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 established protocols^4,5,6. However, the obtained cells display fetal-like phenotypes, with a range of immature characteristics compared to adult ventricular cardiomyocytes^7,8. This can be an obstacle to the application of hPSC-CMs as models of adult heart tissue in drug discovery research and in the development of adult cardiac disease models⁹.

In order to overcome this limitation of phenotypic immaturity, new approaches have been actively investigated to promote cardiomyocyte maturation. Early studies revealed effective pro-maturation properties in neonatal rat cardiomyocytes via cyclic mechanical¹⁰ or electrical stimulation¹¹. Gel compaction and cyclic mechanical stimulation were also shown to improve some aspects of hPSC-CM maturation^12,13, with minimal enhancement of the electrophysiological and calcium handling properties. Therefore, a platform system called "biological wire" (biowire) was devised by providing both structural cues and electrical field stimulation to enhance the maturation of hPSC-CMs¹⁴. This system uses a microfabricated platform to create aligned cardiac tissue that is amenable to electrical field stimulation. This can be used to improve the structural and electrophysiological maturity of hPSC-CMs. Here, we describe the details of making such biowires.

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 others¹⁵).
2. Dispense about 4 mL of SU-8 50 onto the center of a 4 inch silicon wafer. Coat the SU-8 50 evenly on the wafer using a spin coater by spinning at 2,000 revolutions per minute (RPM) for 30 s. Bake the wafer on a 95 °C hotplate for 10 min. Expose the wafer to ultraviolet (UV) light at 200 mJ/cm².
3. Pour about 4 mL of SU-8 2050 onto the center of the wafer and spin at 2,500 RPM for 30 s. Bake on the 95 °C hotplate for 15 min. Cool to room temperature (RT).
4. Repeat step 1.3 two more times.
5. Cover the coated wafer with the first-layer transparency photomask. Expose the wafer to UV light at 270 mJ/cm² to make the first layer. Bake on the 95 °C hotplate for 15 min.
6. Make the second layer of SU-8 2050 by repeating step 1.3 twice.
7. Align the second-layer mask to the features on the first layer using a mask aligner. Then, expose to UV light at 240 mJ/cm2. Bake at 95 °C for 15 min.
   NOTE: The exposure time is determined by the required total UV dose and the UV light output intensity measured on the day prior to use.
8. Develop the wafer by submerging it in propylene glycol monomethyl ether acetate (PGMEA) and shaking at 200 RPM for 30 min on an orbital shaker.
9. Place the master into a Petri dish and carefully pour the PDMS mixture (base to crosslinker at a ratio of 10:1 by weight) on the center of the wafer. Cover all the features and avoid air bubbles.
10. Heat in an oven at 70 °C for 2 h. Use a sharp blade to cut around the edge of the PDMS biowire template (Figure 1A, right). Peel the PDMS from the master. Trim the device with the blade. Put the PDMS biowire template in a sterilization pouch and steam autoclave at 121 °C for 20 min.
11. In a biosafety cabinet (BSC), place the PDMS biowire template into a Petri dish. Use tweezers to hold and mount a piece of sterile surgical silk suture (6-0) in the center of the channel of the PDMS microwell by seating it in the grooves at both ends of the channel (Figure 1B-I).

2. Creation of the Electrical Stimulation Chamber

1. Make a pair of rectangular polycarbonate pieces (length x width x thickness: 2 cm x 0.85 cm x 0.35 cm) and use them as the frame of the electrical stimulation chamber (Figure 2).
2. Drill two 3 mm wide holes 2 cm apart along the center line of each polycarbonate frame piece.
3. Cut two pieces of 1.5 cm long carbon rods. Drill a 1 mm-wide hole through the carbon rod about 3 mm from the end. Thread a platinum wire through the carbon rod hole and tighten the wire by wrapping it around the carbon rod. Attach a clip to the other end of the platinum wire for later connection with the electric stimulator.
4. Insert the carbon rods into the polycarbonate frame pieces. Place the assembled frame with the carbon rods into a well of a six-well plate.
5. Pour 2 mL of PDMS into the well. Submerge the bottom of the polycarbonate frames in PDMS while keeping the PDMS surface level close to but not reaching the bottom of the carbon rods. Heat in an oven at 70 °C for 2 h.
6. Take the electrical stimulation chamber out of the six-well plate and put it in a sterilization pouch for steam sterilization at 121 °C for 20 min.

3. Enzymatic Dissociation of hPSC-CMs

1. Induce hPSC to differentiate into cardiomyocytes using an established protocol⁴.
   NOTE: Briefly, generate cardiomyocytes via established embryoid body (EB) protocols in stem pro medium through the sequential supplementation with bone morphogenetic protein 4, basic fibroblast growth factor, activin A, vascular endothelial growth factor, and Dickkopf homolog 1⁴. Use EBs from day 20-34 of differentiation to make biowires.
2. Collect the EBs from the low attachment plate with a P1,000 pipette tip and transfer to a conical tube. Pellet by centrifugation for 5 min at 125 x g and RT. Discard the supernatant and resuspend the pellet with pre-warmed, 37 °C collagenase type I containing 1% deoxyribonuclease I (DNAse I). Incubate at 37 °C for 2 h for digestion.
3. Add 5 mL of pre-warmed, 37 °C wash medium and centrifuge for 5 min at 125 x g and RT. Discard the supernatant. Resuspend the pellet with 2 mL of trypsin/ethylenediaminetetraacetic acid (EDTA) solution and incubate at 37 °C for 5 min.
4. Add 1 mL of stop medium containing 3% (v/v) DNase I.
5. Attach a 20 G (0.9 mm) needle to a 5 mL syringe and pass the EB suspension through it 3-5 times.
6. Add 7 mL of wash medium and centrifuge at 280 x g for 5 min. Discard the supernatant. Resuspend the pellet in Iscove's modified Dulbecco's medium (IMDM) and store on ice. Determine the cell number with a hemocytometer. Take 0.5 x 10⁶ cells and pellet by centrifugation at 280 x g for 5 min. Remove the supernatant.
7. Pre-cool all the gel components on ice before mixing them in a sterile tube. Mix the following collagen gel components (final concentration): 2.1 mg/mL rat tail collagen type I, 24.9 mM glucose, 23.8 mM NaHCO₃, 14.3 mM NaOH, 10 mM HEPES, and 10% extracellular matrix in 1x M199 medium. Add the collagen and extracellular matrix last. Mix by pipetting up and down.
8. Resuspend 0.5 x 10⁶ cells with 3.5 µL of collagen gel and mix well.
9. Use a P10 pipette tip to add 4 µL of the cell suspension into the 0.5 cm-long channel (Figure 1B-II). Adjust the position of the suture with sterile forceps if necessary.
10. Cover the bottom of the Petri dish with sterile phosphate-buffered saline (PBS), making sure not to touch the gel. Incubate at 37 °C for 30 min.
11. Replace the PBS with 12 mL of culture medium (e.g., stem pro or medium recommended by the cell manufacturer), enough to cover the cells (Figure 1B-III). Culture in an incubator at 37 °C for 1 week. Change the medium every other day.

4. Electrical Stimulation for Biowire Cultivation

1. In the BSC, add 2 mL of PBS to each well of a six-well plate that will house an electrical stimulator chamber. Use sterile tweezers to gently place the autoclaved electrical stimulation device into the well. Discard the PBS from the well.
2. Cover the carbon rods with 5 mL of pre-warmed culture medium. Use sterile tweezers to mount the biowires in the electrical stimulation chamber and orient them perpendicular to the carbon rods (Figure 2).
3. Place the lid on top of the six-well plate, but leave sufficient platinum wires reaching outside the plate to connect with the electrical stimulator. Place the six-well plate in a cell culture incubator at 37 °C and connect the platinum wire to the electrical stimulator.
4. Stimulate the biowires using an electrical stimulator with the following setting: biphasic repeating pulse, 1-ms pulse duration, 3 V/cm, and 1 pulse per second (PPS). Raise the PPS every 24 h to the following values: 1.83, 2.66, 3.49, 4.82, 5.15, and 6. Change the medium every other day.
5. Observe the cardiomyocyte contractility in response to electrical stimulation under a microscope at 10X magnification.
   NOTE: After one week of stimulation, the biowires becomes mature (see the results section).

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