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

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. 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 (Post-Mortem Interval or PMI <4 h) human cerebral cortex that has not been frozen.

1. 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.
2. 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.

2. Homogenization

1. 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.
2. 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.

3. Centrifugation

1. 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).
2. 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.

4. Filtration

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

1. 300 µm mesh: After resuspending 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.
2. 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 4.1 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.

5. Capillary Collection

1. 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.
2. 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.

6. Washing

1. 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.
2. 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).

Strokes	Time
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.

Results

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).

Materials

Name	Company	Catalog Number	Comments
Personal Protective Equipment (PPE)
Diamond Grip Plus Latex Gloves, Microflex Medium	VWR, Radnor, PA, USA	32916-636	PPE
Disposable Protective Labcoats	VWR, Radnor, PA, USA	470146-214	PPE; due to the nature of the human source material, the use of a disposable lab coat is recommended
Face Shield, disposable	Thermo Fisher Scientific, Pittsburgh, PA, USA	19460102	PPE; due to the nature of the human source material, the use of a disposable face shield is recommended
Safety Materials
Clavies High-Temperature Autoclave Bags 8 x 12	Thermo Fisher Scientific, Pittsburgh, PA, USA	01-815-6
Versi Dry Bench Paper 18" x 20"	Thermo Fisher Scientific, Pittsburgh, PA, USA	14-206-32	To cover working areas
VWR Sharps Container Systems	Thermo Fisher Scientific, Pittsburgh, PA, USA	75800-272	For used scalpels
Bleach 8.2% Clorox Germicidal 64 oz.	UK Supply Center, Lexington, KY, USA	323775
Equipment
4 °C Refrigerator	Thermo Fisher Scientific, Pittsburgh, PA, USA	13-986-148
Accume BASIC AB15 pH Meter	Thermo Fisher Scientific, Pittsburgh, PA, USA	AB15
Heidolph RZR 2102 Control	Heidolph, Elk Grove Village, IL, USA	501-21024-01-3
Sorvall LEGEND XTR Centrifuge	Thermo Fisher Scientific, Pittsburgh, PA, USA	75004521
Leica L2 Dissecting Microscope	Leica Microsystems Inc, Buffalo Grove IL, USA		Used to remove meninges
POLYTRON PT2500 Homogenizer	Kinematica AG, Luzern, Switzerland	9158168
Scale P-403	Denver Instrument, Bohemia, NY, USA	191392
Standard mini Stir	Thermo Fisher Scientific, Pittsburgh, PA, USA	1151050
Thermo-Flasks Liquid Nitrogen Dewar	Thermal Scientific, Mansfiled, TX, USA	11-670-4C	Used to freeze the tissue
Voyager Pro Analytical Balance	OHAUS, Parsippany, NJ, USA	VP214CN
ZEISS Axiovert Microcope	Carl Zeiss, Inc Thornwood, NY, USA		Used to check isolated capillaries
Tools and Glassware
Finnpipette II Pipette 1-5 mL	Thermo Fisher Scientific, Pittsburgh, PA, USA	21377823T1	Wash capillaries off filter
Finnpipette II Pipette 100-1,000 µL	Thermo Fisher Scientific, Pittsburgh, PA, USA	21377821T1	Resuspend pellet in BSA
Pipet Boy	Integra, Hudson, NH, USA	739658
50 mL Falcon tubes 25/rack - 500/cs	VWR, Radnor, PA, USA	21008-951
EISCO Scalpel Blades	Thermo Fisher Scientific, Pittsburgh, PA, USA	S95938C	To mince brain tissue
PARAFILM	VWR, Radnor, PA, USA	52858-000	To cover beaker and volumetric flask
Thermo Scientific Finntip Pipet Tips 5 mL	Thermo Fisher Scientific, Pittsburgh, PA, USA	21-377-304	To wash capillaries off filter
60 mL syringe with Luer-Lok	Thermo Fisher Scientific, Pittsburgh, PA, USA	BD309653	Used with connector ring to filter capillaries
Scalpel Handle #4	Fine Science Tools, Foster City, CA, USA	10060-13	Used for mincing
Dumont Forceps #5	Fine Science Tools, Foster City, CA, USA	11251-10	Used to remove meninges
Potter-Elvehjem Tissue Grinder	Thomas Scientific, Swedesboro, NJ, USA	3.43E+28	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