Isolation and Purification of Murine Cardiac Pericytes
August 16th, 2019
11.5K views
We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.
Overview
This article presents an optimized protocol for isolating and purifying murine cardiac pericytes, facilitating research into their biology and therapeutic potential. The method is applicable to various mouse models, including those with genetic alterations or diseases.
Key Study Components
Area of Science
- Neuroscience
- Cardiac Biology
- Cell Isolation Techniques
Background
- Cardiac pericytes play a crucial role in cardiac homeostasis and hemodynamics.
- Understanding their biology is essential for therapeutic advancements.
- This protocol utilizes readily available materials for researchers.
- It can be adapted for various experimental conditions.
Purpose of Study
- To provide a reliable method for isolating cardiac pericytes from murine models.
- To enhance understanding of cardiac pericyte functions in health and disease.
- To support future research into therapeutic applications involving cardiac pericytes.
Methods Used
- Perfusion of the heart with heparinized PBS.
- Collagenase digestion for tissue dissociation.
- Cell counting and viability assessment using FACS.
- Fluorescence-activated cell sorting (FACS) for purification.
Main Results
- Successful isolation of viable cardiac pericytes.
- Protocol applicable to various mouse models.
- Insights gained into the biology of cardiac pericytes.
- Potential for therapeutic exploration in cardiac health.
Conclusions
- The optimized protocol is effective for cardiac pericyte research.
- It opens avenues for understanding cardiac diseases.
- Future studies can leverage this method for therapeutic insights.
Frequently Asked Questions
What are cardiac pericytes?
Cardiac pericytes are contractile cells located around capillaries in the heart, playing a role in vascular stability and function.
Why is it important to isolate cardiac pericytes?
Isolating cardiac pericytes allows researchers to study their specific functions and contributions to heart health and disease.
Can this protocol be used for diseased mouse models?
Yes, the protocol is designed to be applicable to healthy, diseased, or genetically altered mouse models.
What materials are required for this protocol?
The protocol utilizes commonly available materials such as heparinized PBS and collagenase for tissue digestion.
How do you assess cell viability after isolation?
Cell viability is assessed using a cell counter after resuspending the isolated cells in a suitable buffer.
What is FACS and how is it used in this protocol?
FACS, or fluorescence-activated cell sorting, is used to purify isolated cardiac pericytes based on specific markers.
Executive Industry Relevance
Reliable isolation and purification of murine cardiac pericytes enables mechanistic studies of vascular stability and cardiac homeostasis in disease-relevant models. This protocol supports target validation and functional de-risking for cardiovascular research portfolios by providing standardized access to primary pericyte populations. The method's reproducibility and adaptability across mouse models position it as a foundational capability for early discovery and translational research.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of pericyte-specific roles in cardiac physiology and disease models.
- Supports biological de-risking by isolating pure pericyte populations for functional assays.
- Facilitates mechanistic studies to clarify pericyte contributions to vascular integrity.
- Provides a platform for hypothesis-driven target validation in cardiovascular research.
Screening & Assay Development
- Delivers standardized, homogenous pericyte cultures for reproducible downstream assays.
- Enables quantitative assessment of pericyte function in co-culture and barrier integrity assays.
- Supports assay development for compound screening targeting pericyte biology.
- Improves screening readiness by ensuring cell identity and viability through FACS-based purification.
Translational & Preclinical Research
- Aligns with disease-relevant mouse models for translational continuity in cardiovascular studies.
- Enables risk-adjusted advancement decisions by providing primary cells for preclinical validation.
- Supports biomarker discovery and mechanistic de-risking in cardiac vascular research.
Pipeline & Workflow Integration
This protocol integrates into the discovery-to-preclinical continuum by supplying validated cardiac pericytes for mechanistic, screening, and translational workflows.
- Discovery Biology: Provides a robust platform for hypothesis testing and pathway clarification in cardiac vascular research.
- Screening: Ensures assay reproducibility and quantitative outputs through standardized cell preparation and FACS purification.
- Analytics: Delivers high-purity cell populations for comparative functional analyses and quantitative readouts.
- Translational Research: Bridges early discovery with preclinical validation using disease-relevant murine models.
- Enterprise Reuse: Establishes a reusable protocol adaptable to various mouse models and experimental conditions.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in cardiac vascular studies.
- Operational Value: Standardizes pericyte isolation for reproducibility and scalability across research teams.
- Strategic Value: Enables informed go/no-go decisions and capital-efficient portfolio management in cardiovascular R&D.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of cardiovascular targets and models.
Implementation Considerations
- Requires expertise in murine cardiac dissection, enzymatic digestion, and FACS operation.
- Demands access to flow cytometry instrumentation and analytical infrastructure for cell sorting and characterization.
- Necessitates rigorous cross-team standardization of gating strategies and marker panels.
- Adaptable to healthy, diseased, or genetically altered mouse models for broad applicability.
- Cell viability and marker fidelity must be maintained through cold-chain handling and fresh reagent preparation.
Biopharma FAQs
Why does null hypothesis testing matter for FACS-based pericyte isolation?
Null hypothesis testing ensures that observed differences in pericyte marker expression or function are statistically significant, supporting robust target validation and reducing false positives in early discovery.
How does independent variable isolation fit into the cardiac pericyte gating strategy?
Isolating independent variables through sequential gating (e.g., CD31-, CD34-, CD45-, NG2+, CD140b+, CD146+) enables precise identification of pericyte populations, minimizing confounding cell types and increasing mechanistic clarity.
What do quantitative dependent variable measurements enable in pericyte functional assays?
Quantitative measurements, such as cell viability and marker expression levels, enable comparative analyses across experimental conditions and support data-driven decisions in assay development and target prioritization.
Why are replication requirements critical for cross-functional pericyte research?
Replication ensures that pericyte isolation and functional outputs are reproducible across teams and models, facilitating reliable cross-functional collaboration and enterprise-wide data integration.
Which statistical analysis capabilities are required before implementing pericyte isolation protocols?
Robust statistical analysis of gating, viability, and marker expression data is essential to validate cell purity and functional consistency, supporting confident integration into discovery and translational workflows.
Chapters in this video
0:04
Title
0:44
Preparation of Animal, Procurement of Cardiac Tissue, and Dissociation of Heart Tissue
3:28
Preparation of the Crude Cell Mixture for Fluorescence Activating Cell Sorting (FACS)
6:34
Purification of Pericytes from Crude Cell Mixture Using FACS
8:18
Results: Analysis of Crude Cells and Characterization of Primary Isolated Cardiac Pericytes
9:55
Conclusion
