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PANCREAS Publication-derived

Pancreatic Organoids for Disease Modelling

Full SOP with Academic or a plan

Source Loomans et al. · Leiden University Medical Center

Authors: Cindy J.M. Loomans, Nerys Williams Giuliani, Jeetindra Balak, Femke Ringnalda, Léon van Gurp, Meritxell Huch, Sylvia F. Boj, Toshiro Sato, Lennart Kester, Susana M. Chuva de Sousa Lopes, Matthias S. Roost, Susan Bonner-Weir, Marten A. Engelse, Ton J. Rabelink, Harry Heimberg, Robert G.J. Vries, Alexander van Oudenaarden, Françoise Carlotti, Hans Clevers, Eelco J.P. de Koning Duration: 30 days 14 phases Cell source: Human adult pancreatic exocrine tissue (islet-depleted)

Abstract

This protocol describes the expansion of adult human pancreatic organoids from islet-depleted tissue using an ENR-based (EGF/Noggin/R-spondin) medium, yielding organoids enriched for ALDH+ progenitor cells capable of endocrine differentiation. The method includes in vitro differentiation and in vivo transplantation under the kidney capsule to characterize insulin-producing capacity and endocrine maturation.

Cell source
Human adult pancreatic exocrine tissue (islet-depleted)
Application
Disease modeling and developmental characterization of pancreatic progenitor organoids

Protocol overview

53 steps across 14 phases

Phase 1 · Pancreatic Tissue Preparation and Initial Culture Day 0-1
  1. a Obtain islet-depleted pancreatic tissue
  2. b Mechanically dissociate islet-depleted tissue into small clumps
  3. c Plate tissue clumps in Matrigel
Phase 2 · Organoid Expansion Culture Day 1-14+ (passages every 7-14 days)
  1. a Add Expansion Medium to organoid culture
  2. b Refresh expansion culture medium
  3. c Passage organoids
Phase 3 · Cryopreservation of Pancreatic Tissue and Organoids As needed (before passage or for storage)
  1. a Cryopreserve adult pancreatic tissue
  2. b Cryopreserve fetal pancreatic tissue
  3. c Thaw cryopreserved fetal pancreatic tissue
  4. d Mince thawed or fresh fetal pancreatic tissue
Phase 4 · Single-Cell Expansion and Clonal Analysis Day 7-14 (from single cell)
  1. a Dissociate expanded organoids to single cells
  2. b Filter single-cell suspension
  3. c Plate single cells in Matrigel with Rock inhibitor
Phase 5 · Aldefluor Labeling and FACS-Based Cell Sorting Day 7 (organoids ready for sorting)
  1. a Label organoids with Aldefluor™ fluorescent reagent
  2. b Image organoids with Aldefluor label
  3. c Dissociate Aldefluor-labeled organoids to single cells
  4. d Prepare single cells for FACS
  5. e Set FACS gates and sort ALDH populations
Phase 6 · Colony-Forming Unit (CFU) Assay Day 0-7 (post-sorting)
  1. a Plate sorted ALDH-positive and ALDH-negative cells in dilution series
  2. b Culture sorted cells with Rock inhibitor
  3. c Count organoid-forming units
Phase 7 · In Vitro Differentiation of Pancreatic Organoids Day 0-7 (differentiation phase)
  1. a Prepare differentiation medium
  2. b Transfer organoids to differentiation medium
Phase 8 · In Vivo Differentiation via Kidney Capsule Transplantation Day 0-30 (post-transplantation)
  1. a Prepare immunodeficient mice for transplantation
  2. b Optionally induce hyperglycemia in recipient mice
  3. c Prepare organoids for transplantation
  4. d Transplant organoids under the kidney capsule
  5. e Monitor recipient mice and collect grafts
Phase 9 · Cell Growth Kinetics Analysis Days 1, 7, 14, 21, 28, etc. (at each passage)
  1. a Dissociate organoids and count cells
Phase 10 · Gene Expression Analysis via qPCR Post-expansion or post-differentiation
  1. a Extract total RNA from organoids
  2. b Synthesize complementary DNA (cDNA)
  3. c Perform real-time quantitative PCR (qPCR)
Phase 11 · Immunohistochemical Staining and Imaging Post-expansion, post-differentiation, or post-transplantation
  1. a Fix organoids or tissue grafts
  2. b Embed tissue in paraffin
  3. c Section tissue and incubate with primary antibodies
  4. d Incubate with secondary antibodies
  5. e Capture and process fluorescent images
  6. f Perform quantitative image analysis
  7. g Capture brightfield images
Phase 12 · Single-Molecule Fluorescence In Situ Hybridization (smFISH) Post-expansion (e.g., day 7)
  1. a Retrieve organoids from Matrigel
  2. b Wash and fix organoids
  3. c Freeze organoids in OCT compound
  4. d Section frozen samples and perform smFISH
Phase 13 · Human C-Peptide Measurements in Transplanted Mice Post-transplantation (at time of blood collection)
  1. a Perform glucose challenge in normoglycemic mice
  2. b Collect blood from mice
  3. c Prepare plasma samples
  4. d Measure human C-peptide by ELISA
Phase 14 · RNA Sequencing and Bioinformatics Analysis Post-expansion or post-differentiation
  1. a Prepare RNA for sequencing
  2. b Perform CEL-Seq library preparation
  3. c Perform sequencing
  4. d Align sequencing reads to transcriptome
  5. e Normalize and filter sequencing data
  6. f Generate correlations and perform statistical analysis

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Attribution

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

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