Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

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 function^2,3. 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 medium (RPMI 1640).
   3. Prepare purification Medium #3 by adding supplement (50x B27 plus insulin) to 500 mL cardiomyocyte basal medium (RPMI 1640) ....

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.......

Materials

Name	Company	Catalog Number	Comments
12-well plates	Fisher Scientific	08-772-29
3D micro-molds	Microtissues	12-81 format
6-well plates	Fisher Scientific	08-772-1B
AutoMACS Rinsing Solution	Thermo Fisher Scientific	NC9104697
B27 Supplement minus Insulin	Life Technologies	A1895601
B27 Supplement plus Insulin	Life Technologies	17504-044
BD Cytofix	BD Biosciences	554655
BD Matrigel, hESC-qualified matrix	BD Biosciences	354277
Cardiac Troponin T Antibody	Miltenyi	130-120-403
CD144 (VE-Cadherin) MicroBeads	Miltenyi	130-097-857
CD31 Antibody	Miltenyi	130-110-670
CD31 Microbeads	Miltenyi	130-091-935
CHIR-99021	Selleckchem	S2924
DDR2	Santa Cruz Biotechnology	sc-81707
Dead Cell Apoptosis Kit with Annexin V FITC and PI	Thermo Fisher Scientific	V13242
Dispase I	Millipore Sigma	4942086001
DMEM, high glucose (4.5g/L) no glutamine medium		11960044
DMEM/F-12 basal medium	Gibco	11320033
Dulbecco's phosphate buffered saline (DPBS), no calcium, no magnesium	Life Technologies	14190-136
EGM2 BulletKit	Lonza	CC-3124
Fetal bovine serum	Life Technologies	10437
FibroLife Serum-Free Fibroblast LifeFactors Kit	LifeLIne Cell Technology	LS-1010
Glucose free RPMI medium	Life Technologies	11879-020
Goat serum	Life Technologies	16210-064
Human FGF-basic	Thermo Fisher Scientific	13256029
Human VEGF-165	PeproTech	100-20
IWR-1-endo	Selleckchem	S7086
Liberase TL	Millipore Sigma	5401020001
LS Sorting Columns	Miltenyi	130-042-401
MACS BSA Stock solution	Miltenyi	130-091-376
MACS Rinsing Buffer	Miltenyi	130-091-222
MidiMACS Separator	Miltenyi	130-042-302
RPMI medium	Life Technologies	11835055
SB431542	Selleckchem	S1067
TO-PRO 3	Thermo Fisher Scientific	R37170
Triton X-100	Millipore Sigma	X100-100ML
TrypLE Select 10X	Thermo Fisher Scientific	red
Vimentin Alexa Fluor® 488-conjugated Antibody	R&D Systems	IC2105G