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KIDNEY Publication-derived

Flow-enhanced vascularization and maturation of kidney organoids in vitro

Source Homan et al., 2019 · Wyss Institute for Biologically Inspired Engineering, Harvard University; Renal Division, Brigham and Women's Hospital · 10.1038/s41592-019-0325-y

👤 Kimberly A. Homan, Navin Gupta, Katharina T. Kroll, David B. Kolesky, Mark Skylar-Scott, Tomoya Miyoshi, Donald Mau, M. Todd Valerius, Thomas Ferrante, Joseph V. Bonventre, Jennifer A. Lewis, Ryuji Morizane ⏱ 21 days 📋 5 phases 🧫 Human iPSC (BJFF line) and Human ESC (H9)

Abstract

This protocol describes an in vitro method for culturing kidney organoids derived from human pluripotent stem cells on millifluidic chips under controlled fluidic shear stress (FSS). The system greatly expands endogenous endothelial progenitor cell pools and generates vascularized kidney organoids with perfusable lumens, mature podocytes, and tubular compartments, mimicking early-stage embryonic kidney development.

Cell source
Human iPSC (BJFF line) and Human ESC (H9)
Application
Disease modeling, developmental study

Protocol overview

26 steps across 5 phases

Millifluidic chip fabrication and ECM preparation Before organoid seeding
  1. 1 Prepare silicone ink for 3D printing
  2. 2 3D print perfusion chip gasket
  3. 3 Cure and sterilize printed chip
  4. 4 Prepare gelatin solution for ECM
  5. 5 Prepare fibrinogen solution for ECM
  6. 6 Prepare transglutaminase (TG) solution for ECM
  7. 7 Prepare CaCl₂ and thrombin stock solutions for ECM
  8. 8 Mix ECM components before gelation
  9. 9 Rapidly mix ECM with thrombin and cast on chip
  10. 10 Incubate chip with cured ECM
hPSC culture and organoid preparation Days 0–14
  1. 1 Maintain hPSCs in feeder-free culture
  2. 2 Differentiate hPSCs into metanephric mesenchyme
  3. 3 Form pretubular aggregates in suspension culture
Organoid seeding and chip assembly Day 14 (chip seeding)
  1. 1 Seed pretubular aggregates onto ECM on chip
  2. 2 Assemble chip housing and tubing
  3. 3 Connect perfusion circuit and fill chip with media
  4. 4 Equilibrate media reservoir and initiate low-flow perfusion
Flow perfusion culture and organoid maturation Days 14–21 (chip culture)
  1. 1 Transition to high fluidic shear stress (FSS) perfusion
  2. 2 Change media every 2–3 days
  3. 3 Continue high-flow culture through Day 21
Analysis and assessment (Day 21) Day 21
  1. 1 Prepare organoids for immunostaining
  2. 2 Perform immunostaining with primary antibodies
  3. 3 Perform secondary antibody staining and counterstaining
  4. 4 Image organoids using confocal microscopy
  5. 5 Analyze vasculature using Angiotool
  6. 6 Quantify tubule-vascular associations using Imaris

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Homan et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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