Cytodex microcarrier beads

Cytodex microcarrier beads (Sigma-Aldrich, St. Louis, MO) were hydrated and sterilized in phosphate buffer saline (PBS). Beads were prepared for coating by washing repeatedly with 1 mL of EGM-2, with time to settle between washes. Endothelial cells were cultured in T-75 flasks to 80% confluency and rinsed with PBS before being harvested via 0.25% trypsin incubation for 5 min at 37 °C and 5% CO₂. Trypsin was neutralized using DMEM supplemented with 10% FBS. The cellular suspension was centrifuged (200 × G for 5 min) and supernatant was aspirated immediately. The cell pellet was re-suspended in 4 mL of fresh EGM-2. 10,000 microcarrier beads were combined with four million ECs, HUVEC or iPSC-EC, (5 mL total) in an inverted T-25 culture flask. Over a 4 hour incubation period, the culture flask was agitated every 30 minutes to ensure EC coating of beads. After 4 hours, the cell-bead mixture was added to a new T-25 culture flask. Fresh EGM-2 (5 mL) was added to the old flask to remove any remaining beads and transferred to the new culture flask. The total volume (10 mL) was incubated overnight in standard cell culture position.

The sprouting angiogenesis assay was performed as described [29 (link)]. HUVEC were infected with virus 72 hr prior to the start of the assay. 10⁶ HUVEC were coated onto Cytodex microcarrier beads and allowed to settle overnight, then suspended in 2mg/mL fibrinogen (Sigma, Fisher) plus 0.15 units/mL aprotinin (Sigma) in PBS. Upon addition of 0.625 U/mL thrombin (Sigma), the fibrinogen clotted to form a fibrin matrix. NHLF were plated on top of the fibrin, and media (EBM2 supplemented with EGM2 bullet kit) was added and changed every second day.

Approximately 500 Cytodex Microcarrier beads (Sigma Aldrich, St. Louis, MO, USA, C3275) per 1.25 × 10⁴ cells/ml were mixed in FACS tubes (Becton, Dickinson and Company, Allschwill, Switzerland, Falcon T7597-5J) and incubated at 37 °C for 6 h with a mild shaking of the tubes at every one hour. Cells, which were not adhered to the beads, were removed by washing the beads with fresh medium. Cell-coated microbeads were re-suspended in 2.5% bovine collagen 1 (Advanced BioMatrix, San Diego, CA, USA, 5005-B) and seeded in μ-clear 96 well plate (Greiner CELLSTAR®, 655090). Following the polymerization of collagen, the cell coated microbeads were overlaid with fresh medium and treated with appropriate concentrations of growth factors/cytokines or with HGF or with c-Met inhibitors. After 24 h, cells were fixed with 4% paraformaldehyde (PFA) and stained with Hoechst. Images were acquired using an ImageXpress Micro 2 automated microscope (Molecular Devices, LLC, USA) as described for the zone infiltration assay. Cell invasion is expressed as the average of the distance invaded by the cells from the circumference of the bead as measured by our cell dissemination counter software aMDIcs.

Approximately 500 Cytodex Microcarrier beads (Sigma Aldrich C3275) per 1.25 × 10⁴ LA-EGFP-DAOY cells/ml were mixed in FACS tubes (BD Falcon T7597-5 J) and incubated at 37°C for 6 h, followed by incubation under rotation for 18 h. Non-adherent cells were removed. Cell-coated microbeads were resuspended in 2.5% bovine collagen I (5005-B, Advanced BioMatrix) in 96-well plate, after polymerization of collagen overlaid with fresh medium and treated with appropriate concentrations of c-Met inhibitors or HGF. After 24 h, cells were fixed with 4% PFA and stained with Hoechst. Images were acquired using the ImageXpress microscope. The distance between the microbead and the nuclei of the invaded cells was measured using ImageJ software. Velocity was calculated as the distance of displacement/time.