Publication Date
8-1-2023
Journal
Tissue Engineering Part C: Methods
DOI
10.1089/ten.TEC.2023.0009
PMID
37310900
PMCID
PMC10442675
PubMedCentral® Posted Date
8-8-2023
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Keywords
Swine, Humans, Animals, Endocardium, Heart, Human Umbilical Vein Endothelial Cells, Cell Separation, Cells, Cultured, Endothelium, Vascular, endocardial, endothelial cells, cell isolation, behavior, phenotype
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.
Impact statement
Many researchers model cardiovascular disease using tissue engineering to determine disease etiology on the cellular and molecular level. However, researchers usually rely on commercially-available cell lines, which can result in utilizing cells that are not specific to the region of the heart being modeled and could lead to incorrect conclusions. By providing a detailed protocol for isolating and purifying endocardial endothelial cells, we are enabling other researchers to access these cells and model cardiovascular disease states more accurately.
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