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Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

Published: June 15th, 2021



1School of Biological and Health Systems Engineering, Arizona State University, 2Biodesign Virginia G. Piper Center for Personalized Diagnostics, Arizona State University

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium's cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.

Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

Tissue engineering approaches have been widely explored, in recent years, to accompany in vivo clinical findings in regenerative medicine and disease modeling1,2. Significant emphasis has been particularly placed on in vitro cardiac tissue modeling due to the inherent difficulties in sourcing human primary cardiac tissue and producing physiologically relevant in vitro surrogates, limiting the fundamental understanding of the complex mechanisms of cardiovascular diseases (CVDs)1,3. Traditional models have often involved 2D mon....

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Perform all cell handling and reagent preparation within a Biosafety Cabinet. Ensure all surfaces, materials, and equipment that come into contact with cells are sterile (i.e., spray down with 70% ethanol). Cells should be cultured in a humidified 37 °C, 5% CO2 incubator. All hiPSC culture and differentiation is performed in 6-well plates.

1. Microfluidic device creation (approximate duration: 1 week)

  1. Photolithography
    NOTE: The mask, designed.......

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To obtain a highly purified population of CMs from hiPSCs, a modified version involving a combination of the Lian differentiation protocol33 and Tohyama purification steps34 is used (refer to Figure 1A for experimental timeline). The hiPSCs need to be colony-like, ~85% confluent, and evenly spread throughout the culture well 3-4 days after passage, at the onset of CM differentiation (Figure 1B). Specifically, on Da.......

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The formation of an in vitro human cardiac tissue model with enhanced cell-cell interactions and biomimetic 3D structure is imperative for basic cardiovascular research and corresponding clinical applications1. This outlined protocol explains the development of 3D human anisotropic cardiac tissue within a microfluidic device, using co-culture of stem cell-derived CMs with connective CFs encapsulated within a collagen hydrogel, serving to model the complex cell composition and structure of.......

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We would like to thank NSF CAREER Award #1653193, Arizona Biomedical Research Commission (ABRC) New Investigator Award (ADHS18-198872), and the Flinn Foundation Award for providing funding sources for this project. The hiPSC line, SCVI20, was obtained from Joseph C. Wu, MD, PhD at the Stanford Cardiovascular Institute funded by NIH R24 HL117756. The hiPSC line, IMR90-4, was obtained from WiCell Research Institute55,56.


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Name Company Catalog Number Comments
0.65 mL centrifuge tubes VWR 87003-290
1 mm Biopsy punch VWR 95039-090
1.5 mm Biopsy punch VWR 95039-088
15 mL Falcon tubes VWR 89039-670
18x18mm coverslips VWR 16004-308 The coverslips should be No.1, to allow for high magnification imaging
4% paraformaldehyde ThermoFisher 101176-014
6-well flat botttom tissue-culture plates VWR 82050-844
B27 minus insulin LifeTech A1895602
B27 plus insulin LifeTech 17504001
CHIR99021 VWR 10188-030
Collagen I, rat tail Corning 47747-218
DMEM F12 ThermoFisher 11330057
DPBS ThermoFisher 21600069
E8 ThermoFisher A1517001 can also be made in house
EDTA VWR 45001-122
FGM3 VWR 10172-048
GFR-Matrigel VWR 47743-718
Glycine Sigma G8898-500G
Goat serum VWR 10152-212
hESC-Matrigel Corning BD354277
IWP2 Sigma I0536-5MG
Kimwipes VWR 82003-820
MTCS Sigma 440299-1L
NaN3 Sigma S2002-25G
NaOH Sigma S5881-500G
Pen/Strep VWR 15140122
Petri dish (150x15mm) VWR 25384-326
Petri dish (60x15mm) VWR 25384-092
Phenol Red Sigma P3532-5G
RPMI 1640 ThermoFisher MT10040CM
RPMI 1640 minus glucose VWR 45001-110
Silicon Wafers (100mm) University Wafer 1196
Sodium lactate Sigma L4263-100ML
SU8 2075 Microchem Y111074 0500L1GL
SU8 Developer ThermoFisher NC9901158
Sylgard Elastomer Essex Brownell DC-184-1.1
T75 flasks VWR 82050-856
Triton X-100 Sigma T8787-100ML
TrypLE ThermoFisher 12604021
Trypsin-EDTA (0.5%) ThermoFisher 15400054
Tween20 Sigma P9416-50ML
Y-27632 Stem Cell Technologies 72304
EVG620 Aligner EVG
Plasma cleaner PDC-32G Harrick Plasma
Zeiss AxioObserver Z1 microscope Nikon
Leica SP8 Confocal microscope Leica

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