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, 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. 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 debatable^6,7,9,10. 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.

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) into an extension tube.
   4. Connect a 19 G needle to one end of the heparin-filled extension tube and a heparin-containing 60 mL syringe to the other end.
2. Anesthetize a pig according to Institutional policies
   1. Administer an IM injection of atropine (0.05 mg/kg), tiletamine/zolazepam (2-5 mg/kg), and xylazine (2 mg/kg) to induce anesthesia.
      NOTE: adequate anesthesia is achieved once the animal is unconscious and the mandibular muscles are non-rigid.
   2. Lay the animal in the supine position and place rolled-up towels on both sides to provide stability.
   3. Apply an ophthalmic ointment to the eyes to prevent dryness while under anesthesia.
   4. Provide thermal support during anesthesia including blankets, heating pads, and/or heated procedure table.
3. Clean the femoral groin area with betadine scrub solution.
4. Puncture the femoral vein or artery with the 19 G needle and slowly (~1-2 mL/s) draw 50 mL of blood into the syringe.
   NOTE: Drawing too fast can damage the cells.
5. Leaving the needle in place, immediately kink the extension tube and disconnect the 60 mL syringe
6. Slowly transfer the blood to a heparin-containing 50 mL conical tube.
7. Tightly cap the 50 mL conical tube, gently invert a few times to mix, and place on ice.
8. Connect the remaining heparin-containing 60 mL syringe to the extension tube, unkink the extension tube, and slowly draw an additional 50 mL of blood into the syringe.
9. Immediately remove the needle from the artery or vein and slowly draw the remaining blood from the extension tube into the syringe.
10. Disconnect the 60 mL syringe from the extension tube and slowly transfer the blood to the remaining heparin-containing 50 mL conical tube.
11. Tightly cap the 50 mL conical tube, gently invert a few times to mix, and place on ice.
12. Apply pressure hemostasis to the pig's femoral groin area.
13. Recover the pig according to Institutional policies. Continuously monitor the animal until it regains sufficient consciousness to maintain sternal recumbency and return to the regular housing/kennel area.

2. Isolation of mononuclear cells

1. Dilute the blood 1:1 with phosphate-buffered saline (PBS) in four 50 mL conical tubes.
   NOTE: This work needs to be performed in a cell culture hood using an aseptic technique to avoid contaminating the cell culture.
2. Add 25 mL of room temperature ready-to-use density gradient solution to eight 50 mL conical tubes.
3. Very slowly pipette 25 mL of blood/saline solution along the inside of each density gradient solution containing 50 mL conical tube by holding the tube at a sharp angle to the pipette tip.
   NOTE: The blood solution should gently layer on top of the density gradient solution and maintain a well-defined interface.
4. Centrifuge the tubes for 30 min at 560 x g and room temperature (RT).
   NOTE: For best results, disable the centrifuge brake if possible.
5. Use a thin pipette to carefully collect the buffy coat (cloudy layer of mononuclear cells above the density gradient solution and below the clear plasma) from each tube and distribute evenly into two new 50 mL conical tubes. Discard the density gradient solution containing tubes.
   ​NOTE: Collect as much of the buffy coat as possible while collecting as little of the density gradient solution and plasma as possible.

3. Washing and plating of cells

1. Coat a 6-well plate with fibronectin.
   1. Make a stock solution of human plasma fibronectin by diluting it to 1 mg/mL in sterile water. Store the aliquots at -20 °C.
   2. Make 3.6 mL of a working solution of fibronectin by diluting 600 µL of fibronectin stock solution in 3 mL of PBS.
   3. Transfer 600 µL of the fibronectin working solution into each well of a 6-well plate and gently rock until evenly coated.
   4. Wait 30 min for the fibronectin to coat the wells and gently aspirate the solution out of each well.
2. While waiting for the fibronectin to coat, wash the mononuclear cells
   1. Top up the tubes with up to 45 mL of PBS
   2. Centrifuge for 5 min at 560 x g and 4 °C with low brake. Aspirate the supernatant from both tubes.
   3. Resuspend each cell pellet in 25 mL of PBS.
   4. Repeat to wash a second time.
3. Resuspend each cell pellet in 5 mL of PBS and add 15 mL of 0.8% ammonium chloride solution to each tube.
   NOTE: This step is to lyse the remaining red blood cells.
4. Incubate on ice for 10 min.
5. Wash the cells as before by topping up tubes with PBS and centrifuge for 5 min at 560 x g and 4 °C with low brake. Aspirate the supernatant from both tubes.
6. Resuspend each cell pellet in 25 mL of PBS and repeat to wash a second time.
7. Resuspend each cell pellet in 6 mL of EGM-2 culture medium supplemented with 10% fetal bovine serum (FBS) and 1x antibiotic/antimycotic solution containing 10,000 U/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin.
   NOTE: EGM-2 culture medium consists of EBM-2 culture medium supplemented with 200 µL of hydrocortisone, 2 mL of human fibroblast growth factor (hFGF-B), 500 µL of vascular endothelial growth factor (VEGF), 500 µL of recombinant analog of insulin-like growth factor (R₃ IGF-1), 500 µL of ascorbic acid, 500 µL of human epidermal growth factor (hEGF), 500 µL of gentamicin/amphotericin, and 500 µL of heparin per 500 mL.
8. Gently transfer 2 mL of the cell suspension to each well of the fibronectin-coated 6-well plate and gently rock until evenly coated.
   ​NOTE: The goal is to seed as many cells as possible to maximize the number of endothelial colonies that will form.

4. Cell culture

1. Incubate the cells at 37 °C, 5% CO₂, and 100% humidity.
2. On the next day, gently add 1 mL of fresh culture medium to each well.
   NOTE: it is important to be gentle not to disturb cells that are loosely adherent to the fibronectin coating.
3. On the next day, perform a half culture medium change by gently aspirating 1.5 mL of culture medium from each well and replacing it with 1.5 mL of fresh culture medium.
4. On each of the next 5 days, gently perform a full culture medium change to each well (2 mL of fresh culture medium per well).
5. After that, gently change the culture medium three times per week.
6. Regularly visualize the cell cultures under low power (4x objective) light microscopy.
   NOTE: Adherent blood outgrowth endothelial cell (BOEC) colonies will begin to appear in the wells 7-10 days after plating. Non-adherent cell types will be discarded with medium changes, and other adherent cell types will gradually dissipate as the BOEC colonies grow.
7. Passage the BOECs into a fibronectin-coated T-75 flask when ~70%-80% confluent and continue changing culture medium three times per week.
   NOTE: Seeding density can be controlled by passaging colonies into a T25 flask instead. Subsequent passages do not require fibronectin-coating, and culture medium changes can be reduced to two times per week.
8. Cells from passages 1-3 may be used for experiments or transferred to a freezing medium and stored in liquid nitrogen for future use.

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