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INTESTINAL Publication-derived

Colorectal Adenocarcinoma Intestinal Bioprinted and Engineered Tissues

Full SOP with Academic or a plan

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

Authors: Justine Creff, Rémi Courson, Thomas Mangeat, Julie Foncy, Sandrine Souleille, C. Thibault, Arnaud Besson, Laurent Malaquin Duration: 21 days 5 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Creff et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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