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 endothelial cells are removed by gentle trypsinization as according to the described protocol. Remnants of the capillaries can be present after the trypsinization, but will disappear from the flask in the following days (Figure 1, day 4b-6). After removal of the endothelial-layer, pericytes are easily detected with morphology distinct from the endothelial cells. The pericytes present finger-like processes that attach strongly to the flask (Figure 1, day 4b). Subsequently, pericytes are allowed to grow until confluency (Figure 1, day 4b-9) and on day 9, the pericytes have reached approximately 80% confluency and grow in islands. This is in contrast to the endothelial cells that do create a contact inhibited monolayer observed at day 4.
ATP is a well-known endogenous inducer of intracellular Ca2+-signaling11 and was used as an extracellular stimulant to induce cytosolic changes in Ca2+-levels in pericytes. Addition of ATP to the Fura-2 loaded pericytes, as according to the described protocol, resulted in an increase in cytosolic Ca2+-levels measured as the fluorescent ratio as shown in Figure 2. The ATP-induced response occurs immediately after addition of ATP to the pericytes and declines slowly over the measured time period.
Using this protocol for real-time confocal imaging of intracellular Ca2+-responses, pericytes were seeded on coated coverslips, loaded with Cal-520 AM and placed at the confocal microscope. Figure 3 (0 s) shows the pericytes with baseline levels of fluorescence prior to treatment. During live-recording, ATP is added to the pericytes and a strong intracellular Ca2+-response is evident shortly after (64 s). Soon after, the cytosolic Ca2+ compartmentalizes in the cells and a reduction in cell area is visible (189 s). At 300 s. post start of the recordings, the cell area is heavily reduced and the Ca2+-signal has declined to intensity close to the baseline fluorescence.
Figure 1: Culturing of capillaries and isolation of pericytes. Capillaries have been isolated from fresh bovine brain and seeded in culture flasks on day 0. Outgrowth from the bovine brain capillaries and the following isolation of pericytes were followed over days with a light microscope. Day 4a shows the endothelial cell growth prior to treatment with trypsin to remove endothelial cells and day 4b shows the remnants immediately after the treatment. Day 8a shows the focus plane, where any capillary remnants would be visible, whereas day 8b are focused on the plane where the growth of the pericytes is visible. Please click here to view a larger version of this figure.
Figure 2: Representative example of intracellular calcium-measurement using Fura-2 calcium indicator dye. Primary pericytes have been seeded in 96 well plates and loaded with Fura-2 AM in order to visualize changes in intracellular calcium. 10 µM ATP is added to the pericytes at 30 s. and the cytosolic Ca2+-response is measured as the ratio between the two excitation wavelengths; 340 nm and 380 nm over time. Scale bars are defined as standard deviation (N=3, n=1). Please click here to view a larger version of this figure.
Figure 3: Representative example of intracellular calcium live-imaging using Cal-520 calcium indicator dye. Primary pericytes have been seeded in a cell chamber and loaded with Cal-520 in order to visualize changes in intracellular calcium and cell morphology. 600 µM ATP is added to the pericytes and snapshots from different time points during the live-imaging are presented here. Please click here to view a larger version of this figure.
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 Ca2+-imaging using two different methods, depending on the intended outcome.
One of the main issues when isolating primary cells from brain tissue is the limited access to tissue. In several earlier studies, rats and mice are the traditional model animals used, but the brain tissue from these small animals are sparse6,13. Isolation from human brain has also been conducted14; however, due to ethical issues this is not easily accessible. Hence, a high cell yield from an available source is preferred. The number of cell vials obtained from isolation from a single bovine brain outnumbers the amount obtained from either mouse or rat by far, making the bovine tissue advantageous compared to the other mentioned sources. Another advantage of the method described here is the low passage number to obtain a pure culture of pericytes. Passaging of mural cells can lead to de-differentiation15 and therefore, should preferably be avoided. In this protocol, gentle trypsinization to remove the endothelial cell layer is a single step used to isolate the pericytes and therefore also one of the most critical steps described in this protocol. If the trypsinization is prolonged, the pericytes will start to detach from the flask together with the endothelial cells, leading to only a small yield. On the other hand, only allowing a very short trypsinization time can cause an impure culture of pericytes. It is therefore of utmost importance to observe the cells very frequently during the trypsinization step. One should be aware of the morphological differences between pericytes and endothelial cells to be able to distinguish the two cell types during the trypsinization procedure. Although, this step might require some training, the method is less time consuming and inexpensive compared to other methods used to obtain pure pericyte cultures7,8,14. Furthermore, the obtained culture of pericytes here showed the typical "ghost"-like morphology with finger-like processes and expressed several specific markers such as α-SMA, PDGFR-β and Nestin (data not shown here)12, which are well known markers for primary pericytes in culture1.
Here, we also presented a simple method for Ca2+-imaging using fluorescent calcium indicator dyes. Loading of the pericytes with the dyes (Fura-2 and Cal-520) should be performed at RT and not at 37 °C. Several studies have performed loading of AM esters at 37 °C16,17, but loading at this temperature can lead to the esters entering intracellular compartments such as the endoplasmic reticulum. This is not preferable, as the Fura-2 AM can bind to free Ca2+ trapped in the internal stores and thereby result in a lower increase in the fluorescent measurements. Hence, it can cause false results. Although, we have not experienced any major difficulties with loading of the pericytes at RT, if one is experiencing problems anyhow, one might consider optimizing loading by sonication of the loading solution for 5 min in an ultrasound bath prior to loading of the cells.
With the described method, intracellular Ca2+-signaling together with contraction of the pericytes is readily observed during live recordings, which can also be quantified for further analysis. The method has previously been used to make the first demonstration of in vitro Ca2+-signaling and contraction of brain capillary pericytes12. In previous studies various methods have been used to measure and quantify cell contraction as a consequence of extracellular stimulus18,19,20. However, observing the preliminary intracellular response directly followed by contraction of the cells was not a possibility in these studies. In the current study it is possible to observe the intracellular signaling and the following contraction of the cells and both measurements can be quantified in the same experiment. Thus, it eliminates additional experiments, which are more time consuming as well, and shows the direct link between intracellular Ca2+-signaling and the morphological changes.
In conclusion, this study represents a simple and effective method for isolating and culturing primary brain pericytes. In addition, we demonstrate an easy and reproducible method to study intracellular Ca2+-signaling in cultured pericytes. The methods described here should provide other researchers in the field with strong tools to study the pericyte biology and intracellular signaling in pericytes in vitro.
The authors have nothing to disclose.
The authors wish to acknowledge funding from the Lundbeck Foundation Research initiative on Brain Barriers and Drug Delivery (RIBBDD) and Simon Hougners Family Foundation.
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 22×22 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 |