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 peptides⁶, to bio-ink deposition technologies² and bioprinting technologies⁷.

It has been shown that scaffold-free methods can produce comparable tissues as biomaterial-based methods, without the drawbacks of incorporating foreign scaffolding material⁸. Oren Caspi et al. demonstrated that the incorporation of various types of cells enables the generation of highly vascularized human engineered cardiac tissue⁹. Chin et al. developed a 3-D printing method for cardiac patch creation from spheroids. Resulting patches are composed of cardiomyocytes, fibroblasts, and endothelial cells in a 70:15:15 ratio¹⁰. Spheroids have been shown to be effective "building blocks" of scaffold-free cardiac tissue creation, as they are resistant against hypoxia and possess sufficient mechanical integrity for implantation^11,12. Previous studies have demonstrated several fabrication methods for spheroid creation, including the use of the hanging drop method, spinner flasks¹³, microfluidic systems¹⁴, and non-adherent culture surfaces uncoated or coated with agarose micro-molds¹⁵. In this protocol, we use the hanging drop device, which contains micropores for condensing hundreds of spheroids at one time.

This study presents a novel and efficient scaffold-free method for cardiac tissue creation, which includes manually seeding the spheroids into a square mold cavity and incubating the tissue on a shaker for maturation. Under usual static culture conditions, oxygen diffusion is limited to the outer aspects of the tissue construct, resulting in central necrosis. However, with the net mold, all the spheroids seeded into the mold are immersed in media with a constant fluidic motion, allowing for the increased diffusion of nutrients and oxygen. Additionally, this mold-based method allows for the simultaneous creation of different-sized tissue patches with minimal manual effort and the resultant tissue can be easily removed from the mold. This novel method allows for the efficient and reproducible creation of scaffold-free, multilayered cardiac patches.

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 dissociation reagent (see Table of Materials) using an equal volume of Roswell Park Memorial Institute (RPMI) cell media supplemented with B-27 (RPMI/B-27 cell media). Pipette up and down to loosen any adhered cells.
5. Collect the suspended cardiomyocytes with a 10-mL serological pipette and transfer them to a 50-mL conical tube.
6. Centrifuge the cell suspension at 250 x g for 5 min at room temperature to obtain a cell pellet.
7. Resuspend the pellet in 10 mL of RPMI/B-27 cell media.
8. Combine 20 µL of cell suspension with an equal amount of 0.4% Trypan blue solution and mix gently.
9. Use a manual hemocytometer to count and obtain the concentration and cell viability of the cell suspension.

2. Preparation of Fibroblasts

1. Initiate a culture of a human cardiac fibroblast (HCF) (adult ventricular type) cell line as described previously¹⁶.
2. Suspend the HCFs by incubating them with an appropriate amount of trypsin or cell dissociation reagent (see Table of Materials) for 5 min at room temperature¹⁶. For a T175 flask, 10 mL of trypsin or cell dissociation reagent was used.
3. Neutralize the trypsin or cell dissociation reagent (see Table of Materials) using an equal volume of medium, then transfer the sample to a 50-mL conical tube and centrifuge the cell suspension at 250 x g for 5 min at room temperature to obtain a pellet.
4. Resuspend the pellet in 10 mL of fibroblast growth medium (see Table of Materials).
5. Combine 20 µL of the HCF cell suspension with an equal amount of 0.4% trypan blue solution and mix gently.
6. Use an automated cell counter or manual hemocytometer to count and obtain the concentration and cell viability of the new cell suspension.

3. Preparation of Endothelial Cells

1. Initiate a culture of a human umbilical vein endothelial cell (HUVEC) line as described previously¹⁷. Suspend the HUVECs by incubating them with an appropriate amount of trypsin or cell dissociation reagent (see Table of Materials) for 3 min at room temperature¹⁷. Neutralize the trypsin or cell dissociation reagent (see Table of Materials) using an equal volume of medium, then transfer to a 50-mL conical tube and centrifuge the cell suspension at 250 x g for 5 min at room temperature to obtain a pellet.
   NOTE: For a T175 flask, 10 mL of trypsin or cell dissociation reagent was used.
2. Resuspend the pellet in 10 mL of endothelial cell growth medium (see Table of Materials).
3. Combine 20 µL of the HUVEC suspension and stain it with an equal amount of 0.4% Trypan blue solution and mix gently.
4. Use an automated cell counter or a manual hemocytometer to count and obtain the concentration and cell viability of the cell suspension.

4. Creation of Hanging Drop Spheroids

1. Place the hanging drop device which contains 850 micropores (each with a diameter of 350 µm) into sterile 6-well plates.
2. Isolate the three types of cells as described above: hiPSC-CMs, HCFs, and HUVECs. Combine them in a ratio of 70% iPSC-CMs, 15% HCFs, and 15% HUVECs in a 50-mL conical tube with RPMI/B-27 cell media at a concentration of 2,475,000 cells per mL.
3. Dispense 4 mL of the cell suspension (2,475,000 cells per mL) to each well of an ultra-low attachment hanging drop system (with a micropore diameter of 350 µm) seated in a 6-well plate.
4. Spheroids will spontaneously form within 12 h of dispensing the cell suspension into the hanging drop device. Continue to culture for a total of 72 h (3 d) at 37 °C, 5% CO₂, and 95% humidity for the maturation of the spheroids.
5. After 72 h of culture, harvest the spheroids through the pores at the bottom of the hanging drop system by placing the device into a dish with medium and swirling it gently to release the spheroids from the hanging drop device.

5. 3-D Patch Creation Using the Novel Net Mold

1. The base, bottom square plate, bottom net, and 10 layered side nets are assembled to create the novel mold with the aid of a pair of sterile forceps. First, stack and align the base, bottom square plate, and bottom net using the corner posts. Then, stack and layer the 10 side nets in alternating directions to create a net of fine stainless-steel prongs.
2. Flush the filling base with PBS or medium to reduce the surface tension and, then, transfer the assembled mold onto the filling base.
3. Wet the net mold cavity with PBS or medium in the same method. Slowly fill the spheroids into the presupposed cavity of the desired size (2 x 2 x 1 mm, 4 x 4 x 1 mm, or 6 x 6 x 1 mm).
   Note: Make sure that 120% of the anticipated volume of spheroids is fed into the cavity so that the cell aggregates are in tight connection and stay static.
4. Assemble the top net and top square plate onto the corner posts and secure the stoppers and holding tubes to prevent a washout of the spheroids.
5. Transfer the filled net mold system to a 6-well plate with 6 mL of RPMI/B-27 cell media or into a sterile container with enough medium to cover the entire assembled mold.
6. Incubate the 6-well plate with the filled mold system on a swinging shaker for 7 - 10 days at 3,000 - 4,000 x g.
   NOTE: The duration of the incubation is decided by the size of the cardiac patches. With larger patches, the incubation time will need to be increased for cardiac patch maturation.

6. Removal of the Patch from the Novel Net Mold

1. Remove the mold system from the media and place it on the sterilized handling mat in a sterile 10-cm dish.
2. Remove the holding tube, stoppers, the top square plate, and the top net. Carefully slide out the layered side nets one by one with a pair of sterile forceps. After decannulation, an intact cardiac patch is obtained atop the bottom net.
3. Pick up the bottom net with the patch using sterile forceps and transfer the bottom net into a 35-mm dish with 5 mL of RPMI/B27 media. Gently loosen the patch from the bottom net by slowly swirling the net in the dish, or with a sterile cell scraper. After detachment from the bottom net, the cardiac tissue can be cultured with RPMI/B27 media.
4. Continue to incubate the free-floating 3-D cardiac patch in the 35-mm dish. Functional synchronous beating can be observed as early as 24 h after the removal from the mold.
5. After removing the patch from the net mold, observe that its shape is maintained with integrity and the intensity of synchronous beating increases with time.
   NOTE: The 3-D net mold-based cardiac patches exhibited electrical integration of component cardiospheres after decannulation. We observed a beating frequency ranging from 60 to 80 beats per minute that persisted for more than 60 days.

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