Biological tissue cryopreservation method and construction and related application of biological sample library

Abstract

The invention discloses a biological tissue cryopreservation method and construction and related application of a biological sample library, and relates to the field of biology. According to the invention, a biological tissue block is filtered through a 500 [mu] m grid so as to obtain a more uniform sample, and then the tissue is gradually cooled and transferred to liquid nitrogen for preservation. According to the method, the activity of cells in the tissue is effectively maintained, long-distance transportation and storage of the sample are facilitated, the cryopreserved tissue sample can be applied to construction of PDOs, and the success rate exceeds 95%; the cryopreserved sample is also suitable for the construction of a gas-liquid interface culture model, and the culture method can retain the original tumor microenvironment, allows the test of the reaction of anti-tumor drugs including immune checkpoint inhibitors, and provides a new way for the research and development of drugs.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a method for cryopreservation of biological tissues and the construction and related applications of a biological sample library. Background Art

[0002] Biobanking involves the collection and storage of large numbers of biological specimens, along with associated personal and health data, such as health records, family histories, lifestyle information, and genetic data. These valuable resources primarily facilitate health and medical research efforts. In recent years, there has been a marked increase in the establishment of large specimen biobanks, including tumor tissue and other samples. These banks typically store specimens directly under cryogenic conditions, i.e., deep freeze or in liquid nitrogen. Unfortunately, most storage methods kill cells and are limited to use for the extraction of proteins, DNA, or RNA, but cannot be used to culture living cells, such as patient-derived tumor organoids (PDOs), which have great potential for personalized therapy.

[0003] Patient-derived organoids (PDOs) are ex vivo tumor models developed in recent years. They are three-dimensional tumor models derived from tissue subunits or stem cells under specific culture conditions. The PDO model has a fast modeling speed and a high success rate. It can reproduce the key characteristics of the original tumor, such as tissue structure, heterogeneity, and physiological properties. It is an ideal model to simulate the parent tumor in an ex vivo environment. The extensive cancer PDO models that have been successfully established help to better understand the development pattern of cancer and provide an ideal model to explore the interaction between drugs and tumors. Existing research results show that PDO drug sensitivity testing can not only reflect the patient's previous clinical treatment response, but also predict the patient's effective individualized treatment options, providing a new method for drug research and development.

[0004] Currently, most PDO models use fresh tumor specimens as starting materials to ensure the success rate of modeling to a certain extent. However, a portion of patients will experience tumor recurrence or metastasis after treatment; these patients are usually unable to undergo surgery again, and specimen acquisition is often difficult. In addition, recurrent and / or metastatic tumors usually develop rapidly and are resistant to most treatments, requiring the best treatment plan to be determined in the shortest possible time. For these tumor patients, if live tumor specimens are preserved at the time of initial surgery, frozen specimens can be reused to establish PDO models and perform drug sensitivity testing, thereby helping to provide personalized treatment options in a timely manner.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a method for cryopreservation of biological tissues and the construction and related applications of a biological sample bank.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides a method for cryopreservation of biological tissue, which comprises the following steps: preprocessing the biological tissue into a biological tissue block with a diameter of less than 500 μm; placing the biological tissue block in a tissue freezing solution, cooling it down in a gradient manner, and then cryopreserving it in liquid nitrogen; wherein the gradient cooling includes: freezing at -30 to -10°C in the first stage for 0.5 to 5 hours; freezing at -100 to -60°C in the second stage for 10 to 40 hours.

[0009] In a second aspect, an embodiment of the present invention provides a method for constructing a biological sample library, which comprises: preserving biological tissue samples using the biological tissue cryopreservation method described in the above embodiment.

[0010] In a third aspect, embodiments of the present invention provide the use of biological tissues cryopreserved by the cryopreservation method described in the aforementioned embodiments in the construction of organoids.

[0011] In a fourth aspect, an embodiment of the present invention provides the use of biological tissues cryopreserved by the cryopreservation method described in the above embodiments in preparing a gas-liquid interface culture model.

[0012] In a fifth aspect, embodiments of the present invention provide applications of biological tissues cryopreserved by the cryopreservation method described in the preceding embodiments, or organoids or air-liquid interface culture models constructed from the cryopreserved biological tissues, in drug screening or drug efficacy evaluation.

[0013] The present invention has the following beneficial effects:

[0014] The present invention standardizes the technology by filtering small tissue blocks through a 500μm mesh to obtain a more uniform sample, then gradually cools the tissue and transfers it to liquid nitrogen for storage. This method effectively maintains the vitality of cells in the tissue, which is conducive to the long-distance transportation and storage of samples. The frozen tissue samples can be used for the construction of PDOs with a success rate of over 95%; the frozen samples are also suitable for the construction of air-liquid interface culture models. This culture method can preserve the original tumor microenvironment and allow the testing of the response of anti-tumor drugs including immune checkpoint inhibitors, providing a new way for drug research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 Figure 2. Schematic diagram of the workflow for collecting, processing, cryopreserving surgically resected tumor tissue, and culturing organoids. (A) Schematic diagram of the workflow for collecting, processing, cryopreserving surgically resected tumor tissue, and culturing organoids. (B) Bright field microscopy images show the morphology of five pairs of organoids derived from fresh (top row) and frozen (bottom row) human tumor tissues. Scale bar, 100 μm. (C) Bright field microscopy images show the morphology of one pair of fresh (CPDO8, top row) and frozen (F-CPDO8, bottom row) colorectal cancer tissue-derived organoids at different time points during culture. Scale bar, 100 μm. (D) Histological and immunohistochemical images show the structural organization and expression of proliferation markers (Ki-67) and colorectal cancer-related markers (CK20, CDX2, and MUC2) in primary colorectal cancer (CPT16) and its fresh (CPDO16) and frozen (F-CPDO16) tissue-derived organoids. Scale bar, 20 μm.

[0017] Figure 2 Genetic features of parental tumors are recapitulated for organoids derived from fresh and frozen tumor tissues; (A) Comparison of the copy number alteration (CNA) landscape of six pairs of organoids derived from fresh (PDOs) and frozen tumor tissues (F-PDOs) and their parental tumors, with copy number amplifications represented by red shading and copy number deletions represented by blue shading; (B) Heat map comparing copy number alterations (CNAs) of cancer genes in fresh (PDOs) and frozen tumor tissue-derived organoids (F-PDOs) with parental tumors; (C) Heat map comparing somatically mutated cancer genes in fresh (PDOs) and frozen tumor tissue-derived organoids (F-PDOs) with somatically mutated cancer genes in parental tumors; (D) Scatter plot of CNA of primary tumor tissue (green), fresh tumor tissue-derived organoids (dark blue), frozen tumor tissue-derived organoids (red), and germline (grey); copy number is shown on the y-axis, with chromosomes arranged from left to right;

[0018] Figure 3 The drug responses of organoids derived from frozen and fresh tumor tissues are consistent; (A) Heat map shows the IC of 14 cancer therapeutic drugs in 5 pairs of fresh tumor tissue-derived organoids (PDOs) and frozen tumor tissue-derived organoids (F-PDOs). 50Values, the tested drugs and their targets are listed on the right; (B) Comparison of representative drug response curves of a pair of colorectal cancer organoids derived from fresh tissue (CPDO5) and frozen tissue (F-CPDO5); Results are expressed as mean ± SEM; (C) 3D bispectral images showing the drug response of a pair of colorectal cancer organoids derived from fresh (CPDO5) and frozen tissue (F-CPDO5) after 96 hours of treatment with sensitive drugs (neratinib, 1.0 μM) and insensitive drugs (clofarabine, 1.0 μM), purple represents active organoids, while yellow represents dead organoids; (D) Quantification of the cytotoxic effects of drugs on organoids in (D) by 3D live cell imaging; Results are expressed as mean ± SEM (n = 3), ** indicates P < 0.01; ns indicates P > 0.05;

[0019] Figure 4 After long-term culture, organoids derived from frozen tumor tissues retain their morphological structure, histological characteristics and genomic characteristics; (A) Bright field microscopy images show the morphology of fresh colorectal cancer tissue-derived organoids (CPDO3-P5) at passage 5, and the morphology of corresponding frozen tumor tissue-derived organoids at passages 5 (F-CPDO3-P5), 13 (F-CPDO3-P13) and 23 (F-CPDO3-P23); scale bar, 100 μm; (B) Histological and immunohistochemical images show the structural organization and expression of proliferation markers (Ki-67) and colorectal cancer-related markers (CK20, CDX2 and MUC2) of primary colorectal tumors (CPT3), as well as the morphology of fresh tumor tissue-derived organoids at passages 5 (F-CPDO3-P5), 13 (F-CPDO3-P13) and 23 (F-CPDO3-P23). ), organoids derived from frozen tumor tissues at passage 13 (F-CPDO3-P13) and passage 23 (F-CPDO3-P23); scale bar, 20 μm; (C) Comparison of the CNA landscape of two pairs of organoids derived from fresh tumor tissues (PDOs) and frozen tumor tissues of different passages (F-PDOs) and their parental tumors; copy number amplification is indicated by red shading, while copy number deletion is indicated by blue shading; (D) Heatmap compares the CNAs of cancer genes in two pairs of organoids derived from fresh tumor tissues (PDOs) and frozen tumor tissues of different passages (F-PDOs) and their parental tumors; (E) Heatmap compares somatically mutated cancer genes in two pairs of organoids derived from fresh tumor tissues (PDOs) and frozen tumor tissues of different passages (F-PDOs) and their parental tumors;

[0020] Figure 5To identify effective drugs against drug-resistant tumors using organoids derived from frozen tumor tissues of drug-resistant patients; (A) Schematic diagram of the workflow of tumor tissue collection, processing, freezing, organoid culture and high-throughput drug screening of effective drugs against drug-resistant tumors; (B) High-throughput drug screening (using 430 kinase inhibitors) identified 30 drugs that were most sensitive to organoids (F-BPDO1) derived from frozen breast cancer tissue with multidrug resistance; bar graph shows the IC 50 Values; (C) Pie chart showing the signaling pathways targeted by the 30 drugs most sensitive to F-BPDO1; (D) Drug response curves of the three most sensitive drugs for F-BPDO1 (dinaciclib (SCH727965), CUDC-907, and flavopiridol (alvocidib)); (E) 3D bispectral image showing the drug response of F-BPDO1 after 96 hours of treatment with 0.02 μM and 0.05 μM concentrations of dinaciclib; purple represents active organoids, while yellow represents dead organoids; (F) Quantification of the cytotoxic effects of drugs on organoids in (E) by 3D live cell imaging; Results are expressed as mean ± SEM (n = 3); ** indicates P < 0.01; *** indicates P < 0.001;

[0021] Figure 6Cryopreserved tumor tissues can be used for air-liquid interface (ALI) culture and screening of anti-tumor drugs; (A) Schematic diagram showing the workflow of tumor tissue collection, processing and cryopreservation, ALI culture and its application in screening of anti-tumor drugs, including immune checkpoint inhibitors (ICIs); (B) Histological and immunohistochemical images demonstrate that similar to fresh tissues, cryopreserved tissues preserve the structure of the original tumor tissue (HP6654T) after ALI culture and the infiltration of SMA-positive fibroblasts, CD45-positive leukocytes and CD8-positive T lymphocytes; (C) Optical microscopy images of fresh and frozen tumor tissues (HP6654T) cultured in ALI and stained with MTT after treatment with different drugs; (D) MTT was used to analyze the cytotoxic effects of drugs on fresh tumor tissues in Figure (C); Results are expressed as mean ± SEM (n = 3 ), ** indicates P < 0.01, ns indicates P > 0.05; (E) In Figure (C), MTT was used to analyze the cytotoxic effects of drugs on frozen tumor tissues; the results are expressed as mean ± SEM (n = 3), ** indicates P < 0.01; ns indicates P > 0.05; (F) Light microscopic images of fresh and frozen patient colorectal cancer tissues (BPT12) cultured with ALI and treated with different drugs, MTT staining; (G) MTT was used to analyze the cytotoxic effects of drugs on fresh tumor tissues in (F), and the results are expressed as mean ± SEM (n = 3); ** indicates P < 0.01, *** indicates P < 0.001; (H) MTT was used to analyze the cytotoxic effects of drugs on frozen tumor tissues in (F); the results are expressed as mean ± SEM (n = 3), ** indicates P < 0.01, *** indicates P < 0.001;

[0022] Figure 7 The types of tumors in cryopreserved human tissues and the success rate for culturing organoids; (A) The types and numbers of human tumor specimens used in this application; (B) The success rate of culturing PDO in cryopreserved tumor tissues; (C) The duration of cryopreservation of each tumor tissue before culturing organoids. Light blue (kidney cancer) and dark blue (breast cancer, colorectal cancer, and lung cancer) represent tumor tissues that were successfully cultured, while red represents samples that were not successfully cultured.

[0023] Figure 8To establish organoids using cryopreserved mouse tumor biopsy tissue; (A) Schematic diagram of the workflow for collecting, processing, cryopreserving biopsied mouse tumor tissue, and culturing organoids; (B) Bright field microscopy images showing organoid morphology (after 10 days of culture) obtained from fresh (top row), 7-day cryopreserved (middle), and 6-month cryopreserved (bottom row) biopsied mouse tumor tissue; scale bar, 100 μm; (C) Bright field microscopy images showing the morphology of organoids derived from fresh (top row), 7-day cryopreserved (middle), and 6-month cryopreserved (bottom row) biopsied mouse tumor tissue (H6433T) at different time points during the culture process; scale bar, 100 μm;

[0024] Fig. 9 Comparison of morphological and histological features of organoids derived from frozen and fresh tumor tissues; (A) Bright field microscopy images show the morphology of organoids derived from fresh and frozen human tumor tissues; Scale bar, 100 μm; (B) Histological and immunohistochemical images show the expression of structural organization and proliferation markers (Ki-67) and breast cancer-related markers (ER, PR and HER2) of primary breast cancer (BPT8), as well as its fresh tissue-derived (BPDO8) and frozen tissue-derived (F-BPDO8) organoids; Scale bar, 20 μm; (C) Histological and immunohistochemical images show the expression of structural organization and proliferation markers (Ki-67) and colorectal cancer-related markers (CK20, CDX2 and MUC2) of primary colorectal cancer (CPT2) organoids, as well as fresh tissue-derived (CPDO2) and frozen tissue-derived (F-CPDO2) organoids; Scale bar, 20 μm;

[0025] Fig.10 Comparison of genetic characteristics between organoids derived from frozen and fresh tumor tissues and primary lesions; (A) Comparison of copy number alteration (CNA) landscapes of 12 pairs of organoids derived from fresh (PDOs) and frozen tumor tissues (F-PDOs) with their parental tumors, with copy number amplification (CNA) represented by red shading and copy number deletion represented by blue shading; (B) CNA scatter plot of primary breast cancer tissue (BPT2, green), fresh tissue-derived organoids (BPDO2, dark blue), frozen tissue-derived organoids (F-BPDO2, red) and germline (gray), with copy number displayed on the axis; chromosomes are arranged from left to right; (C) CNA scatter plot of primary colorectal cancer tissue (CPT8, green), fresh tissue-derived organoids (CPDO8, dark blue), frozen tissue-derived organoids (F-CPDO8, red) and germline (gray), with copy number displayed on the y-axis and chromosomes arranged from left to right;

[0026] Fig.11Drug response analysis of organoids from frozen and fresh tumor tissues; (A) Comparison of the response curves of 14 drugs in a pair of colorectal cancer organoids from fresh (CPDO5) and frozen tissues (F-CPDO5), the results are expressed as mean ± SEM; (B) 3D bispectral images showing the drug response of a pair of breast cancer organoids from fresh (BPDO7) and frozen tissues (F-BPDO7) after 96 hours of treatment with sensitive drugs (afatinib, 0.1 μM) and insensitive drugs (gemcitabine hydrochloride, 2.0 μM), purple represents active organoids, while yellow represents dead organoids; (C) Quantification of the cytotoxic effects of drugs in (B) on organoids by 3D live cell imaging, the results are expressed as mean ± SEM (n = 3), ** indicates P < 0.01; ns indicates P > 0.05;

[0027] Fig.12 Organoids derived from frozen tumor tissues retain their morphological structure, histological characteristics and genomic characteristics after long-term culture; (A) Bright field microscopy images show the morphology of fresh breast cancer tissue-derived organoids (BPDO5-P5) at the 5th generation, and the morphology of corresponding frozen tumor tissue-derived organoids at the 4th generation (F-BPDO5-P4), the 10th generation (F-BPDO5-P10) and the 19th generation (F-BPDO5-P19), scale bar, 100 μm; (B) Histological and immunohistochemical images showing the expression of structural organization and proliferation markers (Ki-67) and breast cancer-related markers (ER, PR and HER2) in primary breast cancer tissue (BPT5), as well as the 5th generation fresh tumor tissue-derived organoids (BPDO5-P5) and the 4th generation (F-BPDO5-P4), the 10th generation (F-BPDO5-P10) and the 19th generation (F-BPDO5-P19), scale bar, 100 μm. 19th generation (F-BPDO5-P19), scale bar, 20 μm; (C) CNA scatter plots of alterations detected in primary breast cancer tissue (BPT5), fresh tumor tissue-derived organoids at generation 5 (BPDO5-P5), and frozen tumor tissue-derived organoids at generation 4 (F-BPDO5-P4), generation 10 (F-BPDO5-P10), and generation 19 (F-BPDO5-P19), with copy number indicated on the y-axis and chromosomes arranged from left to right; (D) CNA scatter plots of alterations detected in primary colorectal cancer tissue (CPT3), fresh tumor tissue-derived organoids at generation 5 (CPDO3-P5), and frozen tumor tissue-derived organoids at generation 5 (F-CPDO3-P5), generation 13 (F-CPDO3-P13), and generation 23 (F-CPDO3-P23), with copy number indicated on the y-axis and chromosomes arranged from left to right;

[0028] Fig.13Organoids derived from cryopreserved tumor tissue retain their drug responses after long-term culture; (A) Heat map showing the IC of 14 cancer therapeutic drugs in two pairs of fresh tumor tissue-derived organoids (PDOs) and frozen tumor tissue-derived organoids (F-PDOs, three different passages). 50 Values, the tested drugs and their targets are listed on the right; (B) Comparison of the response curves of 14 drugs in the 5th generation fresh colorectal cancer tissue-derived organoids (CPDO3-P5) and their frozen tissue-derived organoids at the 5th generation (F-CPDO3-P5), 13th generation (F-CPDO3-P13) and 23rd generation (F-CPDO3-P23), the results are expressed as mean ± SEM;

[0029] Fig.14 To identify effective drugs against drug-resistant tumors using organoids derived from frozen tumor tissues of drug-resistant patients; (A) High-throughput drug screening (using 430 kinase inhibitors) identified 30 drugs that were most sensitive to organoids derived from frozen breast cancer tissue with multidrug resistance (F-BPDO5). The bar graph shows the IC 50 Values; (B) Pie chart showing the signaling pathways affected by the 30 drugs most sensitive to F-BPDO5; (C) Drug response curves of the three most sensitive drugs to F-BPDO5 (GSK2126458 (GSK458), dinaciclib (SCH727965) and BGT (NVP-BGT226)); (D) 3D dual-spectrum image showing the drug response of F-BPDO5 after 96 hours of treatment with 0.02 μM and 0.05 μM concentrations of dinaciclib, purple represents active organoids, while yellow represents dead organoids; (E) Quantification of the cytotoxic effects of drugs in (D) on organoids by 3D live cell imaging, the results are expressed as mean ± SEM (n = 3), *** indicates P < 0.001;

[0030] Fig.15Cryopreserved tumor tissues can be used for air-liquid interface (ALI) culture and screening of antitumor drugs; (A) Histological and immunohistochemical images demonstrated that after ALI culture, the infiltration of CD4-positive T lymphocytes and F4 / 80-positive macrophages was retained in the cryopreserved tissues (HP6654T), similar to fresh tissues. In addition, the proliferation activity (Ki-67) and expression of PD-L1 were also well preserved; (B) Optical microscopy images of fresh and frozen tumor tissues (HP6403T) cultured by ALI, using different The cytotoxic effects of drugs on fresh tumor tissues were analyzed by MTT staining. (C) In Figure (B), the cytotoxic effects of drugs on fresh tumor tissues were analyzed by MTT staining. The results are expressed as mean ± SEM (n = 3). * indicates P < 0.05, ** indicates P < 0.01, and ns indicates P > 0.05. (D) In ​​Figure (B), the cytotoxic effects of drugs on frozen tumor tissues were analyzed by MTT staining. The results are expressed as mean ± SEM (n = 3). * indicates P < 0.05, ** indicates P < 0.01, and ns indicates P > 0.0 5; (E) Optical microscopy images of fresh and frozen patient colorectal cancer tissues (CPT20) cultured with ALI and treated with different drugs and stained with MTT; (F) The cytotoxic effects of drugs on fresh tumor tissues were analyzed by MTT in (E), and the results are expressed as mean ± SEM (n = 3), * indicates P < 0.05, ** indicates P < 0.01; (G) The cytotoxic effects of drugs on frozen tumor tissues were analyzed by MTT in (E), and the results are expressed as mean ± SEM (n = 3), * indicates P < 0.05 , ** indicates P < 0.01; (H) Optical microscopy images of fresh and frozen patient colorectal cancer tissues (CPT21) cultured in ALI, treated with different drugs, and stained with MTT; (I) The cytotoxic effects of drugs on fresh tumor tissues were analyzed by MTT in (H), and the results are expressed as mean ± SEM (n = 3), ns indicates P > 0.05; (J) The cytotoxic effects of drugs on frozen tumor tissues were analyzed by MTT in (H), and the results are expressed as mean ± SEM (n = 3), ns indicates P > 0.05. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0032] Based on the defects of existing biological tissue preservation technology, the inventors of this application provide a new cryopreservation technology. It has been verified that tumor specimens preserved by this technology maintain their vitality, which is conducive to long-distance transportation and storage of samples, and can still successfully culture PDOs after long-term freezing, with a success rate of up to 95.2%. It is worth noting that even after nearly 4 years of freezing, some tumor samples can still be used for organoid establishment ( Figure 7 C). More importantly, PDOs derived from frozen tissues are highly similar to PDOs derived from fresh tissues in terms of structural characteristics, tumor marker expression, and drug response direction. And PDOs derived from frozen tissues can be routinely passaged and frozen, making them ideal for high-throughput drug screening at any time. In addition, frozen biological tissues are also suitable for air-liquid interface (ALI) culture, which allows the original tumor microenvironment to be preserved, making them a valuable resource for anti-tumor drug response testing, including immune checkpoint inhibitors (ICI). The innovative cryopreservation technology provided in the embodiments of the present application can identify potentially effective drugs for patients, which is conducive to promoting the development of new therapeutic drugs and has a good application prospect for personalized treatment.

[0033] In one aspect, an embodiment of the present invention provides a method for cryopreservation of biological tissue, comprising the following steps:

[0034] Preprocessing biological tissue into biological tissue blocks with a diameter of less than 500 μm;

[0035] The biological tissue block is placed in a tissue freezing solution, and after gradient cooling, it is frozen and stored in liquid nitrogen; wherein the gradient cooling includes: freezing at -30 to -10°C in the first stage for 0.5 to 5 hours; and freezing at -100 to -60°C in the second stage for 10 to 40 hours.

[0036] Compared with larger tissue fragments (2-5 mm), 500 μm filtration allows cells in tissue fragments to fully contact the cryopreservation solution, avoiding or reducing damage during the freezing and recovery process.

[0037] In some embodiments, the pretreatment method comprises: placing the biological tissue on a 500 μm filter in a culture dish containing a culture medium, and crushing the biological tissue until it passes through the 500 μm filter to obtain the biological tissue block with a diameter of less than 500 μm.

[0038] Compared with other existing solutions that directly freeze samples, or omit the first freezing stage or the second freezing stage, the gradient cooling program of the present application is more conducive to improving the survival rate and function of cells after cryopreservation.

[0039] In some embodiments, the freezing temperature of the first stage can be any one of -30, -28, -26, -25, -24, -22, -20, -18, -16, 15, -14, -12, -10°C or a range between any two of them.

[0040] In some embodiments, the freezing time of the first stage can be any one of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 hours or a range between any two of them.

[0041] In some embodiments, the freezing temperature of the second stage can be any one of -100, -90, -85, -80, -75, -70, -60°C or a range between any two of them.

[0042] In some embodiments, the freezing time of the second stage can be any one of 10, 12, 14, 15, 16, 18, 20, 22, 24, and 25 hours, or a range between any two of them.

[0043] In some embodiments, the components of the tissue freezing solution include: dimethyl sulfoxide and FBS.

[0044] In some embodiments, the volume fraction of dimethyl sulfoxide in the tissue freezing solution is 80% to 99%, and the volume fraction (percentage) can specifically be any one of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or a range between any two of them.

[0045] In some embodiments, the volume fraction of the FBS in the tissue freezing solution is 1% to 20%, and the volume fraction can be any one of 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, or a range between any two thereof.

[0046] On the other hand, an embodiment of the present invention provides a method for constructing a biological sample library, which includes: preserving biological tissue samples using the biological tissue cryopreservation method described in any of the above embodiments.

[0047] The present invention has been verified to clearly verify the applicability of this technology (cryopreservation method) in effectively cryopreserving small specimens (including biopsy tissues). This technology can maintain different tumor specimens for long-term freezing while retaining the vitality of tumor cells in the tissue.

[0048] In some embodiments, the biological tissue sample includes: a biological tissue sample obtained by biopsy, puncture or surgical resection.

[0049] This technology has no special restrictions on the type of tumor and can be widely applied to different tumor specimens. In some embodiments, the tumor includes: benign tumors and malignant tumors. The tumor can specifically be breast cancer, colorectal cancer, lung cancer, kidney cancer, thyroid cancer, liver cancer, lung squamous cell carcinoma, esophageal cancer, pancreatic cancer, ovarian cancer, cervical cancer, prostate cancer, testicular cancer, etc.

[0050] On the other hand, an embodiment of the present invention provides the use of biological tissue cryopreserved by the cryopreservation method as described in any of the aforementioned embodiments in the construction of organoids.

[0051] The embodiments of the present application have no special restrictions on the method for constructing organoids, and can be implemented using existing publicly available construction methods. The inventive point of the present application is mainly to use the biological tissue preserved by the cryopreservation method provided in any of the aforementioned embodiments as the original material for the construction of organoids.

[0052] On the other hand, an embodiment of the present invention provides the use of biological tissue cryopreserved by the cryopreservation method as described in any of the above embodiments in preparing an air-liquid interface culture model.

[0053] The embodiments of the present application have no special restrictions on the method for constructing the gas-liquid interface culture model, and can be implemented using the existing disclosed gas-liquid interface culture method. The inventive point of the present application is mainly to use the biological tissue preserved by the cryopreservation method provided in any of the aforementioned embodiments as the original material for the cultivation of the gas-liquid interface culture model.

[0054] On the other hand, an embodiment of the present invention also provides the use of biological tissues cryopreserved by the cryopreservation method as described in any of the preceding embodiments, or organoids or air-liquid interface culture models constructed from the cryopreserved biological tissues in drug screening or drug efficacy evaluation.

[0055] In some embodiments, the drug includes: any one or more of an anti-tumor drug and an immune checkpoint inhibitor. The features and performance of the present invention are further described in detail below in conjunction with the examples.

[0056] Example 1

[0057] 1.1 Human samples

[0058] A total of 42 surgically resected human cancer tissue samples from the Kiang Wu Hospital in Macau were collected, including 2 renal cancer tissue samples, 11 breast cancer tissue samples, 13 lung cancer tissue samples, and 16 colon cancer tissue samples. The ethics committees of the University of Macau and all hospitals involved in this application carefully evaluated and approved the study (approval number: BSERE16-APP010-FHS). This application followed ethical guidelines, and informed consent was obtained from all donors before any samples were collected. The clinical data of the patients were obtained from the medical record system.

[0059] The method for cryopreservation of biological tissue samples includes the following steps:

[0060] Tumor tissue was placed on a 500-micron filter in a culture dish containing culture medium. Using scissors, the tissue pieces were carefully trimmed until they could pass through the filter smoothly to obtain fragments with a diameter of less than 500 microns. A portion of these fresh tissue fragments were used for organ sample culture, and the remaining tissue was suspended in a cryopreservation solution composed of 10% dimethyl sulfoxide (Sigma-Aldrich) and 90% FBS (GIBCO) at a density of approximately 200-300 fragments per tube. The tissue fragments were then divided into cryopreservation tubes for a gradient cooling, which included two hours in a -20°C freezer and then 22 hours in a -80°C freezer; finally transferred to liquid nitrogen for long-term storage. Careful optimization of each processing step is critical to ensure that the cells still have high survival and functionality after cryopreservation.

[0061] 1.2 Mouse samples

[0062] A total of 6 mice were used in the example: H5690T (Br co / co ,Cre-MMTV),H6164T(Br co / co ,Cre-MMTV),HP6403T(Br co / + ,Cre-MMTV),H6433T(Br co / co ,Cre-MMTV),HP6654T(Neu,Cre-MMTV) and H10166T(Fgfr2 pLoxpneo-S525w / + ,P53 co / + ,Cre-MMTV) mice were 129SVEV:Black Swiss:FVB mixed genetic background, with a ratio of approximately 1:1:2. These mice may spontaneously develop mammary tumors at approximately 12-24 months of age. After mice were euthanized by cervical dislocation, tumor tissues were obtained by surgical resection and subsequently processed in the same way as human tumor tissues (refer to step 1.1 for cryopreservation of biological tissue samples). To obtain core needle biological examination tumor tissues, mice were anesthetized in advance and then puncture biopsies were performed using an 18-gauge needle. Approximately 2 cm long biopsies were placed in cryovials containing 1 ml of cryopreservation solution. After gradient cooling (same as gradient cooling in step 1.1), the samples were transferred to liquid nitrogen for long-term storage. All animal experiments were approved by the Animal Ethics Committee of the University of Macau (approval number: UMARE-015-2019).

[0063] 1.3 Tissue digestion

[0064] Fresh tissue pieces can be used directly for tissue digestion without any pretreatment. Frozen tissue requires some preparation steps before digestion, which include thawing the tissue at 37°C and centrifuging the tissue at 400×g for 4 minutes. The resulting tissue pellet is then ready for digestion. To ensure optimal tissue separation, an appropriate amount of digestion buffer is added, which is usually 6-12 ml depending on the size of the tissue. The components of the digestion buffer include: DMEM / F12 medium (Gibco), collagenase type III (Worthington) at a concentration of 300U / mL, hyaluronidase (Sigma-Aldrich) at a concentration of 100U / mL, insulin (Sigma) at a concentration of 5μg / mL, hydrocortisone (Sigma-Aldrich) at a concentration of 500ng / mL, cholera toxin (Sigma-Aldrich) at a concentration of 20ng / mL, EGF (PeproTech) at a concentration of 10ng / mL, and FBS (Gibco) at a concentration of 5%. The tissue digestion process was carried out on a shaker at 37°C for 1 to 2 hours, with the solution being lifted off intermittently until all visible samples disappeared. The resulting cell pellet was centrifuged at 400×g for 4 minutes, then washed once with DMEM containing 5% FBS and centrifuged at 400×g for 4 minutes to obtain a concentrated cell pellet.

[0065] 1.4 Organoid culture, passaging, and cryopreservation

[0066] Organoid culture: The cell pellet was resuspended in 50% cold Matrigel (Corning) and then dropped into a pre-warmed 6-well plate (Corning), 25 μL each time. The drops were incubated in a 37°C and 5% CO2 incubator for 30 minutes before solidification. After solidification, 2.5 mL of organoid culture medium was added to each well, and the culture medium was renewed every 3-6 days according to the observed cell growth rate. Conditioned medium was prepared using the L-WRN cell line. 30% L-WRN conditioned medium was used for the organoid culture medium composition of breast cancer. The culture medium used for the other three cancers (renal cancer, lung cancer, and colon cancer) was: Advanced DMEM / F12 medium supplemented with 30% L-WRN conditioned medium, 5 μM Y-27632, 1.0 μM SB202190, 0.5 μM A83-01, 50 ng / ml EGF, 500 ng / ml hydrocortisone, 10 mM nicotinamide, 1.25 mM acetyl-L-cysteine, 15 mM HEPES, 1×B27, 1×GlutaMAX, 1×insulin-transferrin-selenium-sodium butyrate, 0.5 μg / ml mycin B, 5 μg / ml gentamicin, and 5 μg / ml pramofloxacin.

[0067] Organoid culture passaging: Organoids were passaged every 1-4 weeks, with the frequency of passaging determined by their size and density. The culture medium was removed and the organoids were gently resuspended in cold 0.25% trypsin using a 1 ml pipette. The resulting mixture was incubated at 37°C for 4 min; additional pipetting and incubation were performed as needed. The cells were centrifuged at 400 × g for 4 min, washed and suspended in 6 ml of DMEM containing 5% FBS, and then centrifuged again. Finally, the cells were resuspended in Matrigel at 1:3 to 1:6 depending on their density and growth rate, then seeded into pre-warmed 6-well plates and cultured as described above for organoid culture.

[0068] 1.5. Whole Exome Sequencing

[0069] DNeasy Blood & Tissue Kit (QIAGEN) was used to prepare genomic DNA samples according to the manufacturer's instructions. After hydrodynamic shearing of DNA using the Covaris system, 1 μg of genomic DNA was used for library construction per sample, generating fragment lengths of 180-280 bp. Whole exon capture libraries were prepared using the Agilent SureSelect Human All Exon Kit (Agilent Technologies, CA, USA) according to the manufacturer's recommended protocol. Subsequently, the products were assessed for quality and quantity using the Agilent High Sensitivity DNA Kit on the Agilent 2100 Bioanalyzer. Libraries were clustered on the cBot Cluster Generation System using the TruSeq PE Cluster Kit v4-cBot-HS (Illumina, San Diego, USA) and subsequently sequenced on the Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads.

[0070] 1.6. Whole exome sequencing data analysis

[0071] Quality control of DNA sequencing data was performed using MultiQC (v1.11). Adapter trimming was performed using Trim Galore (v0.6.7). Sequence reads were aligned to human genome build 38 (Hg38) using the Burrows-Wheeler Alignment tool with maximal exactmatches (v0.7.17), followed by repetitive sequence removal using Sambamba (v0.6.6), and indels and base recalibration were performed using the Genome Analysis ToolKit (v4.2.2.0) according to best practice guidelines.

[0072] Somatic SNVs were identified using three independent programs with default parameters: MuTect2 (v4.2.2.0), Strelka2 (v2.9.2), and LANCET (v1.1.0). Paired blood control samples or adjacent control samples were used as reference for somatic mutation identification of each tumor or PDO. The results of each identification program were stored in VCF format and further filtered to obtain the mutations that passed. VCF was converted to MAF using the vcf2maf tool (https: / / github.com / mskcc / vcf2maf) according to the annotation of the reference release of ENSEMBL Variant Effect Predictor. Only mutations detected by at least two identification programs were considered true positives and used for further analysis. To detect somatic CNAs (copy number alterations), BAM files of tumors or PDOs were analyzed by comparing them with reference blood controls or adjacent control samples using CNVkit (v0.9.9).

[0073] 1.7 Drug response test

[0074] Organoid samples were first dissociated into smaller cell clusters and then suspended in culture medium supplemented with 2.5% Matrigel. The resulting cell suspension was plated into 384-well plates coated with collagen I gels, 36 μL per well, corresponding to approximately 1500 cells. 48 hours after seeding, 4 μL of each compound, which had been appropriately diluted, was added to the corresponding wells. A total of 14 drugs were tested, and these compounds were used at their highest concentration of 20 μM and diluted 3-fold to seven concentrations. Each drug had three technical replicates on each of the three plates. When testing a drug plate containing 430 kinase inhibitors, the highest concentration was 10 μM and diluted 3-fold to eight concentrations. No replicates were analyzed for these drug plates. Positive controls used bolretrinib (1 μM), while negative controls used 0.2% dimethyl sulfoxide. After four days of drug incubation, cell viability was assessed using CellTiter-Glo 2.0 reagent (Promega) according to the manufacturer's instructions. Results were normalized to control values ​​and expressed as a percentage of cell viability. IC 50 Values ​​were determined by using GraphPad Prism 7. If some organ samples were resistant to a particular drug, their IC 50 The value will exceed the highest detectable concentration and be assigned to the highest tested drug concentration. Conversely, some organ samples show high sensitivity to a particular drug, and the IC 50 The values ​​exceeded the minimum detectable concentration and were therefore assigned as the lowest tested drug concentration.

[0075] 1.8. Live Cell Imaging of Organoids after Drug Treatment

[0076] The cell pellet was suspended in 50% cold Matrigel (Corning) and then dropped in 25 μL on a pre-warmed 35 mm glass-bottom culture dish with 4 chambers (Cellvis, D35C4-20-0-N). The drops were incubated in a cell culture incubator at 37°C and 5% CO2 for 30 minutes to solidify. After solidification, 0.6 mL of organoid culture medium was added to each chamber. Depending on the cell growth rate, the cells were cultured for 2-3 days, and then drugs were added and cultured for another 4 days. In order to mark living and dead cells (referred to as "imaging medium"), NucRed Dead 647 (one drop per ml; Thermo Fisher) and TO-PRO-3 (1:1,500; Thermo Fisher) were added for fluorescent labeling, respectively. The cells were further incubated for 2 hours for adequate staining. The culture dish was placed in a Nikon A1R confocal system with an incubator (37°C, 5% CO2). The emission spectra of fluorescent real-time dead cell dye (NucRed Dead 647, 638 nm) and live cell dye (TO-PRO-3, 562 nm) were imaged using the λ laser mode. Plan imaging was performed using a PlanApochromat ×20 / 0.8 numerical aperture objective with the following settings: online fingerprint mode, bidirectional scanning, optimal Z stack step size, total Z stack thickness of 200 μm, resolution of 1024 × 1024, and one image was taken every 2.5 μm.

[0077] 1.9 Air-liquid interface (ALI) culture and drug testing

[0078] To prepare the ice-cold collagen solution, solution A (rat collagen I, R&D Systems, 3447-020-01), solution B (10× Ham's F-12), and solution C (100 ml of 0.05 M NaOH and 200 mM HEPES sterile reconstitution buffer containing 2.2 g NaHCO3) were mixed in a volume ratio of 8:1:1. Subsequently, 150 μl of collagen solution was added to the well plate (SPLInsert Standing, 37524) and incubated in a 37°C incubator for 20-30 minutes until the collagen was completely solidified. Then, the tumor tissue (<500 μm) was mixed with the ice-cold collagen solution, and 50 μl of tissue suspension (about 200 pieces) was added to each well. After the gel containing the tissue on top solidified, 400 μl of ALI basal medium or drug-containing medium was added to the outside of the culture well. Next, the culture wells were placed in a humidity-controlled incubator containing 5% CO2 and 37°C for 4 days. At the end of the culture, 200 μL of 50 μg / mL MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) was added to each well and incubated at 37°C for 2 hours. Then, the upper layer containing the tissue was removed and placed in a 96-well plate, and 200 μL of DMSO was added to each well to dissolve the azo coloring substance in each well. The absorbance of each well was read at a wavelength of 490 nm. The ALI basal medium was F-medium, and 500 ml of the medium consisted of 373 ml of DMEM, 125 ml of Ham's F-12 nutrient mixture, 5 μg / ml insulin, 250 ng / ml mycin B, 10 μg / ml gentamicin, 0.1 nmol cholera toxin, 0.125 ng / ml epidermal growth factor, 25 ng / ml hydrocortisone, and 10 μmol Y-27632. To evaluate the antitumor activity of mouse PD-L1 antibody (BioLegend, 124301) or human PD-1 antibody (BeiGene, SFDAS20190045) (Table 3), 500 IU / ml of recombinant mouse IL-2 (PeproTech, 212-12) or 500 IU / ml of recombinant human IL-2 (PeproTech, 200-02) were added to the culture medium to maintain immune cell activity.

[0079] 1.10 Histology and Immunohistochemistry

[0080] Tumor tissues and ALI culture samples were fixed in 4% paraformaldehyde and subsequently embedded in paraffin using standard protocols. For PDOs samples, they were suspended in cold PBS by pipette, centrifuged, and then fixed in 4% paraformaldehyde for 24 h. Subsequently, organ samples were dehydrated and paraffin-embedded. Paraffin sections with a thickness of 5 μm were used for all analyses. Histopathological evaluation was performed using hematoxylin-eosin (H&E) staining, while immunohistochemical analysis was used to detect the expression of tumor markers. The primary antibodies used in this application include anti-ER (Abcam, ab16660, 1:200), anti-PR (Abcam, ab101688, 1:400), anti-HER2 (CST, 2165, 1:200), anti-CK20 (CST, 13063, 1:200), anti-CDX2 (Abcam, ab76541, 1:800), anti-MUC2 (CST, 88686, 1:200), anti-Ki-6 7 (CST, 9449S, 1:500), anti-CD4 (Abcam, ab183685, 1:300), anti-CD8 (CST, 98941, 1:300), anti-CD45 (CST, 70257, 1:400), anti-F4 / 80 (CST, 70076, 1:300), anti-SMA (Abcam, ab124964, 1:300) and anti-PD-L1 (CST, 13684, 1:200). Images were taken using a Nikon microscope (Nikon, ECLIPSE, Ci).

[0081] 1.11 Quantitative and statistical analysis

[0082] All analyses were performed in GraphPad Prism 7 (GraphPad Software, La Jolla, CA) and presented as means ± standard errors of the mean (SEMs) for individual samples. The P values ​​reported in the figures were obtained by unpaired two-tailed t-test. P values ​​< 0.05 were considered statistically significant. Details of the sequence analysis are described in the relevant methods section.

[0083] 2. Results

[0084] 2.1. Establishing organoids from cryopreserved live tumor tissue biobanks

[0085] In order to compare the effect of the cryopreservation method provided in Example 1 on the viability of cells in tissues, the viability of three fresh and frozen colon cancer specimens was compared.

[0086] The results showed that the cell viabilities of fresh tissues of the three specimens (CPT17, CPT18, and CPT19) were 85.65%, 87.9%, and 89.55%, respectively, while the cell viabilities within frozen tissues were 83%, 84.73%, and 85.58%, respectively. This suggests that freezing reduces the cell viability within the tissue, but the reduction is within 5%. In order to study the success rate of culturing PDOs from frozen tumor tissues, 42 surgically removed human cancer tissue samples were used, including 2 renal cancer tissue samples, 11 breast cancer tissue samples, 13 lung cancer tissue samples, and 16 colon cancer tissue samples. PDOs could be successfully established from all of these fresh tumor tissues. These specimens were frozen in liquid nitrogen for 4 to 46 months. Using a laboratory-developed protocol ( Figure 1 The results showed that except for one lung cancer tissue and one colon cancer tissue, PDOs were successfully established in the other 40 frozen tissues, with a success rate of 95.2% (40 / 42) ( Figure 7 Importantly, all nine specimens that had been frozen for more than 40 months were successfully cultured to obtain PDOs ( Figure 7 This achievement is strong evidence that the freezing technology provided by the embodiment of the present application can maintain viable cells in tumor tissue for a long period of time.

[0087] This application also studies the establishment of cryopreservation methods for small samples, such as mouse tumor biopsies, and studies the effect of cryopreservation time on their activity ( Figure 8 (A). Biopsied tumor tissue from four different mice was examined. It was found that after one week and six months of cryopreservation, the tumor tissue could be used in organoid culture and maintained a growth rate similar to that of fresh tissue-derived organoids ( Figure 8 These results suggest that small tissues preserved by this method have no negative impact on cell viability and can also be used for organ sample culture.

[0088] Next, the organoid samples were subjected to morphological analysis. It was found that most breast, lung, and kidney cancer organoid samples showed cystic or solid phenotypes, while one breast cancer organoid sample showed grape-like morphology ( Figure 1 Medium B and Fig. 9 Although most colon cancer organoids had hollow ring-like structures, a few had solid morphology ( Figure 1 Medium B and Fig. 9 (A). The morphology of organoid samples derived from frozen tissue was indistinguishable from that of organoid samples derived from fresh tissue. In addition, the growth patterns of organoid samples derived from fresh and frozen tissue were compared and found to have very similar growth rates ( Figure 1 Middle C).

[0089] In general, this application establishes a protocol for the collection and long-term storage of tumor tissues, which has the key characteristics of tumor tissues and can maintain their viability for subsequent applications, which is usually not considered by current frozen cancer banks. The results of this application show that cryopreserved tissues, even after long periods of appropriate freezing, have a high success rate of organoid culture, and the established organoids are highly consistent with fresh tissue-derived organoids in terms of morphology, structure and growth rate.

[0090] 2.2. Both cryopreserved tissue and fresh tissue-derived PDO (tissue-specific 3D models) well reproduce the histopathological and genetic characteristics of the parent tumor

[0091] The histopathological characteristics of breast and colon cancer organoids were compared with those of the parent tumors. Hematoxylin and eosinophil (H&E) staining results showed that organoids derived from cryopreserved tissues were able to retain the histological characteristics of their parent tumor tissues, similar to those of organoids derived from fresh tissues ( Figure 1 Medium D and Fig. 9 BC). Subsequently, the expression of cell proliferation marker Ki-67, breast cancer-related markers [estrogen receptor (ER), progesterone receptor (PR), and human growth factor receptor 2 (HER2)], and colon cancer-related markers (CK20, CDX2, and MUC-2) in the corresponding organoids and tumor tissues was evaluated. Immunohistochemical staining (IHC) showed that the expression patterns of these markers in organoids and tumor tissues derived from the corresponding fresh / cryopreserved tissues were highly consistent. These results indicate that, like organoids derived from fresh tissues, organoids derived from cryopreserved tissues are able to retain the histopathological characteristics of the original tumor ( Figure 1 Medium D and Fig. 9 Chinese BC).

[0092] Whole exome sequencing (WES) analysis was performed on 12 pairs of cryopreserved / fresh tissue-derived organoids and parent tumors from six breast cancer patients and six colon cancer patients to determine whether cryopreserved tissue-derived organoids could retain the genomic characteristics of their parent tumors like organoids derived from fresh tissue. Genome-wide exon copy number alteration (CNA) analysis showed that in both tumor types, organoids derived from cryopreserved tissues retained the DNA copy number characteristics of the parent tumor to a certain extent, similar to organoids derived from fresh tissue ( Figure 2 Middle A and Fig.10(A). We observed that the number of CNA genes in cryopreserved and fresh tissue-derived organoids was generally similar, but greater than in the corresponding primary tumors. Next, we compared the CAN signatures of cancer-associated genes in organoids and corresponding tumors. Consistent with the genome-wide results, the CNA patterns of cancer-associated genes in fresh tissue-derived organoids were well recapitulated in organoids derived from frozen samples, but were generally greater than in the primary tumors ( Figure 2 (B). We further compared single nucleotide variant (SNV) genes in the parental tumors and organoids. Mutations in tumor-associated genes in the original tissues, such as TP53, PIK3CA, COL11A1, AKT1, and SMAD3 / 4, were largely preserved in both cryopreserved and fresh tissue-derived organoids. Figure 2 CNA distribution scatter plots further confirmed that CNA genes were more abundant in organoids derived from cryopreserved and fresh tissues compared with primary tumor tissues ( Figure 2 Medium D and Fig.10 Chinese BC).

[0093] In general, similar to organoids derived from fresh tissue, organoids derived from cryopreserved tissues can well preserve the histological characteristics, expression of tumor markers, and genomic characteristics of the original tumor. Like organoids derived from fresh tissue, organoids derived from cryopreserved tissues usually present more CNA and SNV genes than primary tumors. This is mainly because cancer organoids are composed entirely of tumor cells, while the original tissue contains other types of cells, such as stromal cells and immune cells, resulting in lower tumor cell purity than organoids.

[0094] Comparison of drug responses in cryopreserved and fresh tumor tissue-derived organoids

[0095] In order to evaluate the feasibility of using frozen tumor tissue-derived organoids to evaluate patient tumor drug responses, this example compared the drug sensitivity of frozen and fresh tumor tissue-derived PDO. This example designed a drug combination containing 14 commonly used clinical treatment drugs (7 chemotherapy drugs and 7 targeted therapy drugs) to test the drug sensitivity of 3 colorectal cancer organoids and 2 breast cancer organoids. Drug sensitivity is expressed as the concentration that inhibits 50% of cells (IC 50 ). The differences in the responses of PDO to different drugs were statistically significant. Among the 7 chemotherapy drugs tested, PDO was more sensitive to epirubicin hydrochloride and doxorubicin hydrochloride, more resistant to gemcitabine hydrochloride, bleomycin sulfate, clofarabine and cytarabine, and had a greater sensitivity difference to topotecan hydrochloride ( Figure 3Middle A). Organoids derived from different individuals are sensitive to neratinib and afatinib, which target HER2 and EGFR, but are resistant to gefitinib and erlotinib, which target EGFR ( Figure 3 Importantly, this example detected a high degree of consistency in the drug response of organoids derived from frozen and fresh tumor tissues from the same individual ( Figure 3 AB and Fig.11 Middle A). This suggests that drug responses in derived organoids are not affected by cryopreservation of tumor tissue. Like organoids derived from fresh tumor tissue, they should be able to represent patient-specific drug responses.

[0096] To confirm the similarity of drug responses between frozen and fresh tumor tissue-derived organoids, two pairs of organoids (CPDO5 / F-CPDO5 and BPDO7 / F-BPDO7) were treated with drugs with different inhibitory effects. After 96 hours of drug treatment, it was observed that CPDO5 / F-CPDO5 showed a relatively high sensitivity to neratinib (1.0 μM) ( Figure 3 After neratinib treatment, only a small number of surviving and smaller organoids were observed, and the proportion of dead organoids was significantly greater than that in the control group ( Figure 3 However, neither CPDO5 nor F-CPDO5 showed sensitivity to clofarabine (1.0 μM) treatment ( Figure 3 After afatinib (0.1 μM) treatment, both BPDO7 and F-BPDO7 showed a significant decrease in the proportion of surviving organoids and a significant increase in the proportion of dead organoids ( Fig.11 However, BPDO7 and F-BPDO7 showed limited response to gemcitabine hydrochloride (2.0 μM) treatment ( Fig.11 These results further confirm that organoids derived from cryopreserved tissues exhibit similar drug responses to those derived from fresh tissues.

[0097] 2.4. Characteristics of organoids after long-term culture of cryopreserved tumor tissue

[0098] Next, the effects of long-term culture on organoids derived from cryopreserved tumor tissue were studied. The colorectal cancer organoids F-CPDO3 and breast cancer organoids F-BPDO5 were passaged for 23 and 19 generations, respectively, for more than one year. After long-term culture, organoids derived from cryopreserved tumor tissue still showed a growth rate similar to that of organoids derived from early passage cryopreserved and fresh tumor tissue. The morphology of these organoids remained unchanged, retaining the morphological structure observed in early passage cryopreserved and fresh tumor tissue-derived organoids ( Figure 4 Middle A and Fig.12Further histological examination and IHC staining revealed no significant changes in the tissue structure or expression of Ki-67, breast cancer-related markers (ER, PR, and HER2), or colorectal cancer-related markers (CK20, CDX2, and MUC-2) in either organoid lineage after long-term culture ( Figure 4 Medium B and Fig.12 (middle B).

[0099] Subsequently, WES analysis was performed on F-CPDO3 and F-BPDO5 of different generations. The results of whole-genome CNA analysis showed that after continuous passage, F-CPDO3 retained the characteristics of fewer CNA genes, while F-BPDO5 retained the characteristics of more CNA genes in the early generations of organoids ( Figure 4 CD and Fig.12 C to D in the middle). Further analysis revealed that the mutation status of cancer-related genes in both organoid lines was well preserved after serial passage. F-CPDO3 showed a greater number of SNV gene mutations, while F-BPDO5 showed a smaller number of SNV gene mutations ( Figure 4 Middle E). The results show that organoids derived from cryopreserved tumor tissue retain the genomic characteristics of early generation organoids after long-term culture.

[0100] In order to evaluate whether long-term culture affects the drug response of cryopreserved tumor tissue-derived organoids, drug sensitivity tests were performed on F-CPDO3 and F-BPDO5 of different passages. The results showed that even after continuous passage to the 19th passage (F-BPDO5) and the 23rd passage (F-CPDO3), the cryopreserved tissue-derived organoids still showed similar drug responses to those of early passage cryopreserved tissue-derived organoids and fresh tissue-derived organoids ( Fig.13 (middle AB).

[0101] In summary, organoids derived from frozen tumor tissues can be passaged in culture for a long time while maintaining morphological structure, histological characteristics, and genomic characteristics similar to those of fresh tissue sources. In addition, their response to drugs does not change significantly during passage.

[0102] 2.5. High-throughput drug screening using organoids derived from cryopreserved tumor tissues of drug-resistant patients

[0103] Drug resistance, including intrinsic resistance and acquired resistance, is one of the main obstacles to cancer treatment and greatly limits the selection and use of cancer drugs. In order to identify drugs that effectively reverse tumor resistance, this example established a drug panel containing 430 kinase inhibitors. High-throughput drug sensitivity screening was performed on two organoids derived from cryopreserved tissues of drug-resistant tumors ( Figure 5(A). F-BPDO5 is derived from neoadjuvant chemotherapy-resistant tumor tissue. Drug screening results showed that F-BPDO5 exhibited resistance to 200 kinase inhibitors (IC 50 ≥10μM), and showed moderate response to 200 kinase inhibitors (2μM <IC 50 <10 μM, showing sensitivity to 30 kinase inhibitors (IC 50 Among the 30 most sensitive kinase inhibitors, 13 targeted the PI3K / Akt / mTOR signaling pathway and 10 targeted the cell cycle signaling pathway ( Fig.14 IC values ​​of the three most sensitive drugs GSK2126458 (GSK458), dinaciclib (SCH727965), and BGT226 (NVP-BGT226) 50 The values ​​were 0.004673μM, 0.011μM and 0.06567μM ( Fig.14 A and C in Figure 2). F-BPDO1 was derived from a HER2+ breast cancer patient who developed multidrug resistance and multi-site systemic metastasis after 7 rounds of treatment with more than 10 different drugs. High-throughput drug screening showed that F-BPDO1 exhibited resistance to most of the tested kinase inhibitors (IC 50 ≥10μM), and showed moderate response to 30 kinase inhibitors (2μM <IC 50 <10 μM, sensitive to only 10 kinase inhibitors (IC 50 Among the 30 most sensitive kinase inhibitors, 10 targeted the PI3K / Akt / mTOR signaling pathway, 7 targeted the cell cycle signaling pathway, and 6 targeted the protein tyrosine kinase signaling pathway ( Figure 5 The IC values ​​of the three most sensitive drugs, dinaciclib (SCH727965), CUDC-907, and flavopiridol (alvocidib) 50 The values ​​were 0.02773 μM, 0.3043 μM and 0.3204 μM respectively ( Figure 5 Interestingly, both F-BPDO1 and F-BPDO5 are highly sensitive to dinaciclib (SCH727965) ( Figure 5 Middle B, D, E and F; Fig.14A, C, D, and E in the figure). Dinaciclib is a novel potent small molecule inhibitor of cyclin-dependent kinases (CDK) 1, CDK2, CDK5, and CDK9 involved in the cell cycle. It has been shown to have good clinical therapeutic effects on refractory chronic lymphocytic leukemia. The results showed that dinaciclib may have broad application prospects in the treatment of multidrug-resistant breast cancer. In summary, pre-frozen living tumor tissue can be revived when needed and used for organoid culture, which can screen effective drugs for patients with refractory tumors such as metastasis / recurrence or be used to develop new drugs. This greatly enhances the application scope of existing biobanks.

[0104] 2.6. Cryopreserved tumor tissue can be used for ALI culture and evaluation of anti-tumor drugs, including ICI

[0105] Organoids can effectively preserve the genomic and transcriptomic features of the original tumor, providing a promising prospect for precision cancer therapy. However, organoid culture leads to the loss of tumor-associated fibroblasts and immune cells. In contrast, the air-liquid interface (ALI) model overcomes this limitation by successfully maintaining the tumor microenvironment, making it suitable for testing anti-tumor drugs, including ICIs. This example successfully revived frozen tumor tissue and used it for ALI culture ( Figure 6 Middle A). SMA-positive fibroblasts and CD45 in ALI model cultured with frozen and fresh tumor tissues. + 、CD4 + 、CD8 + and F4 / 80 + Immune cells are well preserved ( Figure 6 Medium B and Fig.15 Furthermore, the proliferation activity and expression of PD-L1 in cells were effectively restored ( Fig.15 To further explore the feasibility of using frozen tumor tissue for ALI culture and screening of effective anti-tumor drugs, we first compared the drug responses of two pairs of frozen and fresh tumor tissue ALI models. After 96 hours of drug treatment, the frozen and fresh HP6403T tumor tissue ALI models showed highly similar drug responses to the same concentration of BKM120 ( Fig.15 Importantly, both cryopreserved and fresh tumor ALI models generated with HP6654T not only showed comparable drug responses to the targeted drug afatinib, but also showed similar sensitivity to a monoclonal antibody targeting the immune checkpoint PD-L1 ( Figure 6 CE). One pair of human breast cancer tissues and two pairs of human colorectal cancer tissues were further studied. Similar to the mouse tumor samples, cryopreserved samples showed similar drug responses to chemotherapeutic drugs and monoclonal antibodies targeting PD-1 in all three pairs of human tumor tissue ALI models ( Figure 6 FH and Fig.15 In summary, the cryopreserved tumor tissue samples of the present application can be effectively used for ALI culture, and can preserve the cellular components and tumor microenvironment of the original tumor, including fibroblasts, immune cells and tumor cells. This method is very valuable for screening chemotherapy and targeted therapy drugs and for the pharmacodynamic evaluation of ICI.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for cryopreservation of biological tissues, characterized in that: It includes the following steps: Preprocessing biological tissue into biological tissue blocks with a diameter of less than 500 μm; The biological tissue block is placed in a tissue freezing solution, and after gradient cooling, it is frozen and stored in liquid nitrogen; wherein the gradient cooling includes: freezing at -30 to -10°C in the first stage for 0.5 to 5 hours; and freezing at -100 to -60°C in the second stage for 10 to 40 hours.

2. The cryopreservation method according to claim 1, characterized in that The freezing temperature of the first stage is -25 to -15°C; Optionally, the freezing time of the first stage is 1 to 3 hours.

3. The cryopreservation method according to claim 1, characterized in that: The freezing temperature of the second stage is -90 to -70°C; Optionally, the freezing time of the second stage is 15 to 25 hours.

4. The cryopreservation method according to any one of claims 1 to 3, characterized in that: The pretreatment method comprises: placing the biological tissue on a 500 μm filter in a culture dish containing a culture medium, and crushing the biological tissue until it passes through the 500 μm filter to obtain the biological tissue block with a diameter less than 500 μm.

5. The cryopreservation method according to any one of claims 1 to 3, characterized in that: The components of the tissue freezing solution include: dimethyl sulfoxide and FBS.

6. The cryopreservation method according to claim 5, characterized in that: The volume fraction of dimethyl sulfoxide in the tissue freezing solution is 80% to 99%; Optionally, the volume fraction of the FBS in the tissue freezing solution is 1% to 20%.

7. A method for constructing a biological sample library, characterized in that: It includes: The biological tissue sample is preserved by using the biological tissue cryopreservation method described in any one of claims 1 to 6.

8. Use of biological tissue cryopreserved by the cryopreservation method according to any one of claims 1 to 6 in the construction of organoids.

9. Use of biological tissue cryopreserved by the cryopreservation method according to any one of claims 1 to 6 in preparing an air-liquid interface culture model.

10. Use of a biological tissue cryopreserved by the cryopreservation method according to any one of claims 1 to 6, or an organoid or air-liquid interface culture model constructed from the cryopreserved biological tissue in drug screening or drug efficacy evaluation.

Images