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 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. Place the brains in a sterile flow bench and gently…

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