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This is intended as a guide only; for full experimental details please read the reference provided.
Wimmer et al. describe a protocol to generate human blood vessel organoids from H9 ES cells or iPS cells.
Human H9 ES cells or iPS cells were disaggregated then resuspended in differentiation medium 1 containing Y-27632 (50 μM) and plated into one well of an ultra-low attachment six-well plate for cell aggregation. On day 3 cell aggregates were treated with 12 μM CHIR 99021, then on days 5, 7 and 9 BMP4, VEGF-A and FGF-2 were added.
On day 11, differentiation media 2 containing VEGF-A, FGF-2 and SB 431542 (10 μM) was added to increase the yield of endothelial cells and suppress pericyte formation.
On day 13 cell aggregates were embedded in Matrigel:collagen I (1:1) gels and differentiation media 3 was added. Media was changed every second to third day. Approximately day 18, vascular networks were established, extracted from the gels and further cultured in 96-well low-attachment plates. These vascular networks self-assembled into vascular organoids and could be cultured for up to 3 months.
|Differentiation Media 1||Differentiation Media 2||Differentiation Media 3|
|DMEM:F12 medium||DMEM:F12 medium||DMEM:F12 medium|
|20% KOSR||20% KOSR||20% KOSR|
|Glutamax (equivalent to GlutaminePlus (B90210))||Glutamax||Glutamax|
|Y-27632 (Cat.No. 1254)||50 μM||VEGF-A (293-VE)||30 ng ml-1||15% FBS|
|CHIR 99021 (added day 3) (Cat.No. 4423)||20 μM||FGF (233-FB)||30 ng ml-1||VEGF-A (293-VE)||100 ng ml-1|
|BMP-4 (314-BP) (added days 5, 7 and 9)||30 ng ml-1||SB 431542 (Cat.No. 1614)||10 μM||FGF (233-FB)||100 ng ml-1|
|VEGF-A (293-VE) (added days 5, 7 and 9)||30 ng ml-1|
|FGF (233-FB) (added days 5, 7 and 9)||30 ng ml-1|