Tissue protection liquid and application thereof

Abstract

The invention provides a tissue protection solution and application thereof. The tissue protection liquid comprises a basic culture medium; vitamins, wherein the vitamins comprise vitamin E; the antioxidant is prepared from at least one of N-acetylcysteine amide, superoxide dismutase and catalase; and a specific cell factor, wherein the specific cell factor comprises at least one of a ROCK inhibitor Y27632 and insulin. Therefore, when the tissue protection liquid is used for storing and transporting a tissue sample, the loss in the process from tissue sample separation to experiment starting can be reduced, the function and activity of tissue cells are ensured, and the success rate of establishment of various primary tissue cultures is effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a tissue protection solution and application thereof. Background Art

[0002] With the advancement of regenerative medicine and stem cell research, the scientific and clinical value of living tumor tissue is becoming increasingly prominent. The collection and preservation of fresh clinical tumor tissue specimens after ex vivo storage are crucial to specimen quality and directly impact the reliability of subsequent research. During the collection and transportation of tissue samples, selecting the appropriate preservation medium can maintain tissue structure and morphology and cell viability. Improper storage can, conversely, lead to apoptosis of tissue cells.

[0003] Typically, tumor tissue is stored in an appropriate preservation solution at a low temperature of 2-8°C for transportation to reduce enzyme activity within the cells, thereby reducing the rate of cell metabolism, slowing the rate of tissue autolysis, and prolonging the survival time of cells within the tissue after ex vivo. In general, people often use complete culture medium containing serum as an in vitro preservation solution for tumor tissue for short-term preservation of tissue. However, since complete culture medium is used under specific culture conditions and temperatures, it is more suitable for cell growth and proliferation, which is not exactly the same as the purpose of tissue preservation. Especially when it is necessary to preserve and transport samples for a long time in clinical practice, complete cell culture medium cannot meet the requirements, and the serum composition is complex, with differences between different brands and batches. Unknown growth factors in the serum may induce unpredictable differentiation of stem cells, which may have an immeasurable impact on subsequent clinical application research.

[0004] Therefore, clinically, it is necessary to develop a tissue protection solution suitable for long-term low-temperature storage and transportation under closed conditions based on the actual needs of in vitro tissue preservation and transportation. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0006] In its first aspect, the present invention provides a tissue protection solution. According to an embodiment of the present invention, the tissue protection solution comprises: a basal culture medium; vitamins, including vitamin E; an antioxidant, including at least one of N-acetylcysteine amide, superoxide dismutase, and catalase; and specific cytokines, including at least one of the ROCK inhibitor Y27632 and insulin. Thus, when using the tissue protection solution of the present invention to preserve and transport tumor / normal tissue samples, it is possible to reduce losses from tissue sample isolation to the start of the experiment, ensure the function and vitality of tissue cells, and effectively improve the success rate of establishing various types of primary tissue cultures.

[0007] In a second aspect of the present invention, the present invention proposes use of the tissue protection solution described in the first aspect in preserving and / or transporting animal tissue samples.

[0008] In a third aspect, the present invention provides a method for preserving and / or transporting animal tissue samples. According to an embodiment of the present invention, the method comprises placing the animal tissue sample in the tissue protection solution described in the first aspect. As previously mentioned, the tissue protection solution of the present invention can reduce losses from tissue sample isolation to the start of the experiment when preserving and transporting tumor / normal tissue samples, thereby ensuring the function and vitality of tissue cells. Therefore, the method described in the present invention can maintain the original function and vitality of animal tissue cells.

[0009] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. DETAILED DESCRIPTION

[0010] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0011] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0012] In this document, the terms “contain”, “include” or “include” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0013] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0014] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0015] Tissue protection solution

[0016] The present invention provides a tissue protection solution. According to an embodiment of the present invention, the tissue protection solution includes: a basal culture medium; vitamins, including vitamin E; an antioxidant, including at least one of N-acetylcysteine amide, superoxide dismutase, and catalase; and specific cytokines, including at least one of the ROCK inhibitor Y27632 and insulin.

[0017] Among them, vitamins and antioxidants can enhance cell activity, protect proteins and lipids in cell membranes, block harmful free radicals, enhance the self-repair ability of cells, and prevent the oxidation of unsaturated fatty acids in the phospholipids that make up biological membranes; specific cytokines can maintain a stable tissue environment, replenish nutrients needed during tissue transport, and maintain tissue cell activity. Therefore, when using the tissue protection solution of the present invention to store and transport tumor / normal tissue samples, it can reduce losses from tissue sample isolation to the start of the experiment, ensure the function and vitality of tissue cells, and effectively improve the success rate of establishing various primary tissue cultures.

[0018] In some embodiments of the present invention, vitamin E has strong antioxidant properties, which can protect fragile proteins and lipids in cell membranes and prevent the oxidation of unsaturated fatty acids in the phospholipids that make up biological membranes. Therefore, by adding vitamin E to the tissue protection solution, the activity of tissue cells can be improved and the loss of tissue samples during transportation can be reduced.

[0019] In some embodiments of the present invention, N-acetylcysteine amide, superoxide dismutase, and catalase all have strong antioxidant properties, which can further protect the fragile proteins and lipids in the cell membrane and prevent the oxidation of unsaturated fatty acids in the phospholipids that constitute the biological membrane. Therefore, by adding N-acetylcysteine amide, superoxide dismutase, and / or catalase to the tissue protection solution, the activity of tissue cells can be further improved and the loss of tissue samples during transportation can be reduced.

[0020] In some embodiments of the present invention, the ROCK inhibitor Y27632 can reduce tissue cell apoptosis during dissociation and cryopreservation, increase the adhesion properties of tissue cells, and improve the survival rate and function of tissue cells; insulin can support the growth and function of tissue cells through its extensive metabolic regulatory effects.

[0021] It should be noted that Y27632 is a highly selective and potent small molecule inhibitor of the Rho-associated protein kinase p160ROCK. It inhibits the kinase activity of ROCK-1 and ROCK-2 by targeting their catalytic sites. Y27632 significantly reduces cell separation-induced apoptosis, helping to maintain cell survival and function.

[0022] In some embodiments of the present invention, the tissue protection solution may further include at least one of the following additional technical features:

[0023] In some embodiments of the present invention, the concentration of vitamin E in the tissue protection solution is 0-0.1 μM. For example, it can be 0 μM, 0.02 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.08 μM, 0.1 μM, etc., or it can be a range consisting of any of the above values. Thus, by keeping the concentration of vitamin E within the above range, it is possible to prevent the oxidation of polyunsaturated fatty acids in biological membranes (including cell membranes and organelle membranes) and lipoproteins, cytoskeleton, and other proteins, thereby maintaining the integrity and function of cells.

[0024] In some embodiments of the present invention, the concentration of N-acetylcysteine amide in the tissue protection solution is 0-0.1 μM. For example, it can be 0 μM, 0.02 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.08 μM, 0.1 μM, etc., or can be a range consisting of any of the above values. Thus, by ensuring that the concentration of N-acetylcysteine amide is within the above range, cells can be protected from damage by reactive oxygen species (ROS), reducing their damage to cell membranes, proteins, and nucleic acids, and maintaining cell integrity and function.

[0025] In some embodiments of the present invention, the concentration of superoxide dismutase in the tissue protection solution is 0-0.1 μM. For example, it can be 0 μM, 0.02 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.08 μM, 0.1 μM, etc., or it can be a range consisting of any of the above values. Thus, by maintaining the concentration of superoxide dismutase within the above range, cells can be protected from damage by reactive oxygen species, reducing damage to cell membranes, proteins, and nucleic acids, and maintaining cell integrity and function.

[0026] In some embodiments of the present invention, the concentration of catalase in the tissue protection solution is 0-0.1 μM. For example, it can be 0 μM, 0.02 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.08 μM, 0.1 μM, etc., or it can be a range consisting of any of the above values. Thus, by maintaining the concentration of catalase within the above range, cells can be protected from damage by reactive oxygen species, reducing damage to cell membranes, proteins, and nucleic acids, and maintaining cell integrity and function.

[0027] In some embodiments of the present invention, the concentration of the ROCK inhibitor Y27632 in the tissue protection solution is 3.5 μM to 6.5 μM. For example, it can be 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, etc., or any range consisting of the above values. Thus, by maintaining the concentration of the ROCK inhibitor Y27632 within the above range, it helps to reduce apoptosis of tissue cells during cryopreservation and improve the adhesion and survival rate of tissue cells.

[0028] In some embodiments of the present invention, the concentration of insulin in the tissue protection solution is 0-1 μM. For example, it can be 0 μM, 0.2 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.8 μM, 1 μM, etc., or any range of the above values. Thus, by maintaining the insulin concentration within the above range, it is possible to provide tissue cells with necessary nutrients and support their growth and metabolic activity.

[0029] In some embodiments of the present invention, the basal medium is a serum-free tumor organoid basal medium. It should be noted that the basal medium is a conventional medium, for example, a basal medium purchased from Shandong Bozhen Biotechnology Co., Ltd. Thus, the basal medium can provide the nutrients and growth factors required by cells, help maintain the metabolic needs and physiological state of cells, and maintain the activity and function of cells in tissue samples during transportation and short-term storage.

[0030] In some embodiments of the present invention, the tissue protection solution further includes basal nutritional factors and antibiotics. The basal nutritional factors maintain a stable tissue environment, replenish nutrients needed during tissue transport, and maintain tissue cell activity; the antibiotics prevent contamination during tissue transport. Thus, the tissue protection solution can further maintain tissue cell activity and function, preventing or inhibiting potential bacterial infection.

[0031] In some embodiments of the present invention, the basic nutritional factors include at least one of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), transferrin, and bovine serum albumin. Therefore, the addition of these basic nutritional factors helps maintain the metabolic activity of tissue cells and ensures the activity and health of tissue cells during transportation and short-term storage.

[0032] In some embodiments of the present invention, the antibiotic includes at least one of gentamicin, amphotericin, penicillin, and streptomycin. Thus, the tissue protective solution can prevent or inhibit possible bacterial infection, ensure the sterility of tissue samples during storage and transportation, and improve the accuracy and reliability of subsequent experiments.

[0033] In some embodiments of the present invention, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the tissue protection solution is 0-1 μM. For example, it can be 0 μM, 0.2 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.8 μM, 1 μM, etc., or it can be a range consisting of any of the above values. Thus, by keeping the concentration of 4-hydroxyethylpiperazineethanesulfonic acid within the above range, it is possible to maintain pH stability during tissue transport, reduce the stress response of tissue cells caused by pH fluctuations, and thus protect tissue cells from damage.

[0034] In some embodiments of the present invention, the concentration of transferrin in the tissue protection solution is 0-0.1 μM. For example, it can be 0 μM, 0.02 μM, 0.04 μM, 0.05 μM, 0.06 μM, 0.08 μM, 0.1 μM, etc., or it can be a range consisting of any of the above values. Thus, by ensuring that the concentration of transferrin is within the above range, the tissue protection solution can contain the iron required for tissue cell growth, thereby promoting the proliferation of tissue cells.

[0035] In some embodiments of the present invention, the concentration of bovine serum albumin in the tissue protection solution is 20 μM to 50 μM. For example, the concentration may be 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, or any range thereof. Thus, maintaining the concentration of bovine serum albumin within the above range helps ensure the activity and health of tissue cells during transportation and short-term storage.

[0036] In some embodiments of the present invention, the concentration of gentamicin in the tissue protection solution is 0-1 μM. For example, it can be 0 μM, 0.2 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.8 μM, 1 μM, etc., or any range consisting of the above values. Thus, by ensuring that the concentration of gentamicin is within the above range, the risk of contamination of tissue cells during storage and transportation can be reduced, thereby improving the accuracy and reliability of subsequent experiments.

[0037] In some embodiments of the present invention, the concentration of amphotericin in the tissue protection solution is 0-1 μM. For example, it can be 0 μM, 0.2 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.8 μM, 1 μM, etc., or any range consisting of the above values. Thus, by ensuring that the concentration of amphotericin is within the above range, the risk of contamination of tissue cells during storage and transportation can be reduced, thereby improving the accuracy and reliability of subsequent experiments.

[0038] In some embodiments of the present invention, the concentration of penicillin in the tissue protection solution is 0-1 μM. For example, it can be 0 μM, 0.2 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.8 μM, 1 μM, etc., or it can be a range consisting of any of the above values. Thus, by ensuring that the concentration of penicillin is within the above range, the risk of contamination of tissue cells during storage and transportation can be reduced, thereby improving the accuracy and reliability of subsequent experiments.

[0039] In some embodiments of the present invention, the concentration of streptomycin in the tissue protection solution is 0-1 μM. For example, it can be 0 μM, 0.2 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.8 μM, 1 μM, etc., or it can be a range consisting of any of the above values. Thus, by keeping the concentration of streptomycin within the above range, the risk of contamination of tissue cells during storage and transportation can be reduced, thereby improving the accuracy and reliability of subsequent experiments.

[0040] Uses and methods

[0041] The present invention proposes the use of the above-mentioned tissue protection solution in the preservation and / or transportation of animal tissue samples. As previously mentioned, the tissue protection solution of the present invention can provide tissue cells with the necessary nutrients, stabilize cell membranes and intracellular lipids, prevent tissue samples from generating oxidative stress during storage and transportation, protect tissue cells from damage by reactive oxygen species, and maintain cell integrity and function. Therefore, the use of the above-mentioned tissue protection solution in the process of preserving and / or transporting animal tissue samples can provide more comprehensive protection for tissue samples, ensuring that they maintain an optimal biological state during transportation and storage, thereby improving the success rate of subsequent experiments.

[0042] In some embodiments of the present invention, the animal tissue sample is a normal tissue sample or a tumor tissue sample.

[0043] In some embodiments of the present invention, the normal tissue sample or tumor tissue sample is derived from, but is not limited to, heart, brain, kidney, liver, lung, stomach, intestine, spleen, placenta, blood vessel, endometrium, skin, organoid or PDX model.

[0044] The present invention provides a method for preserving and / or transporting animal tissue samples. According to an embodiment of the present invention, the method comprises placing the animal tissue sample in the aforementioned tissue protection solution. This method can provide comprehensive protection for the animal tissue sample, ensuring it maintains its optimal biological state during transportation and preservation, preventing oxidative stress during storage and transport, and maintaining the integrity and function of tissue cells.

[0045] In some embodiments of the present invention, the temperature of the tissue protection solution is 2-8° C. For example, it can be 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., etc., or can be any range of the above values.

[0046] In some embodiments of the present invention, the animal tissue sample is a normal tissue sample or a tumor tissue sample.

[0047] In some embodiments of the present invention, the animal tissue sample can be completely immersed in the tissue protection solution.

[0048] In some embodiments of the present invention, a method for preserving and / or transporting an animal tissue sample comprises the following steps:

[0049] S1. Place the tumor / normal tissue sample in pre-cooled tissue protection solution (2-8°C) within 5 minutes of ex vivo removal. The tissue sample must be completely covered by the tissue protection solution.

[0050] S2. Before placing the tumor / normal tissue sample in the pre-cooled tissue protection solution, the ex vivo tumor / normal tissue sample can be rinsed with sterile PBS solution or physiological saline (e.g., tissues from the digestive system such as the intestine and stomach, and the urogenital system, etc.) to remove blood stains and dirt;

[0051] S3. During transportation, place the tissue protection solution containing tumor / normal tissue samples in a low-temperature environment of 2-8°C (e.g., an insulated box with ice packs);

[0052] S4. After storage or transportation, remove the tumor / normal tissue sample from the tissue protection solution for subsequent experiments, such as using an organoid culture kit to establish tumor or normal tissue organoids.

[0053] The present invention proposes a method for maintaining the original function and vitality of animal tissue cells. According to an embodiment of the present invention, the method includes: placing the animal tissue sample in the above-mentioned tissue protection solution. As mentioned above, the tissue protection solution can provide the required nutrients for tissue cells, stabilize the cell membrane and the lipid part in the cell, improve the self-repair ability of tissue cells, reduce the loss during the transportation of tissue samples, and ensure the function and vitality of tissue cells. Therefore, this method can maintain the original function and vitality of animal tissue cells during the process of preserving and / or transporting animal tissue samples.

[0054] In some embodiments of the present invention, the temperature of the tissue protection solution is 2-8°C.

[0055] In some embodiments of the present invention, the animal tissue sample is a normal tissue sample or a tumor tissue sample.

[0056] In some embodiments of the present invention, the animal tissue sample can be completely immersed in the tissue protection solution.

[0057] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0058] The sources / product numbers of the relevant reagents involved are as follows:

[0059] Basal culture medium: purchased from Shandong Bozhen Biotechnology Co., Ltd., catalog number B213152;

[0060] Vitamin E: purchased from Haoyuan Biopharmaceutical Technology Co., Ltd., catalog number HY-W020044;

[0061] N-acetylcysteine amide: purchased from Beijing Bainuowei Biotechnology Co., Ltd., product number A9165;

[0062] Superoxide dismutase: purchased from Shanghai MacLean Biochemical Technology Co., Ltd., catalog number S6375;

[0063] Catalase: purchased from Jiangsu Xingkeyuan Information Technology Co., Ltd., product number C1345;

[0064] HEPES: purchased from Jiangsu Xingkeyuan Information Technology Co., Ltd., catalog number H109407;

[0065] Transferrin: purchased from Wuhan Heyuan Biotechnology Co., Ltd., catalog number HYC044M01;

[0066] Bovine serum albumin: purchased from Shenzhen Yawei Century Technology Co., Ltd., catalog number 69760;

[0067] Y27632:R&D SYSTEMS TM Y-27632dihydrochloride, TB1254-GMP; purchased from Shanghai Haoyuan, catalog number HY-10583 / CS-0878;

[0068] Insulin: purchased from Tonghua Dongbao Pharmaceutical Co., Ltd., product number Y20209990005;

[0069] Gentamicin: purchased from Shanghai MacLean Biochemical Technology Co., Ltd., catalog number G6064;

[0070] Amphotericin: purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number 1397-89-3;

[0071] Penicillin: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number P433335;

[0072] Streptomycin: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number S432672.

[0073] Example 1

[0074] A tissue protection solution is prepared, the components of which are: basal culture medium; vitamin E at a final concentration of 0.05 μM; N-acetylcysteine amide at a final concentration of 0.05 μM; superoxide dismutase at a final concentration of 0.05 μM; catalase at a final concentration of 0.05 μM; HEPES at a final concentration of 0.5 μM; transferrin at a final concentration of 0.05 μM; bovine serum albumin at a final concentration of 35 μM; Y27632 at a final concentration of 5 μM; insulin at a final concentration of 0.5 μM; gentamicin at a final concentration of 0.5 μM; amphotericin at a final concentration of 0.5 μM; penicillin at a final concentration of 0.25 μM; and streptomycin at a final concentration of 0.25 μM.

[0075] Within 5 minutes of ex vivo removal, human colorectal and renal cancer tissue samples were placed in pre-chilled tissue protection solution (2-8°C) and completely covered with the tissue protection solution. Tumor tissue samples were rinsed with sterile PBS or saline to remove blood and dirt, and then completely covered with the tissue protection solution.

[0076] Specific experimental process (taking human colorectum as an example):

[0077] (1) After the human colorectal tissue preserved in the above-configured tissue protection solution is transported to the laboratory within 72 hours, the relevant operations of the organoid are carried out in the biosafety cabinet in the clean area. After the tissue is transferred to the basal culture medium and washed twice, it is placed in a sterile culture dish containing the basal culture medium, and the sample is recorded before the experiment using a photographic device. Use sterile tweezers to clean up all dead cells and non-epithelial tissue (such as muscle or fat) as much as possible, and use sterile tissue scissors to cut the tissue into pieces of about 0.5-2mm in a culture dish or centrifuge tube. 3 size;

[0078] (2) Transfer the chopped sample into a 15ml centrifuge tube and add 10ml of basal culture medium to wash the sample. Allow the tissue fragments to settle naturally, then discard the supernatant and add 10ml of basal culture medium. Wash 3 to 5 times.

[0079] (3) Discard the supernatant and add 10 ml of pre-chilled basal medium containing 2.5 mM EDTA (ethylenediaminetetraacetic acid) for digestion. Shake at 4°C for 40 min.

[0080] (4) After digestion is complete, transfer the tissue fragments to a new culture dish containing basal medium and wash them once to remove EDTA;

[0081] (5) Use a 10 ml pipette to pipette and resuspend the tissue fragments in a 15 ml centrifuge tube containing cold basal medium. Repeatedly pass the tissue through the pipette tip to generate mechanical shear force to separate the crypts from the basal layer. Take a portion of the suspension for microscopic examination. When a large number of crypt-like structures can be seen, stop pipetting and transfer the supernatant containing the isolated crypts to a new 15 ml centrifuge tube. Centrifuge at 300 g for 3 min.

[0082] (6) Discard the supernatant, resuspend the tissue pellet in 1 ml of basal medium and transfer it to a 1.5 ml centrifuge tube. Take 18 μl of the suspension for microscopic examination and crypt counting. After counting, aspirate the suspension containing the required number of crypts, centrifuge at 300 g for 3 min, discard the supernatant, and place on ice.

[0083] (7) Add the corresponding amount of organoid Matrigel according to the ratio of 50 to 250 crypts per well to 20 to 30 μl of Matrigel, mix well on ice, and place on ice;

[0084] (8) Use a pipette to transfer the mixture of matrix gel and cells to a 24-well cell culture plate. Apply 20-30 μl of the mixed suspension to each well. The mixed suspension must be applied to the center of the bottom of the culture well and should not touch the side walls of the culture well after spreading. Place the culture plate in a 37°C, 5% CO2 cell culture incubator to solidify for 20 minutes. After the matrix gel solidifies and no longer flows, slowly add complete culture medium along the well wall, adding 500 μl to each well. Place the 24-well plate in a 37°C, 5% CO2 cell culture incubator for incubation.

[0085] Change the medium every two days;

[0086] (9) Place the cell culture plate in an incubator for culture. Observe the growth status of the organoids under an inverted microscope every 3 days, take photos under an inverted microscope, and record the morphology and distribution of the organoids in multiple fields of view.

[0087] The experimental process for processing human renal cancer tissue is the same as that for human colorectal cancer.

[0088] Example 2

[0089] A tissue protection solution is prepared, the components of which are: basal culture medium; vitamin E at a final concentration of 0.08 μM; N-acetylcysteine amide at a final concentration of 0.08 μM; superoxide dismutase at a final concentration of 0.08 μM; catalase at a final concentration of 0.08 μM; HEPES at a final concentration of 0.8 μM; transferrin at a final concentration of 0.08 μM; bovine serum albumin at a final concentration of 50 μM; Y27632 at a final concentration of 4 μM; insulin at a final concentration of 0.8 μM; gentamicin at a final concentration of 0.8 μM; amphotericin at a final concentration of 0.8 μM; penicillin at a final concentration of 0.4 μM; and streptomycin at a final concentration of 0.4 μM.

[0090] Specific experimental process (taking human colorectum as an example):

[0091] (1) After the human colorectal tissue preserved in the above-configured tissue protection solution is transported to the laboratory within 72 hours, the relevant operations of the organoid are carried out in the biosafety cabinet in the clean area. After the tissue is transferred to the basal culture medium and washed twice, it is placed in a sterile culture dish containing the basal culture medium, and the sample is recorded before the experiment using a photographic device. Use sterile tweezers to clean up all dead cells and non-epithelial tissue (such as muscle or fat) as much as possible, and use sterile tissue scissors to cut the tissue into pieces of about 0.5-2mm in a culture dish or centrifuge tube. 3 size;

[0092] (2) Transfer the chopped sample into a 15ml centrifuge tube and add 10ml of basal culture medium to wash the sample. Allow the tissue fragments to settle naturally, then discard the supernatant and add 10ml of basal culture medium. Wash 3 to 5 times.

[0093] (3) Discard the supernatant, add 10 ml of pre-chilled basal medium containing 2.5 mM EDTA for digestion, and shake at 4°C for 40 min;

[0094] (4) After digestion is complete, transfer the tissue fragments to a new culture dish containing basal medium and wash them once to remove EDTA;

[0095] (5) Use a 10 ml pipette to pipette and resuspend the tissue fragments in a 15 ml centrifuge tube containing cold basal medium. Repeatedly pass the tissue through the pipette tip to generate mechanical shear force to separate the crypts from the basal layer. Take a portion of the suspension for microscopic examination. When a large number of crypt-like structures can be seen, stop pipetting and transfer the supernatant containing the isolated crypts to a new 15 ml centrifuge tube. Centrifuge at 300 g for 3 min.

[0096] (6) Discard the supernatant, resuspend the tissue pellet in 1 ml of basal medium and transfer it to a 1.5 ml centrifuge tube. Take 18 μl of the suspension for microscopic examination and crypt counting. After counting, aspirate the suspension containing the required number of crypts, centrifuge at 300 g for 3 min, discard the supernatant, and place on ice.

[0097] (7) Add the corresponding amount of organoid Matrigel according to the ratio of 50 to 250 crypts per well to 20 to 30 μl of Matrigel, mix well on ice, and place on ice;

[0098] (8) Use a pipette to transfer the mixture of matrix gel and cells to a 24-well cell culture plate. Apply 20-30 μl of the mixed suspension to each well. The mixed suspension must be applied to the center of the bottom of the culture well and should not touch the side walls of the culture well after spreading. Place the culture plate in a 37°C, 5% CO2 cell culture incubator to solidify for 20 minutes. After the matrix gel solidifies and no longer flows, slowly add complete culture medium along the well wall, adding 500 μl to each well. Place the 24-well plate in a 37°C, 5% CO2 cell culture incubator for incubation.

[0099] Change the medium every two days;

[0100] (9) Place the cell culture plate in an incubator for culture. Observe the growth status of the organoids under an inverted microscope every 3 days, take photos under an inverted microscope, and record the morphology and distribution of the organoids in multiple fields of view.

[0101] The experimental process for processing human renal cancer tissue is the same as that for human colorectal cancer.

[0102] Comparative Example 1

[0103] Human colorectal cancer and human renal cancer tissue samples were preserved according to the method of Example 1, except that Miltenyi Biotec Tissue Preservation Solution (Cat. No.: abs9784-100 mL) was used as the tissue preservation solution.

[0104] Comparative Example 2

[0105] Human colorectal and human renal cancer tissue samples were preserved according to the method of Example 1, except that vitamin E was not added to the tissue protection solution. The specific components of the tissue protection solution were: basal culture medium; N-acetylcysteine amide at a final concentration of 0.05 μM; superoxide dismutase at a final concentration of 0.05 μM; catalase at a final concentration of 0.05 μM; HEPES at a final concentration of 0.5 μM; transferrin at a final concentration of 0.05 μM; bovine serum albumin at a final concentration of 35 μM; Y27632 at a final concentration of 5 μM; insulin at a final concentration of 0.5 μM; gentamicin at a final concentration of 0.5 μM; amphotericin at a final concentration of 0.5 μM; penicillin at a final concentration of 0.25 μM; and streptomycin at a final concentration of 0.25 μM.

[0106] The results of organoid diameter changes after 6 days of continuous culture of primary human colorectal and human renal cancer organoids from Examples 1-2 and Comparative Examples 1-2 are shown in Table 1. The results show that the cell activity of human colorectal and human renal cancer tissue samples preserved in the tissue protection solution of the present invention after primary treatment is higher than that of human colorectal and human renal cancer tissue samples preserved in the tissue protection solution of Comparative Examples 1 and 2.

[0107] Table 1 Diameter (μm) of primary human colorectal and renal cancer organoids grown for 6 days after plating

[0108]

[0109] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0110] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A tissue protection solution, characterized in that: include: basal culture medium; Vitamins, including vitamin E; an antioxidant comprising at least one of N-acetylcysteine amide, superoxide dismutase, and catalase; Specific cytokines, comprising at least one of ROCK inhibitor Y27632 and insulin.

2. The tissue protection solution according to claim 1, characterized in that The concentration of vitamin E in the tissue protection solution is 0-0.1 μM; Optionally, the concentration of N-acetylcysteine amide in the tissue protection solution is 0-0.1 μM; Optionally, the concentration of superoxide dismutase in the tissue protection solution is 0-0.1 μM; Optionally, the concentration of catalase in the tissue protection solution is 0-0.1 μM; Optionally, the concentration of the ROCK inhibitor Y27632 in the tissue protection solution is 3.5 μM-6.5 μM; Optionally, the concentration of insulin in the tissue protection solution is 0-1 μM.

3. The tissue protection solution according to claim 2, characterized in that The basal culture medium is a serum-free tumor organoid basal culture medium.

4. The tissue protection solution according to any one of claims 1 to 3, characterized in that: It further includes basic nutritional factors and antibiotics.

5. The tissue protection solution according to claim 4, characterized in that The basic nutritional factors include at least one of 4-hydroxyethylpiperazineethanesulfonic acid, transferrin and bovine serum albumin; Optionally, the antibiotic comprises at least one of gentamicin, amphotericin, penicillin and streptomycin.

6. The tissue protection solution according to claim 5, characterized in that The concentration of the 4-hydroxyethylpiperazineethanesulfonic acid in the tissue protection solution is 0-1 μM; Optionally, the transferrin concentration in the tissue protection solution is 0-0.1 μM; Optionally, the concentration of the bovine serum albumin in the tissue protection solution is 20 μM-50 μM; Optionally, the concentration of gentamicin in the tissue protection solution is 0-1 μM; Optionally, the concentration of the amphotericin in the tissue protection solution is 0-1 μM; Optionally, the concentration of penicillin in the tissue protection solution is 0-1 μM; Optionally, the concentration of streptomycin in the tissue protection solution is 0-1 μM.

7. Use of the tissue protection solution according to any one of claims 1 to 6 in preserving and / or transporting animal tissue samples.

8. The use according to claim 7, characterized in that The animal tissue sample is a normal tissue sample or a tumor tissue sample.

9. A method for preserving and / or transporting animal tissue samples, characterized in that: include: The animal tissue sample is placed in the tissue protection solution according to any one of claims 1 to 6.

10. The method according to claim 9, characterized in that The temperature of the tissue protection solution is 2-8°C.

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