Format

Send to

Choose Destination
Nat Protoc. 2018 Sep;13(9):2102-2119. doi: 10.1038/s41596-018-0036-3.

PEG-4MAL hydrogels for human organoid generation, culture, and in vivo delivery.

Author information

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. spencejr@umich.edu.
8
Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA. spencejr@umich.edu.
9
Center for Organogenesis, University of Michigan Medical School, Ann Arbor, MI, USA. spencejr@umich.edu.
10
Department of Pathology, University of Michigan, Ann Arbor, MI, USA. anusrat@med.umich.edu.
11
Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA, USA. andres.garcia@me.gatech.edu.
12
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA. andres.garcia@me.gatech.edu.

Abstract

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.

PMID:
30190557
DOI:
10.1038/s41596-018-0036-3

Supplemental Content

Full text links

Icon for Nature Publishing Group
Loading ...
Support Center