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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 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, together with endothelial cells and astrocytes, constitute the NVU1,2,3. The endothelial cells, which form the structural basis of the capillaries, form long cylindrical tubes with a diameter of 5-8 µm. The endothelial cells are sporadically covered with pericytes and surrounded by protrusions from astrocytes; the astrocyte endfeet.
The blood-brain barrier (BBB), situated at the brain capillaries, is the main site for exchange of nutrients, gases and waste products between the brain and the blood. The BBB also protects the brain from endogenous and exogenous neurotoxins and serves as a barrier for the delivery of a large number of drug compounds. The barrier function is a focus area, as well as an obstacle, for drug companies developing central nervous system (CNS) medicines. This has spurred a large interest in investigating the cells of the NVU in culture4. Brain astrocytes and endothelial cells have been cultured and characterized in a number of studies, whereas the studies and protocols for pericyte culture are sparse.
Previously published protocols have described generation of brain capillary pericyte cultures to some degree, using a range of different approaches such as immunopanning5, high- and low-glucose media6, fluorescent-activated cell sorting7, density gradient centrifugation8, etc. Although these methods seem sufficient to obtain cultures of pericytes, some are time consuming, cost expensive and the pericytes obtained might not be ideal due to the number of culture passages that can de-differentiate the pericytes9. Furthermore, the potential of cultured pericytes in in vitro signaling studies has been fairly unexplored until now.
The present work focuses on the generation of pericyte cultures from isolated bovine brain capillaries and the subsequent setup for measurements and imaging studies of changes in intracellular calcium, an important intracellular second messenger. We briefly describe the isolation of capillaries from cortical gray matter (for details see Helms et al.10) and the isolation and culture of pericytes in pure monoculture without contamination with endothelial or glial cells. We then provide a protocol for seeding of pericytes in 96-well plates and loading protocols for the calcium probe Fura-2 AM. Finally, we show how pericytes can be used in real-time confocal imaging in microscope culture chambers and describe the protocols for this.
1. Preparation of buffers and solutions for cell culturing
2. Isolation of capillaries from fresh bovine brain
NOTE: Bovine brain capillaries are isolated and cultured as previously described (Helms et al.10).
3. Seeding and culturing of bovine capillaries
4. Isolation of primary pericytes from bovine brain capillaries
5. Generation and storage of a monoculture of primary bovine pericytes
6. Setting up a pericyte monoculture for experiments
7. Seeding of pericytes in a coated 96-well plate
8. Preparation of buffers and solutions for Ca2+-imaging
9. Loading of pericytes with Fura-2 AM calcium indicator dye in a plate-reader setup
NOTE: All solutions should be at RT before the experiment starts.
10. Well-plate fluorescence reading of pericytes in a plate-reader setup
11. Seeding of pericytes in a coated cell chamber for live imaging
NOTE: Coverslips may also be placed in the bottom of culture wells, coated and seeded with pericytes as described above, and then mounted in the chamber prior to experiments.
12. Loading of pericytes with Cal-520 AM calcium indicator dye for live imaging
NOTE: All solutions should be at RT before the experiment starts.
13. Live imaging of intracellular Ca2+-levels
NOTE: A variety of microscope types can be used for the imaging. Upright or inverted conventional fluorescence microscopes, as well as upright or inverted confocal laser scanning microscopes with appropriate excitation source (488 nm) and emission filters (510-520 nm) can be used. Objectives should be suited for fluorescence and be of a high quality and with high numerical aperture (NA).
Bovine brain capillaries were isolated from fresh brain tissue and Figure 1 presents the capillary seeding and cellular outgrowth over days and subsequent purification of pericytes. The capillaries are fully attached to the flask at day 1 and on day 2 endothelial sprouting has become visible (Figure 1, day 2). After 4 days, the cellular outgrowth is highly distinctive (Figure 1, day 4a) and the ...
In this study, we have presented a method to isolate primary pericytes from bovine brains. The described protocol allows culture of this otherwise rather inaccessible cell type. The subsequently obtained cell culture was a nearly homogenous population of pericytes, with little or no contamination with endothelial cells and glial cells based on cell morphology and protein expression12. Furthermore, we demonstrated a simple and straightforward method to load the pericytes with calcium dyes for ...
The authors declare no competing financial interests.
The authors wish to acknowledge funding from the Lundbeck Foundation Research initiative on Brain Barriers and Drug Delivery (RIBBDD) and Simon Hougners Family Foundation.
Name | Company | Catalog Number | Comments |
ATP | Tocris | 3245 | |
Cal-520 AM | AAT Bioquest | 21130 | |
Cell incubator | Thermo Fisher | ||
Centrifuge | Thermo Fisher | Heraeus Multifuge 3SR+ | Standard large volume centrifuge for spinning down cells |
Collagen IV | Sigma Aldrich | C5533 | |
Confocal laser scanning microscope | Carl Zeiss | Zeiss LSM 510 | Inverted microscope |
Counting chamber | FastRead | 102 | |
Coverslip cell chamber | Airekacells | SC15022 | |
Cremophor EL | Sigma Aldrich | C5135 | Formerly known as Kolliphor EL |
DMSO | Sigma Aldrich | 471267 | |
Dulbecco's Modified Eagles Medium | Sigma Aldrich | D0819 | |
Fetal bovine serum (FBS) | PAA/GE Healthcare | A15-101 | |
Fibronectin | Sigma Aldrich | F1141 | |
Fura-2 AM | Thermo Fisher | F1201 | |
Glass coverslips 22x22 mm | VWR International | 631-0123 | |
HBSS | Gibco | 14065-049 | |
Heparin | Sigma Aldrich | H3149 | |
HEPES | AppliChem Panreac | A1069 | |
Light microscope | Olympus | Olympus CK2 | Upright light microscope with phase contrast |
MEM nonessential amino acids | Sigma Aldrich | M7145 | |
Microplate Reader | BMG LabTech | NOVOstar | |
PBS | Sigma Aldrich | D8537 | Phosphate-buffered saline |
penicillin G sodium/streptomycin sulfate | Sigma Aldrich | P0781 | |
Pluronic F127 | Sigma Aldrich | P2443 | |
Trypsin-EDTA | Sigma Aldrich | T4299 | |
T-75 flask | Sigma Aldrich | CLS3972 | |
96-well plate | Corning incorporated | 3603 |
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