The
in vitro chemosensitivity of fresh surgical specimens of GC was evaluated using the MTT assay as reported by Mossman, with some modifications [
23]. The tissue specimens obtained during surgery were from patients who had given written informed consent. Resected specimens were stored in Hank’s balanced salt solution (Gibco Gaithersburg, MD, USA) that contained 100 IU penicillin, 100 μg streptomycin and 0.25 μg amphotericin B (all from Gibco) per ml. Single-cell suspensions were prepared enzymatically by incubating the specimens for 30 min in 0.5 mg/ml pronase, 0.2 mg/ml collagenase type І and 0.2 mg/ml DNase (all from Sigma). After 2 centrifugations (1000 r/min), the tumor cells were suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, diluted to 1×10
5 cells/ml and 100 μl aliquots were plated into 96 well microplates (Gibco) to give approximately 10
4 cells per well. The drug solutions were dissolved in RPMI 1640 and 100 μl aliquots were added to each well to give final concentrations of 10 μg/ml MMC, 50 μg/ml 5-FU, 25 μg/ml CDDP, 5 μg/ml TAX, or 4 μg/ml ADM. The control wells contained 100 μl of the cell suspension and 100 μl RPMI 1640 containing 10% FBS, and 200 μl RPMI with 10% FBS was used as a blank. The plates were incubated for 48 h at 37°C in a humidified atmosphere containing 95% air and 5% CO
2. 20 μl mixture of 0.4% MTT (Sigma) and 0.1 M sodium succinate (each dissolved in 10 μl phosphate-buffered saline and filtered through a 0.45 μm membrane filter (Millipore, Bedford, MA, USA)), was then added and the plates were incubated for an additional 3 h at 37°C. After the final incubation, 150 μl dimethyl sulfoxide (Gibco) was added to each well to dissolve the MTT-formazan salt and the plates were mechanically shaken for 10 min on a mixer. The optical densities of each well were determined using a model SM-3 easy reader (Tianshi, Beijing, China) at 570 nm. The inhibition rates (IR) were calculated using the formula (1 – A/B) ×100%, where A and B represent the mean absorbance of the drug-treated and control wells, respectively. The effects were regarded as positive when IR values were ≥ 30%.