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

Summary

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.

Abstract

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.

Introduction

Brain capillary pericytes, together with endothelial cells and astrocytes, constitute the NVU^1,2,3. 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 protec....

Protocol

1. Preparation of buffers and solutions for cell culturing

1. Prepare collagen stock solution by dissolving 5 mg of collagen IV from human placenta in 50 mL of PBS overnight at 4 °C. Aliquot the stock solution into 5 mL portions and store at -20 °C.
2. Prepare fibronectin stock solution by dissolving 5 mg of fibronectin in 5 mL of sterile water overnight. Store the fibronectin stocks in aliquots of 500 µL at -20 °C. When thawing, add PBS to a final volume of 50 mL to prepare the work solution and store it at 4 °C.
3. Prepare Dulbecco's Modified Eagle Medium (DMEM) complete medium by adding 50 mL of fetal bovine serum....

Results

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 .......

Discussion

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 .......

Materials

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