A culture medium and culture method for mouse osteosarcoma organoid culture

Abstract

The present invention discloses a culture medium and a culture method for culturing mouse osteosarcoma organoids, and belongs to the field of biomedicine technology. The technical problem to be solved by the present invention is: how to prepare osteosarcoma organoids. In order to solve this technical problem, the present invention provides a culture medium for preparing osteosarcoma organoids. The present invention also provides a method for preparing osteosarcoma organoids, the method comprising mixing osteosarcoma cells with matrix gel and culture medium to obtain an osteosarcoma organoid suspension, culturing the osteosarcoma organoid suspension, and obtaining osteosarcoma organoids. The present invention establishes a workflow and experimental method for mouse osteosarcoma organoid culture, observation, identification, passage, collection, freezing and downstream analysis, providing a more comprehensive tool for the study of the pathogenesis of osteosarcoma and drug screening and development, and also providing a preclinical guiding research platform for the personalized treatment of osteosarcoma patients.

Description

Technical Field

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

[0002] Osteosarcoma is the most common primary malignant bone tumor in adolescents, often nicknamed the "juvenile killer." Its insidious and rapidly progressive course is characterized by high recurrence, metastasis, and chemotherapy resistance. Pathologically, osteosarcoma primarily manifests as pathological and tumorous osteogenesis, with major pathological subtypes including osteoblastic, chondrogenic, and fibroblastic. Despite the current comprehensive treatment strategy of preoperative high-dose chemotherapy followed by surgical resection and limb reconstruction followed by postoperative high-dose chemotherapy, the 5-year survival rate for osteosarcoma is approximately 60-70%, with rates below 30% for recurrent and metastatic patients. Major chemotherapy regimens and overall survival have not significantly improved over the past 40 years. Extensive attempts to increase the dose and type of existing chemotherapy drugs have proven ineffective in further improving patient outcomes. Furthermore, novel therapeutic approaches, such as immunotherapy, have yet to yield beneficial results in osteosarcoma. Therefore, the search for novel mechanisms, pathways, targets, and drugs for the treatment of osteosarcoma, distinct from current cytotoxic chemotherapy, is imperative. 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] Current preclinical models for osteosarcoma anticancer drugs have significant limitations, the most notable being their inability to replicate the in vivo tumor microenvironment. Commonly used osteosarcoma research models include standard osteosarcoma cell lines, primary cultured cell lines derived from tumor specimens, patient-derived xenograft (PDX) models, humanized PDX models, xenogeneic spontaneous tumor models, and transgenic mouse models. Cell lines, among other models, are two-dimensional, in vitro models that have undergone long-term culture and have been increasingly shown in recent years to not fully reflect the true nature of the tumor. Dogs are often used as xenogeneic spontaneous tumor models for osteosarcoma. Their incidence of osteosarcoma is several dozen times higher than that of humans, making them suitable animal models for osteosarcoma research. However, their overall incidence remains low, their incubation period is long, and experimental costs are extremely high. Mice have long been a key model organism for studying human disease pathogenesis and drug testing. Furthermore, the genetic similarity between humans and mice is higher than that between humans and dogs, offering significant advantages. Consequently, many experimental models are designed based on mouse models. The mouse xenograft model is an in vivo environment, which solves the problem that tumor cell lines are in an artificial environment in vitro, but it is a xenogeneic 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 humanized mouse transplant models have been developed based on xenograft models. However, its immune system comes from multiple donors, and the production cost is extremely high. Transgenic mouse models are currently one of the best models that can simulate the occurrence and development of diseases, conduct pathogenesis research and drug trials, but they also have 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 the body.

[0004] Compared with the traditional methods of in vitro cell line culture and PDX models used for pathogenesis research and drug testing, the three-dimensional tumor organoid (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, thus being proven to better maintain the morphological and genetic characteristics of the original tumor. Compared with traditional methods, three-dimensional 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 various 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 studies on mesenchymal sarcomas are rare, and there are 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: 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 (HEPES), 5α-dihydrotestosterone (DHT), and a 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-coiled 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 comprises 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] Furthermore, 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, comprising Matrigel and the above-mentioned composition.

[0017] In a fourth aspect, the present invention provides a reagent for preparing osteosarcoma organoids, wherein the reagent comprises matrigel 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 culture characteristics.

[0027] 3) Pathological staining and immunohistochemical identification.

[0028] Furthermore, in the method described above, microscopic observation reveals osteosarcoma tumor cells growing in clusters accompanied by osteoid-like matrix formation, with the cells exhibiting significant atypia.

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

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

[0031] Furthermore, in the method, 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 survival time in vitro is prolonged, exceeding the doubling time of primary cell culture;

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

[0035] 3. Through genomic sequencing, histopathological analysis and other methods, it was 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 osteosarcoma organoid suspension, the volume of the matrix gel accounts for 60%, the volume of the culture medium accounts for 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 pathogenesis and progression of osteosarcoma, this invention uses tumor tissue from transgenic osteosarcoma mice to develop 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. This 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 a workflow and experimental method 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 timing, 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 Middle A 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 Microscopic observation of osteosarcoma organoids (reference 400 μm).

[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 (referenced to 100μm).

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

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

[0049] Figure 8Osteosarcoma organoids are used for drug testing. The figure above shows the appearance of osteosarcoma organoids under a microscope after being exposed to anti-tumor drugs (C1) at different concentrations (horizontally) and for different times (vertically). It can be seen that the organoids proliferate well without the effect of drugs. After the drug is applied, 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 figure below shows the activity response of different osteosarcoma organoids (DKO-1, -2 and -3) after testing the anti-tumor drugs in this experiment, and it can be seen that the drug effects are consistent.

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

[0051] Figure 10 These are some of the experimental results for screening osteosarcoma organoid culture media, including Figure 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] Figure 11 These are some of the experimental results for screening osteosarcoma organoid culture media, including Figure 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] Figure 12 These are some of the experimental results for screening osteosarcoma organoid culture media, including Figure 12 M and N are the results of organoid culture in the culture medium lacking glutamine, O and P are the results of organoid culture in the culture medium lacking 4-hydroxyethylpiperazineethanesulfonic acid, Q and R are the results of organoid culture in the culture medium lacking 5α-dihydrotestosterone, and S and T are the results of organoid culture in the culture medium lacking Y-27632. DETAILED DESCRIPTION

[0054] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0055] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[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 of collagenase II to 10 ml of DMEM / F12 culture medium containing 5% FBS, to a final concentration of 5 mg / ml of collagenase II;

[0070] Dispase / DNase I mixed digestion solution: Preheat 2 × 960 μL of Dispase solution in a 37°C water bath for at least 10 minutes before use. Immediately before use, add 2 × 40 μL of DNase I solution and mix.

[0071] ②Osteosarcoma organoid culture medium

[0072] The culture medium was based on DMEM / F12 medium (Cytiva HyClone, #SH3026101) containing 5% (mass ratio) R-Spondin (Novus Biologicals, #NBP192357PE) and 10% (mass ratio) Noggin (R&D Systems, #1967NG025), and the following additional components were added:

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

[0074] 3-(6-methyl-2-pyridyl)-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 recovery

[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 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 medium (Sakura, #4583);

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

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

[0095] Other routine histopathology reagents and consumables.

[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 reagent amounts in the experimental method described below are used to dissociate approximately 1.5×1.5 cm mouse limb osteosarcoma tumor tissue and can be adjusted based on the size. Osteosarcoma mice were prepared by the applicant and are 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 experimental requirements and animal ethics, mice were killed by carbon dioxide and osteosarcoma tissues were collected.

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

[0103] 1.3 Dissect the tumor. Using a dissecting microscope, fine forceps, and tweezers, remove any remaining fat, connective tissue, and any scabs 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 further mince. Add a small amount of preheated osteosarcoma digestion medium while cutting. Sometimes the bone-forming 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. Such cases require more digestion medium and up to 30 minutes of additional digestion and incubation. The amount of digestion medium used is: digest 10 mg of minced tissue with a total content of 1 mg of collagenase II in digestion medium.

[0105] 1.5. Place the mixture of minced tissue and digestion medium in a sterile Petri dish and incubate at 37°C on a gentle shaker at 80 rpm for 30-60 minutes. Use a 25ml pipette to agitate the tissue every 5-10 minutes to further digest the tissue, evenly dissociate and mechanically disrupt undigested tissue, and monitor the progress of tissue digestion. If the digestion process is turbid and no minced meat is visible within 60 minutes, digestion is complete 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 500 rpm for 5 minutes at low temperature. Discard the supernatant and collect the precipitate.

[0107] 1.7. Resuspend the pellet from the previous step in 6 ml of cold 0.25% trypsin-EDTA per reaction. Incubate at 4°C for 20-30 minutes. (Note: To minimize cell death, keep the trypsin at ≤4°C before and during use. Use an orbital shaker during the trypsinization step for optimal digestion.) Terminate the digestion reaction by adding pre-chilled HBSS mixed with 2% FBS.

[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 / DNase I digestion solution to the collected pellet. Use a P1000 pipette to pipette the sample until the solution is uniformly translucent and free of visible tissue debris (do not allow digestion to exceed 2 minutes). Finally, add 5 times the reaction volume of DMEM / F12 medium containing 5% FBS to terminate the reaction and obtain a mixed sample.

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

[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, discard the supernatant, and resuspend the pellet in osteosarcoma organoid culture medium to obtain an 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 matrigel in 1.11 to obtain osteosarcoma organoid suspension. For every 100ul of osteosarcoma organoid suspension, the volume content of matrigel is 60% matrigel, 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 from 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) so that the osteosarcoma organoid monomers are evenly distributed on the surface of the culture dish.

[0114] 1.15 Immediately invert the plate containing the osteosarcoma organoids and place it in a 37°C cell culture incubator for at least 20 minutes to solidify. After 20 minutes, remove the plate and cover the organoids with an appropriate amount of osteosarcoma organoid culture medium. For example, for a 6-well plate, use 1.5 to 2 ml of organoid culture medium, while for a 96-well plate, use 100 μL per well.

[0115] 1.16. Osteosarcoma Organoid Culture Medium: The culture medium (a mixture of osteosarcoma organoid culture medium and Matrigel) should be replaced every 48 hours, with 50% of the total volume of the culture dish replaced each time. Observe the growth of osteosarcoma organoids daily under a microscope. Cultures can be maintained for several weeks to months.

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

[0117] 1. The survival time in vitro 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 was confirmed that organoids and primary tumor tissues have a high degree of similarity in biological behavior, gene and protein expression, etc.

[0120] Figure 3 This is an example of low-magnification 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 In center C, a, b, and 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. Osteosarcoma organoids can generally be passaged 3-6 weeks after initial culture. Specific observation should be combined with microscopic observation. Generally, the density of organoid spheres should not exceed 50% of the total organoid size. After 3-6 weeks of growth, organoids can generally reach a diameter of 500 μM.

[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 reagent to dissociate organoids. Mechanically dissociate organoids using a 1000 μL pipette tip. The volume of GCDR solution used should be approximately equal to the volume of osteosarcoma organoid culture medium. For example, use 2 ml of cold GCDR cell dissociation reagent per well of a 6-well plate.

[0126] 2.4. Osteosarcoma organoids can be directly passaged or used for drug experiments at this step. To directly passage, add Matrigel to osteosarcoma organoid culture medium in a 1:3 ratio, pipette thoroughly to mix, and place on ice. Continue with subsequent experiments as in step 1.14.

[0127] 2.5. Collect all solutions and mixtures, centrifuge at low temperature, centrifuge at 80 rpm for 10 minutes, centrifuge 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-chilled 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 organoid pellet for passaging or cryopreservation.

[0130] 2.8 Osteosarcoma organoids can be cryopreserved at this stage. Resuspend the centrifuged fraction in 1 ml of Osteosarcoma Organoid Freezing Buffer and place in a sterile cryovial. Gradually freeze the sample to ≤ -80°C and 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 HistoGel matrix gel to 60±5℃ in water in advance to liquefy it, then cool it 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 from step 2.5 of Example 2, resuspend it in an appropriate amount of HistoGel, mix thoroughly, and repeatedly pipette and swirl. Then, spread it in the tissue fixation box from the previous step. After cooling and solidifying slightly, add 4-6 drops of HistoGel to form a flat layer to encapsulate 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 and fix it overnight.

[0137] 3.1.5 The next day, remove the sample block from the box, wash away 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 an osteosarcoma organoid, as seen under H&E staining (referenced at 100 μm). H&E staining reveals clustered osteosarcoma cells with osteoid-like matrix formation and significant cellular atypia. Figure 6 Comparative analysis of histopathology and immunohistochemical staining of 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 (refer to 100 μm). The antibodies used are indicated in the figure. Osteosarcoma organoids show a high degree of histopathological similarity to primary osteosarcoma tumors.

[0141] Example 4: Recovery and Recultivation of Osteosarcoma Organoids

[0142] 4.1. Remove the frozen organoid cryovial to be thawed and rapidly shake in a 37°C water bath until thawed. Immediately transfer 1 ml of the organoid cryovial into 10 ml of osteosarcoma organoid culture medium by pipetting repeatedly. Centrifuge at 500 RPM for 5 minutes and discard the supernatant.

[0143] 4.2. Resuspend the cells 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 and rapid drug response

[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. 96-well microplate fluorescence analyzer and CellTiter-Glo 3D cell viability assay kit.

[0146] 5.1. Quantitative Detection of Tumor Proliferation Ability in 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 of Example 1 (or step 4.2 of Example 4). After cell counting, the organoid monomers DKO-1, DKO-2, DKO-3, TKO-1, TKO-2, and TKO-3 were plated separately, with 50 μL of osteosarcoma organoid suspension required per well to ensure consistent volume and cell number per well.

[0147] 5.1.2 Immediately invert the plate containing the organoids in an incubator for at least 20 minutes to solidify. After removal, add 100 μL of organoid culture medium to each well and place in a 37°C incubator for incubation. This is marked as Day 1.

[0148] 5.1.3 The next day, observe the growth of the organoids under a microscope to ensure that they are growing well in the 96-well plates. The organoid culture medium should be replaced daily, with 50% of the total volume replaced each time.

[0149] 5.1.4. Cell viability was measured on days 2, 4, 6, 8, 10, and 12 of culture using the CellTiter-Glo 3D Cell Viability Assay Kit according to the kit instructions. Data were collected using the data recording software provided with the 96-well microplate fluorescence analyzer, and analyzed using SPSS 22.0 software. Three independent experiments were performed.

[0150] The test results are as follows Figure 7 shown. Figure 7Quantitative analysis of the proliferation rates of six different osteosarcoma organoids (DKO-1, DKO-2, DKO-3, TKO-1, TKO-2, and TKO-3) shows that after 12 days of analysis, the three osteosarcoma organoids with the DKO genotype proliferated significantly faster than the three osteosarcoma organoids with the TKO genotype (p < 0.01).

[0151] 5.2 Osteosarcoma Organoids for Rapid Quantitative Drug Response Detection

[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 of Example 1 (or step 4.2 of Example 4). After cell counting, the organoid monomers DKO-1, DKO-2, and DKO-3 were plated separately, with 50 μL of osteosarcoma organoid suspension required per well, ensuring consistent volume and cell number per well.

[0153] 5.2.2 Immediately invert the plate containing the organoids in an incubator for at least 20 minutes to solidify. After removal, add 100 μL of organoid culture medium to each well and place in a 37°C constant temperature incubator for incubation. This is marked as Day 1.

[0154] 5.2.3. Observe the organoid growth under a microscope the next day to ensure that the organoids are growing well in the 96-well plates. Change the organoid culture medium once a day, replacing 50% of the total volume each time.

[0155] 5.2.4. Depending on the growth of osteosarcoma organoids, drug testing begins 3-6 days after plating, when the volume of osteosarcoma organoid monomers under the microscope is approximately 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 not be used as test wells to avoid fluorescence interference.

[0156] 5.2.5. Time 0 (0 h) was recorded after drug addition. Samples were taken at 0 h, 24 h, 48 h, 72 h, and 96 h after drug addition to observe organoid growth under a microscope.

[0157] 5.2.6. Cell viability was measured at 0h, 24h, 48h, 72h, and 96h after drug addition using the CellTiter-Glo 3D Cell Viability Assay Kit according to the kit instructions. Data were collected using the data recording software provided with the 96-well microplate fluorescence analyzer, and analyzed using SPSS 22.0 software. Three independent experiments were performed.

[0158] The test results are as follows Figure 8 . Figure 8 The upper middle image shows microscopic images of osteosarcoma organoids after exposure to an anti-cancer drug (C1, Millipore Sigma, #500519) at varying concentrations (horizontally) and durations (ordinately). The organoids proliferate well in the absence of the drug. However, with increasing doses and duration of drug application, the organoids die and fragment, demonstrating that organoid activity is inversely proportional to drug dose and duration. Figure 8 The lower middle figure shows the activity response of three osteosarcoma organoids (DKO-1, -2, and -3) 96 hours after adding 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 of culture medium

[0160] After the osteosarcoma tissue was lysed and cultured for the same period of time ( Figure 9 A and C were cultured for 48 hours, and B and D were cultured for 120 hours. It can be seen that when using DMEM / F12 culture medium, tumor cells adhered to the wall in large quantities, but failed to form spheres effectively or formed very few spheres (AB). However, DMEM / F12 culture medium supplemented with 5% R-Spondin and 10% Noggin was beneficial for tumor cells to form spheres and grow in suspension in large quantities (CD). Figure 9 .

[0161] Figure 10-12These are the experimental results of screening osteosarcoma organoid culture medium. After the osteosarcoma tissue was lysed and primary cultured under the same conditions for the same 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 7 days), it can be seen that tumor organoids grew well under the conditions of the fully 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 further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using 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; Wherein, the Wnt signaling pathway activator is R-Spondin; The organoid cytokine is Noggin; The ROCK inhibitor is Rho-associated coiled-coil protein kinase; The fibroblast growth factor is FGF-10; The basic fibroblast growth factor is FGF-2; 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.

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

3. A reagent for preparing osteosarcoma organoids, comprising Matrigel and the composition according to claim 1.

4. A reagent for preparing osteosarcoma organoids, comprising matrigel and the culture medium according to claim 2.

5. Use of the composition according to claim 1 in preparing osteosarcoma organoids.

6. Use of the culture medium according to claim 2 in preparing osteosarcoma organoids.

7. Use of the reagent according to claim 3 or 4 in preparing osteosarcoma organoids.

8. A method for preparing osteosarcoma organoids, characterized by: The method comprises mixing osteosarcoma cells with the reagent according to claim 3 or 4 to obtain an osteosarcoma organoid suspension, and culturing the osteosarcoma organoid suspension to obtain osteosarcoma organoids.

9. The method according to claim 8, characterized in that: The osteosarcoma cells are mouse osteosarcoma cells.

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