24 multi well plate

To determine the antibacterial properties of the coatings, the duplicates of the catalyst-coated SS, SS/C-N-TiO₂, SS/Ti(N,O)/C-N-TiO₂ and SS/Cr-N/Cr(N,O)/C-N-TiO₂ (size = 1cm × 1cm) were placed in separate wells of a 24 multi-well plate (Corning, New York city, USA). Uncoated stainless steel (SS) was used as a control. Each well was filled with 3 mL of freshly prepared bacterial culture of Bacillus subtilis (SQUMSF005). The initial concentration of the bacterial culture was ~10 Colony Forming Unit per milliliter (~10 CFU/mL). Two similar sets of experiments were conducted; one of which was exposed to the visible light (~30–31.5 Klux, light experiment) and another one was covered with aluminium foil (dark experiment). In both experiments, the multi-well plates were incubated at 37 °C for 48 h. At the beginning (0 h), after 24 h and at the end of the experiment (48 h), 1 mL of the broth culture from each well (both under light and dark conditions) was collected and diluted for 50 times with sterile marine water to determine the number of CFUs. The experiment was conducted in triplicate (n = 3).

Organoid or organotypic culture pre-seeded into droplets of hydrogel (Matrigel Corning or collagen, ThermoFisher Scientific) were cultured onto 13 mm coverslips (VWR International) in 24-well plates (Corning). On the day of the print, coverslips were removed with sterile tweezers, dried with sterile paper and incubated with photosensitive-polymers. For photosensitive hydrogel diffusion, a liquid drop of HCC-PEG or HCC-Gelatin was added onto the 3D culture and incubated at 37 °C for 15 min (Matrigel-based cell cultures) or 4 h (collagen-based cell cultures). The volume ratio between photosensitive hydrogels and solid hydrogel drops was 2:1. The multiphoton microscopes used for 2P-mediated hydrogel crosslinking were Scientifica 2-Photon microscope, Zeiss Examiner.Z1 Multiphoton LSM880 Confocal microscope equipped with Solent Scientific Incubator Chamber and Multiphoton Leica SP8 confocal microscope. After the bioprinting, the coverslip was moved into another 6 mm petri dish (Corning) filled with 10 ml of basal media and incubated at 37 °C for 5 min to remove the un-crosslinked photosensitive-polymer. Finally, the coverslip was put in a 24 multi-well plate (Corning) with 500 μl of organoid or organotypic culture media.