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Published on: August 14, 2019
Isolation and Characterization of Porcine Endocardial Endothelial Cells
Kathleen N Brown1, Hong Kim T Phan1, Elysa L Jui1
1Department of Bioengineering, Rice University, Houston, Texas, USA.
Insights
This study details a protocol for isolating and characterizing endocardial endothelial cells (EECs) from porcine hearts. These EECs exhibit distinct proliferation and migration behaviors compared to human umbilical vein endothelial cells (HUVECs), highlighting their unique properties.
Area of Science:
- Cardiovascular Biology
- Endothelial Cell Research
- Cellular Phenotyping
Background:
- The heart comprises diverse endothelial cell types, including understudied endocardial endothelial cells (EECs).
- EECs line the heart chambers, and their dysfunction is linked to cardiac pathologies.
- Commercial unavailability of EECs necessitates robust isolation and characterization protocols.
Purpose of the Study:
- To establish a protocol for isolating and culturing endocardial endothelial cells (EECs) from porcine hearts.
- To characterize the phenotype and fundamental behaviors of EECs.
- To compare EECs with a standard endothelial cell model, human umbilical vein endothelial cells (HUVECs).
Main Methods:
- Isolation of EECs from porcine hearts using a detailed protocol.
- Establishment of an EEC cell population via cell sorting.
- Comparative analysis of EECs and HUVECs for proliferation, migration, and phenotypic marker expression (CD31, von Willebrand Factor, VE-cadherin).
Main Results:
- Isolated EECs expressed classic endothelial markers (CD31, von Willebrand Factor, VE-cadherin).
- EECs demonstrated significantly higher proliferation rates than HUVECs at 48h and 96h.
- EECs exhibited significantly slower wound closure (migration) compared to HUVECs at 4h, 8h, and 24h.
- EECs maintained CD31 expression over 14 passages, unlike HUVECs which showed reduced expression.
Conclusions:
- The developed protocol successfully isolates and establishes a viable EEC population.
- EECs possess distinct proliferative and migratory characteristics compared to HUVECs.
- EECs maintain their endothelial phenotype more robustly than HUVECs in long-term culture.
- These phenotypic differences underscore the importance of using relevant cell models for cardiac research.
Abstract:
The heart contains diverse endothelial cell types. We sought to characterize the endocardial endothelial cells (EECs), which line the chambers of the heart. EECs are relatively understudied, yet their dysregulation can lead to various cardiac pathologies. Due to the lack of commercial availability of these cells, we reported our protocol for isolating EECs from porcine hearts and for establishing an EEC population through cell sorting. In addition, we compared the EEC phenotype and fundamental behaviors to a well-studied endothelial cell line, human umbilical vein endothelial cells (HUVECs). The EECs stained positively for classic phenotypic markers such as CD31, von Willebrand Factor, and vascular endothelial (VE) cadherin. The EECs proliferated more quickly than HUVECs at 48 h (1310 ± 251 cells vs. 597 ± 130 cells, p = 0.0361) and at 96 h (2873 ± 257 cells vs. 1714 ± 342 cells, p = 0.0002). Yet EECs migrated more slowly than HUVECs to cover a scratch wound at 4 h (5% ± 1% wound closure vs. 25% ± 3% wound closure, p < 0.0001), 8 h (15% ± 4% wound closure vs. 51% ± 12% wound closure, p < 0.0001), and 24 h (70% ± 11% wound closure vs. 90% ± 3% wound closure, p < 0.0001). Finally, the EECs maintained their endothelial phenotype by positive expression of CD31 through more than a dozen passages (three populations of EECs showing 97% ± 1% CD31+ cells in over 14 passages). In contrast, the HUVECs showed significantly reduced CD31 expression over high passages (80% ± 11% CD31+ cells over 14 passages). These important phenotypic differences between EECs and HUVECs highlight the need for researchers to utilize the most relevant cell types when studying or modeling diseases of interest.
