Cellular dynamics of transgenic porcine endothelial cells to inflammatory stimuli in xenotransplantation settings
Cellular dynamics of transgenic porcine endothelial cells to inflammatory stimuli in xenotransplantation settings
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.