Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

A schematic of the BOEC generation protocol is shown in Figure 1.

1. Blood Collection and Density Gradient Centrifugation

1. For each donor, add 3 ml of sodium citrate to each of two 50 ml conical centrifuge tubes. Collect 60 ml of blood by venipuncture and add 30 ml to each tube. Invert gently 2-3 times to mix. Use as little as 40 ml of blood in the protocol, with citrate volumes adjusted accordingly.
   NOTE: It is essential that blood samples are processed within 2 hr of collection. Delayed processing of blood results in a marked reduction in outgrowth colony yield.
2. Prepare new conical tubes containing density gradient centrifugation medium by first inverting the bottle several times to mix. In a sterile hood, add 15 ml of density gradient centrifugation medium per 50 ml conical tube (1 tube for every 10 ml of blood, 6 tubes per donor).
3. Dilute blood 1:1 with Dulbecco's Phosphate-Buffered Saline (DPBS). Tilt the tube containing the density gradient centrifugation medium to an angle of 20° with the work surface. Using a pipette aid and a 25 ml serological pipette, slowly layer 21 ml of diluted blood on top of the medium by gradually adding blood down the wall of the tilted tube.
   NOTE: Doing so will minimize mixing of the blood with the density gradient layer and will produce a tighter buffy coat, as well as an enhanced yield.
4. Centrifuge samples at 400 x g for 35 min at RT with the accelerator and the brake off.
5. During this centrifugation period, begin the collagen coating detailed in steps 2.1 – 2.4. Ensure these steps have been completed before proceeding to step 3.1.

2. Collagen Coating of T-75 Flask and Preparation of Culture Medium

NOTE: Carry out the following steps in a cell culture hood.

1. Prepare a 50 μg/ml collagen solution by diluting stock Type I collagen in 10 ml of 0.02M acetic acid. Example: for collagen stock at a concentration of 4.05 mg/ml, add 123.5 μL of collagen to 10 ml of 0.02 M acetic acid. Sterile filter collagen suspension prior to use.
2. Using a serological pipette, add 7.5 ml of collagen solution to a T-75 cell culture flask. This volume gives 5 μg collagen/cm² (or 375 μg/flask). Coat flask for 1 hr at RT.
3. Prepare the endothelial growth medium used for BOEC generation by supplementing endothelial basal medium with the growth factor supplements provided by the manufacturer. Add all supplements, but do not add serum. To prepare 15 ml of BOEC generation medium, add 2.5 ml of defined fetal bovine serum (FBS) to 12.5 ml of endothelial growth medium containing the growth factor supplements.
4. Proceed with the steps outlined in Section 3. After the 1 hr coating period, aspirate the collagen solution and wash away residual acetic acid by pipetting in 10 ml DPBS. Aspirate off the DPBS and repeat this wash step once more. If the cell suspension obtained during step 3.3 is not ready for plating at this time, add 5mL of BOEC generation medium to the flask to keep the collagen coating from drying-out.

3. Collection and Plating of PBMNCs

1. Following the density gradient centrifugation outlined in step 1.4, carefully collect the buffy coat layer using a sterile plastic transfer pipette. Collection of the buffy coat should yield approximately 20 ml of cell suspension and plasma from each tube. Avoid transferring the density gradient medium.
2. Dilute the mononuclear cell suspension 1:1 in DPBS and invert to mix. Centrifuge at RT for 20 min at 300 x g with brake and accelerator at maximum.
3. Following centrifugation, aspirate the supernatant and resuspend cells by adding 1 ml of BOEC generation medium to each pellet and pipetting up and down repeatedly. Pool cell suspensions and top up total volume to 10 ml with the remaining medium.
4. To get an estimation of total cell number, sample 10 μl of cell suspension and dilute 50x with 490 μl of Turk's solution, which will lyse red blood cells. Count a 10 μl sample of the diluted cell suspension using a haemocytometer.
   NOTE: 60mL of blood should yield approximately 100-150 x 10⁶ white blood cells for plating.
5. Plate entire cell suspension into a single, collagen-coated T-75 flask, top-up medium volume to 15mL/flask and culture at 37 °C in a humidified atmosphere containing 5% CO₂. Cells plated at this time represent passage 0.
   NOTE: It is possible to freeze the isolated PBMNCs prior to BOEC isolation in order to derive the BOECs at a later time or in order to transport the PBMNCs to a different laboratory where the BOECs can be generated. However, it is important to note that freezing PBMNCs could reduce the efficiency of BOEC isolation. See section 8 below for method.

4. Long-term Cell Culture

1. Change medium every 2 days by adding 15 ml of fresh BOEC generation medium (5:1 mixture of endothelial cell growth medium and defined FBS). For weekends, medium changes can be delayed to every 3 days.
   NOTE: Outgrowth colonies should appear between 7 and 14 days of culture.
2. Monitor the BOEC culture flask on days 7-14 for the appearance of outgrowth colonies. Identify colonies as circular groups of cells exhibiting a classic endothelial cobblestone morphology.
3. Once a colony or multiple colonies are identified in the flask, continue with medium changes and allow colonies to grow to approximately 1000 to 2000 cells per colony before passaging. Determine the cell number per colony through a rough visual estimate.
   NOTE: As a guide, it is customary to passage initial outgrowth colonies one week after the identification of the first outgrowth colony.
4. Passage cells by rinsing flasks twice with 10 ml of DPBS. Add 5 ml of 1X trypsin-EDTA and incubate in a 37 °C incubator for 5 min. After 5 min, neutralize trypsin with 10 ml of medium (containing FBS) and bring cells into suspension with repeated pipetting.
5. Centrifuge suspension at 300 x g for 5 min, resuspend in 15 ml of fresh BOEC generation medium and plate entire cell suspension into a new T-75 flask (representing passage 1, P1). No collagen coating is required.
6. Continue medium changes as described above until cells are confluent (roughly 3-5 x 10⁶ cells per flask). Once confluent, passage cells as described in step 4.3.
   NOTE: From passage 2 onwards, cells no longer require BOEC generation medium and can be cultured in endothelial cell growth medium and 10% standard heat-inactivated FBS. Collagen coating can be used as an optional step going forward, as there is evidence to suggest that the presence of collagen can enhance BOEC proliferation rates.
7. For continued passaging, plate no fewer than 750,000 cells per T-75 flask as low cell densities can cause the BOECs to stop proliferating.

5. Freezing and Thawing BOEC Cultures

1. Trypsinize cells as described in section 4.3 and centrifuge at 300 x g for 5 min. Aspirate the supernatant and resuspend in 10 ml of medium. This does not require endothelial cell growth medium; DMEM with 10% standard FBS will be sufficient. Collect a 10 μl sample of the cell suspension to perform a manual cell count using a haemocytometer.
2. Centrifuge again and resuspend at 2×10⁶ cells/ml in ice-cold cryopreservation medium (40% DMEM with 50% FBS and 10% DMSO). Add 0.5 ml (10⁶ cells) to each vial, place vials in an ice cold isopropanol cryopreservation vessel and place in a -80 °C freezer for at least 2 hr before transferring to liquid nitrogen. Do not leave cells at -80 °C for more than 24 hr.
3. To thaw cells, add 10 ml of pre-warmed medium to a 15 ml conical centrifuge tube.
   NOTE: When thawing out passage 1 cells, thaw into endothelial cell growth medium supplemented with 20% defined FBS for the first 2 days after thawing. This leads to a more stable cell isolation going forward.
4. Remove cells from liquid nitrogen and thaw in a 37 °C water bath with gentle agitation until only a small ice crystal is left in the vial. Add the contents of the vial drop-wise to the conical centrifuge tube and spin down at 300 x g for 5 min.
5. Aspirate supernatant and resuspend cell pellet in 10 ml EGM-2MV+10%FBS. Add to T-75 flask and top up medium to 15 ml.

6. Characterization of BOECs by Flow Cytometry

1. Once the BOEC colonies described in Section 4 have been allowed to expand, trypsinize cells as described in section 4.4 and resuspend at a concentration of 10⁶ cells per ml in staining buffer (DPBS with 2% FBS and 2 mM EDTA).
2. Combine 10⁵ cells with fluorochrome-conjugated antibodies directed against CD45, CD14, CD34, CD31 (use all at 1:20 dilution) or VEGFR2 (dilute 1:10) (see Table 1 for full details). Incubate for 30 min at 4 °C in the dark.
3. Wash cells with 1 ml staining buffer and centrifuge at 300 x g for 5 min. Aspirate supernatant and resuspend in 400 μl staining buffer for analysis. Keep cells at 4 °C and protected from light prior to analysis.
4. Perform cytometric analysis on a cytometer equipped to detect the fluorescently conjugated antibodies used in the assay.
5. Use an unstained BOEC sample to identify the cell population on the cytometer by adjusting forward and side scatter voltages, as well as the voltages for the FITC and APC channels.
6. Determine gating thresholds using isotype stained cells for each fluorochrome. Assess positivity for each cell surface marker based on the presence of a peak shift in fluorescence intensity versus isotype control for the fluorochrome being analysed.

7. Characterization of BOECs by Immunofluorescent Microscopy

1. Plate BOECs in a 24-well, flat-bottom tissue culture plate without coverslips at a density of 5 x 10⁴ cells in 500 μl of endothelial cell growth medium + 10% heat-inactivated FBS per well and leave to adhere and grow at 37 °C, 5% CO₂ until cells cover at least 70-80% of the surface area of each well (estimate confluency by visual inspection under a light microsope).
2. Wash cells in each well for 5 min with 1 ml of DPBS.
3. Fix cells O/N at 4 °C with 500 µl of 4% paraformaldehyde solution in DPBS.
4. Wash cells for 3 x 5 min with 1 ml of DPBS per well. Add and remove the DPBS using a pipette and an aspirator, respectively.
5. Permeabilize cells for 3 x 10 min with 0.2% polysorbate 20 in DPBS.
6. Incubate cells for 1 hr at RT in blocking buffer containing 10% FBS in DPBS.
7. Stain cells at 4 °C O/N in 300 µl of antibody dilution buffer (0.1% bovine serum albumin in DPBS) containing primary antibodies directed against CD34 (10 µg/ml), CD144 (1:300) or vWF (1:250). See Table 1 for full details.
8. Wash off unbound primary antibodies for 5 x 10 min with DPBS.
9. Incubate cells for 1 hr at RT with the appropriate fluorescently-labelled secondary antibody, as detailed in Table 1 (all at 1:200).
10. Wash off unbound secondary antibodies for 5 x 10 min with DPBS.
11. Incubate cells with 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) in DPBS for 10 min at RT.
12. Wash cells with DPBS for a further 3 x 5 min.
13. Image cells at 100x magnification using an inverted microscope equipped with an external light source for fluorescence excitation, and capture images using an attached digital camera for subsequent analysis offline.

8. Freezing and Thawing PBMNCs

1. At the end of stage 3.2, following centrifugation, aspirate the medium and manually disrupt the cell pellet with repeated pipetting up and down using a P1000 pipette tip. Add 1 ml of freezing medium, 90% serum and 10% DMSO, making sure to mix gently to produce a cell suspension. Transfer the cell suspension to a cryovial and freeze and thaw as described in Section 5.