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CARDIAC Publication-derived

Bioprinting 3D Microfibrous Scaffolds for Engineering Endothelialized Myocardium and Heart-on-a-Chip

Source Zhang et al., 2016 · Brigham and Women's Hospital, Harvard Medical School · 10.1016/j.biomaterials.2016.09.003

👤 Yu Shrike Zhang, Andrea Arneri, Simone Bersini, Su-Ryon Shin, Kai Zhu, Zahra Goli-Malekabadi, Julio Aleman, Cristina Colosi, Fabio Busignani, Valeria Dell'Erba, Colin Bishop, Thomas Shupe, Danilo Demarchi, Matteo Moretti, Marco Rasponi, Mehmet Remzi Dokmeci, Anthony Atala, Ali Khademhosseini ⏱ 35 days 📋 9 phases 🧫 Human iPSC-derived cardiomyocytes; Neonatal rat cardiomyocytes; Human umbilical vein endothelial cells (HUVECs)

Abstract

A novel 3D bioprinting strategy is presented to fabricate endothelialized myocardial tissues using a composite bioink with dual-step crosslinking. Human umbilical vein endothelial cells encapsulated within bioprinted microfibrous scaffolds migrate to microfiber peripheries to form confluent endothelium, which is then seeded with cardiomyocytes to generate aligned, beating myocardium. The endothelialized-myocardium-on-a-chip platform integrated into a microfluidic perfusion bioreactor enables cardiovascular drug toxicity screening and demonstrates translational potential with human iPSC-derived cardiomyocytes.

Cell source
Human iPSC-derived cardiomyocytes; Neonatal rat cardiomyocytes; Human umbilical vein endothelial cells (HUVECs)
Application
Cardiac tissue engineering, disease modeling, drug screening, cardiovascular toxicity evaluation

Protocol overview

46 steps across 9 phases

Cell Culture Preparation Prior to bioprinting
  1. 1 HUVEC Culture
  2. 2 Neonatal Rat Cardiomyocyte Isolation
  3. 3 Cardiomyocyte Culture Media Preparation
  4. 4 hiPSC-Cardiomyocyte Culture
Bioink Preparation and Coaxial Nozzle Setup Prior to bioprinting
  1. 1 Assemble Coaxial Nozzle
  2. 2 Prepare Bioink Solution
  3. 3 Prepare Crosslinking Solution
  4. 4 Resuspend HUVECs in Bioink
3D Bioprinting of Microfibrous Scaffolds Day 0
  1. 1 Program Bioprinter with Custom G-code
  2. 2 Load Bioink and Crosslinking Solution into Syringes
  3. 3 Begin Bioprinting Process
  4. 4 UV Crosslinking of Scaffolds
  5. 5 Wash Scaffolds
HUVEC Endothelialization (Pre-seeding Phase) Day 0–15
  1. 1 Culture Scaffolds with Encapsulated HUVECs
  2. 2 Monitor HUVEC Migration and Endothelialization
  3. 3 Assess Endothelialization Progress (Optional)
  4. 4 Prepare Endothelialized Scaffolds for Cardiomyocyte Seeding
Cardiomyocyte Seeding and Myocardial Tissue Development Day 15–35
  1. 1 Prepare Cardiomyocyte Suspension
  2. 2 Prepare Hydrophobic PDMS Surface
  3. 3 Seed Cardiomyocytes onto Endothelialized Scaffolds
  4. 4 Transfer to Culture Vessel
  5. 5 Culture Cardiomyocytes on Scaffolds
  6. 6 Monitor Beating and Maturation
Bioreactor Assembly and Perfusion Culture Day 3 onwards (after static culture)
  1. 1 Assemble Microfluidic Bioreactor
  2. 2 Connect Inlet and Outlet Tubing
  3. 3 Set Up Reservoir and Pump
  4. 4 Start Perfusion Culture
  5. 5 Monitor Culture and Change Medium
Drug Screening and Toxicity Assessment Day 18–35 (after tissue maturation)
  1. 1 Prepare Drug Solutions
  2. 2 Establish Control and Treated Groups
  3. 3 Apply Drug Treatments
  4. 4 Monitor Beating Behavior During Drug Exposure
  5. 5 Measure Endothelial Secretion Biomarkers
  6. 6 Assess Cell Viability (Optional)
Immunofluorescence Staining and Analysis Throughout culture (typically at Day 3, 7, 15, 28)
  1. 1 Fix Tissue Constructs
  2. 2 Permeabilize Cells
  3. 3 Block Non-specific Binding
  4. 4 Incubate with Primary Antibodies
  5. 5 Apply Secondary Antibodies
  6. 6 Perform F-actin Staining (Optional)
  7. 7 Stain Nuclei
  8. 8 Mount and Image Samples
Mechanical Characterization of Scaffolds After bioprinting (Day 0–1)
  1. 1 Prepare Scaffolds for Testing
  2. 2 Perform Compressive Stress-Strain Testing
  3. 3 Calculate Elastic Modulus
  4. 4 Analyze Results

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Zhang et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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