Osteosarcoma organ culture medium and gelatin planting method

Abstract

The invention belongs to the field of cell culture, and discloses an osteosarcoma organ culture medium and a gelatin planting method. The following components are added into the osteosarcoma organ-like culture medium: a B27 additive, an N-2 additive, a GlutaMAX additive, casein amino acid, N-acetylcysteine, nicotinamide, vitamin C, sodium pyruvate, human gastrin, Wnt3A, R-spondin 1 protein, Noggin protein, activin A protein, Forskolin, Y-27632, SB202190, CHIR99021, A83-01, h-EGF, FGF-10, HGF, VEGF A, IGF 1, IL-6, cyan-streptomycin and fetal calf serum. The osteosarcoma organoid culture medium provided by the invention can promote organoid cells to maintain relatively high multiplication capacity and maintain self-renewal, so that the amplification efficiency and the modeling success rate of the organoid are greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to an osteosarcoma organoid culture medium and a gel seeding method. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be regarded as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Osteosarcoma, also known as osteogenic sarcoma, is a malignant bone tumor characterized by the direct production of bone-like tissue by tumor cells. Osteosarcoma is common in adolescents, with the most common sites being the epiphysis of the distal femur, proximal tibia, and proximal humerus. It often forms a spindle-shaped tumor that can involve the periosteum, cortical bone, and medullary cavity. The cross-section of the lesion is fish-like, brown-red or gray-red. However, the pathogenesis of osteosarcoma is currently unclear. Osteosarcoma organoids constructed in vitro are an advantageous model for studying osteosarcoma, which is convenient for research and analysis and can play a positive role in the study of its pathogenesis.

[0004] The 3D culture system of organoids provides cells with a matrix similar to the in vivo growth environment. Cells form a certain three-dimensional structure through the connection with the extracellular matrix, which is more similar to the cell growth in vivo. However, at present, osteosarcoma organoid culture has problems such as low success rate, slow expansion speed, and long primary culture process. Therefore, there is an urgent need for an excellent osteosarcoma organoid culture medium and a faster seeding method. Summary of the invention

[0005] In view of the problems in the prior art, the present invention provides an osteosarcoma organoid culture medium, which can increase the probability of successful organoid culture and improve the efficiency of osteosarcoma organoid expansion.

[0006] The present invention also provides a method for seeding osteosarcoma, which reduces the time spent on primary culture of organoids.

[0007] To achieve the above purpose, the present invention adopts the following technical solution.

[0008] A culture medium for osteosarcoma organoids comprising the following components supplemented to a basal medium: B27 Supplement (without vitamin A) 0.5-2×, N-2 Supplement 1×, GlutaMAX Supplement 1-10mM, Casein Amino Acids 100-1000mg / L, N-Acetylcysteine ​​0.5-5mM, Nicotinamide 1-100mM, Vitamin C 0.5-2μM, Sodium Pyruvate 1-5mM, Human Gastrin 1-100nM, Wnt3A 5-20ng / mL, R-spondin 1 Protein 100-1000ng / mL, Noggin Protein 10-500ng / mL, Activin A 10-100ng / mL, Forskolin 1-15μM, Y-27632 5-15μM, SB202190 10-20 nM, CHIR99021 1-10μM, A83-01 0.1-10μM, h-EGF (human epidermal growth factor) 5-500ng / mL, FGF-10 (fibroblast growth factor 10) 20-500ng / mL, HGF (hepatocyte growth factor) 10-30ng / mL, VEGF A (vascular endothelial growth factor A) 10-100ng / mL, IGF 1 (insulin-like growth factor 1) 1-5μg / mL, IL-6 1-2μg / mL, penicillin-streptomycin double antibody 1-2×, serum 1%-10%.

[0009] Preferably, the above osteosarcoma organoid culture medium contains the following components added to the basal culture medium: B27 Supplement (without vitamin A) 1×, N-2 Supplement 1×, GlutaMAX Supplement 8mM, Casein Amino Acids 500-1000mg / L, N-Acetylcysteine ​​0.5-1mM, Nicotinamide 5-15mM, Vitamin C 1-2μM, Sodium Pyruvate 1-5mM, Human Gastrin 50-100nM, Wnt3A 10-15ng / mL, R-spondin 1 Protein 500-1000ng / mL, Noggin Protein 200-500ng / mL, Activin A 80-100ng / mL, Forskolin 8-15μM, Y-2763210-15μM, SB202190 10-20nM, CHIR99021 5-10μM, A83-01 5-10μM, h-EGF (human epidermal growth factor) 5-50ng / mL, FGF-10 (fibroblast growth factor 10) 20-100ng / mL, HGF (hepatocyte growth factor) 10-30ng / mL, VEGF A (vascular endothelial growth factor A) 20-50ng / mL, IGF 1 (insulin-like growth factor 1) 1-5μg / mL, IL-6 1-2μg / mL, penicillin-streptomycin double antibody 1×, serum 10%.

[0010] More preferably, the above osteosarcoma organoid culture medium comprises the following components added to the basal culture medium: B27 Supplement (without vitamin A) 1×, N-2 Supplement 1×, GlutaMAX Supplement 8mM, Casein Amino Acids 600mg / L, N-Acetylcysteine ​​0.5mM, Nicotinamide 10mM, Vitamin C 1μM, Sodium Pyruvate 1mM, Human Gastrin 100nM, Wnt3A 10ng / mL, R-spondin 1 Protein 900ng / mL, Noggin Protein 400ng / mL, Activin A (Activin A Protein) 100ng / mL, Forskolin 10μM, Y-27632 10μM, SB202190 15nM, CHIR99021 5μM, A83-01 9μM, h-EGF (Human Epidermal Growth Factor) 5ng / mL, FGF-10 (Fibroblast Growth Factor 10) 20ng / mL, HGF (Hepatocyte Growth Factor) 25ng / mL, VEGF A (vascular endothelial growth factor A) 50ng / mL, IGF 1 (insulin-like growth factor 1) 5μg / mL, IL-6 1.5μg / mL, penicillin-streptomycin double antibody 1×, serum 10%.

[0011] The basal culture medium is selected from any one of Advanced DMEM / F12, RPMI 1640 and Leibovitz's L-15 culture medium; preferably Advanced DMEM / F12 culture medium.

[0012] The serum is human serum, fetal bovine serum or a serum substitute.

[0013] The above-mentioned culture medium can be prepared by adding each component in a solid or solution state to the basic culture medium and mixing them evenly by conventional methods in the art.

[0014] A primary culture method of osteosarcoma organoids comprises the following steps: (1) Processing the collected osteosarcoma tissue into cell / cell cluster precipitation; (2) Preheat the osteosarcoma organoid culture medium to 37°C. (3) Mixing the cell / cell cluster pellet and liquid matrix gel to obtain a resuspension; (4) Before solidification, the resuspension is added dropwise to the preheated osteosarcoma organoid culture medium to form a gel ball, and primary osteosarcoma organoids are obtained after culture.

[0015] In step (3), the concentration of matrix gel in the resuspension solution is 75-95%.

[0016] In step (4), the volume ratio of the resuspension solution to the osteosarcoma organoid culture medium is 1:(10-50). The volume of the gel ball is 20-40 μL / ball.

[0017] The synergistic effects of the present invention are as follows: Adding Wnt3A, Rspondin 1 protein, Noggin protein, and fibroblast growth factor 10 to the osteosarcoma organoid culture medium can synergistically activate the Wnt signaling pathway of proliferating stem cells, thereby promoting stem cell growth, proliferation, differentiation, and inhibiting apoptosis, and promoting organoid self-renewal; adding recombinant human epidermal growth factor EGF, fetal bovine serum, HGF, vascular endothelial growth factor A, Activin A, insulin-like growth factor 1, and vitamin C to the osteosarcoma organoid culture medium greatly promotes the maintenance of stem cell stemness and cell activity, increasing the success rate of osteosarcoma organoid culture by 30%.

[0018] The present invention has the following advantages: The osteosarcoma organoid culture medium provided by the present invention can promote organoid cells to maintain a high proliferation capacity and maintain self-renewal. The liquid dripping gel method adopted by the present invention reduces the steps of waiting for matrix gel fixation in the common organoid method, greatly shortens the organoid culture time, increases the contact area between the gel droplet and the culture medium, facilitates the material exchange between the organoid and the culture medium, and greatly improves the amplification efficiency and modeling success rate of the organoid. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Organoid morphology obtained with different culture medium formulations; Figure 2 It is the state of the seed glue obtained by different methods. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0021] Example 1 Preparation of osteosarcoma organoid culture medium Prepare osteosarcoma organoid culture medium using the composition in Table 1 as follows: (1) Dissolve SB202190, N-acetylcysteine, A83-01, Y-27632, Forskolin, and CHIR99021 in DMSO to prepare a stock solution; (2) Dissolve sodium pyruvate, nicotinamide, Wnt3A, casamino acid, VEGF A, Activin A, vitamin C, and IGF 1 in sterile water to prepare a stock solution; (3) Dissolve human gastrin, h-EGF, R-spondin1 protein, Noggin protein, FGF-10, IL-6, and HGF in PBS (pH 7.4) containing 0.1% BSA (bovine serum albumin) to prepare a stock solution; (4) According to the formula concentration, add appropriate doses of mother solution to Advanced DMEM / F12 basal medium in sequence, and then add the formula doses of B27 additive (without vitamin A), penicillin-streptomycin dual antibody, HEPES, GlutaMAX additive, Primocin, N-2 additive, and fetal bovine serum to prepare osteosarcoma organoid culture medium; The "×" in B27 additive (without vitamin A), N-2 additive and penicillin-streptomycin dual antibody indicates a multiple of the commonly used / recommended concentration. For example, the initial concentration of commercially purchased N-2 additive is 50×, which means that the initial concentration is 50 times the commonly used / recommended concentration, while the formula is 1×, so it is diluted 50 times.

[0022] Table 1 Osteosarcoma organoid culture medium formula The formulas of osteosarcoma organoid culture medium are shown in Table 1 , and the obtained culture media are numbered as formulas 1-3.

[0023] Example 2 Primary Culture of Osteosarcoma Organoids 1. Preparation of osteosarcoma cells / cell pellets (1) Place the tissue in a 60 mm × 15 mm culture dish and rinse the tissue 3-5 times with PBS containing 2%; (2) Use sterile scissors to cut the above tissue into 1mm pieces. 3 Blocks of left and right sizes; (3) Add 5 times the volume of digestion solution to the shredded tissue and digest in a 37°C incubator for 20 min-50 min; after digestion, add 2 times the volume of PBS to terminate digestion and pass through a 100 μM mesh to obtain a suspension; (4) Centrifuge the suspension at 500 g for 5 min and remove the supernatant to obtain the cell / cell cluster precipitate.

[0024] 2. Primary Culture The cell / cell cluster pellet and the liquid matrix gel are mixed evenly to obtain a resuspension; Adjust the pipette to 40 μL and draw the above resuspension; drop the resuspension into the middle of the well of the well plate, let it stand for 3-5 minutes, and then invert it in a carbon dioxide incubator at 37°C and incubate for about 30 minutes to solidify; After the incubation, the well plate was removed, 1 mL of culture medium with different formulations was added accordingly, and the well plate was placed in a carbon dioxide incubator for further culture for 6 days.

[0025] Table 2 Average number and diameter of organoids obtained with different culture media formulations Images of organoids with different formulations Figure 1 As shown. Using the high-content imaging analysis system, the number and average diameter of organoids were counted and analyzed (Table 2), and it was found that the number of organoids: Formula 1>Formula 2>Formula 3; the average diameter of organoids: Formula 1>Formula 2>Formula 3. Therefore, Formula 1 is better than Formula 2 and Formula 3. It can be seen that the culture medium containing multiple growth factors can significantly improve the reproduction capacity of organoids, and Forskolin has the most significant effect among multiple growth factors.

[0026] Example 3 Primary Culture of Osteosarcoma Organoids Osteosarcoma cell / cell pellet was prepared according to Example 2, and primary osteosarcoma organoids were obtained by culturing according to the following method: 1. Suspension culture (1) Add about 2 mL of osteosarcoma organoid culture medium of formula 1 to the wells of the low-adsorption plate and incubate in a carbon dioxide incubator at 37°C. (2) Mix the cell / cell cluster pellet and liquid matrix gel in a volume ratio of 1:3 to obtain a resuspension; (3) Take 200 μL of the resuspension, let it stand in a biosafety cabinet to warm up, and add it dropwise to the well in step (1) before it solidifies. During the addition process, the resuspension forms 20 μL / piece of gel balls, which are suspended in the culture medium. After it is completely solidified, it is cultured in a carbon dioxide incubator for 6 days.

[0027] 2. Adherence Method Seed Gel Culture (1) Mix the cell / cell cluster pellet and liquid matrix gel in a volume ratio of 1:3 to obtain a resuspension; (2) Adjust the pipette to 40 μL and draw up the above resuspension; drop the resuspension into the middle of the well of the well plate, let it stand for 3-5 minutes, and then invert it in a carbon dioxide incubator at 37°C and incubate for about 30 minutes to solidify; (3) After the incubation, remove the well plate, add 1 mL of culture medium of Formula 1, and place it in a carbon dioxide incubator for further culture for 6 days.

[0028] Table 3 Average number and average diameter of organoids obtained by different methods The above two methods form the seed glue as Figure 2 As shown, the contact surface between the matrix gel of the adhesion method and the culture medium is hemispherical, and the contact surface between the matrix gel of the suspension method and the culture medium is spherical. In comparison, the contact surface between the matrix gel of the suspension method and the culture medium will be larger, which facilitates the exchange of organoids and culture medium substances inside the matrix gel. Cultivated to the 6th day, the organoids used a high-content imaging analysis system to count the successfully cultivated organoids, and analyzed the number and average diameter of the organoids. It was found that the success rate of suspension cultured osteosarcoma organoids was higher than that of the adhesion method, and the number of organoids cultivated by the suspension method was more and the diameter was larger. The two methods were used to count the success rates of cultivation of 10 samples. The suspension method was able to successfully cultivate organoids of 8 samples, while the traditional adhesion was only able to obtain organoids of 6 samples, indicating that the cultivation success rate of the suspension method was higher. Therefore, for the culture medium provided by the present invention, the suspension method is better than the adhesion method.

[0029] Example 4 Primary Culture of Osteosarcoma Organoids 1. Preparation of Osteosarcoma Organoid Culture Medium Prepare osteosarcoma organoid culture medium according to the following recipe: B27 Supplement (without vitamin A) 1×, N-2 Supplement 1×, GlutaMAX Supplement 8mM, Casein Amino Acids 100mg / L, N-Acetylcysteine ​​0.5mM, Nicotinamide 10mM, Vitamin C 1μM, Sodium Pyruvate 1mM, Human Gastrin 100nM, Wnt3A 20ng / mL, R-spondin 1 Protein 900ng / mL, Noggin Protein 500ng / mL, Activin A 100ng / mL, Forskolin 15μM, Y-27632 10μM, SB202190 15nM, CHIR99021 5μM, A83-01 5μM, h-EGF 5ng / mL, FGF-10 20ng / mL, HGF 30ng / mL, VEGF A 50ng / mL, IGF 1 5μg / mL, IL-6 2μg / mL, penicillin / streptomycin 1×, fetal bovine serum 10%.

[0030] Prepare osteosarcoma organoid culture medium as follows: (1) Dissolve SB202190, N-acetylcysteine, A83-01, Y-27632, Forskolin, and CHIR99021 in DMSO to prepare a stock solution; (2) Dissolve sodium pyruvate, nicotinamide, Wnt3A, casamino acid, VEGF A, Activin A, vitamin C, and IGF 1 in sterile water to prepare a stock solution; (3) Dissolve human gastrin, h-EGF, R-spondin1 protein, Noggin protein, FGF-10, IL-6, and HGF in PBS (pH 7.4) containing 0.1% BSA (bovine serum albumin) to prepare a stock solution; (4) According to the formula concentration, add appropriate doses of the mother solution to the Advanced DMEM / F12 basal medium in sequence, and then add the formulated doses of B27 additive (without vitamin A), penicillin / streptomycin double antibody, HEPES, GlutaMAX additive, Primocin, N-2 additive, and fetal bovine serum to prepare the osteosarcoma organoid culture medium.

[0031] 2. Primary Culture of Osteosarcoma Organoids Osteosarcoma cells / cell clusters were prepared according to Example 2, and primary osteosarcoma organoids were obtained by suspension culture according to Example 3. After 6 days of culture, the proliferation rate of the organoids was 40%.

[0032] Example 5 Primary Culture of Osteosarcoma Organoids 1. Preparation of Osteosarcoma Organoid Culture Medium Prepare osteosarcoma organoid culture medium according to the following recipe: B27 Supplement (without vitamin A) 2×, N-2 Supplement 1×, GlutaMAX Supplement 8mM, Casein Amino Acids 500mg / L, N-Acetylcysteine ​​0.5mM, Nicotinamide 10mM, Vitamin C 1μM, Sodium Pyruvate 1mM, Human Gastrin 100nM, Wnt3A 5ng / mL, R-spondin 1 Protein 900ng / mL, Noggin Protein 10ng / mL, Activin A 100ng / mL, Forskolin 1μM, Y-27632 10μM, SB202190 15nM, CHIR99021 5μM, A83-01 9μM, h-EGF 5ng / mL, FGF-10 20ng / mL, HGF 10ng / mL, VEGF A 50ng / mL, IGF 1 1μg / mL, IL-6 1μg / mL, penicillin / streptomycin 1×, fetal bovine serum 10%.

[0033] Prepare osteosarcoma organoid culture medium as follows: (1) Dissolve SB202190, N-acetylcysteine, A83-01, Y-27632, Forskolin, and CHIR99021 in DMSO to prepare a stock solution; (2) Dissolve sodium pyruvate, nicotinamide, Wnt3A, casamino acid, VEGF A, Activin A, vitamin C, and IGF 1 in sterile water to prepare a stock solution; (3) Dissolve human gastrin, h-EGF, R-spondin1 protein, Noggin protein, FGF-10, IL-6, and HGF in PBS (pH 7.4) containing 0.1% BSA (bovine serum albumin) to prepare a stock solution; (4) According to the formula concentration, add appropriate doses of the mother solution to the Advanced DMEM / F12 basal medium in sequence, and then add the formulated doses of B27 additive (without vitamin A), penicillin / streptomycin double antibody, HEPES, GlutaMAX additive, Primocin, N-2 additive, and fetal bovine serum to prepare the osteosarcoma organoid culture medium.

[0034] 2. Primary Culture of Osteosarcoma Organoids Osteosarcoma cells / cell clusters were prepared according to Example 2, and the organoids were cultured in gelatin according to the suspension method in Example 3. After 6 days of culture, the proliferation rate of the organoids was 21%.

[0035] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. An osteosarcoma organoid culture medium, characterized in that Add the following ingredients to the basal medium: B27 supplement without vitamin A 0.5×-2×, N-2 supplement 1×, GlutaMAX supplement 1-10mM, Casamino Acids 100-1000mg / L, N-acetylcysteine ​​0.5-5mM, Nicotinamide 1-100mM, Vitamin C 0.5-2μM, Sodium Pyruvate 1-5mM, Human Gastrin 1-100nM, Wnt3A 5-20ng / mL, R-spondin 1 protein 100-1000ng / mL, Noggin protein 10-500ng / mL, Activin A protein 10-100ng / mL, Forskolin 1-15μM, Y-27632 5-15μM, SB202190 10-20 nM, CHIR99021 1-10μM, A83-01 0.1-10μM, human epidermal growth factor 5-500ng / mL, fibroblast growth factor 10 20-500ng / mL, hepatocyte growth factor 10-30ng / mL, vascular endothelial growth factor A 10-100ng / mL, insulin-like growth factor 1 1-5μg / mL, IL-6 1-2μg / mL, penicillin-streptomycin double antibody 1-2×, serum 1%-10%.

2. The osteosarcoma organoid culture medium according to claim 1, characterized in that The osteosarcoma organoid culture medium comprises the following components added to the basal culture medium: B27 supplement without vitamin A 1×, N-2 supplement 1×, GlutaMAX supplement 8mM, Casamino Acid 500-1000mg / L, N-acetylcysteine ​​0.5-1mM, Nicotinamide 5-15mM, Vitamin C 1-2μM, Sodium Pyruvate 1-5mM, Human Gastrin 50-100nM, Wnt3A 10-15ng / mL, R-spondin 1 protein 500-1000ng / mL, Noggin protein 200-500ng / mL, Activin A protein 80-100ng / mL, Forskolin 8-15μM, Y-27632 10-15μM, SB202190 10-20nM, CHIR99021 5-10μM, A83-01 5-10μM, human epidermal growth factor 5-50ng / mL, fibroblast growth factor 10 20-100ng / mL, hepatocyte growth factor 10-30ng / mL, vascular endothelial growth factor A 20-50ng / mL, insulin-like growth factor 1 1-5μg / mL, IL-6 1-2μg / mL, penicillin-streptomycin double antibody 1×, serum 10%.

3. The osteosarcoma organoid culture medium according to claim 1, characterized in that The osteosarcoma organoid culture medium comprises the following components added to the basal culture medium: Vitamin A-free B27 supplement 1×, N-2 supplement 1×, GlutaMAX supplement 8mM, Casamino Acid 600mg / L, N-acetylcysteine ​​0.5mM, Nicotinamide 10mM, Vitamin C 1μM, Sodium Pyruvate 1mM, Human Gastrin 100nM, Wnt3A 10ng / mL, R-spondin 1 protein 900ng / mL, Noggin protein 400ng / mL, Activin A protein 100ng / mL, Forskolin 10μM, Y-27632 10μM, SB202190 15nM, CHIR99021 5μM, A83-01 9μM, Human Epidermal Growth Factor 5ng / mL, Fibroblast Growth Factor 10 20ng / mL, Hepatocyte Growth Factor 25ng / mL, Vascular Endothelial Growth Factor A 50ng / mL, Insulin-like Growth Factor 1 5μg / mL, IL-6 1.5μg / mL, penicillin-streptomycin double antibody 1×, serum 10%.

4. The osteosarcoma organoid culture medium according to any one of claims 1 to 3, characterized in that The basal culture medium is selected from any one of Advanced DMEM / F12, RPMI 1640 and Leibovitz's L-15 culture medium; The serum is human serum, fetal bovine serum or a serum substitute.

5. The osteosarcoma organoid culture medium according to any one of claims 1 to 3, characterized in that The basic culture medium is Advanced DMEM / F12 culture medium.

6. A primary culture method for osteosarcoma organoids, characterized in that: The following steps are involved: (1) Processing the collected osteosarcoma tissue into cell / cell cluster precipitation; (2) Preheat the osteosarcoma organoid culture medium to 37°C. (3) Mixing the cell / cell cluster pellet and liquid matrix gel to obtain a resuspension; (4) Before solidification, the resuspension is added dropwise to the preheated osteosarcoma organoid culture medium to form a gel ball, and primary osteosarcoma organoids are obtained after culture.

7. The primary culture method according to claim 6, characterized in that: In step (2), the osteosarcoma organoid culture medium is the osteosarcoma organoid culture medium according to any one of claims 1 to 5; In step (3), the concentration of matrix gel in the resuspension solution is 75-95%; In step (4), the volume ratio of the resuspension solution to the osteosarcoma organoid culture medium is 1:(10-50); the volume of the gel ball is 20-40 μL / ball.

Images