Polyacrylamide (PAA) gels were composed of 10% acrylamide (BioRad) and 0.45% bis-acrylamide (BioRad) and 0.5% Irgacure-2959 as a photoinitiator (Advanced Biomatrix). Silanized glass coverslips (Electron Microscopy Sciences) of 9 × 9 mm were placed on top of 19 μl drops of prepolymer solution and cross-linked under UV light at 254 nm for 15 min. PBS was added to the gels and swelled overnight. 0.5 mg/mL Sulfo-SANPAH dissolved in 50 mM HEPES (pH 8.0) was added to gels, and UV light was applied for 25 minutes. Gels were washed 2× in 50 mM HEPES and 2× in PBS on a shaker. Then, 100 μg/ml PureCol (Advanced Biomatrix) diluted in PBS was added to the gels and incubated overnight. The collagen solution was aspirated and 50 mM Tris-HCl was added to the gels and incubated at room temperature for 15 min to quench any remaining sulfo-SANPAH reactive groups. Gels were washed 3× with PBS, and UV light was applied for 30 min to sterilize.
OV90, OVCAR3, or OVCAR8 cells were seeded on gels at a density of 926 000 cells/cm2 in media with 15% serum. After four hours, the media was aspirated to remove any non-adherent cells and cells were washed one time with serum free medium. Using sterile tweezers, the coverslips were carefully placed in a 40 μm cell strainer (Sigma) sitting in a six-well plate filled with 10 ml of serum free medium; 72 h was given for spontaneous detachment to occur. To collect the Sph-CD spheroids, the gels were removed from the filter, and the filter was carefully inverted on top of a 50 ml conical tube, and 4 ml of serum free medium was passed through the filter to bring the spheroids into the tube. The Sph-SC were collected by filtering the medium in the well through a separate 40 μm cell strainer, and in the same manner, the Sph-CD spheroids were also collected. For some experiments, single cells that had detached but not aggregated were collected from the media that had passed through the strainer.
OV90, OVCAR3, or OVCAR8 cells were seeded on gels at a density of 926 000 cells/cm2 in media with 15% serum. After four hours, the media was aspirated to remove any non-adherent cells and cells were washed one time with serum free medium. Using sterile tweezers, the coverslips were carefully placed in a 40 μm cell strainer (Sigma) sitting in a six-well plate filled with 10 ml of serum free medium; 72 h was given for spontaneous detachment to occur. To collect the Sph-CD spheroids, the gels were removed from the filter, and the filter was carefully inverted on top of a 50 ml conical tube, and 4 ml of serum free medium was passed through the filter to bring the spheroids into the tube. The Sph-SC were collected by filtering the medium in the well through a separate 40 μm cell strainer, and in the same manner, the Sph-CD spheroids were also collected. For some experiments, single cells that had detached but not aggregated were collected from the media that had passed through the strainer.
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