Purifying Capillaries From a Porcine Brain

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Purification of Porcine Brain Capillaries

1. Collect 8-10 brains from 5-6-month-old domestic pigs (e.g. from a nearby slaughterhouse) and transport them on ice to the laboratory. We recommend starting the following purification procedure within 2-3 h of termination of the animal.
2. Place the brains in a sterile flow bench and gently wash them with 1 L PBS in a beaker placed on ice.
3. Carefully remove meninges from one brain at a time using fine-tip forceps and transfer the meninges-free brains to another 1 L beaker with PBS placed on ice. Extending the time taken can complicate the removal. The approximate time used for each brain should be 10-15 min.
4. Using a scalpel, scrape off grey matter from one brain at a time and transfer the isolated material to Petri dishes (8.8 cm²) containing 20 mL DMEM Nutrient Mixture F-12 (DMEM/F-12) placed on ice. Isolate as much grey matter as possible without withdrawing white matter material. For initial fragmentation, run the grey matter material through a 50-mL syringe without a needle. Continue for all the brains and pool the collected grey matter material. Extending the time taken can complicate the removal. The approximate time used for each brain should be 10-15 min.
5. Transfer the isolated grey matter material to the grinder tube of a hand-held tissue homogenizer in a ratio of 50/50 with DMEM/F-12 media. Homogenize the material by making 8 up and down strokes with a loose pestle, followed by 8 up and down strokes with a tight pestle. Continue until all isolated material has been homogenized, then transfer the homogenate to a 500-mL bottle and dilute with DMEM/F-12 to approximately 450 mL total volume.
6. Filter the homogenate using a 500-mL blue-cap bottle with filter holder and 140 µm filter-mesh, and isolate capillaries by running the tissue through the filter. Use one filter per 50 mL of homogenate and wash each filter with DMEM/F-12 afterwards.
7. Place the capillary-containing filters in Petri dishes with a digestion solution of trypsin/EDTA (2.5 % trypsin, 0.1 nM EDTA in PBS), collagenase CLS2 (2,000 U/mL) and DNase 1 (3,400 U/mL) in DMEM/F-12. Use 1 Petri dish (8.8 cm², 20 mL solution) per 3 filter meshes.
8. Place the Petri dishes at 37 °C for 1 h on an orbital shaker at 180 rpm or stir them gently every 10 min. After 1 h, wash off the capillaries from the filters with suspension from the Petri dish using a 1 mL pipette.
9. Split the suspension from the 3 dishes into 2 50-mL tubes and stop the digestion by adding 10 mL DMEM/F-12 to each 50-mL tube.
10. Centrifuge the cell-suspensions at 250 x g, 4 °C for 5 min. Aspirate the supernatants and re-suspend each pellet in 10 mL of DMEM/F-12. Add a further 20 mL of DMEM/F12 to each tube. Repeat this centrifuge step twice.
11. Let the tubes cool down on ice for 5 min and transfer the solutions to 2 new 50 mL tubes.
12. Centrifuge the cell-suspensions at 250 x g, 4 °C for 5 minutes, and re-suspend each pellet in 8 – 10 mL (approximately 1 mL/brain used) of freezing solution consisting of 10% DMSO in FBS.
13. Transfer the cell suspension to cryovials, using 1 mL per cryovial. On average, purification results in 1 vial per brain used, on average providing endothelial cells for 12-16 inserts. Place the vials in a freezing box at -80 °C for at least 4 h up to overnight. Afterwards, store the cryovials in a cryotank with liquid nitrogen.
    Caution: Liquid nitrogen has extremely low temperatures. Please wear appropriate protection.