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INTESTINAL Publication-derived

Fabrication of 3D scaffolds reproducing intestinal epithelium topography by high-resolution 3D stereolithography

Source Creff et al., 2019 · LAAS-CNRS, Université de Toulouse, CNRS · 10.1016/j.biomaterials.2019.119404

👤 Justine Creff, Rémi Courson, Thomas Mangeat, Julie Foncy, Sandrine Souleille, C. Thibault, Arnaud Besson, Laurent Malaquin ⏱ 21 days 📋 5 phases 🧫 Caco-2 (human colorectal adenocarcinoma cells)

Abstract

This protocol describes the development of a photopolymerizable PEG-DA/acrylic acid hydrogel combined with high-resolution stereolithography to fabricate 3D scaffolds that reproduce the crypt-villus architecture of the mouse intestinal epithelium. The 5 μm resolution printing enables accurate replication of intestinal topography and supports the culture and differentiation of Caco-2 intestinal epithelial cells for up to 3 weeks.

Cell source
Caco-2 (human colorectal adenocarcinoma cells)
Application
Disease modeling and developmental study of intestinal epithelium

Protocol overview

25 steps across 5 phases

Hydrogel Preparation Before printing
  1. 1 Dissolve photoinitiator
  2. 2 Mix hydrogel components
  3. 3 Perform UV crosslinking
  4. 4 Wash polymerized hydrogels
3D Printing of Intestinal Scaffold Printing day
  1. 1 Prepare STL file and slicing parameters
  2. 2 Prepare substrate for adhesion
  3. 3 Print pedestal for strong adhesion
  4. 4 Print scaffold structure
  5. 5 Develop printed scaffold
  6. 6 Sterilize and wash scaffold
Cell Seeding and Culture Days 0-21
  1. 1 Prepare culture medium for Caco-2 cells
  2. 2 Seed cells on 3D scaffolds
  3. 3 Culture cells in incubator
  4. 4 Change culture medium
Immunofluorescence Analysis At harvest (day 6 or 21)
  1. 1 Fix cells with PFA
  2. 2 Permeabilize cells
  3. 3 Wash and prepare for staining
  4. 4 Apply primary antibodies
  5. 5 Apply phalloidin (alternative to primary antibodies)
  6. 6 Wash after primary antibodies
  7. 7 Apply secondary antibodies
  8. 8 Nuclear staining and final washes
Alkaline Phosphatase Activity Assay At harvest (day 6 or 21)
  1. 1 Rinse cells with PBS
  2. 2 Incubate with Blue AP substrate
  3. 3 Rinse and fix

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Creff et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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