title: Cellular dynamics of transgenic porcine endothelial cells to inflammatory stimuli in xenotransplantation settings
design: Cellular dynamics of transgenic porcine endothelial cells to inflammatory stimuli in xenotransplantation settings
library_name: E02_s
library_construction_protocol: To culture the the porcine endothelial cells using a microfluidic system, 100,000 cells in 100 μl of complete media were seeded in a μ-slide VI 0.4 (Ibidi, 80606) 16,21. Prior to seeding, the μ-slide was coated with 100 μl of 10 μg/ml fibronectin (Merck, FC010) for 30 min at 37 °C. Cells were incubated overnight before being connected to a peristaltic pump (Gilson, Minipuls 3) using sterile silicone tubings to introduce the flow. Flow media consisting of DMEM supplemented with 10% FBS, 1% glutamine, 1%BSA (Sigma, A7030), and 4% dextran (Sigma, 31390), was used as the flow medium. The laminar shear stress was adjusted to 10 dyn/cm2 and maintained for 72 h in a humidified incubator at 37 °C and with 5% CO2. The media was refreshed every day. For simulating xenogeneic activation, cells were perfused with 3 mL of flow media containing 10% pooled normal human serum (NHS) obtained from three healthy donors for 2 h. Additionally, cells were primed with 100 ng/ml recombinant human TNF-α (RnD Systems, 210-TA-020) for 4 h, prior to NHS perfusion, to simulate the inflammatory conditions in xenotransplantation settings.  Total cellular RNA from the all porcine endothelial samples was isolated using the NucleoSpin RNA kit (Macherey-Nagel, 740955) according to the manufacturer's guidelines, followed by quantification of the total RNA with a Quantifluor RNA system (Promega, E3310). Bulk RNA barcoding and sequencing (BRB-seq) was performed by Alithea Genomics. A total of 200 ng of cellular RNA from four independent biological replicates was used to generate BRB-seq libraries,according to the company's pipeline.