Construction method of osteosarcoma-like organ model derived from patient

Abstract

The invention belongs to the field of biological medicine, and particularly provides a construction method of a patient-derived osteosarcoma organ model, which comprises the steps of cleaning, digestion, filtration, centrifugation, resuspension and seed plate culture. The last step comprises the steps that a cell suspension starting plate is placed on a low-adhesion plate to be cultured, constant-temperature culture is kept in a shaking table for 5 days or above, a new complete culture medium is replaced every 1-3 days during the period, and the complete culture medium comprises an ADMEM culture medium, fetal calf serum FBS, double antibodies, Glutmax, N-Ac, Nico, anti-apoptosis compounds Y27632, EGF, FGF2, FGF7, FGF10, RS-1 and Wnt. According to the method, the osteosarcoma organoid model is cultured in a 3D manner in combination with the shaking table and the complete culture medium in a non-stent and non-matrigel state, and mechanical stimulation is generated during culture, so that the consistency of the obtained organoid and a bone-derived tumor microenvironment is higher, the quality of the organoid is higher, and the growth speed of the organoid is higher.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for constructing a patient-derived osteosarcoma organoid model. Background Art

[0002] Osteosarcoma (OS) is a malignant tumor of mesenchymal origin characterized by spindle-shaped cells that produce bone-like tissue. It is one of the most common primary malignant bone tumors, predominantly arising in the metaphysis of the long bones of the limbs, most commonly in the distal femur and proximal tibia, followed by the proximal humerus. Osteosarcoma is most common during childhood and adolescence, with males more likely than females. This is the first peak of osteosarcoma incidence, primarily as primary osteosarcoma. The second peak of incidence occurs in individuals over 60 years of age, primarily as secondary osteosarcomas, such as those following radiation therapy and chronic bone diseases. The incidence of osteosarcoma is approximately 3 to 4.5 cases per million people. Early symptoms primarily include local pain, swelling, and functional limitations, often exacerbated by activity or at night, accompanied by localized bone destruction and mass formation. As the disease progresses, systemic symptoms such as fatigue, weight loss, and anemia may develop. Osteosarcoma is highly invasive, with approximately 10%-15% having metastases at the time of diagnosis. The most common mode of metastasis is hematogenous metastasis, and the most common site of metastasis is the lungs. Currently, surgical treatment and neoadjuvant chemotherapy are the main treatments for osteosarcoma, but the prognosis of osteosarcoma patients has not improved in the past 20-30 years, especially for patients with recurrence and metastasis, where the 5-year survival rate has long remained at 20-30%. In recent years, significant progress has been made in targeted therapies for osteosarcoma. Tyrosinase inhibitors, such as regorafenib and pazopanib, inhibit the activity of receptor tyrosine kinases (RTKs), thereby blocking tumor cell proliferation signals. They have shown good efficacy in clinical trials for osteosarcoma, but more experimental data are still needed to promote their clinical application.

[0003] Due to the heterogeneity of osteosarcoma tissue, previous basic research results based on osteosarcoma cell lines and animal models are difficult to reproduce in clinical osteosarcoma patients, which has largely led to a lag in the level of clinical osteosarcoma diagnosis and treatment.

[0004] Therefore, the use of patient-derived organoids (PDO) models that are more homogeneous with the patient's primary tumor is of far-reaching significance. It can deeply explore the pathogenesis of osteosarcoma. The results of PDO-based research and development can be well replicated in patients. At the same time, it can greatly amplify the limited osteosarcoma samples in clinical patients. PDO can be used instead of clinical patients to screen different drug combinations, thereby selecting the most appropriate drugs, greatly improving effectiveness and reducing drug side effects.

[0005] From another perspective, tumor models are important vehicles for cancer research. Tumor cell line models struggle to replicate the pathophysiological characteristics and internal heterogeneity of parental tumors in vitro, while animal models suffer from issues such as low throughput, long cycles, high costs, and species differences. Compared to tumor cell lines and animal models, tumor organoid models are derived from patients and can better retain the characteristics of parental tumors under in vitro culture conditions. They also have relatively short culture cycles, high throughput, and manageable costs, making them potentially ideal models for conducting basic and clinical cancer research.

[0006] Organoid models can simulate the complex biological processes of human organs in vitro, with greater clinical fidelity than traditional models. This feature enables rapid functional testing of drugs, improving the efficiency of drug discovery and clinical application. In addition, organoids provide a new platform for in vitro gene editing therapies. Using CRISPR-Cas9 and other gene editing tools, researchers can use organoids to model genetic diseases and test treatment strategies, significantly advancing the field of personalized medicine. All of this emphasizes the transformative potential of organoids in biomedical science.

[0007] In cell culture, a scaffold is a three-dimensional (3D) porous or fibrous structure used to mimic the extracellular matrix (ECM) environment of cells in the body, providing physical support, biochemical signals, and spatial structure for cells, promoting cell adhesion, proliferation, differentiation, and tissue formation. Matrigel, a basement membrane matrix extracted from mouse sarcoma cells, primarily mimics the ECM environment and is widely used in cell culture, tissue engineering, and regenerative medicine. Matrigel is a type of scaffold.

[0008] Existing technologies for constructing osteosarcoma organoids mainly include 2D culture and 3D culture. However, the existing 2D culture method cannot simulate the growth environment in the body and the organoids grow slowly. 3D culture generally requires the use of scaffolds or matrix glue, which increases the culture cost of osteosarcoma organoids. In addition, the osteosarcoma organoids obtained by 3D culture in the existing technology lack the mechanical stimulation environment for bone tumor growth, and their culture efficiency is not high.

[0009] For example, invention patent CN202210839348 provides an osteosarcoma organoid model, construction method and application, which requires the use of matrix glue and a scaffold; invention patent application CN202310162592 provides a lung tumor organoid model based on a sodium alginate cryogel scaffold, which requires the use of a scaffold; invention patent application CN202310033244 provides a construction method and application of a gallbladder cancer organoid xenograft tumor model, which requires the use of matrix glue; invention patent CN202010724009 provides a culture method for osteosarcoma organoids and its bone tumor culture medium, which requires the use of a scaffold collagen; invention patent application CN202211664563 provides a single-cell-based osteosarcoma organoid model construction method, wherein planar culture does not require the use of matrix glue, but planar culture is adherent culture, so the organoid model obtained is easy to fall off, and the 3D culture requires the use of matrix glue.

[0010] Therefore, there is a need in the art for a method for constructing an osteosarcoma organoid model that is low-cost and more consistent with the environment in which osteosarcoma grows in the human body. Summary of the Invention

[0011] Therefore, the present invention provides a method for constructing a patient-derived osteosarcoma organoid model, comprising the following steps: step A, washing: washing the selected patient-derived osteosarcoma tissue several times, and optionally chopping the osteosarcoma tissue before and after washing; step B, digestion: adding digestion fluid and double antibody to a container containing osteosarcoma tissue, and using a shaker to digest the osteosarcoma tissue for a period of time so that the osteosarcoma tissue is in a suspension state; step C, filtration: first adding serum to the suspension to terminate digestion, and then filtering the suspension using a cell sieve, retaining the cell filtrate obtained below the cell sieve; step D, centrifugation: placing the cell filtrate in a centrifuge tube and centrifuging, removing the supernatant, and retaining the precipitate obtained by centrifugation; step E, resuspension and plate culture: in the precipitate obtained by centrifugation Complete culture medium is added and the precipitate is resuspended to obtain a cell suspension, which is then plated onto a low-adhesion plate for culture and maintained at a constant temperature in a shaker for more than 5 days, during which new complete culture medium is replaced every 1 to 3 days. The complete culture medium includes ADMEM culture medium, fetal bovine serum (FBS), double antibody, Glutmax, N-Ace, Nico, anti-apoptotic compound Y27632, EGF, FGF2, FGF7, FGF10, RS-1, and Wnt; wherein Glutmax, N-Ace, and Nico are all nutrients, and EGF, FGF2, FGF7, FGF10, RS-1, and Wnt are all growth factors; thus, the patient-derived osteosarcoma organoid model is obtained.

[0012] In a specific embodiment, in step A, the osteosarcoma tissue is washed 2 to 3 times with PBS, and then the osteosarcoma tissue is minced into pieces with a volume of 1 mm. 3 The minced meat inside.

[0013] In a specific embodiment, in step B, the container is a centrifuge tube, the digestion solution includes hyaluronidase, collagenase type I, collagenase type II and DMEM culture medium, the dual antibody includes penicillin and streptomycin, the shaking speed of the shaker in the digestion step is 120-200 rpm, and the digestion time is 0.5-2 hours.

[0014] In a specific embodiment, in step C, after adding serum, the container is shaken up and down several times to terminate the digestion step as soon as possible; then all the suspension is moved to a cell sieve with a pore size of 40 μm using a pipette for filtration, and the tissue above the cell sieve is rinsed with PBS and / or the cell sieve is cleaned. Step C is performed in a sterile operating table.

[0015] In a specific embodiment, in step D, the cell filtrate is transferred to a centrifuge tube using a pipette, the centrifugal speed is 800-1600 rpm, the centrifugal time is 3-10 min, and the precipitate obtained by centrifugation mainly contains osteosarcoma cells and red blood cells.

[0016] In a specific embodiment, after the centrifugation in step D and before the resuspension in step E, a lysis step is further included, i.e., a red blood cell lysis solution is added to the precipitate obtained by centrifugation, and after the precipitate and the red blood cell lysis solution are mixed, they are centrifuged again at 1500-2500 rpm for more than 2 minutes, and the supernatant is removed to obtain a precipitate mainly containing osteosarcoma cells.

[0017] In a specific embodiment, the shaking speed of the shaking incubation in step E is 20-200 rpm, preferably 20-60 rpm; the shaking incubator is a horizontal shaker or a vertical shaker; and the complete culture medium in step E further includes a mycoplasma inhibitor.

[0018] In a specific embodiment, step F is further included after step E, i.e., digesting, centrifuging, resuspending and subculturing the obtained organoid spheres, wherein the subculture uses the same complete culture medium and shaker culture method as step E.

[0019] In a specific embodiment, the organoid spheres obtained by culturing in step E or the organoid spheres obtained by subculture in step F are added to a centrifuge tube together with the culture medium, and the supernatant is removed after centrifugation. The resulting precipitated organoid spheres are resuspended in cell freezing solution and then frozen for storage.

[0020] The present invention also provides a complete culture medium, which is based on ADMEM culture medium and contains 10-20% FBS, 1-2% double antibody, 1-2% Glutmax, 1-3mM N-Ace, 5-150mM Nico, 5-20μM Y27632, 20-120ng / ml EGF, 10-50ng / ml FGF2, 10-30ng / ml FGF7, 50-150ng / ml FGF10, 200-500ng / mL RS-1 and 50-150ng / ml Wnt; the full name of the N-Ace is N-Acetylcysteine, the full name of the Nico is Nicotinamide, and the Wnt is specifically Wnt 3a.

[0021] The beneficial effects of the present invention include at least: the present invention combines shaker culture and complete culture medium in a 3D culture state without a scaffold and matrix glue to culture an osteosarcoma organoid model; the osteosarcoma organoids in the present invention are cultured in a shaker in a suspended state, and are subjected to mechanical stimulation during culture; thus, the organoid model obtained by the present invention is more consistent with the bone-derived tumor microenvironment, thereby having higher quality of osteosarcoma organoids and faster growth rate of the organoid model. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Light microscopy images of first-generation organoids from three patients with osteosarcoma.

[0023] Figure 2 These are H&E staining images of organoid tissue sections and gross photos of tumors implanted under the mouse renal capsule, H&E staining images of tissue sections and human Ki67 staining images.

[0024] Figure 3 Immunofluorescence staining of organoids and tumor tissue sections from the patients from which they were derived.

[0025] Figure 4 A diagram showing the results of single-cell sequencing analysis of osteosarcoma organoids.

[0026] Figure 5 This is an analysis diagram of osteosarcoma organoids and their derived tissue cell subpopulations.

[0027] Figure 6 Heat map of gene expression differences among normal tissue, osteosarcoma organoids, and tumor tissue.

[0028] Figure 7 This is a comparison chart showing whether the same patient has the organoid growth status under the mechanical stimulation of the shaker when cultured on the seed plate. DETAILED DESCRIPTION

[0029] After review and approval by the hospital ethics committee, all patients signed informed consent forms before sample collection. The collected tissues underwent histopathological examination, and the tumor tissue blocks diagnosed as osteosarcoma patients were pathologically confirmed. The osteosarcoma tissue was removed from the fish-like part.

[0030] Unless otherwise specified, various materials used in the present invention can be obtained from commercial sources.

[0031] 1. Establishment of an Osteosarcoma Organoid Model

[0032] Items to prepare: 10 mg / ml hyaluronidase (prepared, stored at -20°C, 1 ml / tube), 20 mg / ml collagenase type I (prepared, stored at -20°C, 1 ml / tube), 20 mg / ml collagenase type II (prepared, stored at -20°C, 1 ml / tube), sterile PBS, pure DMEM.

[0033] 1. Take the selected osteosarcoma tissue (1.0-3.0cm 3 ), and washed 2-3 times with PBS.

[0034] 2. Place the tissue in a 1.5ml centrifuge tube, add a small amount of pure DMEM medium, and cut the tumor into pieces of about 1mm using sterilized scissors. 3 The size of the tissue was adjusted until the osteosarcoma tissue was minced and then transferred to a 15 ml centrifuge tube. If the tissue was too small, it could be digested directly without mincing.

[0035] 3. Add 10 ml of digestion buffer and 100 μl of double-antibody to a 15 ml centrifuge tube. Specifically, the mixture contains: 1 ml collagenase type I + 1 ml hyaluronidase + 1 ml collagenase type II + 7 ml pure DMEM + 100 μl double-antibody. Prepare immediately and digest at 37°C on a shaker at 170 rpm for 30-60 minutes. Observe the digestion process frequently. If the tissue is nearly digested, as indicated by a change from a minced meat to a suspension, stop. If the tissue is too small, reduce the amount of digestion buffer by half.

[0036] 4. On a sterile operating table, add 2 ml of serum to the 15 ml centrifuge tube containing the suspension and mix well to terminate the digestion. Shake the centrifuge tube up and down several times. Use a 1 ml pipette to aspirate the digestion solution containing the cells. Transfer all the digestion solution in the 15 ml centrifuge tube to a cell sieve with a pore size of 40 μm to filter the cells. Place the filtered liquid in a 50 ml tube. The tissue is above the cell sieve and the cell filtrate is below. If there is a lot of tissue remaining above the cell sieve, first aspirate a small amount of PBS solution to rinse the tissue on the cell sieve one or more times to flush more cells into the cell filtrate.

[0037] 5. Wash the cell sieve with 3-5 ml of PBS to allow more cells to be flushed into the cell filtrate.

[0038] 6. Transfer the cell filtrate in the 50ml tube to a 15ml centrifuge tube using a 1ml pipette and centrifuge. If a large amount of PBS was added previously, divide the filtrate into two evenly distributed tubes and centrifuge them simultaneously at 1200 RPM for 5 minutes. Remove the supernatant after centrifugation. The pellet mainly contains osteosarcoma cells and red blood cells.

[0039] 7. If the precipitate is reddish, that means there are many red blood cells, add 5 times the volume of red blood cell lysis buffer and mix well. For example, the amount of red blood cell lysis buffer is 2 ml. Treat at room temperature for 5-10 minutes, centrifuge at 2000RPM for 5 minutes, and remove the supernatant.

[0040] 8. Collect the precipitate, add an appropriate amount (e.g. 1 ml) of complete culture medium to the centrifuge tube for resuspending to obtain a cell suspension, and plate the cell suspension onto a low-adhesion plate (i.e., a low-adhesion cell culture plate) for culture. 100 μl per well of a 96-well plate, culture at 37°C, 45 rpm (horizontally rotating) on a shaker, and culture for more than 7 days. Osteosarcoma organoids are generally passaged in about 5 to 10 days. If culture is continued after 10 days, the organoid model will grow very slowly and the state of the organoid will deteriorate. It is preferred to add a mycoplasma inhibitor to the complete culture medium at a dilution of 1:500.

[0041] During the culture process, the culture medium should generally be replaced every 1–3 days. When the color of the culture medium changes from pink to yellow, it is time to replace the medium. At this time, turn off the shaker and let it sit for about 5 minutes. Allow the organoid spheres to sink to the bottom of the low-adhesion plate wells. Remove the old medium with a pipette and replace with fresh complete medium. As the culture medium is replaced, the organoid spheres that have sunk to the bottom will be observed to grow larger day by day.

[0042] 2. Complete Culture Medium

[0043] The complete medium used in step 8 above is as follows. First, prepare 10 ml of complete medium containing an ADMEM to FBS ratio of 8:2 to 9:1. For example: 9 ml of ADMEM + 1 ml of FBS + double-antibody + Glutmax + N-Ace + Nico + Y27632 + EGF + FGF2 / 7 / 10 + RS-1 + Wnt.

[0044] Among them, the dual antibody and Glutmax = 1:100, that is, the amount of both added is 100μl; N-Ace = 1:400, the amount added is 25μl; Nico = 1:200, the amount added is 50μl; Y27632 = 1:10000, the amount added is 1μl; Mycoplasma inhibitor = 1:500, the amount added is 20μl; EGF+FGF2 / 7 / 10+RS-1+Wnt are all 1:1000, and the amount added is 10μl.

[0045] 3. Passaging and Cryopreservation

[0046] 1. Collect the organoid spheres (including organoid spheres and culture medium) into a 1.5 ml EP tube (a small centrifuge tube), centrifuge at 2000 rpm for 5 minutes, and remove the supernatant to obtain the organoid spheres.

[0047] 2. Add 500 μl of Tryple Express digestion solution and pipette up and down 50 times with a 100 μl pipette. During this step, rinse the pipette tip with serum or culture medium to prevent the organoid spheres from adhering to the pipette tip. Incubate at 37°C for 3-5 minutes, removing the organoid spheres and gently shaking them to monitor the digestion process. Tryple Express digestion solution is commercially available. After digestion, the organoid spheres are transformed into small cells, which can be further subcultured to form more organoid spheres. The resulting organoid spheres are used for osteosarcoma research and drug screening, thereby guiding patient medication use.

[0048] In step B (digestion of tissue) of the present invention, since the patient's tissue is used and its structure is dense, the digestion intensity needs to be very high, but step F (digestion of organoids) does not require too high an intensity.

[0049] 3. Terminate digestion with 200 μl of serum, centrifuge at 2000 rpm for 5 min, and discard the supernatant; the precipitate is the digested organoid cells.

[0050] 4. Add new complete culture medium to resuspend the pellet, transfer it to the wells of a low-adhesion plate and continue shaking culture.

[0051] 5. Alternatively, resuspend the pellet in CellorLab Rapid Cell Freezing Buffer and freeze at -80°C.

[0052] The present invention is further illustrated by the following drawings.

[0053] Figure 1 Light microscopy images of first-generation organoids from three patients with osteosarcoma. Figure 1 In the figure, OS is the abbreviation for osteosarcoma, 439, 451, and 453 are patient numbers, and the last O is the code for the organoid. In the figure, 100μm and 50μm are scales. The figure shows that the first-generation organoids cultured from three osteosarcoma patients using the method of the present invention all grew well, with organoid spheres ranging in diameter from 100 to 200μm. As long as the first-generation organoids are successfully passaged, the quality of the organoids generally improves with each subsequent passage, the organoids grow faster, and the organoid spheres become larger.

[0054] Figure 2The following are H&E staining images of organoid tissue sections, gross photos of tumors implanted under the mouse kidney capsule, H&E staining images of tissue sections, and human Ki67 staining images. Figure A is an H&E staining image of organoid tissue sections. In Figure A, the top two small images are from the patient's osteosarcoma tissue, while the six small images below are from three different organoids of the patient; Figure A shows that the organoids have the same morphology as the tumor tissue of the patient they are from. During the organoid culture and subculture process, 1 to 2 organoid spheres will generally grow in each well of the low-adhesion plate. The top of Figure B is a gross photo of the tumor of the organoid (fifth-generation organoid sphere) implanted under the mouse kidney capsule. The left is a comparison image of a normal mouse kidney. The middle and bottom of Figure B are H&E staining images of tissue sections and human Ki67 staining images, respectively. In Figure B, tissue sections were taken at the junction of the mouse kidney and osteosarcoma. In the center and four images below Figure B, the yellow line represents the junction between the mouse kidney and osteosarcoma. The mouse kidney is at the lower left of the yellow line, and the osteosarcoma is at the upper right. Human Ki67 is a commercially available antibody. Figure B demonstrates cell proliferation in the organoid, confirming that the organoid is human and possesses stem cell properties.

[0055] Figure 3 Immunofluorescence staining of organoids and tumor tissue sections from the patients from which they were derived. Figure 3 In the image, the leftmost column shows immunofluorescence staining of five tumor markers from the same patient. The three columns on the right show immunofluorescence staining of five different organoids from the same patient. The second column shows the nuclear staining first, followed by the five tumor markers in the third column. The fourth column is an overlay of the corresponding images from the second and third columns. Figure 3 This indicates that the expression of organoid tissue markers is consistent with that of the tumor tissue of the patient from whom it was derived.

[0056] Figure 4 The results of single-cell sequencing analysis of osteosarcoma organoids are shown in Figure 2. Figure 4 It can be seen that the diversity of cellular components within osteosarcoma organoids includes osteoblasts, fibroblasts and various immune cells (such as T cells, macrophages, and B cells), reflecting that the organoids retain the cellular components of their source tissues.

[0057] Figure 5 This is an analysis diagram of osteosarcoma organoids and their derived tissue cell subpopulations. Figure 5 The organoid is on the left side of the center, and its source tissue is on the right. Figure 5 The vertical axis is the cell ratio, Figure 5 The color blocks from top to bottom in the middle are T cells, macrophages, fibroblasts, B cells, osteoblasts, and vascular-related cells. Figure 5This demonstrates the consistency between osteosarcoma organoids and the cell subpopulations of their originating tissues, indicating that organoids retain tissue cell subtypes.

[0058] Figure 6 Heat map of gene expression differences among normal tissue, osteosarcoma organoids, and tumor tissue. Figure 6 The three green columns on the left represent the normal tissues of the three patients, the three blue columns in the middle represent the organoids of the three patients, and the three yellow columns on the right represent the tumor tissues of the three patients. Obviously, the gene heatmaps represented by the three columns in the middle and the three columns on the right have a high similarity. Figure 6 It can be seen that compared with normal tissues, from the heat map of the top 25 genes upregulated and downregulated in tumor samples, the gene expression of osteosarcoma organoids is basically consistent with that of their tumor tissue samples.

[0059] Figure 7 This is a comparison chart of whether the same patient has the organoid growth status under the mechanical stimulation of the shaker when cultured on the seed plate. Figure 7 As can be seen, after 7 days of culture under the mechanical stimulation of a shaker, the organoids in the experimental group grew faster than those in the control group. When osteosarcoma organoids were cultured in 3D without a shaker and without a scaffold, organoids could also grow, but the growth rate was slower and the effect was not as good.

[0060] The present invention successfully established a method for culturing osteosarcoma organoids, which does not require matrix glue or scaffolds. At the same time, a shaker is used to provide mechanical stimulation during the organoid culture process, thereby efficiently and successfully constructing an osteosarcoma organoid model.

[0061] The drug screening results developed based on the osteosarcoma organoid model described in the present invention can be well replicated in patients. At the same time, the limited osteosarcoma samples in clinical patients can be amplified in large quantities. The organoid model can be used instead of clinical patients to screen different combinations of drugs, thereby selecting the most appropriate drugs, which can greatly improve effectiveness and reduce drug side effects.

[0062] The present invention belongs to the field of biomedicine and specifically provides a method for constructing a patient-derived osteosarcoma organoid model, comprising the steps of washing, digesting, filtering, centrifuging, resuspending, and plate culturing. The final step involves plating the cell suspension onto a low-adhesion plate for culturing and maintaining it in a shaker at a constant temperature for more than 5 days, during which time new complete culture medium is replaced every 1 to 3 days. The complete culture medium includes ADMEM medium, fetal bovine serum (FBS), double-antibody, Glutmax, N-Ace, Nico, the anti-apoptotic compound Y27632, EGF, FGF2, FGF7, FGF10, RS-1, and Wnt. The present invention utilizes a shaker and complete culture medium in a 3D culture system to produce an osteosarcoma organoid model without a scaffold or matrix gel. The culture provides mechanical stimulation, resulting in a higher consistency between the organoid and the bone-derived tumor microenvironment, resulting in higher quality and faster growth.

[0063] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing a patient-derived osteosarcoma organoid model, characterized in that: The steps include: Step A, washing: washing the selected patient-derived osteosarcoma tissue several times, and optionally mincing the osteosarcoma tissue before and after washing; Step B, digestion: Add digestion solution and double antibody to the container containing osteosarcoma tissue, and use a shaker to digest the osteosarcoma tissue for a period of time to keep the osteosarcoma tissue in a suspension state; Step C, filtration: First, serum is added to the suspension to terminate digestion, and then the suspension is filtered through a cell sieve, and the cell filtrate obtained below the cell sieve is retained; Step D, centrifugation: placing the cell filtrate in a centrifuge tube and centrifuging, removing the supernatant, and retaining the precipitate obtained by centrifugation; Step E, resuspension and plate culture: add complete culture medium to the precipitate obtained by centrifugation, resuspend the precipitate to obtain a cell suspension, and then plate the cell suspension onto a low-adhesion plate for culture, and keep it in a shaker at a constant temperature for more than 5 days, during which time new complete culture medium is replaced every 1 to 3 days, wherein the complete culture medium includes ADMEM culture medium, fetal bovine serum FBS, double antibody, Glutmax, N-Ace, Nico, anti-apoptotic compound Y27632, EGF, FGF2, FGF7, FGF10, RS-1 and Wnt; wherein Glutmax, N-Ace and Nico are all nutrients, and EGF, FGF2, FGF7, FGF10, RS-1 and Wnt are all growth factors; thus, the patient-derived osteosarcoma organoid model is obtained.

2. The method according to claim 1, characterized in that In step A, the osteosarcoma tissue was washed 2-3 times with PBS and then minced into pieces of 1 mm in size. 3 The minced meat inside.

3. The method according to claim 1, characterized in that In step B, the container is a centrifuge tube, the digestion solution includes hyaluronidase, collagenase type I, collagenase type II and DMEM culture medium, the dual antibody includes penicillin and streptomycin, the shaking speed of the shaker in the digestion step is 120-200 rpm, and the digestion time is 0.5-2 hours.

4. The method according to claim 1, wherein In step C, after adding serum, shake the container up and down several times to terminate the digestion step as quickly as possible; then use a pipette to move all the suspension to the top of a cell sieve with a pore size of 40 μm for filtration, and use PBS to rinse the tissue above the cell sieve and / or clean the cell sieve. Step C is performed in a sterile operating table.

5. The method according to claim 1, wherein In step D, the cell filtrate is transferred to a centrifuge tube using a pipette. The centrifugal speed is 800-1600 rpm and the centrifugal time is 3-10 min. The precipitate obtained by centrifugation mainly contains osteosarcoma cells and red blood cells.

6. The method according to claim 1, wherein After the centrifugation in step D and before the resuspension in step E, a lysis step is also included, that is, red blood cell lysis solution is added to the precipitate obtained by centrifugation, and after the precipitate and red blood cell lysis solution are mixed, they are centrifuged again at 1500-2500 rpm for more than 2 minutes. The supernatant is removed and the precipitate obtained mainly contains osteosarcoma cells.

7. The method according to claim 1, characterized in that In step E, the shaking speed of the shaking culture is 20-200 rpm, preferably 20-60 rpm; the shaking culture medium is a horizontal shaking culture medium or a vertical shaking culture medium; and the complete culture medium in step E further includes a mycoplasma inhibitor.

8. The method according to claim 1, characterized in that After step E, the method further includes step F, i.e., digesting, centrifuging, resuspending and subculturing the obtained organoid spheres, wherein the subculture uses the same complete culture medium and shaker culture method as step E.

9. The method according to claim 8, characterized in that The organoid spheres cultured in step E or the organoid spheres subcultured in step F are added to a centrifuge tube together with the culture medium. After centrifugation, the supernatant is removed. The precipitated organoid spheres are resuspended in cell freezing solution and then frozen for storage.

10. A complete culture medium, comprising ADMEM as a base and containing 10-20% FBS, 1-2% bispecific antibody, 1-2% Glutmax, 1-3 mM N-Ace, 5-150 mM Nico, 5-20 μM Y27632, 20-120 ng / ml EGF, 10-50 ng / ml FGF2, 10-30 ng / ml FGF7, 50-150 ng / ml FGF10, 200-500 ng / mL RS-1, and 50-150 ng / ml Wnt; the full name of N-Ace is N-Acetylcysteine, the full name of Nico is Nicotinamide, and the Wnt is specifically Wnt 3a.

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