Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Anika M.S. Hartz; Julia A. Schulz; Brent S. Sokola; Stephanie E. Edelmann; Andrew N. Shen; Ralf G. Rempe; Yu Zhong; Nader El Seblani; Bjoern Bauer

doi:10.3791/57346

JoVE Journal > Neuroscience

Neuroscience

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Published: September 12, 2018

doi:

10.3791/57346

Anika M.S. Hartz¹, Julia A. Schulz², Brent S. Sokola², Stephanie E. Edelmann¹, Andrew N. Shen¹, Ralf G. Rempe², Yu Zhong¹, Nader El Seblani³, Bjoern Bauer²

¹Sanders-Brown Center on Aging, Department of Pharmacology and Nutritional Sciences,University of Kentucky, ²Department of Pharmaceutical Sciences, College of Pharmacy,University of Kentucky, ³Department of Neuroscience,University of Kentucky

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 stroke¹^,²^,³^,⁴^,⁵^,⁶^,⁷. Brain endothelial cells also serve as a barrier to treatment by blocking drug uptake into the brain, e.g., chemotherapy of glioblastoma multiforme following tumor resection⁸^,⁹^,¹⁰. In this regard, isolated human brain capillaries represent a unique ex vivo blood-brain barrier model that closely resembles barrier properties in vivo, which allows for the study of barrier function and dysfunction in health and disease. In this article, we provide a protocol to isolate brain capillaries from human brain at a consistently high capillary quality and yield to study the blood-brain barrier.

In 1969, Siakotos et al.¹¹ were the first to report the isolation of brain capillaries from bovine and human brain tissue using density gradient centrifugation and glass bead column separation. Later, Goldstein et al.¹² improved this method by adding multiple filtration steps to decrease the amount of tissue needed to study brain capillaries isolated from rats, while maintaining the metabolic activity of glucose transport. Since then, researchers optimized the capillary isolation procedure numerous times, improving the method and brain capillary model with each iteration¹³^,¹⁴^,¹⁵. For example, Pardridge et al.¹⁶ isolated bovine capillaries using enzymatic digestion rather than mechanical homogenization, and then subsequently passed a capillary suspension through a 210 µm mesh filter and a glass bead column. These modifications improved the trypan blue exclusion stain of isolated brain capillaries, and thus, increased endothelial cell viability. In the early 1990s, Dallaire et al.¹⁷ isolated bovine and rat capillaries that were clear of neuronal contamination and maintained metabolic activity of γ-glutamyl transpeptidase (γ-GTase) and alkaline phosphatase. In 2000, Miller et al.¹⁸, used isolated rat and porcine brain capillaries in combination with confocal microscopy to show the accumulation of transport substrates into the lumen of capillaries. Subsequently, our laboratory has continued to optimize the brain capillary isolation procedure and we have established transport assays to determine P-glycoprotein (P-gp)¹⁹^,²⁰^,²¹, breast cancer resistance protein (BCRP)²²^,²³, and multi-drug resistance protein 2 (Mrp2)²⁴ transport activity. In 2004, we published two reports where we used isolated rat brain capillaries to investigate various signaling pathways. In Hartz et al.²¹, we found that the peptide endothelin-1 rapidly and reversibly reduced P-gp transport function in brain capillaries by acting through the endothelin receptor B (ET_B) receptor, nitric oxide synthase (NOS), and protein kinase C (PKC). In Bauer et al.¹⁹, we demonstrated expression of the nuclear receptor pregnane X receptor (PXR) and showed PXR-modulation of P-gp expression and transport function in brain capillaries. In experiments with transgenic humanized PXR mice, we expanded this line of research and showed in vivo tightening of the barrier by upregulating P-gp through hPXR activation²⁵. In 2010, Hartz et al.²⁶ used this approach to restore P-gp protein expression and transport activity in transgenic human amyloid precursor protein (hAPP) mice that overexpress hAPP. Moreover, restoring P-gp in hAPP mice significantly reduced amyloid beta (Aβ)₄₀and Aβ₄₂brain levels.

In addition to studying signaling pathways, isolated brain capillaries can be used to determine changes in capillary permeability which we refer to as capillary leakage. In particular, the Texas Red leakage assay is used to assess leakage of the fluorescent dye Texas Red from the capillary lumen over time and these data are then used to analyze leakage rates. Increased capillary leakage rates compared to those from control capillaries indicate changes in the physical integrity of the blood-brain barrier². This is valuable because there are numerous disease states associated with barrier disruption, e.g., epilepsy, multiple sclerosis, Alzheimer's disease, and traumatic brain injury²⁷^,²⁸^,²⁹^,³⁰. Other groups have also utilized isolated capillaries to discern signaling pathways that regulate protein expression and transport activity of proteins³¹^,³²^,³³^,³⁴^,³⁵^,³⁶^,³⁷. Finally, we have continued to optimize this method for the isolation of human brain capillaries and, recently, we showed increased P-gp expression at the human blood-brain barrier in patients with epilepsy compared to seizure-free control individuals³⁸. Taken together, these developments demonstrate that isolated brain capillaries can serve as a versatile model to study barrier function.

Various in vivo, ex vivo, and in vitro blood-brain barrier models have been used in basic research and industrial drug screening, mainly with the goal of testing drug delivery to the brain³⁹^,⁴⁰^,⁴¹^,⁴²^,⁴³^,⁴⁴. In addition to isolated ex vivo brain capillaries, current blood-brain barrier models include in silico models, in vitro cell culture of isolated brain capillary endothelial cells or immortalized cell lines from various species, in vitro culture of human pluripotent stem cells (hPSC) that differentiate into brain capillary endothelial cells, and microfluidic models on a chip.

In silico models are most commonly used in drug development for selecting drug candidates based on predicted absorption, distribution, metabolism, and excretion (ADME) properties. Methods such as quantitative structure-property relationship (QSPR) models and quantitative structure-activity relationship (QSAR) models are popular methods used in high-throughput screening of libraries to predict brain penetration of drug candidates⁴⁵^,⁴⁶. These models are useful to screen molecules for barrier penetration properties.

Betz et al.⁴⁷ established monolayers of cultured brain capillary endothelial cells as an in vitro blood-brain barrier model system. In vitro cell culture models using fresh tissue or immortalized endothelial cell lines such as human cerebral microvessel endothelial cells (hCMECs) can be another high-throughput screening tool for brain penetration or mechanistic studies. However, brain capillary endothelial cell culture models lack the physiologic shear stress of blood flow inside the capillary lumen, are limited in overall biologic complexity, and undergo changes in expression and localization of important barrier components such as tight junction proteins, surface receptors, transporters, enzymes, and ion channels⁴⁸^,⁴⁹^,⁵⁰. Conversely, endothelial monolayers derived from hPSCs, have low sucrose permeability compared to hCMEC/D3 cultures and contain polarized expression of some blood-brain barrier transporters, adhesion molecules, and tight junctions⁵¹^,⁵². However, these cells are also subject to changing properties in the culture, and the system must be validated for its recapitulation of in vivo barrier properties⁵².

Newer trends in blood-brain barrier research include utilizing 3D tissue culture systems to create artificial capillaries, using the organ-on-chip technology to generate microfluidic devices, or utilizing the hollow fiber technology⁵³^,⁵⁴^,⁵⁵. Artificial capillaries, however, have significantly larger diameters (100–200 µm) than brain capillaries (3–7 µm). Hence, the shear forces in vitro do not fully resemble the in vivo situation. This is addressed in "blood-brain-barrier-on-a-chip" microfluidic devices, where artificial membranes form "blood" and "brain" compartments and fluids are pumped through these devices generating microfluidic shear forces. Similarly, co-cultures of endothelial cells in various combinations with astrocytes and vascular smooth muscle cells have also been used with the hollow fiber technology to recreate rheological parameters present under in vivo conditions⁵⁶^,⁵⁷^,⁵⁸. However, it is unclear how well this model reflects other properties of the blood-brain barrier such as transport, metabolism, signaling, and others. These artificial capillary and chip models are suitable for high-throughput screening of drugs, but the cells used to generate these models are also subject to change during culture.

Frozen and fixed brain slices or primary brain capillary endothelial cell cultures are additional models that can be used tostudy the human microvasculature⁵^,⁵⁹^,⁶⁰^,⁶¹. For example, immunohistochemistry of fixed brain tissue is used to determine protein localization and expression in healthy compared to diseased tissue.

In addition to tissue slices and the in vitro models described above, freshly isolated brain capillaries can be utilized to study blood-brain barrier function. Limitations of this isolated capillary model include the difficulty to obtain fresh human brain tissue, absence of astrocytes and neurons, and a relatively time-consuming isolation process. An advantage of the isolated brain capillary model is that this model closely resembles the in vivo situation and, therefore, can be used to characterize barrier function and dysfunction. Importantly, it can also be used to discern signaling mechanisms using a multitude of assays and molecular techniques³^,¹⁹^,⁶²^,⁶³.

Our laboratory has access to both fresh and frozen human brain tissue through the Sanders-Brown Center on Aging (IRB #B15-2602-M)⁶⁴. In this context, autopsies follow a standard protocol, brains are obtained in <4 h, and all procedures conform to NIH Biospecimen Best Practice Guidelines⁶⁵. Given this unique access to human brain tissue, we established and optimized a protocol to isolate brain capillaries from human brain tissue that results in a high yield of intact, viable human brain capillaries. Two common endpoints of interest are to determine the protein expression and activity. In this regard, we and others have established various assays that can be used with isolated brain capillaries to study protein expression and activity levels. These assays include Western blotting, Simple Western assay, enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), zymography, transport activity assays, and capillary leakage assays. These assays allow researchers to study changes in barrier function in human pathologic conditions, determine pathways that govern protein expression and activity, and identify pharmacologic targets for the treatment of blood-brain barrier associated diseases.

Taken together, freshly isolated brain capillaries can serve as a robust and reproducible model of the blood-brain barrier. Especially, this model can be combined with many different assays to determine a wide array of endpoints to study barrier function.

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 to protect laboratory personnel. Working with human tissue in the US requires a biosafety level 2 laboratory as well as safety precautions and training in accordance with NIH Section IV-B-7, OSHA Act of 1970 Clause 5(a)(1) and the user's institutional biological safety program. In general, Institutional Biosafety Committee and/or Institutional Review Board approval must be obtained prior to conducting any research involving human materials (tissue, body fluids). The training is required for all personnel working with human materials and includes basic laboratory safety training, e.g., Chemical Hygiene and Laboratory Safety, as well as specific training on biological safety, hazardous waste, and human blood-borne pathogens. All personnel working with human materials are highly recommended to obtain Hepatitis B vaccinations, prior to working with human materials. Personnel are required to wear specific personal protective equipment while working with human materials, e.g., a cuffed lab coat and a face shield, and wear gloves at all times. All work is performed in a biosafety cabinet (class 2). All equipment that comes in contact with human materials and any waste from human materials is handled appropriately to prevent contamination and/or infection of personnel. All equipment and surfaces are cleaned with 10% bleach and 75% ethanol following each procedure involving human materials. A spill with human materials must be immediately cleaned up. Glassware is autoclaved after each use. Waste, including unfixed human tissue, is collected in a labeled biohazard waste bag and autoclaved. Sharps are collected in a puncture- and leak-proof container labeled as biohazardous. All waste from human materials is disposed according to the institution's biological safety regulations.

NOTE: Our laboratory obtains fresh frontal cortex samples from deceased individuals through the Sanders-Brown Center on Aging (IRB #B15-2602-M). Inclusion criteria are: enrollment in the UK-ADC longitudinal autopsy cohort study and a Post-Mortem Interval (PMI) ≤4 h⁶⁴. Autopsies follow a standard protocol and all procedures conform to NIH Biospecimen Best Practice Guidelines⁶⁵. A short PMI of less than 4 h is of highest importance to ensure capillary viability after isolation. Both fresh and frozen tissue can be used. If freezing is necessary, freshly obtained human brain tissue should be shock-frozen in liquid nitrogen and stored at -80 °C. Fresh or thawed tissue should be stored in isolation buffer (see below) and processed quickly. We find that 10 g of fresh human tissue yields about 100 mg of brain capillaries (wet weight).

1. Setup

Buffer preparation
NOTE: The volume of buffer needed depends on the amount of tissue. All buffer volumes in the following protocol are based on 10 g of human brain cortex tissue.
1. L Isolation Buffer: Use 1.5 L of Dulbecco's phosphate-buffered saline (DPBS; 2.7 mM KCl, 1.47 mM KH₂PO₄, 136.9 mM NaCl, 8.1 mM Na₂HPO₄, 0.9 mM CaCl₂, 0.49 mM MgCl₂) and supplement with 5 mM D-glucose (1.35 g) and 1 mM sodium pyruvate (0.165 g). After adding the glucose and pyruvate, adjust to pH 7.4 with sodium hydroxide. Cool and store the buffer to 4 °C prior to use.
2. Bovine Serum Albumin (BSA): Add 10 g of BSA powder to 1 L of isolation buffer to a final BSA concentration of 1%. Stir slowly to avoid bubbles, adjust to pH 7.4, and store at 4 °C overnight. Immediately prior to use, gently stir; avoid forming bubbles to avoid albumin denaturation.
3. density gradient medium: Weigh 18 g of density gradient medium into a glass bottle and add a magnetic stir bar. Add 60 mL of isolation buffer and shake vigorously for 5 min until all powder is suspended. Store overnight at 4 °C to allow the density gradient medium to dissolve. Stir for 10 min right before use.
4. Store all buffers at 4 °C; keep all tools and buffers on ice during the entire isolation procedure. Stir all buffers before use.
Experimental setup
1. Mount the pestle of the Potter-Elvehjem tissue grinder onto the electronic overhead stirrer. Place the Potter-Elvehjem tissue grinder and the Dounce homogenizer with pestle on ice under the hood. Prepare a 300 µm filter mesh (5 x 5 cm²), fold it to a cone, and insert and attach it to a 50 mL Falcon tube with tape (Figure 1A).
2. Place connecting rings and cell strain filters (pore size: 30 µm) on 50 mL Falcon tubes. Prepare biohazardous waste bags. Place all required equipment in the biosafety cabinet (see Table of Materials).

2. Brain Sample Preparation

NOTE: Figure 1A shows the workflow chart of the entire isolation procedure described below. Human brain tissue can stem from any part of the cortex and can be used fresh or frozen. Frozen brain tissue can be thawed at room temperature (no buffer; ~30 min for 10 g). To achieve comparable results, the brain tissue should be obtained from the same brain region for each experiment. This protocol is optimized for fresh (PMI <4 h) human cerebral cortex that has not been frozen.

Preparation of human brain tissue: Document the weight of the brain tissue. All numbers in the following protocol are appropriate for 10 g of fresh human brain tissue. Place the brain tissue in a 100 mm Petri dish. Carefully remove all the meninges with forceps. Use a scalpel to cut off the white matter.
Mincing of the human brain tissue: Carefully cut up the brain tissue and mince it with a scalpel. Mince for about 5 min (2–3 mm pieces). Transfer the brain tissue to the Potter-Elvehjem tissue grinder. Add 30 mL of isolation buffer.
NOTE: The minced tissue pieces are difficult to see since the brain tissue turns into mush through the mincing process.

3. Homogenization

Potter-Elvehjem tissue grinder (clearance: 150–230 µm): Homogenize each sample with 100 strokes at a homogenizer speed of 50 rpm. Document the time every 25 strokes and the total time needed for 100 strokes. See Table 1 for a proposed homogenization protocol; the total time for homogenizing 10 g of human frontal cortex is about 22 min. Do not stir in air to prevent bubbles.
Dounce homogenizer (clearance: 80–130 µm): Transfer the homogenate to a Dounce homogenizer on ice. Homogenize the suspension with 20 strokes (~6 s/stroke, total of ~2 min). Avoid bubbles.

4. Centrifugation

Distribute the brain homogenate equally into four 50 mL centrifugation tubes and document the total volume of the homogenate. Distribute 50 mL of density gradient buffer into the centrifugation tubes (12.5 mL per tube). Use 10 mL of isolation buffer to rinse the pestle and homogenizer, and distribute into the four centrifugation tubes (~2.5 mL per tube).
Tightly close the centrifuge tubes with caps. Mix the homogenate, density gradient medium, and buffer by vigorously shaking the tubes. Centrifuge at 5,800 x g for 15 min at 4 °C (fixed angle rotor); select a medium deceleration speed to keep the pellet attached to the tube. Discard the supernatant and resuspend each pellet in 2 mL of 1% BSA.

5. Filtration

NOTE: To separate the capillaries from red blood cells and other cell debris, several filtration steps are necessary.

300 µm mesh: After re-suspending the pellet, filter the suspension through the 300 µm mesh. Capillaries are filtered through the mesh, whereas larger vessels and larger brain debris remain on the mesh. Carefully wash the mesh with up to 50 mL of 1% BSA. Discard the mesh.
NOTE: This filtration step clears the capillary suspension from any larger vessels or chunks of brain debris.
30 µM cell strain filter
NOTE: This filtration step separates capillaries from red blood cells and other brain debris.
1. Distribute the capillary filtrate from step 6.1 over the five 30 µm cell strain filters (about 10 mL of capillary filtrate per cell strain filter). Capillaries are held back by this filter, whereas red blood cells, other single cells, and small brain debris pass through the filter and are collected in the filtrate.
2. Wash each filter with 25 mL of 1% BSA. Afterwards, pour all filtrates over the sixth filter to increase the yield. Wash each filter with 50 mL of 1% BSA; keep the cell strain filters with containing the capillaries and discard the filtrate.

6. Capillary Collection

Turn the filters upside down and wash the capillaries with 50 mL of 1% BSA for each filter into 50 mL tubes. Gently apply pressure with the pipet tip of a 5 mL pipettor and move it across the filter to wash off the brain capillaries.
Make sure to wash off all brain capillaries, especially from the rim of the filter. Avoid bubbles since this makes the filtration process more difficult and increases the chance of capillary loss.

7. Washing

After collecting the capillaries, centrifuge all samples at 1,500 x g for 3 min at 4 °C (swinging bucket rotor). Remove the supernatant and re-suspend the pellet in approximately 3 mL of isolation buffer. Combine all resuspended pellets from one sample in a 15 mL conical tube and fill it with isolation buffer. Centrifuge again at 1,500 x g for 3 min at 4 °C and wash two more times.
Document the capillary purity with a microscope (100X magnification) and camera (Figure 1B).
NOTE: The brain capillary yield from 10 g of human brain tissue is usually about 100 mg. The isolated brain capillaries can now be used for experiments, processed (e.g., lysate, membrane isolation), or be flash-frozen and stored at -80 °C in cryotubes for a minimum of 6–12 months (avoid multiple freeze-thaw cycles).

Representative 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, isolated human brain capillaries reveal a tubular, intact structure and morphology. Figure 2A shows a representative transmitted light image of a human brain capillary with an attached pericyte and a red blood cell in the lumen. All of the findings regarding diameter, size, and morphology are in accordance with previous reports on the structure of isolated brain capillaries¹²^,¹⁷^,¹⁸. The isolated human brain capillary in Figure 2B was immunostained for P-gp (green) using C219 as the primary antibody (1 µg/mL); nuclei were counterstained with DAPI (1 µg/mL).

Figure 1: Flowchart for capillary isolation. (A) The pictogram illustrates major steps of the procedure to isolate brain capillaries from fresh human tissue. (B) The picture shows isolated human brain capillaries under a light microscope directly after isolation (100X magnification). Please click here to view a larger version of this figure.

Figure 2: Isolated human brain capillary. (A) A transmitted light image of an isolated human brain capillary. (B) The confocal microscope image shows an isolated human brain capillary immunostained for P-gp (green; C219 1 µg/mL); nuclei were counterstained with DAPI (blue; 1 µg/mL). Please click here to view a larger version of this figure.

Figure 3: Troubleshooting density centrifugation. The pictogram shows the preparation after the density gradient centrifugation. It highlights the effects of too much and too little density gradient medium and how this affects the separation and the resulting capillary pellet. Please click here to view a larger version of this figure.

Figure 4: P-gp protein expression in isolated human brain capillaries. The Western blot shows strong bands for P-gp (1 µg/mL) in isolated human capillaries compared to hCMECD3 cells. β-Actin was used as a loading control (1 µg/µL). Please click here to view a larger version of this figure.

Figure 5: Applications for isolated human brain capillaries. An overview of the most common applications for isolated brain capillaries published in the literature. Isolated capillaries have been used for: 1) Genomics⁸⁵^,⁸⁶, 2) Proteomics³^,³⁸^,⁸⁷^,⁸⁸^,⁸⁹^,⁹⁰^,⁹¹^,⁹²^,⁹⁴^,⁹⁵^,⁹⁶, 3) Functional Proteomics²^,³⁸, and 4) Cellomics⁸²^,⁸³^,⁸⁴^,⁹⁷^,⁹⁸^,⁹⁹. Please click here to view a larger version of this figure.

Strokes	Time [min]
1–25	7–7.5
26–50	5–5.5
51–75	5–5.5
76–100	5–5.5
Total Time:	22–24 min

Table 1: Homogenization protocol. The homogenization protocol for the Potter-Elvehjem tissue grinder to homogenize 10 g of human frontal cortex at a homogenization speed of 50 rpm. Note that the first several strokes require additional time to homogenize the minced tissue. After this initial homogenization, each stroke is 12 s in duration (6 s for downward movement, 6 s for upward movement). Thus, after the initial homogenization, 5 strokes can be accomplished in 1 min, or 25 strokes in 5 min.

Problem	Potential Cause	Solution
No Capillary Pellet	1) Incorrect Ficoll concentration	1) Adjust Ficoll concentration
	2) Incorrect centrifugation speed	2) Adjust centrifugation speed
	3) Incorrect acceleration and/or deceleration speed	3) Adjust acceleration and/or deceleration speed
Low Capillary Yield	1) Meninges blocking filtration steps	1) Remove ALL meninges prior to filtration
	2) Too many capillaries lost during isolation procedure	2) Calculate buffer concentration correctly, rinse pipette tips
	3) Washing capillaries off PluriStrainer filters was insufficient	3) Turn filters over and carefully inspect for capillaries (use microscope)
	4) Excess bubbles during resuspensions	4) Pipette slowly to avoid bubbles
Non-Viable Capillaries	1) Extended post-mortem interval	1) Reduce interval if possible or use snap-frozen brains
	2) Using frozen tissue for the isolation	2) Use fresh tissue
	3) Time of isolation procedure too long	3) Optimize workflow
	4) Equipment/buffers were not kept on ice during the isolation procedure	4) Keep equipment and buffers on ice during the isolation

Table 2: Troubleshooting of common problems. A list of the most common errors and problems that occur during the isolation procedure and how they can be resolved.

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 human frontal cortex tissue. However, it is relatively simple to modify this procedure for: 1) more or less than 10 g of tissue, 2) frozen brain tissue, or 3) brain tissue from a brain region other than the frontal cortex. First, with more or less than 10 g of brain tissue, the necessary volume of the buffers can simply be scaled up or down to the available amount of tissue. Thus, if only 5 g of brain tissue is available, the volume of the buffers should be reduced by half. Second, we describe a capillary isolation that used fresh brain tissue, but frozen tissue may be used if fresh tissue is unavailable³⁸. Third, we used fresh brain tissue taken from the frontal cortex, but capillaries may be isolated from other cortical brain regions if there is enough available tissue. It is also possible to isolate capillaries from non-cortical brain regions (e.g., white matter), but these regions have a different cell composition and capillary density ⁶⁶^,⁶⁷^,⁶⁸. Thus, using tissue from a different brain region would likely require the protocol to be adjusted (e.g., buffer volume, gradient medium density, centrifugation speed, and/or number of filtration steps).

The capillary isolation procedure, while not complex per se, is sensitive to small perturbations or alterations in the protocol. Modifications may result in a diminished capillary yield or reduced capillary viability. Table 2 outlines the most common errors and problems that are encountered during the isolation and lists tips to avoid these errors and solutions for troubleshooting if they occur. The most common problem associated with the procedure is a low capillary yield. The loss of capillaries is often the cumulative sum of small losses at each step and is due to small deviations across the procedure. A critical step in which a large amount of capillaries may be lost is the density centrifugation. An incorrect concentration in the buffer results in an incorrect density to separate the capillaries from cellular debris that reduces the volume of the capillary pellet. Figure 3 shows the consequences of too little or too much density gradient medium in the centrifugation step relative to brain homogenate. Adjusting to the correct the concentration may solve this problem. Note that the acceleration and deceleration speed of the centrifuge can also affect the formation of the brain capillary pellet. Capillaries may also be lost during steps 6–7 if part of the capillary material sticks to the pipette tips. This issue can be addressed by thoroughly rinsing each pipet tip before changing it. During step 7, washing off the capillaries from the cell strain filter may be incomplete and/or capillaries may stick to the rim of the filter. This can be avoided by checking the filter under the microscope followed by additional washing steps. Losing capillaries during each step of the isolation procedure can result in a negligible capillary pellet or not enough capillary material for further processing and experimentation.

Isolating brain capillaries from fresh human tissue represents a unique blood-brain barrier model that closely resembles the in vivo situation. However, several limitations of the technique exist. One challenge is the availability of fresh human tissue. As the optimal PMI is ≤4 h, brain tissue collected at a significantly longer PMI will not be fresh enough for some downstream applications. In some cases, it may be difficult to obtain tissue amounts that are large enough for multiple experimental groups, thereby restricting downstream applications. Thus, isolating fresh capillaries from rodent¹⁹, canine⁶⁹, bovine⁴², or porcine⁷⁰ brain tissue may be more suitable to model the blood-brain barrier. Factors that determine the variability of human brain tissue such as age, sex, ethnicity, disease state, medication history, brain region of sample, and PMI should be taken into consideration when interpreting and publishing data. On an experimental level it is important to note that isolated capillaries still include pericytes, but astrocytic endfeet are removed by the procedure⁴⁴. It needs to be taken into consideration that the model presented here serves as an ex vivo model of the blood-brain barrier (i.e., capillary endothelial cells) but not as a model of the neurovascular unit.

Working with any human tissue always presents an inherent safety risk and researchers must take appropriate precautions during the isolation procedure to avoid infection. Specifically, in the U.S., work with human tissue requires designated laboratory space that is BSL 2-certified and includes a biosafety cabinet (class A2). In addition, staff must use personal protective equipment (i.e., lab coat, gloves, and face shield) and designated equipment for work with human tissue and implement biohazardous waste handling. Implementing these safety measures is time-consuming, cost-intensive, and increases the difficulty of the procedure, especially for inexperienced laboratory personnel.

The blood-brain barrier is highly conserved among organisms with a well-defined CNS⁷¹. Modeling the human blood-brain barrier is difficult because there is complex neurovascular coupling among the cells of the neurovascular unit. An adult human brain has been estimated to have in average about 86 billion neurons and it is thought that almost every neuron has its own capillary in the vicinity to ensure proper supply with oxygen and nutrients⁷²^,⁷³. Capillary endothelial cells constitute the largest surface area of the blood-brain interface (12–18 m² for a healthy adult human). Tight junctions represent an impediment to a wide array of pharmacotherapeutics by blocking paracellular diffusion of solutes. In addition, numerous studies describe barrier dysfunction in neurodegenerative disorders, e.g., Alzheimer's disease⁷⁴, stroke⁷⁵, epilepsy³⁸^,⁷⁶, multiple sclerosis⁷⁷, and traumatic brain injury²⁸^,⁷⁸. Thus, it is imperative to establish models that closely represent the human blood-brain barrier and allow for a better understanding of barrier function in health and disease.

Numerous in vitro cell culture models of the blood-brain barrier exist; for expert reviews on the subject see ⁴¹^,⁴⁹^,⁵¹^,⁶⁹^,⁷⁹^,⁸⁰. Briefly, both freshly isolated brain capillary endothelial cells for primary culture and immortalized brain capillary endothelial cell lines are available. Primary cultures of cerebral microvessel endothelial cells are mostly used from mouse, rat, pig, and cow. However, primary cell cultures are labor-intensive since cells must be freshly isolated. Immortalized brain capillary endothelial cell lines are available from mouse, rat, and human and are less labor-intensive because they can be passaged for longer-term use. However, even immortalized cell lines have a limit on how often they can be passaged before losing their endothelial characteristics. Both primary cells as well as immortalized cells lines are often cultured on plates to model the brain capillary endothelium and measure the barrier permeability and drug transport across the cell monolayer, thereby mimicking blood-to-brain transport⁴¹^,⁸¹^,⁸². The culture media in these models may also be modified or supplemented with astrocytes, pericytes, or other physiologically relevant factors like cAMP⁴¹^,⁸³^,⁸⁴.

The advantage of immortalized cell lines is their relatively easy access and availability. While cultured endothelial cells can reach confluence, they lose endothelial cell properties as they grow side-by-side in a monolayer. For example, cultured hCMECs display reduced expression of transporters like P-gp, tight junction proteins, and display variable permeability to xenobiotics⁴¹. Figure 4 shows a Western blot for P-gp protein expression in hCMEC/D3 cells compared to freshly isolated human capillaries. Despite a 10-fold lower amount of total protein, the signal for P-gp is stronger in isolated human capillaries compared to hCMEC/D3 cells. This indicates that hCMEC/D3 cells lost a significant amount of P-gp protein expression in culture. Moreover, differences in culture media, environment, and equipment affect key measures of barrier integrity, i.e., TEER measurements in Transwell plate assays. Some of these issues can be overcome by utilizing the isolated brain capillary model that more closely represents the human blood-brain barrier in vivo.

Isolated brain capillaries have been used for a wide range of studies, including genomics, proteomics, functional proteomics, and cellomics studies (Figure 5). In addition, many techniques and methods exist to analyze isolated brain capillaries within each of these fields. Notably, the experimental techniques shown in Figure 5 can be used on brain capillaries isolated from a number of sources, including human, bovine, rodent, and porcine tissue, which may facilitate translational research. For example, Li et al.⁸⁵ studied the genomics of the blood-brain barrier using suppression subtractive hybridization by purifying mRNA isolated from rat brain capillaries. Additionally, Ott et al.⁸⁶ used RT-PCR and qRT-PCR to study the regulation of P-gp by PXR. Many proteomic studies utilize Western blotting³, Dot Blot analysis⁸⁷, Simple Western assays³^,³⁸, ELISA⁸⁸^,⁸⁹, immunoprecipitation³, and immunostaining³^,⁹⁰^,⁹¹. To discern transporter trafficking in the brain endothelium, McCaffrey et al.⁹² used subcellular fractionation of isolated brain capillaries. Sánchez del Pino et al.⁹³used isolated bovine endothelial membrane vesicles to discern transporter location and transport direction across the blood-brain barrier. In other proteomic studies, researchers used liquid chromatography and tandem mass spectrometry to quantify transporter proteins⁹⁴^,⁹⁵^,⁹⁶. Functional proteomic studies have utilized transporter and leakage assays²^,³⁸. Hartz et al.² used zymography to determine enzyme activity in isolated brain capillaries. In addition, vast cellomic research using cell culture has generated numerous endothelial cell lines and models of the blood-brain barrier⁸²^,⁸³^,⁸⁴. With these models, common assays used include migration assays⁹⁷^,⁹⁸, viability and toxicity assays⁹⁹, and angiogenesis assays⁹⁸.

Isolated brain capillaries allow for accurate characterization of protein expression and activity and description of signaling pathways at the blood-brain barrier. This is due, in part, to the capillary content of the brain, which is only approximately 1% (v/v). Thus, using whole brain homogenate or brain slices as a substitute for purified capillary endothelial cells will most likely result in a poor signal-to-noise ratio²⁴. In addition, after isolation, brain capillaries are viable for at least 6 h (unpublished data from mouse and rat), which allows studies to discern specific signaling pathways. It is recommended to include a control group from the same preparation.

Representative and translational models of the blood-brain barrier, such as the isolated capillary model discussed in this report, are needed to study barrier function in health and disease. Here we present a protocol to obtain isolated human brain capillaries at a good yield and high quality that can serve as an ex vivo model of the blood-brain barrier. Isolated capillaries retain their original structure and function, which allows using them for a number of post-isolation molecular, biochemical, and physiological assays. Care should be taken when handling human tissue samples, preferably in a BSL 2 or higher setting.

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank and acknowledge Dr. Peter Nelson and Sonya Anderson at the UK-ADC Brain Tissue Bank for providing all human brain tissue samples (NIH grant number: P30 AG028383 from the National Institute on Aging). We thank Matt Hazzard and Tom Dolan, Information Technology Services, Academic Technology and Faculty Engagement, University of Kentucky for graphical assistance. This project was supported by grant number 1R01NS079507 from the National Institute of Neurological Disorders and Stroke (to B.B.) and by grant number 1R01AG039621 from the National Institute on Aging (to A.M.S.H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Neurological Disorders and Stroke or the National Institute on Aging. The authors declare no competing financial interests.

Materials

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 8X12	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 1-5mL	Thermo Fisher Scientific, Pittsburgh, PA, USA	21377823T1	wash capillaries off filter
Finnpipette II Pipette 100-1000 µL	Thermo Fisher Scientific, Pittsburgh, PA, USA	21377821T1	resuspend pellet in BSA
Pipet Boy	Integra, Hudson, NH, USA	739658
50mL 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

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Hartz, A. M., Schulz, J. A., Sokola, B. S., Edelmann, S. E., Shen, A. N., Rempe, R. G., Zhong, Y., Seblani, N. E., Bauer, B. Isolation of Cerebral Capillaries from Fresh Human Brain Tissue. J. Vis. Exp. (139), e57346, doi:10.3791/57346 (2018).

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