Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Summary

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Abstract

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Introduction

The blood-brain barrier is a tightly controlled interface between the blood and brain that determines what goes into and comes out of the brain. Anatomically, endothelial cells compose the blood-brain barrier and forms a complex, continuous capillary network. Physiologically, this capillary network supplies the brain with oxygen and nutrients while simultaneously disposing of carbon dioxide and metabolic waste products. Importantly, evidence supports that the changes to the barrier contribute to numerous pathologies, including Alzheimer's disease, epilepsy, and stroke^1,2,3

Protocol

The information below is based on current safety and regulatory standards at the University of Kentucky, Lexington, KY, USA. As a safety precaution, refer to the institution's biological safety program and the most current regulations and recommendations before working with human tissue.

CAUTION: Human tissue can be a source of blood-borne pathogens, including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and others. Working with human tissue poses the risk of infection from blood-borne pathogens. Therefore, certain regulatory and safety considerations are imperative when working with human tissue....

Results

The isolations from human brain tissue yield a suspension enriched in human brain capillaries (Figure 1B) with small amounts of larger vessels, red blood cells, other single cells, and some cell debris. Some capillaries are branched, and, in some, red blood cells are entrapped in the capillary lumens. The typical capillary has a 3–7 µm diameter and is approximately 100-200 µm long with open lumens; most capillary ends are collapsed. Using confocal microscopy,.......

Discussion

The present protocol describes the isolation of intact and viable human brain capillaries from fresh tissue. In this section, we discuss in detail the following: 1) modifications to the protocol, 2) troubleshooting of common errors, 3) limitations of the technique, 4) the significance of the model with respect to existing and alternative blood-brain barrier models, and 5) potential applications for isolated human brain capillaries.

The protocol described here is optimized for 10 g of fresh hum.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Personal Protective Equipment (PPE)
Diamond Grip Plus Latex Gloves, Microflex Medium	VWR, Radnor, PA, USA	32916-636	PPE
Disposable Protective Labcoats	VWR, Radnor, PA, USA	470146-214	PPE; due to the nature of the human source material, the use of a disposable lab coat is recommended
Face Shield, disposable	Thermo Fisher Scientific, Pittsburgh, PA, USA	19460102	PPE; due to the nature of the human source material, the use of a disposable face shield is recommended
Safety Materials
Clavies High-Temperature Autoclave Bags 8 x 12	Thermo Fisher Scientific, Pittsburgh, PA, USA	01-815-6
Versi Dry Bench Paper 18" x 20"	Thermo Fisher Scientific, Pittsburgh, PA, USA	14-206-32	to cover working areas
VWR Sharps Container Systems	Thermo Fisher Scientific, Pittsburgh, PA, USA	75800-272	for used scalpels
Bleach 8.2% Clorox Germicidal 64 oz.	UK Supply Center, Lexington, KY, USA	323775
Equipment
4 °C Refrigerator	Thermo Fisher Scientific, Pittsburgh, PA, USA	13-986-148
Accume BASIC AB15 pH Meter	Thermo Fisher Scientific, Pittsburgh, PA, USA	AB15
Heidolph RZR 2102 Control	Heidolph, Elk Grove Village, IL, USA	501-21024-01-3
Sorvall LEGEND XTR Centrifuge	Thermo Fisher Scientific, Pittsburgh, PA, USA	75004521
Leica L2 Dissecting Microscope	Leica Microsystems Inc, Buffalo Grove IL, USA		used to remove meninges
POLYTRON PT2500 Homogenizer	Kinematica AG, Luzern, Switzerland	9158168
Scale P-403	Denver Instrument, Bohemia, NY, USA	0191392
Standard mini Stir	Thermo Fisher Scientific, Pittsburgh, PA, USA	1151050
Thermo-Flasks Liquid Nitrogen Dewar	Thermal Scientific, Mansfiled, TX, USA	11-670-4C	used to freeze the tissue?
Voyager Pro Analytical Balance	OHAUS, Parsippany, NJ, USA	VP214CN
ZEISS Axiovert Microcope	Carl Zeiss, Inc Thornwood, NY, USA		used to check isolated capillaries
Tools and Glassware
Finnpipette II Pipette 1-5 mL	Thermo Fisher Scientific, Pittsburgh, PA, USA	21377823T1	wash capillaries off filter
Finnpipette II Pipette 100-1,000 µL	Thermo Fisher Scientific, Pittsburgh, PA, USA	21377821T1	resuspend pellet in BSA
Pipet Boy	Integra, Hudson, NH, USA	739658
50 mL Falcon tubes 25/rack - 500/cs	VWR, Radnor, PA, USA	21008-951
EISCO Scalpel Blades	Thermo Fisher Scientific, Pittsburgh, PA, USA	S95938C	to mince brain tissue
PARAFILM	VWR, Radnor, PA, USA	52858-000	to cover beaker and volumetric flask
Thermo Scientific Finntip Pipet Tips 5 mL	Thermo Fisher Scientific, Pittsburgh, PA, USA	21-377-304	to wash capillaries off filter
60 mL syringe with Luer-Lok	Thermo Fisher Scientific, Pittsburgh, PA, USA	BD309653	used with connector ring to filter capillaries
Scalpel Handle #4	Fine Science Tools, Foster City, CA, USA	10060-13	used for mincing
Dumont Forceps #5	Fine Science Tools, Foster City, CA, USA	11251-10	used to remove meninges
Potter-Elvehjem Tissue Grinder	Thomas Scientific, Swedesboro, NJ, USA	3431E25	50 mL volume, clearance: 150-230 μm
Dounce Homogenizer	VWR, Radnor PA USA	62400-642	15 mL volume, clearance: 80-130 μm
Spectra/Mesh Woven Filters (300 µm)	Spectrum Laboratories, Rancho Dominguez, CA, USA	146424	Used to filter capillary suspension to remove any meninges that may be left
pluriStrainers (pore size: 30 µm)	pluriSelect Life Science, Leipzig, Germany	43-50030-03
Connector Ring	pluriSelect Life Science, Leipzig, Germany	41-50000-03	reuse multiple time
1 L Volumetric Flask			for preparation of Isolation Buffer
1 L Beaker			for preparation of 1% BSA
Stir Bar			for preparation of 1% BSA and Ficoll®
Schott Bottle (60 mL)			for preparation of Ficoll®
Ice Bucket			to keep everything cold
100 mm Petri dish			for mincing of brain tissue
Tissue Culture Cell Scraper	VWR, Radnor, PA, USA	89260-222	to remove supernatant after centrifugation
Chemicals
BSA Fraction V, A-9647	Sigma-Aldrich, St. Louis, MO, USA	A9647-500g	prepare in DPBS with Ca2+ & Mg2+ the day before. Avoid bubbles during preparation. Store in the refrigerator. Slowly stir for 10 min before use.
DPBS with Ca2+ & Mg2+	Hyclone	SH30264.FS	DPBS - part of the Isolation Buffer
Ficoll PM400	Sigma-Aldrich, St. Louis, MO, USA	F4375	Exact measurement is important here. Weigh out in bottle with stir bar. Shake vigurously after adding DPBS. Keep in the fridge O/N. It will be clear in the morning. Stir gently for 10-15 min before use. Keep on ice until use.
Glucose (D-(+) Dextrose)	Sigma-Aldrich, St. Louis, MO, USA	G7528	Glucose (D-(+) Dextrose) Concentration: 5 mM
Sodium Hydroxide Standard Solution	Sigma-Aldrich, St. Louis, MO, USA	71474	to adjust pH of the DPBS
Sodium Pyruvate	Sigma-Aldrich, St. Louis, MO, USA	P2256	Concentration: 1 mM