A method and kit for osteosarcoma organoid culture

Abstract

The application provides a kit for culturing osteosarcoma organoids, the kit comprising a special culture medium, a sample dissociation digestive solution and a digestion termination solution, the special culture medium being based on a basic culture medium and adding dexamethasone, glycerophosphate disodium salt and calcium chloride, the osteosarcoma organoids carrying or not carrying an immune feature. The application also provides a culture method of the osteosarcoma organoids carrying the immune feature. The osteosarcoma organoids are cultured by using the culture method and the kit, the culture period is short, the stability is high, the in situ tumor heterogeneity can be reserved, the tumor microenvironment immune feature is stable, and the kit can be used for high-throughput drug screening, target gene identification, immunotherapy drug screening, oncolytic virus killing experiment, omics research such as single cell sequencing, and animal model construction. The application fills the blank of the interstitial-derived osteosarcoma organoid immune co-culture, and has a wide application prospect in the field of osteosarcoma basic research and clinical diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method and kit for culturing osteosarcoma organoids. Background Technology

[0002] Osteosarcoma is a primary malignant bone tumor that develops from stromal cells. It commonly occurs in the highly vascularized metaphysis, resulting in a high early metastasis rate and poor prognosis. Although the incidence of osteosarcoma is not high, my country has a large population base, and this disease is prevalent among adolescents. Its high malignancy causes immense suffering and stress for patients, their families, and society as a whole. Since suitable gene therapy targets have not yet been found, neoadjuvant chemotherapy combined with limb-sparing surgery is commonly used to treat osteosarcoma clinically. With the continuous development of science and technology, the overall prognosis of osteosarcoma patients has improved significantly, but many patients still experience disease progression and even death due to lung metastasis. Immunotherapy is considered a promising way to fundamentally change cancer treatment. Currently, immune checkpoint inhibitors, represented by anti-programmed death protein-1 (PD-1) / programmed death protein ligand-1 (PD-L1) immunotherapy, are widely used in the clinical treatment of osteosarcoma, but the objective response rate is low. The main reason for this is that existing preclinical disease models cannot accurately reproduce the characteristics of the in vivo osteosarcoma immune microenvironment, thus limiting our understanding of the tumor immune microenvironment and hindering the efficacy of immunotherapy in cancer treatment. Therefore, there is an urgent need to construct new osteosarcoma disease models carrying immune signatures.

[0003] Existing tumor models mainly include tumor cell line models, genetically engineered cell models, mouse xenograft models, and organoid models. Unlike other preclinical tumor models, which have limitations such as long processing times, limited success rates, and difficulty in simulating the characteristics of the patient's tumor microenvironment, tumor organoid models have advantages such as short processing time, high efficiency, and preservation of the in situ tumor microenvironment characteristics. In recent years, co-culture models of tumor organoids and immune cells have been widely used in predicting the efficacy of immunotherapy and evaluating the effects of combined treatments with other therapies, showing excellent drug prediction performance and research potential in many preclinical studies. However, these models are all epithelial-derived tumor organoids, and their culture conditions and methods are not suitable for the culture and immune co-culture of mesenchymal-derived tumor organoids (such as osteosarcoma), resulting in a lack of mesenchymal-derived osteosarcoma organoid models and their immune co-culture models. Currently, the scarcity of mesenchymal-derived osteosarcoma organoid models and their immune co-culture models has led to relatively slow progress in osteosarcoma research, stagnation in clinical treatment methods, and unsatisfactory treatment outcomes. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies and fill the gap in the immune co-culture of mesenchymal tumor organoids, this application provides a kit and method suitable for osteosarcoma organoid culture and immune co-culture of osteosarcoma organoids. The kit has a short culture cycle, high stability, can preserve tumor heterogeneity, and has stable tumor microenvironment and immune microenvironment characteristics.

[0005] The first aspect of this application provides a kit for culturing osteosarcoma organoids, the kit comprising a special culture medium, a sample dissociation digestion solution, and a digestion termination solution, wherein the osteosarcoma organoids may or may not carry immune signatures.

[0006] A second aspect of the present invention provides the use of the kit described in the first aspect in culturing osteosarcoma organoids, said osteosarcoma organoids carrying or not carrying immune signatures.

[0007] The third aspect of this application provides a method for culturing osteosarcoma organoids carrying immune characteristics, wherein immune cells and osteosarcoma single cells are co-cultured using the kit of the first aspect to obtain osteosarcoma organoids carrying immune characteristics.

[0008] The method comprises the following steps: 1) dissociating osteosarcoma tissue using sample dissociation digestion solution to obtain a single-cell suspension; 2) resuspending lymphocytes and osteosarcoma single-cell suspension in a special culture medium in the above kit to obtain a mixed suspension; 3) mixing the mixed suspension with matrix gel and incubating until the matrix gel solidifies; 4) adding the special culture medium in the above kit to culture and obtain osteosarcoma organoids carrying immune characteristics.

[0009] The fourth aspect of the present invention provides a method for culturing osteosarcoma organoids, characterized in that the method comprises the following steps: 1) obtaining an osteosarcoma single-cell suspension; 2) resuspending the osteosarcoma single-cell suspension in a special culture medium in the above-mentioned kit to obtain a cell suspension; 3) mixing the cell suspension obtained in 2) with a matrix gel and incubating until the matrix gel solidifies; 4) adding the special culture medium in the above-mentioned kit to culture and obtain osteosarcoma organoids.

[0010] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0011] (1) Prepare a special culture medium suitable for osteosarcoma organoid culture system carrying immune characteristics. The organoid culture cycle is 5-10 days, which is short and has a high throughput.

[0012] (2) It can ensure the normal expansion of tumor cells and the stability of the tumor microenvironment, and preserve the tumor heterogeneity of patients or mouse xenograft models.

[0013] (3) It can preserve fibroblasts in tumor tissue;

[0014] (4) Through mild tissue digestion, the osteosarcoma organoid culture medium is used to ensure the stable passage of osteosarcoma organs derived from patients or mouse xenograft models.

[0015] (5) The amount of tissue sample required is small, only about 50mg of osteosarcoma solid tumor surgical sample is needed;

[0016] (6) High culture stability. The success rate of in vitro culture of osteosarcoma from qualified patients or mouse xenograft models using this method is as high as 80%.

[0017] (7) By separating peripheral blood lymphocytes from patients or mouse xenograft models and culturing them together with osteosarcoma organoid models cultured in accordance with the method provided in this invention, osteosarcoma organoid models carrying immune microenvironment characteristics can be obtained.

[0018] The osteosarcoma organoids carrying immune signatures obtained using the method provided in this invention, either from patient-derived or mouse xenograft models, can better mimic the tumor microenvironment. These organoids can be used for high-throughput drug screening and target gene identification, co-culture experiments with CAR-T / NK immune cells, oncolytic virus killing experiments, single-cell sequencing and other omics studies, and the construction of animal models, among other applications. It is foreseeable that this osteosarcoma organoid culture method will have broad application prospects in the research and clinical diagnosis and treatment of osteosarcoma solid tumors. Attached Figure Description

[0019] Figure 1 Single cells obtained from processed osteosarcoma tissue derived from patients; scale bar 500 μm, 40x magnification.

[0020] Figure 2 Single cells obtained from mouse xenograft osteosarcoma tissue after processing; scale bar is 500 μm, magnified 40 times.

[0021] Figure 3 Images of single cells obtained from processed osteosarcoma tissue from patients on day 7 (Figure A) and day 14 (Figure B) of organoid culture; scale bar 500 μm, 40x magnification.

[0022] Figure 4 Images of single cells obtained from xenograft osteosarcoma tissue in mice on day 7 (Fig. A) and day 14 (Fig. B) of organoid culture; scale bar 500 μm, 40x magnification.

[0023] Figure 5 To detect the proliferative capacity of constructed osteosarcoma organoids.

[0024] Figure 6This refers to day 3 of culture of osteosarcoma organoids carrying immunogenic characteristics constructed using the method and kit of this invention. Figure 6 A, scale bar 250μm, 100x magnification) and day 7 ( Figure 6 Bright-field image (B, scale bar 500μm, 40x magnification).

[0025] Figure 7 Patient-derived osteosarcoma organoids obtained after passage, day 1 (Figure A) and day 7 (Figure B), scale bar 250 μm, 100x magnification.

[0026] Figure 8 The images show osteosarcoma organoids obtained from mouse xenografts after passage, day 1 (Fig. A, 40x magnification) and day 7 (Fig. B, scale bar 500μm, 40x magnification).

[0027] Figure 9 H&E staining images of patient-derived osteosarcoma organoids; scale bar 100 μm, 200x magnification (Figure A); scale bar 25 μm, 1000x magnification (Figure B).

[0028] Figure 10 Immunohistochemical staining of patient-derived osteosarcoma organoids (Ki67); scale bar 100 μm, 200x magnification (Fig. A); scale bar 25 μm, 1000x magnification (Fig. B).

[0029] Figure 11 (AD) shows the flow cytometry identification results of the osteosarcoma organoid model carrying immune characteristics, and the osteosarcoma organoid model simulating the immune microenvironment characteristics constructed using the culture medium for constructing osteosarcoma organoids carrying immune characteristics provided by the present invention.

[0030] Figure 12 (AB) Comparison of osteosarcoma organoid culture using the osteosarcoma organoid culture medium provided in this patent and the colorectal cancer organoid culture medium described in Comparative Example 1. Figure 12 (A) An osteosarcoma organoid constructed using the osteosarcoma organoid culture medium provided in this patent. Figure 12 (B) Osteosarcoma organoid culture using the colorectal cancer organoid culture medium described in Comparative Example 1, with a large number of cell debris visible to the naked eye. Scale bar is 500 μm, 40x magnification (Figure AB). Detailed Implementation

[0031] This application first provides a kit for culturing osteosarcoma organoids, the kit comprising a special culture medium, a sample dissociation digestion solution, and a digestion termination solution, wherein the special culture medium is based on a basal culture medium and contains dexamethasone, disodium glycerol phosphate, and calcium chloride, and the osteosarcoma organoids may or may not carry immune signatures.

[0032] The term "carrying immune characteristics" refers to osteosarcoma organoids being co-cultured with immune cells, thereby giving the osteosarcoma organoids immune characteristics; the term "not carrying immune characteristics" refers to osteosarcoma organoids being cultured alone without co-culturing with immune cells, and therefore not having immune characteristics.

[0033] In a specific embodiment of this application, the special culture medium further includes penicillin-streptomycin bispecific antibody, insulin-transferrin-selenium, MEM non-essential amino acid solution, fetal bovine serum, ascorbic acid, Y-27632, growth factor, adenine, A83-01, and SB202190.

[0034] In the general case of this application, the basal culture medium includes DMEM / F12 medium and / or 1640 medium; when the osteosarcoma organoids carry immunomarkers, the basal culture medium includes DMEM / F12 medium and 1640 medium, and the special culture medium is supplemented with the basal culture medium to a volume fraction of 100%; when the osteosarcoma organoids do not carry immunomarkers, the basal culture medium is DMEM / F12 medium, and the special culture medium is supplemented with the basal culture medium to a volume fraction of 100%.

[0035] In some embodiments of this application, the growth factors include recombinant human epidermal growth factor protein, recombinant human fibroblast growth factor protein, recombinant human insulin-like growth factor protein, and recombinant human transforming growth factor-β3 protein.

[0036] In some embodiments of this application, the special culture medium further includes additives, including GlutaMAX. TM Additives, N-2 additives and B-27 serum-free additives.

[0037] When the kit is used to culture osteosarcoma organoids carrying immune signatures, i.e. when the kit is used for immune co-culture of osteosarcoma organoids, the special culture medium also includes granulocyte-macrophage colony-stimulating factor, interleukin-4, interleukin-2, CD3 antibody, and CD28 antibody.

[0038] As those skilled in the art will recognize, growth factors refer to cytokines, which are substances in tissue culture that, in addition to normal components such as amino acids, vitamins, glucose, and inorganic salts, can replace serum macromolecules in the culture medium to promote cell growth. They are cytokines with cell growth-stimulating activity, present in platelets, various adult and embryonic tissues, and most cultured cells, exhibiting a certain specificity for different cell types. Growth factors are mostly broadly defined peptide hormones, including insulin, epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and somatostatin (SRIH), etc. In the specific embodiments of this application, the growth factors include recombinant protein EGF, human recombinant protein bFGF, human recombinant protein IGF-1, and human recombinant protein TGF-β3.

[0039] In a specific embodiment of this application, the additive includes GlutaMAX. TM Additives, N-2 additives, and B-27 serum-free additives. Among them, GlutaMAX... TM The additive, L-alanyl-L-glutamine, significantly reduces the accumulation of toxic ammonia, improves cell viability and growth, and remains stable over a wide temperature range. As cells gradually release aminopeptidase, L-alanyl-L-glutamine is hydrolyzed, slowly releasing L-alanine and L-glutamine into the culture medium for absorption by the cells, which can be used for protein production or TCA cycling. N-2 additive is commonly used for neuroblastoma and for the growth and expression of neurons in late mitotic phase in primary cell cultures of the peripheral nervous system (PNS) and central nervous system (CNS). B-27 additive is a serum-free additive containing antioxidants, proteins, vitamins, and fatty acids. Typically, these components, when mixed in optimal proportions, provide B27 additive to support nerve cell culture.

[0040] The basal culture medium includes DMEM / F12 medium and / or 1640 medium. DMEM / F12 and 1640 media can be commercially available products, such as those from Sigma, Thermo Fisher Scientific, and Omniscience, or they can be formulated according to existing recipes, with adjustments made to the amounts of each component as needed. When osteosarcoma organoids are co-cultured with immunoassay, the basal culture medium includes both DMEM / F12 and 1640 media; when osteosarcoma organoids are cultured alone, the basal culture medium is DMEM / F12 medium.

[0041] The penicillin-streptomycin bispecific antibody, a mixture of penicillin and streptomycin, is used for cell culture. After filtration and sterilization, it can be added directly to the cell culture medium. Penicillin and streptomycin can be added separately, or a mixture can be prepared first and then added. Generally, the penicillin concentration in the penicillin-streptomycin bispecific antibody (100×) is 10 KU / ml, and the streptomycin concentration is 10 mg / ml. This solution can be prepared with 0.85% sodium chloride.

[0042] Insulin-transferrin-selenium, as a basal culture medium supplement, can reduce the amount of fetal bovine serum (FBS) required for cell culture. Insulin promotes glucose and amino acid uptake, lipid formation, intracellular transport, and protein and nucleic acid synthesis. Transferrin, as an iron carrier, helps reduce the toxicity levels of oxygen free radicals and peroxides. Selenium (provided as sodium selenite) acts as an antioxidant in the culture medium, serving as a cofactor for glutathione peroxidase and other proteins.

[0043] MEM non-essential amino acid solution is used as an additive in cell culture media to improve cell growth and activity.

[0044] Fetal bovine serum (FBS) refers to the supernatant obtained by puncturing blood from unborn fetuses, followed by natural chromatography, centrifugation, and three-stage 0.1μm microfiltration. It contains high levels of embryonic growth factors, thus providing essential nutrients and growth factors for cell maintenance and growth.

[0045] Dexamethasone, an anti-inflammatory glucocorticoid, has a series of effects on cell survival, cell signal transduction, and gene expression, such as: inhibiting the induction of nitric oxide synthase (IC50 = 5 nM); enhancing the transport of cations in large artery smooth muscle cells by stimulating the Na+-K+ pump; reducing the activity of cyclin A and Cdk2; inhibiting the G1 / S transition in osteocytes; inhibiting the phosphorylation of Rb protein; and inducing apoptosis in human thymocytes. This application creatively incorporates dexamethasone, which can regulate the osteogenic differentiation of mesenchymal stem cells by genes such as Runx2, Noggin, and leucine zipper protein. The concentration used in this method can effectively support the growth of osteosarcoma organoids derived from mesenchymal stem cells.

[0046] Ascorbic acid possesses antioxidant properties, helping to eliminate free radicals and reduce oxidative damage to cells. It participates in collagen synthesis, playing a crucial role in maintaining the normal structure and function of skin, blood vessels, bones, and teeth. Furthermore, ascorbic acid promotes iron absorption and enhances immunity.

[0047] Sodium glycerol phosphate is used as an organophosphate donor to promote the differentiation of mesenchymal stem cells into osteoblasts in culture media.

[0048] Calcium ions in calcium chloride are involved in many important cellular functions, including enzyme activity, attachment, migration, tissue morphology, metabolic processes, signal transduction, replication, and electrochemical reactions in specific cells such as muscle and nerve cells. This application creatively incorporates disodium glycerol phosphate with calcium chloride, which regulates the metabolism of mesenchymal stem cells and osteoblasts, as well as the growth and differentiation of bone marrow-derived mesenchymal stem cells (MSCs). The concentration used in this method effectively supports the growth of osteosarcoma organoids derived from mesenchymal tissue.

[0049] Y-27632 is a small molecule inhibitor of the Rho-associated protein kinase p160ROCK, with a Ki value of 140 nM for p160ROCK, which is 200 times more potent than other kinases, including PKC, cAMP-dependent protein kinases, MLCK, and PAK. Y-27632 inhibits the kinase activity by targeting the catalytic site of ROCK-1 / 2. Therefore, Y-27632 can act on Rho-mediated stress fiber formation, inhibiting G1-S phase cell cycle progression and cytokinesis. Furthermore, Y-27632 can induce selective differentiation of human pluripotent stem cells (hIPSCs) into mesenchymal lineages by regulating epithelial-mesenchymal transition-like processes. Y-27632 can be used to prevent stem cell apoptosis and improve clonogenic efficiency.

[0050] Adenine is a component of nucleic acids and participates in the synthesis of RNA and DNA in organisms. When white blood cells are deficient, it can promote white blood cell proliferation and is used to prevent and treat leukopenia and acute granulocytopenia.

[0051] A83-01 is a potent inhibitor of the TGF-β type I receptor ALK5 (IC50: 12 nm) / ALK4 (IC50: 45 nm) / ALK7 (IC50: 7.5 nm) kinases, exhibiting strong inhibitory activity against ALK5 and suppressing Smad2 / 3 phosphorylation and TGF-β-induced growth. A83-01 has little or no effect on bone morphogenetic protein type I receptors, p38 mitogen-activated protein kinase, or extracellular regulated kinases. Simultaneously, A83-01 inhibits TGF-β-induced epithelial-mesenchymal metastasis, suggesting that A83-01 may contribute to inhibiting the progression of advanced cancer.

[0052] SB202190, as a highly selective cell-permeable p38 MAPK inhibitor, inhibits p38α (IC50: 50 nM) and p38β (IC50: 100 nM). It can induce apoptosis by activating the cpp32-like caspases pathway, with typical apoptotic features such as nuclear condensation or intranuclear chromosomal DNA fragmentation.

[0053] Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony-stimulating factor 2 (CSF2), is a monomeric glycoprotein that functions as a cytokine through the secretion of macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts. It particularly promotes the proliferation and maturation of neutrophils. It participates in many biological responses, including the growth and development of granulocyte and macrophage progenitor cells, the stimulation and initiation of myelocyte and monocyte differentiation, and the chemotaxis of eosinophils.

[0054] Interleukin-4 (IL-4) has numerous biological functions, including stimulating the proliferation of activated B cells and T cells, and differentiating B cells into plasma cells. It is a key regulator of humoral and adaptive immunity. IL-4 induces the conversion of B cell classes to IgE and upregulates the production of MHC class II. IL-4 reduces the production of IL-12 by Th1 cells, macrophages, IFN-γ, and dendritic cells.

[0055] Interleukin-2 is mainly produced by activated T cells and has multi-directional effects. It mainly promotes the growth, proliferation and differentiation of lymphocytes; plays an important role in the body's immune response and antiviral infection; can stimulate the proliferation of T cells that have been activated by specific antigens or mitogenic factors[1]; can activate T cells and promote the production of cytokines; stimulate NK cell proliferation, enhance NK killing activity and produce cytokines, induce LAK cell production; promote B cell proliferation and antibody secretion; and activate macrophages.

[0056] CD3 and CD28 antibodies are used to activate T cells via the αβ-T cell receptor (TCR complex) and promote T cell proliferation in vitro. Using monoclonal anti-CD3 and anti-CD28 antibodies to stimulate T cells generates co-stimulatory signals, which, together with the TCR, achieve antigen-induced activation.

[0057] Recombinant protein EGF is a small peptide composed of 53 amino acids with a molecular weight of 6201 Daltons. It contains three intramolecular ring structures formed by six cysteine ​​residues, creating the receptor-binding region essential for its biological activity. EGF lacks a glycosylation site, making it highly stable, heat- and acid-resistant. It is widely distributed in body fluids and various glands, primarily synthesized by the submandibular gland and duodenum. It has been found in most human body fluids, with particularly high concentrations in breast milk, urine, and semen, but lower concentrations in serum. Recombinant protein EGF has a strong pro-mitotic effect on various tissue cells.

[0058] Human recombinant protein bFGF belongs to the fibroblast growth factor (FGF) family. It interacts with high-affinity transmembrane receptors and influences cell proliferation and tissue angiogenesis.

[0059] Human recombinant protein IGF-1 is a mitotic growth factor primarily produced by the liver. It belongs to the insulin gene family and also contains insulin and relaxin. IGF-I and IGF-II signaling are mediated through the tyrosine kinase type I receptor (IGF-IR), while IGF-II can also be signaled via the IGF-II / mannose-6-phosphate receptor. Mature IGF-1 is produced by the proteolytic hydrolysis of an inactive precursor protein containing N-terminal and C-terminal precursor regions, and is used to stimulate the proliferation and survival of various cells, including muscle, bone, and cartilage tissues.

[0060] Human recombinant protein TGF-β3 is a member of a subset of the TGF-β superfamily that performs many cellular functions. TGF-β3 plays a role in embryogenesis and cell differentiation. It also plays a crucial role in palatogenesis and wound healing. TGF-β3 can directly bind to its type II receptor (TβRII). Three TGF-β isoforms—TGF-β1, 2, and 3—have been identified in mammals; they are structurally and functionally similar. TGF-β3 is important in embryonic development, scarless repair of embryonic injuries, adult wound healing, and tissue homeostasis. It is used to regulate cell migration, angiogenesis, epithelial-mesenchymal transition, apoptosis, immune function regulation, extracellular matrix (ECM) production, and ECM remodeling.

[0061] In the general case described in this application, when the osteosarcoma organoids carry immunogenicity, i.e., when the kit is used for immunoco-culture of osteosarcoma organoids, the final concentrations of each component are as follows:

[0062] The penicillin-streptomycin bispecific antibody in this application can be prepared by adding penicillin and streptomycin separately, or it can be prepared as a penicillin-streptomycin bispecific antibody (100×) first, or ready-made penicillin-streptomycin bispecific antibody (100×) can be used directly. The content of penicillin can be 10 KU / ml, the content of streptomycin can be 10 mg / ml, and then the penicillin-streptomycin bispecific antibody is added. The final concentration of penicillin-streptomycin can be 0.5-1%, specifically 0.5-0.7%, 0.7-0.9%, or 0.9-1.0%.

[0063] The final concentration of insulin-transferrin-selenium is 1.2-1.5% by volume, specifically 1.2-1.3%, 1.3-1.4%, or 1.4-1.5%.

[0064] The final concentration of the MEM non-essential amino acid solution can be 1.2-1.5% by volume, specifically 1.2-1.2%, 1.3-1.4%, or 1.4-1.5%.

[0065] The final concentration of fetal bovine serum is 10-14% by volume, optionally 10-11%, 11-12%, 12-13%, or 13-14%.

[0066] This application invents the addition of dexamethasone to a culture medium, wherein the final concentration of dexamethasone is 10. -7 -10 -6 Specifically, it can be 10 -7 -5×10 -7 It can be 5×10 -7 -10 -6 .

[0067] The final concentration of ascorbic acid is 40-60 μmol / L. Optionally, the final concentration of ascorbic acid can be 40-45 μmol / L, 45-50 μmol / L, 50-55 μmol / L, or 55-60 μmol / L.

[0068] The final concentration of disodium glycerol phosphate can be, for example, 2-4 mmol / L, and in some embodiments, it can be 2-2.5 mmol / L, 2.5-3 mmol / L, 3-3.5 mmol / L, or 3.5-4.0 mmol / L.

[0069] The final concentration of calcium chloride is 0.4-0.6 mmol / L. Specifically, the final concentration of calcium chloride can be 0.4-0.45 mmol / L, 0.45-0.5 mmol / L, 0.5-0.55 mmol / L, or 0.55-0.6 mmol / L.

[0070] The final concentration of Y-27632 can be 5-15 μmol / L. In specific embodiments of this application, the final concentration of Y-27632 can be 5-8 μmol / L, 8-12 μmol / L, or 12-15 μmol / L.

[0071] The final concentration of human recombinant epidermal growth factor protein is 10-15 ng / mL, specifically 10-11 ng / mL, 11-12 ng / mL, 12-13 ng / mL, 13-14 ng / mL, or 14-15 ng / mL.

[0072] The final concentration of human recombinant fibroblast growth factor protein is 20-30 ng / mL, which can be 20-22 ng / mL, 22-24 ng / mL, 24-26 ng / mL, 26-28 ng / mL, or 28-30 ng / mL.

[0073] The final concentration of human recombinant insulin-like growth factor protein is 10-15 ng / mL, specifically 10-11 ng / mL, 11-12 ng / mL, 12-13 ng / mL, 13-14 ng / mL, or 14-15 ng / mL.

[0074] The final concentration of human recombinant transforming growth factor-β3 protein is 5-8 ng / mL. In specific embodiments, it can be 5-6 ng / mL, 6-7 ng / mL, or 7-8 ng / mL.

[0075] The final concentration of adenine can be, for example, 100-150 μmol / L, specifically 100-120 μmol / L, 120-130 μmol / L, or 130-150 μmol / L.

[0076] The final concentration of A83-01 can be 0.2-0.4 μmol / L, typically 0.2-0.3 μmol / L, or 0.3-0.4 μmol / L.

[0077] The final concentration of SB202190 can be 4-6 μmol / L, typically 4-5 μmol / L or 5-6 μmol / L.

[0078] The final concentration of granulocyte-macrophage colony-stimulating factor can be 4-10 ng / mL, for example, optionally 4-6 ng / mL, 6-8 ng / mL, or 8-10 ng / mL.

[0079] The final concentration of interleukin-4 can be 5-10 ng / mL, for example, 5-6 ng / mL, 6-7 ng / mL, 7-8 ng / mL, 8-9 ng / mL, or 9-10 ng / mL.

[0080] The final concentration of interleukin-2 can be 4-10 ng / mL, and in specific embodiments it can be 4-6 ng / mL, 6-8 ng / mL, or 8-10 ng / mL.

[0081] The final concentration of CD3 can be 2-8 μg / mL, and in some embodiments it can be, for example, 2-4 μg / mL, 4-6 μg / mL, or 6-8 μg / mL.

[0082] The final concentration of CD28 can be 5-10 μg / mL, specifically 5-6 μg / mL, 6-7 μg / mL, 7-8 μg / mL, 8-9 μg / mL, or 9-10 μg / mL.

[0083] In a specific embodiment of this application, the GlutaMAX TM The final concentration of the additive is 1.2-1.5%, and specifically, it can be 1.2-1.3%, 1.3-1.4%, or 1.4-1.5%.

[0084] In this application, the final concentration of the N-2 additive is 1.2-1.5%, specifically, it can be 1.2-1.3%, 1.3-1.4%, or 1.4-1.5%.

[0085] In this application, the final concentration of the B-27 additive is 1.2-1.5%, specifically, it can be 1.2-1.3%, 1.3-1.4%, or 1.4-1.5%.

[0086] The basal culture medium is prepared by mixing DMEM / F12 medium and 1640 medium in a certain ratio, with the volume ratio of DMEM / F12 medium to 1640 medium being 1-3:1, for example, 1-1.5:1, 1.5-2:1, 2-2.5:1, or 2.5-3:1; preferably, the volume ratio of DMEM / F12 medium to 1640 medium is 1:1. The basal culture medium is used to supplement the volume of the special culture medium to the target volume after the other components in the special culture medium have been added, i.e., to 100% of the volume fraction.

[0087] In the general case described in this application, when the osteosarcoma organoids do not carry immune signatures, i.e., when the kit is used for the culture of osteosarcoma organoids alone, the final concentrations of each component are as follows:

[0088] The penicillin-streptomycin bispecific antibody in this application can be prepared by adding penicillin and streptomycin separately, or it can be prepared as a penicillin-streptomycin bispecific antibody (100×) first, or ready-made penicillin-streptomycin bispecific antibody (100×) can be used directly. The content of penicillin can be 10 KU / ml, the content of streptomycin can be 10 mg / ml, and then the penicillin-streptomycin bispecific antibody is added. The final concentration can be 0.5-1%, specifically 0.5-0.7%, 0.7-0.9%, or 0.9-1.0%.

[0089] The final concentration of insulin-transferrin-selenium is 0.8-1.2% by volume, specifically 0.8-0.9%, 0.9-1.0%, 1.0-1.1%, or 1.1-1.2%.

[0090] The final concentration of the MEM non-essential amino acid solution can be 0.8-1.2% by volume, specifically 0.8-0.9%, 0.9-1.0%, 1.0-1.1%, or 1.1-1.2%.

[0091] The final concentration of fetal bovine serum is 10-14% by volume, optionally 10-11%, 11-12%, 12-13%, or 13-14%.

[0092] This application invents the addition of dexamethasone to a culture medium, wherein the final concentration of dexamethasone is 10. -6 -10 -5 Specifically, it can be 10 -6 -5×10 -6 It can be 5×10 -6 -10 -5 .

[0093] The final concentration of ascorbic acid is 80-100 μmol / L. Optionally, the final concentration of ascorbic acid can be 80-85 μmol / L, 85-90 μmol / L, 90-95 μmol / L, or 95-100 μmol / L.

[0094] The final concentration of disodium glycerol phosphate can be, for example, 8-10 mmol / L, and in some embodiments, it can be 8-8.5 mmol / L, 8.5-9 mmol / L, 9-9.5 mmol / L, or 9.5-10 mmol / L.

[0095] The final concentration of calcium chloride is 0.8-1 mmol / L. Specifically, the final concentration of calcium chloride can be 0.8-0.85 mmol / L, 0.85-0.9 mmol / L, 0.9-0.95 mmol / L, or 0.95-1 mmol / L.

[0096] The final concentration of Y-27632 can be 5-15 μmol / L. In specific embodiments of this application, the final concentration of Y-27632 can be 5-8 μmol / L, 8-12 μmol / L, or 12-15 μmol / L.

[0097] The final concentration of human recombinant epidermal growth factor protein is 10-20 ng / mL, specifically 10-12 ng / mL, 12-14 ng / mL, 14-16 ng / mL, 16-18 ng / mL, or 18-20 ng / mL.

[0098] The final concentration of human recombinant fibroblast growth factor protein is 20-40 ng / mL, which can be 20-25 ng / mL, 25-30 ng / mL, 30-35 ng / mL, or 35-40 ng / mL.

[0099] The final concentration of human recombinant insulin-like growth factor protein is 10-20 ng / mL, specifically 10-12 ng / mL, 12-14 ng / mL, 14-16 ng / mL, 16-18 ng / mL, or 18-20 ng / mL.

[0100] The final concentration of human recombinant transforming growth factor-β3 protein is 5-10 ng / mL. In specific embodiments, it can be 5-6 ng / mL, 6-7 ng / mL, 7-8 ng / mL, 8-9 ng / mL, or 9-10 ng / mL.

[0101] The final concentration of adenine can be, for example, 150-200 μmol / L, specifically 100-120 μmol / L, 120-140 μmol / L, 140-160 μmol / L, 160-180 μmol / L, or 180-200 μmol / L.

[0102] The final concentration of A83-01 can be 0.25-0.5 μmol / L, typically 0.25-0.3 μmol / L, 0.3-0.35 μmol / L, 0.35-0.4 μmol / L, 0.4-0.45 μmol / L, or 0.45-0.5 μmol / L.

[0103] The final concentration of SB202190 can be 5-10 μmol / L, typically 5-6 μmol / L, 6-7 μmol / L, 7-8 μmol / L, 8-9 μmol / L, or 9-10 μmol / L.

[0104] In a specific embodiment of this application, the GlutaMAX TM The final concentration of the additive is 0.8-1%, specifically optional to be 0.8-0.9% or 0.9-1%.

[0105] In this application, the final concentration of the N-2 additive is 0.8-1.2%, specifically optionally 0.8-0.9%, 0.9-1.0%, 1.0-1.1%, or 1.1-1.2%.

[0106] In this application, the final concentration of the B-27 additive is 0.8-1.2%, specifically optionally, it can be 0.8-0.9%, 0.9-1.0%, 1.0-1.1%, or 1.1-1.2%.

[0107] The basal medium was DMEM / F12 medium. After the above components were added, the special medium was supplemented to 100% by volume with DMEM / F12 medium.

[0108] Adding to 100% volume fraction means adding to the final volume required to prepare the product.

[0109] The sample dissociation digestion solution in the kit of this application is used to dissociate tumor tissue into single cells. In a specific embodiment of this application, the sample dissociation digestion solution is a trypsin-EDTA solution containing phenol red. The trypsin-EDTA solution containing phenol red is prior art. It can be purchased as a ready-made product or prepared according to existing formulas. For example, the trypsin-EDTA solution can be prepared by the user with potassium chloride 400 mg / L, potassium dihydrogen phosphate 60 mg / L, sodium bicarbonate 350 mg / L, sodium chloride 8000 mg / L, sodium dihydrogen phosphate 90 mg / L, D-glucose 1000 mg / L, EDTA 4Na 2H2O 380 mg / L, phenol red 10 mg / L and trypsin 2500 mg / L, and the dosage can be adjusted as needed.

[0110] The digestion termination solution in the kit of this application is used to stop or terminate the digestion reaction of the sample dissociation digestion solution on the tissue. In a specific embodiment of this application, the digestion termination solution includes fetal bovine serum (FBS), penicillin-streptomycin antibiotics, and DMEM culture medium. The FBS is a byproduct of the meat processing industry, made from the embryonic serum of healthy prepartum cows, and contains high levels of embryonic growth-promoting factors (FGFs), thereby providing essential nutrients and growth factors for cell maintenance and growth. The volume fraction of the FBS in the product is 10%–14%. In general, the volume fraction of FBS in the product in this application can be, for example, 10–11%, 11–12%, 12–13%, or 13–14%. In this application, a penicillin-streptomycin bispecific antibody (100×) can be prepared first, wherein the penicillin content can be 10 KU / ml and the streptomycin content can be 10 mg / ml. Then, the penicillin-streptomycin bispecific antibody is added, with a final concentration of 1-5% by volume. In specific embodiments of this application, this concentration can be 1-2%, 2-3%, 3-4%, or 4-5%. The DMEM culture medium is used to replenish the volume of the digestion stop solution to the required volume after the fetal bovine serum and penicillin-streptomycin bispecific antibody have been added according to the specified ratio, i.e., to bring the volume fraction to 100%.

[0111] In some embodiments of this application, the kit further contains one or more of the following: sample preservation solution, organoid cryopreservation solution, and sample cleaning solution.

[0112] The sample preservation solution is used to preserve tumor tissue samples, maintain cell viability, reduce cell metabolism, and stabilize gene expression. In a specific embodiment of this application, the sample preservation solution includes fetal bovine serum, penicillin-streptomycin bispecific antibody, 4-hydroxyethylpiperazine ethanesulfonic acid, and Hank's balanced salt solution. 4-hydroxyethylpiperazine ethanesulfonic acid is a commonly used zwitterionic buffer used to maintain the pH stability of the cultured cell environment. In this application, the final concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 8-10 mmol / L, specifically 8-8.5 mmol / L, 8.5-9.0 mmol / L, 9.0-9.5 mmol / L, or 9.5-10 mmol / L. Additionally, the final concentration of fetal bovine serum is 1-5% by volume, specifically 1-2%, 2-3%, 3-4%, or 4-5%. The penicillin-streptomycin bispecific antibody can be first prepared as a penicillin-streptomycin bispecific antibody (100×), wherein the content of penicillin can be 10 KU / ml and the content of streptomycin can be 10 mg / ml. Then, the penicillin-streptomycin bispecific antibody is added, and the final concentration of the penicillin-streptomycin bispecific antibody is 1-5% by volume. In the specific embodiments of this application, it can be 1-2%, 2-3%, 3-4%, or 4-5%. Hank's balanced salt solution is one of the commonly used phosphate buffers in cell isolation or culture. Its main components are NaCl, KCl, KH2PO4, Na2HPO4, NaHCO4, and glucose. It is used to maintain osmotic pressure, keep pH stable, and provide simple nutrients. In this application, the volume fraction of the sample preservation solution is supplemented to 100% using Hank's balanced salt solution.

[0113] Organoid cryopreservation solution is used to cryopreserve organoids obtained from cell culture, protecting and stabilizing them under low-temperature conditions. The organoid cryopreservation solution includes serum-free cell cryopreservation solution and Y-27632. The serum-free cell cryopreservation solution can be a commercially available solution or a custom-prepared formulation. The final concentration of Y-27632 is 8-12 μmol / L; in specific embodiments of this application, it can be 8-9 μmol / L, 9-10 μmol / L, 10-11 μmol / L, or 11-12 μmol / L. The organoid cryopreservation solution is then replenished to 100% by volume using serum-free cell cryopreservation solution.

[0114] The sample washing solution is used to clean the acquired tumor tissue samples and remove contaminants remaining on the tumor tissue. The sample washing solution includes penicillin-streptomycin antibiotics and phosphate buffer. The penicillin-streptomycin antibiotics can be first prepared as penicillin-streptomycin antibiotics (100×), wherein the penicillin content can be 10 KU / ml and the streptomycin content can be 10 mg / ml. Then, the penicillin-streptomycin antibiotics are added, with a final concentration of 1-5% by volume. In the specific embodiments of this application, it can be 1-2%, 2-3%, 3-4%, or 4-5%. Then, the sample washing solution is replenished to 100% by volume using phosphate buffer.

[0115] The components in this application can be added either directly or by preparing a storage solution. The storage solution refers to a solution with a high concentration prepared beforehand for storage and use; the storage solution is then diluted and added during use.

[0116] All ingredients in this application can be obtained by purchasing existing products and used in accordance with the product instructions or conventional operation in the prior art.

[0117] In this application, the term "final concentration" refers to the concentration of the substance in the system at the end of the operation.

[0118] This application also provides the use of the above-described kit in culturing osteosarcoma organoids carrying or not carrying immune signatures.

[0119] In addition, this application also provides a method for culturing osteosarcoma organoids carrying immune characteristics, wherein immune cells and osteosarcoma single cells are co-cultured using the above-mentioned kit to obtain osteosarcoma organoids carrying immune characteristics.

[0120] Co-culture refers to culturing two or more types of cells (from the same or different tissues) in the same culture system.

[0121] The immune cells refer to cells that participate in or are related to the immune response. These include lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. In this application, the immune cells are preferably monocytes, and more preferably peripheral blood monocytes.

[0122] The method comprises the following steps:

[0123] 1) Obtain a single-cell suspension of osteosarcoma;

[0124] 2) Resuspend the lymphocyte and osteosarcoma single-cell suspensions in the special culture medium in the above kit to obtain a mixed cell suspension;

[0125] 3) Mix the cell suspension and matrix gel, and incubate until the matrix gel solidifies;

[0126] 4) Add the special culture medium from the above kit to culture and obtain osteosarcoma organoids carrying immune characteristics.

[0127] In step 1), specifically, osteosarcoma single-cell suspension can be obtained by dissociating osteosarcoma tissue using sample dissociation digestion solution, and the digestion reaction of the sample dissociation digestion solution can be terminated with digestion termination solution. Under normal circumstances, the volume of digestion termination solution can be 3 times the volume of sample dissociation digestion solution.

[0128] The osteosarcoma tissue can be derived from mammals, such as mice or humans, that have bone tumors or have undergone xenografting. Specifically, when the osteosarcoma tissue is derived from humans, fresh osteosarcoma tissue specimens can be collected from patients with bone tumors in a sterile operating room environment. When the osteosarcoma tissue is derived from mice, the bone tumor tissue sample can be carefully dissected in a sterile environment using pre-autoclaved scalpels, curved forceps, and tissue scissors. After obtaining the bone tumor sample, it can be pretreated, for example, by washing the sample multiple times with 75% ethanol, sample washing solution, and sterile phosphate buffer, and then storing it in a sample preservation solution for later use.

[0129] The ratio of the volume of the sample dissociation digestion solution to the mass of the osteosarcoma tissue can be 0.4-1.0:1 mL / mg; specifically, it can be 0.4-0.6 mL / mg, 0.6-0.8 mL / mg, or 0.8-1.0 mL / mg.

[0130] In step 2), the lymphocytes are peripheral blood lymphocytes, the lymphocytes are from humans or mice, and the ratio of the number of osteosarcoma single cells to the number of lymphocytes is 2-4:1; optionally, it can be 2-2.5:1, 2.5-3:1, 3-3.5-1, or 3.5-4:1.

[0131] In step 3), the matrix gel refers to the basement membrane component extracted from mouse tumor tissue (the basement membrane is a stromal membrane on the basal surface of epithelial cells in an animal). Typically, the main components of matrix gel are laminin, type IV collagen, nestin, and various growth factors, such as epidermal growth factor EFG, platelet-derived growth factor PDGF, nerve growth factor NGF, basic fibroblast growth factor FGF-2, transforming growth factor beta-TGF-beta, and insulin-like growth factor ILGF. The volume ratio of the matrix gel to the mixed cell suspension is 1:3-5. In specific embodiments, it can be 1:3-3.5, 1:3.5-4, 1:4-4.5, or 1:4.5-5. In this application, when culturing osteosarcoma organoids carrying immune characteristics, it is preferable to use Matrigel growth factor-reduced basement membrane matrix, which is free of phenol red and LDEV (lactate dehydrogenase-enhancing virus).

[0132] The incubation refers to the reaction of substances at a certain temperature. The incubation time is 30-60 minutes, optionally 30-40 minutes, 40-50 minutes, or 50-60 minutes. The incubation temperature is a suitable temperature for cell culture; generally, preferably, the suitable temperature for cell culture is 37°C.

[0133] In step 4), the culture time is 7-14 days, optionally 7-9 days, 9-11 days, 11-13 days, or 13-14 days; the culture temperature is a suitable temperature for cell culture, generally, preferably 37℃. During the culture process, the special culture medium can be changed every 1-2 days.

[0134] The osteosarcoma organoids obtained through culture can be cryopreserved in the organoid cryopreservation solution provided in the kit described above.

[0135] In addition, this application also provides a method for culturing osteosarcoma organoids, the method comprising the following steps:

[0136] 1) Obtain a single-cell suspension of osteosarcoma;

[0137] 2) Osteosarcoma single-cell suspension was resuspended in the special culture medium in the above kit to obtain cell suspension;

[0138] 3) Mix the cell suspension obtained in 2) with the matrix gel and incubate until the matrix gel solidifies;

[0139] 4) Add the special culture medium from the above kit to culture and obtain osteosarcoma organoids.

[0140] In step 1), specifically, osteosarcoma single-cell suspension can be obtained by dissociating osteosarcoma tissue using sample dissociation digestion solution, and the digestion reaction of the sample dissociation digestion solution can be terminated with digestion termination solution. Under normal circumstances, the volume of digestion termination solution can be 3 times the volume of sample dissociation digestion solution.

[0141] The osteosarcoma tissue can be derived from mammals, such as mice or humans, that have bone tumors or have undergone xenografting. Specifically, when the osteosarcoma tissue is derived from humans, fresh osteosarcoma tissue specimens can be collected from patients with bone tumors in a sterile operating room environment. When the osteosarcoma tissue is derived from mice, the bone tumor tissue sample can be carefully dissected in a sterile environment using pre-autoclaved scalpels, curved forceps, and tissue scissors. After obtaining the bone tumor sample, it can be pretreated, for example, by washing the sample multiple times with 75% ethanol, sample washing solution, and sterile phosphate buffer, and then storing it in a sample preservation solution for later use.

[0142] The ratio of the volume of the sample dissociation digestion solution to the mass of the osteosarcoma tissue can be 0.4-1.0:1 mL / mg; specifically, it can be 0.4-0.6 mL / mg, 0.6-0.8 mL / mg, or 0.8-1.0 mL / mg.

[0143] In step 3), the volume ratio of the matrix gel to the mixed cell suspension is 1-3:1. In specific embodiments, it can be 1-2:1 or 2-3:1. In this application, when culturing osteosarcoma organoids carrying immune characteristics, it is preferable to use Matrigel growth factor reduced basement membrane matrix, which is free of phenol red and LDEV (lactate dehydrogenase-enhancing virus).

[0144] The incubation refers to the reaction of substances at a certain temperature. The incubation time is 30-60 minutes, optionally 30-40 minutes, 40-50 minutes, or 50-60 minutes. The incubation temperature can be a suitable temperature for cell culture; generally, preferably, the suitable temperature for cell culture is 37°C.

[0145] In step 4), the culture time is 7-14 days, optionally 7-9 days, 9-11 days, 11-13 days, or 13-14 days; the culture temperature is a suitable temperature for cell culture, generally, preferably 37℃. During the culture process, the special culture medium can be changed every 1-2 days.

[0146] The osteosarcoma organoids obtained through culture can be cryopreserved in the organoid cryopreservation solution provided in the kit described above.

[0147] The immune characteristics described in this application refer to the different in situ tumor immune microenvironment characteristics of different osteosarcoma patients. The term "carrying immune characteristics" refers to the process by which organoids, after co-culturing with patient-derived peripheral blood mononuclear cells, acquire relevant immune characteristics. Peripheral blood mononuclear cells (PBMCs) are a mixed cell population of mononuclear cells in peripheral blood (i.e., blood other than bone marrow), including natural killer cells (NK cells), T lymphocytes (70%-90%), and B lymphocytes. They can be further isolated and purified, and are a major source of immune cells.

[0148] The culture method provided in this application is not for disease diagnosis and treatment purposes, but can be used for high-throughput drug screening and target gene identification, CAR-T / NK immune cell co-culture experiments, oncolytic virus killing experiments, single-cell sequencing and other omics research, animal model construction, etc.

[0149] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0150] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0151] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature.

[0152] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0153] Example 1: Preparation of an Osteosarcoma Organoid Culture Kit Carrying Immune Characteristics

[0154] 1. Sample preservation solution

[0155] The specific formula of the sample preservation solution (50 mL) is shown in Table 1.

[0156] Table 1. Sample preservation solution (50 mL)

[0157]

[0158] After the sample preservation solution is prepared, it is aliquoted into 15mL centrifuge tubes, 5mL per tube. The aliquots can be stored at 4℃ for one month.

[0159] 2. Sample cleaning solution

[0160] The specific formula of the sample cleaning solution (50 mL) is shown in Table 2.

[0161] Table 2 Sample cleaning solution (50 mL)

[0162]

[0163] The sample cleaning solution must be prepared and used immediately.

[0164] 3. Digestion Termination Solution

[0165] The specific formula for the digestion termination solution (100 mL) is shown in Table 3.

[0166] Table 3. Digestion Termination Solution (50 mL)

[0167]

[0168] After preparation, the digestion termination solution can be stored at 4°C for one month.

[0169] 4. Special culture medium suitable for osteosarcoma organoids carrying immune signatures

[0170] The specific formulations of the special culture medium for osteosarcoma organoids carrying immune characteristics are shown in Table 4.

[0171] Table 4. Culture medium (50 mL) of osteosarcoma organoids carrying immune signatures.

[0172]

[0173] After the above culture medium is prepared, it is filtered and sterilized using a 0.22μm syringe filter and can be stored at 4℃ for 3-4 weeks.

[0174] The specific formulations of the special culture medium for osteosarcoma organoids that do not carry immune characteristics are shown in Table 5.

[0175] Table 5. Culture medium (50 mL) for osteosarcoma organoids without immune signatures.

[0176]

[0177] 5. Cryopreservation solution for osteosarcoma organoids

[0178] The specific formula for the cryopreservation solution for osteosarcoma organoids is shown in Table 6.

[0179] Table 6. Organ cryopreservation solutions (10 mL)

[0180]

[0181] Osteosarcoma organoid cryopreservation solutions should be prepared and used immediately.

[0182] Example 2: Obtaining patient-derived osteosarcoma in situ tumor specimens

[0183] 1. We collaborate with top-tier hospitals, and the collaboration has undergone formal medical ethics review.

[0184] 2. The attending physician selects patients for enrollment according to the clinical indications specified in the medical guidelines, and selects appropriate samples for in vitro culture based on the clinical indications during the operation. The selection criteria for the samples are: osteosarcoma without chemotherapy, and surgical specimens weighing more than 30mg.

[0185] 3. The attending physician should provide the patient's basic clinical information, including gender, age, present medical history, personal history, family history, smoking history, tumor pathological stage and type, and clinical diagnosis. Patient names, ID numbers, and other privacy-related information will be redacted and replaced with a standardized experiment number. The naming principle for the experiment number is the eight-digit date of sample collection + the order in which patients underwent surgery on the day of sample collection + the letter "T". For example, a sample provided on January 1, 2022, where the patient was the first to undergo surgery that day, would have the experiment number 2022010101T.

[0186] 4. During the operation, a surgeon collects fresh tumor tissue specimens in a sterile operating room environment and places them in a pre-prepared sample preservation solution (see Example 1). After removal from the body, the samples are temporarily stored on ice and transported to the laboratory within 2 hours for further processing.

[0187] Example 3: Obtaining osteosarcoma tumor tissue from mouse xenograft

[0188] 1. We collaborated with the Laboratory Animal Center of the Clinical Medical College, and the collaboration underwent formal ethical review.

[0189] 2. The attending physician shall select suitable mouse xenograft-derived osteosarcoma solid tumor tissue based on the clinical indications and intraoperative pathological results as specified in the medical guidelines. The selection criteria for the sample are: osteosarcoma.

[0190] 3. Tumor-bearing mice were euthanized in a sterile environment using cervical dislocation, and then disinfected by immersion in 75% alcohol. The tumor tissue was carefully dissected using a pre-autoclaved scalpel, curved forceps, and tissue scissors. The tumor tissue was then transferred to a culture dish for further pre-dissection processing.

[0191] 4. Wash the surface of the mouse xenograft-derived osteosarcoma solid tumor tissue sample with 75% ethanol for 15 to 20 seconds; wash the mouse xenograft-derived osteosarcoma solid tumor tissue 10 times with sample cleaning solution, and wash the mouse xenograft-derived osteosarcoma solid tumor tissue 10 times with sterile phosphate buffer solution; then carefully remove impurities, necrotic tissue, adipose tissue, and other components that may affect the primary cell organoid culture from the mouse xenograft-derived osteosarcoma solid tumor tissue sample.

[0192] 5. Place in the prepared sample preservation solution. After removal from the body, store the sample on ice for 2 hours before proceeding to the next step.

[0193] Example 4: Pre-dissociation treatment of osteosarcoma tumor tissue derived from patients or mouse xenograft models

[0194] The following operations must be performed on ice, and the entire operation must be completed within 15 minutes.

[0195] All instruments used in the following operations must be sterilized under high temperature and high pressure (104.0-137.3 kPa, 121.3℃, 30 minutes) and dried before use.

[0196] (1) Weigh the samples obtained in Examples 2 and 3. Clean the sample surface with 75% (volume percentage) ethanol for 15 to 20 seconds.

[0197] (2) The sample was washed 10 times with sample cleaning solution and 10 times with sterile phosphate buffer solution. Using ophthalmic scissors, ophthalmic forceps, scalpel and other instruments, adipose tissue, necrotic tissue and other impurities were removed from the sample.

[0198] (3) Transfer the solid tumor tissue to the prepared sample preservation solution. After the sample is removed from the body, store it on ice for 2 hours before proceeding to the next step.

[0199] Example 5: Dissociation of osteosarcoma solid tumor tissue samples from patients or mouse xenograft models

[0200] All instruments used in the following operations must be sterilized under high temperature and high pressure (104.0-137.3 kPa, 121.3℃, 30 minutes) and dried before use.

[0201] (1) After cleaning the sample from Example 4, cut it into 2-3 mm pieces using ophthalmic scissors. 3 Small pieces on the left and right.

[0202] (2) Using 0.4 mL of the tissue dissociation solution (trypsin-EDTA (0.25%)) per mg of tissue, treat the bone, cartilage, or fibrous tissue samples with the preheated dissociation solution at 37°C. Dissociate the samples at 37°C for 20 minutes to 2 hours. Observe the dissociation of the samples under a microscope every 20 minutes until a large number of single cells are observed.

[0203] (3) Terminate the digestion reaction with a digestion termination solution (3 times the volume) and collect the cell suspension.

[0204] (4) Filter the cell suspension with a 100μm sterile cell filter to remove tissue fragments.

[0205] (5) Centrifuge at 1500g at room temperature for 3 minutes and remove the supernatant. Resuspend the cells in 3 mL of sterile phosphate buffer solution, centrifuge at 1500g at room temperature for 3 minutes and remove the supernatant.

[0206] (6) Resuspend the cell pellet in osteosarcoma organoid culture medium (see Example 1) to obtain a primary osteosarcoma cell suspension, and observe the cell state under a microscope (see Example 1). Figure 1 and Figure 2 ), to perform cell counting.

[0207] Example 6: Culture of osteosarcoma organoids derived from patients or mouse xenograft models

[0208] Osteosarcoma organoids derived from patients or mouse xenograft models were cultured using organoid culture containers (96-well transparent round-bottom ultra-low adsorption microplates) and Matrigel (Matrigel growth factor reduced basement membrane matrix, phenol red-free).

[0209] (1) Initial cell seeding quantity: Taking the organoid culture container as an example, the patient or mouse xenograft osteosarcoma primary cell suspension obtained in Example 5 was used, at a density of 2 × 10⁶ cells per well. 5 Add a suspension of primary osteosarcoma cells from a patient or mouse xenograft at a density of 1,000 cells, and resuspend the cell suspension in the osteosarcoma organoid culture medium.

[0210] (2) Mix the matrix gel and the osteosarcoma organoid culture medium containing the cell suspension at a volume ratio of 3:2. Then, taking the organoid culture container as an example, gently add 50 μL per well into the organoid culture container.

[0211] (3) Let stand at 37℃ and 5% CO2 for 30-60 minutes; take out the organoid culture container, use osteosarcoma organoid culture medium, and gently drop 100-150 μL per well into each well of the primary osteosarcoma cells from the patient or mouse xenograft.

[0212] (4) Culture at 37℃ and 5% CO2, changing the culture medium every 1-2 days until the primary osteosarcoma cells xenografted from the patient or mouse form organoid masses with a diameter of 1000-1500μm.

[0213] Figure 3-4 Images show single cells obtained from processed osteosarcoma tissue from patients, cultured as organoids on days 7 and 14. Figure 5 The proliferative capacity of the constructed osteosarcoma organoids was demonstrated.

[0214] Example 7: Culture of osteosarcoma organoids carrying immune microenvironment characteristics

[0215] (1) Isolate peripheral blood lymphocytes from osteosarcoma patients and culture them in 37°C and 5% CO2 for 1-2 days using 1640 complete culture medium.

[0216] (2) After cleaning the sample obtained in Example 4, cut the cleaned sample into 2-3 mm pieces using ophthalmic scissors. 3 Small pieces on the left and right.

[0217] (3) Using 0.4 mL of the sample dissociation solution per mg of tissue, treat the sample with the preheated tissue dissociation solution at 37°C. Dissociate the sample at 37°C for 20 minutes to 2 hours. Observe the dissociation of the sample under a microscope every 20 minutes until a large number of single cells are observed.

[0218] (4) Terminate the digestion reaction with digestion termination solution (3 times the volume) and collect the cell suspension.

[0219] (5) Filter the cell suspension through a 100μm sterile cell filter to remove tissue fragments. Centrifuge at 1500g at room temperature for 3 minutes and aspirate the supernatant.

[0220] (6) Resuspend the cells in 3 mL of sterile phosphate buffer solution, centrifuge at 1500 g for 3 minutes at room temperature, and remove the supernatant.

[0221] (7) Resuspend the cell pellet in the culture medium used to establish an osteosarcoma organoid culture system carrying immune characteristics, observe the cell state under a microscope, and count the cells.

[0222] Osteosarcoma tumor organoids carrying immune signatures, derived from patients or mouse xenograft models, were cultured using organoid culture containers and a reduced-dose growth factor matrix gel. The culture medium used was the same as the one used in Example 1 for establishing the culture system of osteosarcoma organoids carrying immune signatures. The steps are as follows:

[0223] (8) Initial cell seeding quantity: Taking the organoid culture container as an example, 50 μL of the digested single-cell suspension of the patient or mouse xenograft osteosarcoma was resuspended in the culture medium for establishing an osteosarcoma organoid culture system carrying immune characteristics, and the cells were mixed at a 1:2 ratio, with 2 × 10 cells per well. 5 Prepare for cell-density plating;

[0224] (9) Take the growth factor matrix gel and the above mixed cell suspension at a volume ratio of 1:3, and then gently add 50 μL of the mixture to each well of the organoid culture container.

[0225] (10) Let stand at 37℃ and 5% CO2 for 30-60 minutes; remove the organoid culture container and gently add 100-150 μL of the culture medium used to establish the osteosarcoma organoid culture system with immune characteristics to each well.

[0226] (11) Cultured at 37℃ and 5% CO2, with the culture medium changed every 1-2 days until the xenografted osteosarcoma organoids from the patients or mice form organoid masses with a diameter of 1000-1500μm.

[0227] Example 8: Passaging of osteosarcoma organoids derived from patients or mouse xenograft models

[0228] (1) Gently pipette and collect the bone tumor organoids to be passaged obtained in Example 6 using 4°C sterile phosphate buffer solution (200 μl / well) to dissolve the matrix gel, transfer it to a 15 ml centrifuge tube and let it stand at 4°C for 15 minutes.

[0229] (2) Add 1-2 mL of sterile phosphate buffer solution and gently blow the organoid mass. Centrifuge at 1500 g for 5 minutes at 4℃ and remove the supernatant.

[0230] (3) Add 1-2 mL of organoid digestion solution to the organoid mass and gently mix by blowing. Digest at 37°C and 5% CO2 for 10 minutes until the organoid mass is digested into single cells.

[0231] (4) Terminate the digestion reaction with digestion termination solution (3-4 times the volume) and collect the cell suspension.

[0232] (5) After centrifuging at 1500g at room temperature for 5 minutes, the cell pellet was resuspended in osteosarcoma organoid culture medium (see Example 1), and the cell morphology was observed and the cells were counted under a microscope.

[0233] (6) Use organoid culture containers, with 2 × 10⁶ cells per well. 5 Prepare a suspension of osteosarcoma cells from the patient or mouse xenograft at a density of 1 cell, and resuspend the cell suspension in the osteosarcoma organoid culture medium.

[0234] (7) Take the growth factor matrix gel and the osteosarcoma organoid culture medium containing osteosarcoma cell suspension at a volume ratio of 3:2, mix them evenly, and then gently add 50 μL of each volume to the organoid culture container.

[0235] (8) Let stand at 37℃ and 5% CO2 for 30-60 minutes; take out the organoid culture container and gently add 100-150 μL of osteosarcoma organoid culture medium to each well into which the xenografted osteosarcoma organoids from the patient or mouse have been added.

[0236] (9) Culture at 37℃ and 5% CO2, and change the culture medium every 1-2 days.

[0237] (10) Figure 7 The image shows organoid masses obtained after passage of patient-derived osteosarcoma tissue organoids. Figure 8 The image shows organoid masses obtained after passage of xenografted osteosarcoma tissue from mice.

[0238] Example 9: Cryopreservation of osteosarcoma organoids derived from patients or mouse xenograft models

[0239] Osteosarcoma organoids derived from patients or mouse xenograft models and cultured in 3D can be cryopreserved after 2-3 passages for expansion.

[0240] (1) Take the bone tumor organoid mass obtained in Example 6, gently blow it with 4℃ sterile phosphate buffer solution (200μl / well) and collect the bone tumor organoid mass to dissolve the matrix gel, transfer it to a 15ml centrifuge tube and let it stand at 4℃ for 15 minutes.

[0241] (2) Add 1-2 mL of sterile phosphate buffer solution and gently blow the organoid mass. Centrifuge at 1500 g at room temperature for 5 minutes.

[0242] (3) Using the cell cryopreservation solution, resuspend the organoid cell pellet at 0.5-1 mL / well, store it in cryopreservation tubes, place it at 4°C for slow cooling for 1-2 hours, and then transfer it to liquid nitrogen for long-term storage.

[0243] Example 10: Resuscitation of osteosarcoma organoids derived from patients or mouse xenograft models

[0244] Osteosarcoma organoids from patients or mouse xenograft models, preserved in liquid nitrogen, can be resuscitated.

[0245] (1) Prepare sterile water at 37°C 10 minutes in advance. Take the cryopreservation tube of Example 9 out of liquid nitrogen and thaw the organoids frozen in Example 9 quickly in sterile water at 37°C.

[0246] (2) Centrifuge at 1500g at room temperature for 5 minutes, and slowly remove the supernatant. Resuspend the organoid pellet in osteosarcoma organoid culture medium, observe cell morphology under a microscope and count cells.

[0247] (3) Using an organoid culture container (96-well round-bottom low-absorption plate), the patient or mouse xenograft osteosarcoma primary cell suspension was seeded at a density of 2 × 10⁵ cells per well.

[0248] (4) Further processing of the primary osteosarcoma solid tumor cells from patients or mice after plating: centrifuge at 500g at room temperature for 3 minutes, take the matrix gel (Matrigel growth factor reduced basement membrane matrix, without phenol red) and mix it with the osteosarcoma organoid culture medium at a volume ratio of 3:2, and then gently add 75μL per well to cover the centrifuged osteosarcoma organoids from patients or mice.

[0249] (5) Incubate at 37°C and 5% CO2 for 30-60 minutes; gently add 100-150 μL of osteosarcoma organoid culture medium to each well containing the xenografted osteosarcoma organoids from the patient or mouse. Culture at 37°C and 5% CO2, changing the culture medium every 1-2 days.

[0250] like Figure 3 As shown, the osteosarcoma organoids derived from the patient are surrounded by a large number of fibroblasts.

[0251] Example 11: HE staining identification of osteosarcoma organoids derived from patients or mouse xenograft models

[0252] (1) Take the bone tumor organoids obtained in Example 6, immerse the osteosarcoma organoids cultured to a suitable size in 4% paraformaldehyde, place them on a shaker overnight at room temperature for fixation, and then place the tissue completely infiltrated with 4% paraformaldehyde at 4°C for storage.

[0253] (2) Tissue embedding was performed using a gradient dehydration method. 75% ethanol for 1 h, 85% ethanol for 1 h, 95% ethanol for 1 h, 100% ethanol for 1 h × 2 times, xylene for 1 h × 2 times, paraffin embedding for >2 h; the tissue was cut and embedded in paraffin blocks according to the experimental requirements on a preheated embedding instrument and placed on ice to cool; the paraffin blocks were pre-cooled at -8℃, cut into 4μm thick sections using a tissue sectioner, spread in 43℃ hot water until completely flat, and after removing the sections, they were baked in a 60℃ oven for >1-2 h.

[0254] (3) Dewaxing the sections. Preheat the sections to 60℃ and dewax them in a gradient to water: xylene 10 minutes × 2 times, 100% ethanol 5 minutes × 2 times, 95% ethanol 5 minutes, 85% ethanol 5 minutes, 75% ethanol 5 minutes, phosphate buffer solution 5 minutes × 3 times.

[0255] (4) Add sufficient hematoxylin staining solution evenly to the section to completely cover the tissue, stain for 10 minutes, and wash away excess hematoxylin staining solution with double-distilled water.

[0256] (5) Apply sufficient differentiation solution evenly and completely to the tissue area in the section, differentiate for a few seconds until the tissue on the section fades to a light blue-red color, and wash away excess differentiation solution with double-distilled water.

[0257] (6) Add sufficient blueing solution evenly to the slice to completely cover the tissue area. Let it blue for a few seconds until the tissue on the slice turns blue. Use a double needle to wash away the excess blueing solution.

[0258] (7) Add eosin staining solution evenly to the section to completely cover the tissue area and stain for 3 minutes.

[0259] (8) Dehydrate tissue sections using a gradient dehydration method. 75% ethanol, 5s; 95% ethanol, 2min; anhydrous ethanol, 2min.

[0260] (9) Immerse the dehydrated tissue sections in paraffin solution twice, for 4 minutes each time; then dry the tissue samples in a constant temperature drying oven and mount them with neutral resin.

[0261] HE staining images of organoids from osteosarcoma tissue derived from patients, observed under an optical microscope. Figure 9 As shown in the figure. The results show that the osteosarcoma organoids obtained by using the osteosarcoma organoid culture method provided by this product retain the histopathological characteristics of osteosarcoma.

[0262] Example 12: Immunohistochemical identification of osteosarcoma organoids derived from patients or mouse xenograft models

[0263] (1) The tissue paraffin blocks were prepared according to steps 1-2 of Example 11. The sections were placed in the dewaxing solution and repeatedly immersed for 10 minutes, and the dewaxing solution was changed each time.

[0264] (2) Perform gradient hydration of the sections with ethanol. Anhydrous ethanol, 5 minutes; 95% ethanol, 5 minutes; 75% ethanol, 5 minutes. After completion, place the sections in double-distilled water for hydration for 5 minutes.

[0265] (3) After heating the sodium citrate antigen retrieval solution (pH=6.0) in a microwave oven, fully immerse the hydrated tissue slices in it and continue heating in the microwave oven for 15 minutes.

[0266] (4) After antigen retrieval is completed, the tissue sections are immersed in the antigen retrieval solution and placed at 4°C to cool.

[0267] (5) Gently shake off the residual liquid on the tissue section, block the section with hydrogen peroxide solution (3%), and let it stand at room temperature for 10 minutes.

[0268] (6) Gently wash the slides with 1×TBST buffer solution twice, 5 minutes each time.

[0269] (7) Gently drop the diluted primary antibody onto the selected section area, ensuring that the tissue section area is completely covered. Then place the tissue section in a light-proof and humidified container and incubate overnight at 4°C.

[0270] (8) Remove the primary antibody and gently wash the slide twice with 1×TBST buffer solution, 5 minutes each time.

[0271] (9) Gently drop diluted secondary antibody (horseradish peroxidase labeled) onto the selected section area, ensuring that the tissue section area is completely covered. Then place the tissue section in a light-proof and humidified container and incubate at room temperature for 30 minutes.

[0272] (10) Remove the secondary antibody and gently wash the slide twice with 1×TBST buffer solution for 5 minutes each time.

[0273] (11) Freshly prepared DAB staining solution (stored away from light) is dropped onto the selected tissue section area and stained for 5-10 minutes. The staining effect is observed under a microscope. The staining reaction is terminated by gently washing with double-distilled water.

[0274] (12) Gently wash the slides with 1×TBST buffer solution twice, for 5 minutes each time.

[0275] (13) After placing the slice in the hematoxylin staining solution for a few seconds, gently wash with double-distilled water for 3 minutes.

[0276] Mounted with neutral resin, staining results observed under an optical microscope. Immunohistochemical staining image of organoid culture from patient-derived osteosarcoma tissue (Ki67) is shown below. Figure 10 As shown in the figure. The results show that the osteosarcoma organoids obtained by using the osteosarcoma organoid culture method provided by this product retain their proliferative capacity.

[0277] Example 13: Flow Cytometry Identification of Osteosarcoma Organoids Carrying Immune Signs

[0278] (1) Take the bone tumor organoid mass carrying immune characteristics obtained in Example 7, gently remove the culture medium from the organoid culture container, gently blow and collect the bone tumor organoid mass carrying immune characteristics with 4°C sterile phosphate buffer solution (200 μl / well), dissolve the matrix gel, transfer it to a 15 ml centrifuge tube and let it stand at 4°C for 15 minutes.

[0279] (2) Add 1-2 mL of sterile phosphate buffer solution and gently pipette the bone tumor organoid mass carrying immune characteristics. Centrifuge at 1500g for 5 minutes at 4℃ and remove the supernatant.

[0280] (3) Add 1-2 mL of organoid digestion solution to the organoid mass and gently mix by pipetting. Digest at 37°C and 5% CO2 for 10 minutes until the bone tumor organoid mass is digested into single cells.

[0281] (4) Terminate the digestion reaction with digestion termination solution (3-4 times the volume) and collect the cell suspension.

[0282] (5) After centrifuging at 300g at room temperature for 5 minutes, the cell pellet was resuspended in osteosarcoma organoid culture medium (see Example 1), and the cell morphology was observed and the cells were counted under a microscope.

[0283] (6) Centrifuge at 300g at room temperature for 5 minutes, and resuspend the cell pellet with phosphate buffer to remove the influence of the culture medium.

[0284] (7) Select appropriate fluorescently labeled antibodies to detect immune cell surface markers, such as CD3, CD45, CD4, and CD8. Add the corresponding antibodies to the organoid single-cell suspension sample, mix thoroughly, and incubate for a period of time. Incubate at 4°C in the dark for no less than 30 minutes.

[0285] (8) After centrifuging at 300g at room temperature for 5 minutes, the cell pellet was resuspended in phosphate buffer and washed. The sample was then analyzed using flow cytometry. Appropriate parameters were set to detect fluorescence signals. This included side-scattering (FSC) and forward-scattering (SSC), as well as fluorescence parameters for each antibody. Data were collected and analyzed to identify and quantify different types of immune cells.

[0286] Flow cytometry identification results of osteosarcoma organoid models carrying immune signatures, as follows: Figure 11 As shown in (AD), an osteosarcoma organoid model simulating the immune microenvironment was constructed using the osteosarcoma organoid culture medium carrying immune characteristics provided by this invention. Results show that osteosarcoma organoids carrying immune characteristics obtained by culturing osteosarcoma organoids using the osteosarcoma organoid culture method provided by this product retain the immunosuppressive microenvironment characteristics commonly found in orthotopic osteosarcoma tumors, including CD8+. + The proportion of T cells is low.

[0287] Comparative Example 1: Culture of osteosarcoma organoids using colorectal cancer organoid culture medium.

[0288] Currently, there is a lack of efficient culture media and methods for osteosarcoma organoids. While the culture techniques for epithelial tumor organoids from colorectal cancer and lung cancer are relatively mature, the culture conditions used for these are difficult to apply to osteosarcoma organoid culture. The following uses colorectal cancer organoid culture as a comparative example 1.

[0289] (1) Preparation of colorectal cancer organoid culture medium. This includes basal culture medium Advanced DMEM / F12, specific additives, and sterile water; wherein the volume ratio of the basal culture medium Advanced DMEM / F12 to the sterile water is 99:1; the specific additives include: vitamin A-free B27 (1%, volume percentage); N-acetylcysteine ​​(1 μM); EGF (20 ng / ml); Noggin (50 ng / ml); R-spondin1 (200 ng / ml); Wnt3a (100 ng / ml); Chir99021 (5 μM); Thiazovivin (2 μM); Gastrini (10 ng / ml); and penicillin-streptomycin antibiotics (5 mg / ml).

[0290] (2) Culture method: The osteosarcoma primary cell suspension obtained in Example 5 was cultured using the culture method of Example 6.

[0291] (3) Culture results: Osteosarcoma organoids showed significant cell death and visible cell debris within 7 days. Results are as follows... Figure 12 As shown. Figure 12(A) An osteosarcoma organoid constructed using the osteosarcoma organoid culture medium provided in this patent. Figure 12 (B) Osteosarcoma organoid culture using the colorectal cancer organoid culture medium described in Comparative Example 1 revealed a large number of cell debris visible to the naked eye. This indicates that other existing culture media are not suitable for culturing osteosarcoma organoids.

[0292] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A kit for culturing osteosarcoma organoids, the kit comprising a special culture medium, a sample dissociation digestion solution, and a digestion termination solution, wherein the special culture medium is based on a basal culture medium and supplemented with dexamethasone, disodium glycerol phosphate, and calcium chloride, and the osteosarcoma organoids carry immunomarkers; the immunomarkers are acquired by co-culturing osteosarcoma organoids with immune cells, thereby imparting immunomarkers to the osteosarcoma organoids; the final concentration of dexamethasone is 10... -7 -10 -6 The final concentration of the disodium glycerol phosphate is 2-4 mmol / L, and the final concentration of the calcium chloride is 0.4-0.6 mmol / L.

2. The reagent kit according to claim 1, characterized in that, The kit also has one or more of the following features: A) The special culture medium also includes the following components: penicillin-streptomycin bispecific antibody, insulin-transferrin-selenium, MEM non-essential amino acid solution, fetal bovine serum, ascorbic acid, Y-27632, growth factor, adenine, A83-01 and SB202190; B) The basal culture medium includes DMEM / F12 medium and 1640 medium, and the special culture medium is supplemented with the basal culture medium to a volume fraction of 100%.

3. The reagent kit according to claim 2, characterized in that, The kit also has one or more of the following features: a) The final concentration of the penicillin-streptomycin bispecific antibody is 0.5-1% (v / v), the final concentration of the insulin-transferrin-selenium solution is 1.2-1.5% (v / v), the final concentration of the MEM non-essential amino acid solution is 1.2-1.5% (v / v), the final concentration of the fetal bovine serum is 10-14% (v / v), the final concentration of ascorbic acid is 40-60 μmol / L, the final concentration of Y-27632 is 5-15 μmol / L, the final concentration of adenine is 100-150 μmol / L, the final concentration of A83-01 is 0.2-0.4 μmol / L, and the final concentration of SB202190 is 4-6 μmol / L. b) The special culture medium also includes granulocyte-macrophage colony-stimulating factor, interleukin-4, interleukin-2, CD3 antibody, and CD28 antibody; c) The volume ratio of the DMEM / F12 medium to the 1640 medium is 1-3:

1.

4. The reagent kit according to claim 3, characterized in that, The kit also has one or more of the following features: 1) The growth factors include recombinant human epidermal growth factor protein, recombinant human fibroblast growth factor protein, recombinant human insulin-like growth factor protein, and recombinant human transforming growth factor-β3 protein; 2) The special culture medium also includes additives, including GlutaMAX. TM Additives, N-2 additives and B-27 serum-free additives.

5. The reagent kit according to claim 4, characterized in that, The kit also has one or more of the following features: 1) The final concentration of the recombinant human epidermal growth factor protein is 10-15 ng / mL, the final concentration of the recombinant human fibroblast growth factor protein is 20-30 ng / mL, the final concentration of the recombinant human insulin-like growth factor protein is 10-15 ng / mL, and the final concentration of the recombinant human transforming growth factor-β3 protein is 5-8 ng / mL. 2) The GlutaMax TM The final concentration of the additive is 1.2-1.5% by volume, the final concentration of the N-2 additive is 1.2-1.5% by volume, and the final concentration of the B-27 serum-free additive is 1.2-1.5% by volume. 3) The final concentration of the granulocyte-macrophage colony-stimulating factor is 4-10 ng / mL, the final concentration of interleukin-4 is 5-10 ng / mL, the final concentration of interleukin-2 is 4-10 ng / mL, the final concentration of CD3 is 2-8 μg / mL, and the final concentration of CD28 is 5-10 μg / mL.

6. The reagent kit according to claim 1, characterized in that, The kit also has one or more of the following features: 1) The sample dissociation digest includes a trypsin-EDTA solution containing phenol red; 2) The digestion termination solution includes fetal bovine serum, penicillin-streptomycin antibiotics, and DMEM culture medium; 3) The kit also includes one or more of the following: sample preservation solution, organoid cryopreservation solution, and sample cleaning solution.

7. The reagent kit according to claim 6, characterized in that, The kit also has one or more of the following features: 1) In the digestion termination solution, the final concentration of the fetal bovine serum is 10-14% by volume, the final concentration of the penicillin-streptomycin antibiotic is 1-5% by volume, and the solution is supplemented to 100% by volume using the DMEM / F12 medium; 2) The sample preservation solution includes fetal bovine serum, penicillin-streptomycin antibiotics, 4-hydroxyethylpiperazine ethanethioic acid, and Hank's balanced salt solution; 3) The organoid cryopreservation solution includes serum-free cell cryopreservation solution and Y-27632; 4) The sample washing solution includes penicillin-streptomycin antibiotics and phosphate buffer solution.

8. The reagent kit according to claim 7, characterized in that, The kit also has one or more of the following features: 1) In the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% by volume, the final concentration of the penicillin-streptomycin antibody is 1-5% by volume, the final concentration of the 4-hydroxyethylpiperazine ethanethioic acid is 8-10 mmol / L, and the concentration is supplemented to 100% by volume with Hank's balanced salt solution; 2) In the organoid cryopreservation solution, the final concentration of Y-27632 was 8-12 μmol / L, which was then replenished to 100% by volume with serum-free cell cryopreservation solution; 3) In the sample washing solution, the final concentration of penicillin-streptomycin bispecific antibody is 1-5% by volume, and it is supplemented to 100% by volume with phosphate buffer.

9. A kit for culturing osteosarcoma organoids, the kit comprising a special culture medium, a sample dissociation digestion solution, and a digestion termination solution, wherein the special culture medium is based on a basal culture medium and supplemented with dexamethasone, disodium glycerol phosphate, and calcium chloride; the osteosarcoma organoids do not carry immunomarkers; the osteosarcoma organoids being free of immunomarkers are cultured alone, without co-culturing with immune cells; the final concentration of dexamethasone is 10... -6 -10 -5 The final concentration of the disodium glycerol phosphate is 8-10 mmol / L, and the final concentration of the calcium chloride is 0.8-1.0 mmol / L.

10. The reagent kit according to claim 9, characterized in that, The kit also has one or more of the following features: C) The special culture medium also includes the following components: penicillin-streptomycin bispecific antibody, insulin-transferrin-selenium, MEM non-essential amino acid solution, fetal bovine serum, ascorbic acid, Y-27632, growth factor, adenine, A83-01 and SB202190; D) The basal culture medium is DMEM / F12 medium, and the special culture medium is supplemented with the basal culture medium to a volume fraction of 100%.

11. The kit according to claim 10, characterized in that, The final concentration of the penicillin-streptomycin bispecific antibody is 0.5-1% (v / v), the final concentration of the insulin-transferrin-selenium solution is 0.8-1.2% (v / v), the final concentration of the MEM non-essential amino acid solution is 0.8-1.2% (v / v), the final concentration of the fetal bovine serum is 10-14% (v / v), the final concentration of ascorbic acid is 80-100 μmol / L, the final concentration of Y-27632 is 5-15 μmol / L, the final concentration of adenine is 150-200 μmol / L, the final concentration of A83-01 is 0.25-0.5 μmol / L, and the final concentration of SB202190 is 5-10 μmol / L.

12. The kit according to claim 10, characterized in that, The kit also has one or more of the following features: 1) The growth factors include recombinant human epidermal growth factor protein, recombinant human fibroblast growth factor protein, recombinant human insulin-like growth factor protein, and recombinant human transforming growth factor-β3 protein; 2) The special culture medium also includes additives, including GlutaMAX. TM Additives, N-2 additives and B-27 serum-free additives.

13. The reagent kit according to claim 12, characterized in that, The kit also has one or more of the following features: 1) The final concentration of the recombinant human epidermal growth factor protein is 10-20 ng / mL, the final concentration of the recombinant human fibroblast growth factor protein is 20-40 ng / mL, the final concentration of the recombinant human insulin-like growth factor protein is 10-20 ng / mL, and the final concentration of the recombinant human transforming growth factor-β3 protein is 5-10 ng / mL. 2) The GlutaMax TM The final concentration of the additive is 0.8-1% by volume, the final concentration of the N-2 additive is 0.8-1.2% by volume, and the final concentration of the B-27 serum-free additive is 0.8-1.2% by volume.

14. The kit according to claim 9, characterized in that, The kit also has one or more of the following features: 1) The sample dissociation digest includes a trypsin-EDTA solution containing phenol red; 2) The digestion termination solution includes fetal bovine serum, penicillin-streptomycin antibiotics, and DMEM culture medium; 3) The kit also includes one or more of the following: sample preservation solution, organoid cryopreservation solution, and sample cleaning solution.

15. The kit according to claim 14, characterized in that, The kit also has one or more of the following features: 1) In the digestion termination solution, the final concentration of the fetal bovine serum is 10-14% by volume, the final concentration of the penicillin-streptomycin antibiotic is 1-5% by volume, and the solution is supplemented to 100% by volume using the DMEM / F12 medium; 2) The sample preservation solution includes fetal bovine serum, penicillin-streptomycin antibiotics, 4-hydroxyethylpiperazine ethanethioic acid, and Hank's balanced salt solution; 3) The organoid cryopreservation solution includes serum-free cell cryopreservation solution and Y-27632; 4) The sample washing solution includes penicillin-streptomycin antibiotics and phosphate buffer solution.

16. The reagent kit according to claim 15, characterized in that, The kit also has one or more of the following features: 1) In the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% by volume, the final concentration of the penicillin-streptomycin antibody is 1-5% by volume, the final concentration of the 4-hydroxyethylpiperazine ethanethioic acid is 8-10 mmol / L, and the concentration is supplemented to 100% by volume with Hank's balanced salt solution; 2) In the organoid cryopreservation solution, the final concentration of Y-27632 was 8-12 μmol / L, which was then replenished to 100% by volume with serum-free cell cryopreservation solution; 3) In the sample washing solution, the final concentration of penicillin-streptomycin bispecific antibody is 1-5% by volume, and it is supplemented to 100% by volume with phosphate buffer.

17. Use of the kit according to any one of claims 1-8 in culturing osteosarcoma organoids, wherein the osteosarcoma organoids carry immune signatures.

18. Use of the kit according to any one of claims 9-16 in culturing osteosarcoma organoids, wherein the osteosarcoma organoids do not carry immune signatures.

19. A method for culturing osteosarcoma organoids carrying immune signatures, characterized in that, The method involves co-culturing immune cells and osteosarcoma single cells using the kit described in any one of claims 1-8 to obtain osteosarcoma organoids carrying immune characteristics.

20. A method for culturing osteosarcoma organoids without carrying immune signatures, characterized in that, The method involves co-culturing immune cells and osteosarcoma single cells using the kit described in any one of claims 9-16 to obtain osteosarcoma organoids that do not carry immune characteristics.

21. The method according to claim 19, characterized in that, The method comprises the following steps: Obtain a single-cell suspension of osteosarcoma; The lymphocyte and osteosarcoma single-cell suspensions were resuspended in the special culture medium in the kit according to any one of claims 1-8 to obtain a mixed cell suspension; Mix the cell suspension and matrix gel, and incubate until the matrix gel solidifies; By adding the special culture medium from any one of the kits described in claims 1-8, osteosarcoma organoids carrying immune characteristics can be obtained.

22. The method according to claim 21, characterized in that, The method has one or more of the following characteristics: 11) In step 1), osteosarcoma tissue is dissociated using sample dissociation digestion solution to obtain osteosarcoma single-cell suspension; 21) In step 2), the lymphocytes are derived from humans or mice; and / or the lymphocytes are peripheral blood lymphocytes; 22) In step 2), the ratio of the number of osteosarcoma single cells to the number of lymphocytes is 2-4:1; 31) In step 3), the matrix gel includes Matrigel growth factor reduction basement membrane matrix; 32) In step 3), the volume ratio of the matrix gel to the mixed cell suspension is 1:3-5; 33) In step 3), the incubation time is 30-60 min; and / or the incubation temperature is a suitable temperature for cell culture; 41) In step 4), the culture time is 7-14 days; and / or the culture temperature is a suitable temperature for cell culture.

23. The method according to claim 22, characterized in that, The ratio of the volume of the sample dissociation digestion solution to the mass of the osteosarcoma tissue was 0.4-1.0:1 mL / mg; And / or, the osteosarcoma tissue is derived from humans or mice.

24. A method for culturing osteosarcoma organoids, characterized in that, The method comprises the following steps: S1) Obtain a single-cell suspension of osteosarcoma; S2) Osteosarcoma single-cell suspension is resuspended in the special culture medium in the kit according to any one of claims 9-16 to obtain cell suspension; S3) Mix the cell suspension obtained in S2) with the matrix gel and incubate until the matrix gel solidifies; S4) Add the special culture medium from the kit according to any one of claims 9-16 to culture and obtain osteosarcoma organoids that do not carry immune characteristics.

25. The method according to claim 24, characterized in that, The method has one or more of the following characteristics: In step S1), osteosarcoma tissue is dissociated using sample dissociation digestion solution to obtain osteosarcoma single-cell suspension. S31) The matrix gel mentioned in step S3) includes Matrigel growth factor reduction basement membrane matrix; S32) The volume ratio of the matrix gel to the cell suspension in step S3) is 1-3:1; S33) The incubation time in step S3) is 30-60 min; and / or the incubation temperature is a suitable temperature for cell culture; S41) The culture time in step S4) is 7-14 days; and / or the culture temperature is a suitable temperature for cell culture.

26. The method according to claim 25, characterized in that, The ratio of the volume of the sample dissociation digestion solution to the mass of the osteosarcoma tissue was 0.4-1.0:1 mL / mg; And / or, the osteosarcoma tissue is derived from humans or mice.

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