CN112760282B - Method for culturing bone and soft tissue tumor solid tumor primary cells - Google Patents

Abstract

The invention discloses a method for culturing primary cells of solid tumors of bone and soft tissue. The invention provides a method for culturing primary cells of solid tumors of bones and soft tissues and a matched reagent, and the technical core is as follows: the bone and soft tissue tumor solid tumor tissues are treated by using a mild cell dissociation reagent, so that the activity of tumor cells in the tissues is ensured to the greatest extent; a special serum-free culture medium is prepared, and a suspension culture system is utilized to carry out in-vitro culture on solid tumor cells derived from bone and soft tissue tumors, so that the interference of normal cells is eliminated to the maximum extent while the normal amplification of the tumor cells is ensured. The primary cell culture of the solid tumor of bone and soft tissue tumor obtained by the method can be used for in vitro experiments of various cell levels, next generation sequencing, animal model construction, cell line construction and the like. The culture method has wide application prospect in the research and clinical diagnosis and treatment fields of bone and soft tissue tumor solid tumors.

Description

A method for culturing primary cells of solid tumors of bone and soft tissue tumors

Technical Field

The invention relates to the field of biotechnology, and in particular to a method for culturing primary cells of bone and soft tissue tumor solid tumors.

Background Art

Bone and soft tissue tumors are serious diseases that endanger human health and life. The incidence rate has gradually increased in recent years. Primary malignant bone tumors are more common in adolescents and middle-aged people. The most common ones are osteosarcoma, Ewing sarcoma, chondrosarcoma, malignant fibrous histiocytoma, chordoma, etc. Common soft tissue malignant tumors are synovial sarcoma, fibrosarcoma, liposarcoma, rhabdomyosarcoma, etc. The incidence of bone and soft tissue tumors accounts for about 1% of the incidence of malignant tumors, but there are many types, which makes non-standard diagnosis and treatment very common. Currently, all clinical guidelines recommend the use of clinical trials for the treatment of soft tissue sarcoma.

Although scientific research and medical institutions around the world have made great efforts to study the causes and development of bone and soft tissue tumors, humans still know very little about this disease. Bone and soft tissue tumors are a complex disease. Their occurrence and development is a dynamic process, involving the interaction of many signal molecules, forming a complex molecular regulatory network, and are also affected by external environmental factors. The causes and development of bone and soft tissue tumors have strong individual differences and cannot be generalized. Therefore, using primary cell cultures of bone and soft tissue tumors as models for personalized and precise research is a trend in the field of bone and soft tissue tumor research and even in the field of bone and soft tissue tumor diagnosis and treatment.

Existing primary tumor cell culture technologies mainly include 2D culture, 3D culture, reprogramming culture and other categories. These methods all face problems such as extremely long culture cycle, low culture success rate, and difficulty in removing impurity cells to varying degrees.

Summary of the invention

In order to effectively solve the above technical problems, the present invention provides a new bone and soft tissue tumor solid tumor primary cell culture technology and supporting reagents. The core of the technology is: (1) using a mild cell dissociation reagent to treat bone and soft tissue tumor solid tumor tissues to ensure the vitality of tumor cells in the tissues to the greatest extent; (2) preparing a special serum-free culture medium and using a suspension culture system to culture bone and soft tissue tumor solid tumor primary cells in vitro, ensuring the normal proliferation of tumor cells while maximally eliminating the interference of normal cells.

In a first aspect, the present invention claims a method for culturing primary cells of solid tumors of bone and soft tissue tumors.

The method for culturing primary cells of bone and soft tissue tumor solid tumors claimed in the present invention may include the following steps:

Primary cells of solid tumors of bone and soft tissue tumors were cultured in suspension using primary cell culture medium of solid tumors of bone and soft tissue tumors;

The bone and soft tissue tumor solid tumor primary cell culture medium consists of three antibacterial and antifungal agents (penicillin-streptomycin-amphotericin B), HEPES, GlutaMax, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, human recombinant protein IGF-I, human recombinant protein BDNF, ITS-X (Insulin, Transferrin, Selenium, Ethanolamine Solution), N-acetyl-L-cysteine, N-2 Supplement, Y-27632 and Advanced DMEM/F12 culture medium. The final concentration of penicillin in the three antibiotics of antibacterial and antifungal agents is 100-200 U/mL (such as 100 U/mL); the final concentration of streptomycin in the three antibiotics of antibacterial and antifungal agents is 100-200 μg/mL (such as 100 μg/mL); the final concentration of amphotericin B in the three antibiotics of antibacterial and antifungal agents is 250-250 ng/mL (such as 250 ng/mL); the final concentration of HEPES is 8-12 mM (such as 10 mM); the final concentration of GlutaMax is 0.8-1.2% (such as 1%, % represents volume percentage); the human weight The final concentration of histone bFGF is 10-50 ng/mL; the final concentration of the human recombinant protein HGF is 5-25 ng/mL; the final concentration of the human recombinant protein MSP is 5-25 ng/mL; the final concentration of the human recombinant protein IGF-I is 50-100 ng/mL; the final concentration of the human recombinant protein BDNF is 50-200 ng/mL; the final concentration of ITS-X is 1% (volume percentage); the final concentration of N-acetyl-L-cysteine is 0.5-2 mM; the final concentration of N-2 Supplement is 1% (volume percentage); the final concentration of Y-27632 is 5-20 μM; the remainder is Advanced DMEM/F12 culture medium.

抗真菌剂。所述GlutaMAX为“GlutaMAX^TMSupplement”(如Gibco#35050061，或与其组成相同的其他产品)。所述“GlutaMAX^TMSupplement”的成分为L-alanyl-L-glutamine，是L-glutamine的替代物，浓度为200nM，溶剂为0.85％NaCl溶液。所述N-2 Supplement为“N-2 Supplement(100X)”(如Gibco#17502001，或与其组成相同的其他产品)。所述“N-2 Supplement(100X)”中含有终浓度为1mM的人全铁转铁蛋白(Human Transferrin(Holo))、500mg/L的重组胰岛素全链(InsulinRecombinant Full Chain)、0.63mg/L的孕酮(Progesterone)、10mM的腐胺(Putrescine)、0.52mg/L的亚硒酸盐(Selenite)。所述ITS-X的溶剂为EBSS溶液(Earle's平衡盐溶液)，溶质及浓度如下：胰岛素1g/L；转铁蛋白0.55g/L；亚硒酸钠0.00067g/L；乙醇胺0.2g/L。所述GlutaMAX是一种高级细胞培养添加剂，可直接替代细胞培养基中的L-谷氨酰胺。所述GlutaMAX为“GlutaMAX^TM Supplement”(如Gibco#35050061，或与其组成相同的其他产品)。所述Y-27632为“Y-27632dihydrochloride(一种ATP竞争性的ROCK-I和ROCK-II抑制剂，Ki分别为220nM和300nM)”(如MCE#129830-38-2，或与其组成相同的其他产品)。Furthermore, the triple antibiotic (penicillin-streptomycin-amphotericin B) of the antibacterial and antifungal agent is composed as follows: each milliliter contains 10,000 units of penicillin (base), 10,000 μg of streptomycin (base) and 25 μg of amphotericin B. The triple antibiotic (penicillin-streptomycin-amphotericin B) of the antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100X" (such as Gibco #15240062, or other products with the same composition). The "Antibiotic-Antimycotic, 100X" contains 10,000 units of penicillin (base), 10,000 μg of streptomycin (base) and 25 μg of amphotericin B per milliliter, using penicillin G (sodium salt), streptomycin sulfate and amphotericin B in the form of 0.85% saline solution as

Antifungal agent. The GlutaMAX is "GlutaMAX ^TM Supplement" (such as Gibco #35050061, or other products with the same composition). The ingredient of the "GlutaMAX ^TM Supplement" is L-alanyl-L-glutamine, which is a substitute for L-glutamine, with a concentration of 200nM, and the solvent is 0.85% NaCl solution. The N-2 Supplement is "N-2 Supplement (100X)" (such as Gibco #17502001, or other products with the same composition). The "N-2 Supplement (100X)" contains human transferrin (Human Transferrin (Holo)) with a final concentration of 1mM, 500mg/L of recombinant insulin full chain (Insulin Recombinant Full Chain), 0.63mg/L of progesterone (Progesterone), 10mM of putrescine (Putrescine), and 0.52mg/L of selenite (Selenite). The solvent of the ITS-X is EBSS solution (Earle's balanced salt solution), and the solutes and concentrations are as follows: insulin 1g/L; transferrin 0.55g/L; sodium selenite 0.00067g/L; ethanolamine 0.2g/L. The GlutaMAX is an advanced cell culture additive that can directly replace L-glutamine in cell culture medium. The GlutaMAX is "GlutaMAX ^TM Supplement" (such as Gibco#35050061, or other products with the same composition). The Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor, Ki 220nM and 300nM, respectively)" (such as MCE#129830-38-2, or other products with the same composition).

In a specific embodiment of the present invention, the brand number of the antibacterial and antifungal agent triple antibiotic (penicillin-streptomycin-amphotericin B) is Gibco#15240062; the brand number of the HEPES is Gibco#15630080; the brand number of the GlutaMAX is Gibco#35050061; the brand number of the human recombinant protein bFGF is Peprotech AF-100-18B-50; the brand number of the human recombinant protein HGF is Peprotech AF-100-39-100; the brand number of the human recombinant protein MSP is R&D#352-MS-050; the brand number of the human recombinant protein IGF-I is Peprotech#AF-100-11; the brand number of the human recombinant protein BDNF is Peprotech#450-02; the brand number of the ITS-X is Gibco#51500056; the brand number of the N-acetyl-L-cysteine is Sigma#A9165; the brand number of the N-2 Supplement is Gibco#17502001; the brand number of the Y-27632 is MCE#129830-38-2; the brand number of the Advanced DMEM/F12 culture medium is Gibco#12634010.

The primary cells of bone and soft tissue tumor solid tumors can be obtained by dissociating bone and soft tissue tumor solid tumor tissues with a sample dissociation solution.

The sample dissociation solution consists of collagenase I, collagenase II, collagenase III, collagenase IV and PBS; wherein the final concentration of the collagenase I is 150-250U/mL (such as 200U/mL); the final concentration of the collagenase II is 150-250U/mL (such as 200U/mL); the final concentration of the collagenase III is 250-350U/mL (such as 290U/mL); the final concentration of the collagenase IV is 150-250U/mL (such as 200U/mL); and the remainder is PBS.

The unit U of collagenase (the collagenase I, the collagenase II, the collagenase III or the collagenase IV) is defined by the enzymatic activity of the protease: at 37°C and pH 7.5, 1U of protease is used to treat the collagenase (the collagenase I, the collagenase II, the collagenase III or the collagenase IV) for 5 hours to release 1 μmol of L-leucine.

In a specific embodiment of the present invention, the brand number of the collagenase I is Gibco#17100-017; the brand number of the collagenase II is Gibco#17101-015; the brand number of the collagenase III is Solarbio#C8490; the brand number of the collagenase IV is Gibco#17104-019; and the brand number of the PBS is Gibco#21-040-CVR.

Furthermore, the bone and soft tissue tumor solid tumor tissue can be dissociated with the sample dissociation solution according to a method comprising the following steps: 0.1-0.3 mL (e.g., 0.1 mL) of the sample dissociation solution is used per mg of tissue, the minced bone and soft tissue tumor solid tumor tissue (e.g., cut into small pieces of 0.8-1.2 mm ³ ) is treated with the sample dissociation solution preheated at 37° C., and the sample is dissociated at 37° C. for 15 minutes to 3 hours. The dissociation of the sample is observed under a microscope every 15 minutes until a large number of single cells are observed.

In the method, the bone and soft tissue tumor solid tumor primary cells can be suspended and cultured using the bone and soft tissue tumor solid tumor primary cell culture medium according to a method comprising the following steps: using a cell culture container M, suspending and culturing the bone and soft tissue tumor solid tumor primary cells using the bone and soft tissue tumor solid tumor primary cell culture medium, culturing at 37° C., 5% CO ₂ , and replacing the culture medium every 2-4 days (such as 3 days) until the cells form a mass with a diameter of 80-120 μm (such as 100 μm).

Among them, the initial seeding density can be 10 ⁵ cells/cm ² of the bottom area of the container. Taking a six-well plate as an example, the plate is plated at a density of 10 ⁶ cells per well.

The cell culture container M may be any of the following: (I) a cell culture container made of polystyrene, a cell culture container made of polycarbonate, a cell culture container made of polymethyl methacrylate, a cell culture container made of COC resin, a cell culture container made of cycloolefin polymer or a cell culture container with a low adsorption surface; (II) a cell culture container obtained by modifying the cell culture container in (I) with CYTOP.

Furthermore, the cell culture container is a cell culture dish, a cell culture well plate, or a microplate chip for cell culture (such as the microplate chip shown in FIG. 5 in Example 13), etc.

In (II), the cell culture container in (I) can be modified with CYTOP according to a method comprising the following steps: the cell culture container in (I) is etched with pure oxygen at a power of 20 W and an etching time of 3 minutes; then the surface of the cell culture container is covered with a 1% CYTOP solution, and the 1% CYTOP solution is dried to complete the CYTOP modification.

The composition of the 1% CYTOP solution is as follows: every 100 mL of the 1% CYTOP solution contains 1 mL of CYTOP, and the remainder is fluorinated oil.

The CYTOP is perfluoro (1-butenylvinylether) polymer. The fluorine oil can be fluorine oil with a brand number of 3M#FC40, or other products with the same composition.

In a specific embodiment of the present invention, the brand number of the CYTOP is Asashi glass#CTL-809M; the brand number of the fluoro oil is 3M#FC40.

Furthermore, the method may also include the following steps of pre-dissociation treatment of the bone and soft tissue tumor solid tumor tissue: washing the surface of the bone and soft tissue tumor solid tumor tissue sample with 70-75% (e.g., 75%) ethanol by volume for 10 to 30 seconds; washing the bone and soft tissue tumor solid tumor tissue sample with a sample washing solution for 10-20 times (e.g., 10 times), and washing the bone and soft tissue tumor solid tumor tissue sample with a sterile PBS solution for 5-10 times (e.g., 5 times); and then removing impurities, connective tissue, adipose tissue, necrotic tissue, and other components that affect primary cell culture in the bone and soft tissue tumor solid tumor tissue sample.

The sample cleaning solution is composed of double-antibody P/S (penicillin-streptomycin) and PBS; the final concentration of penicillin in the double-antibody P/S is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-antibody P/S is 100-200μg/mL (such as 100μg/mL); the remainder is PBS.

In a specific embodiment of the present invention, the brand number of the bispecific antibody P/S is Gibco#15140122; the brand number of the PBS is Gibco#21-040-CVR.

The step of pre-dissociation treatment of the bone and soft tissue tumor solid tumor tissue needs to be performed on ice, and the entire operation step needs to be completed within 10 minutes.

Furthermore, the bone and soft tissue tumor solid tumor tissue samples subjected to the pre-dissociation treatment must be isolated from the body within 2 hours and must be kept in a sample preservation solution before the pre-dissociation treatment is performed.

The sample preservation solution is composed of fetal bovine serum, double-antibody P/S (penicillin-streptomycin), HEPES and HBSS (Hank's balanced salt solution); the final concentration of the fetal bovine serum is 1-5% (such as 2%, % represents the volume percentage); the final concentration of penicillin in the double-antibody P/S is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-antibody P/S is 100-200μg/mL (such as 100μg/mL); the final concentration of HEPES is 8-12mM (such as 10mM); the remainder is HBSS.

In a specific embodiment of the present invention, the brand number of the bispecific antibody P/S is Gibco#15140122; the brand number of the PBS is Gibco#21-040-CVR.

Furthermore, in the method, after the bone and soft tissue tumor solid tumor tissue is dissociated with the sample dissociation solution, the following steps may be included: terminating the dissociation reaction with 8-15 times (such as 10 times) the volume of the digestion stop solution, and collecting the cell suspension; filtering the cell suspension with a 100μm or 40μm sterile cell filter to remove tissue debris and adherent cells; centrifuging at 800-1000g (such as 800g) at room temperature for 10-15 minutes (such as 10 minutes), and discarding the supernatant; then resuspending the cells with 3-5mL (such as 5mL) of sterile PBS; centrifuging at 800-1000g (such as 800g) at room temperature for 10-15 minutes (such as 10 minutes), and discarding the supernatant; then resuspending the cell pellet with the bone and soft tissue tumor solid tumor primary cell culture medium, observing the cell state under a microscope, and counting the cells.

The digestion stop solution is composed of fetal bovine serum, double-antibody P/S (penicillin-streptomycin) and DMEM culture medium; the final concentration of the fetal bovine serum is 8-12% (such as 10%, % represents volume percentage); the final concentration of penicillin in the double-antibody P/S is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-antibody P/S is 100-200μg/mL (such as 100μg/mL); the remainder is DMEM culture medium.

In a specific embodiment of the present invention, the brand number of the bispecific antibody P/S is Gibco#15140122; the brand number of the PBS is Gibco#21-040-CVR.

Furthermore, the method may also include the following step: when the primary cells of bone and soft tissue tumor solid tumor form a mass with a diameter of 80-120 μm (such as 100 μm), the primary cells of bone and soft tissue tumor solid tumor are passaged.

The composition of the cell digestion solution used for the passaging is as follows: each 10 mL of the cell digestion solution contains 4-6 mL (such as 5 mL) Accutase, EDTA with a final concentration of 5 mM (i.e. 10 μL 0.5 M EDTA), 1.5-2.5 mL (such as 2 mL) TrypLE Express, and the remainder is PBS.

Furthermore, the Accutase is "StemPro ^TM Accutase ^TM Cell Dissociation Reagent" (such as Gibco#A11105-01, or other products with the same composition). The Accutase is a single-component enzyme, which is dissolved in D-PBS, 0.5mM EDTA solution. The TrypLE Express is "TrypLE ^TM Express Enzyme (1X), no phenol red" (such as Gibco#12604013, or other products with the same composition). The "TrypLE ^TM Express Enzyme (1X) no phenol red" contains 200 mg/L KCl, 200 mg/L KH ₂ PO ₄ , 8000 mg/L NaCl, 2160 mg/L Na ₂ HPO ₄ ·7H ₂ O, 457.6 mg/L EDTA; and also contains recombinant protease.

In a specific embodiment of the present invention, the brand number of the Accutase is Gibco#A11105-01; the brand number of the 0.5M EDTA is Invitrogen#AM9261; the brand number of the TrypLE Express is Gibco#12604013; and the brand number of the PBS is Gibco#21-040-CVR.

Furthermore, the digestion temperature used during the passaging is 37°C.

Furthermore, the digestion stop solution used in the passaging is the digestion stop solution described above.

More specifically, the steps of performing the subculture are as follows: collecting the cell clumps to be subcultured, washing the cell clumps with a sterile PBS solution after centrifugation, centrifuging again, and then resuspending the cell clumps with the cell digestion solution, digesting at 37°C until the cell clumps are digested into single cells, terminating the digestion reaction with the digestion termination solution (the amount of which can be 5-10 times, such as 10 times the volume), and collecting the cell suspension; resuspending the cell precipitate with the bone and soft tissue tumor solid tumor primary cell culture medium after centrifugation, counting, and then suspending and culturing the cells in a culture container with a low adsorption surface (the initial seeding density can be 10 ⁵ cells/cm ² container bottom area, taking a six-well plate as an example, the plate is plated at a density of 10 ⁶ cells per well), and the culture conditions are 37°C and 5% CO _2. All centrifugation in the above subculture steps can be specifically centrifuged at 800-1000g (such as 800g) at room temperature for 10-20 minutes (such as 10 minutes).

Furthermore, the method may also include the step of freezing and/or reviving the primary cells of the bone and soft tissue tumor solid tumors after 2-3 passages and expansions.

The cell freezing solution used for the freezing is composed of Advanced DMEM/F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium, the DMSO and the 1% methylcellulose solution is 20:2:(0.8-1.2), such as 20:2:1; and the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1 g/100 ml.

In a specific embodiment of the present invention, the brand number of the Advanced DMEM/F12 culture medium is Gibco#12634010; the brand number of the DMSO is Sigma#D2438; and the brand number of the methyl cellulose is Sigma#M7027.

Furthermore, the specific steps of the freezing are as follows: collecting the cell clumps to be frozen, washing the cell clumps with a sterile PBS solution after centrifugation, centrifuging again, and then resuspending the cell clumps with the cell digestion solution, digesting at 37°C until the cell clumps are digested into single cells, using the digestion stop solution (the amount can be 5-10 times, such as 10 times the volume) to terminate the digestion reaction, and collecting the cell suspension; after centrifugation, resuspending the cell precipitate with the cell freezing solution at a density of 0.5-2×10 ⁶ /mL (such as 10 ⁶ /mL), freezing overnight in a gradient cooling box, and then transferring to liquid nitrogen for long-term storage. All centrifugation in the above freezing steps can be specifically centrifugation at 800-1000g (such as 800g) at room temperature for 10-20 minutes (such as 10 minutes).

Furthermore, the specific steps of the recovery are as follows: taking out the cryopreservation tube containing the cells to be recovered from liquid nitrogen, and rapidly thawing the cells in sterile water at 37-39°C (such as 37°C); resuspending the cell pellet with the bone and soft tissue tumor solid tumor primary cell culture medium after centrifugation (such as 800-1000g, such as 800g centrifugation at room temperature for 5-10 minutes, such as 10 minutes), and then suspending and culturing the cells in a culture container with a low adsorption surface (the initial seeding density can be 10 ⁵ cells/cm ² container bottom area), and recovering each tube of cells (10 ⁶ cells) to a 3.5 cm culture dish), and the culture conditions are 37°C, 5% CO ₂ .

In a second aspect, the present invention claims a kit of reagents for culturing primary cells of solid tumors of bone and soft tissue tumors.

The complete set of reagents for culturing primary cells of solid tumors of bone and soft tissue tumors provided by the present invention can specifically be composed of the above-mentioned culture medium for primary cells of solid tumors of bone and soft tissue tumors and at least one of the following reagents: the above-mentioned sample dissociation solution, the sample preservation solution, the cell digestion solution, the sample cleaning solution, the digestion termination solution, the cell freezing solution and the 1% CYTOP solution.

The sample preservation solution can be used for temporary preservation of samples after they are removed from the body, and can maintain the activity of cells in the sample for a short period of time after the sample is removed from the body. The sample preservation solution can be stored at 4° C. for 1 month after preparation.

The sample cleaning solution can be used for cleaning and disinfecting samples. The sample cleaning solution needs to be prepared and used immediately.

The sample dissociation solution can be used for sample dissociation, and can dissociate primary cells of bone and soft tissue tumor solid tumors from tissues in the sample. The sample dissociation solution needs to be prepared and used immediately, and the collagenase I, collagenase II, collagenase III and collagenase IV therein can be stored for a long time at -20°C in the form of a stock solution (mother solution), specifically a 10-fold stock solution (mother solution). The 10× collagenase I stock solution is composed of the collagenase I and PBS; wherein the final concentration of the collagenase I is 2000U/mL; the 10× collagenase II stock solution is composed of the collagenase II and PBS; wherein the final concentration of the collagenase II is 2000U/mL; the 10× collagenase III stock solution is composed of the collagenase III and PBS; wherein the final concentration of the collagenase III is 2900U/mL; the 10× collagenase IV stock solution is composed of the collagenase IV and PBS; wherein the final concentration of the collagenase IV is 2000U/mL; and the remainder is PBS. The enzyme activities of the collagenase I, the collagenase II, the collagenase III and the collagenase IV are defined as above.

The cell digestion solution can be used for digestion and passage of cell masses, and can digest bone and soft tissue tumor solid tumor masses into single cells. The cell digestion solution needs to be prepared and used immediately.

The digestion stop solution can be used to stop sample dissociation or cell digestion process. After being prepared, the digestion stop solution can be stored at 4°C for one month.

The bone and soft tissue tumor solid tumor primary cell culture medium can be used for the culture of bone and soft tissue tumor solid tumor primary cells. After the bone and soft tissue tumor solid tumor primary cell culture medium is prepared, it needs to be filtered and sterilized with a 0.22μM syringe filter (Millipore SLGP033RS) and can be stored at 4°C for two weeks. The human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, human recombinant protein IGF-I and human recombinant protein BDNF can be stored in the form of a storage solution (mother solution) at -80°C for a long time, specifically 1000 times the storage solution (mother solution). N-acetyl-L-cysteine and Y-27632 can be stored in the form of a storage solution (mother solution) at -20°C for a long time, specifically 1000 times the storage solution (mother solution).

The 1000× human recombinant protein bFGF stock solution is composed of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20μg/mL, the final concentration of the BSA is 0.01g/mL, and the remainder is PBS. The 1000× human recombinant protein HGF stock solution is composed of human recombinant protein HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20μg/mL, the final concentration of the BSA is 0.01g/mL, and the remainder is PBS. The 1000× human recombinant protein MSP stock solution is composed of human recombinant protein MSP, BSA and PBS, wherein the final concentration of the human recombinant protein MSP is 20μg/mL, the final concentration of the BSA is 0.01g/mL, and the remainder is PBS. The 1000× human recombinant protein IGF-I stock solution is composed of human recombinant protein IGF-I, BSA and PBS, wherein the final concentration of the human recombinant protein IGF-I is 100μg/mL, the final concentration of the BSA is 0.01g/mL, and the remainder is PBS. The 1000× human recombinant protein BDNF stock solution is composed of human recombinant protein BDNF, BSA and PBS, wherein the final concentration of the human recombinant protein BDNF is 100μg/mL, the final concentration of the BSA is 0.01g/mL, and the remainder is PBS. Among the above five 1000-fold stock solutions, the BSA can exist in the form of a 100-fold stock solution (mother solution) (prepared and used immediately), specifically composed of BSA and PBS, wherein the final concentration of BSA (Sigma#A1933) is 0.1g/mL, and the remainder is PBS. In addition, 1000×N-acetyl-L-cysteine stock solution is composed of N-acetyl-L-cysteine and ultrapure water, wherein the concentration of N-acetyl-L-cysteine is 0.5 M, and the remainder is ultrapure water. 1000×Y-27632 is composed of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10 mM, and the remainder is ultrapure water.

The cell freezing solution must be prepared and used immediately. The 1% methylcellulose solution can be stored at 4°C for a long time.

In a third aspect, the present invention claims the use of the above-mentioned set of reagents in culturing primary cells of solid tumors of bone and soft tissue tumors.

The above-mentioned culture medium for primary cells of solid tumors of bone and soft tissue tumors, and its use in culturing primary cells of solid tumors of bone and soft tissue tumors also fall within the protection scope of the present invention.

In the above aspects, the bone and soft tissue tumor solid tumor can be a primary bone and soft tissue tumor solid tumor. The bone and soft tissue tumor solid tumor can specifically be osteosarcoma, synovial sarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing sarcoma, liposarcoma, alveolar soft tissue sarcoma, clear cell sarcoma or fibroma.

In the above aspects, the primary bone and soft tissue tumor solid tumor cells are separated from surgical samples of patients with bone and soft tissue tumor solid tumors. The bone and soft tissue tumor solid tumor tissue specimens obtained from the surgical samples preferably weigh more than 20 mg.

In the present invention, all of the above PBS can be 1×PBS, pH 7.3-7.5, and its specific composition is as follows: the solvent is water, and the solutes and concentrations are: KH ₂ PO ₄ 144 mg/L, NaCl 9000 mg/L, Na ₂ HPO ₄ ·7H ₂ O 795 mg/L.

The present invention provides a method and supporting reagents for extracting and culturing primary cells of bone and soft tissue tumor solid tumors from fresh bone and soft tissue tumor solid tumors. The method has the following advantages:

1. Small amount of tissue samples are required, only about 20 mg of bone and soft tissue tumor solid tumor surgical samples are needed;

2. The culture cycle is short. It only takes 3-10 days to obtain ¹⁰⁷ primary tumor cells of bone and soft tissue tumor solid tumors;

3. High culture stability. The success rate of in vitro culture of qualified bone and soft tissue tumor solid tumor surgical specimens using this method is as high as 70%;

4. The cell purity is high. In the primary cell culture of bone and soft tissue tumor solid tumors obtained by this method, the proportion of tumor cells can reach 70%-95%, and there is little interference from foreign cells.

The primary cell culture of solid tumors of bone and soft tissue tumors obtained by the method of the present invention can be used for in vitro experiments at various cell levels, second-generation sequencing, construction of animal models, construction of cell lines, etc. It can be foreseen that this culture method has broad application prospects in the research and clinical diagnosis and treatment of solid tumors of bone and soft tissue tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows single cells obtained from synovial sarcoma tissue after processing. The scale bar is 100 μm, 100 times magnification.

Figure 2 shows the cell aggregates obtained after primary culture of synovial sarcoma tissue. The scale bar is 100 μm, 100 times magnification.

Figure 3 is a HE staining image of bone and soft tissue sarcoma cells obtained after primary culture of synovial sarcoma tissue. The scale bar is 100 μm, and the magnification is 40 times.

Figure 4 is an immunofluorescence staining image of tumor cell aggregates obtained after primary culture of synovial sarcoma tissue. The scale bar is 100 μm, and the magnification is 40 times.

FIG. 5 is a design diagram of the microplate chip of the present invention.

DETAILED DESCRIPTION

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Example 1: Preparation of reagents for culturing primary cells of bone and soft tissue tumor solid tumors

1. Sample preservation solution (100mL)

The specific formula of the sample preservation solution (100 mL) is shown in Table 1.

Table 1 Sample storage solution (100mL)

After the sample preservation solution is prepared, it is divided into 15mL centrifuge tubes, 5mL per tube. After division, it can be stored at 4℃ for 1 month.

2. Sample cleaning solution (100mL)

The specific formula of the sample cleaning solution (100 mL) is shown in Table 2.

Table 2 Sample cleaning solution (100mL)

The sample cleaning solution must be prepared and used immediately.

3. Sample dissociation solution (10mL)

The specific formula of the sample dissociation solution (10 mL) is shown in Table 3.

Table 3 Sample dissociation solution (10mL)

Note: The sample dissociation solution should be prepared and used immediately.

In Table 3, the preparation of collagenase stock solution is shown in Tables 4 to 7.

Table 4 10× collagenase I stock solution (100 mL)

After the 10× collagenase I stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

Table 5 10× collagenase II stock solution (100 mL)

After the 10× collagenase II stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

Table 6 10× collagenase III stock solution (100 mL)

After the 10× collagenase III stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

Table 7 10× Collagenase IV stock solution (100 mL)

After the 10× collagenase IV stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20°C for a long time.

In Tables 4 to 7, the unit U of collagenase (the collagenase I, the collagenase II, the collagenase III or the collagenase IV) is defined by the enzymatic activity of the protease: at 37°C and pH 7.5, 1U of protease is used to treat the collagenase (the collagenase I, the collagenase II, the collagenase III or the collagenase IV) for 5 hours, and 1 μmol of L-leucine can be released.

4. Cell digestion solution (10 mL)

The specific formula of the cell digestion solution (10 mL) is shown in Table 8.

Table 8 Cell digestion solution (10 mL)

The cell digestion solution is prepared and used immediately.

5. Digestion stop solution (100 mL)

The specific formula of the digestion stop solution (100 mL) is shown in Table 9.

Table 9 Digestion stop solution (100 mL)

After the digestion stop solution is prepared, it can be stored at 4°C for one month.

6. Bone and soft tissue tumor solid tumor primary cell culture medium (100mL)

The specific formula of the bone and soft tissue tumor solid tumor primary cell culture medium (100 mL) is shown in Table 10.

Table 10 Bone and soft tissue tumor solid tumor primary cell culture medium (100mL)

After the preparation of the bone and soft tissue tumor solid tumor primary cell culture medium is completed, it is filtered and sterilized using a 0.22μM syringe filter (Millipore SLGP033RS) and can be stored at 4°C for two weeks.

In Table 10, the preparation of human recombinant protein stock solutions is shown in Tables 12 to 16, the preparation of N-acetyl-L-cysteine stock solutions is shown in Table 17, and the preparation of Y-27632 stock solutions is shown in Table 18. The preparation of 100×BSA solutions required for preparing these stock solutions is shown in Table 11.

Table 11 100×BSA solution (1 mL)

100× BSA solution is prepared and used immediately.

Table 12 1000× human recombinant protein bFGF stock solution (2.5 mL)

After the 1000× human recombinant protein bFGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 13 1000× human recombinant protein HGF stock solution (5 mL)

After the 1000× human recombinant protein HGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 14 1000× human recombinant protein MSP stock solution (2.5 mL)

After the 1000× human recombinant protein MSP stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 15 1000× human recombinant protein IGF-I stock solution (1 mL)

After the 1000× human recombinant protein IGF-I stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 16 1000× human recombinant protein BDNF stock solution (1 mL)

After the 1000× human recombinant protein BDNF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 17 1000×N-acetyl-L-cysteine stock solution (5 mL)

After the 1000× N-acetyl-L-cysteine stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

Table 18 1000×Y-27632 stock solution (3.125 mL)

After the 1000×Y-27632 stock solution is prepared, it is packaged in 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

7. Cell Cryopreservation Solution

The specific formula of the cell freezing solution is shown in Table 19.

Table 19 Cell freezing solution

Cell freezing solution is prepared and used immediately.

In Table 19, the preparation of 1% methylcellulose solution is shown in Table 20.

Table 20 1% methylcellulose solution (10 mL)

1% methylcellulose solution can be stored at 4°C for a long time after preparation.

8. 1% CYTOP solution

Table 21 1% CYTOP solution (100 mL)

After the 1% CYTOP solution is prepared, it can be stored at room temperature for a long time.

Example 2. Obtaining postoperative specimens/biopsy specimens of bone and soft tissue tumors

1. Cooperate with tertiary hospitals, and the cooperation has passed formal medical ethics review.

2. The attending physician selects patients for inclusion in the study according to the clinical indications specified in the medical guidelines, and selects appropriate specimens for in vitro culture based on the intraoperative clinical indications. The sample selection criteria are: primary osteosarcoma, synovial sarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing sarcoma, liposarcoma, alveolar soft tissue sarcoma, clear cell sarcoma, fibroma, and surgical specimens weighing more than 20 mg.

3. The attending physician shall provide the patient's gender, age, medical history, family history, smoking history, pathological staging, clinical diagnosis and other basic clinical information. The patient's name, ID number and other information related to the patient's privacy shall be concealed and replaced with a unified experimental number. The naming principle of the experimental number is the eight-digit date of sample collection + the last four digits of the patient's hospitalization number. For example, for a sample provided on January 1, 2018, the patient's hospitalization number is T001512765, and the sample experimental number is 201801012765.

4. During the operation, the surgeon collects fresh specimens in the sterile environment of the operating room and places them in the sample preservation solution prepared in advance (see Example 1). After the specimen is removed from the body, it is temporarily stored on ice and transported to the laboratory within two hours for the next step of operation.

Example 3. Pre-dissociation treatment of bone and soft tissue tumor solid tumor tissue samples

The following operations need to be performed on ice and the entire operation should be completed within 10 minutes.

The surgical instruments used in the following operations must be sterilized at high temperature and high pressure and dried before use.

1. Weigh the sample.

2. Clean the sample surface with 75% (volume percentage) ethanol for 10 to 30 seconds.

3. Wash the sample 5 times with sample cleaning solution and 5 times with sterile PBS solution.

4. Use ophthalmic scissors, ophthalmic tweezers, scalpel and other instruments to carefully peel off the fat tissue, connective tissue and necrotic tissue in the sample.

Example 4: Dissociation of bone and soft tissue tumor solid tumor tissue samples

The surgical instruments used in the following embodiments must be sterilized at high temperature and high pressure in advance and dried before use.

1. Use ophthalmic scissors to cut the tissue into small pieces of about 1 ^mm3 .

2. Treat the shredded tissue sample with the sample dissociation solution preheated at 37°C at a dosage of 0.1 mL of sample dissociation solution (see Example 1) per mg of tissue, and dissociate the sample at 37°C for 15 minutes to 3 hours. Observe the dissociation of the sample under a microscope every 15 minutes until a large number of single cells are observed.

3. Use 10 times the volume of digestion stop solution (see Example 1) to terminate the dissociation reaction and collect the cell suspension.

4. Filter the cell suspension with a 100 μm sterile cell strainer to remove tissue debris and adherent cells.

5. Centrifuge at 800g for 10 minutes at room temperature and discard the supernatant.

6. Resuspend the cells in 5 mL of sterile PBS, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

7. Resuspend the cell pellet with the bone and soft tissue tumor solid tumor primary cell culture medium (see Example 1), observe the cell state under a microscope, and count the cells.

As shown in Figure 1, in addition to tumor cells, the dissociated single cell suspension is mixed with a large number of other types of cells, such as red blood cells, lymphocytes, fibroblasts, etc. One of the advantages of this method is that in the subsequent culture process, only tumor cells can be amplified in large quantities, while the proportion of other cells gradually decreases or even disappears, and ultimately a high-purity primary tumor cell of bone and soft tissue tumor solid tumor is obtained.

Example 5: Primary cell culture of bone and soft tissue solid tumors

1. Use a low-attachment-surface to perform suspension culture of primary cells of bone and soft tissue tumor solid tumors. The culture medium used is the primary cell culture medium of bone and soft tissue tumor solid tumors in Example 1 (wherein the final concentration of human recombinant protein bFGF is 20 ng/mL; the final concentration of human recombinant protein HGF is 20 ng/mL; the final concentration of human recombinant protein MSP is 20 ng/mL; the final concentration of human recombinant protein IGF-I is 50 ng/mL; the final concentration of human recombinant protein BDNF is 100 ng/mL; the final concentration of N-acetyl-L-cysteine is 1 mM; the final concentration of Y-27632 is 10 μM). Taking a six-well plate as an example, plate the cells at a density of 10 ⁶ cells per well and culture them in a cell culture incubator at 37° C. and 5% CO ₂ .

2. Observe the cell status every day and replace the culture medium every 3 days until the cells form clumps with a diameter of about 100 μm.

As shown in Figure 2, after 3-10 days of culture, tumor cells proliferate to form cell clusters with a diameter of 100 μm, the total number of tumor cells can exceed 10 ⁷ , and the number of other types of cells decreases significantly or even disappears. This method has been tested on a large number of samples, and the success rate of in vitro culture of primary tumor cells of bone and soft tissue tumors can reach 70%.

Example 6: Passaging of primary cells of bone and soft tissue tumor solid tumors

1. Collect the cell clumps in the culture dish, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

2. Wash the cell pellet with sterile PBS solution, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

3. Resuspend the cell mass with cell digestion solution (see Example 1) and digest at 37° C. Observe the digestion of the cell mass under a microscope every 5 minutes until the cell mass is digested into single cells.

4. Use 10 times the volume of digestion stop solution (see Example 1) to terminate the dissociation reaction and collect the cell suspension.

5. Centrifuge at 800g for 10 minutes at room temperature and discard the supernatant.

6. Resuspend the cell pellet with the bone and soft tissue tumor solid tumor primary cell culture medium (see Example 1) and count the cells.

7. Use a low-attachment-surface to culture primary cells of bone and soft tissue tumor solid tumors. The culture medium used is the primary cell culture medium of bone and soft tissue tumor solid tumors in Example 1. Taking a six-well plate as an example, the cells are plated at a density of 10 ⁶ cells per well and cultured in a cell culture incubator at 37°C and 5% CO ₂ .

Example 7. Cryopreservation of primary cells of bone and soft tissue tumor solid tumors

After 2-3 passages of primary cells of solid tumors of bone and soft tissue tumors cultured in suspension, they can be cryopreserved:

1. Collect the cell clumps in the culture dish, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

2. Wash the cell pellet with sterile PBS solution, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

3. Resuspend the cell mass with cell digestion solution (see Example 1) and digest at 37° C. Observe the digestion of the cell mass under a microscope every 15 minutes until the cell mass is digested into single cells.

4. Use 10 times the volume of digestion stop solution (see Example 1) to terminate the dissociation reaction, collect the cell suspension, and count the cells.

5. Centrifuge at 800g for 10 minutes at room temperature and discard the supernatant.

6. Use cell freezing solution (see Example 1) to resuspend the cell pellet at a density of 10 ⁶ /mL, add 1 mL of cell suspension to each 2 mL cryotube, freeze overnight in a gradient cooling box, and then transfer to liquid nitrogen for long-term storage.

Example 8: Recovery of primary cells of bone and soft tissue solid tumors

Primary solid tumor cells of bone and soft tissue tumors stored in liquid nitrogen can be revived:

1. Prepare 37℃ sterile water five minutes in advance.

2. Take out the cryovial from liquid nitrogen and thaw the cells quickly in sterile water at 37°C.

3. Centrifuge at 800g for 10 minutes at room temperature and discard the supernatant.

4. Resuspend the cell pellet with the bone and soft tissue tumor solid tumor primary cell culture medium (see Example 1), use a low adsorption surface to culture the bone and soft tissue tumor solid tumor primary cells, and resuscitate each tube of cells in a 3.5 cm culture dish and culture them in a cell culture incubator at 37° C. and 5% CO ₂ .

Example 9: HE staining and identification of primary cells of bone and soft tissue tumor solid tumors

Description of reagents and consumables used in the following examples:

HE staining kit (Beijing Solebow Biotechnology Co., Ltd., #G1120);

Cationic anti-shedding slides (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.);

Xylene, methanol, and acetone (Beijing Chemical Reagent Company, analytical grade);

Neutral resin glue (Beijing Yili Fine Chemicals Co., Ltd.).

1. Prepare a cell suspension with a concentration of 10 ⁴ /mL by suspending cells, drop 10 μL on a cationic anti-shedding glass slide, and let it dry naturally.

2. Carefully add 50 μL of methanol/acetone mixture (volume ratio 1:1) pre-cooled at 4°C on the air-dried cells, and then place the slide in a 4°C refrigerator for 10 minutes.

3. Take out the slide with fixed cells and let it dry naturally at room temperature.

4. Wash the slides twice with 200 μL PBS.

5. When the water on the slide is slightly dry, add 100μL of hematoxylin staining solution and stain for 1 minute.

6. Aspirate the hematoxylin stain and wash the slides three times with 200 μL of tap water.

7. Add 100 μL differentiation solution and differentiate for 1 min.

8. Aspirate the differentiation solution and wash the slides twice with tap water and once with distilled water.

9. Absorb the moisture on the surface of the slide and add 200 μL of eosin staining solution for 40 seconds.

10. Aspirate the eosin stain and rinse and dehydrate with 75%, 80%, 90%, and 100% ethanol for 20s, 20s, 40s, and 40s respectively.

11. After the ethanol has dried, add 50 μL of xylene to permeabilize the cells.

12. After xylene is completely dry, add a drop of neutral resin glue, seal the slide with a coverslip, observe under a microscope and take pictures.

Figure 3 shows the HE staining effect of primary tumor cells of bone and soft tissue tumor solid tumors obtained by in vitro culture. It can be seen that these cells generally have tumor cell characteristics such as high nuclear-cytoplasmic ratio, dark nuclear staining, intranuclear chromatin condensation, multiple nuclei, and uneven cell size.

Example 10: Immunofluorescence staining of primary cells of bone and soft tissue tumor solid tumors

The reagents used in the following examples are described:

Paraformaldehyde (Beijing Chemical Reagent Company, analytical grade) was prepared by dissolving paraformaldehyde powder in ultrapure water to prepare a 4% (4 g/100 mL) paraformaldehyde solution;

Methanol and dimethyl sulfoxide (Beijing Chemical Reagent Company, analytical grade);

Hydrogen peroxide (Beijing Chemical Reagent Company, 35%);

Methanol, dimethyl sulfoxide, and 35% hydrogen peroxide were mixed in a ratio of 4:4:1 (volume ratio) to prepare Dan's rinse solution;

Bovine serum albumin (Sigma, #A1933) was dissolved in PBS to prepare a 3% (3 g/100 mL) BSA solution;

Primary antibody for immunofluorescence (Abcam, #ab92547);

immunofluorescence secondary antibody (CST, #4408);

Hoechst stain (Beijing Solebow Biotechnology Co., Ltd., #C0021);

Immunofluorescence staining of primary cell aggregates of solid tumors of bone and soft tissue tumors was performed as follows, and the primary antibody was an antibody against Vimentin.

1. Collect the cell clumps in the culture dish, wash them with PBS, resuspend the cell pellet with 4% paraformaldehyde, and fix them at 4℃ overnight.

2. Centrifuge at 800 g and discard the supernatant. Resuspend the cell pellet with pre-cooled methanol solution and place on ice for 1 hour.

3. Centrifuge at 800 g and discard the supernatant. Resuspend the cell pellet in Dan's washing solution and leave it at room temperature for 2 hours.

4. Centrifuge at 800 g and discard the supernatant. Wash the cells with 75%, 50%, and 25% (volume percentage) methanol solutions diluted with PBS, respectively, for 10 minutes each time.

5. Centrifuge at 800 g and discard the supernatant. Suspend the cell pellet with 3% BSA solution and block at room temperature for 2 hours.

6. Dilute the primary antibody with 3% BSA solution at a ratio of 1:500, resuspend the cell pellet with antibody diluent (3% BSA solution), and incubate the primary antibody at 4°C overnight.

7. Centrifuge at 800 g and discard the supernatant. Wash the cell pellet with PBS solution 5 times, 20 minutes each time.

8. Dilute the secondary antibody with 3% BSA solution at a ratio of 1:2000, and resuspend the cell pellet with antibody diluent (3% BSA solution) and incubate the secondary antibody at room temperature for 2 hours.

9. Centrifuge at 800 g and discard the supernatant. Wash the cell pellet with PBS solution 5 times, 20 minutes each time.

10. Add 100× Hoechst dye solution at a volume ratio of 1/100 and stain at room temperature for 20 minutes.

11. Wash the cell pellet twice with PBS solution, 10 minutes each time. Observe the staining of the cell aggregates using a laser confocal microscope.

Figure 4 shows the effect of immunofluorescence staining of primary tumor cell clusters of bone and soft tissue sarcoma cultured in vitro. It can be seen that the cells that make up the cell clusters are all Vimentin positive, which is consistent with the patient's pathological results, confirming that the method can culture tumor cells with higher purity.

Example 12: In vitro culture of primary tumor cells of different types of bone and soft tissue tumors

The operation method and process of primary culture of all samples in this example are completely consistent (refer to the above description), and only the pathological types of the samples are different. The conditions of each sample tested are shown in Table 22.

Table 21 In vitro culture of primary tumor cells of bone and soft tissue sarcomas of various pathological types

It can be seen that this method can achieve a very high success rate for in vitro culture of primary tumor cells in various types of bone and soft tissue sarcoma solid tumor samples.

Example 12: Cultivation of primary tumor cells of bone and soft tissue solid tumors using CYTOP-modified cell culture consumables

The operation methods and procedures for primary culture of all samples in this example are completely consistent (refer to the above description), the modification methods of CYTOP are completely consistent, and only the materials of cell culture consumables are different (Table 23).

The CYTOP modification method is as follows: first, the cell culture container is etched with pure oxygen, the etching condition is 20W power, and the etching time is 3 minutes. Then, an appropriate amount (taking a 96-well plate as an example, 20 μL per well, the appropriate amount means completely covering the bottom of the culture dish) of 1% CYTOP solution is used to cover the surface of the culture dish or culture plate, and the CYTOP solution can be used after it is completely dried.

Table 22 Effects of CYTOP-modified consumables on primary tumor cell culture of bone and soft tissue tumor solid tumors

Note: Polystyrene (abbreviated as PS).

It can be seen from Table 22 that: It can be seen that the success rate of sample culture can be greatly improved after CYTOP modification.

Example 13: Microplate chip processing

In this embodiment, the microplate chip used for culturing primary cells of bone and soft tissue tumor solid tumors of the present invention is obtained by injection molding using PMMA material (or materials such as PS, PC, COC, COP, LAS, etc.). The chip can be used for primary cell culture of bone and soft tissue tumor solid tumors and in vitro drug sensitivity detection experiments. The design drawing of the microplate chip is shown in Figure 5.

In the actual application process, PMMA material (or PS, PC, COC, COP, LAS and other materials) is used to prepare the structure of the microplate chip shown in the design drawing of Figure 5, and then the surface is CYTOP modified by the above-mentioned CYTOP modification method (see Example 11) to obtain the microplate chip that can be used for primary cell culture of solid tumors of bone and soft tissue tumors.

Claims (20)

1. A method for culturing primary cells of solid tumors of bone and soft tissue tumors, comprising the following steps: suspending and culturing primary cells of solid tumors of bone and soft tissue tumors using a culture medium for primary cells of solid tumors of bone and soft tissue tumors; The bone and soft tissue tumor solid tumor primary cell culture medium is composed of three antibacterial and antifungal agents, HEPES, GlutaMax, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, human recombinant protein IGF-I, human recombinant protein BDNF, ITS-X, N-acetyl-L-cysteine, N-2 Supplement, Y-27632 and Advanced DMEM/F12 culture medium; wherein the final concentration of penicillin in the three antibacterial and antifungal agents is 100-200U/mL; the final concentration of streptomycin in the three antibacterial and antifungal agents is 100-200µg/mL; the final concentration of amphotericin in the three antibacterial and antifungal agents is 100-200µg/mL. The final concentration of B is 250ng/mL; the final concentration of HEPES is 8-12mM; the final concentration of GlutaMax is 0.8-1.2% by volume; the final concentration of the human recombinant protein bFGF is 20ng/mL; the final concentration of the human recombinant protein HGF is 20 ng/mL; the final concentration of the human recombinant protein MSP is 20ng/mL; the final concentration of the human recombinant protein IGF-I is 50ng/mL; the final concentration of the human recombinant protein BDNF is 100ng/mL; the final concentration of ITS-X is 1% by volume; the final concentration of N-acetyl-L-cysteine is 1mM; the final concentration of N-2 Supplement is 1% by volume; the final concentration of Y-27632 is 10μM; the remainder is Advanced DMEM/F12 culture medium. 2. The method according to claim 1, characterized in that: the primary cells of bone and soft tissue tumor solid tumors are obtained by dissociating bone and soft tissue tumor solid tumor tissues with a sample dissociation solution; The sample dissociation solution consists of collagenase I, collagenase II, collagenase III, collagenase IV and PBS; wherein the final concentration of the collagenase I in the sample dissociation solution is 150-250 U/mL; the final concentration of the collagenase II in the sample dissociation solution is 150-250 U/mL; the final concentration of the collagenase III in the sample dissociation solution is 250-350 U/mL; the final concentration of the collagenase IV in the sample dissociation solution is 150-250 U/mL; and the remainder is PBS. 3. The method according to claim 2 is characterized in that the bone and soft tissue tumor solid tumor tissue is dissociated with the sample dissociation solution according to a method comprising the following steps: the minced bone and soft tissue tumor solid tumor tissue is treated with the sample dissociation solution preheated at 37°C at a dosage of 0.1-0.3 mL of the sample dissociation solution per mg of tissue, and the sample dissociation is performed at 37°C for 15 minutes to 3 hours. 4. The method according to claim 1, characterized in that: in the method, the primary cells of bone and soft tissue tumor solid tumor are suspended and cultured with the primary cell culture medium of bone and soft tissue tumor solid tumor according to the method comprising the following steps: using a cell culture container M, suspending and culturing the primary cells of bone and soft tissue tumor solid tumor with the primary cell culture medium of bone and soft tissue tumor solid tumor, culturing at 37°C and 5% CO2, and replacing the culture medium every 2-4 days; The cell culture container M is any of the following: (I) a cell culture container made of polystyrene, a cell culture container made of polycarbonate, a cell culture container made of polymethyl methacrylate, a cell culture container made of COC resin, a cell culture container made of cycloolefin polymer or a cell culture container with a low adsorption surface; (II) a cell culture container obtained by modifying the cell culture container in (I) with CYTOP. 5 . The method according to claim 4 , wherein the cell culture container is a cell culture dish, a cell culture well plate or a microplate chip for cell culture. 6. The method according to claim 4, characterized in that: in said (II), the cell culture container in said (I) is subjected to CYTOP modification according to a method comprising the following steps: the cell culture container in said (I) is subjected to pure oxygen etching, the etching conditions being a power of 20 W and an etching time of 3 minutes; then the surface of the cell culture container is covered with a 1% CYTOP solution, and the 1% CYTOP solution is dried to complete the CYTOP modification. 7. The method according to claim 6, characterized in that the composition of the 1% CYTOP solution is as follows: every 100 mL of the 1% CYTOP solution contains 1 mL of CYTOP, and the remainder is fluorinated oil. 8. The method according to any one of claims 1 to 7, characterized in that: the method further comprises the following steps of pre-dissociation treatment of the bone and soft tissue tumor solid tumor tissue: washing the surface of the bone and soft tissue tumor solid tumor tissue sample with 70-75% by volume ethanol; washing the bone and soft tissue tumor solid tumor tissue sample with a sample washing solution and a sterile PBS solution in sequence; The sample cleaning solution is composed of dual-antibody P/S and PBS; wherein the final concentration of penicillin in the dual-antibody P/S is 100-200U/mL; the final concentration of streptomycin in the dual-antibody P/S is 100-200µg/mL; and the remainder is PBS. 9. The method according to claim 8, characterized in that: the bone and soft tissue tumor solid tumor tissue samples subjected to the pre-dissociation treatment are isolated from the body within 2 hours and are kept in a sample preservation solution before the pre-dissociation treatment; The sample preservation solution consists of fetal bovine serum, double-antibody P/S, HEPES and HBSS; wherein the final concentration of the fetal bovine serum is 1-5% by volume; the final concentration of penicillin in the double-antibody P/S is 100-200U/mL; the final concentration of streptomycin in the double-antibody P/S is 100-200µg/mL; the final concentration of HEPES is 8-12 mM; and the remainder is HBSS. 10. The method according to any one of claims 1 to 7, characterized in that: in the method, after the bone and soft tissue tumor solid tumor tissue is dissociated with the sample dissociation solution, the method further comprises the following steps: terminating the dissociation reaction with a digestion stop solution and collecting the cell suspension; filtering the cell suspension to remove tissue debris and adherent cells; resuspending the cells with sterile PBS after centrifugation; centrifuging again, and then resuspending the cell precipitate with the bone and soft tissue tumor solid tumor primary cell culture medium; The digestion stop solution is composed of fetal bovine serum, double-antibody P/S and DMEM culture medium; wherein the final concentration of the fetal bovine serum is 8-12% by volume; the final concentration of penicillin in the double-antibody P/S is 100-200U/mL; the final concentration of streptomycin in the double-antibody P/S is 100-200µg/mL; and the remainder is DMEM culture medium. 11. The method according to any one of claims 1 to 7, characterized in that: in the process of culturing the bone and soft tissue tumor solid tumor primary cells using the bone and soft tissue tumor solid tumor primary cell culture medium, it also includes the following step: when the bone and soft tissue tumor solid tumor primary cells form a mass with a diameter of 80-120 μm, the bone and soft tissue tumor solid tumor primary cells are passaged. 12. The method according to claim 11, characterized in that: the cell digestion solution used in the subculturing is composed of: 4-6 mL Accutase, EDTA with a final concentration of 5 mM, 1.5-2.5 mL TripLE Express, and the balance is PBS per 10 mL of the cell digestion solution. 13. The method according to claim 11, characterized in that the digestion stop solution used in the subculturing is the digestion stop solution described in claim 10. 14. The method according to claim 11, characterized in that: the method further comprises the step of freezing and/or reviving the primary cells of the bone and soft tissue tumor solid tumors after 2-3 passages and amplifications. 15. The method according to claim 14, characterized in that: the cell freezing solution used for the freezing is composed of Advanced DMEM/F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium, the DMSO and the 1% methylcellulose solution is 20:2:(0.8-1.2); and the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1 g/100 ml. 16. The method according to any one of claims 1-7, characterized in that the bone and soft tissue tumors are osteosarcoma, synovial sarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing sarcoma, liposarcoma, alveolar soft tissue sarcoma, clear cell sarcoma or fibroma. 17. A set of reagents for culturing primary cells of solid tumors of bone and soft tissue tumors, comprising the culture medium for primary cells of solid tumors of bone and soft tissue tumors as claimed in claim 1 and at least one of the following reagents: the sample dissociation solution as claimed in claim 2, the sample preservation solution as claimed in claim 9, the cell digestion solution as claimed in claim 12, the sample cleaning solution as claimed in claim 8, the digestion termination solution as claimed in claim 10, the cell freezing solution as claimed in claim 15, and the 1% CYTOP solution as claimed in claim 7. 18. The reagent set according to claim 17, characterized in that the bone and soft tissue tumors are osteosarcoma, synovial sarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing sarcoma, liposarcoma, alveolar soft tissue sarcoma, clear cell sarcoma or fibroma. 19. Use of the reagent set according to claim 17 in culturing primary cells of solid tumors of bone and soft tissue tumors. 20. The use according to claim 19, characterized in that the bone and soft tissue tumors are osteosarcoma, synovial sarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing sarcoma, liposarcoma, alveolar soft tissue sarcoma, clear cell sarcoma or fibroma.

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