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CARDIAC Publication-derived

Scalable production of homogeneous cardiac organoids derived from human pluripotent stem cells

Source Moriwaki et al., 2023 · Department of Cardiology, Keio University School of Medicine · 10.1016/j.crmeth.2023.100666

👤 Taijun Moriwaki, Hidenori Tani, Kotaro Haga, Yuika Morita-Umei, Yusuke Soma, Tomohiko C. Umei, Otoya Sekine, Kaworu Takatsuna, Yoshikazu Kishino, Hideaki Kanazawa, Jun Fujita, Keiichi Fukuda, Shugo Tohyama, Masaki Ieda ⏱ 10 days 📋 10 phases 🧫 Human iPSC

Abstract

This protocol describes a scalable 'suction method' for producing large quantities of highly homogeneous hiPSC-derived cardiac spheroids and cardiac organoids using a porous ceramic substratum. The method enables production of approximately 40,000 homogeneous spheroids at once and facilitates fabrication of fused organoids with localized atrial and ventricular tissue, applicable to drug discovery and cardiac regenerative therapy.

Cell source
Human iPSC
Application
Disease modeling, Drug screening, Regenerative therapy

Protocol overview

49 steps across 10 phases

Differentiation of hiPSCs to Cardiomyocytes Days 0-10
  1. 1 Prepare Matrigel-coated culture plates
  2. 2 Seed hiPSCs on coated plates
  3. 3 Day 0 - Initiate cardiac differentiation
  4. 4 Day 1 - First medium change
  5. 5 Day 3 - Second differentiation phase
  6. 6 Day 6 - Replace with maturation medium
  7. 7 Day 7 - Switch to MEMα medium
  8. 8 Day 10 - Harvest and plate cardiomyocytes
  9. 9 Metabolic purification of cardiomyocytes
  10. 10 Harvest and freeze purified cardiomyocytes
Assembly of Suction Device Pre-experiment setup
  1. 1 Assemble suction apparatus
  2. 2 Prepare vacuum vessel
Suction Method for Spheroid Production (Small-Scale Substratum) Days 0-2
  1. 1 Prepare ceramic substratum
  2. 2 Prepare cell suspension
  3. 3 Seed cells on substratum using suction method
  4. 4 Transfer substratum to culture plates
  5. 5 Culture spheroids for 2 days
Suction Method for Large-Scale Spheroid Production Days 0-2
  1. 1 Prepare large-scale ceramic substratum
  2. 2 Prepare large-volume hiPSC-CM suspension
  3. 3 Apply suction to large-scale substratum
  4. 4 Transfer to culture vessel and incubate
  5. 5 Culture for 2 days
Production of Cardiac Organoids with Mixed Cell Types Days 0-2
  1. 1 Prepare mixed cell suspension for organoids
  2. 2 Seed cells on substratum using suction method
  3. 3 Transfer and incubate organoids
  4. 4 Culture organoids for 2 days
Production of Fused Cardiac Organoids with Localized Atrial and Ventricular Tissue Days 0-2
  1. 1 Prepare atrial and ventricular cardiomyocytes
  2. 2 First aspiration: seed atrial cardiomyocytes
  3. 3 Second aspiration: add ventricular cardiomyocytes
  4. 4 Transfer fused organoids to culture plates
  5. 5 Culture and verify organoid composition
Functional Assessment - Beating Rate Analysis Day 2-3
  1. 1 Harvest spheroids from substratum
  2. 2 Incubate spheroids for beating assessment
  3. 3 Analyze beating profiles
Drug Response Testing (Optional) Day 2-3
  1. 1 Baseline beating rate recording
  2. 2 Add isoproterenol at defined concentrations
  3. 3 Record beating rate response
Quality Control - Morphological Characterization Day 2
  1. 1 Harvest spheroids for imaging
  2. 2 Bright-field microscopy imaging
  3. 3 Calculate spheroid metrics
  4. 4 3D imaging for sphericity assessment
Quality Control - Immunostaining and Marker Verification Day 2
  1. 1 Fix spheroids
  2. 2 Cryoprotection and embedding
  3. 3 Prepare cryosections
  4. 4 Antigen retrieval
  5. 5 Blocking and primary antibody incubation
  6. 6 Secondary antibody incubation
  7. 7 Nuclear staining and mounting
  8. 8 Fluorescence microscopy imaging

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Moriwaki et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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