Supporting Information. Monitoring Tumor Response to Anticancer Drugs. Using Stable Three-Dimensional Culture in a. Recyclable Microfluidic Platform

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1 Supporting Information Monitoring Tumor Response to Anticancer Drugs Using Stable Three-Dimensional Culture in a Recyclable Microfluidic Platform Wenming Liu,*,, Juan Xu, Tianbao Li, Lei Zhao, Chao Ma, Shaofei Shen, and Jinyi Wang*,, College of Science and College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi , China Abstract Supporting Information includes all additional information as noted in the manuscript. S1

2 Materials and Reagents. RTV 615 PDMS prepolymer and curing agent were purchased from Momentive Performance Materials (Waterford, NY, USA). Surface-oxidized silicon wafers were purchased from Shanghai Xiangjing Electronic Technology Ltd. (Shanghai, China). AZ 50XT photoresist and developer were obtained from AZ Electronic Materials (Somerville, NJ, USA) and SU photoresist and developer were obtained from Microchem (Newton, MA, USA). Vincristine (VCR) and bleomycin (BLM) were purchased from Melonepharma Co., Ltd (Dalian, China). 3,3 -dioctadecyloxacarbocyanine perchlorate (DiO), Pluronic F127, Hoechst 33258, fluorescein diacetate (FDA) and propidium iodide (PI) were obtained from Sigma-Aldrich (MO, USA). Dulbecco s modified Eagle s medium (DMEM), fetal bovine serum (FBS) and tetramethylrhodamine (TRITC)-phalloidin were purchased from Gibco/Life Technologies (CA, USA). The DEVD-NucView 488 Caspase-3 assay kit and the JC-1 (5,5,6,6 -tetrachloro-1,1,3,3 tetraethylbenzimidazolylcarbocyanine iodide) mitochondrial membrane potential detection kit were purchased from Biotium, Inc. (Hayward, CA, USA). All solvents and other chemicals of analytical reagent grade were purchased from local commercial suppliers, unless otherwise stated. All solutions were prepared using ultra-purified water supplied by a Milli-Q system (Millipore ). Microfluidic Device Design. The microfluidic device in this study was composed of four layers (Figure 1B and Figures S1 and S2): the fluidic layer (channel: 200 μm to 800μm wide and 100 μm high; chamber: 3500 μm wide, 5500 μm long, and 100 μm high; pillar: 90 μm in diameter and 100 μm high), the control layer (channel: 25 µm to 100 μm wide and 20 μm high, and PµS: 100 µm to 150 μm wide and 40 μm high), the supporting layer and a glass slide. In detail, the fluidic layer, with a micrometer-scaled fluid network, contained eight individual chambers for tumor cell staying, and was organized in a 4 2 geometry. Forty-five micropillars were commonly set in for the prevention of chamber subsidence. One inlet and eight outlets were used to perform cell loading, chamber purging, and waste exclusion. In the control layer, one water-filled channel network incorporated 360 PµSs in their middles or terminals, and was pressured by a compressed nitrogen source to form three-dimensional microbarriers in the microchambers. Forty-five PµSs in each chamber could S2

3 effectively regulate the trapping and releasing of cell samples. The networking of the functional components simplifies the actuation sequence during the trapping process. Finally, the supporting layer patterned on the glass slide was employed for the seal of the channel networks in the control layer. Device Fabrication. The microfluidic devices were fabricated using multilayer soft lithography. 1 3 Briefly, fluidic mold was made from a 100 μm thick SU photoresist patterned on a silicon wafer using photolithography, and control mold was made by sequentially introducing a 20-μm thick SU photoresist for the control channels and a 40-μm thick AZ 50XT photoresist for the pneumatic microstructure (PµS) arrays on the same silicon wafer. Next, PDMS was poured onto the molds to make chips after all molds were exposed to trimethylchlorosilane vapor for 3 min. PDMS mixture with a ratio of 5:1 was used to yield a 3 mm thick fluidic layer of the device, whereas PDMS with a ratio of 20:1, 25:1, or 30:1, was spin-coated onto the control mold at 1200 rpm for 65 s to obtain a thin control layer. After both of these layers were cured in an oven at 80 C for 45 min, the thick fluidic layer was peeled off from the fluidic mold and holes were punched. The fluidic layer was trimmed, cleaned, and aligned onto the thin control layer. After baking at 80 C for 12 h, the assembled layers were peeled off from the control mold and a hole for control channels was punched. Finally, the assembled layers were placed on a glass slide, which was spin-coated with PDMS (ratio 10:1, 1000 rpm for 60 s, and cured at 80 C for 10 min). The PDMS device was ready for use after baking at 80 C for 72 h. Cell Culture. Human glioma (U251) cells were routinely cultured in Dulbecco s modified Eagle s medium (DMEM) supplemented with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 C in a humidified atmosphere with 5% CO 2. Cells were normally passaged at a ratio of 1:3 every 3 days to maintain their exponential growth phase. Before use, cells were harvested by trypsinization with 0.25% trypsin (Invitrogen) in Ca 2+ - and Mg 2+ -free Hanks balanced salt solution at 37 C. Trypsinization was stopped upon the addition of fresh supplemented DMEM, and cell suspensions were centrifuged at 800 rpm for 3 min. The cells were then resuspended in supplemented DMEM for use. S3

4 Cell Viability and Chemotherapy Evaluation. Cell viability was evaluated using a FDA/PI staining protocol. After removing the growth medium from cells and rinsing with PBS, the FDA/PI (10 µg/ml of each in PBS) staining solution was introduced (10 µl/min) into the chambers, and the staining process was performed for 15 min at 37 C. PBS was then introduced for 10 min as a final rinse. In these procedures, the dead cells were stained red by the fluorescent dye PI, whereas the living cells were stained green by the fluorescent dye FDA. For quantitative analysis of chemotherapy, cell death in 3D tumor was calculated as Chemotherapy (%) = ( A t A s A t + A d A s A s A t ) 100 (1) where A t is the total area of 3D tumor before drug treatment; A s is the area of 3D tumor after drug treatment and A d is the area of dead cells in 3D tumor after drug treatment. Cell Staining. For cytoskeleton presentation, 3D tumors in the device were fixed in 4% paraformaldehyde in PBS for 30 minutes and permeabilized in 0.3% Triton X-100 in PBS for 30 minutes at room temperature. The fixed tumors were then incubated with TRITC-phalloidin (100 nm) in PBS solution at 4 C for overnight. Nuclei were counterstained using a H33258 (0.5 µg/ml in PBS) staining solution. In addition, tumor cells were pre-stained selectively for cell tracking using DiO fluorescent dye (10 µμ in DMEM medium, 10 min), following a wash step with PBS. S4

5 Figure S1. AutoCAD schematic of the microfluidic device. S5

6 Figure S2. The actual microfluidic device. Different food dyes were used to visualize microfluidic components: green for fluidic channels and chambers, red for control channels and PµSs. Figure S3. Simulated flow condition in the device with either off switch of PµSs (left) or on switch of PµSs (right). Finite element analysis was conducted using ESI-CFD software (V2010.0, ESI CFD Inc., Huntsville, AL, USA) to evaluate the flow profile in the device. The initial fluid state at the fluidic inlet in the device was defined as 1. The results showed that the perfusion was equal in all chambers, suggesting that 8 chambers in the device were exposed to the same flow conditions. The flow condition in the activated of PµSs for tumor culture is the same suggesting any potential effects of flow-induced shear stress on cellular activities were identical across all conditions. S6

7 Figure S4. Array-like cell trapping in PµSs-based microfluidics at different flow rates, i.e., 5 µl/min (A), 25 µl/min (B) and 50 µl/min (C). The loading time was 10 min for each cell trapping. Figure S5. PµSs-based trapping in the devices at a flow rate of 10 µl/min. (A) Cell trapping in different microfluidic devices. Seven devices named device 1, 2, 3, 4, 5, 6, and 7 were used here. (B) Inter-device reproducibility of cell trapping. A trapping comparison of each device (device 2, 3, 4, 5, 6, and 7) with device 1 was presented. The results suggest the quantitative stability of PµSs-based trapping in the devices. S7

8 Figure S6. Inter-device reproducibility of cell trapping in different devices for investigating device reusability. Six devices (device 1, 2, 3, 4, 5, and 6) were used for this experiment. A trapping comparison of each device (device 2, 3, 4, 5, and 6) with device 1 was presented. The reproducibility was over 90% suggesting that PµSs-based microfluidic devices can be used for repeatable cell trapping. Figure S7. U251 tumor culture and maintenance using PµS array. (A) Tumor growth. (B) Optical image of array-like 3D tumors after 10 days in culture using 10% FBS-supplemented medium. S8

9 Figure S8. Size comparison of tumors after 10 days in culture using 5%, 10%, and 20% FBS-supplemented medium. *P < Figure S9. Optical and fluorescent images of FDA/PI-stained 3D tumors after 10 days in culture using 5% FBS-supplemented medium. S9

10 Figure S10. Growth rate of 3D tumors derived by different numbers of trapped U251 cells. Figure S11. Repeatable tumor culture in single devices. (A) Tumor size in differnt repeated cultures. (B) Tumor size in differnt devices (device 1, 2, 3, and 4). The result showed the 3D tumors between each two cultures (A) and devices (B) were size-similar, suggesting that size-controllable tumor formation could be achieved in each cultivation in single devices. S10

11 Figure S12. Viability in the repeated cultures in single devices. The result showed the tumors in each repeated culture was highly viable. Figure S13. Cytotoxicity evaluation of vincristine (A) and bleomycin (B) to U251 tumor cells using 3D culture in PµSs-based microfluidic devices and 2D culture in 96-well plates. The standard MTT protocol was used when doing 2D chemotherapy analysis (Wang, J.; Wan, Z.; Liu, W.; Li, L.; Ren, L.; Wang, X.; Sun, P.; Zhao, H.; Tu, Q.; Zhang, Z.; Song, N.; Zhang, L. Biosens. Bioelectron. 2009, 25, ). *P < 0.05 vs. 3D tumor culture. The difference of drug-resistance between 3D and 2D culture was more significant in anticancer drug treatments with low concentrations than in that with high concentrations. S11

12 Figure S14. Fluorescent images of a JC-1 stained U251 tumor with 5 µg/ml VCR treatment, corresponding Figure 5A and 5B. Figure S15. Ratio change of JC-1 aggregates to its monomers during 5 µg/ml VCR treatment to U251 tumors (n=122). S12

13 Figure S16. Fluorescent images of a JC-1 stained U251 tumor with 25 µg/ml BLM treatment, corresponding Figure 5C. Figure S17. Ratio change of JC-1 aggregates to its monomers during 25 µg/ml BLM treatment to U251 tumors (n=134). S13

14 Movie Captions Movie S1. Cell trapping using an activated PµS array in a microfluidic device. Movie S2. Repeatable cell trappings in a single PµSs-based microfluidic device. S14

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