Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies
May 27th, 2020
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.
Overview
This study details a protocol for isolating and culturing primary brain capillary pericytes, key regulators of the blood-brain barrier and vascular function. By using this protocol, researchers can investigate intracellular calcium signaling in a nearly homogenous population of pericytes, enabling insights into pericyte biology.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Vascular Biology
Background
- Pericytes play a crucial role in maintaining blood-brain barrier integrity.
- Understanding calcium signaling in pericytes is essential for elucidating their regulatory functions.
- The methodology provides a means to study pericyte physiology in vitro.
Purpose of Study
- To isolate and culture brain capillary pericytes for subsequent studies.
- To facilitate the investigation of intracellular calcium signaling.
- To provide a reliable protocol for future pericyte research.
Methods Used
- Utilized a cell culture approach for isolating brain capillary pericytes.
- Involved thawing capillary samples, centrifugation, and adherence to coated flasks.
- Calcium imaging employed Fura-2 AM dye to assess calcium dynamics in cultured cells.
- The protocol includes specific timelines for cell adherence, confluency, and imaging.
Main Results
- The pericytes achieved approximately 80% confluency within nine days.
- Addition of ATP resulted in a significant increase in cytosolic calcium levels, observed immediately upon application.
- Detailed observations of cellular outgrowth and the detachment of endothelial cells were reported.
Conclusions
- This protocol enables the effective study of pericyte biology and intracellular signaling.
- Understanding calcium signaling mechanisms could have implications for vascular and neural health.
Frequently Asked Questions
Executive Industry Relevance
Reliable isolation and culture of brain capillary pericytes with quantitative calcium imaging enables mechanistic interrogation of neurovascular signaling pathways. This capability supports early discovery efforts targeting the neurovascular unit and de-risks hypotheses around pericyte-mediated regulation of blood-brain barrier function. The method provides a standardized platform for evaluating pericyte responses to pharmacological agonists, informing target validation and translational research in neurovascular disorders.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct measurement of pericyte calcium signaling in response to defined agonists.
- Supports mechanistic de-risking of neurovascular targets by isolating pericyte-specific responses.
- Facilitates functional validation of pericyte involvement in capillary tone and barrier regulation.
Screening & Assay Development
- Provides a nearly homogenous pericyte culture system for reproducible calcium imaging assays.
- Standardizes quantitative readouts of intracellular calcium dynamics using Fura-2 AM and confocal imaging.
- Enables scalable plate-based measurements suitable for compound screening and comparative studies.
Translational & Preclinical Research
- Aligns in vitro pericyte responses with disease-relevant neurovascular signaling pathways.
- Supports continuity from cellular mechanism studies to preclinical models of brain pathologies.
- De-risks translational hypotheses by providing direct evidence of pericyte contractility and signaling.
Pipeline & Workflow Integration
This method integrates into the discovery continuum from early mechanistic studies to preclinical target validation for neurovascular drug discovery.
- Discovery Biology: Enables hypothesis testing of pericyte-mediated signaling and contractility in the neurovascular unit.
- Screening: Delivers reproducible, quantitative calcium imaging outputs for compound evaluation.
- Analytics: Provides real-time measurement of cytosolic calcium changes in response to pharmacological stimuli.
- Translational Research: Bridges in vitro pericyte biology with disease-relevant neurovascular mechanisms.
- Enterprise Reuse: Establishes a reusable platform for pericyte functional assays across neurovascular research programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in pericyte target validation and mechanistic studies.
- Operational Value: Standardizes pericyte isolation and calcium imaging for reproducibility and scalability.
- Strategic Value: Informs go/no-go decisions for neurovascular targets by providing quantitative functional data.
- Portfolio Impact: Supports risk-adjusted prioritization of neurovascular discovery programs.
Implementation Considerations
- Requires expertise in primary cell culture and live-cell calcium imaging.
- Needs access to confocal microscopy and plate reader instrumentation for quantitative analysis.
- Demands rigorous standardization of cell isolation and dye loading protocols for reproducibility.
- May require adaptation for pericytes from different species or brain regions.
- Dependent on maintaining cell viability and purity throughout the workflow.
Biopharma FAQs
Chapters in this video
0:04
Introduction
0:41
Isolation of Primary Pericytes from Bovine Brain Capillaries
3:01
Loading of Pericytes with Calcium Indicator Dye in a Plate Reader Setup
5:18
Live Imaging of Intracellular Ca2+ Levels
7:11
Results: Pericyte Isolation and Calcium Measurements
9:02
Conclusion
