Data_Cassie-Baxter Surfaces for Reversible, Barrier-Free Integration of Microfluidics and 3D Cell Culture

PEGDA hydrogel was made by diluting a mixture of 25 wt% of PEGDA 3500 powder (Sigma-Aldrich), 35mM of TEA(triethanolamine) (Acros), and 35 mM of VP(1-vinyl-2-pyrrolidinone) (Sigma-Aldrich) in PBS (Sigma-Aldrich). Gelatin-Methacryloyl hydrogel was made by mixing 3 wt% GelMA (BioBots), 1 wt% PEGDA powder, 35 mM TEA, and 35 mM VP in PBS. The high concentration collagen solution (70 vol%) was made by mixing F12 basic medium (Sigma-Aldrich), 10x DMEM (Sigma-Aldrich), 1 M NaOH (Sigma-Aldrich) and collagen (Bovine collagen type I, 3.18 mg/mL, Angio-Proteomie) in a 680:43:4:434 ratio. For low concentration collagen (37 vol%), the respective ratio was 340:43:4:434. Agarose hydrogel was made by dissolving LMP agarose powder (Promega) in 77°C DI water at 1.5 wt%. Matrigel (Corning) was prepared on ice. The contact angle measurements were conducted by applying a 10 µL drop of each liquid solution on a flat PDMS surface and using an optical goniometer to read the contact angle. In a separate set of experiments designed to demonstrate dynamic solute exchange between the hydrogel and fluid in the microchannels, a liquid suspension of 1.5 wt% agarose in DI water was pipetted into the open reservoir of the microfluidic device. After solidification of the agarose, water containing 1.0 wt% rhodamine was introduced into the inlet using a syringe pump at a constant volumetric flowrate of 25 µL/min. Time-lapse images were taken every 30 seconds; fluorescent intensity in the resulting images was measured using NIS-Elements software in order to track the rhodamine diffusion from the channels into the agarose hydrogel. The experiments were repeated with a solution of 2,000 kDa FITC-dextran (Sigma-Aldrich) at 0.6 mg/mL in DI water at the same flowrate. The PDMS microfluidic devices were sterilized prior to use with cells using a two-step process. First, each device, including the fluidic pathways and cell reservoir, were rinsed with 70% ethanol (Sigma-Aldrich) and then placed in a 60 oven for 20 minutes to dry. Then, immediately prior to introducing the cell/hydrogel suspension, each device was exposed to UV light (254 nm wavelength) in a class II biosafety cabinet for 30 minutes. For all cell experiments, MDA-MB-231 Luc-GFP cells were suspended in a collagen solution or Matrigel at a concentration of 40,000 cells/mL. The collagen solution was made by mixing F12 basic medium (Sigma-Aldrich), 10x DMEM (Sigma-Aldrich), 0.2M NaOH (Sigma-Aldrich) and collagen (Bovine collagen type I, 3.18 mg/mL, Angio-Proteomie) in a 680:43:43:434 ratio on ice. For cell culture in the microfluidic devices, 120 µL of the hydrogel-cell suspension was carefully pipetted into the cell culture reservoir and then cured in the incubator for 40 minutes at 37°C. The reservoir dimensions are 5mm x 5mm x5 mm, and the device can be operated with the reservoir partially or fully filled with hydrogel-cell suspension. Once the collagen was cured, a removable lid was added to each device and sealed by using a syringe to draw a vacuum through the vacuum port. Media was continuously supplied through the microfluidic inlet ports at a rate of 2 µL/min using a syringe pump. To obtain the proliferation rate, luciferase was measured using an IVIS imaging system (Xenogen IVIS 50) on days 0-4, and the relative population was calculated based on the number of cells on day 0.(6) Colony sizes were measured using dark field imaging (TI-E Nikon microscope).