Table 2.
Angiogenesis Assay Using Hydrogel in Induced Pluripotent Stem Cell–Endothelial Cells and Human Umbilical Vein Endothelial Cells
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Dispense hydrogel | 3 μL | 4°C |
| 2 | Centrifugation | 30 s | 200 g for 30 s |
| 3 | UV polymerization | 8 min | 302 nm; 2 × 15 W bulbs; 9 cm |
| 4 | Dispense PBS | 20 μL | Sterile |
| 5 | Incubation time | 24 h | 37°C, 5% CO2 |
| 6 | Serum starve cells | 24 h | Basal media w/0.5% FBS |
| 7 | Wash hydrogel | 20 μL | Sterile PBS |
| 8 | Equilibrate hydrogel | 20 μL | Starvation media |
| 9 | Incubate hydrogel | 30 min | 37°C, 5% CO2 |
| 10 | Wash hydrogel | 20 μL | Sterile PBS |
| 11 | Cells stained | 2 mL | CellMask Green |
| 12 | Library compounds | 10 μL | 2 × concentration stock |
| 13 | Plate cells | 10 μL | 3.5 × 103 |
| iCell endothelial cells | |||
| 6.125 × 103 | |||
| HUVECs | |||
| 14 | Incubation time | 24 h | 37°C, 5% CO2 |
| 15 | Dispense fixation solution | 6 μL | 16% formaldehyde in water |
| 16 | Acquire images | 4 × Objective | With FITC filter |
Step Notes
1. Plate: 384-well Small Volume™, LoBase, PS, μclear, black, TC plates. Dispense SP-105 hydrogel on ice.
6. Serum starvation is only needed with the iCell endothelial cells.
9. Treatment of hydrogel is only needed with the iCell endothelial cells.
7, 10. For washing of the hydrogel, dispense 15 μL of sterile PBS to each well resulting in a meniscus above the well. Flip the plate over onto a sponge cloth and add weight to apply pressure then incubate for 1 min. Additional tapping may be required for removal of liquid in the well. Add 20 μL of sterile PBS and repeat washing.
8. Starvation media: Basal media with 0.5% FBS and 25 ng/mL VEGF.
11. For staining of the cells, cells were counted and desired number of cells transferred to a centrifuge tube. The cell suspension was centrifuged at 200 g for 5 min. Supernatant was discarded. The cell pellet was resuspended in 2 mL of FluoroBrite DMEM with 1:1,000 CellMask Green dye. Cells were incubated for 10 min at 37°C, 5% CO2. Following incubation, 12 mL of FluoroBrite DMEM was added to cell suspension and spun at 200 g for 5 min. The resulting cell pellet was resuspended in 2 × assay media.
For iCell endothelial cell 2 × assay media: VascuLife Basal Medium with 10 ng/mL FGF, 100 μg/mL ascorbic acid, 2 μg/mL hydrocortisone hemisuccinate, 8 mM l-glutamine, 30 ng/mL IGF-1, 10 ng/mL EGF, 10 ng/mL VEGF, 1.5 U/mL heparin sulfate, and 20% iCell endothelial cells media supplement.
For HUVEC 2 × assay media: Medium 199 containing the EGM™-2 BulletKits™ at 2 × concentration, also the VEGF component was replaced with 12.5 ng/mL VEGF from R&D Systems.
13. Final DMSO concentration on assay plate: 0.5%.
16. ImageXpress was used for image acquisition.
FBS, fetal bovine serum; PBS, phosphate-buffered saline.