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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Abstract

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Introduction

Drug discovery and disease modeling in the field of cardiovascular research face several challenges due to a lack of clinically relevant samples and inadequate translational tools1. Highly complex pre-clinical models or oversimplified in vitro single-cell models do not exhibit pathophysiological conditions in a reproducible manner. Therefore, several miniaturized tissue-engineered platforms have evolved to help bridge the gap, with the goal of achieving a balance between ease of application in a high-throughput manner and faithful recapitulation of tissue function2,3. With the ....

Protocol

1. Medium, reagent, culture plate preparation

  1. Cell wash solution for cell culture: Use 1x phosphate buffered saline (PBS) or Hanks balanced salt solution (HBSS) without calcium or magnesium.
  2. Cardiomyocyte differentiation media
    1. Prepare differentiation Medium #1 by adding 10 mL supplement (50x B27 plus insulin) to 500 mL cardiomyocyte basal medium (RPMI 1640).
    2. Prepare differentiation Medium #2 by adding 10 mL supplement (50x B27 minus insulin) to 500 mL cardiomyocyte basal mediu.......

Representative Results

Immunostaining and flow cytometry characterization of iPSC-derived CMs, ECs, and CFs
To generate cardiac microtissues composed of iPSC-CMs, iPSC-ECs, and iPSC-CFs, all three cell types are differentiated and characterized individually. In vitro differentiation of iPSCs to iPSC-CMs has improved over the past several years. However, the yield and purity of iPSC-CMs differ from line to line. The current protocol yields over 75% pure iPSC-CMs that spontaneously start beating around day 9 (

Discussion

To generate cardiac microtissues from pre-differentiated iPSC-CMs, iPSC-ECs, and iPSC-CFs, it is essential to obtain a highly pure culture for better control of cell numbers after contact-inhibited cell compaction within the cardiac microtissues. Recently, Giacomelli et. al.18 have demonstrated the fabrication of cardiac microtissues using iPSC-CMs, iPSC-ECs, and iPSC-CFs. Cardiac microtissues generated using the described method consist of ~5,000 cells (70% iPSC-CMs, 15% iPSC-ECs, and 15% iPSC-CF.......

Acknowledgements

We thank Dr. Amanda Chase for her helpful feedback on the manuscript. Funding support was provided by the Tobacco-Related Disease Research Program (TRDRP) of the University of California, T29FT0380 (D.T.) and 27IR-0012 (J.C.W.); American Heart Association 20POST35210896 (H.K.) and 17MERIT33610009 (J.C.W.); and National Institutes of Health (NIH) R01 HL126527, R01 HL123968, R01 HL150693, R01 HL141851, and NIH UH3 TR002588 (J.C.W).

....

Materials

NameCompanyCatalog NumberComments
12-well platesFisher Scientific08-772-29
3D micro-moldsMicrotissues12-81 format
6-well platesFisher Scientific08-772-1B
AutoMACS Rinsing SolutionThermo Fisher ScientificNC9104697
B27 Supplement minus InsulinLife TechnologiesA1895601
B27 Supplement plus InsulinLife Technologies17504-044
BD CytofixBD Biosciences554655
BD Matrigel, hESC-qualified matrixBD Biosciences354277
Cardiac Troponin T AntibodyMiltenyi130-120-403
CD144 (VE-Cadherin) MicroBeadsMiltenyi130-097-857
CD31 AntibodyMiltenyi130-110-670
CD31 MicrobeadsMiltenyi130-091-935
CHIR-99021SelleckchemS2924
DDR2Santa Cruz Biotechnologysc-81707
Dead Cell Apoptosis Kit with Annexin V FITC and PIThermo Fisher ScientificV13242
Dispase IMillipore Sigma4942086001
DMEM, high glucose (4.5g/L) no glutamine medium11960044
DMEM/F-12 basal mediumGibco11320033
Dulbecco's phosphate buffered saline (DPBS), no calcium, no magnesiumLife Technologies14190-136
EGM2 BulletKitLonzaCC-3124
Fetal bovine serumLife Technologies10437
FibroLife Serum-Free Fibroblast LifeFactors KitLifeLIne Cell TechnologyLS-1010
Glucose free RPMI mediumLife Technologies11879-020
Goat serumLife Technologies16210-064
Human FGF-basicThermo Fisher Scientific13256029
Human VEGF-165PeproTech100-20
IWR-1-endoSelleckchemS7086
Liberase TLMillipore Sigma5401020001
LS Sorting ColumnsMiltenyi130-042-401
MACS BSA Stock solutionMiltenyi130-091-376
MACS Rinsing BufferMiltenyi130-091-222
MidiMACS SeparatorMiltenyi130-042-302
RPMI mediumLife Technologies11835055
SB431542SelleckchemS1067
TO-PRO 3Thermo Fisher ScientificR37170
Triton X-100Millipore SigmaX100-100ML
TrypLE Select 10XThermo Fisher Scientificred
Vimentin Alexa Fluor® 488-conjugated AntibodyR&D SystemsIC2105G

References

  1. Neofytou, E., O'Brien, C. G., Couture, L. A., Wu, J. C. Hurdles to clinical translation of human induced pluripotent stem cells. Journal of Clinical Investigation. 125 (7), 2551-2557 (2015).
  2. Lancaster, M. A., Knoblich, J. A.

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