Name: isolation of cerebral capillaries from fresh human brain tissue
Uploaded: 2018-09-12T13:00:00.000+00:00
Duration: 6 min 35 s
Description: Watch this Scientific Journal Video about Isolation of Cerebral Capillaries from Fresh Human Brain Tissue at JoVE.com

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.

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&#39;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&#39;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&#39;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) &#8804;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 &#176;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
<ol>
	<li>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.
	<ol>
		<li>L Isolation Buffer: Use 1.5 L of Dulbecco&#39;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 &#176;C prior to use.</li>
		<li>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 &#176;C overnight. Immediately prior to use, gently stir; avoid forming bubbles to avoid albumin denaturation.</li>
		<li>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 &#176;C to allow the density gradient medium to dissolve. Stir for 10 min right before use.</li>
		<li>Store all buffers at 4 &#176;C; keep all tools and buffers on ice during the entire isolation procedure. Stir all buffers before use.</li>
	</ol>
	</li>
	<li>Experimental setup
	<ol>
		<li>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 &#181;m 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).</li>
		<li>Place connecting rings and cell strain filters (pore size: 30 &#181;m) on 50 mL Falcon tubes. Prepare biohazardous waste bags. Place all required equipment in the biosafety cabinet (see Table of Materials).</li>
	</ol>
	</li>
</ol>
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 &#60;4 h) human cerebral cortex that has not been frozen.
<ol>
	<li>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.</li>
	<li>Mincing of the human brain tissue: Carefully cut up the brain tissue and mince it with a scalpel. Mince for about 5 min (2&#8211;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.</li>
</ol>
3. Homogenization
<ol>
	<li>Potter-Elvehjem tissue grinder (clearance: 150&#8211;230 &#181;m): 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.</li>
	<li>Dounce homogenizer (clearance: 80&#8211;130 &#181;m): Transfer the homogenate to a Dounce homogenizer on ice. Homogenize the suspension with 20 strokes (~6 s/stroke, total of ~2 min). Avoid bubbles.</li>
</ol>
4. Centrifugation
<ol>
	<li>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).</li>
	<li>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 &#176;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.</li>
</ol>
5. Filtration
NOTE: To separate the capillaries from red blood cells and other cell debris, several filtration steps are necessary.
<ol>
	<li>300 &#181;m mesh: After re-suspending the pellet, filter the suspension through the 300 &#181;m 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.</li>
	<li>30 &#181;M cell strain filter 
	NOTE: This filtration step separates capillaries from red blood cells and other brain debris.
	<ol>
		<li>Distribute the capillary filtrate from step 6.1 over the five 30 &#181;m 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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
6. Capillary Collection
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>
7. Washing
<ol>
	<li>After collecting the capillaries, centrifuge all samples at 1,500 x g for 3 min at 4 &#176;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 &#176;C and wash two more times.</li>
	<li>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 &#176;C in cryotubes for a minimum of 6&#8211;12 months (avoid multiple freeze-thaw cycles).</li>
</ol>

The authors have nothing to disclose.

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...

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&#39;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 &#181;m 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 &#947;-glutamyl transpeptidase (&#947;-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 (ETB) 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 (A&#946;)40and A&#946;42brain 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&#39;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&#8211;200 &#181;m) than brain capillaries (3&#8211;7 &#181;m). Hence, the shear forces in vitro do not fully resemble the in vivo situation. This is addressed in &#34;blood-brain-barrier-on-a-chip&#34; microfluidic devices, where artificial membranes form &#34;blood&#34; and &#34;brain&#34; 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 &#60;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.

<table><tbody><tr><td>&lt;strong&gt;Personal Protective Equipment (PPE)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Diamond Grip Plus Latex Gloves, Microflex Medium</td><td>VWR, Radnor, PA, USA</td><td>32916-636</td><td>PPE</td></tr><tr><td>Disposable Protective Labcoats</td><td>VWR, Radnor, PA, USA</td><td>470146-214</td><td>PPE; due to the nature of the human source material, the use of a disposable lab coat is recommended</td></tr><tr><td>Face Shield, disposable</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>19460102</td><td>PPE; due to the nature of the human source material, the use of a disposable face shield is recommended</td></tr><tr><td>Safety Materials</td><td></td><td></td><td></td></tr><tr><td>Clavies High-Temperature Autoclave Bags 8 x 12</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>01-815-6</td><td></td></tr><tr><td>Versi Dry Bench Paper 18&quot; x 20&quot;</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>14-206-32</td><td>to cover working areas</td></tr><tr><td>VWR Sharps Container Systems</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>75800-272</td><td>for used scalpels</td></tr><tr><td>Bleach 8.2% Clorox Germicidal 64 oz.</td><td>UK Supply Center, Lexington, KY, USA</td><td>323775</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>4 &amp;deg;C Refrigerator</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>13-986-148</td><td></td></tr><tr><td>Accume BASIC AB15 pH Meter</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>AB15</td><td></td></tr><tr><td>Heidolph RZR 2102 Control</td><td>Heidolph, Elk Grove Village, IL, USA</td><td>501-21024-01-3</td><td></td></tr><tr><td>Sorvall LEGEND XTR Centrifuge</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>75004521</td><td></td></tr><tr><td>Leica L2 Dissecting Microscope</td><td>Leica Microsystems Inc, Buffalo Grove IL, USA</td><td></td><td>used to remove meninges</td></tr><tr><td>POLYTRON PT2500 Homogenizer</td><td>Kinematica AG, Luzern, Switzerland</td><td>9158168</td><td></td></tr><tr><td>Scale P-403</td><td>Denver Instrument, Bohemia, NY, USA</td><td>0191392</td><td></td></tr><tr><td>Standard mini Stir</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>1151050</td><td></td></tr><tr><td>Thermo-Flasks Liquid Nitrogen Dewar</td><td>Thermal Scientific, Mansfiled, TX, USA</td><td>11-670-4C</td><td>used to freeze the tissue?</td></tr><tr><td>Voyager Pro Analytical Balance</td><td>OHAUS, Parsippany, NJ, USA</td><td>VP214CN</td><td></td></tr><tr><td>ZEISS Axiovert Microcope</td><td>Carl Zeiss, Inc Thornwood, NY, USA</td><td></td><td>used to check isolated capillaries</td></tr><tr><td>&lt;strong&gt;Tools and Glassware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Finnpipette II Pipette 1-5 mL</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>21377823T1</td><td>wash capillaries off filter</td></tr><tr><td>Finnpipette II Pipette 100-1,000 &amp;micro;L</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>21377821T1</td><td>resuspend pellet in BSA</td></tr><tr><td>Pipet Boy</td><td>Integra, Hudson, NH, USA</td><td>739658</td><td></td></tr><tr><td>50 mL Falcon tubes 25/rack - 500/cs</td><td>VWR, Radnor, PA, USA</td><td>21008-951</td><td></td></tr><tr><td>EISCO Scalpel Blades</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>S95938C</td><td>to mince brain tissue</td></tr><tr><td>PARAFILM</td><td>VWR, Radnor, PA, USA</td><td>52858-000</td><td>to cover beaker and volumetric flask</td></tr><tr><td>Thermo Scientific Finntip Pipet Tips 5 mL</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>21-377-304</td><td>to wash capillaries off filter</td></tr><tr><td>60 mL syringe with Luer-Lok</td><td>Thermo Fisher Scientific, Pittsburgh, PA, USA</td><td>BD309653</td><td>used with connector ring to filter capillaries</td></tr><tr><td>Scalpel Handle #4</td><td>Fine Science Tools, Foster City, CA, USA</td><td>10060-13</td><td>used for mincing</td></tr><tr><td>Dumont Forceps #5</td><td>Fine Science Tools, Foster City, CA, USA</td><td>11251-10</td><td>used to remove meninges</td></tr><tr><td>Potter-Elvehjem Tissue Grinder</td><td>Thomas Scientific, Swedesboro, NJ, USA</td><td>3431E25</td><td>50 mL volume, clearance: 150-230 &amp;mu;m</td></tr><tr><td>Dounce Homogenizer</td><td>VWR, Radnor PA USA</td><td>62400-642</td><td>15 mL volume, clearance: 80-130 &amp;mu;m</td></tr><tr><td>Spectra/Mesh Woven Filters (300 &amp;micro;m)</td><td>Spectrum Laboratories, Rancho Dominguez, CA, USA</td><td>146424</td><td>Used to filter capillary suspension to remove any meninges that may be left</td></tr><tr><td>pluriStrainers (pore size: 30 &amp;micro;m)</td><td>pluriSelect Life Science, Leipzig, Germany</td><td>43-50030-03</td><td></td></tr><tr><td>Connector Ring</td><td>pluriSelect Life Science, Leipzig, Germany</td><td>41-50000-03</td><td>reuse multiple time</td></tr><tr><td>1 L Volumetric Flask</td><td></td><td></td><td>for preparation of Isolation Buffer</td></tr><tr><td>1 L Beaker</td><td></td><td></td><td>for preparation of 1% BSA</td></tr><tr><td>Stir Bar</td><td></td><td></td><td>for preparation of 1% BSA and Ficoll&lt;sup&gt;&amp;reg;&lt;/sup&gt;</td></tr><tr><td>Schott Bottle (60 mL)</td><td></td><td></td><td>for preparation of Ficoll&lt;sup&gt;&amp;reg;&lt;/sup&gt;</td></tr><tr><td>Ice Bucket</td><td></td><td></td><td>to keep everything cold</td></tr><tr><td>100 mm Petri dish</td><td></td><td></td><td>for mincing of brain tissue</td></tr><tr><td>Tissue Culture Cell Scraper</td><td>VWR, Radnor, PA, USA</td><td>89260-222</td><td>to remove supernatant after centrifugation</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BSA Fraction V, A-9647</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>A9647-500g</td><td>prepare in DPBS with Ca&lt;sup&gt;2+&lt;/sup&gt; &amp;amp; Mg&lt;sup&gt;2+&lt;/sup&gt; the day before. Avoid bubbles during preparation. Store in the refrigerator. Slowly stir for 10 min before use.</td></tr><tr><td>DPBS with Ca&lt;sup&gt;2+&lt;/sup&gt; &amp;amp; Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Hyclone</td><td>SH30264.FS</td><td>DPBS - part of the Isolation Buffer</td></tr><tr><td>Ficoll PM400</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>F4375</td><td>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.</td></tr><tr><td>Glucose (D-(+) Dextrose)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>G7528</td><td>Glucose (D-(+) Dextrose) Concentration: 5 mM</td></tr><tr><td>Sodium Hydroxide Standard Solution</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>71474</td><td>to adjust pH of the DPBS</td></tr><tr><td>Sodium Pyruvate</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>P2256</td><td>Concentration: 1 mM</td></tr></tbody></table>

isolation of cerebral capillaries from fresh human brain tissue

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.

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&#8211;7 &#181;m diameter and is approximately 100-200 &#181;m long with open lumens; most capillary ends are collapsed. Using conf...

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Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

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.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

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Research

JoVE Journal

Neuroscience

12.5K Views.  University of Kentucky. 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.

Video: Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Biology

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Isolation of Cerebral Capillaries from Human Brain: An Optimized Method to Extract Cerebral Capillaries from Human Brain Tissue

Source: Hartz, A. M. S. et al. <a href="https://www.jove.com/video/57346" target="_blank">Isolation of Cerebral Capillaries from Fresh Human Brain Tissue.</a> J. Vis. Exp. (2018)
This video presents the isolation of cerebral capillaries from a fresh human brain tissue. The isolated capillaries can then be used to generate an ex vivo blood brain barrier model.

Source: Hartz, A. M. S. et al. Isolation of Cerebral Capillaries from Fresh Human Brain Tissue. J. Vis. Exp. (2018)
This video presents the isolation of ...

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

Ex vivo studies

Isolation and Culture of Primary Brain Pericytes: A Procedure to Isolate and Culture Pericytes from Bovine Brain Capillaries Without Endothelial Cell Contamination

Source: H&#248;rlyck, S. et al. <a href="https://www.jove.com/video/61253" target="_blank">Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.</a>&#160;J. Vis. Exp. (2020)
In this video, we demonstrate the procedure to isolate and culture pericytes from bovine brain capillaries without endothelial cell contamination.

Source: H&#248;rlyck, S. et al. Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies.&#160;J. Vis. Exp. ...

In vitro studies

Purifying Capillaries From a Porcine Brain

Source: Nielsen, S. S. E., et al. <a href="https://app.jove.com/v/56277">Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells.</a> 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.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Purification of Porcine Brain ...

Neuronal Culture Techniques

Advanced Neuronal Models

A Method for the Isolation of Neurovascular Units from Rat Brain

Source: Brzica, H., et al,. <a href="https://app.jove.com/v/57698">A simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels.</a> J. Vis. Exp. (2018).
This video demonstrates the process of isolating brain microvessels from euthanized rats. It includes the dissection of the brain, cleaning of the tissue, and mechanical digestion, followed by repeated centrifugation to isolate the pure fraction of microvessels.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. The procedural flow for the protocol ...