3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

This study aims to develop biometric 3D cardiac tissues using melt electric scaffolding and human iPSC-derived cardiac cells to improve disease modeling, drug testing, and regenerative medicine applications. Human-induced report in stem cell cardiomyocytes remain immature, limiting their functionality. Additionally, it is challenging to reproduce the severe complexity required to model cardiac tissue in 3D.

This model better mimics the native myocardium, enabling complex 3D extracellular matrix interactions for disease modeling, drug testing, and patient-specific applications. It offers a more relevant alternative to 2D cultures and animal models. Going forward, our research will focus on investigating cardiomycology models, enhancing 3D tissue maturation protocols, and developing larger constructs for preclinical myocardial infarction therapies in large animal models such as pigs.

To begin, connect the syringe to a pressurized nitrogen supply pipe and introduce it inside the heating chamber. Turn on the melt electrospinning writing equipment and set the temperature regulators to 80 degrees Celsius for the chamber and 65 degrees Celsius for the nozzle. After 30 minutes, move the collector plate until the print head is positioned at one edge of the plate or any desired location.

Adjust the distance between the heating chamber and the collector plate manually to 10 millimeters in the z-plane. Close the door of the equipment, which automatically connects the electric field supply. Set the voltage to seven kilovolts in nitrogen pressure to two bars for extrusion through the 23-gauge tip.

Load the design G Code in the software to print scaffolds with a square pattern geometry. Adjust the collector speed to 1080 millimeters per minute. Then press the Cycle Start button to initiate printing.

After printing is finished, carefully remove the scaffold from the collector. Cut the printed mesh using a six-millimeter diameter punch to obtain the final scaffolds for tissue fabrication. Treat the scaffolds for five minutes with oxygen plasma.

Sterilize the meshes by immersing them in 70%ethanol for 30 minutes. Wash extensively with sterile distilled water for 30 minutes, then leave them to dry. After detaching the human iPSC cardiomyocytes, re-suspend the cell pellet in the tissue generation medium and count the cells using the Neubauer chamber.

Similarly, after detaching the human iPSC cardiac fibroblasts, re-suspend the cell pellet in the tissue generation medium and count the cells. Mix the needed total cells in a new tube and refer to the contents as Cell Mix. And centrifuge at 300G for five minutes at room temperature.

Then re-suspend the cell mix in the required volume of tissue generation medium. To generate Hydrogel Mix, add the required volume of fibrinogen to the tube at room temperature and mix carefully. Seed the Hydrogel Mix onto a polytetrafluoroethylene surface to prevent fibrin adhesion to the plate.

Pipette half the volume of the tissue, leaving it as a drop. Place a polycaprolactone, or PCL, scaffold on top of each drop and add the remaining volume onto the scaffold. Now, add the required amount of thrombin and quickly mix the hydrogel, carefully avoiding bubbles.

Incubate the tissues at 37 degrees Celsius for one hour to complete the fibrin polymerization. Gently pick up each tissue by the edge using sterile tweezers and transfer them to 12 well plates containing tissue generation medium supplemented with aprotinin. Incubate the tissues at 37 degrees Celsius for 24 hours.

On the following day, refresh the medium with two milliliters of tissue maintenance medium, removing KSR and Y-27 residues. Seeding of an eight to two mixture of human iPSC cardiomyocytes and human iPSC cardiac fibroblasts into fibrin hydrogels results in uniform cell distribution across the scaffold pores within one hour after polymerization. Confocal immunofluorescence images showed a mixed cell distribution of the cells through the 3D tissue interacting with the PCL scaffold with a majority of human iPSC cardiomyocytes stained for sarcomeric actinin interspaced with vimentin-labeled human iPSC cardiac fibroblasts.

Human iPSC cardiomyocytes exhibit a well-organized sarcomere structure by regularly-spaced sarcomeric actinin protein. Cells began spontaneous beating by day two with a mean beating frequency of 30 beats per minute at day seven, showing stabilized contraction throughout the entire mesh. A gradual decline was observed over time, reaching 17 BPM by day 14.

Despite this, metabolic activity remained stable between day seven and day 14, confirming sustained cell viability. Finally, track point analysis of the beating tissues showed a contraction speed of 38 micrometers per second and a contraction amplitude of 29 micrometers.