Name: 3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells (Video) | JoVE Journal
Uploaded: 2025-03-28T14:45:00.000+00:00
Duration: 6 min 17 s
Description: Learn melt electrospinning writing to create 3D myocardial tissues using polycaprolactone scaffolds and hiPSC-derived cardiac cells. This method advances cardiac tissue engineering for improved disease modeling and drug testing.

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This research was funded by the H2020 research and innovation program under grant agreements No 874827 (BRAV<img alt="Equation 1" src="/files/ftp_upload/67847/67847eq01.jpg" style="margin: auto;" />); Ministerio de Ciencia y Universidades (Spain) through projects PLEC2021-008127 (CARDIOPRINT), PID2022-142562OB-I00 (VOLVAD) and PID2022-142807OA-I00 (INVESTTRA) funded by MICIU/AEI /10.13039/501100011033 and by the European Union NextGenerationEU/PRTR; Gobierno de Navarra Proyectos Estrat&#233;gicos IMPRIMED (0011-1411-2021-000096) and BIOHEART (0011-1411-2022-000071); Gobierno de Navarra Proyectos Colaborativos BIOGEN (PC020-021-022) and Gobierno de Navarra Salud GN32/2023. Figure 1A, Figure 2A, and Figure 4A are created with BioRender.com.

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The authors have no conflicts of interest.

To generate a 3D composite cardiac mini-tissue model that replicates the native characteristics of the human myocardium, several essential factors must be considered. These can be grouped into three main points: (1) optimizing the yield and purity of the cells for tissue fabrication, (2) printing the fibrillar scaffold to mimic the 3D mechano-environment, and (3) integrating the fibrin hydrogel in the fabrication process.
Some of the key steps to ensure a successful differentiation process inc...

Fabrication of functional human cardiac-engineered tissues holds great promise for high-impact applications. These span from the development of modeling drug cardiotoxicity and human cardiac disease to the generation of therapeutic tissues of relevant sizes1. Although the last 15 years have witnessed significant advances in the field, the fabrication of highly mimetic human myocardium is currently thwarted by difficulties in reproducing the complex structural and mechanical organization of its natural counterpart2.
On the biological side, the revolutionary cell reprogramming technology opened up the possibility of developing patient-specific therapeutic cells. Currently, human induced pluripotent stem cells (hiPSCs) are the only source of human cardiomyocytes in a personalized context. A high yield of cardiomyocytes can be obtained following robust differentiation protocols3,4. A key issue is the degree of maturation as human hiPSC-derived cardiomyocytes (hiPSC-CMs) currently used display an immature phenotype under conventional 2D differentiation conditions5. This is tied to the fact that the structural organization of the cardiac tissue is very complex, absolutely different from conventional 2D cultures. Also, the myocardium is comprised of different cardiovascular cells, including non-myocytes such as cardiac fibroblasts, smooth muscle, and endothelial cells, orderly arranged through a specific 3D structure, allowing efficient blood pumping6,7. Thus, highly structured human cardiac mini tissues composed of all the main cell types are needed to generate physiologically relevant biomimetic tissues.
The application of novel bio-fabrication approaches can break this stalemate. Amongst these, melt electrospinning writing (MEW), an advanced 3D printing technology is able to supply high precision and resolution. In MEW, an electrical field is used to deposit a molten polymer in the form of fibers in the size range of the cell, an order of magnitude smaller than conventional fused deposition modeling (FDM) 3D printing, resulting in highly defined scaffold structures8,9. MEW has been shown capable of translating a complex in silico design into a printed matrix and tuning physical properties in 3D to match those of cardiac tissue10. Using the MEW technology, it is possible to print polymeric meshes with different shapes and structures that, combined with soft cell-friendly hydrogels, generate composite tissues mimicking the micro- and macro-mechanical environment of the native adult myocardium11,12. Modifying the design of the MEW 3D scaffold has been shown to alter the resulting functionality (calcium transients)10, establishing the grounds for understanding the form-function relationship on this promising 3D-engineered system.
Here, a detailed protocol is provided for the fabrication of contractile human cardiac mini tissues from hiPSC-CMs and human iPSC-derived cardiac fibroblasts (hiPSC-CFs) and their combination within a fibrin hydrogel reinforced with 3D MEW printed structures. The addition of fibroblasts has been widely used in different 3D-engineered systems to enhance hiPSC-CMs survival and maintain tissue structure, but its use in 3D-MEW systems has not yet been explored13. The technology described here provides a versatile platform for researchers aiming to develop accurate cardiac tissue models with applications such as understanding disease mechanisms, preclinical toxicology, and drug screening. This model is suited for assessing the cardiotoxicity of established drugs such as anthracyclines (e.g., doxorubicin), tyrosine kinase inhibitors, and emerging therapies like chimeric antigen receptor T (CAR-T) cells, which have been associated with cardiac dysfunction14. Furthermore, the model holds significant potential in advancing regenerative medicine by enabling the testing of biomaterials, bioinks, and cell-based therapies aimed at improving cardiac function and restoring myocardial tissue in conditions such as myocardial infarction.

<table><tbody><tr><td>&amp;micro;-Slide 8 Well chamber coverslip (IbiTreat)</td><td>IBIDI</td><td>80826</td><td></td></tr><tr><td>0.1% Gelatin Solution (Embryomax)</td><td>Merck Millipore</td><td>ES-006-B</td><td></td></tr><tr><td>10% formalin</td><td>Sigma Aldrich</td><td>HT501128</td><td></td></tr><tr><td>12-well plates</td><td>Costar/Corning</td><td>3513</td><td></td></tr><tr><td>6-well plates</td><td>Costar/Corning</td><td>3506</td><td></td></tr><tr><td>Advanced DMEM 1x (ADMEM)</td><td>Gibco</td><td>12491015</td><td></td></tr><tr><td>Aprotinin from bovine lung</td><td>Sigma Aldrich</td><td>A1153</td><td></td></tr><tr><td>B-27 SUPLEMENT, PLUS INSULIN (50x)&amp;nbsp;</td><td>Life Technologies</td><td>A317504044</td><td></td></tr><tr><td>B27 SUPPLEMENT, MINUS INSULIN (50x)</td><td>Life Technologies</td><td>A1895601</td><td></td></tr><tr><td>Biopsy punch (6mm diameter)</td><td>Medical</td><td>BP-60F</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma Aldrich</td><td>A9647</td><td></td></tr><tr><td>CHIR-99021&amp;nbsp;</td><td>AXON MEDCHEM</td><td>AXON1386</td><td></td></tr><tr><td>Confocal Laser Scanning Microscope</td><td>Zeiss</td><td>LSM 800</td><td></td></tr><tr><td>Culture flask 175 cm</td><td>Greiner Bio-One</td><td>660175</td><td></td></tr><tr><td>Culture flask 75 cm</td><td>Falcon</td><td>353136</td><td></td></tr><tr><td>Cytometer</td><td>Beckman Coulter</td><td>CytoFlex&amp;nbsp;</td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>Sigma Aldrich</td><td>D9542</td><td></td></tr><tr><td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A-21202&amp;nbsp;</td><td></td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td><td>Invitrogen</td><td>A-21207&amp;nbsp;</td><td></td></tr><tr><td>DPBS (Mg2+, Ca2+ free)</td><td>Gibco</td><td>A314190094</td><td></td></tr><tr><td>EDTA 0.5M pH 8.0</td><td>Life Technologies</td><td>AM9260G</td><td></td></tr><tr><td>ESSENTIAL 8 MEDIUM KIT</td><td>Life Technologies</td><td>A1517001</td><td></td></tr><tr><td>FGF2 (Recombinant Human FGF-basic 154 a.a.)</td><td>Peprotech</td><td>100-18B</td><td></td></tr><tr><td>Fibrinogen from bovine plasma</td><td>Sigma Aldrich</td><td>F8630</td><td></td></tr><tr><td>Fibroblast Growth Medium 3 KIT (FGM3)</td><td>PromoCell</td><td>C-23130</td><td></td></tr><tr><td>Knockout Serum Replacement (KSR)</td><td>Life Technologies</td><td>10828028</td><td></td></tr><tr><td>Lactate (Sodium L-lactate)</td><td>Sigma Aldrich</td><td>71718</td><td></td></tr><tr><td>Matrigel Growth Factor Reduced (MGFR) Basement Membrane Matrix, LDEV-free</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>MEW 23-G needle</td><td>Nordson</td><td>7018302</td><td></td></tr><tr><td>MEW printer</td><td>QUT, Queensland University of Technology</td><td></td><td></td></tr><tr><td>MEW syringe</td><td>Nordson</td><td>7012072</td><td></td></tr><tr><td>Mouse Anti-Cardiac Troponin T Monoclonal Antibody</td><td>Invitrogen</td><td>MA5-12960&amp;nbsp;</td><td></td></tr><tr><td>Mouse Anti-DDR2 monoclonal antibody</td><td>Sigma Aldrich</td><td>SAB5300116</td><td></td></tr><tr><td>Mouse Anti-&amp;alpha;-actinin (sarcomeric) Monoclonal Antibody</td><td>Sigma Aldrich</td><td>A7811&amp;nbsp;</td><td></td></tr><tr><td>PENICILLIN - STREPTOMYCIN</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>Poly &amp;epsilon;-caprolactone (PCL), medical grade</td><td>Corbion</td><td>PURASORB&amp;reg; PC 12</td><td></td></tr><tr><td>Rabbit Anti-Vimentin Recombinant Monoclonal Antibody [EPR3776] - Cytoskeleton Marker</td><td>Abcam</td><td>Ab29547&amp;nbsp;</td><td></td></tr><tr><td>Retinoic Acid&amp;nbsp;</td><td>Sigma Aldrich</td><td>R2625</td><td></td></tr><tr><td>ROCK inhibitor Y-27632 (10mg)</td><td>Fisher Scientific</td><td>HB2297</td><td></td></tr><tr><td>RPMI 1640 (L-glutamine)</td><td>Gibco</td><td>21875034</td><td></td></tr><tr><td>RPMI 1640 (no phenol red)</td><td>Gibco</td><td>32404014</td><td></td></tr><tr><td>RPMI no glucose</td><td>Gibco</td><td>11879020</td><td></td></tr><tr><td>SB431542</td><td>SELLECK CHEMICALS</td><td>S1067</td><td></td></tr><tr><td>Spectrophotometer&amp;nbsp;</td><td>BMG Labtech&amp;nbsp;</td><td>SPECTROstar Nano</td><td></td></tr><tr><td>Thrombin (human alpha thrombin, Factor IIa)</td><td>Enzyme Research Lab</td><td>HT 1002a</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T8787</td><td></td></tr><tr><td>TrypLE Express</td><td>Life Technologies</td><td>12604021</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P2287</td><td></td></tr><tr><td>Wnt-C59</td><td>AXON MEDCHEM</td><td>AXON2287</td><td></td></tr></tbody></table>

3d human myocardial tissue generation using melt electrospinning writing of polycaprolactone scaffolds and hipsc-derived cardiac cells

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

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

Characterization of 2D hiPSC-derived cardiac cells 
In order to generate multi-phenotypic cardiac tissues, hiPSC-CMs and hiPSC-CFs are independently differentiated and characterized in vitro. With appropriate optimization and strict maintenance, the following protocol will result in a hiPSC-CMs yield of over 80% around day 9 of differentiation, giving rise to spontaneously beating cells (Figure 1A). Furthermore, purity enrichment by metabolic selection largely el...

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.

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and ...

3d-human-myocardial-tissue-generation-using-melt-electrospinning

Research

JoVE Journal

Bioengineering

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

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

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

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The presented method offers an innovative way for engineering biomimetic fiber structures in three-dimensional (3D) scaffolds (e.g., heart valve leaflets). 3D-printed, conductive geometries were used to determine shape and dimensions. Fiber orientation and characteristics were individually adjustable for each layer. Multiple samples could be manufactured in one setup.

Combining 3D-Printing and Electrospinning to Manufacture Biomimetic Heart Valve Leaflets

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

Learn melt electrospinning writing to create 3D myocardial tissues using polycaprolactone scaffolds and hiPSC-derived cardiac cells. This method advances cardiac tissue engineering for improved disease modeling and drug testing.

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells (Video) | JoVE Journal

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

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