Purifying Capillaries From a Porcine Brain

Abstract

Source: Nielsen, S. S. E., et al. Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells. J. Vis. Exp. (2017).

This video demonstrates the isolation of capillaries from fresh porcine brains. The process involves removing the meninges, isolating the grey matter from the brain tissue, homogenizing and filtering the grey matter to isolate the capillaries, and finally using enzymatic digestion to purify the capillaries further.

Protocol

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.

Materials

DMEM/F-12	Lonza	BE12-719F
Fetal bovine serum (FBS)	Gibco Invitrogen	10-270-106
Trypsin/EDTA	Gibco Invitrogen	15090-046
DMSO	Sigma-Aldrich	34896
PBS	Sigma-Aldrich	D8537
DNAse 1	Sigma-Aldrich	D4513
Collagenase CLS2	Sigma-Aldrich	C6885
ddH2O			Made with Elga System
15 ml centrifuge tubes	Cellstar	188271
50 ml centrifuge tubes	Cellstar	227261
Petri dishes	Thermo Scientific	150350
Cryo vials	Thermo Scientific	377224
500 ml bottle	Thermo Scientific	159910/159920
Scalpels	Swann-Morten	REF0211	Type 24
Tissue homogenizer	Sigma	D9188
140 μm filters	MERCK	NY4H04700
Fine-tip curved forceps	KLS Martin	12-409-12-07
Broad tip forceps	VWR	82027-390
Filter holder	MERCK Milipore	Swinnex-47
50 ml syringe	Braun	4617509F