A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

Summary

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

Abstract

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

Introduction

The goal of current cardiac tissue engineering is to develop a therapy to replace or repair the structure and function of injured myocardial tissue¹. Methods to create 3-D cardiac tissue models exhibiting the important contractile and electrophysiological properties of native cardiac tissue have been rapidly expanding^2,3. A variety of strategies have been explored and used in studies^4,5. These methods range from the use of specific synthetic and natural bioactive hydrogels, such as gelatin, collagen, fibrin, and peptides

Protocol

1. Preparation of Cardiomyocytes

1. Coat 6-well plates with basement membrane matrix and culture human-induced pluripotent stem cells (hiPSCs) as previously described¹⁷.
2. Differentiate hiPSCs into hiPSC-CMs using previously described methods¹⁸.
3. At 16 - 18 d post-differentiation, suspend the cardiomyocytes by rinsing each well with 2 mL of 1x phosphate-buffered saline (PBS) without calcium or magnesium, followed by incubation with 1 mL/well of trypsin or cell dissociation reagent (see Table of Materials) for 5 min at room temperature.
4. Neutralize the trypsin or cell di....

Results

In our experiments, we utilized a cell suspension of 70% iPSC-CMs, 15% HCFs, and 15% HUVECs in RPMI/B-27 cell media at a concentration of 2,475,000 cells per mL. After creating the cell suspension, we dispensed 4 mL of the cell suspension to each well of an ultra-low attachment hanging drop system, as described in step 4.3 of the protocol. The use of the hanging drop system resulted in the spontaneous formation of hundreds of beating spheroids after 3 days of culture at 37 °C, 5% CO_2.......

Discussion

The significance of this method lies in its reproducibility and the effectiveness of the resultant multilayered cardiac tissue. In the field of cardiac tissue engineering, one of the current goals is to identify a method to construct beating, multilayered, and functional 3-D cardiac patches. We report an efficient and reproducible method of creating multilayered cardiac tissues by direct manual seeding of spheroids composed of cardiomyocytes, endothelial cells, and fibroblasts into a novel net mold. The net mold used in .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Human Cardiac fibroblasts (HCF)	Sciencell	6310
FM-2 Consists of Basal Medium	Sciencell	2331	HCF culture medium
Human umbilical vein endothelial cells (HUVEC)	Lonza	CC-2935
EGM+Bullet Kit	Lonza	CC5035	HUVEC culture medium
E8 media	Invitrogen	A1517001	HiPSC culture medium
Geltrex	Invitrogen	A1413202
TrypLE Express Enzyme (1X)	Thermo Fisher	12604013	Trypsin and Cell dissociation reagent
RPMI media	Invitrogen	11875093	RPMI media with B-27 supplement is hiPSC-CM culture medium
B-27 supplement (50x)	Thermo Fisher	17504044	RPMI media with B-27 supplement is hiPSC-CM culture medium
Trypan Blue Solution, 0.4%	Thermo Fisher	15250061
Novel net mold	TissueByNet Co.,Ltd	NM25-1
Hanging drop plate	Kuraray Co.,Ltd	MPc350
6 well plates	Sigma-Aldrich	CLS-3516