Porcine serum

The study was conducted using RPE-choroid-sclera explants from freshly enucleated porcine eyes obtained from a local slaughterhouse. After the extraocular tissues were removed, the eyes were immersed in a disinfection solution (povidone iodine, diluted to 2%) for about 5 minutes, rinsed with sterilized phosphate-buffered saline without calcium and magnesium (PBS(−). The anterior part of the eye was resected through the circular incision at approximately 5 mm behind the corneal limbus, followed by the removal of the lens and the vitreous. A round piece of tissue composed of the retina-RPE-choroid-sclera were extracted using a trepan with a 12 mm diameter and a scalpel. This tissue explant was immersed in the sterilized PBS(−) and the neural retina was carefully removed from the RPE layer. The remained RPE-choroid-sclera explants were maintained in Dulbecco’s Modified Eagles Medium (DMEM; high glucose, Sigma Aldrich, St. Louis, MO, USA) supplemented by 10% porcine serum, 1 mM sodium pyruvate, 100 unit/mL penicillin and 0.1 mg/mL streptomycin (Sigma Aldrich) at 37 °C in 5% CO₂ incubator. After incubating the tissues for 24 h, the laser irradiation was conducted.

For cultivation of chicken PBMCs over several days, different sera were tested as cell culture supplements. Porcine serum (Sigma-Aldrich, St. Louis, MO, USA), chicken serum (Gibco™, ThermoFisher Scientific, Waltham, MA, USA), or fetal calf serum (FCS, Gibco™, ThermoFisher Scientific, Waltham, MA, USA) was added to the cells cultured in T25 flasks in a concentration of 10% to the RPMI-1640 medium. Immune cells were cultured for 3 days. After 24 h and 72 h, cells were subjected to flow cytometric measurement.

We used IPEC-J2 cells (DSMZ, Braunschweig, Germany), a non-transformed cell line that stems from porcine jejunal epithelia, to analyze whether the changes in epithelial barrier function in PP tissue also occur in a cell culture model. Cells were seeded on semi-permeable cell culture inserts (Millipore, Darmstadt, Germany) at 10⁵ cells per filter. Dulbecco’s MEM/Ham’s F12 with 3.15 g/L glucose and 2 mM stable glutamine (Biochrom, Berlin, Germany) was used and supplemented with 10% porcine serum and 1% penicillin/streptomycin (Sigma Aldrich, Munich, Germany). Medium was changed every 2–3 days, and filters were filled with 500 μl apically and 1 ml basolaterally. By using a chopstick electrode and an epithelial Volt/Ohm Meter (EVOM, World Precision Instruments, Sarasota, FL, United States), we measured the TER immediately before each media exchange, and values were corrected with the media and blank values used in our experimental settings. Once the cells had built up a confluent monolayer, resulting in consistent TER values, the incubation experiments were started. We added 5000 U/ml TNF (PeproTech, Hamburg, Germany) to the basolateral side of the cell filters, and TER was recorded for up to 10 h. Cell passages between 8 and 13 were used for experiments.