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

Abstract

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.

Introduction

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 hemostasis^1,2,3,4. Human umbilical vein endothelial cells are a widely studied cell type to assess the functionality of endothelial cells. However, the patient-specific batch variability, inconsiste....

Protocol

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

1. Prepare materials.
   1. Dilute heparin solution to 100 U/mL in sterile saline.
   2. Add 3-4 mL of heparin solution to each of two 50 mL conical tubes and two 60 mL syringes.
   3. Draw undiluted heparin solution (1,000 U/mL) in....

Results

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.......

Discussion

BOECs are a powerful tool that may be used in various scientific and therapeutic approaches^7,8,16. BOECs have been used to analyze EC gene expression to elucidate the key factors responsible for the development of vascular diseases and cancer^5,19,20,21. BOECs have also been used in therapeutic applicat.......

Materials

List of materials used in this article

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