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CARDIAC Publication-derived

Generation of human vascularized and chambered cardiac organoids (vaschamcardioids) for cardiac disease modelling and drug evaluation

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

👤 Jingsi Yang, Wei Lei, Yang Xiao, Shuai Tan, Jiani Yang, Yingjiong Lin, Zhuangzhuang Yang, Dandan Zhao, Chunxiang Zhang, Zhenya Shen, Shijun Hu ⏱ 72 days 📋 17 phases 🧫 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

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

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Yang et al., 2024. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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