Sep 09, 2025
Generating vessel organoids (VOs) from hiPSCs - A Detailed, Step-By-Step Guide
- Nicole Pek1,2
- 1Center for Stem Cell and Organoid Medicine, Cincinnati Children's Hospital Medical Center;
- 2College of Medicine, University of Cincinnati
Protocol Citation: Nicole Pek 2025. Generating vessel organoids (VOs) from hiPSCs - A Detailed, Step-By-Step Guide. protocols.io https://dx.doi.org/10.17504/protocols.io.261gekejog47/v1
Manuscript citation:
Wimmer RA, Leopoldi A, Aichinger M, Kerjaschki D, Penninger JM. Generation of blood vessel organoids from
human pluripotent stem cells. Nat Protoc. 2019 Nov;14(11):3082-3100. doi: 10.1038/s41596-019-0213-z. Epub 2019 Sep 25. PMID: 31554955.
Wimmer RA, Leopoldi A, Aichinger M, Wick N, Hantusch B, Novatchkova M, Taubenschmid J, Hämmerle M, Esk C, Bagley JA, Lindenhofer D, Chen G, Boehm M, Agu CA, Yang F, Fu B, Zuber J, Knoblich JA, Kerjaschki D, Penninger JM. Human blood vessel organoids as a model of diabetic vasculopathy. Nature. 2019 Jan;565(7740):505-510. doi: 10.1038/s41586-018-0858-8. Epub 2019 Jan 16. PMID: 30651639; PMCID:
PMC7116578.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 08, 2025
Last Modified: September 09, 2025
Protocol Integer ID: 226706
Keywords: organoids, vessels, endothelial, pericytes, vascular, generating vessel organoid, derived vessel organoid, vessel organoid, vascularizing other organoid type, human vascular development, dimensional vascular model system, functional vessel, source of functional vessel, other organoid type, step guide for vo generation, generating vo, vo generation
Disclaimer
The published hiPSC-derived vessel organoid protocol was adapted with slight modifications, and all essential molecular and cellular validations were completed.
Abstract
hiPSC-derived vessel organoids (VOs) serve as a three-dimensional vascular model system to study human vascular development and disease. Recently, VOs were even used as a source of functional vessels for vascularizing other organoid types. Furthermore, with NIH's recent emphasis on prioritizing human-based model systems, there is an increased interest in utilizing VOs in both basic and pre-clinical research. Generating VOs from hiPSCs is a labor-intensive, technically challenging, multi-step process. Improper handling can lead to variability in VO size and quality. Here, I provide a detailed, step-by-step guide for VO generation from hiPSCs, including images of expected morphologies and practical tips to improve outcomes.
Image Attribution
Image is prepared by Nicole Pek
Materials
Reagents:
| Name | Company | Catalog no. | |
| KnockOut™ DMEM/F-12 | Gibco | 12660012 | |
| KnockOut™ Serum Replacement | Gibco | 10828028 | |
| GlutaMAX™ Supplement | Gibco | 35050061 | |
| MEM Non-Essential Amino Acids Solution (100X) | Gibco | 11140050 | |
| Antibiotic-Antimycotic (100X) | Gibco | 15240062 | |
| β-Mercaptoethanol, Molecular Biology Grade | MilliporeSigma | 444203-250ML | |
| Anti-adherence Rinsing Solution | STEMCELL Technologies | 07010 | |
| Y-27632 | Tocris | 1254 | |
| UltraPure™ 0.5M EDTA, pH 8.0 | Gibco | 15575020 | |
| Gibco™ DPBS, no calcium, no magnesium | Gibco | 14190144 | |
| Trypan Blue Solution, 0.4% | Trypan Blue Solution, 0.4% | 15250061 | |
| DMEM/F12 | Gibco | 11320033 | |
| Neurobasal™ Medium | Gibco | 21103049 | |
| B-27™ Supplement (50X), serum free | Gibco | 17504044 | |
| N-2 Supplement (100X) | Gibco | 17502001 | |
| CHIR99201 | Selleckchem | S2924 | |
| Recombinant Human BMP-4 Protein | R&D Systems | 314-BP-050/CF | |
| Forskolin | Sigma-Aldrich | F3917-10MG | |
| VEGFA-165 | GeminiBio | 300-196P-100 | |
| Sodium hydroxide solution | Sigma-Aldrich | S2770 | |
| DMEM, powder, high glucose | Gibco | 12100046 | |
| Ham's F-12 Nutrient Mix | Gibco | 11765054 | |
| Sodium Bicarbonate 7.5% solution | Gibco | 5080094 | |
| HEPES (1M) | Gibco | 15630080 | |
| PureCol® | Advanced BioMatrix | 5005 | |
| Corning® Matrigel® Matrix for Organoid Culture, Phenol Red-free, LDEV-free, 10 mL | Corning | 356255 | |
| StemPro™-34 SFM (1X) | Gibco | 10639011 | |
| FGF2 | GeminiBio | 300-112P-100 | |
| Fetal Bovine Serum, qualified, United States | Gibco | 26140079 |
Plasticware/ tools:
| Name | Company | Catalog no. | |
| AggreWell 400 | STEMCELL Technologies | 34415 | |
| Flowmi cell strainers 40 micron | SP Bel-Art | H13680-0040 | |
| LevGo™ smartSpatula™ Disposable Polypropylene Spatula | Fisher Scientific | 18-001-022 | |
| ART™ Wide Bore Filtered Pipette Tips | Thermo Fisher Scientific | 2079GPK | |
| Costar® 6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates | Corning | 3471 | |
| Costar® 12-well Clear TC-treated Multiple Well Plates | Corning | 3513 | |
| Corning® 100 mm TC-treated Culture Dish | Corning | 430167 | |
| Corning™ 96-Well Clear Ultra Low Attachment Microplates | Corning | 7007 | |
| 27G needles | BD Biosciences | 301629 |
Equipment:
- Centrifuge
- Incubator
- Tissue culture hood
- Orbital shaker
- Light microscope/ dissection microscope
Troubleshooting
Embryoid Body (EB) Formation (Day 0)
Prepare Aggregation media*.
Recipe For Aggregation media:
| Components | Working Concentration | |
| KO DMEM/F12 Media | - | |
| KO Serum Replacement | 20% | |
| GlutaMAX | 1% | |
| NEAAs | 1% | |
| Antibiotic-Antimycotic | 1X | |
| β-mercapethanol | 55μM |
*Aggregation media can be stored at 4°c for up to 2 weeks.
Optional: Pass the media through a 0.2µm filter to remove residual particles and enhance sterility.
Prepare Wells* of the AggreWellTM 400 Plate (24 well-plate).
*Use only the wells required; any unused wells can be saved for future use.
Coat each well* of AggreWellTM 400 with 500μL of Anti-Adherence Rinsing Solution.
*Only add rinsing solution to wells that are needed.
Centrifuge AggreWellTM 400 plate at 2000g, 5-10mins to remove bubbles from microwells.
Observe wells of AggreWellTM 400 plate under microscope to ensure no bubbles are present in the wells.
Aspirate Anti-adhesion solution from wells and add 1ml DMEM/F12 media per well.
*Be careful not to touch the bottom of the wells when aspirating - this may result in damage to the microwells of the AggreWellTM plates.
Aspirate DMEM/F12 media.
*Be careful not to touch the bottom of the wells when aspirating - this may result in damage to the microwells of the AggreWellTM plates.
Add 1ml of Aggregate media (supplemented with 20μM* of ROCK inhibitor Y-27632) to each well.
*Concentration may vary depending individual hiPSC line, please optimize accordingly. If more than one hiPSC lines are utilized, determine an optimal concentration that would work for all lines.
Prepare hiPSCs For EB Formation.
To ensure high-quality EBs, use hiPSCs at 70–80% confluency and avoid over-confluent cultures. Additionally, check morphology of hiPSCs before EB formation to determine cell quality; i.e. if hiPSCs are spontaneously differentiating,
Trypsinize hiPSCs into single cells using 0.5mM EDTA (in DPBS)*.
*There are many ways to trypsinize hiPSCs, use whichever method that has been established in your lab.
Incubate cells at 37°c for 5 mins*.
*Check cells under microscope to make sure that cells are starting to lift off the plate.
Add 1ml of DMEM/F12 media to each well of cells and gently titrate to flush cells.
Transfer cell mixture into a conical tube and spin cells down at 200g for 5 mins.
Aspirate supernatant and resuspend the cell pellet gently* with 1ml Aggregation media (supplemented with Y-27632).
*hiPSCs are fragile at this stage, gentle tritation helps ensure high cell viability.
Pass the cell suspension through the 40µm FlowmiTM cell strainers into a new conical tube to obtain a single-cell suspension of hiPSCs.
Perform cell count:
a. To a fresh microtube, add 10µl of cells and 10µl of Tryphan blue. Mix cells gently.
b. Transfer 10µl of the cell-tryphan blue mixture to a hemocytometer.
Transfer 1.2x106 hiPSCs to each well of the AggreWellTM 400 plate*.
*This results in approximately 1,000 cells per EB.
Important Note: Only proceed when >90% of cell viability is achieved to ensure optimal quality of EB.
Top up with Aggregation media (supplemented with Y-27632) to a final volume of 2ml per well.
Centrifuge plate at 100g for 3 mins*.
*Ensure the plate is balanced to allow even distribution of cells into each microwell, resulting in uniform EB size.
Remove plate containing cells carefully and place plate into a 37°c incubator. Let cells form EBs overnight.
Next day, check that EBs with smooth and round borders are formed in each microwell (Fig. 1). If not, please see 'Troubleshooting' section. Only proceed to the next step when 'good quality' EBs were achieved.
Fig. 1 - Example of 'good quality' EBs.
Mesoderm Differentiation (Day 1-5)
Prepare N2B27 media*.
Recipe for N2B27 media:
| Components | Working Concentration | |
| Neurobasal media | - | |
| DMEM/F12 media | - | |
| B27 Supplement | 1% | |
| N2 Supplement | 0.5% | |
| GlutaMAX | 0.5% | |
| Antibiotic-Antimycotic | 1X | |
| β-mercaptoethanol | 55µM |
*N2B27 media can be stored at 4°c for up to 2 weeks.
Highly recommended: Pass the media through a 0.2µm filter to remove residual particles and enhance sterility.
Pro tip: If not using the entire 500ml of N2B27 media, you may freeze them as smaller aliquots e.g. 40ml in 50ml conical tubes.
Mesoderm Differentiation 1 (Day 1-3)
Prepare 'MD1' media*
Recipe for 'MD1' media:
| Components | Working Concentration | |
| N2B27 media | - | |
| CHIR99021** | 12µM | |
| BMP4 | 30ng/ml |
*Prepare 'MD1' media fresh.
** CHIR99021 concentration may require optimization for different hiPSC lines. If multiple hiPSC lines are used, find a common concentration that would work for all the lines.
Cells that did not form EBs tend to aggregate and form an 'odd-shaped' cluster outside of the microwells. Carefully remove it using a P200 pipette.
Aspirate aggregation media from each well carefully to avoid disturbing the EBs and add 1ml of N2B27 media to each well.
Gently tritrate to resuspend EBs in the N2B27 media and transfer EB mixture to a conical tube. Allow EBs to settle to the bottom of the tube by gravitation.
Wash each well of the AggreWellTM plate that was used with 1ml of sterile DPBS. Aspirate DPBS and seal AggreWellTM plate with parafilm for future use.
Once EBs have settled to the bottom of the conical tube, aspirate supernatant carefully and resuspend EBs in 'MD1' media.
Transfer EBs to an ultra-low attachment (ULA) 6 well-plate.
* 1 well of EBs should be transferred to 3 wells of the ULA 6-well plate to avoid over-crowding
Top up each well with 'MD1 media'. The final volume of 'MD1' media per well should be 3ml.
Place EB-containing plate on an orbital shaker* in a 37°c incubator. No media change is required from days 1 to 3.
*Every orbital shaker is slightly different, optimal shaking rate should be sufficiently fast to prevent fusion of EBs/aggregates but not too fast such that the EBs/ aggregates are sheared due to excessive force.
Mesoderm Differentiation 2 (Day 3-5)
Prepare 'MD2' media*
Recipe for 'MD2' media:
| Components | Working Concentration | |
| N2B27 media | - | |
| Forskolin | 2µM | |
| VEGFA-165 | 100ng/ml |
*Prepare 'MD2' media fresh.
On day 3, gently transfer aggregates (Fig. 2) to a conical tube. Allow aggregates to settle to the bottom of the tube by gravitation.
Fig. 2 - Example of day 3 mesodermal aggregates.
Carefully aspirate supernatant and wash aggregates once with 1ml of DMEM/F12 media. Carefully aspirate supernatant.
Resuspend aggregates in 'MD2' media and gently transfer aggregates back to the ULA 6-well plate.
Top up each well with 'MD2 media'. The final volume of 'MD2' media per well should be 3ml.
Place aggregate-containing plate back on an orbital shaker in a 37°c incubator. No media change is required from days 3 to 5.
Extracellular Matrix Embedding (Day 5-10)
Prepare Collagen 1 Solution (2mg/ml).
Recipe for Collagen 1 solution*:
| Components | Working Concentration | |
| NaOH | 11.3% v/v | |
| DMEM (10X) | 4.7% v/v | |
| F-12 media | 6.9% v/v | |
| NaHCO3 | 0.7% v/v | |
| GlutaMAX | 0.5% v/v | |
| HEPES | 0.9% v/v | |
| PureCol® | 50% v/v |
*Keep solution on ice at all times.
Important Note: Aggregates are embedding in wells of a 12-well plate and each well contains about 40-60 aggregates. Determine the number of wells needed for aggregate embedding in order to calculate the volume of Collagen 1 solution and Matrigel needed.
Prepare Collagen 1-Matrigel (Col-MG) solution.
Recipe for Collagen 1-Matrigel solution*:
| Components | Working Concentration | Example 1 (6.4ml for 6 wells) | Example 2 (13ml for 12 wells) | |
| Collagen 1 solution | 75% | 4.8ml | 9.75ml | |
| Organoid Matrigel®** | 25% | 1.6ml | 3.25ml |
*Keep solution on ice at all times.
**Thaw Organoid Matrigel overnight in 4°c before use.
Embed day 5 aggregates into a Col-MG sandwich.
Prepare the first layer of the Col-MG sandwich by pipetting 500µL of Col-MG solution prepared in step 9 into each well of a 12-well plate.
Place plate into a 37°c incubator and allow Col-MG solution to solidify for 1-1.5h.
Gently transfer day 5 aggregates (Fig. 3) from the ULA 6-well plate to a conical tube. Allow aggregates to settle to the bottom of the tube by gravitation.
Fig. 3 - Example of day 5 vascular aggregates.
Aspirate supernatant and wash aggregates once with 1ml of DMEM/F12 media.
Aspirate supernatant and if needed, use a P200 to carefully remove excess media without disturbing the aggregates.
Add the cold Col-MG solution to the aggregates* and place the tube of aggregate-ECM mixture on ice.
*The volume of Col-MG solution to add depends on the number of wells that will be used for embedding e.g. 2.2 ml of Col-MG will be added if 4 wells will be used (~0.2ml excess is to account for pipetting errors).
Remove plate containing the first layer of the Col-MG sandwich out from the incubator and quickly but carefully transfer 500µL of aggregate-ECM mixture to each of the wells*.
*Shaking the plate after transferring the aggregate-ECM mixture into the wells does not help to distribute aggregates within the wells. Instead do this - dispense aggregate-ECM mixture in a drop-wise manner into the wells.
Immediately place plate back into the 37°c incubator and allow the second layer of Col-MG solution to solidify for 1.5-2h.
Prepare the StemProTM-34 complete media*.
Recipe for StemProTM-34 complete media:
| Components | Working Concentration | |
| StemPro-34 SFM media | - | |
| StemPro-34 Supplement | - | |
| GlutaMAX | 1% | |
| Antibiotic-Antimycotic | 1X |
*StemProTM-34 complete media can be stored at 4°c for up to 2 weeks.
Highly recommended: Pass the media through a 0.2µm filter to remove residual particles and enhance sterility.
Pro tip: If not using the entire 500ml of StemProTM-34 complete media, you may freeze them as smaller aliquots e.g. 40ml in 50ml conical tubes.
Prepare the Blood Vessel Induction (BVI) media*.
Recipe for BVI media:
| Components | Working Concentration | |
| StemPro-34 complete media | - | |
| FBS | 15% | |
| FGF2 | 100ng/ml | |
| VEGFA-165 | 100ng/ml |
*BVI media can be stored at 4°c for up to 2 weeks
Optional: Pass the media through a 0.2µm filter to remove residual particles and enhance sterility.
Once the second layer of Col-MG solution has solidified completely, gently add 1ml of pre-warmed BVI media to each well.
Place plate containing embedding aggregates and BVI media back into the 37°c incubator and leave it overnight.
The next day, small sprouts should be seen forming around each embedded aggregate (Fig. 4).
Fig. 4 - Example of vascular sprout formation 24h after aggregate embedding.
Change with fresh pre-warmed BVI media* once every 2 days until day 10.
*When changing media, aspirate media gently so as not to disturb the delicate Col-MG sandwich. When dispensing fresh media into the wells, do it gently and slowly to prevent accidentally flushing the sandwich out from the well plate.
Pro tip: Avoid aspirating all the media, as this may dislodge the delicate Col-MG sandwich. When adding fresh media, dispense along the wall of the well rather than directly onto the sandwich.
Micro-dissection of Vascular Sprouts (Day 10-12)
Place a light microscope or a dissection microscope into the tissue culture hood*.
*Clean microscope thoroughly with 70% ethanol and if possible, UV-sterilize microscopes in the tissue culture hood before use.
Gently dislodge Col-MG sandwich consisting of vascular sprouts from well plate using a plastic spatula. Transfer sandwich to the lid of a 10cm tissue culture plate, place on the microscope, and micro-dissect out the vascular sprouts* (Fig. 5) using a pair of sterile 27G syringe needles.
*Avoid dissecting out too much excess Col-MG matrix
Fig. 5 - Outline indicating the boundary for microdissection of vascular sprouts.
Prepare a new ULA 6-well plate and add 3ml of fresh BVI media into each well.
Transfer micro-dissected vascular sprouts into the wells of the ULA 6-well plate. Each well can hold up to 40-60 vascular sprouts to prevent over-crowding.
Place plate containing the vascular sprouts on the orbital shaker in a 37°c incubator. Leave dissected vascular sprouts on the shaker* for 2 days.
*While on the shaker, any excess Col-MG attached to the sprouts would be dislodged.
Formation of Vessel Organoids (Day 12-15)
Over the 2-day shaking period, vascular sprouts gradually round up to form vessel organoids.
Transfer individual vessel organoids into each well of a ULA 96-well plate using a wide-bore P1000 pipette tip.
Add 100µL of fresh BVI media each well and place organoid-containing plate back into the 37°c incubator (no shaking).
Change fresh BVI media once every 2 days until day 15. At day 15, vessel organoids should appear round, dense, with smooth borders (Fig. 6). The average size of a typical day 15 VO is about ~1mm.
Fig. 6 - Example of a day 15 vessel organoid.
Troubleshooting
Problem: EBs did not form smooth, round edges.
In the event EBs do not form smooth and round edges (Fig. 7), this is an indication of unsuccessful EB formation. Do not proceed with subsequent steps.
There are numerous factors that could result in unsuccessful EB formation. Some factors include low cell viability during EB formation, low quality of hiPSCs, un-optimized Y-27632 concentration, old aggregation media, and incorrect preparation of aggregation media.
Fig. 7 - Example of a unsuccessful EB formation.
Problem: EBs fell apart/ disintegrated when differentiating in 'MD1' media between days 1 to 3.
Low-quality EBs typically fail to differentiate efficiently. However, if high-quality EBs disintegrate when cultured in MD1 media, possible causes include incorrect preparation of N2B27 media, old N2B27 media, old small molecules (CHIR99021, BMP4), incorrect preparation of CHIR99021, un-optimized CHIR99021 concentration), or orbital shaking rate is too high.
Problem: Day 5 aggregates had an elongated shaped.
We expect Day 5 aggregates to be spherical shaped, with rougher edges compared to day 3 aggregates. However, elongated day 5 aggregates could indicate fusion of aggregates. Undesired aggregate fusion may result from insufficient shaking speed or excessive aggregate density per well.
Problem: Vascular sprouts did not form well.
Vascular sprouts form when vascular progenitors in the day 5 aggregates begin differentiating to form vascular cells such as endothelial cells and pericytes. The Col-MG sandwich provides the extracellular support for the vascular cells to further grow and form vessel networks. Poor vascular sprout formation could point to possible reasons such as inefficient mesoderm differentiation, inefficient vascular differentiation, incorrect preparation of StemPro-34 complete media or BVI media, old media, incorrect preparation of FGF2/ VEGFA-165, poor quality FBS.
Problem: Col-MG sandwich dislodged from wells upon addition of BVI media.
After forming the second layer, the Col-MG sandwich is very delicate. The Col-MG sandwich may dislodge from wells during BVI media addition if the second layer did not solidify completely, the BVI media was not pre-warmed, or the media was added too forcefully.
Problem: Vascular sprouts fused and overlapped too much.
During the course of day 5-10, vascular sprouts will continue to grow and expand. Therefore it is important to ensure that the day 5 aggregates are well-distributed within the wells during the embedding step and the wells are not over-crowded. Excessive fusion or overlap of vascular sprouts may result from poor aggregate distribution or from seeding too many aggregates per well.
FAQs
Q: Should I micro-dissect out vascular sprouts that have merged with one another?
A: It is unavoidable that some aggregates would have been embedded close to one another and so eventually merging as the aggregates grow in size and form vascular sprouts. If the sprouts have merged and overlapped extensively, I would avoid micro-dissecting them to ensure better consistency in vessel organoid size. However if the sprouts overlap slightly, I would still micro-dissect them out as individual sprouts
Q: Do I have to use all 24 wells of the AggreWellTM 400 plate?
A: No. Just use the wells that you need, wash the used wells with sterile DPBS, and seal the plate with parafilm for further use.
Q: How many EBs and therefore aggregates can I make from 1 well of the AggreWellTM 400 (24-well plate)?
A: There are about 1200 microwells in each well of the AggreWellTM 400 (24-well plate), in theory, 1200 EBs can be made from each well. However, some EBs/ aggregates would be lost during the different transfer and media changing steps. Some aggregates may also be lost during mesoderm differentiation.
Protocol references
Wimmer RA, Leopoldi A, Aichinger M, Kerjaschki D, Penninger JM. Generation of blood vessel organoids from
human pluripotent stem cells. Nat Protoc. 2019 Nov;14(11):3082-3100. doi: 10.1038/s41596-019-0213-z. Epub 2019 Sep 25. PMID: 31554955.
Wimmer RA, Leopoldi A, Aichinger M, Wick N, Hantusch B, Novatchkova M, Taubenschmid J, Hämmerle M, Esk C, Bagley JA, Lindenhofer D, Chen G, Boehm M, Agu CA, Yang F, Fu B, Zuber J, Knoblich JA, Kerjaschki D, Penninger JM. Human blood vessel organoids as a model of diabetic vasculopathy. Nature. 2019 Jan;565(7740):505-510. doi: 10.1038/s41586-018-0858-8. Epub 2019 Jan 16. PMID: 30651639; PMCID:
PMC7116578.
Acknowledgements
We acknowledge the original authors and publishers of the hiPSC-derived vessel organoid protocol.