Method for culturing gynaecological tumour primary cells and matching culture medium

A method for culturing primary gynecological tumor cells, comprising the step of:

• (1) obtaining primary gynecological solid tumor cells or primary tumor cells in gynecological tumor pleural effusions and ascites samples;
• wherein the primary gynecological solid tumor cells are obtained by dissociating gynecological solid tumor tissue with the sample dissociation solution;
• the primary tumor cells in gynecological tumor pleural effusions and ascites samples are isolated from the gynecological tumor pleural effusions and ascites samples with the cell isolation buffer;
• wherein the sample dissociation solution comprises:
• a collagenase I having a final concentration of 150-250 U/mL,
• a collagenase III having a final concentration of 250-350 U/mL,
• a collagenase IV having a final concentration of 150-250 U/mL,
• and a phosphate-buffered saline (PBS) as a remainder of the sample dissociation solution;
• the cell isolation buffer comprises:
• two antibiotics penicillin-streptomycin (P/S);
• wherein, the final concentration of penicillin in the two antibiotics P/S is 100-200 U/mL;
• the final concentration of streptomycin in the two antibiotics P/S is 100-200 μg/mL;
• heparin sodium having a final concentration of 10 IU/mL, and
• PBS as a remainder of the cell isolation buffer;
• (2) conducting a suspension culture with a culture medium on the primary gynecological solid tumor cells or primary tumor cells in gynecological tumor pleural effusions and ascites samples obtained in step (1);
• wherein the culture medium consists of:
• a solution containing three antibiotic and antimycotic agents,
• 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
• L-alanyl-L-glutamine,
• human recombinant protein epidermal growth factor (EGF),
• human recombinant protein basic fibroblast growth factor (bFGF),
• human recombinant protein hepatocyte growth factor (HGF),
• human recombinant protein macrophage-stimulating protein (MSP),
• N-acetyl-L-cysteine,
• N-2 Supplement,
• Y-27632,
• progesterone,
• β-estradiol, and
• a basic culture medium for human cell culture;
• wherein,
• a final concentration of penicillin in the solution containing three antibiotic and antimycotic agents is 100-200 U/mL;
• a final concentration of streptomycin in the solution containing three antibiotic and antimycotic agents is 100-200 μg/mL;
• a final concentration of amphotericin B in the solution containing three antibiotic and antimycotic agents is 250-250 ng/ml;
• a final concentration of HEPES is 8-12 mM;
• a final concentration of L-alanyl-L-glutamine is 0.8-12% (v/v);
• 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 MSP is 5-25 ng/ml;
• a final concentration of N-acetyl-L-cysteine is 0.5-2 mM;
• a final concentration of N-2 Supplement is 1% (v/v);
• a final concentration of Y-27632 is 5-20 μM;
• a final concentration of progesterone is 50-100 nM;
• and
• a final concentration of β-estradiol is 10-50 nM.