Isolation of Cerebral Capillaries from Human Brain: An Optimized Method to Extract Cerebral Capillaries from Human Brain Tissue
Published: April 30, 2023
Abstract
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 cerebral capillaries from a fresh human brain tissue. The isolated capillaries can then be used to generate an ex vivo blood brain barrier model.
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 u…
Representative 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).
Declarações
The authors have nothing to disclose.
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 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 |
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Isolation of Cerebral Capillaries from Human Brain: An Optimized Method to Extract Cerebral Capillaries from Human Brain Tissue. J. Vis. Exp. (Pending Publication), e20682, doi: (2023).