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INTESTINAL Publication-derived

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

Source Cruz-Acuña et al., 2018 · Georgia Institute of Technology; University of Michigan · 10.1038/s41596-018-0036-3

👤 Ricardo Cruz-Acuña, Miguel Quirós, Sha Huang, Dorothée Siuda, Jason R. Spence, Asma Nusrat, Andrés J. García ⏱ 21 days 📋 5 phases 🧫 Human iPSC, Human ESC

Abstract

This protocol describes the synthesis and use of fully defined PEG-4MAL synthetic hydrogels to generate, culture, and deliver human pluripotent stem cell-derived organoids. The modular hydrogels replace Matrigel, offering tunable biochemical and biophysical properties to support robust in vitro organoid development and in vivo transplantation with minimal immune response.

Cell source
Human iPSC, Human ESC
Application
Organoid generation and culture; in vivo delivery to intestinal wounds

Protocol overview

18 steps across 5 phases

Preparation of hydrogel precursor solutions Day 0 (1-2 h before encapsulation)
  1. 1 Equilibrate hydrogel components to room temperature
  2. 2 Weigh hydrogel precursors
  3. 3 Dissolve peptides in HEPES buffer
  4. 4 Filter peptide and buffer solutions
  5. 5 Dissolve PEG-4MAL macromer
  6. 6 Generate functionalized PEG-4MAL macromer and incubate
Preparation of biological suspensions Day 0 (concurrent with Phase 1)
  1. 7 Prepare spheroid or organoid suspension
Hydrogel casting and organoid encapsulation Day 0 (2.5–3.5 h total)
  1. 8 Mix functionalized PEG-4MAL with biological suspension
  2. 9 Dispense cross-linker into culture wells
  3. 10 Pipette functionalized PEG-4MAL and cell mixture into cross-linker
  4. 11 Incubate hydrogels for cross-linking
  5. 12 Overlay hydrogels with culture medium
Organoid expansion and passaging Days 1–21+ (passaging every 7–10 d)
  1. 13 Monitor organoid growth and passage when ready
In vivo delivery to colonic mucosal wounds Day 0 (Day -1 for wounding; ~5 h for delivery on Day 1)
  1. 13 Induce colonic mucosal wounds (Day -1)
  2. 14 Prepare organoids for injection (Day 0, concurrent with wounding day +1 h)
  3. 15 Assemble custom injection device and load solutions
  4. 16 Mix organoids with functionalized PEG-4MAL and load syringe
  5. 17 Perform colonoscopic injection into wound

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Cruz-Acuña et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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