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INTESTINAL Publication-derived

Small Intestinal Bioprinted and Engineered Tissues for Disease Modelling

Full SOP with Academic or a plan

Source Schlieve et al., 2017 · The Saban Research Institute at Children's Hospital Los Angeles · 10.1016/j.stemcr.2017.07.017

Authors: Christopher R. Schlieve, Kathryn L. Fowler, Matthew Thornton, Sha Huang, Ibrahim Hajjali, Xiaogang Hou, Brendan Grubbs, Jason R. Spence, Tracy C. Grikscheit Duration: 105 days 9 phases Cell source: Human iPSC, Human ESC

Abstract

This protocol describes the derivation of human pluripotent stem cell (hPSC)-derived enteric neural crest cells (ENCCs) and their co-implantation with human intestinal organoids (HIOs) to generate tissue-engineered small intestine (ENCC-HIO-TESI) with a functional enteric nervous system. The co-implantation approach establishes mature ganglia, diverse neuronal subtypes, and neuroepithelial connections in vivo, restoring neuron-dependent contractility for potential therapeutic treatment of enteric neuropathies.

Cell source
Human iPSC, Human ESC
Application
Disease modeling; Tissue engineering of functional small intestine with enteric nervous system restoration

Protocol overview

45 steps across 9 phases

Phase 1 · hPSC Maintenance and HIO Generation Days 0-35
  1. a Maintain hPSCs on Matrigel substrate
  2. b Direct differentiate hPSCs into intestinal organoids (HIOs)
Phase 2 · ENCC Derivation and Neurosphere Formation Days 0-15
  1. a Derive ENCCs from unmodified hPSCs
  2. b Plate unsorted ENCCs into ultra-low attachment plates
  3. c Culture ENCCs in neurosphere medium for 4 days
Phase 3 · Scaffold Seeding and Surgical Implantation Day 15
  1. a Prepare biodegradable scaffolds
  2. b Seed scaffolds with HIOs and ENCC neurospheres
  3. c Implant constructs into NOD/SCID mice omentum
Phase 4 · In Vivo Maturation Days 15-105 (3 months)
  1. a Allow implants to mature in vivo
  2. b Perform surgical explantation
Phase 5 · Tissue Fixation and Histological Processing Day 105 (post-explantation)
  1. a Fix implants in buffered formalin
  2. b Embed fixed tissues in paraffin
  3. c Section paraffin blocks
  4. d Perform H&E staining
Phase 6 · Immunofluorescence Analysis Post-fixation
  1. a Deparaffinize and rehydrate tissue sections
  2. b Perform antigen retrieval
  3. c Block non-specific binding
  4. d Apply primary antibodies
  5. e Wash and apply secondary antibodies
  6. f Mount and image sections
  7. g Quantify immunostaining
Phase 7 · Light-Sheet and Confocal Microscopy Analysis Post-fixation
  1. a Prepare tissue for light-sheet microscopy
  2. b Freeze-thaw cycle for permeabilization
  3. c Rehydrate and block tissue
  4. d Incubate with primary antibodies
  5. e Apply secondary antibodies
  6. f Clear tissue
  7. g Perform light-sheet microscopy
  8. h Perform confocal microscopy
Phase 8 · Live Video Imaging of Mechanical Contractility Post-explantation (same day preferred)
  1. a Equilibrate explants in imaging medium
  2. b Baseline contractility imaging
  3. c Test ICC-dependent contractility with methylene blue
  4. d Test neuron-dependent contractility with tetrodotoxin (TTX)
  5. e Image control explants without pharmacological treatment
  6. f Process and analyze videos
Phase 9 · RNA-Seq and Transcriptome Analysis Post-explantation
  1. a Extract total RNA from tissue samples
  2. b Quantify RNA concentration
  3. c Prepare cDNA libraries for RNA-seq
  4. d Perform deep sequencing
  5. e Align reads to human genome reference
  6. f Normalize data and remove batch effects
  7. g Identify differentially expressed genes
  8. h Perform gene ontology and pathway analysis
  9. i Analyze epithelial cell type genes
  10. j Deposit data in public repository

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Attribution

This SOP was written by Organthis based on the published method in Schlieve et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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