Electronic Supplementary Information (ESI) for Lab on a Chip Electronic Supplementary Information Construction of oxygen and chemical concentration gradients in a single microfluidic device for studying tumor cell drug interactions in a dynamic hypoxia microenvironment Lei Wang, a Wenming Liu, b Yaolei Wang, b Jian-chun Wang, b Qin Tu, b Rui Liu, b and Jinyi Wang* ab a College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China b College of Science, Northwest A&F University, Yangling, Shaanxi, 712100, China Abstract. This supplementary information provides all the additional information and a more detailed discussion of the current study. 1
Fig. S1. The optical (A to C) and fluorescence (A to C ) images of AO/PI staining A549 cells after treatment with culture medium containing 4 g L -1 Na 2 SO 3 (A and A ); culture medium containing 4.5 g L -1 Na 2 SO 4 (B and B ); and fresh culture medium (C and C ) for 48 h. The results indicate that the cells were all viable and highly adherent, which suggests that Na 2 SO 4, the reaction product between Na 2 SO 3 and O 2, has low cytotoxicity. 2
Fig. S2. Na 2 SO 3 -aided and PtOEP-visualized oxygen control in the 900 μm microchannel. Na 2 SO 3 was tested at 0.2, 1, 2, and 4 g L -1. Na 2 SO 3 (4 g L -1 ) was chosen for the subsequent study based on the total free oxygen in the solution, clear visualization, and convenient phosphorescence imaging. Air and N 2 (pressure, 20 psi) were used for the negative and the positive control of oxygen-free condition, respectively. The flow rate was 2 μl min -1. 3
Fig. S3. Oxygen distribution dynamics after stopping the 10 μl min -1 oxygen gradient flow in the 900 μm microchannel. The result show that the oxygen gradient can disappear within 16 min. 4
Fig. S4. The phosphorescent images of oxygen gradients in the 600 μm and 300 μm channels at various flow rates (0.05 µl min -1 to 10 μl min -1 ). The results suggest that the decrease in both channel width and flow rate decreases the phosphorescent gradient, which indicates an increase in the minimum oxygen concentration in the microfluidic gradient. 5
Fig. S5. Comparison of the simulated and experimental results of oxygen gradient on the bottom surface of the 900 µm microfluidic channel at 2 µl min -1. The result shows that the oxygen distributions from both the simulated and experimental investigations are similar. 6
Fig. S6. Oxygen gradient profiles in different treated 900 µm channels. The FN modification did not significantly influence the oxygen gradient distribution, similar to that in the unmodified PDMS channel. 7
Fig. S7. Oxygen gradient profiles of the upstream, midstream and downstream of the cell culture chamber. The results show that there were not obvious changes of the oxygen gradient between the upstream, midstream and downstream of the cell culture chamber. 8
Fig. S8. Oxygen gradient profiles before and after cell introduction in the chamber. A comparative test was performed to monitor the cell-involved oxygen gradient dynamics using phosphorescence- and fluorescence-visualized evaluation. The oxygen gradient was measured after 2 h incubation of A549 cells in the chamber, as well as before cell seeding. The visualized (A) and quantified (B) results show that oxygen distribution before and after cell seeding was similar. 9
Fig. S9. Microfluidic cultivation of A549 cells and HeLa cells in the 900 µm chamber. (A) A549 cells after the seeding process. (B) HeLa cells after culturing for 48 h. (C) Fluorescence staining of AO/PI staining A549 cells after culturing for 24 h. (D) Fluorescence staining of AO/PI staining HeLa cells after culturing for 24 h. The AO/PI staining method was used in this test: green for viable cells; red for dead cells. The result shows that the two cell lines were successfully cultured in the chambers with good adhesion and proliferation. 10
Fig. S10. Viability of A549 cells (A) and HeLa cells (B) after culturing for 24 h in normoxic condition or oxygen gradient condition. The results show that the number of viable cells in the low oxygen area (Z2) was lower than those in both Z1 and Z3, which indicate slight inhibition of cell proliferation by the hypoxic microenvironment. 11