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KIDNEY Publication-derived

Kidney Organ-on-Chip Models for Drug Screening

Full SOP with Academic or a plan

Source Schutgens et al., 2019 · Hubrecht Institute—Royal Netherlands Academy of Arts and Sciences, Utrecht, the Netherlands; Department of Nephrology and Hypertension, University Medical Centre Utrecht, Utrecht, the Netherlands · 10.1038/s41587-019-0048-8

Authors: Frans Schutgens, Maarten B Rookmaaker, Thanasis Margaritis, Anne Rios, Carola Ammerlaan, Jitske Jansen, Linda Gijzen, Marianne Vormann, Annelotte Vonk, Marco Viveen, Fjodor Yousef Yengej, Sepide Derakhshan, Karin M. de Winter-de Groot, Benedetta Artegiani, Ruben van Boxtel, Edwin Cuppen, Antoni P. A. Hendrickx, Marry M. van den Heuvel-Eibrink, Ellen Heitzer, Henriette Lanz, Jeffrey Beekman, Jean-Luc Murk, Rosalinde Masereeuw, Frank Holstege, Jarno Drost, Marianne C Verhaar, Hans Clevers Duration: 30 days 9 phases Cell source: Human Adult Kidney Tissue, Human Urine

Abstract

This protocol describes the long-term culture of primary kidney tubular epithelial organoids ('tubuloids') from human adult kidney tissue and urine samples. The tubuloids can be expanded for at least 20 passages while maintaining chromosomal stability and represent multiple nephron segments (proximal tubule, loop of Henle, distal tubule, collecting duct). These cultures are applied to model infectious, malignant, and hereditary kidney diseases including BK virus infection, Wilms tumor, and cystic fibrosis.

Cell source
Human Adult Kidney Tissue, Human Urine
Application
Disease modeling, Drug screening, Personalized medicine

Protocol overview

38 steps across 9 phases

Phase 1 · Tissue Preparation and Tubuloid Initiation Day 0-6
  1. a Tissue digestion with collagenase
  2. b Resuspend tubular fragments in Matrigel
  3. c Prepare tubuloid culture medium
  4. d Add culture medium and incubate
Phase 2 · Tubuloid Expansion and Passage Day 7-ongoing (minimum 20 passages)
  1. a Prepare tubuloids for passaging
  2. b Enzymatic dissociation of tubuloids
  3. c Centrifuge and resuspend cells
  4. d Seed tubuloids at split ratio
Phase 3 · Tubuloid Culture from Urine Day 0-ongoing (10-15 passages)
  1. a Collect and process urine sample
  2. b First centrifugation and washing
  3. c Second centrifugation and Matrigel embedding
  4. d Culture in expansion medium
Phase 4 · BK Virus Infection (Disease Modeling) Day 0-30
  1. a Prepare tubuloids for viral infection
  2. b Expose tubuloids to BK virus
  3. c Wash and plate infected tubuloids
  4. d Monitor viral infection and collect samples
  5. e Test viral infectivity (optional)
Phase 5 · Antiviral Treatment with Cidofovir (Drug Efficacy Testing) Day 0-7
  1. a Prepare BK virus-infected tubuloids
  2. b Add cidofovir (CDV) at varied concentrations
  3. c Refresh culture medium with CDV
  4. d Harvest and quantify viral copies
Phase 6 · Tumoroid Culture from Wilms Tumor Tissue Day 0-14 per passage
  1. a Digest tumor tissue with collagenase
  2. b Resuspend tumor fragments in Matrigel
  3. c Add tumoroid culture medium
  4. d Passage tumoroids at altered split ratio
Phase 7 · Cystic Fibrosis Drug Response Assay (Forskolin Swelling) Day 0-3
  1. a Prepare CF tubuloids for swelling assay
  2. b Pre-incubate tubuloids with CFTR-restoring drug (optional)
  3. c Add forskolin (FSK) at indicated concentrations
  4. d Quantify swelling response
Phase 8 · Tubuloids-on-a-Chip (Organ-on-Chip Culture) Day 0-7
  1. a Prepare OrganoPlate microfluidic chips
  2. b Seed tubuloid-derived cells
  3. c Allow cell attachment in static culture
  4. d Initiate medium perfusion
  5. e Confirm tube formation and polarization
Phase 9 · Functional Assays on Organ-on-Chip: Barrier Integrity and Transporter Activity Day 7 onwards
  1. a Barrier integrity assay with FITC-dextran
  2. b P-glycoprotein (P-gp) transporter assay
  3. c Trans-epithelial transport assay (rhodamine 123)
  4. d Calculate apparent permeability

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Attribution

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

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