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PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery
Open accessAbstract
In vitro differentiation of human pluripotent stem cell (hPSC)-derived organoids (HOs) facilitates the production of multicellular three-dimensional structures analogous to native human tissues. Most current methods for the generation of HOs rely on Matrigel, a poorly defined basement membrane derivative secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, limiting the potential use of HOs for regenerative medicine applications. Here, we describe a protocol for the synthesis of a fully defined, synthetic hydrogel that supports the generation and culture of HOs. Modular, cell-encapsulating hydrogels are formed from a four-armed poly(ethylene glycol) macromer that has maleimide groups at each terminus (PEG-4MAL) and is conjugated to cysteine-containing adhesive peptides and cross-linked via protease-degradable peptides. The protocol also includes guidelines for the localized in vivo delivery of PEG-4MAL hydrogel-encapsulated HOs to injured mouse colon. The PEG-4MAL hydrogel supports the engraftment of the HOs and accelerates colonic wound repair. This culture and delivery strategy can thus be used to develop HO-based therapies to treat injury and disease. Hydrogel and tissue preparation and subsequent encapsulation can be performed within 2.5-3.5 h. Once HOs have been cultured in synthetic hydrogels for at least 14 d, they can be prepared and delivered to the mouse colon in under 5 h.
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“Intriguingly, when PEG was crosslinked with dithiothreitol to inhibit matrix degradation, organoid viability at seven days was poor as measured by live-dead staining.”
From Towards organoid culture without Matrigel (2021) · cites this paper as [164]
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“Organoid viability was reduced at high PEG density and matrix stiffness;”
From Towards organoid culture without Matrigel (2021) · cites this paper as [164]
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“RGD- or AG73 (CGGRKRLQVQLSIRT)-functionalized PEG matrices promoted greater organoid viability than laminin-derived IKVAV- or type I collagen-derived GFOGER peptides163,164.”
From Towards organoid culture without Matrigel (2021) · cites this paper as [164]
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“Cruz-Acuna and colleagues also found that PEG-based materials may need to be soft and degradable in order to support differentiated intestinal organoids.”
From Towards organoid culture without Matrigel (2021) · cites this paper as [164]
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Self-citation
Metadata
- Authors
- Cruz-Acuña, R., Quirós, M., Huang, S., Siuda, D., Spence, J. R., Nusrat, A., & García, A. J.
- Affiliations
- University of Michigan (US)
- Corresponding author
- Spence, Jason R.
- Nusrat, Asma
- Venue
- Nature Protocols
- Published
- 2018-09
- Volume
- 13
- Issue
- 9
- Pages
- 2102-2119
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC7240347
- OpenAlex
- W2892378739
- Cited by
- 168
- References
- 30
- Citations by year
- 2026: 16 · 2025: 26 · 2024: 16 · 2023: 24 · 2022: 26 · 2021: 30 · 2020: 16 · 2019: 14
- FWCI
- 5.67
- Type
- journal article
- Language
- en
- Update notices
- correction (2018-10-24)
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (UL1TR000454)
- National Heart, Lung, and Blood Institute (R01HL119215)
- National Institute of Allergy and Infectious Diseases (U19AI116482)
- National Institute of Diabetes and Digestive and Kidney Diseases (U24DK085532, P30DK034933, R01DK055679, R56DK089763, R29DK055679, R01DK059888, U01DK085532, U01DK103141, R01DK089763)
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR062368, R01AR062920)
- National Center for Advancing Translational Sciences
- Topics
- Physical Sciences › Materials Science › Biomaterials › Hydrogels: synthesis, properties, applications
- Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Keywords
- human organoids, pluripotent stem cell differentiation, synthetic hydrogels, hydrogel encapsulation, in vivo delivery, tissue regeneration
- MeSH terms
- Animals, Colon, Humans, Organoids, Organ Transplantation, Hydrogels, Models, Animal, Pluripotent Stem Cells, Tissue Culture Techniques, Mice
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