US 2022/0177852 A1Published application

Method for culturing primary cells of gastric cancer and gallbladder and bile duct cancer, and supporting reagents

AssigneeGenex Health Co., Ltd

InventorsShenyi YIN, Hanshuo ZHANG

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Abstract

A method for culturing primary cells of gastric cancer and gallbladder cancer and cholangiocarcinoma and auxiliary reagents. A method for culturing primary cells of gastric cancer and gallbladder cancer and cholangiocarcinoma and auxiliary reagents. The core of the technology is that: (1) the solid tumor tissues of gastric cancer and gallbladder cancer and cholangiocarcinoma are treated with a mild cell dissociation reagent, and the primary tumor cells of gallbladder cancer and cholangiocarcinoma in a bile sample are isolated by a mild method to ensure the vitality of cancer cells to the greatest extent; (2) a special serum-free medium is prepared, and tumor cells of gastric cancer and gallbladder cancer and cholangiocarcinoma are cultured in vitro by a suspension culture system to eliminate the interference of normal cells to the greatest extent while ensuring normal amplification of cancer cells.

Claims19

Dependent claims

A primary cell culture medium for gastric cancer and/or gallbladder cancer and cholangiocarcinoma, is composed of antibiotic-antimycotic, HEPES, GlutaMax, non-essential amino acids, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein Wnt-3a, human recombinant protein Noggin, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, Nicotinamide, N-2 Supplement, cortisol, B27, ITS-X, Gastrin 1, Y-27632 and Advanced DMEM/F12 culture medium;

wherein, the final concentration of penicillin in the antibiotic-antimycotic is 100-200 U/mL;
the final concentration of streptomycin in the antibiotic-antimycotic is 100-200 μg/mL;
the final concentration of amphotericin B in the antibiotic-antimycotic is 250-250 ng/mL;
the final concentration of the HEPES is 8-12 mM;
the final concentration of the GlutaMax is 0.8-1.2% (volume percentage);
the concentration of glycine in the non-essential amino acids is 80-120 μM;
the concentration of L-alanine in the non-essential amino acids is 80-120 μM;
the concentration of L-asparagine in the non-essential amino acids is 80-120 μM;
the concentration of L-aspartic acid in the non-essential amino acids is 80-120 μM;
the concentration of L-glutamic acid in the non-essential amino acids is 80-120 μM;
the concentration of L-proline in the non-essential amino acids is 80-120 μM;
the concentration of L-serine in the non-essential amino acids is 80-120 μM;
the final concentration of the human recombinant protein EGF is 10-100 ng/mL;
the final concentration of the human recombinant protein bFGF is 10-50 ng/mL;
the final concentration of the human recombinant protein HGF is 5-25 ng/mL;
the final concentration of the human recombinant protein FGF-10 is 5-25 ng/mL;
the final concentration of the human recombinant protein Wnt-3a is 200-300 ng/mL;
the final concentration of the human recombinant protein Noggin is 100-200 ng/mL;
the final concentration of the SB202190 is 5-10 μM;
the final concentration of the A83-01 is 0.25-1.25 μM;
the final concentration of Primocin is 1% (volume percentage);
the final concentration of the N-acetyl-L-cysteine is 0.5-2 mM;
the final concentration of the Nicotinamide is 5-10 mM;
the final concentration of the N-2 Supplement is 1% (volume percentage);
the final concentration of the cortisol is 20-50 ng/mL;
the final concentration of the B27 is 1.5-2.5% (volume percentage);
the final concentration of the ITS-X is 0.8-1.2% (volume percentage);
the final concentration of the Gastrin 1 is 8-12 nM;
the final concentration of the Y-27632 is 5-20 μM;
and the rest is the Advanced DMEM/F12 medium.

27 more elements

A kit of reagents for culturing primary cells of gastric cancer and/or gallbladder cancer and cholangiocarcinoma, which is any of the following:

(A1) Consisting of the culture medium and all or part of the following:
sample dissociation solution, sample preservation solution and sample washing solution;
The sample dissociation solution is composed of collagenase I, collagenase II, collagenase IV and PBS;
wherein the final concentration of the collagenase I is 150-250 U/mL;
the final concentration of the collagenase II is 150-250 U/mL;
the final concentration of the collagenase IV is 50-150 U/mL;
and the rest is PBS;
The sample preservation solution is composed of fetal bovine serum, antibiotic-antimycotic, HEPES and HBSS;
wherein the final concentration of the fetal bovine serum is 1-5% (volume percentage);
the final concentration of penicillin in the antibiotic-antimycotic is 100-200 U/mL;
the final concentration of streptomycin in the antibiotic-antimycotic is 100-200 μm/mL;
the final concentration of amphotericin B in the antibiotic-antimycotic is 250-500 ng/mL;
the final concentration of the HEPES is 8-12 mM;
and the rest is HBSS;
The sample washing solution is composed of antibiotic-antimycotic and PBS;
wherein the final concentration of penicillin in the antibiotic-antimycotic
is 100-200 U/mL;
the final concentration of streptomycin in the antibiotic-antimycotic is 100-200 μg/mL;
the final concentration of amphotericin B in the antibiotic-antimycotic is 250-500 ng/mL;
and the rest is PBS;
(A2) Consisting of the culture medium and the cell isolation buffer;
The cell isolation buffer is composed of P/S, heparin sodium and PBS;
wherein the final concentration of penicillin in the P/S is 100-200 U/mL;
the final concentration of streptomycin in the P/S is 100-200 μg/mL;
the final concentration of the heparin sodium is 10 IU/mL;
and the rest is PBS;
(A3) Consisting of (A1) and all or part of the following reagents:
cell digestion solution, digestion termination solution and cell cryopreserving solution;
(A4) Consisting of (A2) and all or part of the following reagents:
cell digestion solution, digestion termination solution and cell cryopreserving solution;
The composition of the cell digestion solution is as follows:
each 10 mL of the cell digestion solution contains 4-6 mL of Accutase, EDTA with a final concentration of 5 mM and 1.5-2.5 mL of TrypLE Express, and the rest is PBS;
The digestion termination solution is composed of fetal bovine serum, antibiotic-antimycotic and DMEM culture medium;
wherein the final concentration of the fetal bovine serum is 8-12% (volume percentage);
the final concentration of penicillin in the antibiotic-antimycotic is 100-200 U/mL;
the final concentration of streptomycin in the antibiotic-antimycotic is 100-200 μg/mL;
the final concentration of amphotericin B in the antibiotic-antimycotic is 250-500 ng/mL;
and the rest is the DMEM culture medium;
The cell cryopreserving solution is composed of Advanced DMEM/F12 medium, DMSO and 1% methylcellulose solution;
wherein the volume ratio of the Advanced DMEM/F12 medium, the DMSO and the 1% methylcellulose solution is 20:2:
(0.8-1.2);
the 1% methylcellulose solution is an aqueous solution of methylcellulose with a concentration of 1 g/100 ml.

39 more elements

A method for culturing primary cells of gastric cancer and/or gallbladder cancer and cholangiocarcinoma is either method A or method B:

Method A:
A method for culturing primary cells in solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma, comprising the following steps:
(a1) dissociating solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma with the sample dissociation solution, to obtain primary cells in solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma;
(a2) suspension-culturing the dissociated primary cells in solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma in step (a1) with the medium;
Method B:
A method for culturing primary tumor cells in a bile sample of gallbladder cancer and cholangiocarcinoma, comprising the following steps:
(b1) separating primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma, to obtain primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma;
(b2) suspension-culturing the primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma separated in step (b1) with the medium.

5 more elements

Dependencies are inferred from the claim text.