Media was placed up to the midline of the construct simulating the more nutrient rich blood stream relative to the tissue environment in this case a material-air interface. After each time point the media was removed surrounding the constructs and CFU/mL counts were generated for all replicates. One well without constructs but with media was inoculated with bacteria as a positive growth control. The materials were then subjected to a thorough sterile saline wash to eliminate any unattached bacteria and imaged with confocal microscopy.
After each time point (2 4 6 hrs) bacteria-seeded constructs i.e. GelMA alginate and decellularized porcine tissue were incubated in staining media supplemented with 4 mmol/L calcein AM and 2 mmol/L ethidium-homodimer for 20 min at 37˚C. Each of the constructs were then washed with PBS solution and covered with glass coverslips for conducting confocal microscopic analysis. Confocal fluorescence microscopy (Zeiss LSM 900 Zeiss GmBH) was used for taking high-resolution 3D images and the bacterial count was analyzed at three time points (n=3 per construct per biological replicate per time point) using ImageJ software (National Institutes of Health).
After each time point (2 4 6 hrs) bacteria-seeded constructs i.e. GelMA alginate and decellularized porcine tissue were incubated in staining media supplemented with 4 mmol/L calcein AM and 2 mmol/L ethidium-homodimer for 20 min at 37˚C. Each of the constructs were then washed with PBS solution and covered with glass coverslips for conducting confocal microscopic analysis. Confocal fluorescence microscopy (Zeiss LSM 900 Zeiss GmBH) was used for taking high-resolution 3D images and the bacterial count was analyzed at three time points (n=3 per construct per biological replicate per time point) using ImageJ software (National Institutes of Health).