Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Summary

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

Abstract

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

Introduction

Cell-based therapy is one of the most promising and investigated strategies to achieve cardiac repair/regeneration. It has been aided by cardiac tissue engineering and the co-delivery of biomaterials^1,2. Most available cell sources have been studied in animal models for their potentially beneficial effects on damaged, diseased, or aged hearts³. In particular, significant efforts have been made to use human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CM), a potentially unlimited autologous cell source for cardiac tissue engineering. hPSC-CMs can be produced using several es....

Protocol

1. Master Design and Fabrication

NOTE: Use soft lithography for device fabrication. Make a two-layer SU-8 master for polydimethylsiloxane (PDMS) molding.

1. Design the device using a design and drafting software (Figure 1A, left). Draw each layer of the master separately. Print the device design on two photomasks at 20,000 dpi, corresponding to the two layers of the master¹⁵. Set the device pattern as transparent and the surrounding area as dark; the masks will serve as the template to optically transfer the device pattern onto SU-8 by lithography (as described by others^15<....

Results

The rational for the use of a suture in the biowires is to serve as a template for the formation of 3D constructs that align in one axis and mimic the shape of cardiac fibers. We show that after seven days of culture in the biowire, cells remodeled the gel around the suture (Figure 3A). The cells assembled along the axis of the suture to form aligned cardiac tissue (Figure 3). After 7 days of preculture, the biowires were subjected to 7 days of electrical.......

Discussion

This manuscript describes the setup and implementation of the engineered platform, biowire, to improve the maturation of hPSC-CMs. The device can be made in standard microfabrication facilities, and biowires can be produced with common cell culture techniques and an electrical stimulator.

To our knowledge, there is no reported method similar to biowires. This strategy reveals that improved maturation properties were dependent on the electrical stimulation regimen, as evidenced by the greater m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
L-Ascorbic acid	Sigma	A-4544	hPSC-CM culture media componet
AutoCAD	Autodesk, Inc		Software to design device
Carbon rods, Ø 3 mm			Electrical stimulator chamber component
Collagen, type 1, rat tail	BD Biosciences	354249	Collagen gel: 2.1 mg/mL of rat tail collagen type I in 24.9 mM glucose, 23.8 mM NaHCO3, 14.3 mM NaOH, 10 mM HEPES, in 1x M199 media with 10 % of growth factor-reduced Matrigel.
Collagenase type I	Sigma	C0130	0.2% collagenase type I (w/v) and 20% FBS (v/v) in PBS with Ca2+ and Mg2+. Sterilize with 0.22 μm filter and make 12 mL aliquots. Store at -20 °C.
Deoxyribonuclease I (DNase I)	EMD Millipore	260913-25MU	Make 1 mg/mL DNase I stock solution in water. Filter sterile and store 0.5 mL aliquots at −20 °C
Drill & drill bits (Ø 1 mm and 2 mm)	Dremel		Drill holes in polycarbonate frames
Electrical stimulator	Grass	s88x
Fetal bovine serum (FBS)	WISENT Inc.	080-450
D-(+)-Glucose	Sigma	G5767	Collagen gel component
L-Glutamine	Thermo Fisher Scientific	25030081
H2O	MilliQ		18.2 MΩ·cm at 25 °C, ultrapure, to make all solutions
HEPES	Sigma	H4034	Collagen gel component
Hot plate	Torrey Pines	HS40
Iscove's Modified Dulbecco's Medium(IMDM)	Thermo Fisher Scientific	12440053
Mask aligner	EVG	EVG 620
Matrigel, growth factor reduced	Corning	354230	Collagen gel component
Medium 199 (M199)	Thermo Fisher Scientific	11825015	Collagen gel component
Monothioglycerol (MTG)	Sigma	M-6145	hPSC-CM culture media componet
Orbital shaker	VWR	89032-088
Penicillin/Streptomycin (P/S)	Thermo Fisher Scientific	15070063
Phosphate-buffered saline (PBS) with Ca2+ and Mg2+	Thermo Fisher Scientific	14040133
Plate (6-well)	Corning	353046
Plate (6-well), low attachment	Corning	3471
Platinum wires, 0.2 mm			Electrical stimulator chamber component
Polydimethylsiloxane (PDMS)	Dow Corning	Sylgard 184
Propylene glycol monomethyl ether acetate (PGMEA)	Doe & Ingalls Inc.		To develop the wafer
Pouch, peel-open	Convertors	92308	For steam sterilization
Silicon wafer, 4 inch	UniversityWafer Inc.
Sodium bicarbonate (NaHCO3)	Sigma	S5761	Collagen gel component
Sodium hydroxide	Sigma	S8045	Collagen gel component
Sprin coater	Specialty Coating Systems	G3P-8
StemPro-34 culture medium	Thermo Fisher Scientific	10639011	hPSC-CM culture medium. To make 50 mL, add 1.3 mL supplement, 500 μL of 100× L-Glutamine, 250 μL of 30 mg/mL transferrin, 500 μl of 5 mg/mL ascorbic acid, 150 μL of 26 μl /2 mL monothioglycerol (MTG), and 500 μL (1 %) penicillin/streptomycin.
Stop media			Wash medium:FBS (1:1)
SU-8 50	MicroChem Corp.		photoresist, master component
SU-8 2050	MicroChem Corp.		photoresist, master component
Transferrin	Roche	10-652-202	hPSC-CM culture media componet
Trypsin/EDTA, 0.25%	Thermo Fisher Scientific	25200056	hPSC-CM culture media componet
Wash medium			IMDM containing 1% Penicillin/Streptomycin