, sodium pyruvate: 30 mg / L, glutamine: 500 mg / L, nicotinamide: 2.4 g / L, N-acetylcysteine: 0.8 g / L, β-mercaptoethanol: 4.7 mg / L, sodium selenite: 1.7 mg / L, β-glycerophosphate: 3.4 g / L, cycloastragalol: 50 μg / L, Y-27632: 1.2 mg / L, hydrocortisone: 1.0 mg / L, ascorbic acid: 0.2 mg / L, vitamin B12: 1.0 mg / L, vitamin B6: 100 μg / L, vitamin B2: 0.1 mg / L, and the remainder was DMEM basal medium.

[0055] Example 4

[0056] A method for cryopreservation and thawing of primary tissues includes the following steps:

[0057] Primary tissue collection: Fresh mouse liver tissue was surgically removed and cut into 5mm×5mm×5mm pieces. After being removed from the body, the tissue was placed in a sample tube containing primary tissue preservation solution as soon as possible and stored at 4℃ or transported via cold chain.

[0058] Primary tissue cryopreservation: Samples were transported to a clean cell room and operated in a biosafety cabinet. Tissues were transferred to culture dishes, and the cryopreservation solution prepared in Example 1 was added to ensure the tissue surface was moist. Using a clean scalpel, the tissues were divided into tissue fragments with a diameter of 1 ± 0.1 mm. Three to five tissue fragments were grouped into cryovials containing 1 ml of tissue cryopreservation solution. The cryovials were gently shaken at 4°C for 20 minutes to equilibrate, then placed directly into a -80°C freezer overnight. After freezing, the cryovials were placed in liquid nitrogen for long-term storage.

[0059] Primary tissue thawing: Remove the cryovial containing the tissue to be thawed from liquid nitrogen and quickly place it in a 37°C water bath to gently thaw until only a single ice crystal remains after the cryopreservation solution has melted. Remove the cryovial from the freezer. In a laminar flow hood, remove the tissue and place it in a 10cm diameter sterile culture dish. Rinse three times with PBS containing antibiotics and antibiotics. Then transfer the tissue block to a centrifuge tube containing tissue basal culture medium (Advanced DMEM / F12 or DMEM / F12). The thawed primary tissue can be processed using the same methods as fresh tissue.

[0060] Example 5

[0061] A method for cryopreservation, thawing, and culture of organoids, comprising the following steps:

[0062] Organoid collection: For the C57 mouse small intestinal organoids being cultured, remove the culture medium from the well plate containing the organoids, add cold PBS, place on ice for at least half an hour to gel dissolve, and use a pre-chilled pipette tip to collect all the organoids into a 15 ml centrifuge tube. Then centrifuge at 4°C, 1300 rpm for 5 min, remove the supernatant, add cold PBS again, tighten the centrifuge tube cap, gently invert to mix, centrifuge at 4°C, 1300 rpm for 5 min, and collect the organoids.

[0063] Organoid cryopreservation: Add an appropriate volume of the tissue / organoid cryopreservation solution prepared in Example 1 to a 15ml centrifuge tube containing collected organoids. Gently resuspend the organoid precipitate with a low-absorption pipette tip. Then, aliquot the suspension into cell cryopreservation tubes, gently shake to equilibrate at 4°C for 10 minutes, and place directly into a -80°C freezer. After overnight storage, place the cryopreservation tubes in liquid nitrogen for long-term preservation.

[0064] Organoid resuscitation: Before the experiment, PBS was pre-cooled on ice. The pipette tip in contact with the matrix gel was frozen at -20°C for 1 hour. The organoid cryopreservation tubes were taken out from liquid nitrogen or -80°C and quickly placed in a 37°C water bath to thaw gently until the cryopreservation solution thawed down to the point where only one ice crystal remained. All the solution was collected into a 15 mL centrifuge tube. 2 mL of routine organoid culture medium was added to rinse the cryopreservation tube once. The liquid was collected into the centrifuge tube and centrifuged at 4°C, 1300 rpm for 5 min. The supernatant was then removed.

[0065] Organoid culture: Add 2 ml of organoid culture medium to a centrifuge tube, slowly resuspend the organoids, centrifuge at 1300 rpm for 5 min at 4°C, discard part of the supernatant, add 50 μl-200 μl of matrix gel on ice, and slowly mix with a pre-chilled 200 μl pipette tip, being careful not to generate air bubbles. Then, drop approximately 70 μl of the cell-containing matrix gel into a preheated 24-well low-adsorption cell culture plate. Place the culture plate in a 37°C, 5% CO2 incubator and heat for 30 min. After the gel solidifies, remove the culture plate and add 700 μl of preheated organoid culture medium to each well. Place the culture plate in a 37°C, 5% CO2 incubator for incubation. Change the culture medium every 2-3 days. When adding the culture medium, point the pipette tip towards the side wall and add it slowly to avoid damaging the matrix gel. Observe and photograph the organoids daily to understand the initial cell number, cell proliferation rate, organoid morphology, and microbial contamination.

[0066] Comparative Example 1

[0067] Referring to Example 1, a cryopreservation solution was prepared without adding cycloastragalool, while keeping other components and contents unchanged.

[0068] Comparative Example 2

[0069] Prepare a standard DMSO-fetal bovine serum cryopreservation solution for later use. The specific formula is as follows: 100 g / L dimethyl sulfoxide, 100 g / L fetal bovine serum, and DMEM / F12 basal culture medium to a final volume of 1000 mL.

[0070] Comparative Example 3

[0071] Referring to the primary tissue cryopreservation and thawing method in Example 4, the freshly collected surgically removed mouse liver tissue was cryopreserved and thawed using the cryopreservation solution prepared in Comparative Example 1.

[0072] Comparative Example 4

[0073] Referring to the organoid cryopreservation, thawing, and culture method in Example 5, the cryopreservation solution prepared in Comparative Example 1 was used to cryopreserve, thaw, and culture C57 mouse small intestinal organoids.

[0074] Comparative Example 5

[0075] Referring to the primary tissue cryopreservation and thawing method in Example 4, the cryopreservation solution prepared in Comparative Example 2 was used to cryopreserve and thaw the collected fresh mouse liver tissue that had been surgically removed.

[0076] Comparative Example 6

[0077] Referring to the organoid cryopreservation, thawing, and culture method in Example 5, the cryopreservation solution prepared in Comparative Example 2 was used to cryopreserve, thaw, and culture C57 mouse small intestinal organoids.

[0078] Comparative Example 7

[0079] Referring to Example 1, without adding Y-27632, and keeping other components and contents unchanged, a cryopreservation solution was prepared.

[0080] Comparative Example 8

[0081] Referring to Example 1, without adding CAG and Y-27632, and keeping other components and contents unchanged, a cryopreservation solution was prepared.

[0082] Comparative Example 9

[0083] Referring to Example 1, the concentrations of cycloastragalool (2.4 mg / L) and Y-27632 (1.2 mg / L) were adjusted, while other components and their contents remained unchanged, to prepare a cryopreservation solution.

[0084] Comparative Example 10

[0085] Referring to Example 1, the concentrations of cycloastragalool were adjusted to 1.2 mg / L and Y-27632 to 2.4 mg / L, while other components and their contents remained unchanged, to prepare a cryopreservation solution.

[0086] Comparative Example 11

[0087] Referring to Example 1, the concentrations of cycloastragalool (2.4 mg / L) and Y-27632 (0.8 mg / L) were adjusted, while other components and their contents remained unchanged, to prepare a cryopreservation solution.

[0088] Comparative Example 12

[0089] Referring to Example 1, the concentrations of cycloastragalool were adjusted to 1.2 mg / L and Y-27632 to 2.5 mg / L, while other components and their contents remained unchanged, to prepare a cryopreservation solution.

[0090] Cell viability assay after thawing cryopreserved primary tissues

[0091] Mouse liver tissues from Examples 4 and 3 & 5 of this invention, which were cryopreserved for one month and then revived, were digested into single cells using conventional methods. Cell counting was performed using a cell counter, and cell viability was determined using trypan blue staining. The results are shown in Table 1, indicating that the cell viability using the cryopreservation solution of this invention was significantly higher than that of the control examples.

[0092] Table 1

[0093] Example 4 Comparative Example 3 Comparative Example 5 Cell viability 93％ 80％ 68％

[0094] Organoid resuscitation survival rate determination

[0095] The organoid survival rate of organoids (C57 mouse small intestinal organoids) cryopreserved for one month, thawed, and cultured in Example 5 and Control Examples 4 and 6 of this invention was determined. The method was to cryopreserve the organoids when they grew to 100-200 μm, count the organoids under a microscope during cryopreservation (without staining, using a manual counter), and freeze 300 organoids per tube. After thawing, they were cultured routinely for one day and then manually counted under a microscope. The organoid survival rate was calculated as (number of surviving organoids after thawing / 300) × 100%. The results are shown in Table 2, indicating that the organoid survival rate using the cryopreservation solution of this invention was significantly higher than that of the control examples.

[0096] Table 2

[0097] Example 5 Comparative Example 4 Comparative Example 6 organoid survival rate 92％ 79％ 70％

[0098] Synergistic effect verification experiment

[0099] Mouse liver tissue was cryopreserved using the method in Example 4. The results are shown in Table 3, indicating that the survival rate was improved when CAG and Y-27632 coexisted, proving that the synergistic gain far exceeded the contribution of a single component.

[0100] Table 3

[0101] Group Example 4 Comparative Example 3 Comparative Example 7 Comparative Example 8 Cell viability 93％ 80％ 52％ 32％

[0102] Following the methods of Examples 4 and 5, the cryopreservation solutions prepared in Comparative Examples 9, 10, 11, and 12 were used to determine cell viability in surgically removed fresh mouse liver tissue and organoid viability in C57 mouse small intestinal organoids. The results are shown in Table 4. The results indicate that cycloastragaloyl alcohol (CAG) and Y-27632 at a concentration ratio of 1:0.5 to 1:2 can exert a significant synergistic protective effect on tissues and organoids. When CAG:Y-27632 = 1:0.75 (2.4 mg / L: 1.8 mg / L), the synergistic effect of CAG and Y-27632 is the most significant, with a survival rate of 87% for mouse liver tissue after resuscitation and a survival rate of 92% for mouse small intestinal organoids.

[0103] Table 4

[0104]

[0105] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A cryopreservation solution for primary tissues or organoids, characterized in that, Formulated from the following components: dimethyl sulfoxide, dextran-20, primary cell antibiotics, penicillin-streptomysin, bovine serum albumin, insulin, transferrin, growth hormone, fibroblast growth factor, human platelet-derived growth factor, bone morphogenetic protein 4, human epidermal growth factor, insulin-like growth factor, hepatocyte growth factor, L-glutamate, alanine, glycine, L-aspartic acid, L-proline, serine, sodium pyruvate, glutamine, nicotinamide, N-acetylcysteine, β-mercaptoethanol, sodium selenite, β-glycerophosphate, cycloastragaloyl alcohol, Y-27632, hydrocortisone, ascorbic acid, vitamin B12, vitamin B6, and vitamin C. Vitamin B2, DMEM basal medium, with the following concentrations: dimethyl sulfoxide (DMSO) at 5–8 g / L, dextran-20 at 5–8 g / L, primary cell antibiotic at 0.05–0.15 g / L, penicillin-streptomysin at 0.5–1.5 g / L, bovine serum albumin at 2–3 mg / L, insulin at 5–20 mg / L, transferrin at 0.02–0.2 mg / L, growth hormone at 1–5 ng / mL, fibroblast growth factor at 5–30 ng / mL, human platelet-20 μg / mL, and bone morphogenetic protein 4 at 0. The dosages are as follows: 1–0.2 ng / mL for human epidermal growth factor (HGF), 10–20 ng / mL for insulin-like growth factor (IGF), 50–200 ng / mL for hepatocyte growth factor (HGF), 10–30 ng / mL for L-glutamate, 5–20 mg / L for alanine, 5–30 mg / L for glycine, 2–10 mg / L for L-aspartic acid, 5–20 mg / L for L-proline, 5–20 mg / L for serine, 30–70 mg / L for sodium pyruvate, 100–500 mg / L for glutamine, and 0.6–2.4 g / L for nicotinamide. The dosage of acetylcysteine ​​is 70–700 mg / L, β-mercaptoethanol is 1.5–4.5 mg / L, sodium selenite is 1.7–8.5 mg / L, β-glycerophosphate is 0.3–3 g / L, cycloastragalool is 0.05–5 mg / L, Y-27632 is 1.2–2.4 mg / L, hydrocortisone is 0.2–1.0 mg / L, ascorbic acid is 0.2–0.5 mg / L, vitamin B12 is 0.2–1.0 mg / L, vitamin B6 is 0.01–0.1 mg / L, vitamin B2 is 0.1–0.8 mg / L, and the remainder is DMEM basal medium.

2. The cryopreservation solution for primary tissues or organoids according to claim 1, characterized in that, The dosages of dimethyl sulfoxide (DMSO) are 6 g / L, dextran-20 is 6 g / L, the primary cell antibiotic is 0.1 g / L, penicillin-streptomysin is 1 g / L, bovine serum albumin is 2.5 mg / L, insulin is 12 mg / L, transferrin is 0.11 mg / L, growth hormone is 3 μg / L, and fibroblast growth factor is... The dosages were as follows: 17.5 μg / L for [specific drug], 12.5 mg / L for human platelet-like growth factor, 0.15 μg / L for bone morphogenetic protein 4, 15 μg / L for human epidermal growth factor, 125 μg / L for insulin-like growth factor, 20 μg / L for hepatocyte growth factor, 12.5 mg / L for L-glutamate, 17.5 mg / L for alanine, and [specific dosages for glycine]. The dosages are as follows: L-aspartic acid 12.5 mg / L, L-proline 12.5 mg / L, serine 12.5 mg / L, sodium pyruvate 50 mg / L, glutamine 300 mg / L, nicotinamide 1.5 g / L, N-acetylcysteine ​​448 mg / L, β-mercaptoethanol 3.1 mg / L, and sodium selenite 5 mg / L. The concentrations of β-glycerophosphate (1.8 g / L), cycloastragalool (2.4 mg / L), Y-27632 (1.8 mg / L), hydrocortisone (0.6 mg / L), ascorbic acid (0.35 mg / L), vitamin B12 (0.6 mg / L), vitamin B6 (55 μg / L), vitamin B2 (0.45 mg / L), and the remainder is DMEM basal medium.

3. The cryopreservation solution for primary tissues or organoids according to claim 1, characterized in that, The dosages of dimethyl sulfoxide (DMSO) are 8 g / L, dextran-20 is 8 g / L, primary cell antibiotic is 0.15 g / L, penicillin-streptomysin is 1.5 g / L, bovine serum albumin is 2 mg / L, insulin is 5 mg / L, transferrin is 0.02 mg / L, growth hormone is 5 μg / L, and fibroblast... The dosages of the following drugs were used: human cytokinase growth factor (CGF) 5 μg / L, human platelet-like growth factor (PGF) 5 mg / L, bone morphogenetic protein 4 (BMP-4) 0.1 μg / L, human epidermal growth factor (HGF) 20 μg / L, insulin-like growth factor (IGF) 200 μg / L, hepatocyte growth factor (HGF) 10 μg / L, L-glutamate 5 mg / L, alanine 30 mg / L, and glycine 2 mg / L. The dosages are as follows: L-aspartic acid 20 mg / L, L-proline 5 mg / L, serine 20 mg / L, sodium pyruvate 70 mg / L, glutamine 100 mg / L, nicotinamide 0.6 g / L, N-acetylcysteine ​​80 mg / L, β-mercaptoethanol 1.5 mg / L, and sodium selenite 8.5 mg / L. The dosage of β-glycerophosphate was 0.3 g / L, the dosage of cycloastragalool was 5 mg / L, the dosage of Y-27632 was 2.47 mg / L, the dosage of hydrocortisone was 0.2 mg / L, the dosage of ascorbic acid was 0.5 mg / L, the dosage of vitamin B12 was 0.2 mg / L, the dosage of vitamin B6 was 10 μg / L, the dosage of vitamin B2 was 0.8 mg / L, and the balance was DMEM basal medium.

4. The cryopreservation solution for primary tissues or organoids according to claim 1, characterized in that, The dosages of dimethyl sulfoxide (DMSO) are 5 g / L, dextran-20 is 5 g / L, primary cell antibiotic is 0.05 g / L, penicillin-streptomysin is 0.5 g / L, bovine serum albumin is 3 mg / L, insulin is 20 mg / L, transferrin is 0.2 mg / L, growth hormone is 1 μg / L, and fibroblasts... The dosage of growth factors was 30 μg / L, human platelet-derived growth factor was 20 mg / L, bone morphogenetic protein 4 was 0.2 μg / L, human epidermal growth factor was 10 μg / L, insulin-like growth factor was 50 μg / L, hepatocyte growth factor was 30 μg / L, L-glutamate was 20 mg / L, alanine was 5 mg / L, and glycine was 10 mg / L. The dosages are as follows: L-aspartic acid 5 mg / L, L-proline 20 mg / L, serine 5 mg / L, sodium pyruvate 30 mg / L, glutamine 500 mg / L, nicotinamide 2.4 g / L, N-acetylcysteine ​​0.8 g / L, β-mercaptoethanol 4.7 mg / L, and sodium selenite 1.7 mg / L. The dosage of β-glycerophosphate was 3.4 g / L, the dosage of cycloastragalool was 50 μg / L, the dosage of Y-27632 was 1.2 mg / L, the dosage of hydrocortisone was 1.0 mg / L, the dosage of ascorbic acid was 0.2 mg / L, the dosage of vitamin B12 was 1.0 mg / L, the dosage of vitamin B6 was 100 μg / L, the dosage of vitamin B2 was 0.1 mg / L, and the balance was DMEM basal medium.

5. A method for cryopreservation and thawing of primary tissues, characterized in that, The cryopreservation and thawing method includes the following steps: Primary tissue collection: Collect fresh tissue removed by surgery, cut it into 5mm×5mm×5mm pieces, avoiding necrotic and ulcerated areas, and place the tissue into a sample tube containing primary tissue preservation solution as soon as possible after removal from the body, and store it at 4℃ or transport it via cold chain. Primary tissue cryopreservation: The sample is transported to the ultra-clean cell room and operated in a biosafety cabinet. The tissue is transferred to a culture dish, and any one of the primary tissue or organoid cryopreservation solutions of claims 1 to 4 is added to ensure that the tissue surface is moist. The tissue is divided using a clean scalpel blade and placed into cryovials containing cryopreservation solution. The tissue is gently shaken to equilibrate and placed directly into the freezer. After overnight storage, the cryovials are placed in liquid nitrogen for long-term preservation. Primary tissue thawing: Remove the cryovial containing the tissue to be thawed from liquid nitrogen and quickly place it in a constant temperature water bath to gently thaw until only a single ice crystal remains after the cryopreservation solution has melted. Remove the cryovial and extract the tissue in a clean bench. Place the tissue in a sterile culture dish and rinse with PBS containing antibiotics. Then transfer the tissue block to a centrifuge tube containing tissue basal culture medium. After thawing, the primary tissue is processed using the same methods as fresh tissue.

6. The method for cryopreservation and thawing of primary tissues according to claim 5, characterized in that, In the primary tissue cryopreservation step, the tissue is divided into tissue fragments with a diameter of 1±0.1mm using a clean surgical blade. The fragments are then placed in cryovials containing 1ml of cryopreservation solution in groups of 3-5 tissue fragments. The tissues are gently shaken at 4℃ for 20 minutes to equilibrate, and then placed directly into a -80℃ freezer. In the primary tissue resuscitation procedure: the cryovial containing the tissue to be resuscitated is removed from liquid nitrogen and quickly placed in a 37°C constant temperature water bath to gently thaw until only a single ice crystal remains after the cryopreservation solution has melted. The cryovial is then removed, and the tissue is taken out in a laminar flow hood and placed in a 10cm diameter sterile culture dish. The tissue is rinsed three times with PBS containing double antibodies. The tissue block is then transferred to a centrifuge tube containing Advanced DMEM / F12 or DMEM / F12. The resuscitated primary tissue is then processed using the same methods as fresh tissue.

7. A method for cryopreservation, thawing, and culture of organoids, characterized in that, The cryopreservation, thawing, and culture include the following steps: Organoid collection: Remove the culture medium from the well plate containing organoids, add cold PBS, place on ice to dissolve the gel, use a pre-chilled pipette tip to collect all the organoids into a centrifuge tube, centrifuge, remove the supernatant, add cold PBS again, tighten the centrifuge tube cap, gently invert to mix, centrifuge, and collect the organoids. Organoid cryopreservation: Add primary tissue / organoid cryopreservation solution to the centrifuge tubes containing collected organoids, gently resuspend the organoid precipitate with a low-absorption pipette tip, then aliquot the suspension into cell cryopreservation tubes, shake to equilibrate, place in a freezer, and after overnight storage, place the cryopreservation tubes in liquid nitrogen for long-term preservation. Organoid resuscitation: Remove the cryopreservation tubes from liquid nitrogen and quickly place them in a water bath to thaw gently until only one ice crystal remains. Collect all the solution into centrifuge tubes, add organoid culture medium to rinse the cryopreservation tubes, collect the liquid into the centrifuge tubes, centrifuge, and remove the supernatant. Organoid culture: Add organoid culture medium to centrifuge tubes, slowly resuspend the organoids, centrifuge, discard part of the supernatant, add matrix gel on ice, and slowly mix with a pre-cooled pipette tip, being careful not to generate air bubbles. Then, drop the cell-containing matrix gel onto a preheated low-adsorption cell culture plate. Place the culture plate in a CO2 incubator and heat until the gel solidifies. Remove the culture plate, add preheated organoid culture medium to each well, and place the culture plate in a CO2 incubator for incubation. Change the culture medium every 2-3 days. Observe and photograph the organoids daily to understand the initial cell number, cell proliferation rate, organoid morphology, and microbial contamination.

8. The method for cryopreservation, thawing, and culture of organoids according to claim 7, characterized in that, The cryopreservation, thawing, and culture include the following steps: Organoid collection: Remove the culture medium from the well plate containing organoids, add cold PBS, place on ice for at least half an hour to gel dissolve, and use a pre-chilled pipette tip to collect all organoids into a 15 ml centrifuge tube. Centrifuge at 4°C, 1300 rpm for 5 min, remove the supernatant, add cold PBS again, tighten the centrifuge tube cap, gently invert to mix, centrifuge at 4°C, 1300 rpm for 5 min, and collect the organoids. Organoid cryopreservation: Add an appropriate volume of tissue / organoid cryopreservation solution to a 15ml centrifuge tube containing collected organoids, gently resuspend the organoid precipitate with a low-absorption pipette tip, then aliquot the suspension into cell cryopreservation tubes, gently shake to equilibrate at 4℃ for 10min, and place directly into a -80℃ freezer. After overnight storage, place the cryopreservation tubes in liquid nitrogen for long-term preservation. Organoid resuscitation: Before the experiment, PBS was pre-cooled on ice. The pipette tip in contact with the matrix gel was frozen at -20°C for 1 hour. The organoid cryopreservation tubes were taken out from liquid nitrogen or -80°C and quickly placed in a 37°C water bath to thaw gently until the cryopreservation solution thawed down to the point where only one ice crystal remained. All the solution was collected into a 15 mL centrifuge tube. 2 mL of routine organoid culture medium was added to rinse the cryopreservation tube once. The liquid was collected into the centrifuge tube and centrifuged at 4°C, 1300 rpm for 5 min. The supernatant was then removed. Organoid culture: Add 2 ml of organoid culture medium to a centrifuge tube, slowly resuspend the organoids, centrifuge at 1300 rpm for 5 min at 4°C, discard part of the supernatant, add 50 μl-200 μl of matrix gel on ice, and slowly mix with a pre-chilled 200 μl pipette tip, being careful not to generate air bubbles. Then, drop approximately 70 μl of the cell-containing matrix gel into a preheated 24-well low-adsorption cell culture plate. Place the culture plate in a 37°C, 5% CO2 incubator and heat for 30 min. After the gel solidifies, remove the culture plate and add 700 μl of preheated organoid culture medium to each well. Place the culture plate in a 37°C, 5% CO2 incubator and incubate. Change the culture medium every 2-3 days. When adding the culture medium, point the pipette tip towards the side wall and add slowly to avoid damaging the matrix gel. Observe and photograph the organoids daily to understand the initial cell number, cell proliferation rate, organoid morphology, and microbial contamination.

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