Generating Monocyte Derived Endothelial-like Cells for Vascular Regeneration

Abstract

A major limitation in engineering vascular grafts is the lack of proper endothelium to prevent thrombosis and subsequent graft failure. Obtaining endothelial cells from patients’ vasculature is intrusive and requires extensive culture time. Here we present an alternative strategy wherein abundant and easily accessible monocytes from peripheral blood are cultured and differentiated towards an endothelial like state capable of preventing thrombosis through production of nitric oxide and formation of endothelial adherens junctions. Considering the plethora of monocytes present within peripheral blood, this method provides a robust alternative to generating endothelial cells required for vascular graft production.

1. Introduction

Endothelial cells are an essential component of the vasculature, without which tissue engineered vessels thrombose and fail. Thus, obtaining endothelial cells either prior to or after implantation is a major obstacle. In adult humans, endothelial cells do not proliferate from anastomotic ends of implanted vessels [1] and must come from circulating endothelial progenitors (EPCs). However, EPCs are exceedingly rare within human blood, representing less than 0.01% of mononuclear cells in the blood [2]. However, we have shown that vascular endothelial growth factor (VEGF) immobilized vascular grafts can capture circulating cells expressing the VEGF receptors, namely monocytes, and that these cells can then differentiate towards an endothelial phenotype capable of maintaining patency [3–5]. Monocytes greatly outnumber potential EPCs at nearly 20% of peripheral blood mononuclear cells.

Previous studies have shown that monocytes can express endothelial genes after in-vitro culture [6–11] but lack VE-cadherin positive adherens junctions and the ability to form tubes, indicating lack of endothelial (EC) functionality. To address these drawbacks, we developed a strategy to coax monocytes (MC) to differentiate into functional EC like cells in-vitro [12] and within this chapter we describe the methodology to differentiate this plentiful cell source into the crucial endothelial component of the vasculature.

2. Materials

All cells obtained from blood is following an approved IRB protocol or by purchasing platelet apheresis byproduct from a medical institution and further cell isolation.

2.1. Cell Media

All media used in this protocol are based on complete endothelial growth media (EGM2, Lonza; Allendale, NJ) with all supplements except for fetal bovine serum (FBS). The media is then supplemented with additional VEGF at a concentration of 50ng/mL (see Note 1) and macrophage colony stimulating factor (MCSF) at 1ng/mL (Thermo Fisher; Grand Island, NY). Serum is replaced with 20% autologous platelet rich plasma (PRP) (see Note 2).

1. Attachment media: Endothelial growth media supplemented with 10μM Y-27632 (Sigma-Millipore; St. Louis, MO).

2. Activation media: Endothelial growth media supplemented with 10μM CHIR-99021 (Sigma-Millipore).

3. Platelet Rich Plasma: Blood is collected with anti-thrombotic agents such as heparin at 10U/mL blood Freshly drawn human blood is separated into the cellular component and plasma components by simple centrifugation at 1500RPM for 10min with no brake applied. Carefully separate the top, yellow appearing plasma from the bottom red cellular component. Save both components. PRP can be frozen and thawed once with a shelf life of 1–2 months at −20°C.

2.2. Culture Surface Preparation

Fibronectin Surface: Fibronectin is dissolved at 1mg/mL in phosphate buffered saline (PBS). Non-tissue culture plates (hydrophobic) are coated for 12hr overnight at 4°C with gentle rocking.

2.3. Other materials:

Red blood cell (RBC) lysis buffer (155mM NH₄Cl, 12mM NaHCO₃, 0.1mM EDTA), chloroform, shaking platform (capable of 40–150 rotations per minute).

2.4. Shear Plate Preparation:

The following is adapted from a similar procedure in White et al. [13]

1. Using a 10cm² diameter dish as a culture surface and a 3–5cm² diameter dish as an insert ring, attach the smaller dish to the larger dish.

2. Dip the smaller dish into a bath of chloroform so that the edge is submerged.

3. Quickly place the treated edge of the smaller dish into the middle of the larger dish so that the smaller dish is centered onto the larger dish. Apply pressure as the chloroform seals the two plates together.

3. Methods

3.1. Monocyte Isolation

1. Obtain freshly drawn blood with 10U/mL heparin to prevent thrombosis OR obtain the leftover platelet apheresis cone (see Note 3). 50mL peripheral blood yields ×10×10⁶ monocytes and 20–25mL of PRP (see Note 4). Keep the blood at room temperature to aid in component separation. Cold temperatures inhibit the separation.

2. Centrifuge the blood at 1500RPM for 10min at room temperature with no brake. This separates the PRP from the cellular component containing monocytes.

3. Carefully remove the yellow layer containing PRP and use this to prepare endothelial generation media; the remaining PRP can be frozen for future use.

4. The remaining red component containing RBC and white blood cells (WBC) is approximately half the initial volume. Use 1x RBC lysis buffer (or see Note 5) to lyse red blood cells for 5–10min with occasional mixing by inverting the tube. Proper lysis is observed when the red component appears to darken to a very dark red color (see Note 6).

5. Centrifuge the lysed product at 1350RPM for 10min with no brake at room temperature.

6. Carefully remove the supernatant- the cell pellet will appear white. Repeat the lysis procedure one time for complete lysis.

7. Resuspend cell pellet in PBS to wash the cells. Centrifuge the washed cells at 1350RPM for 5min.

8. Repeat the wash until the supernatant is clear.

9. The resulting cells are all WBC- to obtain pure (90%+) monocytes it is recommended to use adherence-based isolation. Seed cells on fibronectin coated dishes at 2–3×10⁶ cells per cm² in endothelial growth media without serum or PRP. Serum inhibits initial adhesion. Incubate for 1hr at 37°C in a 5% CO₂ incubator.

10. Carefully remove unbound cells and wash the plate with pre-warmed PBS to further remove unbound cells.

11. Monocyte purity can be verified with flow cytometry. A typical panel to determine purity would include the following: CD45, CD14, CD16, and CCR2. Such a panel would distinguish total monocytes as well as the subtypes: classical (CD14++/CD16−/CCR2+), non-classical (CD14−/CD16+/CCR2−), and intermediate (CD14+/CD16+/CCR2+) [14]. Inclusion of VEGFR2/KDR, CD133, CD144, and CD34 can determine if there is contaminating endothelial cells.

3.2. Monocyte culture and differentiation under static conditions: See Figure 1 for schematic of time-course.

Figure1:

Schematic of monocyte differentiation.

1. Immediately after adherence isolation add endothelial attachment media consisting of EGM2 containing Y-27632 (10uM) and PRP (20%).

2. Leave cells in the humidified incubator for 3 days without disturbing. Attachment and spreading occurs slowly during this time.

3. Replace media with endothelial activation media on Day 3 consisting of EGM2 with CHIR99021 (10uM) (without Y-27632). Leave media for 2 days (see Note 7).

4. Replace media with endothelial growth media (20% PRP, 1ng/mL MCSF, 50ng/mL VEGF) and continue to culture for 7 to 10 days until endothelial like cobblestone morphology begins to appear. Replace media every other day.

5. Monocyte derived endothelial cells (MC-EC) can be further cultured under shear conditions that induce maturation.

3.3. MC-EC culture under shear conditions

1. MC-EC are detached from the surface using 0.1% Trypsin in PBS and quenched with FBS once all the cells are detached.

2. Collect detached cells and centrifuge for 5min at 1350RPM (300g).

3. Resuspend cell pellet in endothelial attachment media to allow cells to reattach with ease to the new surface.

4. Seed MC-EC at <80% confluency in the shear plates as prepared above.

5. Allow cells to spread overnight before removing endothelial attachment media and replacing with endothelial basal media.

6. Place shear plate on shaker device at the lowest setting possible to allow for around 20–40 RPM (see Note 8).

   1. Shear strength is then changed by changing the rotation speed. We adapted this procedure with an orbital shaker of 1.9 cm radius. Shear was determined by the equation:

      $$\tau =r\sqrt{\eta \rho (2\pi f{)}^3}$$
      Where $\tau$ is the desired shear (dynes/cm²), r is the radius of the orbital shaker, η is the viscosity (poise), ρ is the density (g/mL), and f is the rotations speed in rounds per second. Using this equation and converting to rotations per minute, approximately 29, 84, and 133 rotations per minute equated to 1, 5, and 10 dynes/cm² of shear, respectively.

7. Shear is slowly ramped up from 1 to 10 dynes/cm² over 2 days and maintained at that level for 3 days. Medium is replaced every other day during shear studies. Representative images of MC-EC after maturation under shear is depicted in Figure 2.

8. Cells are now ready for use in vessel engineering.

Figure 2:

Maturation of monocyte derived endothelial cells under shear. Cells were fixed after 9 days of differentiation and 5 days of shear and assessed by immunocytochemistry for the endothelial markers CD144 and CD31 and monocyte markers CD14 and CD163. White arrows indicate direction of shear.

4. Notes

1. VEGF can be produced as a recombinant protein as described[4]. Optimal concentration varies between 50 –100ng/mL.

2. PRP is best when autologous, but success has also been achieved with other human PRP and other non-human sources such as ovine. Low PRP concentrations increase attachment at the expense of survival. 10–20% PRP is best.

3. The cones are a component of platelet apheresis used in platelet donation. These cones accumulate WBC during the procedure and are a rich source of cells, often containing up to 1×10⁹ mononuclear cells.

4. 50mL of peripheral blood generates enough PRP to make at least 100mL of endothelial media if using 20% PRP. This volume of PRP can be adjusted down to 10% if PRP is a limiting reagent.

5. Alternatively, peripheral blood can be processed using histopaque-1077 or other density-based methods to separate mononuclear cells out of the whole blood.

6. The concentration of the RBC lysis buffer components must remain near 1x- concentrations outside of this can inhibit lysis. Incomplete lysis is noticeable when the solution color remains lighter red.

7. During the CHIR activation step the cells may round up and detach. Carefully collect unbound cells at the end of activation and centrifuge them down at 1350RPM for 5min. Re-seed these cells back onto the surface they came from in the presence of Y-27632. This will allow rounded up cells to re-spread if necessary. Y-27632 may be left in the media for up to 7 days if needed before cobblestone morphology is apparent.

8. During the shear process, cells may detach. Ideally, the MC-EC will proliferate into the empty spaces.