CN112760282B

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

Info

Publication number: CN112760282B
Application number: CN201911065585.4A
Authority: CN
Inventors: 尹申意; 张函槊
Original assignee: Genex Health Co Ltd
Current assignee: Beijing Cornerstone Medical Laboratory Co ltd
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2023-03-24
Anticipated expiration: 2039-11-04
Also published as: CN112760282A

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

Method for culturing bone and soft tissue tumor solid tumor primary cells

Technical Field

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

Background

Bone and soft tissue tumors are diseases seriously harming human health and life, the incidence rate is gradually increased in recent years, primary malignant bone tumors are often found in teenagers and middle-aged people, and the primary malignant bone tumors are commonly osteosarcoma, ewing sarcoma, chondrosarcoma, malignant fibrous histiocytoma, chordoma and the like. Common soft tissue malignancies are synovial sarcoma, fibrosarcoma, liposarcoma, rhabdomyosarcoma, and the like. The incidence rate of bone and soft tissue tumors accounts for about 1 percent of the incidence rate of malignant tumors, but the types of the bone and soft tissue tumors are more, so that the non-standard diagnosis and treatment are common, and the current clinical guidelines recommend the treatment mode of soft tissue sarcoma by clinical trials.

Although research and medical institutions in all countries of the world have a great deal of investment in research on the etiology and development process of bone and soft tissue tumors, human beings still have little knowledge of the disease. Bone and soft tissue tumors are a complex disease, the occurrence and development of which are dynamic processes, and the interaction of a plurality of signal molecules is involved, so that a complex molecular regulation network is formed and is influenced by external environmental factors. The etiology, occurrence and development process of bone and soft tissue tumor have strong individual difference, which cannot be determined in a whole. Therefore, the trend of performing individual accurate research by using the primary cell culture of the bone and soft tissue tumor as a model is the research field of the bone and soft tissue tumor and even the diagnosis and treatment field of the bone and soft tissue tumor.

The existing primary tumor cell culture technology mainly comprises 2D culture, 3D culture, reprogramming culture and the like, and the methods all face the problems of extremely long culture period, low culture success rate, difficult removal of mixed cells and the like in different degrees.

Disclosure of Invention

In order to effectively solve the technical problems, the invention provides a novel primary cell culture technology of solid tumor of bone and soft tissue tumor and a matched reagent, and the core of the technology is as follows: (1) The mild cell dissociation reagent is used for treating the bone and soft tissue tumor solid tumor tissues, so that the activity of tumor cells in the tissues is ensured to the greatest extent; (2) Preparing a special serum-free culture medium, and culturing the primary solid tumor cells of the bone and soft tissue tumors in vitro by using a suspension culture system, so that the interference of normal cells is eliminated to the maximum extent while the normal amplification of the tumor cells is ensured.

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

The method for culturing the primary cells of the solid tumors of the bone and the soft tissue as claimed by the invention can comprise the following steps:

performing suspension culture on the primary cells of the solid tumors of the bone and the soft tissue by using a primary cell culture medium of the solid tumors of the bone and the soft tissue;

the primary cell culture medium for the bone and soft tissue tumor solid tumors consists of antibacterial antifungal agents, namely three-antibody (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-acetyl-L-cysteine), N-2 Supplement, Y-27632 and Advanced DMEM/F12 culture medium. Wherein the final concentration of penicillin in the antimicrobial antifungal agent triantion is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the three-component antibacterial antifungal agent is 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the antimicrobial antifungal agent triantion is 250-250ng/mL (e.g., 250 ng/mL); the final concentration of HEPES is 8-12mM (e.g., 10 mM); the final concentration of GlutaMax is 0.8-1.2% (e.g., 1%,% represents volume percent); the final concentration of the human recombinant protein bFGF is 10-50ng/mL; the final concentration of the human recombinant protein HGF is 5-25ng/mL; the final concentration of the human recombinant protein MSP is 5-25ng/mL; the final concentration of the human recombinant protein IGF-I is 50-100ng/mL; the final concentration of the human recombinant protein BDNF is 50-200ng/mL; the final concentration of ITS-X is 1% (volume percent); the final concentration of the N-acetyl-L-cysteine (N-acetyl-L-cysteine) is 0.5-2mM; the final concentration of the N-2 Supplement is 1 percent (volume percentage); the final concentration of the Y-27632 is 5-20 mu M; the balance is Advanced DMEM/F12 medium.

Further, the composition of the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is as follows: each ml contains 10000 units of penicillin (base), 10000. Mu.g of streptomycin (base) and 25. Mu.g of amphotericin B. The antimicrobial antifungal agent triantibody (penicillin-streptomycin-amphotericin B) is "antibacterial-antibacterial, 100X" (e.g., gibco #15240062, or other products of the same composition). The "Antibiotic-Antibiotic, 100X" contained 10000 units of penicillin (base), 10000. Mu.g of streptomycin (base) and 25. Mu.g of amphotericin B per ml, using penicillin G (sodium salt), streptomycin sulfate and amphotericin B in the form of 0.85% saline as the active ingredients

An antifungal agent. The GlutaMAX is GlutaMAX ^TM Supplement "(e.g., gibco #35050061, or other products of the same composition). The "GlutaMAX ^TM Supplement "was L-allyl-L-glutamine as a substitute for L-glutamine at a concentration of 200nM and a solvent of 0.85% NaCl solution. The N-2 Supplement is "N-2 Supplement (100X)" (e.g., gibco #17502001, or other products having the same composition as the N-2 Supplement). The "N-2 Supplement (100X)" contains Human total Transferrin (Human Transferrin (Holo)) at a final concentration of 1mM, recombinant Insulin whole Chain (Insulin Recombinant Full Chain) at 500mg/L, progesterone (Progesterone) at 0.63mg/L, putrescine (Putrescine) at 10mM, and selenious acid at 0.52mg/LAcid salt (Selenite). The ITS-X solvent is EBSS solution (Earle's balanced salt solution), and the solute and the concentration are as follows: 1g/L of insulin; 0.55g/L of transferrin; 0.00067g/L sodium selenite; ethanolamine 0.2g/L. The GlutaMAX is a high-grade cell culture additive and can directly replace L-glutamine in a cell culture medium. The GlutaMAX is GlutaMAX ^TM Supplement "(e.g., gibco #35050061, or other products of the same composition). Y-27632 is "Y-27632dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor with Ki of 220nM and 300nM, respectively)" (e.g. MCE #129830-38-2, or other products of the same composition).

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

The bone and soft tissue tumor solid tumor primary cells can be obtained by dissociating bone and soft tissue tumor solid tumor tissues by using sample dissociation liquid.

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

Wherein the unit U of collagenase (said collagenase I, said collagenase II, said collagenase III or said collagenase IV) is defined by the enzymatic activity of a protease: 1 μmol of L-leucine can be released by treating collagenase (said collagenase I, said collagenase II, said collagenase III or said collagenase IV) with 1U of protease at 37 ℃ and pH 7.5 for 5 hours.

In a specific embodiment of the invention, said collagenase I is branded under the brand name Gibco #17100-017; the brand goods number of the collagenase II is Gibco #17101-015; the brand code of collagenase III is Solarbio # C8490; the brand goods number of the collagenase IV is Gibco #17104-019; the PBS was sold under the brand name Gibco #21-040-CVR.

Further, the bone and the solid tumor tissue of the soft tissue tumor can be dissociated by the sample dissociation fluid according to the method comprising the following steps: shearing the bone and soft tissue into solid tumor tissue (such as 0.8-1.2 mm) according to the dosage of 0.1-0.3mL (such as 0.1 mL) of the sample dissociation solution per mg of tissue ³ Small pieces of (a) were treated with the sample dissociation solution preheated at 37 ℃ in advance, and sample dissociation was performed at 37 ℃ for 15 minutes to 3 hours. The dissociation of the samples was observed under the microscope every 15 minutes until a large number of single cells were observed.

In the method, the bone and soft tissue tumor solid tumor primary cells can be cultured in suspension with the bone and soft tissue tumor solid tumor primary cell culture medium according to a method comprising the following steps: culturing in suspension said bone and soft tissue tumor solid tumor primary cells using said bone and soft tissue tumor solid tumor primary cell culture medium using a cell culture vessel M at 37 ℃,5% ₂ Culturing is carried out under conditions in which the medium is changed every 2-4 days (e.g., 3 days) until the cells form a mass of 80-120 μm (e.g., 100 μm) in diameter.

Wherein the initial seeding density may be 10 ⁵ Per cm ² Bottom area of the container, e.g. six-well plate, 10 per well ⁶ Density of individual cells was plated.

Wherein, the cell culture container M can be any one 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 cyclic olefin polymer, or a cell culture container with a low adsorption surface; (II) the cell culture vessel of (I) is subjected to CYTOP modification.

Further, the cell culture vessel is a cell culture dish, a cell culture well plate, a micro well plate chip for cell culture (such as the micro well plate chip shown in fig. 5 in example 13), or the like.

In the (II), the cell culture vessel in the (I) may be subjected to CYTOP modification according to a method comprising the steps of: carrying out pure oxygen etching on the cell culture container in the step (I), wherein the etching condition is that the power is 20W, and the etching time is 3 minutes; then covering the cell culture vessel surface with 1-percent CYTOP solution, air drying the 1-percent CYTOP solution to complete the CYTOP modification.

Wherein the 1% CYTOP solution has the following composition: each 100mL of the 1-cent CYTOP solution contained 1mL of CYTOP, the balance being fluoro oil.

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

In a particular embodiment of the invention, the CYTOP brand code is specifically asahi glass # CTL-809M; the brand goods number of the fluorine oil is specifically 3M # FC40.

Further, the method may further comprise the step of performing dissociation pretreatment on the bone and the soft tissue tumor solid tumor tissue as follows: cleaning the surface of the solid tumor tissue sample of the bone and soft tissue tumor with ethanol with the volume percentage of 70-75 percent (such as 75 percent) for 10-30 seconds; washing the bone and soft tissue tumor solid tumor tissue sample with a sample washing solution 10-20 times (e.g., 10 times), and washing the bone and soft tissue tumor solid tumor tissue sample with a sterile PBS solution 5-10 times (e.g., 5 times); then removing impurities, connective tissues, fat tissues, necrotic tissues and other components influencing primary cell culture in the solid tumor tissue samples of the bone and soft tissue tumors.

Wherein the sample washing solution consists of double-antibody P/S (penicillin-streptomycin) and PBS; wherein 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 100-200 mug/mL (such as 100 mug/mL); the balance being PBS.

In a specific embodiment of the invention, the brand of the dual-antibody P/S is Gibco #15140122; the PBS was branded under Gibco #21-040-CVR.

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

Furthermore, the bone and soft tissue tumor solid tumor tissue sample subjected to the pre-dissociation treatment needs to be isolated within 2 hours and stored in a sample storage solution before the pre-dissociation treatment.

Wherein the sample preservation solution consists of fetal calf serum, double-antibody P/S (penicillin-streptomycin), HEPES and HBSS (Hank' S balanced salt solution); wherein the final concentration of fetal calf serum is 1-5% (such as 2%,% represents volume percentage content); the final concentration of penicillin in the double-resistant P/S is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL (such as 100 mug/mL); the final concentration of HEPES is 8-12mM (e.g., 10 mM); the balance being HBSS.

Further, in the method, after the dissociation treatment of the bone and the soft tissue tumor solid tumor tissue by the sample dissociation liquid, the method further comprises the following steps: terminating the dissociation reaction with 8-15 (e.g., 10) times the volume of the digestion stop solution, and collecting the cell suspension; filtering the cell suspension with a 100 μm or 40 μm sterile cell strainer to remove tissue debris and adherent cells; centrifuging 800-1000g (such as 800 g) at room temperature for 10-15 min (such as 10 min), and discarding the supernatant; then resuspend the cells in 3-5mL (e.g., 5 mL) sterile PBS; centrifuging at 800-1000g (such as 800 g) at room temperature for 10-15 min (such as 10 min), and discarding the supernatant; then, the primary cell culture medium of the bone and soft tissue tumor solid tumor is used for resuspending cell pellets, and the cell state is observed under a microscope for cell counting.

Wherein the digestion stop solution consists of fetal calf serum, double-antibody P/S (penicillin-streptomycin) and a DMEM medium; wherein the final concentration of fetal calf serum is 8-12% (such as 10%,% represents volume percentage content); the final concentration of penicillin in the double-resistant P/S is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL (such as 100 mug/mL); the balance is DMEM medium.

Further, in the method, the following steps may be further included: and (3) when the primary cells of the solid tumor of the bone and the soft tissue tumor form a mass with the diameter of 80-120 mu m (such as 100 mu m), carrying out passage on the primary cells of the solid tumor of the bone and the soft tissue tumor.

Wherein, the cell digestive juice adopted when the passage is carried out consists of the following components: each 10mL of the cell digest contained 4-6mL (e.g., 5 mL) of Accutase, a final concentration of 5mM EDTA (i.e., 10. Mu.L of 0.5M EDTA), 1.5-2.5mL (e.g., 2 mL) of TrypLE Express, and the balance PBS.

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

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

Further, the digestion temperature used for the passage was 37 ℃.

Further, the digestion stop solution used in the passage is the digestion stop solution described above.

More specifically, the step of performing said passaging is carried out: collecting cell mass to be passaged, washing the cell mass with sterile PBS solution after centrifugation, then centrifuging, then suspending the cell mass with the cell digestive juice again, digesting at 37 ℃ until the cell mass is digested into single cells, stopping digestion reaction with the digestion stop solution (the dosage can be 5-10 times, such as 10 times of volume), and collecting cell suspension; resuspending the cell pellet with the bone and soft tissue tumor solid tumor primary cell culture medium after centrifugation, counting, and then suspension culturing the cells using a culture vessel with a low adsorption surface (initial seeding density can be 10) ⁵ Per cm ² Bottom area of the container, e.g. six-well plate, 10 per well ⁶ Density plating of individual cells), culture conditions 37 ℃,5% CO ₂ . All the centrifugation in the above passaging step may be specifically carried out at 800 to 1000g (e.g., 800 g) at room temperature for 10 to 20 minutes (e.g., 10 minutes).

Further, the method can also comprise the step of freezing and/or reviving the primary bone and soft tissue tumor solid tumor cells after the expansion of 2-3 passages.

Wherein, the cell freezing solution adopted in the freezing process consists of an Advanced DMEM/F12 culture medium, DMSO and a 1% methyl cellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium, the DMSO and the 1% methylcellulose solution is 20 (0.8-1.2), such as 20; the 1% methylcellulose solution is an aqueous solution of methylcellulose having a concentration of 1g/100 ml.

In a specific embodiment of the invention, the brand of Advanced DMEM/F12 medium is Gibco #12634010; the brand code of the DMSO is Sigma # D2438; the brand code of the methylcellulose is Sigma # M7027.

Further, the specific steps of the cryopreservation are as follows: collecting cell mass to be frozen, centrifuging, washing the cell mass with sterile PBS solution, centrifuging, suspending the cell mass with the cell digestive juice, digesting at 37 deg.C until the cell mass is digested into single cell, terminating the digestion reaction with the digestion terminating solution (the dosage can be 5-10 times, such as 10 times of volume), and collecting cell suspension; centrifuging, and freezing the cells at 0.5-2 × 10 ⁶ /mL (e.g., 10) ⁶ mL), and transferring the cell sediment to liquid nitrogen for long-term storage after the cell sediment is frozen and stored overnight by a gradient cooling box. All the centrifugation in the above freezing step may be specifically 800-1000g (e.g., 800 g) at room temperature for 10-20 minutes (e.g., 10 minutes).

Further, the specific steps of performing the resuscitation are: taking out the freezing tube containing the cells to be rescued from the liquid nitrogen, and rapidly thawing the cells in sterile water at 37-39 deg.C (such as 37 deg.C); after centrifugation (e.g., 800-1000g, e.g., 800g for 5-10 minutes, e.g., 10 minutes at room temperature), the cell pellet is resuspended in a primary cell culture medium of the bone and soft tissue tumor solid tumor, and the cells are then resuspended in a culture vessel having a low adsorption surface (initial seeding density can be 10) ⁵ Per cm ² Bottom area of container), per tube of cells (10) ⁶ Individual) revived to 3.5cm petri dish) at 37 ℃ under culture conditions, 5% CO ₂ 。

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

The kit for culturing the primary cells of the solid tumors of the bone and the soft tissue provided by the invention specifically comprises the culture medium for the primary cells of the solid tumors of the bone and the soft tissue and at least one of the following reagents: the sample dissociation solution, the sample preservation solution, the cell digestion solution, the sample washing solution, the digestion stop solution, the cell cryopreservation solution and the 1% CYTOP solution described above.

The sample preservation solution can be used for temporarily preserving a sample after the sample is separated, and can maintain the activity of cells in the sample in a short time after the sample is separated. The sample preservation solution can be preserved for 1 month at 4 ℃ after being prepared.

The sample washing solution can be used for washing and disinfecting a sample. The sample cleaning solution needs to be ready for use.

The sample dissociation liquid can be used for dissociation of a sample, and can dissociate bones and soft tissue tumor solid tumor primary cells in the sample from tissues. The sample dissociation solution should be prepared as it is, wherein collagenase I, collagenase II, collagenase III and collagenase IV can be stored in a stock solution (mother solution) at-20 deg.C for a long time, specifically 10 times of the stock solution (mother solution). The 10 × collagenase I stock consists of the collagenase I and PBS; wherein the final concentration of collagenase I is 2000U/mL; a 10 × collagenase II stock solution consists of the collagenase II and PBS; wherein the final concentration of collagenase II is 2000U/mL; a 10 × collagenase III stock consists of the collagenase III and PBS; wherein the final concentration of collagenase III is 2900U/mL; the 10 × collagenase IV stock consists of the collagenase IV and PBS; wherein the final concentration of collagenase IV is 2000U/mL; the balance being PBS. The enzyme activities of collagenase I, collagenase II, collagenase III and collagenase IV are defined above.

The cell digestive juice can be used for digesting and passaging cell masses, and can digest bone and soft tissue tumor solid tumor mass into single cells. The cell digestive juice is required to be prepared in situ.

The digestion stop solution can be used for stopping the dissociation of the sample or the digestion process of the cells. The prepared digestion stop solution can be stored for one month at 4 ℃.

The bone and soft tissue tumor solid tumor primary cell culture medium can be used for culturing bone and soft tissue tumor solid tumor primary cells. The primary cell culture medium of the solid tumor of the bone and soft tissue tumor needs to be sterilized by filtration by a 0.22 mu M needle filter (Millipore SLGP033 RS) after being prepared, and can be stored for two weeks at 4 ℃. The human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein MSP, the human recombinant protein IGF-I and the human recombinant protein BDNF can be stored in a stock solution (mother solution) form at-80 ℃ for a long time, and particularly can be stored in a 1000-fold stock solution (mother solution). N-acetyl-L-cysteine and Y-27632 can be stored in a stock solution (mother solution) form at-20 ℃ for a long time, and specifically can be 1000 times of the stock solution (mother solution).

The stock solution of 1000 Xhuman recombinant protein bFGF consists of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. The 1000 Xhuman recombinant protein HGF stock solution consists of human recombinant proteins HGF, BSA and PBS, wherein the final concentration of the human recombinant proteins HGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. The stock solution of 1000 Xhuman recombinant protein MSP consists of human recombinant protein MSP, BSA and PBS, wherein the final concentration of the human recombinant protein MSP is 20 μ g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. The 1000 × stock solution of human recombinant protein IGF-I consists of human recombinant protein IGF-I, BSA and PBS, wherein the final concentration of the human recombinant protein IGF-I is 100 μ g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. The 1000 Xhuman recombinant protein BDNF stock solution consists of human recombinant proteins BDNF, BSA and PBS, wherein the final concentration of the human recombinant protein BDNF is 100 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. In the five 1000-fold stock solutions, the BSA can be present (ready for formulation) in the form of 100-fold stock solution (mother liquor), and specifically consists of BSA and PBS, wherein the final concentration of BSA (Sigma # A1933) is 0.1g/mL, and the balance is PBS. In addition, the 1000 XN-acetyl-L-cysteine stock solution consists of N-acetyl-L-cysteine and ultrapure water, wherein the concentration of the N-acetyl-L-cysteine is 0.5M, and the balance is the ultrapure water. 1000 XY-27632 consists of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10mM, and the balance is ultrapure water.

The cell freezing medium needs to be prepared at present. Wherein the 1% methylcellulose solution can be stored for a long period of time at 4 ℃.

In a third aspect, the invention claims the use of a kit as described hereinbefore for culturing primary cells of solid tumors of bone and soft tissue tumors.

The primary cell culture medium for the solid tumor of the bone and soft tissue tumor and the application thereof in culturing the primary cell of the solid tumor of the bone and soft tissue tumor also belong to the protection scope of the invention.

In the above aspects, the bone and soft tissue tumor solid tumor may be a primary bone and soft tissue tumor solid tumor. The solid tumor of the bone and soft tissue tumor can be osteosarcoma, synovial sarcoma, rhabdomyosarcoma, leiomyosarcoma, ewing sarcoma, liposarcoma, acinar soft tissue sarcoma, hyaline cell sarcoma or fibroma.

In each of the above aspects, the bone and soft tissue tumor solid tumor primary cells are isolated from a surgical sample of a patient having a bone and soft tissue tumor solid tumor. Wherein, the bone and soft tissue tumor solid tumor tissue specimen obtained from the operation sample preferably has the weight of more than 20mg.

In the present invention, all of the above PBS's may be 1 XPBS, pH7.3-7.5. The concrete composition is as follows: the solvent is water, and the solute and the concentration are as follows: KH (Perkin Elmer) ₂ PO ₄ 144mg/L，NaCl 9000mg/L，Na ₂ HPO ₄ ·7H ₂ O 795mg/L。

The invention provides a method for extracting primary cells of cultured bones and soft tissue tumor solid tumors from fresh bones and soft tissue tumor solid tumors and a matched reagent, and the method has the following advantages:

1. the dosage of the tissue sample is less, and only about 20mg of bone and soft tissue tumor solid tumor operation sample is needed;

2. the culture period is short, and only 3-10 days are needed to obtain 10 ⁷ Bone and soft tissue tumor solid tumor primary tumor cells of an order of magnitude;

3. the culture stability is high, and the success rate of in vitro culture of qualified bone and soft tissue tumor solid tumor operation specimens by using the method is up to 70 percent;

4. the cell purity is high, the ratio of tumor cells in the primary cell culture of the bone and soft tissue tumor solid tumor obtained by the method can reach 70-95%, and the interference of mixed cells is less.

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, next generation sequencing, animal model construction, cell line construction and the like of various cell levels. It is expected that the culture method has wide application prospect in the research and clinical diagnosis and treatment fields of bone and soft tissue tumor solid tumors.

Drawings

FIG. 1 shows a single cell obtained from synovial sarcoma tissue after treatment. The scale is 100 μm,100 times magnification.

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

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

FIG. 4 is an immunofluorescence staining pattern of tumor cell masses obtained after primary culture of synovial sarcoma tissue. The scale is 100 μm,40 times magnification.

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

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of reagents for culturing bone and Soft tissue tumor solid tumor Primary cells

1. Sample preservation solution (100 mL)

The specific formulation of the specimen preservation solution (100 mL) is shown in table 1.

TABLE 1 sample preservation solution (100 mL)

After the preparation of the sample preservation solution is completed, the sample preservation solution is subpackaged by 15mL centrifuge tubes, and each tube is 5mL. Can be stored at 4 deg.C for 1 month after subpackaging.

2. Sample cleaning solution (100 mL)

The specific formulation of the sample rinse (100 mL) is shown in table 2.

TABLE 2 sample rinse (100 mL)

The sample cleaning solution needs to be prepared for use.

3. Sample dissociation liquid (10 mL)

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

TABLE 3 sample dissociation solution (10 mL)

Note: the sample dissociation liquid is prepared for use.

In table 3, the formulation of collagenase stocks is shown in tables 4-7.

TABLE 4 10 Xcollagenase I stock solution (100 mL)

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After preparing the 10 Xcollagenase I stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

TABLE 5 10 Xcollagenase II stock solution (100 mL)

After preparing the 10 Xcollagenase II stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

TABLE 6 10 collagenase III stock solution (100 mL)

After preparing the 10 Xcollagenase III stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

TABLE 7 collagenase IV stock solution (100 mL)

The 10 Xcollagenase IV stock solution was prepared and dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

In tables 4-7, the unit U of collagenase (said collagenase I, said collagenase II, said collagenase III or said collagenase IV) is defined by the enzyme activity of the protease: 1 μmol of L-leucine can be released by treating collagenase (said collagenase I, said collagenase II, said collagenase III or said collagenase IV) with 1U of protease at 37 ℃ and pH 7.5 for 5 hours.

4. Cell digestive juice (10 mL)

The specific formulation of the cell digest (10 mL) is shown in Table 8.

TABLE 8 cell digest (10 mL)

The cell digestive juice is prepared for use.

5. Digestive stop solution (100 mL)

The specific formulation of the digestion-stopping solution (100 mL) is shown in Table 9.

TABLE 9 digestive stop solution (100 mL)

The digestion stop solution can be stored for one month at 4 ℃ after being prepared.

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

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

TABLE 10 bone and Soft tissue tumors solid tumors Primary cell culture Medium (100 mL)

After the preparation of the primary cell culture medium for solid tumors of bone and soft tissue, the cells were sterilized by filtration using a 0.22 μ M syringe filter (Millipore SLGP033 RS) and stored at 4 ℃ for two weeks.

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

TABLE 11 100 XBSA solution (1 mL)

The 100 × BSA solution is ready for use.

TABLE 12 1000 Xhuman recombinant protein bFGF stock solution (2.5 mL)

After 1000 Xhuman recombinant protein bFGF stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with the volume of 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 13 1000 Xhuman recombinant protein HGF stock solution (5 mL)

1000 Xthe human recombinant protein HGF stock solution is prepared and then subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved for a long time at the temperature of minus 80 ℃.

TABLE 14 1000 Xhuman recombinant protein MSP stock solution (2.5 mL)

1000 Xthe human recombinant protein MSP stock solution is prepared and then subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved for a long time at the temperature of minus 80 ℃.

TABLE 15 1000 Xhuman recombinant protein IGF-I stock solutions (1 mL)

After 1000 Xhuman recombinant protein IGF-I stock solution is prepared, the stock solution is subpackaged by a 1.5mL sterile centrifuge tube, and the stock solution can be stored at the temperature of 80 ℃ below zero for a long time.

TABLE 16 1000 Xhuman recombinant protein BDNF stock solution (1 mL)

After 1000 Xhuman recombinant protein BDNF stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with the volume of 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 17 1000 XN-acetyl-L-cysteine stock solutions (5 mL)

The stock solution of 1000 XN-acetyl-L-cysteine is prepared and then is subpackaged by a sterile centrifuge tube with 0.5mL, and the stock solution can be preserved at the temperature of minus 20 ℃ for a long time.

TABLE 18 1000 XY-27632 stock solution (3.125 mL)

After the stock solution is prepared, 1000 XY-27632 is subpackaged by a sterile centrifuge tube of 0.5mL, and the stock solution can be preserved for a long time at the temperature of minus 20 ℃.

7. Cell cryopreservation liquid

The specific formulation of the cell culture medium is shown in Table 19.

TABLE 19 cell cryopreservation solution

The cell frozen stock solution is prepared for use.

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

TABLE 20 1% methylcellulose solution (10 mL)

The 1% methyl cellulose solution can be stored for a long time at 4 ℃ after being prepared.

8. 1% of CYTOP solution

TABLE 21 The CYTOP solution (100 mL)

1% of CYTOP solution, and can be stored at room temperature for a long period of time.

Example 2 obtaining of post-operative/biopsy puncture specimens of solid tumors of bone and Soft tissue

1. In cooperation with the Hospital, the cooperative development passed a formal medical ethical examination.

2. The attending physician selects patients to be grouped according to clinical indications specified by medical guidelines and selects appropriate samples for in vitro culture according to the clinical indications in surgery, the selection criteria of the samples are as follows: primary osteosarcoma, synovial sarcoma, rhabdomyosarcoma, leiomyosarcoma, ewing sarcoma, liposarcoma, acinar soft tissue sarcoma, clear cell sarcoma, fibroma, and a sample with a surgical specimen weight of more than 20mg.

3. The primary physician provides basic clinical information such as sex, age, medical history, family history, smoking history, pathological staging, clinical diagnosis, etc. of the patient. The name, the identification card number and other information of the patient related to the privacy of the patient are hidden and replaced by a uniform experiment number, and the naming principle of the experiment number is eight-digit numerical date of the collected sample plus four digits after the patient is hospitalized. For example, if the sample is provided on day 1 of 2018, month 1, and the patient is hospitalized with T001512765, the sample is tested with 201801012765.

4. During the operation, fresh specimens are collected by the surgeon in a sterile environment in the operating room and placed in a previously prepared specimen preservation solution (see example 1). The samples were kept temporarily on ice after being isolated and transported to the laboratory within two hours for further processing.

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

The following operations required working on ice and the entire procedure required completion within 10 minutes.

The surgical instruments used in the following operations all need to be sterilized in advance at high temperature and high pressure and can be used after being dried.

1. The samples were weighed.

2. The sample surface was rinsed with 75% (volume percent) ethanol for 10 to 30 seconds.

3. The samples were washed 5 times with sample wash and 5 times with sterile PBS solution.

4. The fat tissue, connective tissue and necrotic tissue in the sample are carefully stripped off with the aid of an ophthalmic scissors, an ophthalmic forceps, a scalpel and the like.

Example 4 bone and Soft tissue tumor solid tumor tissue sample dissociation

The surgical instruments used in the following examples were sterilized at high temperature and high pressure in advance and dried before use.

1. Cutting the tissue into pieces of 1mm by using an ophthalmic scissors ³ The left and right small blocks.

2. The minced tissue samples were treated with a pre-heated sample dissociation solution at 37 ℃ for a dissociation time of 15 minutes to 3 hours at 37 ℃ in an amount of 0.1mL of sample dissociation solution (see example 1) per mg of tissue. The dissociation of the samples was observed under the microscope every 15 minutes until a large number of single cells were observed.

3. The dissociation reaction was stopped with 10-fold volume of a digestion stop solution (see example 1), and the cell suspension was collected.

4. The cell suspension was filtered through a 100 μm sterile cell strainer to remove tissue debris and adherent cells.

5. 800g were centrifuged at room temperature for 10 minutes and the supernatant discarded.

6. The cells were resuspended in 5mL sterile PBS, 800g centrifuged at room temperature for 10 minutes, and the supernatant discarded.

7. The cell pellet was resuspended in bone and soft tissue tumor solid tumor primary cell culture medium (see example 1), and the cell status was observed under a microscope for cell counting.

As shown in FIG. 1, the dissociated single cell suspension contains a large amount of various types of cells, such as erythrocytes, lymphocytes, and fibroblasts, in addition to tumor cells. One of the advantages of the method is that in the subsequent culture process, only the tumor cells can be greatly expanded, while the proportion of other cells is gradually reduced or even disappears, and finally the primary tumor cells of the bone and soft tissue tumor solid tumors with higher purity are obtained.

Example 5 bone and Soft tissue tumor solid tumor Primary cell culture

1. Performing bone and soft tissue tumor solid tumor primary cell suspension culture by using a low-adsorption surface (low-adsorption-surface), wherein the used culture medium is the bone and soft tissue tumor solid tumor primary cell culture medium in example 1 (wherein the final concentration of the human recombinant protein bFGF is 20ng/mL; the final concentration of the human recombinant protein HGF is 20ng/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 the N-acetyl-L-cysteine is 1mM, the final concentration of Y-27632 is 10 mu M), and taking a six-well plate as an example, 10 mu M is taken for each well ⁶ Density plating of individual cells, 37 ℃,5% CO ₂ The culture was carried out in a cell culture incubator under the conditions.

2. The cell status was observed every day, and the medium was changed every 3 days until the cells formed clumps of about 100 μm in diameter.

As shown in FIG. 2, after 3-10 days of culture, the tumor cells were extensively expanded to a size of 100 μm in diameterThe total number of tumor cells can exceed 10 ⁷ The number of other types of cells is significantly reduced or even eliminated. Through a large number of sample tests, the success rate of the in vitro culture of the primary tumor cells of the bone and soft tissue tumor solid tumors can reach 70 percent.

Example 6 passage of bone and Soft tissue tumor solid tumor Primary cells

1. The cell pellet was collected from the dish, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

2. The cell pellet was washed with sterile PBS solution, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

3. The cell pellet was resuspended in cell digest (see example 1) and digested at 37 ℃. The digestion of the cell pellet was observed under a microscope every 5 minutes until the cell pellet was digested into single cells.

4. The dissociation reaction was stopped with 10 volumes of a digestion stop solution (see example 1) and the cell suspension was collected.

6. The cell pellet was resuspended in bone and soft tissue tumor solid tumor primary cell culture medium (see example 1) and the cells counted.

7. Culturing the primary cells of the solid tumor of bone and soft tissue by using a low-adsorption surface (low-adsorption-surface), wherein the culture medium is the primary cell culture medium of the solid tumor of bone and soft tissue in example 1, and the culture medium is a six-well plate, and each well is 10 ⁶ Density plating of individual cells, 37 ℃,5% CO ₂ The culture was carried out in a cell culture incubator under the conditions.

Example 7 cryopreservation of bone and Soft tissue tumor solid tumor Primary cells

After the primary cells of the solid tumor of the bone and soft tissue cultured in suspension are subjected to passage amplification for 2-3 times, the primary cells can be frozen:

1. the cell pellet was collected from the culture dish, centrifuged at 800g for 10 minutes at room temperature, and the supernatant was discarded.

3. The cell pellet was resuspended in cell digest (see example 1) and digested at 37 ℃. The digestion of the cell pellet was observed under a microscope every 15 minutes until the cell pellet was digested into single cells.

4. The dissociation reaction was stopped with 10 volumes of digestion stop solution (see example 1), and the cell suspension was collected and counted.

6. Cell cryopreservation (see example 1) at 10 ⁶ Resuspending the cell sediment at a density of/mL, freezing 1mL of cell suspension in each tube of a 2mL freezing tube, freezing overnight by using a gradient cooling box, and transferring the cell sediment into liquid nitrogen for long-term storage.

Example 8 recovery of solid tumor Primary cells from bone and Soft tissue tumors

Bone and soft tissue tumor solid tumor primary cells preserved in liquid nitrogen can be revived:

1. sterile water at 37 ℃ was prepared five minutes in advance.

2. The vial was removed from the liquid nitrogen and the cells were rapidly thawed in sterile water at 37 ℃.

3. 800g were centrifuged at room temperature for 10 minutes and the supernatant discarded.

4. Resuspending the cell pellet with bone and soft tissue tumor solid tumor primary cell culture medium (see example 1), culturing the bone and soft tissue tumor solid tumor primary cells using a low adsorption surface, resuscitating the cells per tube into a 3.5cm petri dish, 37 ℃,5% CO ₂ The culture was carried out in a cell culture incubator under the conditions.

Example 9 HE staining identification of bone and Soft tissue tumor solid tumor Primary cells

The reagent consumables used in the following examples are illustrated:

HE staining kit (beijing solibao biotechnology limited, # G1120);

cation anticreep slide (Beijing China fir Jinqiao Biotech limited);

xylene, methanol, acetone (beijing chemical reagent, analytical pure);

neutral resin adhesive (fine chemicals, gmbH, beijing).

1. Suspension cells were made to a concentration of 10 ⁴ And dripping 10 mu L of cell suspension on the cation anti-falling glass slide, and naturally drying.

2. 50. Mu.L of a methanol/acetone mixture (volume ratio 1.

3. And taking out the cell-fixed slide, and naturally drying at room temperature.

4. Slides were washed twice with 200 μ L PBS.

5. When the water on the slide is slightly dry, 100 mu L of hematoxylin staining solution is added for staining for 1mins.

6. The hematoxylin stain was aspirated and the slides were washed 3 times with 200 μ L of tap water.

7. 100 mu L of differentiation solution is added dropwise for differentiation for 1mins.

8. The differentiation medium was aspirated off, and the slides were washed sequentially 2 times with tap water and 1 time with distilled water.

9. The water on the surface of the slide was removed by suction, and 200. Mu.L of eosin dye was added dropwise for 40s.

10. Absorbing eosin dye solution, rinsing and dehydrating with 75%, 80%, 90% and 100% ethanol for 20s, 40s and 40s.

11. After the ethanol was dried, 50. Mu.L of xylene was added dropwise for cell permeation.

12. After xylene is completely dried, a drop of neutral resin adhesive is added dropwise, and the piece is mounted by a cover glass, observed under a microscope and photographed.

FIG. 3 shows the HE staining effect of primary tumor cells of solid tumors of bone and soft tissue obtained by in vitro culture, and it can be seen that these cells generally have the characteristics of high nuclear mass ratio, deep nuclear staining, chromatin condensation in the nucleus, multinuclear, and uneven cell size.

Example 10 immunofluorescence staining identification of solid tumor Primary cells of bone and Soft tissue tumors

The reagents used in the following examples are illustrative:

paraformaldehyde (Beijing chemical reagent company, analytical pure) was dissolved in ultrapure water to prepare a 4% (4 g/100 mL) paraformaldehyde solution;

methanol, dimethyl sulfoxide (Beijing chemical reagent company, analytical pure);

hydrogen peroxide (beijing chemicals, 35%);

mixing methanol, dimethyl sulfoxide and 35% hydrogen peroxide according to the proportion of 4;

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

immunofluorescence primary anti-antibody (Abcam, # ab 92547);

immunofluorescent secondary antibody (CST, # 4408);

hoechst dye liquor (Beijing Sorleibao Biotech limited, # C0021);

the primary cell mass of the solid tumor of bone and soft tissue is subjected to immunofluorescence staining according to the following steps, and the primary antibody is an anti-Vimentin antibody.

1. The cell pellet was collected from the dish, washed once with PBS, resuspended in 4% paraformaldehyde and fixed overnight at 4 ℃.

2. The supernatant was discarded by centrifugation at 800g, and the cell pellet was resuspended in a precooled methanol solution and placed on ice for 1 hour.

3. Centrifuging at 800g, discarding supernatant, resuspending cell pellet with Dan's rinsing solution, and standing at room temperature for 2 hr.

4. The supernatant was discarded by centrifugation at 800g, and the cells were washed sequentially with 75%, 50%, 25% (volume percent) methanol diluted with PBS for 10 minutes each.

5. The supernatant was centrifuged at 800g, suspended in 3% BSA solution, and the cell pellet was blocked at room temperature for 2 hours.

6. According to the following steps: 500% by weight of BSA solution, 3%, and resuspending the cell pellet with antibody dilution (3% by weight of BSA solution), 4 ℃ primary antibody overnight.

7. The supernatant was discarded by centrifugation at 800g, and the cell pellet was washed 5 times with PBS solution for 20 minutes each.

8. According to the proportion of 1:2000, the secondary antibody was diluted with 3% BSA solution, and the cell pellet was resuspended in antibody diluent (3% BSA solution), and the secondary antibody was incubated at room temperature for 2 hours.

9. The supernatant was discarded by centrifugation at 800g, and the cell pellet was washed 5 times with PBS solution for 20 minutes each.

10. 100 Xhoechst dye solution was added in a volume ratio of 1/100, and the mixture was dyed at room temperature for 20 minutes.

11. The cell pellet was washed 2 times with PBS solution for 10 minutes each. The staining of the cell mass was observed using a confocal laser microscope.

FIG. 4 shows the effect of immunofluorescence staining of bone and primary tumor cell masses of soft tissue sarcoma cultured in vitro, and it can be seen that the cells constituting the cell masses are all Vimentin positive, consistent with the pathological results of patients, confirming that the tumor cells cultured by the method have higher purity.

Example 12 in vitro culture of solid tumor cells of different types of bone and Soft tissue tumors

The procedures of all primary culture procedures of the samples in this example are completely identical (see the above description), and only the pathological types of the samples are different. The samples tested are shown in Table 22.

TABLE 21 in vitro culture of primary tumor cells of various pathological types of bone and soft tissue sarcomas

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

Example 12 bone and Soft tissue tumor solid tumor Primary tumor cell culture with CYTOP modified cell culture consumables

In this example, the procedures of all primary cultures were identical (see the above description), the CYTOP modification method was identical, and only the materials of the cell culture consumables were different (table 23).

The CYTOP modification method comprises the following steps: firstly, pure oxygen etching is carried out on the cell culture container, the etching condition is 20W, and the etching time is 3 minutes. Then 1% of the CYTOP solution is used to cover the culture dish or the surface of the culture plate, using the appropriate amount (20. Mu.L per well, appropriate amount means complete coverage of the bottom of the culture dish, in the case of 96-well plates), after which the CYTOP solution is completely air-dried.

TABLE 22 Effect of CYTOP-modified consumables on bone and soft tissue tumor solid tumor Primary tumor cell culture

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Note: polystyrene (Polystyrene, abbreviated PS).

As can be seen from table 22: 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, a method of injection molding is used, and a PMMA material (or PS, PC, COC, COP, LAS, etc.) is used to process the microplate chip for culturing the primary cells of bone and soft tissue tumor solid tumors of the present invention. The chip can be used for primary cell culture of solid tumor of bone and soft tissue tumor and in vitro drug sensitivity detection experiment. The microplate chip design is shown in FIG. 5.

In the practical application process, the 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 FIG. 5, and then the surface of the microplate chip is subjected to CYTOP modification by the CYTOP modification method (see example 11), so that the microplate chip which can be used for culturing the primary cells of the bone and soft tissue tumor solid tumors is obtained.

Claims

1. A method for culturing bone and soft tissue tumor solid tumor primary cells comprises the following steps: performing suspension culture on the primary cells of the solid tumors of the bone and the soft tissue by using a primary cell culture medium of the solid tumors of the bone and the soft tissue;

the primary cell culture medium for the bone and soft tissue tumor solid tumors consists of a culture medium of an antibacterial antifungal agent tri-antibody, 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; wherein the final concentration of penicillin in the three antibiotics of the antibacterial antifungal agent is 100-200U/mL; the final concentration of streptomycin in the three antibiotics of the antibacterial antifungal agent is 100-200 mug/mL; the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250ng/mL; the final concentration of the 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 20ng/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% volume percentage; the final concentration of the N-acetyl-L-cysteine is 1mM; the final concentration of the N-2 Supplement is 1 volume percent; the final concentration of the Y-27632 is 10 mu M; the balance is Advanced DMEM/F12 medium.

2. The method of claim 1, wherein: the primary cells of the bone and soft tissue tumor solid tumors are obtained by dissociating the bone and soft tissue tumor solid tumor tissues by using sample dissociation liquid;

the sample dissociation liquid consists of collagenase I, collagenase II, collagenase III, collagenase IV and PBS; wherein the final concentration of the collagenase I in the dissociation liquid of the sample is 150-250U/mL; the final concentration of the collagenase II in the dissociation liquid of the sample is 150-250U/mL; the final concentration of the collagenase III in the dissociation liquid of the sample is 250-350U/mL; the final concentration of the collagenase IV in the dissociation liquid of the sample is 150-250U/mL; the balance being PBS.

3. The method of claim 2, wherein: the method comprises the following steps of dissociating the bone and the solid tumor tissue of the soft tissue tumor by using the sample dissociation liquid: treating the sheared bone and soft tissue tumor solid tumor tissues by using the sample dissociation liquid preheated at 37 ℃ in advance according to the dosage of 0.1-0.3mL of the sample dissociation liquid per mg of tissues, and dissociating the samples at 37 ℃ for 15 minutes to 3 hours.

4. The method of claim 1, wherein: in the method, the primary cells of the solid tumor of the bone and soft tissue tumor are cultured in a suspension manner by using the primary cell culture medium of the solid tumor of the bone and soft tissue tumor according to the following steps: using a cell culture container M, performing suspension culture on the bone and soft tissue tumor solid tumor primary cells by using the bone and soft tissue tumor solid tumor primary cell culture medium, performing culture at 37 ℃ under the condition of 5% CO2, and replacing the culture medium every 2-4 days;

the cell culture vessel M is any one of: (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) subjecting the cell culture vessel of (I) to CYTOP modification.

5. The method of claim 4, wherein: the cell culture container is a cell culture dish, a cell culture pore plate or a micropore plate chip for cell culture.

6. The method of claim 4, wherein: in said (II), the cell culture vessel of said (I) is CYTOP-modified according to a method comprising the steps of: carrying out pure oxygen etching on the cell culture container in the step (I), wherein the etching condition is that the power is 20W, and the etching time is 3 minutes; the cell culture vessel surface was then covered with a 1% CYTOP solution and the CYTOP modification was completed by air drying the 1% CYTOP solution.

7. The method of claim 6, wherein: the composition of the 1% CYTOP solution was as follows: each 100mL of the 1% CYTOP solution contained 1mL of CYTOP, with the balance being fluoro oil.

8. The method according to any one of claims 1-7, wherein: the method also comprises the following steps of carrying out dissociation pretreatment on the bone and soft tissue tumor solid tumor tissues: cleaning the surfaces of the bone and soft tissue tumor solid tumor tissue samples by using ethanol with the volume percentage of 70-75%; sequentially cleaning the bone and soft tissue tumor solid tumor tissue samples by using a sample cleaning solution and a sterile PBS solution;

the sample cleaning solution consists of double-antibody P/S and PBS; wherein 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 100-200 mug/mL; the balance being PBS.

9. The method of claim 8, wherein: the in vitro time of the bone and soft tissue tumor solid tumor tissue sample subjected to the dissociation pretreatment is less than 2 hours, and the bone and soft tissue tumor solid tumor tissue sample is preserved in a sample preservation solution before the dissociation pretreatment;

the sample preservation solution consists of fetal calf serum, double-antibody P/S, HEPES and HBSS; wherein the final concentration of the fetal calf serum is 1-5% volume percentage content; 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 100-200 mug/mL; the final concentration of the HEPES is 8-12mM; the balance being HBSS.

10. The method according to any one of claims 1-7, wherein: in the method, after the dissociation treatment of the bone and the soft tissue tumor solid tumor tissue by using the sample dissociation liquid, the method further comprises the following steps: terminating the dissociation reaction by using a digestion termination solution, and collecting cell suspension; filtering the cell suspension to remove tissue debris and adherent cells; resuspending the cells with sterile PBS after centrifugation; re-centrifuging, and then re-suspending the cell pellet with the primary cell culture medium for the bone and soft tissue tumor solid tumors;

the digestion stop solution consists of fetal calf serum, double-antibody P/S and a DMEM medium; wherein the final concentration of the fetal calf serum is 8-12% volume percentage content; 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 100-200 mug/mL; the balance is DMEM medium.

11. The method according to any one of claims 1-7, wherein: in the process of culturing the primary cells of the solid tumor of the bone and soft tissue tumor by using the primary fine culture medium of the solid tumor of the bone and soft tissue tumor, the method also comprises the following steps: and when the primary cells of the bone and soft tissue tumor solid tumors form a mass with the diameter of 80-120 mu m, carrying out passage on the primary cells of the bone and soft tissue tumor solid tumors.

12. The method of claim 11, wherein: the cell digest used for the passage had the following composition: every 10mL of the cell digestive juice contains 4-6mLACCUTase, EDTA with the final concentration of 5mM, 1.5-2.5mL of TrypLE Express and the balance of PBS.

13. The method of claim 11, wherein: the use of a digestion stop solution for said passaging is a digestion stop solution according to claim 10.

14. The method of claim 11, wherein: the method also comprises the step of freezing and/or reviving the primary solid tumor cells of the bone and soft tissue tumor after the expansion of 2-3 passages.

15. The method of claim 14, wherein: the cell freezing solution adopted during freezing is composed of an Advanced DMEM/F12 culture medium, DMSO and a 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium to the DMSO to the 1% methylcellulose solution is 20 (0.8-1.2); the 1% methylcellulose solution is an aqueous solution of methylcellulose having a concentration of 1g/100 ml.

16. The method according to any one of claims 1-7, wherein: the bone and soft tissue tumor is osteosarcoma, synovial sarcoma, rhabdomyosarcoma, leiomyosarcoma, ewing sarcoma, liposarcoma, acinar soft tissue sarcoma, hyaline cell sarcoma or fibroma.

17. A kit for culturing primary cells of solid tumors of bone and soft tissue, which comprises the culture medium of the primary cells of solid tumors of bone and soft tissue according to claim 1 and at least one of the following reagents: the sample dissociation solution according to claim 2, the sample storage solution according to claim 9, the cell digestion solution according to claim 12, the sample washing solution according to claim 8, the digestion stop solution according to claim 10, the cell cryopreservation solution according to claim 15, and the 1% CYTOP solution according to claim 7.

18. The kit of claim 17, wherein: the bone and soft tissue tumor is osteosarcoma, synovial sarcoma, rhabdomyosarcoma, leiomyosarcoma, ewing sarcoma, liposarcoma, acinar soft tissue sarcoma, hyaline cell sarcoma or fibroma.

19. Use of a kit according to claim 17 for culturing bone and soft tissue tumor solid tumor primary cells.

20. Use according to claim 19, characterized in that: the bone and soft tissue tumor is osteosarcoma, synovial sarcoma, rhabdomyosarcoma, leiomyosarcoma, ewing sarcoma, liposarcoma, acinar soft tissue sarcoma, hyaline cell sarcoma or fibroma.