PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery
- Nat Protoc. 2018 Sep;13(9):2102-2119. doi: 10.1038/s41596-018-0036-3.
- 1. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
- 2. Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA, USA.
- 3. Department of Pathology, University of Michigan, Ann Arbor, MI, USA.
- 4. Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
- 5. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA.
- 6. Center for Organogenesis, University of Michigan Medical School, Ann Arbor, MI, USA.
- 7. Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA. [email protected].
- 8. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA. [email protected].
- 9. Center for Organogenesis, University of Michigan Medical School, Ann Arbor, MI, USA. [email protected].
- 10. Department of Pathology, University of Michigan, Ann Arbor, MI, USA. [email protected].
- 11. Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA, USA. [email protected].
- 12. George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA. [email protected].
- # Contributed equally.
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.