Buffer for tissue separation and preservation and its application, and method for culturing tumor organoids

Abstract

The present invention discloses a buffer for tissue separation and / or preservation and its application, and a method for culturing tumor organoids, and relates to the field of organoid culture. The buffer is used to preserve tumor tissue samples, which are of liquid origin; the buffer includes: a basal culture medium and specific additive factors, and the specific additive factors include: superoxide dismutase, catalase, penicillin, and streptomycin; based on the volume of the basal culture medium, the concentration of superoxide dismutase is 1 to 100 μg / mL, the concentration of catalase is 50 to 500 μg / mL, the concentration of penicillin is 50 to 200 U / mL, and the concentration of streptomycin is 50 to 200 μg / mL. The tissue preservation solution of the present invention is used for the transportation and protection of liquid-derived tumor tissue samples, and has a high tumor cell activity retention rate. It is used to construct tumor organoids with a high success rate and a large number, which can meet the needs of clinical detection and disease research.

Description

Technical Field

[0001] The present invention relates to the field of organoid culture, and specifically relates to a buffer for tissue separation and preservation, its application, and a method for culturing tumor organoids. Background Art

[0002] The transportation of liquid-derived tumor tissue samples is extremely important in biological experiments. A convenient and efficient in vitro storage and transportation method is an essential part of it. Common liquid tumor samples include malignant effusion samples and peripheral blood samples. Malignant effusion is a physiological fluid that accumulates in the bodies of patients with malignant tumors. Usually, when malignant tumor cells grow and spread in the body, they invade the surrounding tissues and organs and damage the normal tissue structure, resulting in excessive accumulation of body fluid (serous fluid). These malignant cells can further spread through the blood circulation or lymphatic system, and thus accumulate in the cavities or organs of tumor patients. Malignant effusion mainly includes malignant pleural effusion, malignant ascites, malignant pericardial effusion, and malignant cerebrospinal fluid. It is a common pathophysiological phenomenon of cancer. Its liquid usually contains malignant tumor cells, proteins, cell debris, white blood cells, red blood cells, extracellular fluid, and various bioactive molecules. Its generation and accumulation are closely related to the development and progression of tumors, and are crucial for the diagnosis and treatment of cancer. Circulating tumor cells (CTCs) are tumor cells that detach from primary tumors or metastatic tumors and enter the blood circulation. The presence of CTCs in the blood is a sign of the invasiveness and metastasis of tumor cells. These cells evade the surveillance of the immune system through various mechanisms in the blood circulation and finally colonize in distal tissues to form metastatic tumors. The presence and quantity of CTCs are closely related to the metastasis and recurrence of tumors. Therefore, the detection and monitoring of CTCs have become an important means to evaluate the progression and treatment effect of cancer.

[0003] Tumor organoids are a biological model, mainly using the tumor tissues of patients for three-dimensional culture in vitro to simulate the biological characteristics of in vivo tumor tissues. Tumor organoids can simulate the microenvironment of tumor cell growth in vitro, which has great significance for studying the occurrence, development, and drug response of tumors.

[0004] Currently, there are few reports on protective solutions that can be used for the separation and long-term transportation of liquid-derived tumor tissue samples. The number of tumor stem cells isolated therefrom and the number of tumor organoids constructed are small, and the success rate is low. Therefore, studying a solution suitable for the separation and transportation protection of liquid-derived tumor tissue samples is of great significance for ensuring the activity of tumor stem cells, improving the success rate and yield of tumor organoid construction. 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. To this end, the present invention provides a buffer for tissue separation and / or preservation and its application. The buffer of the present invention is used for the transport protection of tumor tissue samples from liquid sources, with a high tumor cell activity retention rate. Using it to construct tumor organoids has a high success rate, a large number, and can meet the needs of clinical detection and disease research.

[0006] In the first aspect of the present invention, the present invention proposes a buffer. According to an embodiment of the present invention, the buffer is used to preserve a tumor tissue sample, and the tumor tissue sample is from a liquid source; the buffer includes: a basal medium and a specific additive factor, and the specific additive factor includes: superoxide dismutase, catalase, penicillin, and streptomycin.

[0007] According to an embodiment of the present invention, based on the volume of the basal medium, the concentration of superoxide dismutase is 1 - 100 μg / mL, the concentration of catalase is 50 - 500 μg / mL, the concentration of penicillin is 50 - 200 U / mL, and the concentration of streptomycin is 50 - 200 μg / mL.

[0008] Exemplarily, the concentration of superoxide dismutase can be 1 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL.

[0009] Exemplarily, the concentration of catalase can be 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL.

[0010] Exemplarily, the concentration of penicillin can be 50 U / mL, 75 U / mL, 100 U / mL, 125 U / mL, 150 U / mL, 175 U / mL, 200 U / mL.

[0011] Exemplarily, the concentration of streptomycin can be 50 μg / mL, 75 μg / mL, 100 μg / mL, 125 μg / mL, 150 μg / mL, 175 μg / mL, 200 μg / mL.

[0012] The buffer according to the embodiment of the present invention is used for the separation and transport protection of liquid-source tumor tissue samples, with a high tumor cell activity retention rate. Using it to construct tumor organoids has a high success rate, a large number, and can meet the needs of clinical detection and disease research.

[0013] According to the embodiment of the present invention, based on the volume of the basal medium, the concentration of superoxide dismutase is 20 μg / mL, the concentration of catalase is 300 μg / mL, the concentration of penicillin is 100 U / mL, and the concentration of streptomycin is 100 μg / mL. Thereby, the high activity of tumor cells during transportation and storage is further maintained.

[0014] According to the embodiment of the present invention, the specific additive factor further includes: vitamin E; based on the volume of the basal medium, the concentration of vitamin E is 1.25 - 100 nM. The inventors found through a large number of experiments that the three components of superoxide dismutase, catalase, and vitamin E can synergistically enhance the effect and further improve the retention rate of tumor cell activity.

[0015] Exemplarily, the concentration of vitamin E can be 1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM. According to the embodiment of the present invention, the specific additive factor further includes: transferrin, recombinant human albumin or human serum albumin, Y27632, gentamicin, amphotericin.

[0016] According to the embodiment of the present invention, based on the volume of the basal medium, the concentration of transferrin is 1 - 100 nM, the concentration of recombinant human albumin or human serum albumin is 0.025 - 10 mg / mL, the concentration of Y27632 is 0.5 - 50 μM, the concentration of gentamicin is 1 - 100 μg / mL, and the concentration of amphotericin is 10 - 1000 ng / mL.

[0017] Exemplarily, the concentration of transferrin can be 1 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM.

[0018] Exemplarily, the concentration of the recombinant human albumin or human blood albumin can be 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL.

[0019] Exemplarily, the concentration of the Y27632 can be 0.5 μM, 1 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM.

[0020] Exemplarily, the concentration of the gentamicin can be 1 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL.

[0021] Exemplarily, the concentration of the amphotericin can be 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1000 ng / mL.

[0022] The buffer according to the embodiment of the present invention can provide comprehensive protection and support for tumor tissue samples from liquid sources to improve cell survival rate and maintain cell function.

[0023] According to the embodiment of the present invention, based on the volume of the basal medium, the concentration of vitamin E is 25 nM, the concentration of transferrin is 20 nM, the concentration of the recombinant human albumin or human blood albumin is 0.5 mg / mL, the concentration of Y27632 is 10 μM, the concentration of gentamicin is 10 μg / mL, and the concentration of amphotericin is 250 ng / mL. Thereby, the high survival rate and high activity of tumor cells during transportation and preservation are further maintained.

[0024] According to the embodiment of the present invention, the specific additive factor further includes: N-acetylcysteine amide; based on the volume of the basal medium, the concentration of N-acetylcysteine amide is 0.0625 - 10 mM. Thereby, the survival and function of tumor cells are further maintained, and cell death is reduced.

[0025] Exemplarily, the concentration of the N-acetylcysteine amide can be 0.0625 mM, 0.13 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM.

[0026] According to an embodiment of the present invention, the concentration of the N-acetylcysteine amide is 1.25 mM.

[0027] According to an embodiment of the present invention, the buffer further comprises a hydrogen ion buffer of 5 - 50 mM. Thus, the buffer according to the embodiment of the present invention can provide a more stable environment for tumor tissue samples from liquid sources.

[0028] Exemplarily, the concentration of the hydrogen ion buffer can be 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM.

[0029] According to an embodiment of the present invention, the hydrogen ion buffer is HEPES and the concentration is 10 mM. Thus, the buffer according to the embodiment of the present invention can further reduce the stress response of tumor cells during storage and transportation, reduce cell damage, and extend the storage time.

[0030] According to an embodiment of the present invention, the basal medium is DMEM / F12. The inventors have found through research that the basal medium DMEM / F12 and the above-mentioned specific additive factors can act synergistically to provide a nutritionally suitable environment for various tumor cells. Thus, the buffer according to the embodiment of the present invention can be applicable to various tumor cells and is helpful for maintaining their cell viability and functions.

[0031] According to an embodiment of the present invention, the tumor tissue sample is from at least one of malignant effusion, peripheral blood, bone marrow extract, bronchoalveolar lavage fluid, urine, thoracic or abdominal puncture fluid.

[0032] According to an embodiment of the present invention, the malignant effusion includes at least one of malignant pleural effusion, malignant peritoneal effusion, malignant pericardial effusion, and malignant cerebrospinal fluid.

[0033] In this text, the term "malignant pleural effusion" refers to the accumulation of fluid in the pleural cavity caused by the metastasis of lung cancer, breast cancer, or other cancers to the pleura. The term "malignant ascites" is equivalent to "ascites", which is commonly seen in the accumulation of fluid in the abdominal cavity caused by ovarian cancer, gastric cancer, liver cancer, etc. The term "malignant pericardial effusion" refers to the accumulation of fluid in the pericardial cavity when specific cancers such as lung cancer and breast cancer metastasize to the pericardium. The term "malignant cerebrospinal fluid" refers to the presence of tumor cells in the cerebrospinal fluid due to brain tumors or cancers that have metastasized to the meninges. Tumor cells (circulating tumor cells, CTCs) enter the bloodstream after detaching from the primary tumor or metastatic tumor, and thus peripheral blood may contain tumor cells. In some cases, such as in patients with leukemia or certain types of lymphoma, bone marrow fluid may contain tumor cells. In lung cancer or other lung diseases, fluid samples obtained by bronchoalveolar lavage through a bronchoscope may contain tumor cells. In some urinary system tumors (such as bladder cancer), urine may contain tumor cells. Thoracentesis or abdominocentesis of tumor patients may also contain tumor cells.

[0034] In a second aspect of the present invention, the present invention provides the use of the aforementioned buffer in the preservation of tumor tissue samples, wherein the tumor tissue samples are derived from fluids.

[0035] Those skilled in the art can understand that the features and advantages described above for the buffer also apply to this application and will not be elaborated herein.

[0036] According to an embodiment of the present invention, the tumor tissue samples are derived from at least one of malignant effusions, peripheral blood, bone marrow extracts, bronchoalveolar lavage fluids, urine, thoracentesis or abdominocentesis fluids.

[0037] According to an embodiment of the present invention, the malignant effusions include at least one of malignant pleural effusion, malignant ascites, malignant pericardial effusion, and malignant cerebrospinal fluid.

[0038] According to an embodiment of the present invention, the tumor tissue samples are used for tumor organoid culture.

[0039] According to an embodiment of the present invention, the tumor organoids include at least one of lung adenocarcinoma organoids, lung cancer organoids, breast cancer organoids, colorectal cancer organoids, ovarian cancer organoids, gastric cancer organoids, liver cancer organoids, brain tumor organoids, bladder cancer organoids, and meningioma organoids.

[0040] In a third aspect of the present invention, the present invention provides a method for culturing tumor organoids. According to an embodiment of the present invention, it includes: before subjecting the tumor tissue samples to organoid culture treatment, preserving them in the aforementioned buffer, wherein the tumor tissue samples are derived from fluids. The method according to the embodiment of the present invention has a high success rate, a large quantity, and good experimental repeatability.

[0041] Those skilled in the art can understand that the features and advantages described above for the buffer also apply to this method and will not be elaborated here.

[0042] According to an embodiment of the present invention, the tumor tissue sample is from at least one of malignant effusion, peripheral blood, bone marrow extract, bronchoalveolar lavage fluid, urine, thoracic or abdominal puncture fluid.

[0043] According to an embodiment of the present invention, the malignant effusion includes at least one of pleural effusion, ascites, pericardial effusion, and cerebrospinal fluid.

[0044] According to an embodiment of the present invention, the tumor organoids include at least one of lung adenocarcinoma organoids, lung cancer organoids, breast cancer organoids, colorectal cancer organoids, ovarian cancer organoids, gastric cancer organoids, liver cancer organoids, brain tumor organoids, bladder cancer organoids, and meningioma organoids.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] 1. The buffer for tissue separation and / or preservation of the present invention is applicable to short-term storage or transportation of primary liquid-source tumor tissue samples as well as tissue separation, and can effectively avoid phenomena such as apoptosis and necrosis of samples during separation, storage, or transportation, and maintain the biological functions and vitality of tumor cells, especially tumor stem cells.

[0047] 2. The buffer for tissue separation and / or preservation of the present invention can maintain the vitality of most cells in the liquid-source tumor tissue sample at 4°C for more than 48 hours, significantly reduce the activity loss during the process from sample separation to the start of the experiment, and effectively improve the success rate and the number of organs in constructing and culturing tumor organoids using various primary liquid tumor tissues.

[0048] 3. The buffer for tissue separation and / or preservation of the present invention can also be used for resuspension and washing during the subsequent processing of tumor tissue samples.

[0049] The additional aspects and advantages of the present invention will be partly given in the following description, partly become obvious from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The above and / or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, where:

[0051] Figure 1Schematic diagram of the flow chart for the isolation, enrichment, and organoid culture of tumor cells in liquid-source tumor tissue samples of the embodiments of the present invention; among them, (A) is the schematic diagram of the flow chart for the isolation, enrichment, and organoid culture of tumor cells in malignant effusion; (B) is the schematic diagram of the flow chart for the isolation, enrichment, and organoid culture of tumor cells in peripheral blood samples.

[0052] Figure 2 Bright-field optical microscope image of lung adenocarcinoma organoids cultured from tumor samples derived from pleural effusion in Experimental Group 1 of the embodiments of the present invention; among them, (A) are lung adenocarcinoma organoids on the 0th day of culture; (B) are lung adenocarcinoma organoids on the 3rd day of culture; (C) are lung adenocarcinoma organoids on the 5th day of culture; (D) are lung adenocarcinoma organoids on the 7th day of culture; (E) are lung adenocarcinoma organoids on the 9th day of culture; (F) are lung adenocarcinoma organoids on the 12th day of culture; scale bar: 100 μm.

[0053] Figure 3 Bright-field optical microscope image of lung adenocarcinoma organoids cultured from tumor samples derived from pleural effusion in Control Group 1 of the embodiments of the present invention; among them, (A) are lung adenocarcinoma organoids on the 0th day of culture; (B) are lung adenocarcinoma organoids on the 3rd day of culture; (C) are lung adenocarcinoma organoids on the 5th day of culture; (D) are lung adenocarcinoma organoids on the 7th day of culture; (E) are lung adenocarcinoma organoids on the 9th day of culture; (F) are lung adenocarcinoma organoids on the 12th day of culture; scale bar: 100 μm.

[0054] Figure 4 Immunohistochemical staining images of lung adenocarcinoma organoids in Experimental Group 1 of the embodiments of the present invention; among them, (A) is the staining result of immunohistochemical marker Ki67; (B) is the staining result of immunohistochemical marker TTF-1; (C) is the staining result of immunohistochemical marker Napsin A, scale bar: 50 μm.

[0055] Figure 5 Bright-field optical microscope image of breast cancer organoids cultured from tumor samples derived from pleural effusion in Experimental Group 2 of the embodiments of the present invention; among them, (A) are breast cancer organoids on the 0th day of culture; (B) are breast cancer organoids on the 3rd day of culture; (C) are breast cancer organoids on the 6th day of culture; (D) are breast cancer organoids on the 10th day of culture; (E) are breast cancer organoids on the 14th day of culture; (F) are breast cancer organoids on the 17th day of culture; scale bar: 100 μm.

[0056] Figure 6 Immunohistochemical images of breast cancer organoids cultured from tumor samples derived from pleural effusion in Experimental Group 2 of the embodiments of the present invention; among them, (A) is the staining result of immunohistochemical marker Ki67; (B) is the staining result of immunohistochemical marker ER; (C) is the staining result of immunohistochemical marker HER2; scale bar: 100 μm.

[0057] Figure 7 This is a bright-field optical microscopy image of colorectal cancer organoids cultured from tumor samples of peripheral blood origin in Example 3 of the present invention. Among them, (A) shows colorectal cancer organoids on the 0th day of culture; (B) shows colorectal cancer organoids on the 3rd day of culture; (C) shows colorectal cancer organoids on the 5th day of culture; (D) shows colorectal cancer organoids on the 7th day of culture; (E) shows colorectal cancer organoids on the 9th day of culture; (F) shows colorectal cancer organoids on the 12th day of culture; Scale bar: 100 μm;

[0058] Figure 8 This is an immunohistochemistry image of colorectal cancer organoids cultured from tumor samples of peripheral blood origin in Example 3 of the present invention. Among them, (A) shows the staining result of immunohistochemical marker Ki67; (B) shows the staining result of immunohistochemical marker CDX2; (C) shows the staining result of immunohistochemical marker CK20; Scale bar: 100 μm. Detailed implementation manners

[0059] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention.

[0060] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "plurality" is two or more.

[0061] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0062] Terms and definitions

[0063] To make it easier to understand the present invention, 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 meanings commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0064] In this document, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0065] In this article, the term "DMEM / F12" is a mixture of two culture media, Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Medium, and is commercially available.

[0066] In some specific embodiments of the present invention, the buffer is formulated as follows: based on the volume of the aforementioned basal medium, it contains 1-100 μg / mL of superoxide dismutase, 50-500 μg / mL of catalase, 50-200 U / mL of penicillin, and 50-200 μg / mL of streptomycin. Through a large number of experiments, the inventors determined the basic components and concentrations of this buffer. Each concentration component synergistically enhances the effect, can effectively protect tumor cells, reduce cell damage and death during separation, transportation, and preservation, and maintain the high activity of cells. Due to the effective maintenance of the activity of tumor cells, the success rate of constructing tumor organoids from liquid-derived tumor tissue samples separated or preserved using the buffer of the present invention is significantly increased, and the number of obtained organoids is significantly increased, which can meet the needs of clinical detection and disease research, and provides an important tool for the diagnosis, treatment, and research of cancer.

[0067] In a specific embodiment of the present invention, the buffer is formulated as follows: based on the volume of the aforementioned basal medium, it contains 20 μg / mL of superoxide dismutase, 300 μg / mL of catalase, 100 U / mL of penicillin, 100 μg / mL of streptomycin, 10 mM of HEPES, and the balance of the DMEM / F12 basal medium.

[0068] In some specific embodiments of the present invention, the buffer is formulated as follows: based on the volume of the aforementioned basal medium, it contains 1-100 μg / mL of superoxide dismutase, 50-500 μg / mL of catalase, 1.25-100 nM of vitamin E, 50-200 U / mL of penicillin, 50-200 μg / mL of streptomycin, and the balance of the basal medium.

[0069] In a specific embodiment of the present invention, the buffer is formulated as follows: based on the volume of the aforementioned basal medium, it contains 20 μg / mL of superoxide dismutase, 300 μg / mL of catalase, 25 nM of vitamin E, 100 U / mL of penicillin, 100 μg / mL of streptomycin, 10 mM of HEPES, and the balance of the DMEM / F12 basal medium.

[0070] In some specific embodiments of the present invention, the buffer formulation is as follows: based on the volume of the aforementioned basal medium, it contains 1 - 100 μg / mL of superoxide dismutase, 50 - 500 μg / mL of catalase, 1.25 - 100 nM of vitamin E, 1 - 100 nM of transferrin, 0.025 - 10 mg / mL of recombinant human albumin or human serum albumin, 0.5 - 50 μM of Y27632, 50 - 200 U / mL of penicillin, 50 - 200 μg / mL of streptomycin, 1 - 100 μg / mL of gentamicin, 10 - 1000 ng / mL of amphotericin, and the balance of the basal medium.

[0071] In a specific embodiment of the present invention, the buffer formulation is as follows: based on the volume of the aforementioned basal medium, it contains 20 μg / mL of superoxide dismutase, 300 μg / mL of catalase, 25 nM of vitamin E, 20 nM of transferrin, 0.5 mg / mL of recombinant human albumin, 10 μM of Y27632, 100 U / mL of penicillin, 100 μg / mL of streptomycin, 10 μg / mL of gentamicin, 250 ng / mL of amphotericin, 10 mM of HEPES, and the balance of the DMEM / F12 basal medium.

[0072] In some specific embodiments of the present invention, the buffer formulation is as follows: based on the volume of the aforementioned basal medium, it contains 0.0625 - 10 mM of N-acetylcysteine amide, 1 - 100 μg / mL of superoxide dismutase, 50 - 500 μg / mL of catalase, 1.25 - 100 nM of vitamin E, 1 - 100 nM of transferrin, 0.025 - 10 mg / mL of recombinant human albumin or human serum albumin, 0.5 - 50 μM of Y27632, 50 - 200 U / mL of penicillin, 50 - 200 μg / mL of streptomycin, 1 - 100 μg / mL of gentamicin, 10 - 1000 ng / mL of amphotericin, and the balance of the basal medium.

[0073] In a specific embodiment of the present invention, the buffer formulation is as follows: based on the volume of the aforementioned basal medium, it contains 1.25 mM of N-acetylcysteine amide, 20 μg / mL of superoxide dismutase, 300 μg / mL of catalase, 25 nM of vitamin E, 20 nM of transferrin, 0.5 mg / mL of recombinant human albumin, 10 μM of Y27632, 100 U / mL of penicillin, 100 μg / mL of streptomycin, 10 μg / mL of gentamicin, 250 ng / mL of amphotericin, 10 mM of HEPES, and the balance of the DMEM / F12 basal medium.

[0074] Within the above concentration ranges, the components of the buffer according to the embodiments of the present invention can act synergistically to meet the culture requirements of different types of cells, further improving the success rate and quantity of constructing tumor organoids using liquid-derived tumor tissue samples, and enhancing the repeatability of experiments. Within the above concentration ranges, the N-acetylcysteine amide concentration of the aforementioned buffer can meet the antioxidant requirements of different cell types; the vitamin E concentration within the above concentration ranges helps to fine-tune the intracellular redox balance; the transferrin concentration within the above concentration ranges can ensure the effective supply of iron ions; recombinant human albumin or human serum albumin within the above concentration ranges can provide necessary nutritional support for cells; Y27632 within the above concentration ranges helps to precisely control cell behavior and improve survival rate; gentamicin and amphotericin within the above concentration ranges can provide effective antibacterial and antifungal protection for cells. Thus, the buffer according to the embodiments of the present invention can provide comprehensive protection and support for liquid-derived tumor tissue samples to improve cell survival rate and maintain cell function.

[0075] The inventors investigated the effects of different buffers on the cell viability in liquid-derived tumor tissue samples. The investigation methods and results are as follows:

[0076] Buffer A formulation: Based on the volume of DMEM / F12 basal medium, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 μM Y27632, 10 μg / mL gentamicin, 250 ng / mL amphotericin, and 10 mM HEPES; 20 μg / mL superoxide dismutase, 300 μg / mL catalase; the balance of DMEM / F12 basal medium.

[0077] Buffer B formulation: Based on the volume of DMEM / F12 basal medium, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 μM Y27632, 10 μg / mL gentamicin, 250 ng / mL amphotericin, and 10 mM HEPES; 20 μg / mL superoxide dismutase, 300 μg / mL catalase, 25 nM vitamin E; the balance of DMEM / F12 basal medium.

[0078] Formulation of Buffer C: Based on the volume of DMEM / F12 basal medium, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 μM Y27632, 10 μg / mL gentamicin, 250 ng / mL amphotericin, and 10 mM HEPES; 20 μg / mL superoxide dismutase, 300 μg / mL catalase, 25 nM vitamin E, 20 nM transferrin, 0.5 mg / mL recombinant human albumin; the balance is DMEM / F12 basal medium.

[0079] Formulation of Buffer D: Based on the volume of DMEM / F12 basal medium, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 μM Y27632, 10 μg / mL gentamicin, 250 ng / mL amphotericin, and 10 mM HEPES; 1.25 mM N-acetylcysteine amide, 20 μg / mL superoxide dismutase, 300 μg / mL catalase, 25 nM vitamin E, 20 nM transferrin, 0.5 mg / mL recombinant human albumin; the balance is DMEM / F12 basal medium.

[0080] Formulation of Buffer E: Based on the volume of DMEM / F12 basal medium, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 μM Y27632, 10 μg / mL gentamicin, 250 ng / mL amphotericin, and 10 mM HEPES; the balance is DMEM / F12 basal medium.

[0081] Investigation method: Divide the tumor tissue fluid (pleural effusion sample fluid of colorectal cancer and lung adenocarcinoma tissues) of the same sample into 5 equal parts, and place them into equal amounts of Buffer A - Buffer E respectively. After storing in an environment at 4°C for 48 h, perform centrifugal washing, stain with trypan blue, and perform cell counting.

[0082] Investigation results: Through trypan blue staining, observe and count the number and proportion of live cells in the two groups to judge the effect of different buffers on cell viability.

[0083] Table 1 Effects of Different Buffers on Cell Activity in Tumor Tissue Samples from Liquid Sources

[0084]

[0085] The results showed that buffers containing specific additive factors such as superoxide dismutase, catalase, vitamin E, transferrin or recombinant human albumin could significantly improve the cell viability in tumor tissue samples from liquid sources. Thereby, the survival rate and the retention rate of cell viability of tumor stem cells with self-proliferation ability in tumor tissue samples from liquid sources were further increased. Therefore, when using the samples separated or preserved with the aforementioned buffer to construct tumor organoids, the success rate and the number of organoids were further increased.

[0086] The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in the art or according to the product specifications are followed. The main reagents used and their sources are shown in Table 2. Reagents not included in Table 2 and instruments without indicating the manufacturer are all conventional products that can be obtained through commercial purchase.

[0087] Table 2 Manufacturers and Catalog Numbers of Main Reagents

[0088]

[0089] Unless otherwise specified, in the embodiments of the present invention, the buffer formula and preparation method for transporting tumor tissue samples from liquid sources are as follows: Add 1.25 mM of N-acetylcysteine amide, 20 μg / mL of superoxide dismutase, 300 μg / mL of catalase, 25 nM of vitamin E, 10 mM of HEPES, 20 nM of transferrin, 0.5 mg / mL of recombinant human albumin, 10 μM of Y27632, 10 μg / mL of gentamicin, 250 ng / mL of amphotericin, 100 U / mL of penicillin, and 100 μg / mL of streptomycin to Advanced DMEM / F12 medium.

[0090] Example 1: Preservation of Tumor Tissue Samples from Pleural Effusion and Construction of Lung Adenocarcinoma Organoids

[0091] Reference Figure 1 , in this example, the construction and culture of lung adenocarcinoma organoids derived from tumor tissue samples of pleural effusion were carried out according to the following experimental steps:

[0092] (1) Add the pleural effusion samples of lung adenocarcinoma patients to the pre-cooled tissue preservation solution of the present invention (experimental group) and the control group tissue preservation solution (Miltenyi Biotec tissue preservation solution, catalog number: abs9784 - 100mL) respectively. After mixing, send them to the laboratory for pretreatment within 12 h.

[0093] (2) Centrifuge the obtained effusion sample at 300 g for 5 min in a centrifuge, discard the supernatant, wash it once with the transport solution, and then centrifuge it again at 300 g for 5 min, and discard the supernatant.

[0094] (3) Resuspend the precipitate with 4 mL of 40% Percoll solution.

[0095] (4) Add 4 mL of 70% Percoll solution to a 15 mL centrifuge tube, aspirate the 40% Percoll resuspended precipitate in step 3, and gently drip it onto the upper layer of the 70% Percoll solution to form an obvious liquid level boundary; aspirate 20% Percoll solution and gently drip it onto the upper layer of the 40% Percoll solution, avoiding mixing the two, to form an obvious boundary.

[0096] (5) Perform density gradient centrifugation on the 15 mL centrifuge tube, with the centrifuge accelerating and decelerating 3 times, at a rotation speed of 600 g for 20 min; after centrifugation, aspirate the interface between the 20% Percoll solution and the 40% Percoll solution, add an appropriate amount of transport solution for washing, centrifuge at 300 g for 3 min, and discard the supernatant.

[0097] (6) Take an appropriate amount of Matrigel to resuspend the cell mass precipitate, and then use a pipette to inoculate the gel mixed with cells into a 24-well plate, about 30 μL per well.

[0098] (7) Take an appropriate amount of Matrigel and mix it with the precipitate, inoculate it into a 24-well plate, place the culture plate in a CO 2 incubator and let it stand for 15 - 20 min. After it has fully solidified, add the lung adenocarcinoma tumor organoid medium for culture. Observe the growth status of the lung adenocarcinoma organoids under an inverted optical microscope ( Figure 2 、 Figure 3 ); Take the lung adenocarcinoma organoids cultured for 7 days with stable passage three times for immunohistochemical staining ( Figure 4 ). In this example, immunohistochemical staining was performed on three lung adenocarcinoma biomarkers, Ki67, TTF-1, and Napsin A, to determine whether the obtained organoids have the characteristics of lung adenocarcinoma organoids.

[0099] In this example, the culture conditions for lung adenocarcinoma organoids are as follows in the table:

[0100]

[0101] The results showed that: (1) Compared with the control group, when using the tissue preservation solution of the present invention to preserve and transport tumor tissue liquid samples for constructing and culturing lung adenocarcinoma organoids, the success rate of organoids was significantly improved, with a large number and uniform morphology; (2) The results of immunohistochemical staining showed that for the lung adenocarcinoma organoids constructed and cultured from the tumor tissue liquid samples preserved and transported using the tissue preservation solution of the present invention, the expression abundances of the lung adenocarcinoma biomarkers Ki67, TTF-1, and Napsin A were high, and they could be used for clinical detection and disease research of lung adenocarcinoma.

[0102] Example 2: Preservation of tumor tissue samples derived from pleural effusion and construction of breast cancer organoids

[0103] Reference Figure 1 , in this example, the construction and culture of pleural effusion - breast cancer organoids were carried out according to the following experimental steps:

[0104] (1) Add the pleural effusion samples of breast cancer patients into the pre-cooled tissue preservation solution of the present invention (experimental group) respectively, mix well and send them to the laboratory for pretreatment within 12 h;

[0105] (2) Centrifuge the obtained effusion samples at 300 g for 5 min in a centrifuge, discard the supernatant, wash once with the transport solution, and then centrifuge at 300 g for 5 min again, discard the supernatant;

[0106] (3) Resuspend the precipitate with 4 mL of 40% Percoll solution;

[0107] (4) Add 4 mL of 70% Percoll solution into a 15 mL centrifuge tube, aspirate the 40% Percoll precipitate resuspension in step 3, and gently drip it onto the upper layer of the 70% Percoll solution to form an obvious liquid interface; aspirate 20% Percoll solution and gently drip it onto the upper layer of the 40% Percoll solution to avoid mixing them and form an obvious boundary;

[0108] (5) Perform density gradient centrifugation on the 15 mL centrifuge tube, with the centrifuge accelerating from 3 to 3, the rotation speed of 600 g, and centrifuge for 20 min; after centrifugation, aspirate the interface between the 20% Percoll solution and the 40% Percoll solution, add an appropriate amount of transport solution for washing, centrifuge at 300 g for 3 min, and discard the supernatant;

[0109] (6) Take an appropriate amount of Matrigel to resuspend the cell mass precipitate, and then use a pipette to inoculate the gel mixed with cells into a 24-well plate, about 30 μL per well;

[0110] (7) Take an appropriate amount of Matrigel and mix it with the precipitate, inoculate it into a 24-well plate, and place the culture plate in CO 2Let it stand still in the incubator for 15 - 20 min. After it has fully solidified, add breast cancer tumor organoid medium for culturing. Observe the growth status of breast cancer organoids under an inverted optical microscope as Figure 5 shown; take the breast cancer organoids cultured for 7 days for immunohistochemical staining ( Figure 6 ). In this example, immunohistochemical staining is performed on three breast cancer biomarkers, Ki67, ER, and HER2, to determine whether the obtained organoids have the characteristics of breast cancer organoids.

[0111] In this example, the culture conditions for breast cancer cell organoids are as follows in the table:

[0112]

[0113] The results show that: (1) When using the tissue preservation solution of the present invention to preserve and transport tumor tissue liquid samples to construct and culture breast cancer organoids, the success rate of organoids is significantly improved and the quantity is significantly increased; (2) The immunohistochemical staining results show that for the breast cancer organoids constructed and cultured from the tumor tissue liquid samples preserved and transported using the tissue preservation solution of the present invention, the breast cancer biomarkers Ki67, ER, and HER2 have high expression abundances and can be used for breast cancer clinical detection and disease research.

[0114] Example 3: Preservation of peripheral blood tumor tissue samples of colorectal cancer patients and construction of colorectal cancer organoids

[0115] Refer to Figure 1 , in this example, the separation of circulating tumor cells from colorectal cancer patients and the construction and culture of organoids are carried out according to the following experimental steps:

[0116] (1) Add the pre - cooled tissue preservation solution of the present invention (experimental group) to the peripheral blood samples of colorectal cancer patients for preservation, and send them to the laboratory for pretreatment within 12 h;

[0117] (2) Add an appropriate amount of Ficoll separation solution to the bottom of a new 15 mL centrifuge tube, tilt the centrifuge tube to an angle of 30 - 45 degrees, and slowly suck the peripheral blood and CTCs mixed solution and add it to the upper layer of the Ficoll separation solution.

[0118] (3) Transfer the centrifuge tube to a high - speed refrigerated centrifuge, set the acceleration for speed increase to 9, the acceleration for speed decrease to 1, and centrifuge at 600g for 30 min;

[0119] (4) After gradient centrifugation, suck the milky white cell suspension in the second layer from the bottom up and transfer it to a 15 mL centrifuge tube;

[0120] (5) Add 3 mL - 5 mL of transport medium to the centrifuge tube to resuspend the cells. Use Invitrogen Dynabeads CD45 to adsorb immune cells. Collect the unadsorbed cell suspension in a 15 mL centrifuge tube and centrifuge at a low speed of 300 g for 3 minutes.

[0121] (7) Take an appropriate amount of Matrigel to resuspend the cell mass precipitate, and then use a pipette to inoculate the gel mixed with cells into a 24-well plate, about 30 μL per well.

[0122] (8) Take an appropriate amount of Matrigel and mix it with the precipitate, then inoculate it into a 24-well plate. Place the culture plate in a CO 2 incubator and let it stand for 15 - 20 minutes. After it solidifies sufficiently, add colorectal cancer organoid medium for culture. Observe the growth status of colorectal cancer organoids under an inverted light microscope as Figure 7 shown. Take colorectal cancer organoids cultured for 7 days for immunohistochemical staining ( Figure 8 ). In this example, immunohistochemical staining was performed on three colorectal cancer biomarkers, Ki67, CDX2, and CK20, to determine whether the obtained organoids have the characteristics of colorectal cancer organoids.

[0123] In this example, the culture conditions for colorectal cancer cell organoids are as follows in the table:

[0124]

[0125] The results show that: (1) Using the tissue preservation solution of the present invention to preserve and transport tumor tissue liquid samples to construct and culture colorectal cancer organoids, the success rate of organoids is significantly improved and the number is significantly increased; (2) The immunohistochemical staining results show that for colorectal cancer organoids constructed and cultured using the tumor tissue liquid samples preserved and transported by the tissue preservation solution of the present invention, the colorectal cancer biomarkers Ki67, CDX 2 and CK20 have high expression abundances and can be used for clinical detection and disease research of colorectal cancer.

[0126] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A buffer for tissue separation and / or preservation, characterized in that The buffer is used to store tumor tissue samples, the tumor tissue samples are of liquid origin, and the tumor tissue samples are from at least one of malignant effusion, peripheral blood, bone marrow aspirate, alveolar lavage fluid, urine, and thoracic or abdominal puncture aspirate; The buffer comprises: a basal culture medium, a specific additive factor, and a hydrogen ion buffer, wherein the specific additive factor comprises: superoxide dismutase, catalase, penicillin, streptomycin, vitamin E, transferrin, recombinant human albumin or human serum albumin, Y27632, gentamicin, amphotericin, and N-acetylcysteine ​​amide; Based on the volume of the basal culture medium, the concentration of the superoxide dismutase is 20-40 μg / mL, the concentration of the catalase is 250-350 μg / mL, the concentration of the penicillin is 50-200 U / mL, the concentration of the streptomycin is 50-200 μg / mL, the concentration of the vitamin E is 20-30 nM, the concentration of the transferrin is 1-100 nM, the concentration of the recombinant human albumin or human serum albumin is 0.025-10 mg / mL, the concentration of the Y27632 is 0.5-50 μM, the concentration of the gentamicin is 1-100 μg / mL, the concentration of the amphotericin is 10-1000 ng / mL, and the concentration of the N-acetylcysteine ​​amide is 0.0625-10 mM.

2. The buffer according to claim 1, characterized in that Based on the volume of the basal medium, the concentration of the superoxide dismutase was 20 μg / mL, the concentration of the catalase was 300 μg / mL, the concentration of the penicillin was 100 U / mL, and the concentration of the streptomycin was 100 μg / mL.

3. The buffer according to claim 1, characterized in that Based on the volume of the basal medium, the concentration of vitamin E is 25 nM.

4. The buffer according to claim 1, characterized in that Based on the volume of the basal culture medium, the concentration of transferrin is 20 nM, the concentration of recombinant human albumin or human serum albumin is 0.5 mg / mL, the concentration of Y27632 is 10 μM, the concentration of gentamicin is 10 μg / mL, and the concentration of amphotericin is 250 ng / mL.

5. The buffer according to claim 1, characterized in that The concentration of N-acetylcysteine ​​amide was 1.25 mM.

6. The buffer according to claim 1, characterized in that The concentration of the hydrogen ion buffer is 5-50 mM.

7. The buffer according to claim 6, characterized in that The hydrogen ion buffer is HEPES, and the concentration is 10 mM.

8. The buffer according to any one of claims 1 to 5, characterized in that The basic culture medium is DMEM / F12.

9. Use of the buffer according to any one of claims 1 to 8 in the preservation of tumor tissue samples, wherein the tumor tissue samples are of liquid origin; The tumor tissue sample is from at least one of malignant effusion, peripheral blood, bone marrow aspirate, bronchoalveolar lavage fluid, urine, and thoracic or abdominal puncture fluid.

10. The use according to claim 9, characterized in that: The tumor tissue sample is used for tumor organoid culture.

11. The use according to claim 10, characterized in that: The tumor organoids include: at least one of lung adenocarcinoma organoids, lung cancer organoids, breast cancer organoids, colorectal cancer organoids, ovarian cancer organoids, gastric cancer organoids, liver cancer organoids, brain tumor organoids, bladder cancer organoids, and meningeal cancer organoids.

12. A method for culturing tumor organoids, characterized in that: include: Before the tumor tissue sample is subjected to the organoid culture treatment, the sample is stored in the buffer according to any one of claims 1 to 8, wherein the tumor tissue sample is of liquid origin; The tumor tissue sample is from at least one of malignant effusion, peripheral blood, bone marrow aspirate, bronchoalveolar lavage fluid, urine, and thoracic or abdominal puncture fluid.

13. The method according to claim 12, characterized in that The tumor organoids include: at least one of lung adenocarcinoma organoids, lung cancer organoids, breast cancer organoids, colorectal cancer organoids, ovarian cancer organoids, gastric cancer organoids, liver cancer organoids, brain tumor organoids, bladder cancer organoids, and meningeal cancer organoids.

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