METHOD FOR CULTURING COLORECTAL CANCER SOLID TUMOR PRIMARY CELLS AND COLORECTAL CANCER ASCITES PRIMARY TUMOR CELLS AND SUPPORTING REAGENTS

Abstract

A method for culturing colorectal cancer solid tumor primary cells and colorectal cancer ascites primary tumor cells and supporting reagents. A method for culturing colorectal cancer solid tumor primary cells and colorectal cancer ascites primary tumor cells and supporting reagents. Colorectal cancer solid tumor tissues are treated with mild cell dissociation reagents, and colorectal cancer cells are isolated from ascites with a mild method, thereby ensuring the vitality of cancer cells to the greatest extent. A special serum-free medium is prepared, and colorectal cancer solid tumor-derived tumor cells are cultured in vitro with a suspension culture system to ensure normal expansion of the cancer cells while eliminating the interference of normal cells to the greatest extent. The colorectal cancer primary cell culture obtained by the method are usable for in vitro experiments, second-generation sequencing, building of animal models, and building of cell lines at multiple cell levels.

Claims

1. A medium for culturing colorectal cancer primary cells, consisting of a ternary antibacterial and antifungal agent, HEPES, GlutaMax, a human recombinant protein EGF, a human recombinant protein bFGF, a human recombinant protein HGF, a human recombinant protein Wnt-3a, a human recombinant protein Noggin, SB202190, A83-01, Primocin™, N-acetyl-L-cysteine, Nicotinamide, N-2 Supplement, cortisol, B27, ITS-X, Y-27632 and an Advanced DMEM/F12 medium; wherein a final concentration of penicillin in the ternary antibacterial and antifungal agent is 100-200 U/mL; a final concentration of streptomycin in the ternary antibacterial and antifungal agent is 100-200 μg/mL; a final concentration of amphotericin B in the ternary antibacterial and antifungal agent is 100-250 ng/mL; a final concentration of the HEPES is 8-12 mM; a final concentration of the GlutaMax is 0.8-1.2% (volume percentage); a final concentration of the human recombinant protein EGF is 10-100 ng/mL; a final concentration of the human recombinant protein bFGF is 10-50 ng/mL; a final concentration of the human recombinant protein HGF is 5-25 ng/mL; a final concentration of the human recombinant protein Wnt-3a is 200-300 ng/mL; a final concentration of the human recombinant protein Noggin is 100-200 ng/mL; a final concentration of the SB202190 is 5-10 μM; a final concentration of the A83-01 is 0.25-1.25 μM; a final concentration of the Primocin™ is 1% (volume percentage); a final concentration of the N-acetyl-L-cysteine is 0.5-2 mM; a final concentration of the Nicotinamide is 5-10 mM; a final concentration of the N-2 Supplement is 1% (volume percentage); a final concentration of the cortisol is 20-50 ng/mL; a final concentration of the B27 is 1.5-2.5% (volume percentage); a final concentration of the ITS-X is 0.8-1.2% (volume percentage); a final concentration of the Y-27632 is 5-20 μM; and the balance is the Advanced DMEM/F12 medium.

2. A set of reagents for culturing colorectal cancer primary cells is any of the following: (A1) that consisting of the medium according to claim 1 and all or part of following reagents: a sample dissociation solution, a sample preservation solution and a sample washing solution; the sample dissociation solution is composed of a collagenase I, a collagenase II, a collagenase IV and a PBS; wherein, a final concentration of the collagenase I in the sample dissociation solution is 150-250 U/mL; a final concentration of the collagenase II in the sample dissociation solution is 150-250 U/mL; a final concentration of the collagenase IV in the sample dissociation solution is 50-150 U/mL; and the balance is the PBS; the sample preservation solution is composed of a fetal bovine serum, a ternary antibacterial and antifungal agent, an HEPES and an HBSS; wherein, a final concentration of the fetal bovine serum in the sample preservation solution is 1-5% (volume percentage); a final concentration of penicillin in the ternary antibacterial and antifungal agent in the sample preservation solution is 100-200 U/mL; a final concentration of streptomycin in the ternary antibacterial and antifungal agent in the sample preservation solution is 100-200 μg/mL; a final concentration of amphotericin B in the ternary antibacterial and antifungal agent in the sample preservation solution is 250-500 ng/mL; a final concentration of the HEPES in the sample preservation solution is 8-12 mM; and the balance is the HBSS; the sample washing solution is composed of a ternary antibacterial and antifungal agent and a PBS; wherein, a final concentration of penicillin in the ternary antibacterial and antifungal agent in the sample washing solution is 100-200 U/mL; a final concentration of streptomycin in the ternary antibacterial and antifungal agent in the sample washing solution is 100-200 μg/mL; a final concentration of amphotericin B in the ternary antibacterial and antifungal agent in the sample washing solution is 250-500 ng/mL; and the balance is the PBS; (A2) that consisting of the medium according to claim 1 and a cell isolation buffer; the cell isolation buffer is composed of a ternary antibacterial and antifungal agent, heparin sodium and a PBS; wherein, a final concentration of penicillin in the ternary antibacterial and antifungal agent is 100-200 U/mL; a final concentration of streptomycin in the ternary antibacterial and antifungal agent is 100-200 μg/mL; a final concentration of amphotericin B in the ternary antibacterial and antifungal agent is 250-500 ng/mL; a final concentration of the heparin sodium is 10 IU/mL; and the balance is the PBS; (A3) that consisting of the (A1) and all or part of the following reagents: a cell dissociation solution, a dissociation stop solution and a cell cryopreservation solution; and (A4) that consisting of the (A2) and all or part of the following reagents: a cell dissociation solution, a dissociation stop solution and a cell cryopreservation solution; the composition of the cell dissociation solution is as follows: every 10 mL of the cell dissociation solution contains 4-6 mL of Accutase, EDTA with a final concentration of 5 mM, 1.5-2.5 mL of TrypLE Express, and the balance of PBS; the dissociation stop solution is composed of a fetal bovine serum, a ternary antibacterial and antifungal agent and a dulbecco's modified eagle medium (DMEM); wherein, a final concentration of the fetal bovine serum in the dissociation stop solution is 8-12% (volume percentage); a final concentration of penicillin in the ternary antibacterial and antifungal agent in the dissociation stop solution is 100-200 U/mL; a final concentration of streptomycin in the ternary antibacterial and antifungal agent in the dissociation stop solution is 100-200 μg/mL; a final concentration of amphotericin B in the ternary antibacterial and antifungal agent in the dissociation stop solution is 250-500 ng/mL; and the balance is the DMEM; the cell cryopreservation solution is composed of an Advanced DMEM/F12 medium, a DMSO and a 1% methyl cellulose solution; wherein, a volume ratio of the Advanced DMEM/F12 medium, the DMSO and the 1% methyl cellulose solution is 20:2:(0.8-1.2); and the 1% methyl cellulose solution is a methyl cellulose aqueous solution with a concentration of 1 g/100 ml.

3. (canceled)

4. A method for culturing colorectal cancer primary cells is method A or method B: method A: a method for culturing colorectal cancer solid tumor primary cells comprises the following steps: (a1) dissociating colorectal cancer solid tumor tissues with the sample dissociation solution according to claim 2 to obtain colorectal cancer solid tumor primary cells; and (a2) performing suspension culture with the medium according to claim 2 on the colorectal cancer solid tumor primary cells dissociated in (a1); method B: a method for culturing colorectal cancer ascites primary tumor cells comprises the following steps: (b1) isolating colorectal cancer ascites primary tumor cells from colorectal cancer ascites; and (b2) performing suspension culture with the culture medium according to claim 2 on the colorectal cancer ascites primary tumor cells isolated in (b1).

5. The method according to claim 4, wherein (a1), the colorectal cancer solid tumor tissues are dissociated with the sample dissociation solution according to a method comprising the following steps: treating the cut colorectal cancer solid tumor tissues with the sample dissociation solution preheated at 37° C. according to an amount of 0.1-0.3 mL of the sample dissociation solution per mg of tissues, and dissociating the sample at 37° C. for 15 minutes to 3 hours; and in (b1), the colorectal cancer ascites primary tumor cells are isolated from the colorectal cancer ascites according to a method comprising the following steps: suspending cells in the colorectal cancer ascites with the cell isolation buffer according to claim 4, and then obtaining the colorectal cancer ascites primary tumor cells by means of density gradient centrifugation.

6. The method according to claim 4, wherein (a2), suspension culture is performed with the medium on the colorectal cancer solid tumor primary cells according to a method comprising the following steps: performing suspension culture in a cell culture vessel M with the medium on the colorectal cancer solid tumor primary cells at 37° C. and 5% CO.sub.2, and changing the medium every 2-4 days; in (b2), suspension culture is performed with the medium on the colorectal cancer ascites primary tumor cells according to a method comprising the following steps: performing suspension culture in a cell culture vessel M with the medium on the colorectal cancer ascites primary tumor cells at 37° C. and 5% CO.sub.2, and changing the medium every 2-4 days; and the cell culture vessel M is any of the following: (I) a cell culture vessel made of polystyrene, a cell culture vessel made of polycarbonate, a cell culture vessel made of polymethyl methacrylate, a cell culture vessel made of COC resin, a cell culture vessel made of cyclic olefin polymer, or a cell culture vessel with a low-attachment-surface; and (II) a cell culture vessel modified from the cell culture vessel in (I) by a CYTOP.

7. The method according to claim 6, wherein (II), the cell culture vessel in (I) is modified by the CYTOP according to a method comprising the following steps: etching the cell culture vessel in (I) with pure oxygen under a power of 20 W for 3 minutes; and then covering the surface of the cell culture vessel with a 1% CYTOP solution, and drying the 1% CYTOP solution in the air to complete the CYTOP modification; and 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 balance of fluorocarbon oil.

8. The method according to claim 4, wherein before (a1), the following steps of treating the colorectal cancer solid tumor tissues before dissociation are further comprised: washing the surface of a colorectal cancer solid tumor tissue sample with ethanol having a volume percentage of 70-75%; and sequentially washing the colorectal cancer solid tumor tissue sample with the sample washing solution according to claim 4 and a sterile PBS solution.

9. The method according to claim 8, wherein the in vitro time for treating the colorectal cancer solid tumor tissue sample before dissociation is within 2 hours, and the sample is preserved in the sample preservation solution according to claim 8 all the time prior to the treatment before dissociation.

10. The method according to claim 4, wherein (a1), after the colorectal cancer solid tumor tissues are dissociated with the sample dissociation solution, the following steps are further comprised: stopping the dissociation reaction with the dissociation stop solution according to claim 4, and collecting a cell suspension; filtering the cell suspension to remove tissue debris and adherent cells; re-suspending cells with a sterile PBS after centrifugation; centrifuging again, and then re-suspending a cell precipitate with the medium according to claim 4.

11. The method according to claim 4, wherein (a2), the following step is further comprised: when the colorectal cancer solid tumor primary cells form masses with a diameter of 50-80 passaging the colorectal cancer solid tumor primary cells; and in (b2), the following step is further comprised: when the colorectal cancer ascites primary tumor cells form masses with a diameter of 50-80 μm, passaging the colorectal cancer ascites primary tumor cells.

12. The method according to claim 11, wherein a cell dissociation solution used during the passaging is the cell dissociation solution according to claim 11.

13. The method according to claim 11, wherein a dissociation stop solution used during the passaging is the dissociation stop solution according to claim 11.

14. The method according to claim 4, wherein the method further comprises a step of cryopreserving and/or resuscitating the colorectal cancer solid tumor primary cells or the colorectal cancer ascites primary tumor cells after 2-3 passaging expansions; and a cell cryopreservation solution used during the cryopreserving is the cell cryopreservation solution according to claim 4.

15. Any of the following reagents: (C1) a colorectal cancer solid tumor tissue sample dissociation solution, which is the sample dissociation solution according to claim 2; (C2) a colorectal cancer solid tumor tissue sample preservation solution, which is the sample preservation solution according to claim 2; and (C3) a colorectal cancer ascites cell isolation buffer, which is the cell isolation buffer according to claim 2.

16. (canceled)

17. (canceled)

18. The method according to claim 4, wherein the colorectal cancer is primary colorectal cancer.

19. The method according to claim 4, wherein the colorectal cancer is colorectal cancer or colorectal cancer metastasis lesion.

20. The method according to claim 4, wherein the colorectal cancer primary cells are colorectal cancer solid tumor primary cells or colorectal cancer ascites primary tumor cells.

21. The method according to claim 4, wherein the colorectal cancer primary cells are isolated from surgical samples, colonoscopy puncture samples or ascites samples of colorectal cancer patients.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0114] FIG. 1 shows single cells obtained after treatment of colorectal cancer tissues. The scale is 100 μm, 100 times magnification.

[0115] FIG. 2 shows cell masses obtained after primary culture of colorectal cancer tissues. The scale is 100 μm, 100 times magnification.

[0116] FIG. 3 shows an HE staining map of a colorectal cancer cell mass section obtained after primary culture of colorectal cancer tissues. The scale is 100 μm, 200 times magnification.

[0117] FIG. 4 shows an immunofluorescent staining map of cancer cell masses obtained after primary culture of colorectal cancer tissues. The scale is 50 μm, 200 times magnification.

[0118] FIG. 5 shows that copy number variations of colorectal cancer primary cell cultures of respective generations (P1, P2, P3, P4, P5) and primary colorectal cancer tumor tissues (Tumor) are highly identical after copy number variation (CNV) analysis according to sequencing results.

[0119] FIG. 6 shows single cells isolated from a colorectal cancer ascites sample. The scale is 100 μm.

[0120] FIG. 7 shows cell masses obtained after culture of primary tumor cells in a colorectal cancer ascites sample. The scale is 200 μm.

[0121] FIG. 8 shows an HE staining map of colorectal cancer cells obtained after primary culture of a colorectal cancer ascites sample.

[0122] FIG. 9 shows an immunofluorescent staining map of cancer cell masses obtained after primary culture of a colorectal cancer ascites sample.

[0123] FIG. 10 shows that copy number variations of colorectal cancer ascites primary cell cultures of respective generations (P1, P2, P3, P4, P5) and cancer cells in colorectal cancer ascites are highly identical after copy number variation (CNV) analysis according to sequencing results.

[0124] FIG. 11 shows results of in vitro drug sensitivity test using colorectal cancer ascites primary tumor cells cultured according to the present invention.

[0125] FIG. 12 is a design diagram of a microplate chip according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0126] The following embodiments facilitate better understanding of the present invention, but do not limit the present invention. The experimental methods in the following embodiments are conventional methods, unless otherwise specified. The test materials used in the following embodiments are purchased from conventional biochemical reagent stores, unless otherwise specified.

Embodiment 1. Preparation of Reagents for Culturing Colorectal Cancer Solid Tumor Primary Cells

[0127] 1. Sample Preservation Solution (100 mL)

[0128] A specific formula of the sample preservation solution (100 mL) was shown in Table 1.

TABLE-US-00001 TABLE 1 Sample preservation solution (100 mL) Final Reagent Brand number Amount concentration Fetal bovine serum Gibco#16000-044 2 mL 2% Ternary antibacterial Gibco#15240062 1 mL 1% and antifungal agent HEPES Gibco#15630080 1 mL 10 mM HBSS Gibco#14170161 Made up to 100 mL

[0129] After preparation, the sample preservation solution was dispensed in 15 mL centrifuge tubes, 5 mL per tube. The sample preservation solution can be preserved at 4° C. for 1 month after being dispensed.

[0130] 2. Sample Washing Solution (100 mL)

[0131] A specific formula of the sample washing solution (100 mL) was shown in Table 2.

TABLE-US-00002 TABLE 2 Sample washing solution (100 mL) Final Reagent Brand number Amount concentration Ternary antibacterial Gibco#15240062 1 mL 1% and antifungal agent PBS Gibco#21-040-CVR Made up to 100 mL

[0132] The sample washing solution was prepared for current use.

[0133] 3. Sample Dissociation Solution (10 mL)

[0134] A specific formula of the sample dissociation solution (10 mL) was shown in Table 3.

TABLE-US-00003 TABLE 3 Sample dissociation solution (10 mL) Final Reagent Brand number Amount concentration 10× collagenase I 10× stock solution 1 mL 200 U/mL 10× collagenase II 10× stock solution 1 mL 200 U/mL 20× collagenase IV 20× stock solution 0.5 mL   100 U/mL PBS Gibco#21-040-CVR Made up to 10 mL Note: the sample dissociation solution was prepared for current use.

[0135] In Table 3, the preparations of the collagenase stock solutions were shown in Tables 4-6.

TABLE-US-00004 TABLE 4 10× collagenase I stock solution (100 mL) Final Reagent Brand number Amount concentration Collagenase I Gibco#17100-017 1 g 2,000 U/mL PBS Gibco#21-040-CVR Made up to 100 mL

[0136] After preparation, the 10× collagenase I stock solution was dispensed in 1.5 mL sterile centrifuge tubes, 1 mL per tube. The stock solution can be preserved for a long term at −20° C.

TABLE-US-00005 TABLE 5 10× collagenase II stock solution (100 mL) Final Reagent Brand number Amount concentration Collagenase II Gibco#17101-015 1 g 2,000 U/mL PBS Gibco#21-040-CVR Made up to 100 mL

[0137] After preparation, the 10× collagenase II stock solution was dispensed in 1.5 mL sterile centrifuge tubes, 1 mL per tube. The stock solution can be preserved for a long term at −20° C.

TABLE-US-00006 TABLE 6 20× collagenase IV stock solution (100 mL) Final Reagent Brand number Amount concentration Collagenase IV Gibco#17104-019 1 g 2,000 U/mL PBS Gibco#21-040-CVR Made up to 100 mL

[0138] After preparation, the 20× collagenase IV stock solution was dispensed in 1.5 mL sterile centrifuge tubes, 1 mL per tube. The stock solution can be preserved for a long term at −20° C.

[0139] In Tables 4, 5 and 6, the unit U of the collagenase (the collagenase I or the collagenase IV) was defined by the enzyme activity of a protease: the collagenase (the collagenase I or the collagenase IV) was treated with 1 U of the protease for 5 hours at 37° C. and pH 7.5, and 1 μmol of L-leucine can be released.

[0140] 4. Cell Dissociation Solution (10 mL)

[0141] A specific formula of the cell dissociation solution (10 mL) was shown in Table 7.

TABLE-US-00007 TABLE 7 Cell dissociation solution (10 mL) Final Reagent Brand number Amount concentration Accutase Gibco#A11105-01 5 mL 0.5M EDTA Invitrogen#AM9261 10 μL 5 mM TrypLE Express Gibco#12604013 2 mL PBS Gibco#21-040-CVR Made up to 10 mL

[0142] The cell dissociation solution was prepared for current use.

[0143] 5. Dissociation Stop Solution (100 mL)

[0144] A specific formula of the dissociation stop solution (100 mL) was shown in Table 8.

TABLE-US-00008 TABLE 8 Dissociation stop solution (100 mL) Final Reagent Brand number Amount concentration Fetal bovine serum Gibco#16000-044 10 mL 10% Ternary antibacterial Gibco#15240062  1 mL  1% and antifungal agent DMEM Gibco#11965-092 Made up to 100 mL

[0145] The dissociation stop solution can be preserved at 4° C. for one month after preparation.

[0146] 6. Colorectal Cancer Solid Tumor Primary Cell Medium (100 mL)

[0147] A specific formula of the colorectal cancer solid tumor primary cell medium (100 mL) was shown in Table 9.

TABLE-US-00009 TABLE 9 Colorectal cancer solid tumor primary cell medium (100 mL) Final Reagent Brand number Amount concentration Ternary antibacterial Gibco#15240062 1 mL 1% and antifungal agent HEPES Gibco#15630080 1 mL 10 mM GlutaMax Gibco#35050061 1 mL 1% 1,000× human recombinant 1,000× stock solution 50-500 μL 10-100 ng/mL protein EGF 1,000× human recombinant 1,000× stock solution 50-250 μL 10-50 ng/mL protein bFGF 1,000× human recombinant 1,000× stock solution 25-125 μL 5-25 ng/mL protein HGF 1,000× human recombinant 1,000× stock solution 100-150 μL 200-300 ng/mL protein Wnt-3a 1,000× human recombinant 1,000× stock solution 100-200 μL 100-200 ng/mL protein Noggin SB202190 1,000× stock solution 50-100 μL 5-10 μM A83-01 100,000× stock solution 1-5 μL 0.25-1.25 μM Primocin ™ Invivogene#ant-pm-1 1 mL 1% N-acetyl-L-cysteine 1,000× stock solution 100-400 μL 0.5-2 mM Nicotinamide 1,000× stock solution 100-200 μL 5-10 mM N-2 Supplement Gibco#17502001 1 mL 1% 1,000× cortisol 1,000× stock solution 80-200 μL 20-50 ng/mL B27 Gibco#12587010 2 mL 2% ITS-X Gibco#51500056 1 mL 1% 1,000× Y-27632 1,000× stock solution 50-200 μL 5-20 μM Advanced DMEM/F12 medium Gibco#12634010 Made up to 100 mL

[0148] After preparation, the colorectal cancer solid tumor primary cell medium was filtered and sterilized with a 0.22 μM syringe filter (Millipore SLGP033RS), and can be preserved at 4° C. for two weeks.

[0149] In Table 9, the preparations of the human recombinant protein stock solutions were shown in Tables 11-15, the preparation of the SB202190 stock solution was shown in Table 16, the preparation of the A83-01 stock solution was shown in Table 17, the preparation of the N-acetyl-L-cysteine stock solution was shown in Table 18, the preparation of the Nicotinamide stock solution was shown in Table 19, the preparation of the cortisol stock solution was shown in Table 20, and the preparation of the Y-27632 stock solution was shown in Table 21. The preparation of a 100×BSA solution required when these stock solutions were prepared was shown in Table 10.

TABLE-US-00010 TABLE 10 100× BSA solution (1 mL) Final Reagent Brand number Amount concentration BSA Sigma#A1933 0.1 g 0.1 g/mL PBS Gibco#21-040-CVR Made up to 1 mL

[0150] The 100×BSA solution was prepared for current use.

TABLE-US-00011 TABLE 11 1,000× human recombinant protein EGF stock solution (5 mL) Final Reagent Brand number Amount concentration Human recombinant Peprotech AF-100- 100 μg 20 μg/mL protein EGF 15-100 100× BSA solution 500 μL 0.01 g/mL PBS Gibco#21-040-CVR Made up to 5 mL

[0151] After preparation, the 1,000× human recombinant protein EGF stock solution was dispensed in 1.5 mL sterile centrifuge tubes, and the stock solution can be preserved for a long term at −80° C.

TABLE-US-00012 TABLE 12 1,000× human recombinant protein bFGF stock solution (2.5 mL) Final Reagent Brand number Amount concentration Human recombinant Peprotech AF-100- 50 μg 20 μg/mL protein bEGF 18B-50 100× BSA solution 250 μL 0.01 g/mL PBS Gibco#21-040-CVR Made up to 2.5 mL

[0152] After preparation, the 1,000× human recombinant protein bEGF stock solution was dispensed in 1.5 mL sterile centrifuge tubes, and the stock solution can be preserved for a long term at −80° C.

TABLE-US-00013 TABLE 13 1,000× human recombinant protein HGF stock solution (5 mL) Final Reagent Brand number Amount concentration Human recombinant Peprotech AF-100- 100 μg 20 μg/mL protein HGF 39-100 100× BSA solution 500 μL 0.01 g/mL PBS Gibco#21-040-CVR Made up to 5 mL

[0153] After preparation, the 1,000× human recombinant protein HGF stock solution was dispensed in 1.5 mL sterile centrifuge tubes, and the stock solution can be preserved for a long term at −80° C.

TABLE-US-00014 TABLE 14 1,000× human recombinant protein Wnt-3a stock solution (2.5 mL) Final Reagent Brand number Amount concentration Human recombinant R&D 5036-WN-500 500 μg 200 μg/mL protein Wnt-3a 100× BSA solution 250 μL 0.01 g/mL PBS Gibco#21-040-CVR Made up to 2.5 mL

[0154] After preparation, the 1,000× human recombinant protein Wnt-3a stock solution was dispensed in 1.5 mL sterile centrifuge tubes, and the stock solution can be preserved for a long term at −80° C.

TABLE-US-00015 TABLE 15 1,000× human recombinant protein Noggin stock solution (5 mL) Final Reagent Brand number Amount concentration Human recombinant Shanghai 500 μg 100 μg/mL protein Noggin Novoprotein#C018 100× BSA solution 500 μL 0.01 g/mL PBS Gibco#21-040-CVR Made up to 5 mL

[0155] After preparation, the 1,000× human recombinant protein Noggin stock solution was dispensed in 1.5 mL sterile centrifuge tubes, and the stock solution can be preserved for a long term at −80° C.

TABLE-US-00016 TABLE 16 1,000× SB202190 stock solution (1.51 mL) Final Reagent Brand number Amount concentration SB202190 Sigma#S7067 5 mg 10 mM DMSO Made up to 1.51 mL

[0156] After preparation, the 1,000× SB202190 stock solution was dispensed in 0.5 mL sterile centrifuge tubes, and the stock solution can be preserved for a long term at −20° C.

TABLE-US-00017 TABLE 17 100,000× A83-01 stock solution (1.05 mL) Final Reagent Brand number Amount concentration A83-01 Tocris#2939 10 mg 25 mM DMSO Made up to 1.05 mL

[0157] After preparation, the 100,000×A83-01 stock solution was dispensed in 0.5 mL sterile centrifuge tubes, and the stock solution can be preserved for a long term at −20° C.

TABLE-US-00018 TABLE 18 1,000× N-acetyl-L-cysteine stock solution (5 mL) Final Reagent Brand number Amount concentration N-acetyl-L-cysteine Sigma#A9165 0.41 g 0.5M Ultra-pure water Made up to 5 mL

[0158] After preparation, the 1,000× N-acetyl-L-cysteine stock solution was dispensed in 0.5 mL sterile centrifuge tubes, and the stock solution can be preserved for a long term at −20° C.

TABLE-US-00019 TABLE 19 1,000× Nicotinamide stock solution (4 mL) Final Reagent Brand number Amount concentration Nicotinamide Sigma#N0636 2.44 g 5M Ultra-pure water Made up to 4 mL

[0159] After preparation, the 1,000× Nicotinamide stock solution was dispensed in 0.5 mL sterile centrifuge tubes, and the stock solution can be preserved for a lone term at −20° C.

TABLE-US-00020 TABLE 20 1,000× cortisol stock solution (100 mL) Final Reagent Brand number Amount concentration Cortisol Sigma#H0888 2.5 mg 25 μg/mL Absolute ethanol Sigma#E7023 5 mL 5% Ultra-pure water Made up to 100 mL

[0160] After preparation, the 1,000× cortisol stock solution was dispensed in 1.5 mL sterile centrifuge tubes, and the stock solution can be preserved for a long term at −20° C.

TABLE-US-00021 TABLE 21 1,000× Y-27632 stock solution (3.125 mL) Final Reagent Brand number Amount concentration Y-27632 MCE#129830-38-2 10 mg 10 mM Ultra-pure water Made up to 3.125 mL

[0161] After preparation, the 1,000× Y-27632 stock solution was dispensed in 0.5 mL sterile centrifuge tubes, and the stock solution can be preserved for a long term at −20° C.

[0162] 7. Cell Cryopreservation Solution

[0163] A specific formula of the cell cryopreservation solution was shown in Table 22.

TABLE-US-00022 TABLE 22 Cell cryopreservation solution Reagent Brand number Amount Advanced DMEM/F12 medium Gibco#12634010 20 mL  DMSO Sigma#D2438 2 mL 1% methyl cellulose solution 1 mL

[0164] The cell cryopreservation solution was prepared for current use.

[0165] In Table 22, the preparation of the 1% methyl cellulose solution was shown in Table 23.

TABLE-US-00023 TABLE 23 1% methyl cellulose solution (10 mL) Final Reagent Brand number Amount concentration Methyl cellulose Sigma#M7027 0.1 g 10 g/L Ultra-pure water Made up to 10 mL

[0166] The 1% methyl cellulose solution can be preserved for a long time at 4° C. after preparation.

Embodiment 2. Obtaining of Postoperative Samples of Colorectal Cancer Solid Tumors

[0167] 1. Cooperation with a top three hospital, the cooperation had passed the formal medical ethics review.

[0168] 2. Attending physicians selected participating patients according to the clinical indications specified in the medical guidelines, and selected appropriate samples according to the intraoperative clinical indications for in vitro culture. The selection criteria for samples were: primary colorectal cancer, pathological stage: stage II, stage III or stage IV, colorectal cancer or colorectal cancer metastasis lesions of various pathological types, and colorectal cancer surgical samples with a weight of more than 20 mg.

[0169] 3. The attending physicians provided basic clinical information such as patient's gender, age, medical history, family history, smoking history, pathological stage type, and clinical diagnosis. Information related to patient's privacy, such as patient's name and ID number, was concealed and replaced with a uniform experiment number. The naming principle of the experiment number was the eight-digit date of the collected sample+the last four digits of the patient's hospitalization number. For example, if a sample was provided on Jan. 1, 2018 and the patient's hospitalization number was T001512765, the experiment number of the sample was 201801012765.

[0170] 4. During operation, surgeons collected fresh samples in a sterile environment of an operating room and placed same in the prepared sample preservation solution (Table 1). The samples were temporarily preserved on ice after being isolated, and transported to a laboratory within two hours for next operation.

Embodiment 3. Treatment of Colorectal Cancer Solid Tumor Tissue Samples Before Dissociation

[0171] The following operations were performed on ice, and the entire operation steps were completed within 10 minutes.

[0172] The surgical equipment used in the following operations was sterilized in advance at a high temperature and under a high pressure, and can be used after drying.

[0173] 1. Weighing of samples.

[0174] 2. Surfaces of the samples were washed with 75% (volume percentage) ethanol for 10 to 30 seconds.

[0175] 3. The samples were washed 10 times with the sample washing solution (Table 2) and 5 times with a sterile PBS solution.

[0176] 4. Adipose tissues, connective tissues, and necrotic tissues in the samples were carefully peeled off with equipment such as ophthalmic scissors, ophthalmic tweezers, and scalpels.

Embodiment 4. Dissociation of Colorectal Cancer Solid Tumor Tissue Samples

[0177] The surgical equipment used in the following embodiment was sterilized in advance at a high temperature and under a high pressure, and can be used after drying.

[0178] 1. Tissues were cut into small pieces of about 1 mm.sup.3 with ophthalmic scissors.

[0179] 2. According to an amount of 0.1 mL of the sample dissociation solution (Table 3) per mg of tissues, the cut tissue samples were treated with the sample dissociation solution preheated at 37° C., and dissociated at 37° C. for 15 minutes to 3 hours. The dissociation of the samples was observed under a microscope every 15 minutes until a large number of single cells were observed.

[0180] 3. The dissociation reaction was stopped with 10 times the volume of the dissociation stop solution (Table 8), and a cell suspension was collected.

[0181] 4. The cell suspension was filtered with a 40 μm sterile cell strainer to remove tissue debris and adherent cells.

[0182] 5. 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0183] 6. Cells were re-suspended with 5 mL of a sterile PBS, 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0184] 7. The cell precipitate was re-suspended with the colorectal cancer solid tumor primary cell medium (Table 9), cell statuses were observed under a microscope, and cells were counted.

[0185] As shown in FIG. 1, in the single cell suspension obtained by dissociation, in addition to tumor cells, there were also a large number of other cells of various types, such as red blood cells, lymphocytes, and fibrocytes. One of the advantages of this method was that in the subsequent culture process, only the cancer cells can be expanded in a large amount, while other cells gradually decreased or even disappeared, and finally high-purity colorectal cancer primary tumor cells were obtained.

Embodiment 5. Culture of Colorectal Cancer Solid Tumor Primary Cells

[0186] 1. Suspension culture was performed on the colorectal cancer primary cells by using a low-attachment-surface. The medium used was the colorectal cancer solid tumor primary cell medium (Table 9) in Embodiment 1. Taking a six-pore plank as an example, the cells were planked with a density of 10.sup.6 cells per pore and cultured in a cell incubator at 37° C. and 5% CO.sub.2.

[0187] 2. Cell states were observed every day, and the medium was changed every 3 days until the cells formed masses with a diameter of about 80 μm.

[0188] As shown in FIG. 2, after 3-10 days of culture, the cancer cells were expanded in a large amount to form cell masses with a diameter of 80 μm. The total number of the tumor cells can exceed 10.sup.7, and other types of cells were significantly reduced or even disappeared. This method has been tested on a large number of samples, showing that the success rate of in vitro culture of the colorectal cancer solid tumor primary tumor cells can reach 80%.

Embodiment 6. Passage of Colorectal Cancer Solid Tumor Primary Cells

[0189] 1. Cell masses in a culture dish were collected, 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0190] 2. The cell masses were washed with a sterile PBS solution, 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0191] 3. The cell masses were re-suspended with the cell dissociation solution (Table 7) and dissociated at 37° C. The dissociation of the cell masses was observed under a microscope every 5 minutes, until the cell masses were dissociated into individual cells.

[0192] 4. The dissociation reaction was stopped with 10 times the volume of the dissociation stop solution (Table 8), and a cell suspension was collected.

[0193] 5. 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded. 6. The cell precipitate was re-suspended with the colorectal cancer solid tumor primary cell medium (Table 9), and cells were counted.

[0194] 7. The colorectal cancer primary cells were cultured by using a low-attachment-surface. The medium used was the colorectal cancer solid tumor primary cell medium (Table 9) in Embodiment 1. Taking a six-pore plank as an example, the cells were planked with a density of 10.sup.6 cells per pore and cultured in a cell incubator at 37° C. and 5% CO.sub.2.

Embodiment 7. Cryopreservation of Colorectal Cancer Solid Tumor Primary Cells

[0195] The colorectal cancer solid tumor primary cells subjected to the suspension culture can be cryopreserved after 2-3 passaging expansions:

[0196] 1. Cell masses in a culture dish were collected, 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0197] 2. The cell masses were washed with a sterile PBS solution, 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0198] 3. The cell masses were re-suspended with the cell dissociation solution (Table 7) and dissociated at 37° C. The dissociation of the cell masses was observed under a microscope every 15 minutes, until the cell masses were dissociated into individual cells.

[0199] 4. The dissociation reaction was stopped with 10 times the volume of the dissociation stop solution (Table 8), a cell suspension was collected, and cells were counted.

[0200] 5. 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0201] 6. The cell precipitate was re-suspended with the cell cryopreservation solution (Table 22) according to a density of 10.sup.6 cells/mL, and the cell suspension was dispensed in 2 mL cryopreservation tubes according to 1 ml of the cell suspension per tube, cryopreserved with a gradient cooling box overnight and then transferred to liquid nitrogen for long-term preservation.

Embodiment 8. Resuscitation of Colorectal Cancer Solid Tumor Primary Cells

[0202] The colorectal cancer solid tumor primary cells preserved in the liquid nitrogen can be resuscitated:

[0203] 1. 37° C. sterile water was prepared five minutes in advance.

[0204] 2. The cryopreservation tube was taken out of the liquid nitrogen, and the cells were quickly thawed in the 37° C. sterile water.

[0205] 3. 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0206] 4. The cell precipitate was re-suspended with the colorectal cancer solid tumor primary cell medium (Table 9), the colorectal cancer primary cells were cultured by using a low-attachment-surface, each tube of the cells was resuscitated to a 3.5 cm culture dish, and the cells were cultured in a cell incubator at 37° C. and 5% CO.sub.2.

Embodiment 9. HE Staining Identification of Colorectal Cancer Solid Tumor Primary Cells

[0207] Description of Reagent Consumables Used in the Following Embodiment:

[0208] HE staining kit (Beijing Solarbio Science & Technology Co., Ltd., #G1120);

[0209] Cationic anti-dropping slide (Beijing ZSGB-Bio Co., Ltd.);

[0210] Xylene, methanol, acetone (Beijing Chemical Reagent Company, analytically pure);

[0211] Neutral resin glue (Beijing Yili Fine Chemical Co., Ltd.).

[0212] 1. 800 g of a cell suspension were centrifuged, cells were collected, and cultured with the colorectal cancer ascites primary tumor cell medium (Table 9, where a final concentration of the human recombinant protein EGF was 20 ng/mL; a final concentration of the human recombinant protein bFGF was 20 ng/mL; a final concentration of the human recombinant protein HGF was 10 ng/mL; a final concentration of the human recombinant protein Wnt-3a was 200 ng/mL; a final concentration of the human recombinant protein Noggin was 100 ng/mL; a final concentration of the SB202190 was 10 μM; a final concentration of the A83-01 was 1 μM; a final concentration of the N-acetyl-L-cysteine was 1 mM; a final concentration of the Nicotinamide was 10 mM; a final concentration of the cortisol was 20 ng/mL; a final concentration of the Y-27632 was 10 μM) to obtain suspended colorectal cancer solid tumor primary cell masses, and the cell masses were immobilized with 4% paraformaldehyde. The cell mass precipitate was embedded in paraffin and sectioned with a thickness of 5 μm.

[0213] 2. The paraffin sections were soaked in a xylene solution and incubated at room temperature for 5 minutes to remove the paraffin, and after 3 times, the sections were rinsed twice with deionized water.

[0214] 3. The sections were immersed in absolute ethanol and incubated at room temperature for 10 minutes, and this process was repeated twice.

[0215] 4. Ginger sections were immersed in 95% ethanol and incubated at room temperature for 10 minutes, and after twice, the sections were rinsed twice with deionized water.

[0216] 5. When the water on the slide was slightly dry, 100 μL of hematoxylin staining solution was added for staining 1 minute.

[0217] 6. The hematoxylin staining solution was sucked away, and the slide was washed with tap water 3 times.

[0218] 7. 100 μL of differentiation solution was dripped for differentiating 1 minute. 8. The differentiation solution was sucked away, and the slide was sequentially washed twice with tap water and once with distilled water.

[0219] 9. The water on the surface of the slide was sucked away, and 200 μL of eosin staining solution was added for staining 40 seconds.

[0220] 10. The eosin staining solution was sucked away, and the slide was sequentially rinsed with 75%, 80%, 90%, and 100% ethanol and dewatered for 20 seconds, 20 seconds, 40 seconds, and 40 seconds.

[0221] 11. After the ethanol was dry, 50 μL of xylene was dripped for cell permeation.

[0222] 12. After the xylene was completely dry, a drop of neutral resin glue was dripped, the slide was sealed with a cover glass, the cells were observed under a microscope and pictures were taken.

[0223] FIG. 3 shows an HE staining effect map of colorectal cancer solid tumor primary tumor cells cultured in vitro. It can be seen that these cells generally had cancer cell characteristics such as high nuclear-cytoplasmic ratio, dark nuclear staining, intranuclear chromatin agglutination, multinucleation, and uneven cell size, and dozens to hundreds of tumor cells aggregated to form tumor cell masses with certain three-dimensional structures.

Embodiment 10. Immunofluorescent Staining Identification of Colorectal Cancer Solid Tumor Primary Cells

[0224] Description of Reagents Used in the Following Embodiment:

[0225] Paraformaldehyde (Beijing Chemical Reagent Company, analytically pure), paraformaldehyde powder was dissolved in ultra-pure water to prepare a 4% (4 g/100 mL) paraformaldehyde solution;

[0226] Methanol, dimethyl sulfoxide (Beijing Chemical Reagent Company, analytically pure);

[0227] Hydrogen peroxide (Beijing Chemical Reagent Company, 35%);

[0228] Methanol, dimethyl sulfoxide, and 35% hydrogen peroxide were mixed in a ratio of 4:4:1 (volume ratio) to prepare a DANCLAN rinsing solution;

[0229] Bovine serum albumin (Sigma, #A1933), the bovine serum albumin was dissolved in a PBS solution to prepare a 3% (3 g/100 mL) BSA solution;

[0230] Immunofluorescent primary antibody (Abeam, #ab17139);

[0231] Immunofluorescent secondary antibody (CST, #4408);

[0232] Hoechst staining solution (Beijing Solarbio Science & Technology Co., Ltd., #C0021);

[0233] Immunofluorescent staining was performed on the colorectal cancer solid tumor primary cell masses cultured with the colorectal cancer ascites primary tumor cell medium (Table 9, where the final concentration of the human recombinant protein EGF was 20 ng/mL; the final concentration of the human recombinant protein bFGF was 20 ng/mL; the final concentration of the human recombinant protein HGF was 10 ng/mL; the final concentration of the human recombinant protein Wnt-3a was 200 ng/mL; the final concentration of the human recombinant protein Noggin was 100 ng/mL; the final concentration of the SB202190 was 10 μM; the final concentration of the A83-01 was 1 μM; the final concentration of the N-acetyl-L-cysteine was 1 mM; the final concentration of the Nicotinamide was 10 mM; the final concentration of the cortisol was 20 ng/mL; the final concentration of the Y-27632 was 10 μM) according to the following steps. The primary antibody was CK8+CK18, which characterized epithelial cells.

[0234] 1. The cell masses in the culture dish were collected and washed once with a PBS, and the cell precipitate was re-suspended with 4% paraformaldehyde and immobilized at 4° C. overnight.

[0235] 2. 800 g was centrifuged, the supernatant was discarded, and the cell precipitate was re-suspended with a pre-cooled methanol solution and placed on ice for 1 hour.

[0236] 3. 800 g was centrifuged, the supernatant was discarded, and the cell precipitate was re-suspended with a DANCLAN rinsing solution and placed at room temperature for 2 hours.

[0237] 4. 800 g was centrifuged, the supernatant was discarded, and the cells were washed sequentially with 75%, 50%, and 25% (volume percentage) methanol solutions diluted with a PBS for 10 minutes each time.

[0238] 5. 800 g was centrifuged, the supernatant was discarded, and the cell precipitate was suspended with a 3% BSA solution and sealed at room temperature for 2 hours.

[0239] 6. The primary antibody was diluted with a 3% BSA solution in a ratio of 1:500, the cell precipitate was re-suspended with the antibody diluent (3% BSA solution), and the primary antibody was placed at 4° C. overnight

[0240] 7. 800 g was centrifuged, the supernatant was discarded, and the cell precipitate was washed with a PBS solution 5 times, 20 minutes each time.

[0241] 8. The secondary antibody was diluted with a 3% BSA solution in a ratio of 1:2,000, the cell precipitate was re-suspended with the antibody diluent (3% BSA solution), and the secondary antibody was placed at room temperature for 2 hours.

[0242] 9. 800 g was centrifuged, the supernatant was discarded, and the cell precipitate was washed with a PBS solution 5 times, 20 minutes each time.

[0243] 10. A 100× Hoechst staining solution was added in a volume ratio of 1/100, and staining was performed at room temperature for 20 minutes.

[0244] 11. The cell precipitate was washed twice with a PBS solution for 10 minutes each time. The staining of the cell masses was observed with a laser confocal microscope.

[0245] FIG. 4 shows an immunofluorescent staining effect map of colorectal cancer solid tumor primary tumor cell masses cultured in vitro. It can be seen that the cells constituting the cell masses were all CK8/CK18 positive and were of epithelial origin, which confirmed that highly pure tumor cells were obtained by this culture method. The primary cultures of 20 colorectal cancer samples were identified by immunofluorescent staining. The statistical results showed that the proportion of tumor cells among the colorectal cancer solid tumor primary cells obtained by this method reached 72%-95% (Table 24).

TABLE-US-00024 TABLE 24 Immunofluorescent staining identification of primary cultures of colorectal cancer samples CK8/CK18 Serial number Sample number positive rate 1 20170613FDZ5181 84.5% 2 20170613ZPW3709 85.5% 3 20170623QJS8039 87.5% 4 20170627GYL9278 93.9% 5 20170627XHH2582 79.0% 6 20170627XHH2582 74.5% 7 20170628SY6545 83.6% 8 20170711JP8251 89.5% 9 20170719LWH7572 83.6% 10 20170720YXL2334 73.5% 11 20170721DZK0501 78.6% 12 20170726GYJ1907 87.0% 13 20170807WH1465 89.7% 14 20170822HMR2096 88.0% 15 20170828LYJ7774 83.0% 16 20170830GXJ6131 80.2% 17 20170905LJY4875 88.9% 18 20170912XZG1390 90.8% 19 20170926ZCL9071 72.2% 20 20171010SJ9221 85.1%

Embodiment 11. Primary Cell Cultures and Primary Tumor Tissues of Colorectal Cancer Solid Tumors

[0246] The DNA extraction process mentioned in the following embodiment was performed by means of a Tiangen blood/tissue/cell genome extraction kit (DP304).

[0247] The library building process mentioned in the following embodiment was performed by means of an NEB DNA sequencing library building kit (E7645).

[0248] The high-throughput sequencing mentioned in the following embodiment referred to an Illumina HiSeq X-ten sequencing platform.

[0249] 1. Colorectal cancer solid tumor samples were taken, 10 mg of a colorectal cancer solid tumor sample was first taken for DNA extraction, library building and whole genome high-throughput sequencing (WGS) with a sequencing depth of 30× before in vitro culture operation, and the remaining solid tumor samples were used for in vitro culture of colorectal cancer primary cells.

[0250] 2. After colorectal cancer tissues were treated and cultured for a period of time, cell masses with a diameter of more than 80 μm were formed and recorded as P0 generation cells, and then recorded as P1, P2, . . . , Pn according to the number of passages. 10.sup.6 cells were taken from each of the P1, P2, P3, and P4 generations of colorectal cancer primary tumor cell cultures for DNA extraction, library building and whole genome high-throughput sequencing (WGS) with a sequencing depth of 30×.

[0251] 3. Copy number variation (CNV) analysis was performed on each group of sequencing results to compare copy number variations between the primary colorectal cancer tumor tissues and the colorectal cancer primary cell cultures of respective generations. As shown in FIG. 5, the copy number variations of the colorectal cancer solid tumor primary cell cultures of respective generations (P1, P2, P3, P4, P5) were highly consistent with the copy number variation of the primary colorectal cancer tumor tissues (Tumor), so the colorectal cancer solid tumor primary cells obtained by this method can represent the truth of patient's primary tumors.

Embodiment 12. Comparison of Success Rates of Culturing Colorectal Cancer Solid Tumor Primary Cells with Different Primary Cell Culture Media

[0252] In this embodiment, the operation method and process of primary culture of all samples were completely the same (refer to the above), and only the formulas of the culture media were different. The various primary cell culture media for testing were shown in Table 25. The scheme D was the formula used in the present invention, and details were shown in Table 9.

TABLE-US-00025 TABLE 25 Formulas of primary cell culture media for testing (100 mL) Culture Final medium Reagent Brand number Amount concentration Scheme A mTeSR ™1 medium Stemcell#05850 100 mL Scheme B Alveolar Epithelial Cell Medium ScienCell#3201 100 mL Scheme C Double antibodies P/S Gibco#15140122 1 mL 1% HEPES Gibco#15630080 1 mL 10 mM 1,000× human recombinant 1,000× stock 250 μL 50 ng/mL protein EGF solution 1,000× human recombinant 1,000× stock 100 μL 20 ng/mL protein bFGF solution DMEM/F12 medium Gibco#14170161 Made up to 100 mL Scheme D See Table 9

[0253] After preparation, the primary cell culture media were filtered and sterilized with a 0.22 μM syringe filter (Millipore SLGP033RS), and can be preserved at 4° C. for two weeks.

[0254] Each of the four primary cell medium schemes treated 20 colorectal cancer solid tumor tissue samples. Sample treatment and culture operations were performed according to the methods described in Embodiments 3, 4, and 5. After culture for 10 days, the success rates of colorectal cancer solid tumor primary cell culture were counted as shown in Table 26:

TABLE-US-00026 TABLE 26 Culture statuses of different culture media Primary cell medium scheme Culture success rate Scheme A 0% (0/20) Scheme B 10% (2/20)  Scheme C 20% (4/20)  Scheme D 80% (16/20)

[0255] It can be seen that the primary cell culture media had a great impact on the success rate of colorectal cancer primary cell culture, and the colorectal cancer solid tumor primary cell medium (Table 9) used in the present invention can stimulate the proliferation of cancer cells in the colorectal cancer solid tumor tissue samples to the greatest extent, and improve the success rate of colorectal cancer solid tumor primary cell culture.

Embodiment 13. Comparison of Success Rates of Culturing Colorectal Cancer Solid Tumor Primary Cells with Different Sample Preservation Solutions

[0256] In this embodiment, the operation method and process of primary culture of all samples were completely the same (refer to the above), and only the formulas of the sample preservation solutions were different. The various sample preservation solutions for testing were shown in Table 27. The scheme E was the formula used in the present invention, and details were shown in Table 1.

TABLE-US-00027 TABLE 27 Formulas of sample preservation solutions for testing (100 mL) Sample preservation Final solution Scheme Reagent Brand number Amount concentration Scheme A HBSS Gibco#14170161 100 mL Scheme B Double antibodies P/S Gibco#15140122 1 mL 1% HBSS Gibco#14170161 Made up to 100 mL Scheme C Double antibodies P/S Gibco#15140122 1 mL 1% DMEM Gibco#11965-092 Made up to 100 mL Scheme D Ternary antibacterial Gibco#15240062 1 mL 1% and antifungal agent Fetal bovine serum Gibco#16000-044 10 mL 10%  DMEM Gibco#11965-092 Made up to 100 mL Scheme E See Table 1

[0257] After preparation, the various sample preservation solutions in the above table were dispensed in 15 mL centrifuge tubes, 5 mL per tube. The sample preservation solutions can be preserved at 4° C. for 1 month after being dispensed.

[0258] Each of the five sample preservation solution schemes treated 20 colorectal cancer solid tumor samples. The samples were temporarily preserved in the sample preservation solutions at 4° C. after being isolated. After the samples were isolated for 2 hours, sample treatment and culture operations were performed according to the methods described in Embodiments 3, 4, and 5. After culture for 10 days, the success rates of colorectal cancer solid tumor primary cell culture were counted as shown in Table 28:

TABLE-US-00028 TABLE 28 Culture statuses of different sample preservation solutions Bacteria/fungi Sample preservation Culture success contamination solution rate rate Scheme A 30% (6/20)  75% (15/20) Scheme B 55% (11/20) 15% (3/20)  Scheme C 60% (12/20) 20% (4/20)  Scheme D 75% (15/20) 0% (0/20) Scheme E 80% (16/20) 0% (0/20)

[0259] It can be seen that the formulas of the sample preservation solutions had a great impact on the success rate of colorectal cancer solid tumor primary cell culture, and the sample preservation solution (Table 1) used in the present invention can protect the activity of cancer cells in the colorectal cancer solid tumor tissue samples to the greatest extent, and improve the success rate of culture.

Embodiment 14. Comparison of Success Rates of Culturing Colorectal Cancer Solid Tumor Primary Cells with Different Sample Dissociation Solutions

[0260] In this embodiment, the operation method and process of primary culture of all samples were completely the same (refer to the above), and only the formulas of the sample dissociation solutions were different. The various sample dissociation solutions for testing were shown in Table 29. The scheme D was the formula used in the present invention, and details were shown in Table 3.

TABLE-US-00029 TABLE 29 Formulas of sample dissociation solutions for testing (10 mL) Sample dissociation Final solution Reagent Brand number Amount concentration Scheme A Trypsin Gibco#25200056 10 mL Scheme B 0.5M EDTA Invitrogen#AM9261 10 μL 5 mM DNAse Thermo#EN0521 62.5 μL 62.5 U/mL TrypLE ™ Express Gibco#12604013 Made up to 10 mL Scheme C 20× collagenase IV 20× stock solution 1 mL 200 U/mL DNAse Thermo#EN0521 62.5 μL 62.5 U/mL PBS Gibco#21-040-CVR Made up to 10 mL Scheme D See Table 3

[0261] The sample dissociation solutions were prepared for current use.

[0262] 20 samples of colorectal cancer solid tumor tissue blocks with a weight of more than 100 mg were selected, averagely divided into four parts, and respectively treated and cultured with the above-mentioned four sample dissociation solutions according to the methods described in Embodiments 3, 4, and 5. After culture for 10 days, the success rates of colorectal cancer solid tumor primary cell culture were counted as shown in Table 30:

TABLE-US-00030 TABLE 30 Culture statuses of different sample dissociation solutions Serial Scheme Scheme Scheme Scheme number Sample number A B C D 1 20170621HYF0118 Failure Failure Success Success 2 20170627LSX2279 Failure Success Success Success 3 20170627SXL9673 Failure Failure Failure Success 4 20170628ZSQ7313 Failure Success Success Success 5 20170629YZY3072 Failure Failure Failure Success 6 20170629GLR1929 Failure Failure Failure Failure 7 20170707WY9660 Failure Success Success Success 8 20170717ZJ0831 Failure Failure Failure Success 9 20170720GL0285 Failure Failure Failure Success 10 20170726GXW4931 Failure Failure Success Success 11 20170727MJC5252 Failure Failure Success Success 12 20170801LQS1498 Failure Success Success Success 13 20170807ZJZ9161 Failure Success Success Success 14 20170808ZFG8438 Failure Failure Failure Failure 15 20170810LSL0445 Failure Failure Failure Failure 16 20170810WJ5078 Failure Failure Failure Success 17 20170816ZBH6938 Success Success Success Success 18 20170821GY0193 Failure Failure Failure Success 19 20170828WYQ1401 Failure Failure Success Success 20 20170904ZQR5422 Failure Failure Success Success Summary 5% 25% 55% 85% (1/20) (6/20) (9/20) (17/20)

[0263] It can be seen that the formulas of the sample dissociation solutions had a great impact on the success rate of colorectal cancer solid tumor primary cell culture, and the sample dissociation solution (Table 3) used in the present invention can dissociate cancer cells in the colorectal cancer solid tumor tissues to the greatest extent, and improve the success rate of colorectal cancer solid tumor primary cell culture.

Embodiment 15. Comparison of Success Rates of Passing Colorectal Cancer Solid Tumor Primary Cells with Different Cell Dissociation Solutions

[0264] In this embodiment, the operation method and process of primary cell passage of all samples were completely the same (refer to the above), and only the formulas of the cell dissociation solutions were different. The various cell dissociation solutions for testing were shown in Table 31. The scheme D was the formula used in the present invention, and details were shown in Table 7.

TABLE-US-00031 TABLE 31 Formulas of cell dissociation solutions for testing (10 mL) Cell dissociation Final solution Reagent Brand number Amount concentration Scheme A Trypsin Gibco#25200056 10 mL Scheme B 0.5M EDTA Invitrogen#AM9261 10 μL 5 mM TrypLE ™ Express Gibco#12604013 Made up to 10 mL Scheme C 10× collagenase I 10× stock solution 1 mL 200 U/mL 10× collagenase II 10× stock solution 1 mL 200 U/mL 20× collagenase IV 20× stock solution 0.5 mL 100 U/mL PBS Gibco#21-040-CVR Made up to 10 mL Scheme D See Table 7

[0265] The cell dissociation solutions were prepared for current use.

[0266] 20 colorectal cancer solid tumor primary cell samples that had been successfully cultured were selected, and the cultured colorectal cancer solid tumor primary cells were continuously passaged with the above-mentioned four cell dissociation solutions according to the method described in Embodiment 6. When cancer cells expanded to form cell masses with a diameter of 80 μm, the cancer cells were passaged (not more than 10 times), and the maximum number of passages was recorded. The statistical results were shown in Table 32:

TABLE-US-00032 TABLE 32 Culture statuses of different cell dissociation solutions Serial Scheme Scheme Scheme Scheme number Sample number A B C D 1 20170621HYF0118 1 2 5 8 2 20170623QJS8039 1 3 7 10 3 20170627GYL9278 1 3 5 10 4 20170627LSX2279 1 4 9 10 5 20170627SXL9673 1 5 5 10 6 20170627XHH2582 1 5 6 10 7 20170627XHH2582 1 3 6 8 8 20170628SY6545 1 2 8 8 9 20170628ZSQ7313 2 5 9 10 10 20170629GLR1929 1 5 6 8 11 20170629YZY3072 1 2 8 9 12 20170707WY9660 1 4 6 8 13 20170711JP8251 1 5 5 8 14 20170717ZJ0831 1 4 7 9 15 20170719LWH7572 1 5 7 10 16 20170720GL0285 1 5 7 9 17 20170720YXL2334 1 2 9 9 18 20170721DZK0501 1 3 9 10 19 20170726GXW4931 1 4 5 8 20 20170816ZBH6938 2 6 10 10 Summary of possible passages 1-2 2-6 5-10 8 times times times times or more

[0267] It can be seen that the formulas of the cell dissociation solutions had a great impact on the success rate of passage of the colorectal cancer solid tumor primary cells. The cell dissociation solution (Table 7) used in the present invention can gently dissociate cancer cells in the cell masses, so that the samples can be continuously passaged while maintaining the activity of the colorectal cancer solid tumor primary cells.

Embodiment 16. Preparation of Reagents for Culturing Colorectal Cancer Ascites Primary Tumor Cells

[0268] 1. Cell Isolation Buffer (100 mL)

[0269] A specific formula of the cell isolation buffer (100 mL) was shown in Table 33:

TABLE-US-00033 TABLE 33 Cell isolation buffer (100 mL) Final Reagent Brand number Amount concentration Ternary antibacterial Gibco#15240062 1 mL 1% and antifungal agent 1,000× heparin sodium 1,000× stock 0.1 mL 10 IU/mL solution solution PBS Gibco#21-040-CVR Made up to 100 mL

[0270] The cell isolation buffer can be preserved at 4° C. for 1 month after preparation.

[0271] In Table 33, the preparation of the heparin sodium solution was shown in Table 34.

TABLE-US-00034 TABLE 34 1,000× heparin sodium (1 mL) Final Reagent Brand number Amount concentration Heparin sodium Solarbio#H8270 10 kIU 10 kIU/mL Ultra-pure water Made up to 1 mL

[0272] The 1,000× heparin sodium solution was prepared for current use.

[0273] 2. Cell Dissociation Solution (10 mL)

[0274] A specific formula of the cell dissociation solution (10 mL) was shown in Table 7 (same as the formula of the cell dissociation solution used for colorectal cancer solid tumor primary cell culture).

[0275] 3. Dissociation Stop Solution (100 mL)

[0276] A specific formula of the dissociation stop solution (100 mL) was shown in Table 8 (same as the formula of the dissociation stop solution used for colorectal cancer solid tumor primary cell culture).

[0277] 4. Colorectal Cancer Ascites Primary Tumor Cell Medium (100 mL)

[0278] A specific formula of the colorectal cancer ascites primary tumor cell medium (100 mL) was shown in Table 9 (same as the formula of the colorectal cancer solid tumor primary cell medium used for colorectal cancer solid tumor primary cell culture).

[0279] 5. Cell Cryopreservation Solution

[0280] A specific formula of the cell cryopreservation solution was shown in Table 22 (same as the formula of the cell cryopreservation solution used for colorectal cancer solid tumor primary cell culture).

Embodiment 17. Obtaining of Colorectal Cancer Ascites Samples

[0281] 1. Cooperation with a top three hospital, the cooperation had passed the formal medical ethics review.

[0282] 2. Attending physicians selected participating patients according to the clinical indications specified in the medical guidelines, and selected appropriate samples according to the intraoperative clinical indications for in vitro culture. The selection criteria for samples were: primary colorectal cancer, the pathological stage was stage IV, the pathological type was colorectal cancer or colorectal cancer metastasis lesion, the patients had malignant ascites required to be discharged, and the discharge volume was not less than 50 mL.

[0283] 3. The attending physicians provided basic clinical information such as patient's gender, age, medical history, family history, smoking history, pathological stage type, and clinical diagnosis. Information related to patient's privacy, such as patient's name and ID number, was concealed and replaced with a uniform experiment number. The naming principle of the experiment number was the eight-digit date of the collected sample+the last four digits of the patient's hospitalization number. For example, if a sample was provided on Jan. 1, 2018 and the patient's hospitalization number was T001512765, the experiment number of the sample was 201801012765. 4. The attending physicians collected ascites samples with sterile equipment and vessels. The samples were temporarily preserved on ice after being isolated, and transported to a laboratory within 72 hours for next operation.

Embodiment 18. Pretreatment of Colorectal Cancer Ascites Samples

[0284] The Following Embodiment was Operated on Ice:

[0285] 1. The colorectal cancer ascites samples were placed on ice for about 30 minutes, so that clots and large insoluble solids in the samples precipitated to bottoms of sample tubes.

[0286] 2. The supernatant was carefully transferred to 50 mL sterile centrifuge tubes.

[0287] 3. 2,000 g was centrifuged at normal temperature for 5 minutes, and the supernatant was discarded.

[0288] 4. The cell precipitate was re-suspended with the cell isolation buffer (Table 33), 2,000 g was centrifuged at normal temperature for 5 minutes, and the supernatant was discarded.

[0289] 5. The cell precipitate was re-suspended with the cell isolation buffer (Table 33), and the concentration of cells was adjusted to 10.sup.7-10.sup.8 cells/mL.

Embodiment 19. Density Gradient Centrifugation of Colorectal Cancer Ascites Samples

[0290] 1. A Ficoll cell isolation solution (MP #50494) with the same volume as the cell suspension was taken with a 50 mL sterile centrifuge tube.

[0291] 2. The cell suspension was carefully added to the upper layer of the cell isolation solution to form a clear interface between the two.

[0292] 3. 2,000 g was horizontally centrifuged at normal temperature for 20 minutes.

[0293] 4. Albuginea in the middle layer was sucked into a new tube.

[0294] 5. The cell precipitate was re-suspended with 20 mL of sterile PBS, 1,500 g was centrifuged at normal temperature for 10 minutes, and the supernatant was discarded.

[0295] 6. The cell precipitate was re-suspended with the colorectal cancer ascites primary tumor cell medium (Table 9), cell statuses were observed under a microscope, and cells were counted.

[0296] As shown in FIG. 6, in the single cell suspension obtained by isolation from the colorectal cancer ascites samples, in addition to tumor cells, there were also a large number of other cells of various types, such as red blood cells, lymphocytes, and fibrocytes. One of the advantages of this method was that in the subsequent culture process, only the cancer cells can be expanded in a large amount, while other cells gradually decreased or even disappeared, and finally high-purity colorectal cancer primary tumor cells were obtained.

Embodiment 20. Culture of Primary Tumor Cells Derived from Colorectal Cancer Ascites

[0297] 1. Suspension culture was performed on the colorectal cancer ascites primary tumor cells by using a low-attachment-surface. Taking a six-pore plank as an example, the cells were planked with a density of 10.sup.6 cells per pore and cultured in a cell incubator at 37° C. and 5% CO.sub.2.

[0298] 2. Cell statuses were observed every day, and the medium was changed every 3 days until the cells formed masses with a diameter of about 80 μm.

[0299] As shown in FIG. 7, after 3-10 days of culture, the cancer cells were expanded in a large amount to form cell masses with a diameter of 80 μm. The total number of the tumor cells can exceed 10.sup.7, and other types of cells were significantly reduced or even disappeared. This method has been tested on a large number of samples, showing that the success rate of in vitro culture of the primary tumor cells derived from colorectal cancer ascites can reach 80%.

Embodiment 21. Passage of Primary Tumor Cells Derived from Colorectal Cancer Ascites

[0300] 1. Cell masses in a culture dish were collected, 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0301] 2. The cell masses were washed with a sterile PBS solution, 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0302] 3. The cell masses were re-suspended with the cell dissociation solution (Table 7) and dissociated at 37° C. The dissociation of the cell masses was observed under a microscope every 5 minutes, until the cell masses were dissociated into individual cells.

[0303] 4. The dissociation reaction was stopped with 10 times the volume of the dissociation stop solution (Table 8), and a cell suspension was collected.

[0304] 5. 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded. 6. The cell precipitate was re-suspended with the colorectal cancer ascites primary tumor cell medium (Table 9), and cells were counted.

[0305] 7. The colorectal cancer primary cells were cultured by using a low-attachment-surface. Taking a six-pore plank as an example, the cells were planked with a density of 10.sup.6 cells per pore and cultured in a cell incubator at 37° C. and 5% CO.sub.2.

Embodiment 22. Cryopreservation of Primary Tumor Cells Derived from Colorectal Cancer Ascites

[0306] The colorectal cancer ascites primary tumor cells subjected to the suspension culture can be cryopreserved after 2-3 passaging expansions:

[0307] 1. Cell masses in a culture dish were collected, 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0308] 2. The cell masses were washed with a sterile PBS solution, 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0309] 3. The cell masses were re-suspended with the cell dissociation solution (Table 7) and dissociated at 37° C. The dissociation of the cell masses was observed under a microscope every 5 minutes, until the cell masses were dissociated into individual cells.

[0310] 4. The reaction was stopped with 10 times the volume of the dissociation stop solution (Table 8), a cell suspension was collected, and cells were counted.

[0311] 5. 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0312] 6. The cell precipitate was re-suspended with the cell cryopreservation solution (Table 22) according to a density of 10.sup.6 cells/mL, and the cell suspension was dispensed in 2 mL cryopreservation tubes according to 1 ml of the cell suspension per tube, cryopreserved with a gradient cooling box overnight and then transferred to liquid nitrogen for long-term preservation.

Embodiment 23. Resuscitation of Primary Tumor Cells Derived from Colorectal Cancer Ascites

[0313] The primary tumor cells derived from colorectal cancer ascites, preserved in the liquid nitrogen, can be resuscitated:

[0314] 1. 37° C. sterile water was prepared five minutes in advance.

[0315] 2. The cryopreservation tube was taken out of the liquid nitrogen, and the cells were quickly thawed in the 37° C. sterile water.

[0316] 3. 800 g was centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

[0317] 4. The cell precipitate was re-suspended with the colorectal cancer ascites primary tumor cell medium (Table 9), the primary tumor cells derived from colorectal cancer ascites were cultured by using a low-attachment-surface, each tube of the cells was resuscitated to a 3.5 cm culture dish, and the cells were cultured in a cell incubator at 37° C. and 5% CO.sub.2.

Embodiment 24. HE Staining Identification of Primary Tumor Cells Derived from Colorectal Cancer Ascites

[0318] Description of Reagent Consumables Used in the Following Embodiment:

[0319] HE staining kit (Beijing Solarbio Science & Technology Co., Ltd., #G1120);

[0320] Cationic anti-dropping slide (Beijing ZSGB-Bio Co., Ltd.);

[0321] Xylene, methanol, acetone (Beijing Chemical Reagent Company, analytically pure);

[0322] Neutral resin glue (Beijing Yili Fine Chemical Co., Ltd.).

[0323] 1. A cell suspension with a concentration of 10.sup.4 cells/mL was prepared from suspended primary tumor cells derived from colorectal cancer ascites, which were cultured with the colorectal cancer ascites primary tumor cell medium (Table 9, where the final concentration of the human recombinant protein EGF was 20 ng/mL; the final concentration of the human recombinant protein bFGF was 20 ng/mL; the final concentration of the human recombinant protein HGF was 10 ng/mL; the final concentration of the human recombinant protein Wnt-3a was 200 ng/mL; the final concentration of the human recombinant protein Noggin was 100 ng/mL; the final concentration of the SB202190 was 10 μM; the final concentration of the A83-01 was 1 μM; the final concentration of the N-acetyl-L-cysteine was 1 mM; the final concentration of the Nicotinamide was 10 mM; the final concentration of the cortisol was 20 ng/mL; the final concentration of the Y-27632 was 10 μM), and 10 μL was dripped onto a cationic anti-dropping slide and dried naturally.

[0324] 2. 50 μL of a mixed solution of methanol/acetone (volume ratio 1:1) pre-cooled at 4° C. was carefully dripped onto the air-dried cells, and then the slide was placed in a 4° C. refrigerator and immobilized for 10 minutes.

[0325] 3. The slide with immobilized cells was taken out and dried naturally at room temperature.

[0326] 4. The slide was washed twice with 200 μL of PBS.

[0327] 5. When the water on the slide was slightly dry, 100 μL of hematoxylin staining solution was added for staining 1 minute.

[0328] 6. The hematoxylin staining solution was sucked away, and the slide was washed with 200 μL of tap water 3 times.

[0329] 7. 100 μL of differentiation solution was dripped for differentiating 1 minute.

[0330] 8. The differentiation solution was sucked away, and the slide was sequentially washed twice with tap water and once with distilled water.

[0331] 9. The water on the surface of the slide was sucked away, and 200 μL of eosin staining solution was added for staining 40 seconds.

[0332] 10. The eosin staining solution was sucked away, and the slide was sequentially rinsed with 75%, 80%, 90%, and 100% ethanol and dewatered for 20 seconds, 20 seconds, 40 seconds, and 40 seconds.

[0333] 11. After the ethanol was dry, 50 μL of xylene was dripped for cell permeation.

[0334] 12. After the xylene was completely dry, a drop of neutral resin glue was dripped, the slide was sealed with a cover glass, the cells were observed under a microscope and pictures were taken.

[0335] FIG. 8 shows an HE staining effect map of primary tumor cells derived from colorectal cancer ascites cultured in vitro. It can be seen that these cells generally had cancer cell characteristics such as high nuclear-cytoplasmic ratio, dark nuclear staining, intranuclear chromatin agglutination, multinucleation, and uneven cell size.

Embodiment 25. Immunofluorescent Staining Identification of Primary Tumor Cells Derived from Colorectal Cancer Ascites

[0336] Description of Reagents Used in the Following Embodiment:

[0337] Paraformaldehyde (Beijing Chemical Reagent Company, analytically pure), paraformaldehyde powder was dissolved in ultra-pure water to prepare a 4% paraformaldehyde solution;

[0338] Methanol, dimethyl sulfoxide (Beijing Chemical Reagent Company, analytically pure);

[0339] Hydrogen peroxide (Beijing Chemical Reagent Company, 35%);

[0340] Methanol, dimethyl sulfoxide, and 35% hydrogen peroxide were mixed in a ratio of 4:4:1 to prepare a DANCLAN rinsing solution;

[0341] Bovine serum albumin (Sigma, #A1933), the bovine serum albumin was dissolved in a PBS solution to prepare a 3% BSA solution;

[0342] Immunofluorescent primary antibody (Abeam, #ab17139);

[0343] Immunofluorescent secondary antibody (CST, #4408);

[0344] Hoechst staining solution (Beijing Solarbio Science & Technology Co., Ltd., #C0021);

[0345] Immunofluorescent staining was performed on the colorectal cancer ascites derived primary tumor cell masses cultured with the colorectal cancer ascites primary tumor cell medium (Table 9, where the final concentration of the human recombinant protein EGF was 20 ng/mL; the final concentration of the human recombinant protein bFGF was 20 ng/mL; the final concentration of the human recombinant protein HGF was 10 ng/mL; the final concentration of the human recombinant protein Wnt-3a was 200 ng/mL; the final concentration of the human recombinant protein Noggin was 100 ng/mL; the final concentration of the SB202190 was 10 μM; the final concentration of the A83-01 was 1 μM; the final concentration of the N-acetyl-L-cysteine was 1 mM; the final concentration of the Nicotinamide was 10 mM; the final concentration of the cortisol was 20 ng/mL; the final concentration of the Y-27632 was 10 μM) according to the following steps. The primary antibody was CK8+CK18, which characterized epithelial cells.

[0346] 1. The ascites derived primary tumor cell masses in the culture dish were collected and washed once with a PBS, and the cell precipitate was re-suspended with 4% paraformaldehyde and immobilized at 4° C. overnight.

[0347] 2. 800 g was centrifuged, the supernatant was discarded, and the cell precipitate was re-suspended with a pre-cooled methanol solution and placed on ice for 1 hour.

[0348] 3. 800 g was centrifuged, the supernatant was discarded, and the cell precipitate was re-suspended with a DANCLAN rinsing solution and placed at room temperature for 2 hours.

[0349] 4. 800 g was centrifuged, the supernatant was discarded, and the cells were washed sequentially with 75%, 50%, and 25% methanol solutions diluted with a PBS for 10 minutes each time.

[0350] 5. 800 g was centrifuged, the supernatant was discarded, and the cell precipitate was suspended with a 3% BSA solution and sealed at room temperature for 2 hours.

[0351] 6. The primary antibody was diluted with a 3% BSA solution in a ratio of 1:500, the cell precipitate was re-suspended with the antibody diluent, and the primary antibody was placed at 4° C. overnight

[0352] 7. 800 g was centrifuged, the supernatant was discarded, and the cell precipitate was washed with a PBS solution 5 times, 20 minutes each time.

[0353] 8. The secondary antibody was diluted with a 3% BSA solution in a ratio of 1:2,000, the cell precipitate was re-suspended with the antibody diluent, and the secondary antibody was placed at room temperature for 2 hours.

[0354] 9. 800 g was centrifuged, the supernatant was discarded, and the cell precipitate was washed with a PBS solution 5 times, 20 minutes each time.

[0355] 10. A 100× Hoechst staining solution was added in a volume ratio of 1/100, and staining was performed at room temperature for 20 minutes.

[0356] 11. The cell precipitate was washed twice with a PBS solution for 10 minutes each time. The staining of the cell masses was observed with a laser confocal microscope.

[0357] FIG. 9 shows an immunofluorescent staining effect map of colorectal cancer ascites derived primary tumor cell masses cultured in vitro. It can be seen that the cells constituting the cell masses were all CK8/CK18 positive and were of epithelial origin, which confirmed that highly pure tumor cells were obtained by this culture method. 20 colorectal cancer ascites primary cultures were identified by immunofluorescent staining. The statistical results showed that the proportion of tumor cells among the colorectal cancer ascites derived primary tumor cells obtained by this method reached 72%-91% (Table 35).

TABLE-US-00035 TABLE 35 Immunofluorescent staining identification of primary culture of colorectal cancer ascites CK8/CK18 Serial number Sample number positive rate 1 20180722ZQH3005 86% 2 20181217ZZY1386 84% 3 20180530GW6307 78% 4 20180522HC6914 76% 5 20180508LY0054 74% 6 20181106NLC6799 85% 7 20180423CXC9250 87% 8 20180423WFY4087 85% 9 20180424ZZH5496 88% 10 20181220WF2858 72% 11 20181008JF4076 77% 12 20180530FKX7348 78% 13 20180703QWG0065 81% 14 20180918JJB0734 75% 15 20180531LWY1184 90% 16 20181101LZY1263 77% 17 20180713QZG1394 91% 18 20180711DHD6099 80% 19 20180716CL8052 74% 20 20180702QCF9653 86%

Embodiment 26. Colorectal Cancer Ascites Derived Primary Tumor Cell Cultures and Primary Tumor Tissues

[0358] The DNA extraction process mentioned in the following embodiment was performed by means of a Tiangen blood/tissue/cell genome extraction kit (DP304).

[0359] The library building process mentioned in the following embodiment was performed by means of an NEB DNA sequencing library building kit (E7645).

[0360] The high-throughput sequencing mentioned in the following embodiment referred to an Illumina HiSeq X-ten sequencing platform.

[0361] 1. Colorectal cancer ascites samples were taken, 20 mL of a colorectal cancer ascites sample was first taken for centrifugation before in vitro culture operation to obtain an ascites cell precipitate, DNA extraction, library building and whole genome high-throughput sequencing (WGS) with a sequencing depth of 30× were performed, and the remaining colorectal cancer ascites samples were used for in vitro culture of colorectal cancer ascites primary tumor cells (the specific method refers to the previous embodiment).

[0362] 2. The colorectal cancer ascites samples treated were cultured with the colorectal cancer ascites primary tumor cell medium (Table 9, where the final concentration of the human recombinant protein EGF was 20 ng/mL; the final concentration of the human recombinant protein bFGF was 20 ng/mL; the final concentration of the human recombinant protein HGF was 10 ng/mL; the final concentration of the human recombinant protein Wnt-3a was 200 ng/mL; the final concentration of the human recombinant protein Noggin was 100 ng/mL; the final concentration of the SB202190 was 10 μM; the final concentration of the A83-01 was 1 μM; the final concentration of the N-acetyl-L-cysteine was 1 mM; the final concentration of the Nicotinamide was 10 mM; the final concentration of the cortisol was 20 ng/mL; the final concentration of the Y-27632 was 10 μM) for a period of time to form cell masses with a diameter of more than 80 μm, recorded as P0 generation cells, and subsequent cell masses were sequentially recorded as P1, P2, . . . , Pn according to the number of passages. 10.sup.6 cells were taken from each of the P1, P2, P3, P4, and P5 generations of colorectal cancer ascites derived primary tumor cell cultures for DNA extraction, library building and whole genome high-throughput sequencing (WGS) with a sequencing depth of 30×.

[0363] 3. Copy number variation (CNV) analysis was performed on each group of sequencing results to compare copy number variations between the cancer cells in the colorectal cancer ascites and the colorectal cancer ascites primary cell cultures of respective generations. As shown in FIG. 10, the copy number variations of the colorectal cancer ascites primary cell cultures of respective generations (P1, P2, P3, P4, P5) were highly consistent with the copy number variation of the cancer cells in the colorectal cancer ascites, so the colorectal cancer ascites derived primary tumor cells obtained by this method can represent the truth of cancer cells in patient's ascites.

Embodiment 27. Comparison of Success Rates of Culturing Colorectal Cancer Ascites Derived Primary Tumor Cells with Different Primary Cell Culture Media

[0364] In this embodiment, the operation method and process of primary culture of all samples were completely the same (refer to the above), and only the formulas of the culture media were different. The various primary cell culture media for testing were shown in Table 36. Scheme D was the formula used in the present invention, as shown in Table 9 (where the final concentration of the human recombinant protein EGF was 40 ng/mL; the final concentration of the human recombinant protein bFGF was 20 ng/mL; the final concentration of the human recombinant protein HGF was 20 ng/mL; the final concentration of the human recombinant protein Wnt-3a was 300 ng/mL; the final concentration of the human recombinant protein Noggin was 150 ng/mL; the final concentration of the SB202190 was 10 μM; the final concentration of the A83-01 was 0.5 μM; the final concentration of the N-acetyl-L-cysteine was 1 mM; the final concentration of the Nicotinamide was 10 mM; the final concentration of the cortisol was 20 ng/mL; and the final concentration of the Y-27632 was 5 μM).

TABLE-US-00036 TABLE 36 Formulas of primary cell culture media for testing (100 mL) Culture Final medium Reagent Brand number Amount concentration Scheme A mTeSR ™1 medium Stemcell#05850 100 mL Scheme B Alveolar Epithelial Cell ScienCell#3201 100 mL Medium Scheme C Double antibodies P/S Gibco#15140122 1 mL 1% HEPES Gibco#15630080 1 mL 10 mM 1,000× human recombinant 1,000× stock solution 250 μL 50 ng/mL protein EGF 1,000× human recombinant 1,000× stock solution 100 μL 20 ng/mL protein bFGF DMEM/F12 medium Gibco#14170161 Made up to 100 mL Scheme D See Table 9

[0365] After preparation, the primary cell culture media were filtered and sterilized with a 0.22 μM syringe filter (Millipore SLGP033RS), and can be preserved at 4° C. for two weeks.

[0366] Each of the four primary cell medium schemes treated 20 colorectal cancer ascites samples. Sample treatment and culture operations were performed according to the methods described in Embodiments 18, 19, and 20. After culture for 10 days, the success rates of colorectal cancer ascites primary tumor cell culture were counted as shown in Table 37:

TABLE-US-00037 TABLE 37 Culture statuses of different culture media Primary cell medium scheme Culture success rate Scheme A 5% (1/20) Scheme B 5% (1/20) Scheme C 25% (5/20)  Scheme D 75% (15/20)

[0367] It can be seen that the primary cell culture media had a great impact on the success rate of colorectal cancer ascites primary cell culture, and the colorectal cancer ascites primary tumor cell medium (Table 9) used in the present invention can stimulate the proliferation of cancer cells in the colorectal cancer ascites samples to the greatest extent, and improve the success rate of colorectal cancer ascites primary tumor cell culture.

Embodiment 28. Comparison of Success Rates of Passaging Colorectal Cancer Ascites Primary Tumor Cells with Different Cell Dissociation Solutions

[0368] In this embodiment, the operation method and process of primary cell passage of all samples were completely the same (refer to the above), and only the formulas of the cell dissociation solutions were different. The various cell dissociation solutions for testing were shown in Table 38. The scheme D was the formula used in the present invention, and details were shown in Table 7.

TABLE-US-00038 TABLE 38 Formulas of cell dissociation solutions for testing (10 mL) Cell dissociation Final solution Reagent Brand number Amount concentration Scheme A Trypsin Gibco#25200056 10 mL Scheme B 0.5M EDTA Invitrogen#AM9261 10 μL 5 mM TrypLE ™ Express Gibco#12604013 Made up to 10 mL Scheme C 10× collagenase I 10× stock solution 1 mL 200 U/mL 10× collagenase II 10× stock solution 1 mL 200 U/mL 20× collagenase IV 20× stock solution 0.5 mL 100 U/mL PBS Gibco#21-040-CVR Made up to 10 mL Scheme D See Table 7

[0369] The cell dissociation solutions were prepared for current use.

[0370] 20 colorectal cancer ascites samples that had been successfully cultured were selected, and the cultured colorectal cancer ascites primary tumor cells were continuously passaged with the above-mentioned four cell dissociation solutions according to the method described in Embodiment 21. When cancer cells expanded to form cell masses with a diameter of 80 μm, the cancer cells were passaged (not more than 10 times), and the maximum number of passages was recorded. The statistical results were shown in Table 39:

TABLE-US-00039 TABLE 39 Culture statuses of different cell dissociation solutions Serial Scheme Scheme Scheme Scheme number Sample number A B C D 1 20180712WSS9991 1 4 6 10 2 20180713LCW0205 0 2 5 9 3 20180717WDJ3774 2 4 7 9 4 20181010JJF5094 1 3 5 9 5 20180726ZKY6366 1 3 4 7 6 20180803LYB6630 2 4 6 10 7 20180816WC7596 1 3 6 9 8 20180817HX7778 1 4 7 10 9 20180829ZK1173 0 4 7 9 10 20180816FCG1697 0 2 7 10 11 20180815YJ3423 1 3 5 7 12 20180827ZXC3723 0 1 4 6 13 20181106SBF4195 1 2 4 7 14 20180904ZZC5555 0 3 5 7 15 20180905DP7828 2 4 7 10 16 20181105LXP8013 2 4 6 10 17 20181029JZH0836 0 0 2 6 18 20181031XLJ1029 1 3 6 9 19 20180911CZY1078 1 4 7 10 20 20180920SD1350 1 4 7 10 Summary of possible passages 0-2 0-4 2-7 6 times times times times or more

[0371] It can be seen that the formulas of the cell dissociation solutions had a great impact on the success rate of passage of the colorectal cancer ascites primary tumor cells. The cell dissociation solution (Table 7) used in the present invention can gently dissociate cancer cells in the cell masses, so that the samples can be continuously passaged while maintaining the activity of the colorectal cancer ascites primary tumor cells.

Embodiment 29. Culture of Colorectal Cancer Ascites Primary Tumor Cells with Cell Culture Consumables of Different Materials

[0372] In this embodiment, the operation method and process of primary culture of all samples were completely the same (refer to the above), and only the materials of cell culture consumables (unmodified) were different (Table 40).

TABLE-US-00040 TABLE 40 Effects of unmodified culture consumables of different materials on culture of colorectal cancer ascites primary tumor cells Serial number Sample number PS PC PMMA COC COP LAS 1 20180913GW1496 Failure Failure Failure Failure Failure Failure 2 20181010LTT5115 Failure Failure Failure Failure Failure Failure 3 20180920CQ6010 Failure Failure Failure Failure Failure Success 4 20181010CWY0293 Success Failure Success Success Success Success 5 20181122WYF0822 Failure Failure Success Success Failure Success 6 20181113THC1098 Failure Failure Failure Failure Failure Success 7 20181012HZY1873 Failure Failure Failure Failure Failure Success 8 20181022SML2256 Success Success Success Success Failure Success 9 20181227ZMX2706 Failure Failure Failure Success Success Success 10 20181101DHY3948 Failure Failure Success Failure Failure Success Success rate 2/10 1/10 4/10 4/10 2/10 8/10 Note: Polystyrene (PS), Polycarbonate (PC), poly-methyl methacrylate (PMMA), COC resin, Cyclo Olefin Polymer (COP), low-attachment-surface (LAS).

[0373] It can be seen from Table 40 that the culture consumables of different materials had a certain impact on the success rate of colorectal cancer ascites primary tumor cell culture, and the low-attachment-surface (LAS) had the highest success rate.

Embodiment 30. Culture of Colorectal Cancer Ascites Primary Tumor Cells with CYTOP-Modified Cell Culture Consumables

[0374] In this embodiment, the operation method and process of primary culture of all samples were completely the same (refer to the above), the modification methods of CYTOP were completely the same, and only the materials of cell culture consumables were different (Table 41).

[0375] The method of CYTOP modification was as follows: the cell culture vessel was first etched with pure oxygen under a power of 20 W for 3 minutes. Then, the surface of the culture dish or culture plate was covered with an appropriate amount (taking a 96-pore plank as an example, 20 μL per pore, the appropriate amount meant to completely cover the bottom of the culture dish) of a 1% CYTOP solution (see Table 42 for the formula), and the culture dish or culture plate can be used after the CYTOP solution was completely dried.

TABLE-US-00041 TABLE 41 Effects of CYTOP-modified culture consumables of different materials on culture of colorectal cancer ascites primary tumor cells Serial number Sample number PS PC PMMA COC COP LAS 1 20181022DJ4142 Success Success Success Success Success Success 2 20181030FB4172 Success Success Success Success Success Success 3 20181113YLS4256 Success Success Success Success Success Success 4 20181101QCF4672 Success Success Success Success Success Success 5 20181109YCH6089 Failure Failure Failure Failure Failure Failure 6 20181130YX6531 Success Success Success Success Success Success 7 20181031XYS6782 Success Success Success Success Success Success 8 20181212ZWP6888 Success Success Success Success Success Success 9 20181108HYX7042 Success Success Success Success Success Success 10 20181109WTC7313 Failure Failure Failure Failure Failure Failure Success rate 8/10 8/10 8/10 8/10 8/10 8/10

[0376] It can be seen from Table 41 that the CYTOP modification can effectively improve the culture success rates of various materials.

TABLE-US-00042 TABLE 42 1% CYTOP solution (100 mL) Final Reagent Brand number Amount concentration CYTOP Asashi glass#CTL-809M 1 mL 1% Fluorine oil 3M#FC40 Made up to 100 mL

[0377] The 1% CYTOP solution can be preserved for a long term at normal temperature after preparation.

Embodiment 31. Drug Sensitivity Test Using Colorectal Cancer Ascites Primary Tumor Cells

[0378] The chemotherapy drugs 5-Fluorouracil, Oxaliplatin, and Irinotecan used in this embodiment were all Selleck products.

[0379] The Celltiter-Glo cell viability test kit mentioned in this embodiment was a Promega product. In vitro drug sensitivity test was performed using the colorectal cancer ascites primary tumor cells cultured according to the present invention: the primary cells were seeded with a density of 10.sup.5 cells/pore by means of a standard-sized 96-pore low-attachment cell culture plate, and 5 drug concentration gradients were provided for each drug, where n=3. After the drug was added, the cells were incubated for 7 days at 37° C. and 5% CO.sub.2. After the drug action was over, the viability of the cells in each pore was tested with the Celltiter-Glo cell viability test kit. The experimental results were shown in FIG. 11. The results showed that the colorectal cancer ascites primary tumor cells obtained by this method can be used for in vitro drug sensitivity test.

Embodiment 32. Microplate Chip Processing

[0380] In this embodiment, a microplate chip for culturing the colorectal cancer primary cells of the present invention was obtained by means of injection molding from a PMMA material (or a material such as PS, PC, COC, COP, or LAS). The chip can be used for colorectal cancer primary cell culture and in vitro drug sensitivity test. The design drawing of the microplate chip was shown in FIG. 12.

[0381] In the actual application process, the structure of the microplate chip shown in the design drawing FIG. 12 was obtained from the PMMA material (or a material such as PS, PC, COC, COP, or LAS), and then the surface of the microplate chip was modified by means of the above-mentioned CYTOP modification method (see Embodiment 30) to obtain the microplate chip for culturing the colorectal cancer primary cells here.

Embodiment 33. Culture of Colorectal Cancer Primary Tumor Cells from Micro Colonoscopy Puncture Samples

[0382] In this embodiment, the operation method and process of primary culture of all samples were completely the same as the culture method of the colorectal cancer solid tumor primary cells (refer to the above), only the sources of the samples were different, and the samples were unified into two colonoscopy samples (Table 43).

TABLE-US-00043 TABLE 43 Culture statuses of colorectal cancer primary cells from colonoscopy puncture sample Serial number Sample number Culture status 1 20181115WLF8886 Failure 2 20181115LWW8966 Failure 3 20181115ZXQ9002 Success 4 20181123QLP9291 Success 5 20181122HYJ0804 Success 6 20181203FFZ1845 Success 7 20181114LWT1950 Success 8 20181115MFH2041 Success 9 20181129LLW2643 Success 10 20181129YLL2937 Success Success rate 8/10

[0383] It can be seen from Table 43 that the micro samples obtained by colonoscopy puncture can still be subjected to colorectal cancer primary cell culture with a high success rate.

INDUSTRIAL APPLICATION

[0384] The present invention provides a method for extracting and culturing colorectal cancer primary tumor cells from fresh colorectal cancer solid tumor tissues or colorectal cancer ascites and supporting reagents. The method has the following advantages: for the colorectal cancer solid tumor tissues, the amount of a tissue sample is small, and only about 20 mg of colorectal cancer surgery sample is required. For the colorectal cancer ascites, samples are readily obtainable, the ascites discharged during conventional therapy of colorectal cancer patients is used to the greatest extent, and patients have no additional trauma and pain; the amount of a sample is small, and only about 50 mL of colorectal cancer ascites sample is required; the samples do not need to be immediately treated after collection, and the cell viability of more than 90% can be guaranteed after the samples isolated are treated according to this method within 72 hours. The culture cycle is short, and a 10.sup.7 magnitude order of colorectal cancer primary tumor cells can be obtained only within 3-10 days. The culture stability is high, and the success rates of in vitro culture of qualified colorectal cancer solid tumor and ascites samples by this method are as high as 70%-80%. The cell purity is high, and in the colorectal cancer primary cell culture obtained by this method, the proportion of cancer cells can reach 70%-95% and the interference of parenchyma cells is little. The colorectal cancer primary cell culture obtained by the method of the present invention can be used for in vitro experiments, second-generation sequencing, building of animal models, and building of cell lines at multiple cell levels. It is foreseeable that this culture method has broad application prospects in the fields of research and clinical diagnosis and treatment of colorectal cancer.