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KIDNEY Publication-derived

Kidney Spheroids for Drug Screening

Full SOP with Academic or a plan

Source Freedman et al., 2015 · Brigham and Women's Hospital, Harvard Medical School · 10.1038/ncomms9715

Authors: Benjamin S. Freedman, Craig R. Brooks, Albert Q. Lam, Hongxia Fu, Ryuji Morizane, Vishesh Agrawal, Abdelaziz F. Saad, Michelle K. Li, Michael R. Hughes, Ryan Vander Werff, Derek T. Peters, Junjie Lu, Anna Baccei, Andrew M. Siedlecki, M. Todd Valerius, Kiran Musunuru, Kelly M. McNagny, Theodore I. Steinman, Jing Zhou, Paul H. Lerou, Joseph V. Bonventre 8 phases Cell source: Human iPSC, Human ESC

Abstract

This protocol describes the generation of three-dimensional kidney organoids from human pluripotent stem cells (hPSCs) by first forming cavitated epiblast spheroids in Matrigel sandwich cultures, followed by differentiation into segmented, nephron-like structures containing proximal tubules, podocytes, and endothelial cells. The method enables tissue-specific phenotyping including nephrotoxicity responses and disease modeling via CRISPR/Cas9 genome editing.

Cell source
Human iPSC, Human ESC
Application
Disease modeling, Drug screening

Protocol overview

38 steps across 8 phases

Phase 1 · hPSC Maintenance and Preparation d-7 to d0
  1. a Maintain hPSCs on feeder-free conditions
  2. b Dissociate and prepare hPSCs for 3D culture
Phase 2 · Epiblast Spheroid Formation d0 to d3
  1. a Prepare GelTrex-coated wells
  2. b Plate dissociated hPSCs
  3. c Apply thin GelTrex sandwich overlay
  4. d Change media to remove Y27632
  5. e Monitor cavity formation
Phase 3 · Kidney Organoid Differentiation - GSK3β Inhibition d3 to d10
  1. a Prepare spheroid cultures for differentiation
  2. b Apply CHIR99021 GSK3β inhibition
  3. c Change to B27-supplemented medium (without CHIR99021)
  4. d Continue RB medium culture with media changes
Phase 4 · Kidney Organoid Maturation and Characterization d10 to d21+
  1. a Observe tubular organoid formation
  2. b Maintain long-term culture
  3. c Perform immunofluorescence characterization (optional milestone)
Phase 5 · Immunofluorescence Staining and Microscopy Variable (fixed timepoints)
  1. a Fix organoids while preserving 3D architecture
  2. b Wash fixed cultures
  3. c Permeabilization and blocking
  4. d Incubate with primary antibodies
  5. e Wash to remove unbound primary antibody
  6. f Incubate with secondary antibodies and DAPI
  7. g Wash to remove unbound secondary antibody
  8. h Mount and image
Phase 6 · Functional Assays - Transport and Toxicity (Optional) d14 to d21
  1. a Rhodamine-dextran (RD) uptake assay
  2. b Lucifer yellow (LY) uptake assay
  3. c Methotrexate (MTX) uptake assay
  4. d Wash-out and chase experiments
  5. e Nephrotoxicity assessment - Cisplatin or Gentamicin treatment
  6. f KIM-1 immunofluorescence
Phase 7 · CRISPR/Cas9 Genome Editing (Optional - Disease Modeling) d-14 to d-1 (pre-culture; prior to differentiation)
  1. a Prepare CRISPR/Cas9 constructs
  2. b Transfect Cas9 and guide RNA into hPSCs
  3. c Flow cytometric enrichment of Cas9-expressing cells
  4. d Clonal expansion and screening
  5. e Validate knockout clones
Phase 8 · Disease Modeling - PKD Cystogenesis (PKD1/PKD2 Knockouts) d0 to d58+
  1. a Perform spheroid and organoid differentiation on PKD knockout lines
  2. b Monitor for cyst formation
  3. c Characterize cysts morphologically
  4. d Fix and characterize cysts by immunofluorescence
  5. e Quantify cyst formation frequency

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Attribution

This SOP was written by Organthis based on the published method in Freedman et al., 2015. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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