Preparation of Human Primary Colon Tissue‐Derived Organoid Using Air Liquid Interface Culture

Tatsuya Usui1, Masashi Sakurai2, Koji Umata3, Hideyuki Yamawaki4, Takashi Ohama3, Koichi Sato3

1 Laboratory of Veterinary Pharmacology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, 2 Laboratory of Veterinary Pathology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, 3 Laboratory of Veterinary Pharmacology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, 4 Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Aomori
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 22.6
DOI:  10.1002/cptx.40
Online Posting Date:  February, 2018
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In vitro analysis of intestinal epithelium has been hindered by a lack of suitable culture systems useful for gastrointestinal research. To overcome the problem, an air liquid interface (ALI) method using a collagen gel was established to culture three‚Äźdimensional primary cells containing both primary epithelial and mesenchymal components from mouse gastrointestinal tissues. ALI organoids accurately recapitulate organ structures, multilineage differentiation, and physiology. Since ALI organoids from human tissues have not been produced, we modified the previous protocol for mouse ALI organoid culture to establish the culture system of ALI organoids from normal and tumor colorectal tissues of human patients. The current unit presents a protocol for preparation of the ALI organoid culture from normal and tumor colorectal tissues of human patients. ALI organoid culture from human tissues might be useful for examining not only resistance to chemotherapy in a tumor microenvironment but also toxic effects on organoids. ¬© 2018 by John Wiley & Sons, Inc.

Keywords: organoid; human tissue; air liquid interface culture

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Three‐Dimensional Colon Tissue Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of Three‐Dimensional Colon Tissue Culture

  • Collagen cocktail solution (see preparation of collagen gel matrix)
  • Solution A: Cellmatrix I‐A, pH 3.0 (Nitta Gelatin)
  • Solution B: 10× concentrated sterile culture medium (Ham's F‐12, Sigma‐Aldrich, cat. no. D8900)
  • Solution C: Sterile reconstitution buffer (2.2 g NaHCO 3 in 100 ml of 0.05 N NaOH and 200 mM HEPES)
  • Culture medium (see recipe):
  • B‐27 supplement (Thermo Fisher Scientific, cat. no. 17504044)
  • 1 M Nicotinamide (Sigma‐Aldrich, cat. no. N3376)
  • 0.5 M NAcetylLcysteine (Sigma‐Aldrich, cat. no. A7250)
  • EGF (PeproTech, cat. no. AF‐100‐15)
  • A83‐01 (Adooq Bioscience, cat. no. A12358)
  • Noggin (PeproTech, cat. no. 120‐10C)
  • R‐spondin 1 (PeproTech, cat. no. 120‐38)
  • Wnt‐3A (R&D Systems, cat. no. 5036‐WN‐010)
  • Y‐27632 (Cayman, cat. no. 10005583)
  • SB202190 (Cayman, cat. no. 13067)
  • Penicillin‐Streptomycin (Thermo Fisher Scientific, cat. no. 15140122)
  • 10 mM HEPES
  • GlutaMax (Thermo Fisher Scientific, cat. no. 35050061)
  • Primocin (Nacalai tesque, cat. no. 14860)
  • Advanced Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific, cat. no. 12634)
  • Tissue preservation buffer (see recipe)
  • Ice
  • HEPES buffered saline (HBS; alternatively, it is possible to use phosphate buffered saline.)
  • Human colon tissue piece (normal or malignant)
  • Collagenase IV (Worthington, cat. no. LS004210)
  • 37°C water bath
  • 6‐cm culture dish
  • 10‐cm culture dish
  • 35‐mm insert well (PICM03050, Millicell‐CM, Millipore)
  • 37°C, 5% CO 2 incubator
  • Small sterile scissor
  • 15‐ml tube
  • Sterile metal mesh (>100 μm)
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Literature Cited

Literature Cited
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