# Anti-Cancer Drug Screening with Microfluidic Technology
## 4 Combinatorial Drug Screening on a Microscale Sample
### 2D Cell Cultures  
In this way, a radial microfluidic concentration gradient generator of two model anticancer drugs was developed [58]. They tested 65 combinatorial compounds over human uterine cervix cancer (HeLa) cells This compact platform with fewer inlets and splitting  
Figure 3: Combinatorial drug screening using (**a**) a plug-based microfluidic device using braille valves designed on pancreatic cancer biopsies. Reproduced with permission from Ref. [56] under an open access Creative Common CC BY license. (**b**) a programmable membrane-valve-based microfluidic chip with PDMS-based 3D culture chamber. Reproduced with permission from Ref. [57] under an open access Creative Common CC BY license. and mixing steps and a large range of predictable concentration gradients is suitable for high-throughput drug screening. Another combinatorial drug screening microchip generates up to 100 combinatorial concentrations from two input drugs [59]. However, this system has not been applied for screening studies with cells. Similarly, [PERSON] et al. described a programmable microfluidic cell array for combinatorial drug screening on PC3 prostate cancer cells either sequentially or simultaneously [60]. In their device, 64 pair-wise concentration combinations of either doxorubicin or mitoxantrone with TRAIL (TNF-alpha related apoptosis inducing ligand) are tested on cells cultured in 64 independent cell culture chambers. This microfluidic device uses two concentration gradient generators to deliver different concentrations of two drugs to an 8 \(\times\) 8 chamber array with two micropumps for the sequential drug treatment. The advantage of this system is no need for complex multi-layer structures or continuous medium perfusion. However, it should be programmed to individually control the opening and closing of the array of valves, which is a formidable task. This platform has also been tested for curcumin/TRAIL combinational chemotherapy in human prostate cancer PC3 cells [61]. [PERSON] et al. presented a plug-based microfluidic device for combinatorial drug screening of multiple drugs on pancreatic cancer biopsies [56]. They used braille valves designed to generate up to 1140 plugs per biopsy by 10 different drugs and their pairwise combinations. This is an effective tool for rapid and personalized drug screening on small biopsy samples. [PERSON] et al. presented a print-to-screen (P2S) platform for high-throughput screening combinatorial chemotherapy [62]. They tested 165 different drug combinations (two or three drugs) of 10 clinically used anti-cancer drugs against ovarian cancer cells (SKOV-3) for a proof-of-concept study. The essential part of their presented P2S platform is the multi-channel microfluidic cartridge with the duty of printing a combinatorial droplet array. The cartridge compromises a pluggable microfluidic chip and an adapter to align the microfluidic chip with the dot-matrix pinhead. The assembly of the cartridge components is in a plug-and-play manner, and the adapter can be modified for other modular designs. Triplicate sets of all drug combinations generated in a 23 \(\times\) 23 array format was spotted onto a gel array on a PDMS substrate. Then, a sheet of the cell embedded gel was laid on the top of the printed drugs array. Their results showed that the highly cytotoxic compounds over the studied cells were combinations of three drugs. [PERSON] et al. fabricated a plug-and-play, drug-on-pillar platform for combination drug screening using a microfluidic pneumatic drug printing platform [63]. They nano formulated chemotherapeutic drugs, which are more stable for storage, shipping, and subsequent rehydration [64]. MDA-MB-231 triple-negative human breast cancer cells were cultured in a 1536-well plate. Then, a 48 \(\times\) 32 printed drug array was laid on top of the cell culture plate.