The Effects Of CO₂-Independent Cell Culture System On The Proliferation Of Bladder Cancer Cell T-24

Objective To evaluate the effects of CO₂-independent culture cell system on the prolilferation of bladder cancer cell T-24. Whether there are significant differences in CO₂-dependent and CO₂-independent cell culture systems.Methods Bladder cancer cell T-24 was cultured in CO₂-independent culture cell system or CO₂-dependent culture cell system at 37℃, in 5% carbon dioxide incubator and 37℃in air incubator. The pH value of these two culture cell systems were measured 7 days. Differences of the cell viability were assayed with MTS and colony forming assay. The cell cycle and apoptosis ratio of T-24 cells treated in the two culture cell systems were tested throuth FCM. The expression of survivin mRNA and caspase-3 mRNAof T-24 cells culturing with two system were detected by RT-PCR. The expression of survivin and caspase-3 proteins of T-24 cells culturing with two system were detected by western bolt.Results 1. pH value: The pH of CO₂-independent culture cell system at 37℃in air incubator and the pH of CO₂-dependent culture cell system at 37℃ in 5% carbon dioxide incubator were about 7.4 in 7 day(sP>0.05). However, the pH of CO₂-dependent culture cell system at 37℃in air incubator and the pH of CO₂-dependent culture cell system at 37℃in 5% carbon dioxide incubator were different(P<0.05)in 7 days. The pH value of CO₂-dependent culture cell system at 37℃in air incubator was 8.8.2. Cell clone numbers: The clone numbers of T-24 cell cultured with CO₂-independent culture cell system at 37℃in air incubator(324±11) and CO₂-dependent culture cell system at 37℃in 5% carbon dioxide incubato(r320±15)were not different(P>0.05). But, the clone numbers of T-24 cell cultured with CO₂-dependent culture cell system at 37℃in air incubator(66±6) and CO₂-dependent culture cell system at 37℃in 5% carbon dioxide incubator the clone numbers were differen(tP<0.05). The clone numbers of T-24 cell cultured with CO₂-dependent culture cell system at 37℃in air incubator decreased obviously.3. Cell proliferation curve: The proliferation of T-24 cells cultured with CO₂-independent culture cell system at 37℃in air incubator and CO₂-dependent culture cell system at 37℃in 5% carbon dioxide incubator were not different(P>0.05). However, the proliferation of T-24 cells cultured with CO₂-dependent culture cell system at 37℃in air incubator and CO₂-dependent culture cell system at 37℃in 5% carbon dioxide incubator the cell proliferation were different(P<0.05). The cell proliferation of T-24 cells cultured with CO₂-dependent culture cell system at 37℃in air incubator decreased obviously.4. The apoptosis ratio: The apoptosis ratio of T-24 cells cultured with CO₂-independent culture cell system at 37℃in air incubator and CO₂-dependent culture cell system at 37℃in 5% carbon dioxide incubator were not different(P>0.05). However, the apoptosis ratio of T-24 cells cultured with CO₂-dependent culture cell system at 37℃in air incubator and CO₂-dependent culture cell system at 37℃in 5% carbon dioxide incubator were different(P<0.05).The numbers of apoptotic T-24 cells cultured with CO₂-dependent culture cell system at 37℃in air incubator increased obviously.5. RT-PCR and Western blot: The expression of survivin and caspase-3 of T-24 cells cultured with CO₂-independent culture cell system at 37℃in air incubator and CO₂-dependent culture cell system at 37℃in 5% carbon dioxide incubator were not different(P>0.05). However, the expression of caspase-3 of T-24 cells cultured with CO₂-dependent culture cell system at 37℃in air incubator and CO₂-dependent culture cell system at 37℃in 5% carbon dioxide incubator were different(P<0.05). The expression of caspase-3 of T-24 cells cultured with CO₂-dependent culture cell system at 37℃in air incubator increased obviously, but the expression of survivin did not significantly change.Conclusion CO₂-independent culture cell system is parallel to CO₂-dependent culture cell system on the sides of cell proliferation and gene expression.