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PANCREAS Publication-derived

Pancreatic Organoids for Regenerative Medicine

Full SOP with Academic or a plan

Source Loomans et al., 2018 · Hubrecht Institute/KNAW and University Medical Center Utrecht · 10.1016/j.stemcr.2018.02.005

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: 140 days 6 phases Cell source: Human adult pancreatic ductal tissue (islet-depleted exocrine tissue)

Abstract

This protocol describes a 3D Matrigel-based organoid culture system that enables long-term expansion of adult human pancreatic tissue into budding organoid structures. The organoids contain ALDH-high progenitor cells that express pancreatic markers (PDX1, PTF1A, CPA1, MYC) and can differentiate toward insulin-producing cells both in vitro and in vivo after transplantation under the kidney capsule of immunodeficient mice.

Cell source
Human adult pancreatic ductal tissue (islet-depleted exocrine tissue)
Application
Disease modeling and regenerative medicine for diabetes; expansion of pancreatic progenitor cells with endocrine differentiation potential

Protocol overview

31 steps across 6 phases

Phase 1 · Tissue Preparation and Organoid Initiation Day 0-1
  1. a Obtain islet-depleted pancreatic tissue
  2. b Mechanically dissociate tissue into small clumps
  3. c Embed tissue clumps in Matrigel
Phase 2 · Organoid Expansion (Passage 0-10) Day 1-140+
  1. a Add expansion medium
  2. b Culture organoids in standard incubator
  3. c Monitor organoid morphology and budding
  4. d Refresh expansion medium every 2-3 days
  5. e Passage organoids every 7-14 days
  6. f Document passage number and growth rate
  7. g Maintain cultures for at least 10 passages
Phase 3 · Progenitor Cell Identification and Sorting (Day 7 Expansion Organoids) Day 7 (P0)
  1. a Disperse organoids into single cells
  2. b Label cells with Aldefluor reagent
  3. c Prepare FACS gates using DEAB inhibitor control
  4. d Perform FACS sorting
  5. e Plate sorted cells in Matrigel for colony-forming assay
  6. f Count colonies and calculate colony-forming efficiency
Phase 4 · In Vitro Differentiation toward Endocrine Lineage Day 8-18 (following 7-day expansion)
  1. a Retrieve organoids from expansion culture
  2. b Transfer organoids to low-attachment differentiation plates
  3. c Prepare differentiation medium
  4. d Culture organoids in differentiation medium for 7-18 days
  5. e Monitor endocrine marker expression
Phase 5 · In Vivo Differentiation via Transplantation under Kidney Capsule Day 15-30 (post-transplant)
  1. a Prepare organoids for transplantation
  2. b Prepare immunodeficient mice
  3. c Transplant organoids under kidney capsule
  4. d Monitor blood glucose and graft function post-transplant
  5. e Harvest grafts and assess endocrine differentiation
  6. f Expected outcome at 1 month post-transplant
Phase 6 · Cryopreservation and Recovery of Pancreatic Tissue (Optional) Day 0 or any passage
  1. a Prepare tissue for cryopreservation
  2. b Controlled freezing to –80°C
  3. c Thaw and recover cryopreserved tissue
  4. d Validate cryopreserved tissue functionality

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Attribution

This SOP was written by Organthis based on the published method in Loomans et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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