Rotamax 120

Floating test method performed by Huanbutta et al. [29 (link)] was adapted with slight modification. Floating duration time was measured for floatable solid dosage forms using a shaker (Rotamax 120, Heidolph Instruments, Schwabach, Germany). Each sample was added to 400 mL of water in a beaker which was shaken at 100 RPM to inhibit the adhesion of to the beaker.

Biofilms were formed in vitro, as previously described [29 (link)], with minor modifications. Briefly, single bacterial colonies from overnight S. aureus cultures were suspended in 4 mL of saline solution (0.9% NaCl), in order to reach an optical density of 0.5 (±0.05) at 600 nm in a cell density meter (Ultraspec 10, Amersham Biosciences, Little Chalfont, UK). The suspension (0.5 mL) was then used to inoculate 9.5 mL of sterile Iscove’s modified Dulbecco’s medium (IMDM; Gibco, Bleiswijk, The Netherlands) in an acoustically compatible OptiCell^TM cell culture chamber (Nunc^TM, Thermo Fisher Scientific, Wiesbaden, Germany). Inoculated OptiCells were placed in an incubator at 37 °C for 24 h, first statically for 3 h, in order to allow for bacterial adherence to the gas-permeable, polystyrene film bottom membrane, and then dynamically on a rotary platform shaker at 150 rpm (Rotamax 120, Heidolph Instruments, Schwabach, Germany).

A model protein BSA (0.08, 0.12, and 0.16 μmol) and a model antibiotic small molecular ornidazole (ORN, 18, 36, and 54 μmol) were separately loaded in the MPLE gel by following two different loading methods for comparison. The first method was absorption-based drug loading, which was performed on the gel surface by soaking the ORN antibiotic (3.953 mg, 7.906 mg and 11.860 mg) in 4 mL of PBS (pH 7.4) solution in separate 20 mL glass vials. Afterwards, the dried 3D printed MPLE scaffold samples (4 layers, honeycomb printed structures) were immersed in the above solutions and placed in an orbital shaker (Rotamax 120, Heidolph, Germany) for 24 h. The ORN-loaded MPLE scaffold was then transferred to a lyophilizer kept at − 70 °C for 3 days. The second loading method was direct incorporation of target drugs into the MPLE scaffolds. In this regard, the ORN and BSA were separately incorporated in the LP-capped gel, followed by mixing PEGDA (33.6 wt%) and Irgacure 2959 (3 wt.%) powders by a sonicator. Afterwards, the mixture was directly 3D printed and irradiated under UV light to obtain the drug-loaded MPLE scaffolds.

Protoplasts were isolated from leaves with petioles of 2‒4-week-old in vitro grown plants following the procedure described by Grzebelus et al. [14 (link)]. Briefly, 1 g of plant material was cut into pieces, pre-treated in 8 ml of the plasmolysis solution (0.5 M mannitol; Sigma) and then incubated in 8 ml of the enzyme solution composed of 1% (w/v) cellulase Onozuka R-10 (Duchefa), 0.1% (w/v) pectolyase Y-23 (Duchefa), 20 mM MES [2-(N-Morpholino)ethanesulfonic acid] (Sigma), 5 mM CaCl₂ (Sigma), and 0.6 M mannitol for 16 h on a gyratory shaker (30 rpm; Rotamax 120, Heidolph Instruments, Germany) at 26 ± 2 ℃ in the dark. Then the protoplasts were separated from undigested tissues by filtration through a nylon mesh (80–100 µm; Millipore, USA) and centrifuged (100 g for 5 min; MPW-223e, MPR Med Instruments, Poland; rotor type: MPR no 12,485). The pellet was resuspended in 8 ml of solution containing 0.5 M sucrose and 1 mM MES and overlaid with 2 ml of the W5 solution [70 (link)] for gradient centrifugation (145 g for 10 min). Viable protoplasts localized in the interphase between sucrose and W5 solution were transferred into a fresh tube, washed two times by centrifugation in W5 solution (100 g for 5 min each) and left on ice until further processing.

HUVECs transfected with siRNAs or OE-HUVECs were plated in a 6-well plate and on an orbital shaker (Rotamax120, Heidolph Instruments) at rpm rates previously determined to generate laminar shear stress of 1, 5, or 12 DYNES/cm². Morphological changes upon flow were confirmed in a μ-Slide VI^0.4 (Ibidi) using a pump system (Ibidi). RNA-Seq, qPCR, and Western blot experiments were performed using the orbital shaker.

The viability of all cell cultures was evaluated with the MTT-assay, 24 h post-transfection with mRNA and B18R. First, cell culture medium was removed and replaced by fresh culture medium including 5 mg/mL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent (Sigma-Aldrich, St. Louis, MO, USA) at a ratio of 1:9 (MTT reagent: culture medium). Next, the cells were incubated for 2 h at 37 °C in a humidified atmosphere containing 5% CO₂. After incubation, the solution in the wells was removed and 100 µL DMSO (Sigma-Aldrich, St. Louis, MO, USA) was added to the cells to dissolve the formazan crystals. The well-plate was covered with aluminum foil and positioned on an orbital shaker (Rotamax 120, Heidolph, Germany) for 30 min at 1200 rpm. Lastly, the absorbance was measured at 590 nm and 690 nm (background) with a VICTOR3 1420-012 multilabel microplate reader (Perkin Elmer, Groningen, The Netherlands).