Name: characterization of blood outgrowth endothelial cells (boec) from porcine peripheral blood
Uploaded: 2022-01-06T13:00:00
Description: Watch this Scientific Journal Video about Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood at JoVE.com

The authors wish to acknowledge funding from NIH/NHLBI R00 HL129068.

All animal studies were approved by the respective Institutional Animal Care and Use Committees (IACUC) at the Medical College of Wisconsin and Mayo Clinic.
NOTE: In this study, Yorkshire/Landrace/Duroc cross domestic pigs (Sus domesticus), male and female, 40-80 kg, 3-6 months old, were used.
1. Collection of porcine peripheral blood
<ol>
	<li>Prepare materials.
	<ol>
		<li>Dilute heparin solution to 100 U/mL in sterile saline.</li>
		<l...

<ol>
	<li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+of+the+endothelium:+II.+Representative+vascular+beds.">Phenotypic heterogeneity of the endothelium: II. Representative vascular beds.</a> Circulation Research. 100 (2), 174-190 (2007).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+of+the+endothelium:+I.+Structure,+function,+and+mechanisms.">Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms.</a> Circulation Research. 100 (2), 158-173 (2007).</li><li>Pober, J. 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BOECs are a powerful tool that may be used in various scientific and therapeutic approaches7,8,16. BOECs have been used to analyze EC gene expression to elucidate the key factors responsible for the development of vascular diseases and cancer5,19,20,21. BOECs have also been used in therapeutic applicat...

The endothelium is a highly complex, dynamic structure and a vital component of the vascular wall. It lines the inner surface of blood vessels to provide a physical interface between circulating blood and surrounding tissues. This heterogeneous structure is known to perform various functions such as angiogenesis, inflammation, vasoregulation, and hemostasis1,2,3,4. Human umbilical vein endothelial cells are a widely studied cell type to assess the functionality of endothelial cells. However, the patient-specific batch variability, inconsistent phenotype, and minimum splitting efficiency suggest a need to determine a cell source that could improve upon all of these features5.
Obtaining a homogenous population of primary endothelial cells can be technically challenging, and primary endothelial cells do not possess high proliferative capacity6.&#160;Hence, to study vascular regeneration and evaluate pathophysiological processes, various groups have tried to obtain and assess different types of endothelial cells derived from peripheral blood, e.g., endothelial progenitor cells (EPCs) or blood outgrowth endothelial cells (BOECs)6,7,8,9. The spindle-shaped early EPCs originate from hematopoietic stem cells (HSCs) and have limited growth potency and limited angiogenic ability to produce mature endothelial cells. Furthermore, they closely resemble inflammatory monocytes. In addition, their capacity to further differentiate into functional, proliferating, mature endothelial cells is still debatable6,7,9,10.&#160;The continuous culture of peripheral blood mononuclear cells (PBMCs) can give rise to a secondary population of cells known as late-outgrowth EPCs, BOECs, or endothelial colony-forming cells (ECFCs)6,7,9,10. Medina et al. in 2018, acknowledged the limitations of EPCs, the ambiguity of their nomenclature, along with a general lack of concordance with many distinct cell types continuously grouped under EPCs11. In contrast, BOECs have become recognized for their role in vascular repair, health and disease, and cell therapy. Further study and therapeutic use of these cells will rely on protocols to consistently derive these cell types from circulating progenitor cells.
Primary cells such as BOECs can be used as a surrogate to obtain highly proliferative mature endothelial cells6. BOECs are phenotypically distinct from early EPCs and exhibit typical endothelial features such as cobblestone morphology and expression of adherens junctions and caveolae12. Gene profiling by Hebbel et al.13,14,15 found that BOECs or ECFCs are the true endothelial cells as they promote microvascular and large vessel formation. Thus, BOECs can be used as a tool to evaluate pathophysiological processes and genetic variation16. They are also considered an excellent cell source for cell therapy for vascular regeneration17. Hence, a standardized protocol to consistently derive these highly proliferative cells is essential.
While BOECs provide a powerful tool for studying human pathophysiological and genetic variation, a more homogenous source of BOECs may provide more robust and reliable experimental and therapeutic outcomes. Superior homogeneity can be achieved by using xenogeneic cell sources derived from genetically similar animals raised in similar conditions18. While xenogeneic cell sources are prone to eliciting a host immune response, immunomodulation strategies are being developed with the goal of generating immunocompatible animals and animal products, including cells. Pigs, in particular, are an abundant source of peripheral blood and are commonly used to study medical devices and other therapies due to anatomical and physiological similarities to humans. Hence, this study refines the protocol for the isolation and expansion of highly proliferative BOECs from porcine peripheral blood.The protocol detailed below is a straightforward and reliable method to obtain a large number of BOECs from a relatively small volume of blood. The cultures can be expanded through several passages to generate millions of cells from a single blood sample.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>19 G needle</td>
			<td>Covidien</td>
			<td>1188818112</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL syringes</td>
			<td>Covidien</td>
			<td>8881560125</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride solution (0.8%)</td>
			<td>Stemcell Technologies</td>
			<td>07850</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic/antimycotic solution (100x)</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75-253-839</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 culture medium</td>
			<td>Lonza Walkersville</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Extension tube</td>
			<td>Hanna Pharmaceutical Supply Co.</td>
			<td>03382C6227</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Atlas Biologicals</td>
			<td>F-0500-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque 1077</td>
			<td>Cytiva</td>
			<td>17144003</td>
			<td>Density gradient solution</td>
		</tr>
		<tr>
			<td>Heparin sodium injection (1,000 units/mL)</td>
			<td>Pfizer</td>
			<td>00069-0058-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Human plasma fibronectin</td>
			<td>Gibco</td>
			<td>33016-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette set</td>
			<td>Eppendorf</td>
			<td>2231300004</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td>Gibco</td>
			<td>15230-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin pipette</td>
			<td>Celltreat Scientific</td>
			<td>229280</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of blood outgrowth endothelial cells (boec) from porcine peripheral blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, inflammation, and homeostasis. The endothelium also plays an important role in pathophysiologies such as atherosclerosis, hypertension, and diabetes. Endothelial cells form the inner lining of blood and lymphatic vessels and display heterogeneity in structure and function. Various groups have evaluated the functionality of endothelial cells derived from human peripheral blood with a focus on endothelial progenitor cells derived from hematopoietic stem cells or mature blood outgrowth endothelial cells (or endothelial colony-forming cells). These cells provide an autologous resource for therapeutics and disease modeling. Xenogeneic cells may provide an alternative source of therapeutics due to their availability and homogeneity achieved by using genetically similar animals raised in similar conditions. Hence, a robust protocol for the isolation and expansion of highly proliferative blood outgrowth endothelial cells from porcine peripheral blood has been presented. These cells can be used for numerous applications such as cardiovascular tissue engineering, cell therapy, disease modeling, drug screening, studying endothelial cell biology, and in vitro co-cultures to investigate inflammatory and coagulation responses in xenotransplantation.

Morphology of the cultured cells was observed from the start of the culture until BOEC colonies were observed (Figure 1). A smaller population of adherent cells started to attach to the culture dishes and grow, while non-adherent cells were removed with culture medium changes (Figure 1B). Colonies first appeared on day 6 as a collection of endothelial-like cells proliferating radially outward from a central point (Figure 1D). As the...

Watch this Scientific Journal Video about Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood at JoVE.com

Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, ...

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