Evaporation-based Microfluidic Production of. Oil-free Cell-Containing Hydrogel Particles

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1 Supporting Information Evaporation-based Microfluidic Production of Oil-free Cell-Containing Hydrogel Particles Rong Fan, a Kubra Naqvi, b Krishna Patel, c Jun Sun d and Jiandi Wan *a a Microsystems Engineering and b College of Science, Rochester Institute of Technology, Rochester, NY 14623, USA. c Webster Schroeder High School, Webster, NY 14580, USA. d Department of Biochemistry, Rush University, Chicago, IL 60612, USA. Cell Culture and Maintenance. HCT116 human colon cancer cells (kindly provided by Jun Sun) 1 were cultured in DMEM (Life Technologies, CA) containing 10 % (v/v) fetal bovine serum (Life Technologies), 1 % (v/v) penicillin/streptomycin (Life Technologies). The cells were maintained in a T-25 culture flask and incubated in an incubator at 37 C with 5 % CO 2. For cell encapsulation, cells cultured in the T-25 culture flask were washed by PBS solution and trypsinized for 5 mins to detach cells from the culture flask. The cell suspension was then centrifuged and re-dispersed in a DMEM for cell encapsulation in microfluidics. Because dead cells were not attached on the culture flask and were washed away by PBS solution, the viability of cells in the suspension (checked under a microscope) is approximately the same for all microfluidic experiments. For cell culture in Extracel hydrogel particles, a 35 mm petri dish (In Vitro Scientific) was used to collect hydrogel particles with encapsulated cells, which were then incubated in an incubator. The number of cells in each hydrogel particle throughout the whole 1

2 petri dish was counted under a Leica microscope (DMI 6000, Leica Microsystems) from day 0 to day 4. To study the proliferation of single cells in particles, a time-lapse video of cell growth was recorded by using a camera (C B-H, Hamamatsu) coupled to the Leica microscope. To study the effect of TNF-α (Sigma-Aldrich) on the cell viability, HCT116 cells or encapsulated HCT116 cells were cultured in different concentrations of TNF-α (0.1, 1.0, 10, 50, 100 ng ml -1 ) at 37 C in an incubator. TNF-α was diluted in DMEM culture medium containing 10 % FBS and 1 % P-S. Normal culture medium without TNF-α added was used as control. The viability of the cells under influence of TNF-α is characterized by N/N %, where N is the number of living cells measured every day by counting, and N 0 is the number of living cells at Day 0. To distinguish the live and dead cells during cell number counting, we examine the morphologic changes of cells using a microscope when they are cultured under a 2D petri dish or culture flask. When cells are encapsulated in particles, on the other hand, videos are recorded to examine both the cell movement in the particle and morphological changes to identify live or dead cells. Microfluidic Generation of Droplets and Microparticles with Encapsulated HCT116 Cells. Flow-focusing microfluidic devices were fabricated in poly(dimethylsiloxane) according to the established soft lithography technique. 2 The height of the microchannel is 37 µm everywhere. The widths of aqueous inlet channels are 100 µm; the widths of oil and main channels are 150 µm; the width of the orifice is 50 µm. In the experiment, 1 ml monomer solution and 1mL crosslinker solution were loaded into two 1 ml syringes (Med Lab Supply), which were connected to the inlets of the microfluidic device via polyethylene tubing (Scientific Commodities Inc, 0.015" (0.38 mm) I.D " (1.09 mm) O.D.). A syringe pump (KD Scientific) was then used to inject the solution to the microfluidic device. Oil was introduced from the side channel via another syringe pump (New Era) as the continuous phase. The typical 2

3 flow rates of both aqueous phases and the oil phase varied from 3 to 5 µl min -1, and 30 to 50 µl min -1, respectively, which give a generating efficiency of 500 droplets per second. The generation process of droplets and cell encapsulation were monitored directly by a high-speed video camera (Phantom, Ametek) mounted on the microscope (AmScope). The size of more than 100 droplets was analyzed by using Image J. Calculation for number of cells in drops. Volume of drops: Because drops with a diameter larger than the height of the microfluidic channel (37 µm) are ellipsoidal, the volume of such drops can be calculated by the following equation: where a, b, and c are the three elliptic radii. In our case, a = b = 50/2 µm = 25 µm, c = 37/2 µm = 18.5 µm, which gives ml. Theoretical Poisson distribution of cells in drops: where P(x) is the fraction of droplets expected to contain x cells, and λ is the average number of cells per droplet. For a concentration of cells in the aqueous phase of cells ml -1, theoretical λ will be the product of droplet volume and concentration of cells in the droplet phase, which gives Theoretical Poisson distribution curve (Fig. S1) for the number of cells per drops is then generated using the Poisson function available in Microsoft Excel

4 FIG. S1. Theoretical Poisson distribution of cells in drops. FIG. S2. Evaporation-induced change of weight percentage (W/W %) of the mixture solution of droplets and HFE 7500 oil. W 0 is the weight of the mixture at t = 0 min, and W is the weight at t = n mins, where n = Incubation temperature: 37 C. 4

5 FIG. S3. (a) Image of a layer of hydrogel particles floating on top of the HFE 7500 oil phase after centrifuge (scale bar = 5 mm). (b) Image of a layer of aggregated hydrogel particles sticking to a filter paper after filtration (Scale bar = 50 µm). FIG. S4. Proliferation rate of HCT116 cells cultured in petri dish. The number of cells increases 8 folds within 120 hours, indicating that the percentage of living cells double every 1.6 days on average. 5

6 REFERENCE 1 J. Sun, M. E. Hobert, Y. Duan, A. S. Rao, T. C. He, E. B. Chang, and J. L. Madara, Am. J. Physiol. Gastrointest. Liver Physiol. 289, G129 (2005). 2 D. C. Duffy, J. C. McDonald, O. J. Schueller, and G. M. Whitesides, Anal. Chem. 70, 4974 (1998). 6

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