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Cultivation and characterization of coronary microvascular endothelial cells: a novel porcine model using micropigs
Edward K Johnson1, Margaret E Schelling, Ian J Quitadamo
1School of Molecular Biosciences, College of Education, Washington State University, Pullman, 99164, USA.
Abstract:
Coronary microvascular endothelial cells (CMECs) play an important role in many physiological processes. Porcine CMECs from large breed pigs have been isolated and successfully cultured. However, because micropigs offer research advantages over large breed pigs, micropig CMEC (MPCMEC) cultures may be useful as an alternative in vitro porcine model for cardiovascular studies. We isolated MPCMECs from six Panepinto micropigs using a simplified technique and developed a system for their successful culture. MPCMECs were isolated by collagenase digestion of left ventricular samples obtained using sterile techniques. Primary isolates of MPCMECs grew steadily in complete DMEM supplemented with 20% FBS, 4 mM MgSO(4), and 500 microM dibutyryl cAMP and reached confluence in 7-10 days. Endothelial origin was demonstrated by rapid (4-h) uptake of acetylated low-density lipoprotein, immunostaining for the presence of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), von Willebrand factor (vWf)-related antigen, vascular endothelial cadherin (VE-cadherin), endothelial nitric oxide synthase (eNOS), and by positive staining using two fluorescein isothiocyanate-labeled endothelial-specific lectins, Dolichos biflorus agglutinin and Ulex europaeus agglutinin-1. MPCMECs also exhibited immunostaining for alpha-smooth muscle actin. MPCMECs were successfully subcultured in the absence of dibutyryl cAMP and continued to express PECAM-1 and vWf, but not eNOS, to passage six. The typical morphology of subconfluent MPCMECs consisted of elongated cells that grew in a swirling, herringbone pattern.
Insights
Researchers successfully cultured micropig coronary microvascular endothelial cells (CMECs), establishing a valuable in vitro model for cardiovascular research. This new porcine model offers advantages for studying heart conditions.
Area of Science:
- Cardiovascular Biology
- Cell Biology
- In Vitro Models
Background:
- Coronary microvascular endothelial cells (CMECs) are crucial for cardiovascular homeostasis.
- Existing porcine models often utilize large breed pigs, but micropigs offer distinct research advantages.
- A need exists for a reliable in vitro porcine model using micropig CMECs for cardiovascular studies.
Purpose of the Study:
- To isolate and establish a successful culture system for micropig coronary microvascular endothelial cells (MPCMECs).
- To characterize the isolated MPCMECs and confirm their endothelial origin and characteristics.
- To evaluate the potential of MPCMECs as an alternative in vitro model for cardiovascular research.
Main Methods:
- Micropig coronary microvascular endothelial cells (MPCMECs) were isolated from Panepinto micropigs via collagenase digestion of left ventricular tissue.
- Cells were cultured in DMEM supplemented with FBS, MgSO(4), and dibutyryl cAMP, with characterization using acetylated LDL uptake and immunostaining.
- Immunostaining confirmed endothelial markers such as PECAM-1, vWf, VE-cadherin, eNOS, and specific lectins. Alpha-smooth muscle actin was also assessed.
Main Results:
- A simplified technique successfully isolated and cultured MPCMECs, achieving confluence in 7-10 days.
- Endothelial origin was confirmed by rapid acetylated LDL uptake and positive immunostaining for PECAM-1, vWf, VE-cadherin, eNOS, and endothelial lectins.
- MPCMECs could be subcultured without dibutyryl cAMP, maintaining PECAM-1 and vWf expression up to passage six, and exhibited characteristic elongated morphology.
Conclusions:
- A robust method for isolating and culturing micropig coronary microvascular endothelial cells (MPCMECs) has been established.
- MPCMECs serve as a viable and potentially advantageous in vitro model for cardiovascular research compared to large breed pigs.
- These cells maintain key endothelial characteristics through multiple passages, supporting their utility in experimental settings.