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CARDIAC Publication-derived

Cardiac Organoids for Toxicity Testing with Vasculature

Full SOP with Academic or a plan

Source Yang et al., 2024 · Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, Soochow University, Suzhou, China · 10.1111/cpr.13631

Authors: Jingsi Yang, Wei Lei, Yang Xiao, Shuai Tan, Jiani Yang, Yingjiong Lin, Zhuangzhuang Yang, Dandan Zhao, Chunxiang Zhang, Zhenya Shen, Shijun Hu Duration: 72 days 17 phases Cell source: Human iPSC

Abstract

A three-step method for robust generation of vascularized and chambered cardiac organoids (vaschamcardioids) from hiPSCs, combining vascular sphere derivation with hiPSC-derived cardiomyocytes. The resulting vcCOs display spontaneous beating (~90% ratio), chamber-like structures, vascular networks, and multi-cellular composition suitable for modeling cardiac injury, fibrosis, and drug toxicity assessment.

Cell source
Human iPSC
Application
Disease modeling and drug evaluation

Protocol overview

65 steps across 17 phases

Phase 1 · hiPSC Culture and Maintenance Ongoing
  1. a Culture hiPSCs in PSCeasy medium
  2. b Passage hiPSCs at confluence
Phase 2 · Cardiomyocyte Differentiation Day 0-9
  1. a Initiate cardiac differentiation with CHIR99021
  2. b Inhibit Wnt signaling with Wnt-C59
  3. c Continue differentiation post-Wnt-C59 withdrawal
Phase 3 · Cardiomyocyte Purification Day 9-12
  1. a Passage cardiomyocytes onto gelatin-coated dishes
  2. b Culture in glucose-free medium for 3 days
Phase 4 · Vascular Sphere Generation Day 0-8
  1. a Dissociate hiPSCs and prepare aggregate medium
  2. b Aggregate hiPSCs in ultra-low attachment plates
  3. c Induce vascular lineage with CHIR99021
  4. d Apply vascular growth factor cocktail
Phase 5 · vaschamcardioid (vcCO) Assembly and Culture Day 8-15+
  1. a Co-culture vascular spheres with cardiomyocytes
  2. b Monitor spheroid aggregation
  3. c Culture vcCOs for maturation
Phase 6 · Cryoinjury and Captopril Treatment (Disease Modeling) Day 25 post-differentiation start; 3-day recovery
  1. a Prepare sterile cryoinjury needle
  2. b Perform cryoinjury on vcCOs
  3. c Restore culture medium with or without captopril
Phase 7 · Doxorubicin Toxicity Assessment 24-72 hours
  1. a Treat vcCOs with doxorubicin doses
  2. b Assess beating frequency and ratio
Phase 8 · Whole-Mount Immunofluorescence Staining Variable (post-harvest)
  1. a Collect and fix vcCOs
  2. b Wash and permeabilize
  3. c Incubate with primary antibodies
  4. d Apply secondary antibodies
  5. e Counterstain and mount
Phase 9 · Immunofluorescence on Frozen Sections Variable (post-harvest)
  1. a Fix and dehydrate vcCOs
  2. b Freeze in OCT compound
  3. c Slice cryostat sections
  4. d Block and stain sections
  5. e Apply secondary antibodies and counterstain
  6. f Image and analyze sections
Phase 10 · Three-Dimensional (3D) Imaging of Whole Organoids Variable (post-harvest)
  1. a Fix organoids for iDISCO clearing
  2. b Apply iDISCO clearing protocol
  3. c Optically clear organoids
  4. d Image using light-sheet microscopy
  5. e Process raw images and 3D reconstruction
Phase 11 · Calcium Transient Assay Variable (post-harvest or live)
  1. a Load vcCOs with fluorescent calcium indicator
  2. b Wash and mount organoids
  3. c Acquire and analyze calcium transients
Phase 12 · Single-Cell RNA Sequencing (scRNA-seq) Variable (post-harvest)
  1. a Prepare single-cell suspension
  2. b Assess cell viability
  3. c Generate GEMs using 10× Genomics platform
  4. d Prepare scRNA-seq libraries
  5. e Sequence libraries
  6. f Align reads and quantify gene expression
  7. g Perform downstream analysis with Seurat
  8. h Annotate cell types
  9. i Perform enrichment analyses
  10. j Compare with reference datasets
Phase 13 · Flow Cytometric Analysis Variable (post-harvest)
  1. a Digest and filter cells
  2. b Stain with antibody markers
  3. c Analyze by flow cytometry
  4. d Analyze data
Phase 14 · Enzyme-Linked Immunosorbent Assay (ELISA) for cTnT Variable (post-harvest)
  1. a Collect culture supernatant
  2. b Measure cTnT by ELISA
  3. c Analyze data
Phase 15 · TUNEL Apoptosis Assay Variable (post-harvest)
  1. a Prepare cryostat sections
  2. b Apply TUNEL assay solution
  3. c Counterstain and visualize
Phase 16 · Quantitative Real-Time PCR (qPCR) Variable (post-harvest)
  1. a Extract total RNA
  2. b Synthesize cDNA
  3. c Perform qPCR
  4. d Analyze qPCR data
Phase 17 · Cytotoxicity Assays (CCK8, CellTiter-Glo, Annexin V) 24-72 hours post-treatment
  1. a Perform CCK8 cell viability assay
  2. b Perform CellTiter-Glo ATP assay
  3. c Detect apoptosis by Annexin V-FITC

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Attribution

This SOP was written by Organthis based on the published method in Yang et al., 2024. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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