Temozolomide tmz

After 7 days of spheroid culture in the brain cancer chip, a combination of drugs was applied to the chip. Bevacizumab, trade name Avastin (BEV, Roche, Basel, Switzerland) was prepared at a concentration of 7.5 µM in EGM-2 media^{36 (link)}. Temozolomide (TMZ, Sigma Aldrich, St. Louis, MO) was dissolved in dimethysulfoxide (DMSO) to prepare a solution of 10 mM TMZ^{37 (link)}. This solution was further diluted to 600 µM TMZ in EGM-2 media. Cell culture media was removed from the chip and replaced by adding 7.5 µM BEV to the left inlet and 600 µM TMZ to the right inlet 100 µL at a time, repeated 4 times^{38 (link)–40 (link)}. Drug administration occurred only once, and cells were left in the brain cancer chip for 7 days following drug administration. Control (non-drug treated) brain cancer chips were maintained under the same conditions.

Cells were plated in 96-well plates at 1×10⁴ cells per well after transient transfection or adherence of stable transfected cells. After 24 h, the cells were treated with different concentrations of imatinib (Novartis, Basel, Switzerland), etoposide (VP-16) (Sigma Chemical Co., St. Louis, MO) and temozolomide (TMZ) (Sigma Chemical Co., St. Louis, MO), each at four concentrations ranging from 50 to 200 μg/ml for 48 h. The range of drug concentrations were based on earlier studies and aimed at obtaining IC50 values both for highly sensitive and resistant cases. The absorbance at 450 nm was measured after incubation with 10 μl of CCK-8 reagent (Dojindo, Molecular Technologies, Dojindo, Japan) for four hours. After shaking for one min, the spectrophotometric absorbance of the samples was determined by using Ultra Multifunctional Microplate Reader (Tecan) at 450 nm. The assay was conducted in five replicate wells for each sample and three parallel experiments were performed. For cell viability analysis, cells were plated in 96-well plates at 2×10³ per well in a final volume of 100 μl. And cells were incubated with 50 μg/ml TMZ for 24, 48, 72, 96 and 120 h. Cell growth were determined with CCK-8 according to the manufacturer's instructions.