Culture medium and culture method for mouse osteosarcoma organoid culture

Abstract

The invention discloses a culture medium for mouse osteosarcoma organoid culture and a culture method, and belongs to the technical field of biological medicine. The technical problem to be solved by the invention is how to prepare osteosarcoma organs. In order to solve the technical problem, the invention provides the culture medium for preparing the osteosarcoma organoid. The invention also provides a method for preparing the osteosarcoma organ, which comprises the following steps: mixing osteosarcoma cells with matrigel and a culture medium to obtain an osteosarcoma organ suspension, and culturing the osteosarcoma organ suspension to obtain the osteosarcoma organ. According to the invention, a working process and a test method of mouse osteosarcoma organoid culture, observation, identification, passage, collection, cryopreservation and downstream analysis are established, a more comprehensive tool is provided for research of osteosarcoma pathogenesis and drug screening and development, and a preclinical guidance research platform is also provided for personalized treatment of osteosarcoma patients.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a culture medium and a culture method for mouse osteosarcoma organoid culture. Background Art

[0002] Osteosarcoma is the most common primary malignant bone tumor in adolescents and is known as the "juvenile killer". It has a secretive course and progresses very quickly, and is prone to recurrence, metastasis, and chemotherapy resistance. Pathologically, osteosarcoma mainly manifests as pathological and tumorous osteogenesis, and the main pathological subtypes are osteoblastic, chondrogenic, and fibroblastic. Although osteosarcoma currently adopts a comprehensive treatment strategy of preoperative high-dose chemotherapy-surgical resection, limb reconstruction-postoperative high-dose chemotherapy, the 5-year survival rate of patients is about 60-70%, which is less than 30% in patients with recurrence and metastasis. The main chemotherapy regimens and overall survival of patients have not been effectively improved in the past nearly 40 years. Attempts to increase the dose and type of existing chemotherapy drugs have proven difficult to further improve patient prognosis. On the other hand, attempts to use new treatment ideas, such as immunotherapy, have not yet achieved beneficial effects in osteosarcoma. Therefore, it is imperative to find new mechanisms, new pathways, new targets, and new drugs for the treatment of osteosarcoma that are different from the current cytotoxic chemotherapy drugs. Given the extremely low incidence of osteosarcoma in the population, constructing a biological model that can represent the characteristics of the tumor is of great significance both for studying the pathogenesis and for the exploration and development of new drugs.

[0003] The current preclinical models for osteosarcoma anticancer drugs have significant limitations. The most significant problem is that they cannot restore the real survival microenvironment of the tumor in the body. Commonly used osteosarcoma research models include standard osteosarcoma cell lines, primary cultured cell lines of tumor specimens, xenograft models (Patient Derived Xenograft, PDX), humanized PDX models, xenogeneic spontaneous tumor models, transgenic mouse models, etc. Among them, cell lines are two-dimensional in vitro environments that have been cultured for a long time. In recent years, it has been increasingly proven that they cannot fully reflect the true situation of the tumor. Dogs are often used as xenogeneic spontaneous tumor models for osteosarcoma. Their incidence of osteosarcoma is dozens of times that of humans, and they can be used as animal models for studying osteosarcoma, but their integrated incidence is still very low, and the incubation period is long, and the experimental cost is extremely high. Mice have long been used as one of the most important model organisms for studying the pathogenesis of human diseases and drug testing. At the same time, the genetic similarity between humans and mice is higher than that between humans and dogs, which has significant advantages. Therefore, various experimental models are often designed based on mouse models. The mouse xenograft model is an in vivo environment, which solves the problem that the tumor cell line is an in vitro artificial environment, but it is a heterogeneous in vivo immune interaction environment. In recent years, it is believed that the immune system plays an increasingly important role in the occurrence and development of tumors, so a humanized mouse transplant model has been developed based on the xenograft model. However, its immune system comes from multiple donors, and the production cost is extremely high. The transgenic mouse model is currently one of the best models that can simulate the occurrence and development of the disease, conduct pathogenesis research and drug trials, but it also has the problem of high economic and time costs. It can be seen that there is an urgent need for an experimental model that is low-cost, easy to operate, can achieve high-throughput detection, and can fully simulate the real environment of tumor occurrence and development in vivo.

[0004] Compared with the traditional methods of in vitro cell line culture and PDX models for pathogenesis research and drug testing, the 3D tumor organoid, a revolutionary preclinical tumor research model that has emerged in the past few years, preserves the stem cell components of the primary tumor. The three-dimensional culture method is widely believed to simulate the in vivo growth environment and maintains the tumor tissue structure after long-term in vitro culture, which has been proven to better maintain the morphological and genetic characteristics of the original tumor. Compared with traditional methods, 3D tumor organoids are more representative of the original tumor tissue, and are easy to operate and have low research costs. It is suitable for efficient and accurate research on the mechanism and effect of a variety of anti-tumor drugs ( Figure 1 ). However, in the past few years, the vast majority of research on tumor organoids has focused on epithelial cancers, such as adenocarcinomas, while research on mesenchymal sarcomas has rarely been reported, and there have been no reports on mouse osteosarcoma. Summary of the invention

[0005] The technical problem to be solved by the present invention is: how to prepare osteosarcoma organoids.

[0006] To solve this technical problem, in the first aspect, the present invention provides a composition for preparing osteosarcoma organoids, which consists of the following components: Wnt signaling pathway activator, organoid cytokine, epidermal growth factor, ALK5 inhibitor, fibroblast growth factor, basic fibroblast growth factor, B27, N-acetyl-L-cysteine, glutamine, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 5α-dihydrotestosterone (DHT), and ROCK inhibitor.

[0007] Furthermore, in the above-mentioned composition, the ratio of the composition is as follows: 50 g of the Wnt signaling pathway activator, 100 g of the organoid cytokine, 5 μg of the epidermal growth factor, 500 nmol of the ALK5 inhibitor, 10 μg of the fibroblast growth factor, 1 μg of the basic fibroblast growth factor, 20 mL of B27 (50×), 1.25 mmol of N-acetyl-L-cysteine, 1 mmol of glutamine, 5 mmol of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 1 nmol of 5α-dihydrotestosterone (DHT), and 5 mmol of the ROCK inhibitor.

[0008] Furthermore, in the above composition, the Wnt signaling pathway activator is R-Spondin, and the organoid cytokine is Noggin.

[0009] The ROCK inhibitor may be Rho-associated coiled-coil protein kinase (Y-27632).

[0010] The fibroblast growth factor may be FGF-10.

[0011] The basic fibroblast growth factor may be FGF-2.

[0012] In a second aspect, the present invention provides a culture medium for preparing osteosarcoma organoids, wherein the culture medium contains an animal cell basal culture medium and the above-mentioned composition.

[0013] Furthermore, the animal cell basal culture medium may be DMEM / F12 culture medium.

[0014] Further, the culture medium may be composed of the above-mentioned composition and the DMEM / F12 culture medium. In one embodiment of the present invention, in a total volume of 1L of culture medium, the Wnt signaling pathway activator is 50g, the organoid cytokine is 100g, the epidermal growth factor is 5μg, the ALK5 inhibitor is 500nmol, the fibroblast growth factor is 10μg, the basic fibroblast growth factor is 1μg, the B27 (50×) is 20mL, the N-acetyl-L-cysteine ​​is 1.25mmol, the glutamine is 1mmol, the 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) is 5mmol, the 5α-dihydrotestosterone (DHT) is 1nmol, the ROCK inhibitor is 5mmol, and the rest is DMEM / F12 culture medium.

[0015] Furthermore, the culture medium also contains antibiotics. In one embodiment of the present invention, the antibiotics are a mixture of penicillin and streptomycin.

[0016] In a third aspect, the present invention provides a reagent for preparing osteosarcoma organoids, wherein the reagent comprises matrix gel and the above composition.

[0017] In a fourth aspect, the present invention provides a reagent for preparing osteosarcoma organoids, wherein the reagent comprises matrix gel and the above-mentioned culture medium.

[0018] Furthermore, in the above reagent, the matrix gel is a three-dimensional matrix.

[0019] In one embodiment of the present invention, in the above reagent, the volume of the matrix gel accounts for 60%, and the volume of the culture medium accounts for 40%.

[0020] In a fifth aspect, the present invention provides use of the above-mentioned composition in preparing osteosarcoma organoids.

[0021] In a sixth aspect, the present invention provides use of the above-mentioned culture medium in preparing osteosarcoma organoids.

[0022] In a seventh aspect, the present invention provides the use of the above-mentioned reagent in preparing osteosarcoma organoids.

[0023] In an eighth aspect, the present invention provides a method for preparing osteosarcoma organoids, comprising mixing osteosarcoma cells with the above-mentioned reagent to obtain an osteosarcoma organoid suspension, and culturing the osteosarcoma organoid suspension to obtain osteosarcoma organoids.

[0024] Furthermore, in the method, the identification method of osteosarcoma organoid formation includes any one of the following:

[0025] 1) Cell morphology identification;

[0026] 2) Cultivation and identification of cell characteristics.

[0027] 3) Pathological staining and immunohistochemical identification.

[0028] Furthermore, in the method described above, osteosarcoma tumor cells growing in clusters with osteoid-like matrix formation can be seen under a microscope, and the cells have significant heteromorphism.

[0029] Furthermore, in the method described above, the prepared organoids have a prolonged in vitro survival time, exceeding the doubling time of primary cell culture, and can be passaged and preserved.

[0030] Furthermore, in the method described above, the cell morphology of the osteosarcoma organoids is highly consistent with that of the primary osteosarcoma tumor by H&E staining.

[0031] Furthermore, in the method described above, the expression levels of PCNA, Alkaline phosphatase, p27, and Osteocalcin proteins in the osteosarcoma organoids are highly similar to those in the primary osteosarcoma tumor.

[0032] Furthermore, in the method, the identification criteria for osteosarcoma organoid formation are as follows:

[0033] 1. The in vitro survival time is prolonged, exceeding the doubling time of primary cell culture;

[0034] 2. It can be passed down and preserved;

[0035] 3. Through genome sequencing, histopathological analysis and other methods, it is confirmed that the histopathological characteristics of organoids are highly similar to those of primary tumor tissues.

[0036] In one embodiment of the present invention, in the above-mentioned osteosarcoma organoid suspension, the volume proportion of the matrix gel is 60%, the volume proportion of the culture medium is 40%, and the number of the osteosarcoma cells is 6000-8000.

[0037] In the present invention, the osteosarcoma organoid may be a mouse osteosarcoma organoid.

[0038] The beneficial technical effects achieved by the present invention are as follows:

[0039] 1. Based on the occurrence and development mechanism of osteosarcoma, the present invention uses the tumor tissue of transgenic osteosarcoma mice to invent a method for culturing mouse osteosarcoma organoids. This technology preserves the tumor stem cell components and supporting cells of the primary tumor.

[0040] 2. The present invention also provides an organoid culture medium that can highly simulate the in vivo growth environment of osteosarcoma. The culture medium achieves efficient and rapid in vivo tumor culture by adding extracellular matrix components and osteosarcoma organoid culture fluid containing various growth factors required for tumor growth.

[0041] 3. At the same time, the present invention has established the workflow and experimental methods for the culture, observation, identification, passage, collection, freezing and downstream analysis of mouse osteosarcoma organoids. At the same time, the preliminary research of the present invention has also established specific experimental conditions including culture medium composition, matrix gel concentration, cell density, observation and collection time, and established a standardized experimental method ( Figure 1 ). It provides a more comprehensive tool for the study of the pathogenesis of osteosarcoma and drug screening and development, and also provides a preclinical guiding research platform for the personalized treatment of osteosarcoma patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of osteosarcoma organoids.

[0043] Figure 2 This is a schematic diagram of osteosarcoma organoid culture. Figure 2 A in the middle shows osteosarcoma organoid spheres laid in a 6-well plate. Figure 2 Middle B is a 6-well plate with osteosarcoma organoid monomers and organoid culture medium. Figure 2 Middle C is an example of osteosarcoma organoid monomers plated in a 96-well plate.

[0044] Figure 3 This is a microscopic observation of osteosarcoma organoids (reference 400μm).

[0045] Figure 4 These are microscopic observations of typical osteosarcoma organoids at different time points.

[0046] Figure 5 This is a typical histopathological analysis of osteosarcoma organoids, shown under the H&E staining microscope (reference 100μm).

[0047] Figure 6 Comparative analysis of histopathology and immunohistochemical staining between typical osteosarcoma organoids and primary osteosarcoma tumors.

[0048] Figure 7 Quantitative analysis of the proliferation rates of different osteosarcoma organoids.

[0049] Figure 8Osteosarcoma organoids are used for drug testing. The picture above shows the appearance of osteosarcoma organoids under a microscope after receiving anti-tumor drugs (C1) at different concentrations (horizontal axis) and different times (vertical axis). It can be seen that the organoids proliferate well without the effect of drugs. After taking the drugs, the organoids die and break into fragments. It can be seen that the activity of the organoids is inversely proportional to the drug dose and duration of action. The picture below shows the activity response of different osteosarcoma organoids (DKO-1, -2 and -3) after testing anti-tumor drugs in this experiment. It can be seen that the drug effect is consistent.

[0050] Fig. 9 These are some of the experimental results for screening osteosarcoma organoid culture media, including Fig. 9 A and B are the results of culture in DMEM / F12 medium, and C and D are the results of culture in the presence of R-Spondin and 10% Noggin.

[0051] Fig.10 These are some of the experimental results for screening osteosarcoma organoid culture media, including Fig.10 A and B are the results of culture in organoid culture medium, C and D are the results of culture in organoid culture medium lacking fibroblast growth factor (FGF-10 / FGF-2), and E and F are the results of culture in organoid culture medium lacking epidermal growth factor.

[0052] Fig.11 These are some of the experimental results for screening osteosarcoma organoid culture media, including Fig.11 G and H show the results of organoid culture in the absence of A 83-01, I and J show the results of organoid culture in the absence of B27, and K and L show the results of organoid culture in the absence of N-acetyl-L-cysteine.

[0053] Fig.12 These are some of the experimental results for screening osteosarcoma organoid culture media, including Fig.12 M and N in the middle are the results of organoid culture medium lacking glutamine, O and P are the results of organoid culture medium lacking 4-hydroxyethylpiperazineethanesulfonic acid, Q and R are the results of organoid culture medium lacking 5α-dihydrotestosterone, and S and T are the results of organoid culture medium lacking Y-27632. DETAILED DESCRIPTION

[0054] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0055] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0056] The reagents used in the following examples are as follows:

[0057] ① Reagents required for preparation and culture of osteosarcoma organoids

[0058] Phosphate buffered saline (PBS), wherein the cold PBS solution is a PBS solution refrigerated at 4°C (Gibco, #10-010-023);

[0059] Collagenase II (Warthington, #LS004202);

[0060] DMEM / F12 culture medium (Cytiva HyClone, #SH3026101);

[0061] 0.25% Trypsin-EDTA (STEMCELL Technologies, #07901);

[0062] Fetal bovine serum (FBS) (Gibco, #A4766801);

[0063] Dispase solution, 5U / mL (STEMCELL Technologies, #07913);

[0064] DNase I solution, 1 mg / mL (STEMCELL Technologies, #07900);

[0065] 0.4% Trypan blue solution (Corning, #MT25900CI);

[0066] Red blood cell lysis buffer (Invitrogen, #50-112-9751);

[0067] Matrigel (Corning, #356255);

[0068] HBSS solution, 1× (Cytiva HyClone, #SH3003002);

[0069] Osteosarcoma digestion medium (per 10 ml): add 50 mg collagenase II to 10 ml of DMEM / F12 culture medium containing 5% FBS, and the final concentration of collagenase II is 5 mg / ml;

[0070] Dispase / DNase I mixed digestion solution: 2 × 960 μL Dispase solution was preheated in a 37°C water bath at least 10 minutes before use. Before use, 2 × 40 μL DNase I solution was added and mixed.

[0071] ②Osteosarcoma organoid culture medium

[0072] Based on DMEM / F12 culture medium (Cytiva HyClone, #SH3026101) containing 5% (mass ratio) R-Spondin (Novus Biologicals, #NBP192357PE), 10% (mass ratio) Noggin (R&D Systems, #1967NG025), and additionally adding the following ingredients:

[0073] Epidermal growth factor (EGF), 5 ng / ml (Millipore Sigma, #GF155);

[0074] 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A 83-01), 500 nM (Tocris, #29-395-0);

[0075] mouse fibroblast growth factor 10 (FGF-10), 10 ng / ml (R&D Systems, #6224FG025CF);

[0076] mouse fibroblast growth factor 2 (FGF-2), 1 ng / ml (Invitrogen, #PIRP8626);

[0077] B27, 1×(Gibco, #A3582801);

[0078] N-acetyl-L-cysteine, 1.25 mM (Fisher, #O1049-25);

[0079] Glutamine, 1 mM (Thermo Scientific, #AC128275000);

[0080] 4-Hydroxyethylpiperazineethanesulfonic acid, 5 mM (Sigma-Aldrich, #NC0229916);

[0081] 5α-dihydrotestosterone, 1 nM (MilliporeSigma, #01-001-193);

[0082] Rho-associated coiled-coil protein kinase (Y-27632), 5 mM (STEMCELL, #NC0791122);

[0083] Penicillin-streptomycin mixture, 1× (Corning, #30001CI).

[0084] ③ Osteosarcoma organoid preservation and resuscitation

[0085] GCDR Cell Dissociation Reagent (GCDR) (STEMCELL Technologies, #07174);

[0086] Cell recovery solution (Corning, #354253);

[0087] Dimethyl sulfoxide 1× (Tocris Bioscience, #31-765-ML);

[0088] The composition ratio of the osteosarcoma organoid freezing solution is: 70% (volume ratio) FBS, 20% (volume ratio) osteosarcoma organoid culture medium and 10% (volume ratio) 1× dimethyl sulfoxide solution.

[0089] ④ Histopathological analysis of osteosarcoma organoids

[0090] 10% neutral buffered formalin solution (Epredia, #22-046-361);

[0091] 4% paraformaldehyde (PFA) in PBS (Biyuntian, #P0099);

[0092] Tissue-Tek OCT tissue embedding solution (Sakura, #4583);

[0093] HistoGel matrix gel (Thermo Scientific, #HG-4000-144);

[0094] HistoScreen tissue fixation cassette (Epredia, Fisher, #B851000);

[0095] Other routine reagents and consumables for histopathology.

[0096] ⑤Quantitative evaluation of osteosarcoma organoid growth and drug response

[0097] CellTiter-Glo 3D Cell Viability Assay Kit (Promega #G9682);

[0098] 96-well microplate fluorimeter (Promega, #GM3500).

[0099] Example 1. Preparation and culture of osteosarcoma organoids

[0100] The amount of reagents in the experimental method described below is used to dissociate mouse limb osteosarcoma tumor tissue of approximately 1.5×1.5 cm, and can be adjusted according to the size. Mice with osteosarcoma were prepared by the applicant and disclosed in the document “Wang J, Aldahamsheh O, Ferrena A, et al. The interaction of SKP2 with p27 enhances the progression and stemness of osteosarcoma. Ann NY Acad Sci. 2021; 1490(1): 90-104. doi: 10.1111 / nyas.14578”.

[0101] 1.1. According to the experimental requirements and animal ethics, mice were killed by carbon dioxide and osteosarcoma tissues were collected.

[0102] 1.2. Following the principles of animal aseptic surgery, harvest the entire osteosarcoma tissue, weigh it, determine the volume of reagents required for the reaction based on the volume and mass, then transfer it to a sterile culture dish filled with cold PBS solution and wash it three times, then discard the washing solution.

[0103] 1.3 Dissect the tumor. Using a dissecting microscope, fine forceps and tweezers, remove the remaining fat, connective tissue and any scab or debris, and wash again with cold PBS.

[0104] 1.4. Use a scalpel to cut the tissue into small pieces in a culture dish and mince further. Add a small amount of preheated osteosarcoma digestion medium while cutting. Sometimes the osteogenic part of the tumor is difficult to mince. In this case, it is necessary to use a tungsten steel scalpel for strong cutting, or add digestion medium to the tissue grinding dish and grind it. This type requires more digestion medium and up to 30 minutes of additional digestion and incubation. The amount of digestion medium is: digest 10 mg of minced tissue with a total content of 1 mg of collagenase II.

[0105] 1.5. Place the mixture of chopped tissue and digestion medium in a sterile culture dish, keep it at 37°C, and incubate it on a gentle shaker at 80 rpm for 30-60 minutes. During this period, use a 25 ml pipette to blow and beat every 5-10 minutes to assist in further digestion of the tissue, evenly dissociate and mechanically destroy the undigested tissue, and check the tissue digestion at the same time. If there is no visible minced meat within 60 minutes of the digestion process and it is turbid, it indicates that the digestion has been completed and the reaction should be terminated.

[0106] 1.6. Transfer all the minced meat and tissue fluid to a centrifuge tube, centrifuge the digested tissue at a low temperature of 500 rpm for 5 minutes, discard the supernatant and collect the precipitate.

[0107] 1.7. Use 6 ml cold 0.25% trypsin-EDTA per reaction system to resuspend the pellet obtained in the previous step. Incubate at 4°C for 20-30 minutes. (Note: To minimize cell death, keep trypsin at ≤4°C before and during use. An orbital shaker can be used during the trypsin digestion step for optimal digestion. Finally, add pre-cooled HBSS solution mixed with 2% FBS to terminate the digestion reaction.

[0108] 1.8. Centrifuge at 500 rpm for 5 minutes, discard the supernatant and collect the precipitate sample.

[0109] 1.9. Add 2 ml of preheated Dispase / DNaseI mixed digestion solution to the collected precipitate sample. Use a P1000 pipette to blow the sample until the solution is uniform and translucent, with no visible tissue fragments (do not allow digestion to last more than 2 minutes). Finally, add 5 times the reaction volume of DMEM / F12 culture medium containing 5% FBS to terminate the reaction and obtain a mixed sample.

[0110] 1.10. Filter the mixture sample obtained in the previous step through a 40μm cell strainer and transfer the cell suspension into a new 50ml centrifuge tube. Centrifuge at 200rpm for 5 minutes and discard the supernatant. Repeat the operation and resuspend the precipitate in an appropriate amount of ice PBS solution.

[0111] 1.11. Add 1× red blood cell lysis buffer, incubate on ice with shaking for 3-5 minutes, centrifuge at 200 rpm for 5 minutes and discard the supernatant, resuspend the precipitate in osteosarcoma organoid culture medium to obtain osteosarcoma cell suspension.

[0112] 1.12. Take a small amount of cell suspension, stain it with trypan blue, and then count the cells.

[0113] 1.13. Prepare osteosarcoma organoid suspension: Mix the osteosarcoma cell suspension, osteosarcoma organoid culture medium and matrix gel in 1.11 to obtain osteosarcoma organoid suspension. In each 100ul of osteosarcoma organoid suspension, the volume content of matrix gel is 60% matrix gel, and the content of osteosarcoma cells is 6000-8000. Place the osteosarcoma organoid suspension on ice. 1.14. Use a 200μL pipette to make osteosarcoma organoid spheres (organoid spheres in English). Each monomer is made of 15-20μL of osteosarcoma organoid suspension. For example, for a 6-well plate, each well contains a total of 100μL of organoid mixed solution (for a 96-well plate, 50μL is used per well) to evenly distribute the osteosarcoma organoid monomers on the surface of the culture dish.

[0114] 1.15. Immediately invert the well plate with osteosarcoma organoid monomers in a 37°C cell culture incubator for more than 20 minutes to solidify. Take it out after 20 minutes and cover the organoid monomers with an appropriate amount of osteosarcoma organoid culture medium. For example: for a 6-well plate, use 1.5ml to 2ml of organoid culture medium, and for a 96-well plate, use 100μL per well.

[0115] 1.16. Osteosarcoma organoid culture medium (liquid obtained by mixing osteosarcoma organoid culture medium and matrix gel) is replaced every 48 hours, and 50% of the total liquid volume in the culture dish is replaced each time. The growth of osteosarcoma organoids is observed under a microscope every day. The culture can be sustained for several weeks to several months.

[0116] Among them, the criteria for osteosarcoma organoid formation are as follows:

[0117] 1. The in vitro survival time is prolonged, exceeding the doubling time of primary cell culture;

[0118] 2. It can be passed down and preserved;

[0119] 3. Through genome sequencing, histopathological analysis and other methods, it is confirmed that organoids and primary tumor tissues have a high degree of similarity in biological behavior, gene and protein expression and other aspects.

[0120] Figure 3 This is an example of low-power observation of osteosarcoma organoids cultured for 21 days (as shown by the arrows in the figure, the organoids grow in clusters and are distributed in the culture medium). Figure 4 These are high-power microscopic observations of typical osteosarcoma organoids at different time points. Figure 4 A, B, and C are observation images of several different osteosarcoma organoids at different time points as shown in the figure (reference 100 μm). Figure 4 a, b, and c in center C are three adjacent osteosarcoma organoids.

[0121] Using the above organoid culture method, six organoid models from different osteosarcoma mice were representatively cultured, named DKO-1, DKO-2, DKO-3 and TKO-1, TKO-2, TKO-3. Among them, the genotypes of osteosarcoma mice from DKO-1, DKO-2, and DKO-3 were Osx1-Cre; Trp53 lox / lox ; Rb1 lox / lox The genotype of osteosarcoma mice from TKO-1, TKO-2, and TKO-3 is Osx1-Cre; Rb1 lox / lox ;Trp53 lox / lox ;SKP2 - / - .

[0122] Example 2: Passaging and cryopreservation of osteosarcoma organoids

[0123] 2.1. The passaging of osteosarcoma organoids can generally be carried out 3-6 weeks after the first culture. The specific details need to be combined with microscopic observation. Generally, the density of the organoid sphere is required not to exceed 50% of the entire organoid monomer. After 3-6 weeks of growth, the diameter of the organoid can generally reach 500μM.

[0124] 2.2. Aspirate and discard the culture medium in each well, wash twice with cold PBS solution, and discard the PBS solution.

[0125] 2.3. Add cold GCDR cell dissociation agent to dissociate organoid monomers, and use a 1000μL pipette tip to mechanically separate organoid monomers. The amount of GCDR solution used is approximately equal to the amount of osteosarcoma organoid culture medium used, for example: use 2ml of cold GCDR cell dissociation agent per well of a 6-well plate.

[0126] 2.4. Osteosarcoma organoids can be directly passaged or used for drug experiments at this step. The direct passage method is to add matrix gel and osteosarcoma organoid culture medium in a ratio of 1:3, mix thoroughly by pipetting, place on ice, and continue with subsequent experiments according to step 1.14.

[0127] 2.5. Collect all solutions and mixtures, centrifuge at low temperature, centrifuge at 80 rpm for 10 minutes, and then at 1000 rpm for 5 minutes, discard the supernatant, and collect the centrifuged samples.

[0128] 2.6. Add Dispase / DNaseI mixed digestion solution, pipette on ice for 2-4 minutes, and add pre-cooled HBSS solution mixed with 2% FBS to terminate the digestion reaction.

[0129] 2.7. Filter the cell suspension using a 40 μm cell strainer. Centrifuge at 400 rpm for 5 minutes at low temperature, discard the supernatant, and use the organic pellet for passaging or cryopreservation.

[0130] 2.8 Osteosarcoma organoids can be cryopreserved at this stage. Resuspend the centrifuged components with 1 ml of osteosarcoma organoid cryopreservation solution and place in a sterile cryotube. Gradually freeze the sample to ≤-80°C and further transfer to a liquid nitrogen tank for long-term storage.

[0131] Example 3. Histopathological analysis of osteosarcoma organoids

[0132] 3.1 Experimental Methods

[0133] 3.1.1. Heat the HistoGel matrix gel to 60±5℃ in water in advance to liquefy it, and then cool it down to 50±5℃.

[0134] 3.1.2. Use a 1000P pipette to place 4-6 drops of HistoGel matrix gel in the HistoScreen tissue fixation box, spread a layer of HistoGel matrix gel, and wait for low-temperature solidification.

[0135] 3.1.3. Take the centrifuged sample in step 2.5 of Example 2, resuspend it in an appropriate amount of HistoGel matrix gel, mix it thoroughly, blow it repeatedly, and spread it in the tissue fixation box in the previous step. After it is naturally cooled and slightly solidified, add 4-6 drops of HistoGel matrix gel on it to make it a flat layer to wrap the organoid sample.

[0136] 3.1.4. After the sample block in the tissue fixation box is completely dry, immerse the entire tissue box in formalin solution for overnight fixation.

[0137] 3.1.5 The next day, take the sample block out of the box, wash off the formalin solution, embed the sample block in paraffin, and perform routine histopathological sectioning and analysis.

[0138] 3.1.6. For immunohistochemical staining, standard procedures were followed after sectioning. The relevant antibodies used for immunohistochemistry are as follows: PCNA (Vector Labs, SP6), p27 (BD Biosciences, #610242), Anti-alkalinephosphatase (Abcam, ab354), Anti-osteocalcin (Abcam, ab13420). SKP2 (Proteintech, #15010-1-AP).

[0139] 3.2 Experimental Results

[0140] The experimental results are as follows Figure 5 and Figure 6 shown. Figure 5 This is a typical histopathological analysis of osteosarcoma organoids, shown under H&E staining (reference 100μm). H&E staining shows osteosarcoma tumor cells growing in clusters with osteoid-like matrix formation, and the cells have significant heteromorphism. Figure 6 Comparative analysis of histopathology and immunohistochemical staining between typical osteosarcoma organoids and primary osteosarcoma tumors. Figure 6 A in the middle is the primary tumor of transgenic osteosarcoma mice. Figure 6 B is an osteosarcoma organoid (reference 100 μm). The antibodies used are shown in the figure. It can be seen that the osteosarcoma organoid is highly similar to the primary osteosarcoma tumor in terms of histopathology.

[0141] Example 4. Recovery and re-cultivation of osteosarcoma organoids

[0142] 4.1. Take out the frozen organoid cryopreservation tube to be thawed, shake rapidly in a 37°C water bath, and heat until thawed. Immediately transfer 1 ml of organoid cryopreservation solution to 10 ml of osteosarcoma organoid culture medium pre-filled, and pipette repeatedly. Centrifuge at 500 RPM for 5 minutes and discard the supernatant.

[0143] 4.2. Resuspend in an appropriate amount of osteosarcoma organoid culture medium, take a small amount of cell suspension, stain with trypan blue, and count the cells.

[0144] Example 5: Osteosarcoma organoids for quantitative detection of tumor proliferation ability and rapid drug response quantitative detection

[0145] This method can be used to quantitatively detect the proliferation rate of osteosarcoma organoids and detect the inhibitory rate of drugs on osteosarcoma organoids. This reaction system is based on GloMax 96-well microplate fluorometer and CellTiter-Glo 3D cell viability assay kit.

[0146] 5.1. Quantitative detection of tumor proliferation ability of osteosarcoma organoids 5.1.1. Using a light-proof 96-well plate, the osteosarcoma organoids required for the experiment were cultured for 72 hours starting from step 1.16 in Example 1 (or step 4.2 in Example 4). After cell counting, the organoid monomers DKO-1, DKO-2, DKO-3, TKO-1, TKO-2, and TKO-3 were plated separately, and 50 μL of osteosarcoma organoid suspension was required in each well to ensure that the volume and cell number of each well remained consistent.

[0147] 5.1.2. Immediately invert the well plate with organoid monomers in the incubator for more than 20 minutes to solidify. After taking it out, add 100 μL of organoid culture medium to each well and place it in a 37°C constant temperature incubator for culture. This is recorded as day 1.

[0148] 5.1.3. Observe the growth of organoids under a microscope the next day to ensure that the organoids in the 96-well plate grow well. The organoid culture medium is replaced once a day, with 50% of the total volume replaced each time.

[0149] 5.1.4. Cell viability was measured on the 2nd, 4th, 6th, 8th, 10th and 12th day of culture using the CellTiter-Glo 3D Cell Viability Assay Kit according to the instructions of the kit. The data recording software provided by the 96-well microplate fluorescence analyzer was used for data statistics, and SPSS22.0 software was used for data analysis. Three independent experiments were set up.

[0150] The test results are as follows Figure 7 shown. Figure 7The results are the quantitative analysis of the proliferation rate of 6 different osteosarcoma organoids (DKO-1, DKO-2, DKO-3, TKO-1, TKO-2, TKO-3). The results show that after 12 days of experimental analysis, the proliferation of the 3 osteosarcoma organoids with the DKO genotype was significantly faster than that of the 3 osteosarcoma organoids with the TKO genotype (p < 0.01).

[0151] 5.2 Osteosarcoma organoids for rapid quantitative drug response testing

[0152] 5.2.1. Using a light-proof 96-well plate, the osteosarcoma organoids required for the experiment were cultured for 72 hours starting from step 1.16 in Example 1 (or step 4.2 in Example 4). After cell counting, the organoid monomers DKO-1, DKO-2, and DKO-3 were plated separately, and 50 μL of osteosarcoma organoid suspension was required in each well to ensure that the volume and cell number of each well remained consistent.

[0153] 5.2.2. Immediately invert the well plate with organoid monomers in the incubator for more than 20 minutes to solidify. After taking it out, add 100 μL of organoid culture medium to each well and place it in a 37°C constant temperature incubator for culture. This is recorded as day 1.

[0154] 5.2.3. Observe the growth of organoids under a microscope the next day to ensure that the organoids in the 96-well plate grow well. The organoid culture medium is replaced once a day, with 50% of the total volume replaced each time.

[0155] 5.2.4. According to the growth of osteosarcoma organoids, drug testing starts 3-6 days after plating, when the volume of osteosarcoma organoid monomers under the microscope is about 100 μm. The drug to be tested needs to be diluted into the organoid culture medium and then added to the organoid culture to ensure that its final concentration is consistent during the culture process. In this embodiment, the drug to be tested is C1, purchased from MilliporeSigma, #500519 (CAS#432001-69-9). The concentrations used in the experiment were 0 μM, 0.75 μM, 1.5 μM, 3 μM, 6 μM and 12 μM, respectively, and dimethyl sulfoxide (DMSO) was added in equal amounts to the control group. Each concentration was repeated 3 times. It should be noted that when using a 96-well plate, the four adjacent side alternating wells should be avoided as test wells to avoid fluorescence interference.

[0156] 5.2.5. The time after drug addition was recorded as time 0 (0h). Samples were taken at 0h, 24h, 48h, 72h, and 96h after drug addition to observe the growth of organoids under a microscope.

[0157] 5.2.6. At 0h, 24h, 48h, 72h, and 96h after adding the drug, the CellTiter-Glo 3D Cell Viability Assay Kit was used to measure the cell viability according to the instructions of the kit. GloMax The data recording software provided by the 96-well microplate fluorescence analyzer was used for data statistics, and SPSS22.0 software was used for data analysis. Three independent experiments were set up.

[0158] The test results are as follows Figure 8 . Figure 8 The upper middle picture shows the appearance of osteosarcoma organoids under a microscope after receiving anti-tumor drugs (C1, Millipore Sigma, #500519) at different concentrations (horizontal axis) and different times (vertical axis). It can be seen that the organoids proliferate well without the effect of drugs. After the drug is used, with the increase of drug dosage and the extension of drug treatment time, the organoids die and break into fragments. It can be seen that the activity of the organoids is inversely proportional to the drug dosage and the duration of action. Figure 8 The lower middle figure shows the activity response of three osteosarcoma organoids (DKO-1, -2 and -3) 96 hours after the addition of anti-tumor drugs. It can be seen that the drug effects are consistent. Through experimental analysis, it can be seen that the IC50 of drug C1 in osteosarcoma organoids is 0.47±0.19μM.

[0159] Example 6: Optimization process of culture medium

[0160] After the osteosarcoma tissue was lysed and cultured for the same period of time ( Fig. 9 A and C are cultured for 48 hours, B and D are cultured for 120 hours) It can be seen that when using DMEM / F12 culture medium, tumor cells adhere to the wall in large quantities, fail to effectively form spheres or form very few spheres (AB), while DMEM / F12 culture medium supplemented with 5% R-Spondin and 10% Noggin is conducive to tumor cells forming spheres and growing in suspension (CD). The results are shown in Fig. 9 .

[0161] Figure 10-12These are the experimental results for screening osteosarcoma organoid culture medium. After the osteosarcoma tissue was lysed and cultured under the same conditions for the same period of time (A, C, E, G, I, K, M, O, Q, S are 24 hours after culture, and B, D, F, H, J, L, N, P, R, T are the 7th day), it can be seen that tumor organoids grew well under the conditions of the full-type classified organ culture medium (AB). In comparison, organoid culture medium lacking fibroblast growth factor (FGF-10 / FGF-2) (CD), organoid culture medium lacking epidermal growth factor (EF), organoid culture medium lacking A 83-01 (GH), organoid culture medium lacking B27 (IJ), organoid culture medium lacking N-acetyl-L-cysteine ​​(KL), organoid culture medium lacking glutamine (MN), organoid culture medium lacking 4-hydroxyethylpiperazineethanesulfonic acid (OP), organoid culture medium lacking 5α-dihydrotestosterone (QR), and organoid culture medium lacking Y-27632 (ST) are all not conducive to promoting organoid proliferation.

[0162] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A composition for preparing osteosarcoma organoids, characterized in that: The composition consists of the following components: a Wnt signaling pathway activator, an organoid cytokine, an epidermal growth factor, an ALK5 inhibitor, a fibroblast growth factor, a basic fibroblast growth factor, B27, N-acetyl-L-cysteine, glutamine, 4-hydroxyethylpiperazineethanesulfonic acid, 5α-dihydrotestosterone, and a ROCK inhibitor.

2. The composition according to claim 1, characterized in that: The composition has the following ratio: 50 g of the Wnt signaling pathway activator, 100 g of the organoid cytokine, 5 μg of the epidermal growth factor, 500 nmol of the ALK5 inhibitor, 10 μg of the fibroblast growth factor, 1 μg of the basic fibroblast growth factor, 20 mL of B27, 1.25 mmol of N-acetyl-L-cysteine, 1 mmol of glutamine, 5 mmol of 4-hydroxyethylpiperazineethanesulfonic acid, 1 nmol of 5α-dihydrotestosterone, and 5 mmol of the ROCK inhibitor.

3. A culture medium for preparing osteosarcoma organoids, comprising an animal cell basal medium and the composition according to claim 1 or 2.

4. A reagent for preparing osteosarcoma organoids, comprising matrix gel and the composition according to claim 1 or 2.

5. A reagent for preparing osteosarcoma organoids, comprising matrigel and the culture medium according to claim 3.

6. Use of the composition according to claim 1 or 2 in preparing osteosarcoma organoids.

7. Use of the culture medium according to claim 3 in preparing osteosarcoma organoids.

8. Use of the reagent according to claim 4 or 5 in preparing osteosarcoma organoids.

9. A method for preparing osteosarcoma organoids, characterized in that: The method comprises mixing osteosarcoma cells with the reagent according to claim 4 or 5 to obtain an osteosarcoma organoid suspension, and culturing the osteosarcoma organoid suspension to obtain osteosarcoma organoids.

10. The method according to claim 9, characterized in that: The osteosarcoma cells are mouse osteosarcoma cells.

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