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. S., Tellides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Participation+of+blood+vessel+cells+in+human+adaptive+immune+responses.">Participation of blood vessel cells in human adaptive immune responses.</a> Trends in Immunology. 33 (1), 49-57 (2012).</li><li>Navarro, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+cell+protein+C+receptor:+its+role+in+thrombosis.">The endothelial cell protein C receptor: its role in thrombosis.</a> Thrombosis Research. 128 (5), 410-416 (2011).</li><li>Hasstedt, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecule+1:+a+novel+risk+factor+for+venous+thrombosis.">Cell adhesion molecule 1: a novel risk factor for venous thrombosis.</a> Blood. 114 (14), 3084-3091 (2009).</li><li>Ormiston, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+culture+of+blood+outgrowth+endothelial+cells+from+human+peripheral+blood.">Generation and culture of blood outgrowth endothelial cells from human peripheral blood.</a> Journal of Visualized Experiments: JoVE. (106), e53384 (2015).</li><li>Lin, Y., Weisdorf, D. J., Solovey, A., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+circulating+endothelial+cells+and+endothelial+outgrowth+from+blood.">Origins of circulating endothelial cells and endothelial outgrowth from blood.</a> Journal of Clinical Investigation. 105 (1), 71-77 (2000).</li><li>Martin-Ramirez, J., Hofman, M., Biggelaar, M. V. D., Hebbel, R. P., Voorberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+outgrowth+endothelial+cells+from+peripheral+blood.">Establishment of outgrowth endothelial cells from peripheral blood.</a> Nature Protocols. 7 (9), 1709-1715 (2012).</li><li>Gulati, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+origin+and+function+of+cells+with+endothelial+phenotype+obtained+from+adult+human+blood.">Diverse origin and function of cells with endothelial phenotype obtained from adult human blood.</a> Circulation Research. 93 (11), 1023-1025 (2003).</li><li>Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+endothelial+cells:+utility+from+ambiguity.">Blood endothelial cells: utility from ambiguity.</a> The Journal of Clinical Investigation. 127 (5), 1613-1615 (2017).</li><li>Medina, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitors:+A+consensus+statement+on+nomenclature.">Endothelial progenitors: A consensus statement on nomenclature.</a> Stem Cells Translational Medicine. 6 (5), 1316-1320 (2018).</li><li>Medina, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+endothelial+progenitor+cell+(EPC)+subtypes+reveals+two+distinct+cell+populations+with+different+identities.">Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities.</a> BMC Medical Genomics. 3, 18 (2010).</li><li>Jiang, A., Pan, W., Milbauer, L. C., Shyr, Y., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+question+based+on+cross-platform+microarray+data+normalization:+are+BOEC+more+like+large+vessel+or+microvascular+endothelial+cells+or+neither+of+them.">A practical question based on cross-platform microarray data normalization: are BOEC more like large vessel or microvascular endothelial cells or neither of them.</a> Journal of Bioinformatics and Computational Biology. 5 (4), 875-893 (2007).</li><li>Pan, W., Shen, X., Jiang, A., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semi-supervised+learning+via+penalized+mixture+model+with+application+to+microarray+sample+classification.">Semi-supervised learning via penalized mixture model with application to microarray sample classification.</a> Bioinformatics. 22 (19), 2388-2395 (2006).</li><li>Hirschi, K. K., Ingram, D. A., Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+identity,+phenotype,+and+fate+of+endothelial+progenitor+cells.">Assessing identity, phenotype, and fate of endothelial progenitor cells.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 28 (9), 1584-1595 (2008).</li><li>Fernandez, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+outgrowth+endothelial+cells+from+hereditary+haemorrhagic+telangiectasia+patients+reveal+abnormalities+compatible+with+vascular+lesions.">Blood outgrowth endothelial cells from hereditary haemorrhagic telangiectasia patients reveal abnormalities compatible with vascular lesions.</a> Cardiovascular Research. 68 (2), 235-248 (2005).</li><li>Critser, P. J., Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+colony-forming+cell+role+in+neoangiogenesis+and+tissue+repair.">Endothelial colony-forming cell role in neoangiogenesis and tissue repair.</a> Current Opinion in Organ Transplantation. 15 (1), 68-72 (2010).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+primary+aortic+endothelial+cells+from+miniature+pigs.">Isolation and culture of primary aortic endothelial cells from miniature pigs.</a> Journal of Visualized Experiments: JoVE. (150), e59673 (2019).</li><li>Chang Milbauer, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+endothelial+systems+biology+of+sickle+stroke+risk.">Genetic endothelial systems biology of sickle stroke risk.</a> Blood. 111 (7), 3872-3879 (2008).</li><li>Wei, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+endothelial+cell+gene+expression+by+African+Americans+versusCaucasian+Americans:+a+possible+contribution+to+health+disparity+in+vascular+disease+and+cancer.">Differential endothelial cell gene expression by African Americans versusCaucasian Americans: a possible contribution to health disparity in vascular disease and cancer.</a> BMC Medicine. 9 (1), 2 (2011).</li><li>Hasstedt, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecule+1:+a+novel+risk+factor+for+venous+thrombosis.">Cell adhesion molecule 1: a novel risk factor for venous thrombosis.</a> Blood, The Journal of the American Society of Hematology. 114 (14), 3084-3091 (2009).</li><li>Milbauer, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+outgrowth+endothelial+cell+migration+and+trapping+in+vivo:+a+window+into+gene+therapy.">Blood outgrowth endothelial cell migration and trapping in vivo: a window into gene therapy.</a> Translational Research. 153 (4), 179-189 (2009).</li><li>Matsui, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+gene+therapy+for+hemophilia+A+that+enhances+safe+delivery+and+sustained+in+vivo+factor+VIII+expression+from+lentivirally+engineered+endothelial+progenitors.">Ex vivo gene therapy for hemophilia A that enhances safe delivery and sustained in vivo factor VIII expression from lentivirally engineered endothelial progenitors.</a> Stem Cells. 25 (10), 2660-2669 (2007).</li><li>De Meyer, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+correction+of+von+Willebrand+disease+type+3+blood-derived+endothelial+cells+with+lentiviral+vectors+expressing+von+Willebrand+factor.">Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor.</a> Blood. 107 (12), 4728-4736 (2006).</li><li>Bodempudi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+outgrowth+endothelial+cell-based+systemic+delivery+of+antiangiogenic+gene+therapy+for+solid+tumors.">Blood outgrowth endothelial cell-based systemic delivery of antiangiogenic gene therapy for solid tumors.</a> Cancer Gene Therapy. 17 (12), 855-863 (2010).</li><li>Dudek, A. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+inhibition+of+tumour+angiogenesis+by+endothelial+cell-based+gene+therapy.">Systemic inhibition of tumour angiogenesis by endothelial cell-based gene therapy.</a> British Journal of Cancer. 97 (4), 513-522 (2007).</li><li>Moubarik, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplanted+late+outgrowth+endothelial+progenitor+cells+as+cell+therapy+product+for+stroke.">Transplanted late outgrowth endothelial progenitor cells as cell therapy product for stroke.</a> Stem Cell Reviews and Reports. 7 (1), 208-220 (2011).</li><li>Pislaru Sorin, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+forces+enable+rapid+endothelialization+of+synthetic+vascular+grafts.">Magnetic forces enable rapid endothelialization of synthetic vascular grafts.</a> Circulation. 114 (1), 314 (2006).</li><li>Satyananda, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+concepts+of+immune+modulation+in+xenotransplantation.">New concepts of immune modulation in xenotransplantation.</a> Transplantation. 96 (11), 937-945 (2013).</li><li>Klymiuk, N., Aigner, B., Brem, G., Wolf, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+modification+of+pigs+as+organ+donors+for+xenotransplantation.">Genetic modification of pigs as organ donors for xenotransplantation.</a> Molecular Reproduction and Development. 77 (3), 209-221 (2010).</li><li>Ryczek, N., Hryhorowicz, M., Zeyland, J., Lipi&#324;ski, D., S&#322;omski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas+technology+in+pig-to-human+xenotransplantation+research.">CRISPR/Cas technology in pig-to-human xenotransplantation research.</a> International Journal of Molecular Sciences. 22 (6), 3196 (2021).</li><li>Cooper, D. 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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, ...

The protocol provides highly proliferative blood outgrowth endothelial cells that can be used for various tissue engineering, cell therapy, and disease modeling investigations. This technique consistently yields many cells from a single blood draw. For this technique to be successful, it is crucial to quickly get the cells into the culture medium and incubator environment as soon as possible.First, dilute heparin solution to 100 units per milliliter in sterile saline. Add three to four milliliters of heparin solution to each of two 50-milliliter conical tubes and two 60-milliliter syringes. Then, draw undiluted 1, 000 units per milliliter heparin solution into an extension tube.Connect a 19-gauge needle to one end of the heparin-filled extension tube, and a heparin-containing 60 milliliter syringe to the other end of the extension tube. Next, clean the femoral groin area of an anesthetized pig with a Betadine scrub solution. Puncture the femoral vein or artery with a 19-gauge needle and at one to two milliliters per second, slowly draw 50 milliliters of blood into the syringe.Leaving the needle in place, immediately kink the extension tube and disconnect the 60 milliliter syringe. Slowly transfer the blood to a heparin-containing 50-milliliter conical tube. Tightly cap the conical tube, gently invert the tube a few times to mix, and place it on ice.Connect the remaining heparin-containing 60-milliliter syringe to the extension tube. Unkink the extension tube, and slowly draw additional 50 milliliter blood into the syringe. Immediately remove the needle from the artery or vein and slowly draw the remaining blood from the extension tube into the syringe.Disconnect the 60-milliliter syringe from the extension tube and slowly transfer the blood to the remaining heparin-containing 50-milliliter conical tube. Tightly cap the 50-milliliter conical tube, gently invert a few times to mix, and place on ice. Apply pressure hemostasis to the pig's femoral groin area.Dilute the blood one-to-one with PBS in four 50-milliliter conical tubes. Add 25 milliliters of room temperature, ready-to-use density gradient solution to eight 50-milliliter conical tubes. Slowly pipette 25 milliliters of blood saline solution inside each 50-milliliter conical tube containing density gradient solution by holding the tube at a sharp angle to the pipette tip.Centrifuge the tubes for 30 minutes at 560 g and room temperature. Carefully collect the buffy coat, which is the cloudy layer of mononuclear cells above the density gradient solution and below the clear plasma, from each tube with a thin pipette and distribute it evenly into two new 50-milliliter conical tubes. Discard the density gradient solution containing tubes.To coat a six-well plate with fibronectin, make a stock solution of human plasma fibronectin by diluting it to one milligram per milliliter in sterile water. Make 3.6 milliliters of a working solution of fibronectin by diluting 600 microliters of fibronectin stock solution in three milliliters of PBS. Transfer 600 microliters of the fibronectin working solution into each well of a six-well plate and gently rock until evenly coated.And gently aspirate the solution out of each well. To wash the mononuclear cells while waiting for the fibronectin to coat, top up the tubes with up to 45 milliliters of PBS, centrifuge for five minutes at 560 X g and four degrees Celsius with low break. Aspirate the supernatant from both tubes.Resuspend each cell pellet in 25 milliliters of PBS. Resuspend each cell pellet in five milliliters of PBS and add 15 milliliters of 0.8%ammonium chloride solution to each tube. Wash the cells as before by adding PBS.Aspirate the supernatant from both tubes. Resuspend each cell pellet in 25 milliliters of PBS. Resuspend each cell pellet in six milliliters of EGM-2 culture medium supplemented with 10%FBS and 1X antibiotic-antimycotic solution.Gently transfer two milliliters of the cell suspension to each well of the fibronectin coated six-well plate and gently rock until evenly coated. 24 hours after seeding the cells, gently add one milliliter of fresh culture medium to each well. The following day, gently aspirate 1.5 milliliters of culture medium from each well, and replace it with 1.5 milliliters of fresh culture medium.For the next five days, gently change the culture medium with two milliliters of fresh culture medium per well. Regularly visualize the cell cultures under low power 4X objective light microscopy. Morphology of the cultured cells was observed from the start of the culture until BOEC colonies were observed.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. Colonies first appeared on day six as a collection of endothelial-like cells proliferating radially outward from a central point. As the culture progressed, cell colonies became more dense and displayed a cobblestone morphology similar to mature endothelial cells.Two-dimensional phase contrast microscopy was used to image tube formation in serum-free medium and complete growth medium. Cells in both media conditions underwent morphological differentiation and rapidly organized into extensive networks of capillary tube-like structures. BOECs were further characterized by the expression of mature endothelial cell marker CD31 or platelet endothelial cell adhesion molecule one.BOECs showed uniform expression of CD31. Furthermore, flow cytometry analysis of BOECs with CD14 AF 700 antibody compared to positive control confirmed the absence of EPCs, as the cell stained negative for monocyte marker CD14 compared to the positive control group of PBMCs. Positive CD14 staining shown in blue, and unstained cells shown in red.During blood collection, it is essential to collect as much of the buffy coat layer as possible while collecting as little of the adjacent layers as possible. This technique has enabled researchers to study new strategies for endothelializing implantable cardiovascular devices using a pig model with autologous blood outgrowth endothelial cells.

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Research

JoVE Journal

Bioengineering

1.6K 次观看.  Medical College of Wisconsin & Marquette University. 该方案提供高度增殖的血液生长内皮细胞，可用于各种组织工程、细胞治疗和疾病建模研究。这种技术始终如一地从一次抽血中产生许多细胞。为了使该技术取得成功，尽快将细胞快速放入培养基和培养箱环境中至关重要。首先，在无菌盐水中将肝素溶液稀释至每毫升100单位。向两个 50 毫升锥形管和两个 60 毫升注射器中各添加三至四毫升肝素溶液。然后，将未稀释的?...