Generating Vascular Organoids

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.

https://resources.tocris.com/images/protocols/vascular-organoids-bme-workflow.jpg

Cocktails

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  
NEAA   NEAA   NEAA  
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        
https://resources.tocris.com/images/protocols/vascular-organoids-protocol-timeline.jpg

Reference

Wimmer et al. (2019) Human blood vessel organoids as a model of diabetic vasculopathy. Nature 565 505 PMID: 30651639

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