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

Summary

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.

Abstract

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.

Introduction

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 protocols^3,4. 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 pumping^6,7. 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 structures^8,9. 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 myocardium^11,12. 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.

Protocol

The details of the reagents and the equipment used in this study are listed in the Table of Materials.

1. Media and reagent setup

1. Pre-coating of cell culture plates
   1. Prepare Matrigel Growth Factor Reduced (MGFr) stock aliquots. Defrost one vial of MGFr overnight in the fridge on ice. In a sterile hood, using refrigerated tips and keeping the stock and tubes on ice at all times, make aliquots of suitable volume by diluting the stock 1:1 in cold RPMI 1640 L-glutamine (RPMI).
      NOTE: For hiPSC culture and the seeding of hiPSC-cardiomyocytes (hiPSC-CMs), different dilutions of MGFr are used. The MGFr coating dilution for hiPSCs varies depending on the specific cell line. However, once the cardiomyocyte stage is reached, 1:80 dilution is used regardless of the cell line.
   2. For coating cell culture plates, slowly defrost the required number of MGFr aliquots on ice and further dilute them in cold RPMI, 1:180 for hiPSC culture (100 µL of MGFr in 9 mL of RPMI) and 1:80 for hiPSC-CMs replating (100 µL of MGFr in 4 mL of RPMI).
      1. Immediately add the diluted MGFr at a volume of 1 mL per well for 6-well plates (hiPSC maintenance) and 0.5 mL per well for 12-well plates (hiPSC-CMs replating). Store the plates at 4 °C before use (up to 1 week).
         NOTE: MGFr-coated plates should be warmed at 37 °C for 30 min before cell seeding. Before plating the cells, aspirate the liquid from the MGFr-coated plate.
   3. For hiPSC-CFs culture, coat T75 or T175 flasks with 0.1% gelatin for at least 15 min at room temperature (RT) prior to use, at a volume of 5 mL for T75 or 10 mL for T175 flasks. After incubation, aspirate the liquid before cell seeding.
2. Cell culture media
   1. For hiPSC culture, use Complete Essential 8 Medium (E8 medium) by adding Essential 8 Supplement (50x) to Essential 8 Basal Medium.
   2. For cardiomyocyte differentiation, prepare:
      1. RPMI/B27 without insulin (-INS medium): Mix 500 mL of RPMI and 10 mL of B27 without insulin supplement; RPMI/B27 (B27 medium): Mix 500 mL of RPMI and 10 mL of B27 supplement.
   3. For cardiomyocyte purification (Lactate medium), add 2 mL of 1 M lactate into 500 mL of glucose-free RPMI 1640. The medium can be stored at 4 °C for up to 1 month.
   4. For cardiomyocyte replating (Replating Medium), supplement B27 medium with 10% Knock-out serum replacement (KSR) and 10 µM of Y27.
      NOTE: KSR is a defined, serum-free formulation that replaces FBS in ESC and iPSC culture protocols.
   5. For fibroblast differentiation and maintenance (Day 11 onwards), prepare the complete Fibroblast Growth Medium 3 (FGM3) as specified by the manufacturer. Add its supplement pack (0.1 mL/mL fetal calf serum, 5 µg/mL recombinant human insulin, and 1 ng/mL human basic fibroblast growth factor, FGF2) to the FGM3 basal medium.
      NOTE: To promote fibroblast proliferation, increasing the FGF2 concentration to 10 ng/mL is recommended.
   6. For 3D cardiac tissue generation and maintenance use, prepare:
      1. Tissue Generation Medium: Supplement B27 medium with 1% Penicillin/Streptomycin (P/S), 10% KSR and 10 µM of Y27. Tissue Maintenance Medium: Prepare B27 medium with 1% P/S and aprotinin (0.1% (wt/vol); 33 µg/mL).
         NOTE: Aprotinin allows fibrin gel integrity during the time in culture, preventing its degradation and maintaining the cells within the hydrogel. Therefore, it is highly recommended that it be added to the medium on the same day the tissue is generated.
3. Small molecule and growth factor stock solutions
   1. CHIR-99021 (CHIR): For the GSK3 inhibitor and Wnt signaling activator, prepare a 10 mM stock solution by dissolving it in DMSO. Store aliquots at -20 °C for up to 6 months.
      NOTE: The hiPSC lines may respond to CHIR treatment differently. Optimization of CHIR concentration may be required. 6-10 µM CHIR testing is recommended.
   2. C59, Wnt inhibitor: Prepare a 10 mM stock solution by dissolving it in DMSO. Store aliquots at -20 °C for up to 6 months.
   3. Y-27632, ROCK inhibitor (Y27): Prepare a 10 mM stock by dissolving it in DMSO. Store aliquots at -20 °C for up to 1 month. The final concentration depends on the target cell type. Use 2 µM (1/5000) for hiPSCs and 10 µM (1/1000) for hiPSC-CMs, hiPSC-CFs, and tissue generation.
      NOTE: Y27 promotes cell survival by suppressing cell death in suspension during detachment. Use it when harvesting to avoid excessive cell death.
   4. Retinoic acid: Prepare a 30 mM stock solution by dissolving it in chloroform. Store the aliquots at -20 °C for up to 2 years.
   5. FGF2 (Recombinant Human Fibroblast Growth Factor, also FGF-b, promotes fibroblast proliferation): Prepare a 50 µg/mL stock solution by dissolving it in sterile water. Store the aliquots at -20 °C for up to 6 months.
   6. SB431542 (SB), TGFß1 signaling inhibitor: Prepare a 10 mM stock solution by dissolving it in DMSO. Store the aliquots at -20 °C for up to 6 months.
      NOTE: Add it to fibroblast medium to maintain CFs state and prevent myofibroblast transition (the pathological phenotype arising during fibrosis) in 2D culture.
4. Stock solutions for fibrin hydrogel
   1. Fibrinogen: Prepare a 200 mg/mL stock solution. First, grind the lumps of fibrinogen to a fine powder using sterile tools inside a cell hood. Add warm 0.9% (wt/vol) NaCl solution and keep it at 37 °C until fully dissolved. Store the aliquots at -80 °C up to a year; at 4 °C for 1 week.
      NOTE: Because of its viscosity at 4 °C, defrost it and keep it at RT sometime before the tissue generation step so it can be pipetted accurately. If an aliquot cannot be easily pipetted when reused, it is recommended to discard it and use a new one.
   2. Thrombin: Prepare a 100 U/mL stock solution by dissolving thrombin in sterile 60% PBS + 40% water. Store the aliquots at -20 °C for up to a year at 4 °C for up to 6 months.
      NOTE: Prior to use, defrost it and keep it on ice until the tissue generation step is completed.
   3. Aprotinin: Prepare a 33 mg/mL stock solution by dissolving aprotinin in sterile water (100 mg of powder in 3.04 mL of water). Store the aliquots at -20 °C for up to a year.
5. Solutions for analysis
   1. FACS buffer (cell cytometry): Prepare a PBS solution (Mg²⁺ and Ca²⁺-free) plus 2.5 mM of EDTA and 5% (v/v) FBS.

2. Human iPSC culture and passage

NOTE: The procedures reported here have been performed with several hiPSC lines, including UCSFi001-A (male, kind gift of Professor Bruce Conklin, David J. Gladstone Institutes), ESi044-A (male), ESi007-A (female) and ESi044-C (female) healthy lines and ESi107-A (cardiac amyloidosis female diseased line) with minor adaptations pertaining hiPSC culture medium, passaging dilution and CHIR concentration. The protocol thereof contains the details specific to the use of UCSFi001-A. All cell culture incubations in this protocol are performed at 37 °C, 5% CO_2, and 96% humidity conditions.

1. Culture hiPSC line on 1:180 MGFr pre-coated 6-well plates. Maintain them on the E8 medium to ensure pluripotency.
   NOTE: To prevent spontaneous differentiation, it is critical to avoid overgrowth of cultures. Therefore, perform hiPSC passage when the cells are 80%-90% confluent.
2. For cell passage, aspirate the old medium, wash wells twice with 1 mL of 0.5 mM EDTA solution diluted in PBS (Mg²⁺- and Ca²⁺-free) per well, and incubate for 7 min in EDTA/PBS at RT to disrupt inter-cellular adhesions.
3. Aspirate the EDTA/PBS and harvest the cells, detaching them by vigorous pipetting with a 1000 µL-micropipette with 1 mL of E8 medium over the surface of the well until the cells are detached. Collect them in a sterile centrifuge tube.
   NOTE: Avoid pipetting more than 10-15 times, as it will increase hiPSC death.
4. From this volume, make 1:15-1:20 dilutions and seed the cells on MGF-coated 6-well plates in E8 medium + 2 µM of Y27. Maintain Y27 only the first 24 h after seeding to avoid toxicity by over-exposure.
   NOTE: Adjust the splitting ratio for other hiPSC lines to maintain the cells undifferentiated and on a healthy morphology for 4-5 days.
5. After 24 h, aspirate old medium and add fresh room temperature E8 medium enough for two days (usually 3-4 mL). After that, change the medium each day for 3-4 days.
   NOTE: Passage frequency depends on each cell line; avoid reaching 100% of confluency. hiPSCs should grow in large, flat, and compact colonies with polygonal geometry, smoothed edges, and a high nucleus/cytoplasm ratio.

3. Human cardiomyocyte differentiation

NOTE: For cardiomyocyte generation from hiPSCs (hiPSC-CMs), the described protocol is based on the monolayer differentiation methodology used by Lian et al.^3,15 and Burridge et al.⁴. With adequate maintenance, hiPSC can be differentiated over 30 consecutive passages with high differentiation efficiency. Signs of abnormal behavior are detected by spontaneous differentiation or consecutive failure of more than 4 differentiations. Regular controls, including mycoplasma testing, are recommended.

1. To initiate cardiac differentiation, harvest hiPSCs at 80%-90% confluence as described in the cell passage step above (step 2). Seed cells in 1:180 MGFr-coated 12-well plates adjusting split dilutions to achieve compact cell monolayers at 90% of confluency after 48-72 h of seeding. For this cell line, make 1:10 dilutions, and cells will achieve confluency at 72 h. Change E8 medium daily.
   NOTE: in this protocol, dilutions have been adjusted by volume, not by the number of cells, to avoid user-dependent variability. If the monolayer state is not achieved within 72 h after plating, it is highly probable that the differentiation process will not succeed. In such a case, it is advisable to discard the attempt and restart, ensuring optimal passaging efficiency.
2. When monolayers are stabilized, the differentiation process begins, considered henceforth as day 0. Then, change the medium to -INS medium supplemented with 8 µM of CHIR, the Wnt signaling activator, and incubate the plate for 24 h at 37 °C (1 mL per well).
   NOTE: Titrate CHIR concentration between 6-12 µM for each iPSC line for efficient mesoderm induction. In this step, a high level of cell death is expected, being a sign of an optimal process.
3. Day 1: On the next day, aspirate the medium from each well and wash the cells with RPMI w/o supplements (0.5 mL per well). Then, add 1.5 mL of fresh -INS medium and incubate cells for 2 days.
   NOTE: Washing steps are performed during all media changes to remove debris, dead cells, and supplement residues.
4. Day 3: To induce cardiac-lineage specification, replace medium with 1.5 mL of -INS medium supplemented with 5 µM of Wnt inhibitor C59 for 48 h.
   NOTE: Here, the GSK3 complex is formed, and ß-catenin is degraded, leading to cardiac mesodermal lineage. Some cell death is also observable after this step.
5. Day 5: Wash and replace with fresh -INS medium for 48 h at a volume of 1.5 mL per well, to promote expansion of cardiac progenitors.
6. Day 7: From this day onwards, cells are maintained in the B27 medium, with media change every 2-3 days.
   NOTE: Adjust media quantities to 2.5 mL per well to skip changing medium during the weekend, but only once this state has been reached. Normally, hiPSC-CMs start to spontaneously beat on days 7-9. First, contraction will be observed as isolated and uncoordinated clusters, but in a few days, high-purity cultures should form a uniform beating monolayer.
7. Once obtained, these contractile monolayers (usually at day 10) are subjected to a metabolic selection process consisting of 2 cycles of 72 h in Lactate medium separated by a replating step to eliminate non-cardiomyocyte cells and enrich the purity of the culture.
8. Day 10, first purification step (1^st Lactate): Wash the beating cells with Lactate medium (0.5 mL per well) and incubate them with 1 mL per well of Lactate medium for 72 h.
   NOTE: The washing step must be done with Lactate medium or the basal glucose-free RPMI 1640 to completely remove all traces of the previous glucose medium (B27).
9. Day 13: Wash and let cells recover from the first purification round with 1.5 mL per well of B27 medium for 2 days.
10. Day 15 (replating step): Between the two cycles of purification, dissociate monolayers by washing with EDTA/PBS and incubating with TrypLE for 10 min at 37 °C (0.5 mL per well).
    NOTE: The time of incubation depends on each cell line.
11. Detach the cells using a 1000 µL-micropipette and recover them with 0.5 mL per well of B27 medium supplemented with 10% KSR (v/v) in a sterile centrifuge tube.
    NOTE: Cardiomyocytes are sensitive to shear stress and strong pipetting, so try to avoid repeated pipetting steps. Other options for a lower-damage hiPSC-CM dissociation could be using TrypLE 10x or collagenase with DNase. Optimize the incubation time to ensure it is sufficient for cell detachment without causing cellular damage or requiring excessive pipetting, which could compromise cell integrity.
12. Centrifuge 10 min at 100 x g at RT and replate them in 1:80 MGFr-coated 12-well plates with Replating Medium. Seed them in 1 mL per well.
    NOTE: This will remove excess dead cells and matrix deposited during the differentiation, as these can later have a negative impact on tissue generation.
13. Day 16: After 24 h of replating, remove Y27 and KSR and keep cells in the B27 medium before the next purification step (usually 48-72 h).
14. Day 18: Second purification step (2^nd Lactate). As in the first cycle, wash and incubate the cells with Lactate medium for 72 h (1 mL per well).
    NOTE: Purification cycles can enhance culture purity by up to 30%, as observed by flow cytometry analysis of the cardiomyocyte marker cTNT (cardiac troponin T) (not shown). This process primarily reduces fibroblast and remaining hiPSC contamination. Although the minimum acceptable purity level required to proceed with tissue generation without compromising yield has not been determined, it is recommended to use cultures with a purity of at least 85%-90%. This level of purity supports improved cardiomyocyte attachment, enhances the contractile strength of the final tissue, and supports reproducibility between experiments.
15. Day 21: When lactate purification is completed, maintain purified cardiomyocytes in the B27 medium until their use, with frequent media changes (every 2-3 days).
    NOTE: Delay in usage will decrease cell yield as they will be more difficult to detach. Hence, it is advisable to use them between the day when the second lactate purification is finished and less than an additional week.

4. Human cardiac fibroblast differentiation

NOTE: To obtain human cardiac fibroblasts (hiPSC-CFs) from hiPSCs, the following protocol is based on the two-phase methodology used by Zhang et al.¹⁶. The first step is to obtain epicardial cells (hiPSC-EpiCs) by reactivating Wnt signaling pathway after cardiogenic mesoderm induction. Then, hiPSC-EpiCs are exposed to vascular development inhibitors and FGF2 to obtain cardiac fibroblasts in 18 days.

1. Ensure hiPSC maintenance, harvesting, and seeding density as described in the hiPSC-CMs differentiation protocol. Culture hiPSCs on 1:180 MGFr-coated 12-well plates to start the differentiation.
2. When compact hiPSC monolayers are achieved (Day 0), add -INS medium supplemented with 5 µM of CHIR (1.5 mL per well) for 48 h.
   NOTE: Titrate CHIR concentration for each hiPSC line for efficient mesoderm induction.
3. Day 2: Aspirate and discard the medium, wash the cells with RPMI, and replace it with 1.5 mL of fresh -INS medium for 24 h.
   NOTE: As in the hiPSC-CMs protocol, rinse cells during any media change; depending on each step, use the corresponding unsupplemented basal medium.
4. Day 3: Incubate cells with -INS medium containing 5 µM of C59 (Wnt inhibitor) for 48 h (1.5 mL per well).
5. Day 5: For replating of cardiac mesodermal cells, detach the cells by washing with EDTA/PBS and incubating with TrypLE for 5 min at 37 °C (0.5 mL per well). Collect the cells in 0.5 mL per well of Advance DMEM basal medium (ADMEM) and centrifuge for 5 min at 300 x g at RT.
6. Seed them in 1:180 MGFr pre-coated 12-well plates (1 mL per well) at a density of 20,000 cells/cm². For replating, use ADMEM supplemented with 5 µM of CHIR, 2 µM of retinoic acid, 10% KSR (v/v), and 10 µM of Y27.
7. Day 6: At 24 h after replating, refresh medium to remove Y27 and diminish KSR to 2%. Maintain the same CHIR and retinoic acid concentrations for 48 h more to achieve the development of an epicardial phenotype.
8. Day 8: Culture cells with ADMEM supplemented plus 2% of KSR for 72 h to promote the generation of epicardial monolayers.
   NOTE: hiPSC-EpiCs appear to be flat or cubic-shaped large cells grouped into single colonies. At this timepoint, when performing the first rounds of differentiation, typical epicardial cells markers such as WT1 (nuclear antigen), ZO1 (cytoplasmic membrane antigen), and TCF21 (cytoplasmic antigen) could be analyzed by FACS (flow cell cytometry) or IF (immunofluorescence staining) (not shown).
9. Day 11: For replating epicardial cells, dissociate hiPSC-EpiCs washing with EDTA/PBS and incubate with TrypLE for 5 min at 37 °C (0.5 mL per well). Collect the cells in FGM3 medium (0.5 mL per well) and centrifuge for 5 min at 300 x g at RT.
10. Replate hiPSC-EpiCs in 0.1% gelatin-coated 12-well plates at a cell density of 10,000 cells/cm². Use FGM3 medium supplemented with 10 µM of SB (a TGFß1 signaling inhibitor to avoid myofibroblast transition, usually during on-plastic culture) and 10 µM of Y27 (only for the first 24 h).
11. The following day, refresh fibroblast medium (FGM3 + 10 µM of SB) every 2 days until hiPSC-CFs are obtained, making a total of around 18 days for the whole process.
    NOTE: hiPSC-CFs should present a spindle-shaped morphology. Here, the expression of the fibroblast marker DDR2 should be analyzed by FACS and IF techniques (see steps 7.1-7.2).
12. Once the hiPSC-CFs culture is confluent, perform 1:3 cell passages in gelatin pre-coated T75 or T175 flasks. Cells reach 90% confluence in 4-6 days approximately. To detach hiPSC-CFs, use the TrypLE harvesting protocol described for hiPSC-EpiCs replating; here, Y27 is not required for passage.
13. Maintain hiPSC-CFs in FGM3 medium + 10 µM of SB until their use or freeze them at low passage at a cell density of 1-3 million cells per cryotube.
    NOTE: It is recommended to maintain hiPSC-CFs for a maximum of 6 passages before thawing new cells, as when cultured in plastic flasks long-term, the cells begin to differentiate into a more contractile myofibroblast phenotype.

5. Fabrication of Melt Electrospinning Writing (MEW) scaffolds

NOTE: This protocol uses medical grade poly ε-caprolactone (PCL) homopolymer to print fibrillar scaffolds, using a MEW printer specially designed for this purpose by QUT, Queensland University of Technology¹⁰.

1. Prepare a PCL polymer stock for printing. Charge PCL granules into a 3 mL plastic Luer-lock syringe attached to a 23 G needle and melt them at 80 °C for 2 h in the oven, gently pressing the piston to remove any bubbles whilst the polymer is melted. Prepare several syringes and store them at RT, as PCL solidifies quickly.
2. On the day of printing, introduce the syringe inside the heating chamber and connect it to a pressurized N₂ supply pipe. Turn on MEW equipment, set temperature regulators at 80/65 °C (chamber/nozzle), and keep the syringe for 30 min to ensure proper melting of the polymer.
3. Underneath the syringe, there is the collector plate, motorized and movable in the XY directions by compatible (Mach3) software. Move the collector plate (computer cursors control) until the printhead is placed onto one edge of the plate or at any desired location, and adjust the distance between the heating chamber and the collector plate (collector distance) manually to 10 mm (Z plane).
   NOTE: This distance must be experimentally tested to attain a given fiber diameter. The shorter the collector distance, the thicker the fiber, and vice versa.
4. Close the door of the equipment, which automatically connects the electric field supply. Set voltage at 7 kV and N₂ pressure at 2 bars so it can extrude through the 23 G tip when printing.
   NOTE: By supplying a high voltage, a potential difference is created between the tip of the syringe and the collector plate (made from stainless steel). Then, the drop accumulated at the nozzle by the supplied pressure becomes electrostatically charged, generating the Taylor cone, which drives towards the collector plate. Voltage and pressure can be tuned to modify the final fiber dimensions. High-pressure parameters will extrude thicker fibers, requiring increased voltage to stabilize the fiber. The software is based on computer numerical control (CNC), so scaffold geometries are written as a G code and fed to the program, which controls the X-Y movement and the speed of the collector's plate motor. Therefore, before printing the definitive scaffolds, it is recommended to print any G code as a previous step to get a stable jet that builds defined fibers without any coiling or whipping appearance. For that, optimize the parameters by small changes each time, i.e., 0.1 bar for air pressure, 0.1 kV for voltage, and 60-100 mm/min for collector speed; this will ensure a Taylor cone behavior of the jet.
5. Select the designed G-code in the software to print scaffolds with a square pattern geometry. This example G code prints 15-layer square meshes of 6 cm x 6 cm with square-shaped pores of 0.5 mm x 0.5 mm.
6. To print precisely deposited fibers (i.e., overlapping fibers), adjust the collector speed to 1080 mm/min.
   NOTE: Adjust voltage, pressure, collector speed, and collector distance parameters on each MEW equipment to define precise fiber diameters (µm). Beyond the influence of the previously mentioned parameters, increasing the collector speed results in thinner fibers, while decreasing it produces thicker fibers. The parameter settings in this protocol enable the printing of fibers with diameters of 10-15 microns.
7. Press the START button in the software to start printing. Carefully remove the scaffold from the collector after the printing is finished.
   NOTE: For easier handling of the scaffold after printing, it is recommended to print it on a glass piece larger than the scaffold, secured to the collector base with adhesive tape.
8. Cut the printed mesh with a 6 mm diameter punch to get the final scaffolds for tissue fabrication.
9. As PCL meshes are highly hydrophobic, treat them for 5 min with O₂/Argon plasma to increase their hydrophilicity and promote interaction with fibrin and cells.
   NOTE: Optionally, a 0.1 N of NaOH treatment for 15 min, followed by extensive PBS washes, also works.
10. Sterilize meshes by immersion in 70% ethanol for 30 min, wash extensively with sterile distilled water for 30 min, and leave to dry.
    NOTE: Do this process in a sterile Petri dish and inside a sterile hood. Do not completely dry out the meshes until they are used to avoid their adhesion to the plate and consequent deformation.

6. Fibrin mini tissue generation and maintenance

NOTE: The generation of human myocardial 3D mini tissues relies on the encapsulation of hiPSC-derived cardiac cells within fibrin hydrogels combined with MEW scaffolds that provide fibrillary support. The following protocol has been adapted from the engineering design approaches used by Breckwoldt et al.¹⁷and Ronaldson-Bouchard et al.¹⁸.

1. Detach hiPSC-CMs as described above in the replating step (3.10-3.11) of the hiPSC-CMs differentiation protocol (TrypLE harvesting). Resuspend the cell pellet in the Tissue Generation Medium to count the cells in the Neubauer Chamber.
2. Detach hiPSC-CFs as in the hiPSC-CMs harvesting step with TrypLE. Use 3 mL of TrypLE for T75, 5 mL for T175 flasks, and the same volume for inactivating TrypLE incubation. Finally resuspend the cell pellet in Tissue Generation Medium, as for CMs, as they are going to be mixed and cultured in the same media.
3. Count the cells in a Neubauer Chamber. Calculate the exact total number of cells needed to seed all tissues.
   NOTE: This protocol employs 1 million cells per tissue, consisting of 80% CMs and 20% CFs, i.e. 800,000 CMs and 200,000 CFs. Other quantities, such as totals of 1.5 and 3 million per tissue, are also achievable with practice.
4. Mix and pellet the needed total cells in a new tube (Cell Mix), 5 min at 300 x g at RT. Ensure that the pellet is completely dry and keep it on ice until seeding. Calculate the total volume of hydrogel required for seeding all tissues.
   NOTE: This protocol requires 35 µL per tissue (for a scaffold size of 6 mm in diameter) to a final density close to 30 million cells/mL, but it can be increased, as already mentioned in step 6.4 above. Each fibrin hydrogel consists of 6 mg/mL fibrinogen plus 5 U/mL thrombin together with the Tissue Generation Medium, in which cells are resuspended. For a 35 µL hydrogel, the Fibrinogen/Thrombin ratio is 1.05 µL/1.8 µL. A 10% extra final volume of hydrogel is recommended to avoid volume loss due to pipetting errors. Example: for 10 tissues, resuspend the cells in 350 µL + 10%, i.e., 385 µL as a total end volume.
5. Resuspend the Cell Mix in the required volume of Tissue Generation Medium. Mix carefully, avoiding the formation of bubbles.
   NOTE: For 10 tissues, resuspend 10 million cells (80 CMs:20 CFs) in 374.5 µL of Tissue Generation Medium (total volume of hydrogels minus the total amount of fibrinogen required for total tissues, i.e. 10.5 µL).
6. Add the required volume of fibrinogen and mix carefully. For 10 tissues, add 10.5 µL fibrinogen to the Cell Mix, generating the Hydrogel Mix. Keep it at RT.
   NOTE: It is essential to avoid excessively repeated pipetting so that shear forces don't affect hiPSC-CMs viability. It is recommended to cut a piece off the tip of the pipette tip with a sterile scalpel and then resuspend the Hydrogel Mix with it to reduce shear damage.
7. To avoid fibrin adhesion to the plate, seed Hydrogel Mix in a polytetrafluoroethylene (PTFE) surface. Pipette half the volume of a tissue (17.5 µL), which will remain as a drop, and do it for the total number of tissues.
   NOTE: Do not make more than 10 tissues at a time, as they can dry out.
8. Place a PCL scaffold on top of each drop and add the remaining volume (17.5 µL). Ensure that the scaffold is completely immersed.
   NOTE: Be prepared for pipetting with both hands, as the reaction is very fast. One tissue at a time.
9. Add the required amount of thrombin (1.8 µL) with your non-dominant hand and quickly mix the hydrogel with the dominant hand (pipetting at least 2-3 times).
   NOTE: Preferably mix by pipetting less than total volume, to avoid bubble formation (30 µL). If an air bubble is formed, prick it quickly with a needle.
10. Repeat the previous step for each tissue.
    NOTE: It is advisable to practice this technique without cells until good handling is ensured.
11. Incubate the tissues at 37 °C for 1 h to complete the fibrin polymerization.
12. Gently pick up each tissue by the edge with sterile tweezers and place them in 12-well plates with 2 mL per well of the Tissue Generation Medium supplemented with 33 µg/mL of aprotinin. Place one tissue per well.
    NOTE: Do not pick them up by the center of the hydrogel so that cells can be damaged by mechanical force. If necessary, use a spatula.
13. Incubate the tissues for 24 h at 37 °C.
    NOTE: It is critical to maintain aprotinin in the tissue culture medium from the day of generation onwards, as it has been observed that omitting aprotinin during the first 24 h, even if added later, leads to partial cell detachment from the mesh and hydrogel. This compromises the full cell coverage necessary for the integrity of the final tissue.
14. On the next day, refresh the medium with 2 mL of the Tissue Maintenance Medium, removing KSR and Y27 residues. Change the medium every other day from there on.

7. Systematic analysis of the cardiac differentiation potential of hiPSC and mini tissue function

1. Flow cytometry analysis
   NOTE: Cells are analyzed using the "Fluorescence-activated Cell Sorting" (FACS) cytometry modality to determine the efficiency of hiPSC differentiation. All steps are performed under room temperature conditions.
   1. Dissociate cell cultures into single-cell suspension (TrypLE harvesting), both hiPSC-CMs and CFs separately.
   2. Resuspend the pellet at 250,000 cells/mL in FACS buffer and dispense at least 1 mL per tube. Incubate for 30 min to avoid non-specific antibody binding.
   3. Centrifuge cells at 300 x g at RT for 5 min and discard the supernatant.
      NOTE: All centrifugation steps are performed under these conditions.
   4. To detect intracellular antigens, fix and permeabilize cell membranes with a cell permeabilization kit. First, incubate the cells with Reactive A (Fix) for 30 min and centrifuge (as in step 7.1.3).
   5. Then, incubate cells with Reactive B (Perm) plus the primary antibody for 30 min. Use 100 µL of reactive per tube.
      1. For hiPSC-CMs, use mouse cTNT antibody (cardiac T troponin, marker of CM) at 1/200 dilution. For hiPSC-CFs, use mouse DDR2 antibody (collagen receptor, marker of CF) at 1/500 dilution.
         NOTE: To stop all reactions, add 1 mL of FACS buffer to each tube before centrifugation.
   6. Incubate for 15 min with anti-mouse Alexa-Fluor 488 secondary antibody diluted 1/100 in FACS buffer.
   7. Wash 3 times with PBS, centrifuging between washes (following the same conditions as in step 7.1.3), and finally resuspend the cell pellet in 400 µL of PBS. Analyse in a cytometer or a similar device.
2. Immunofluorescence staining analysis (IF)
   1. For 2D cardiac cell cultures, seed both cell types separately on 1:80 MGFr or 0.1% gelatin pre-coated slide-based culture chamber well system. Plate approximately 80,000 cells/cm² to reach enough cell confluency for the analysis. For 3D mini tissues, gently transfer them with tweezers to a 2 mL microcentrifuge tube.
   2. Discard the cell medium and wash cells or tissues with PBS twice. Fix the samples with 10% formalin (v/v) at RT; 15 min for slide chamber and 1 h for mini tissues.
      CAUTION: Formalin is hazardous if inhaled and must be handled in a fume hood.
   3. Discard the fixation buffer and wash 5 min with PBS thrice. Store the samples at 4 °C in PBS until their use.
   4. To permeabilize the cell membrane, incubate the cells with 0.1% Triton X-100 (v/v) for 10 min or 3D tissues with 1% Tween-20 for 15 min at RT. Then, wash 5 min with PBS thrice.
   5. To reduce non-specific antibody bindings, treat the samples with a 3% (v/v) bovine serum albumin (BSA) solution for 30 min at RT.
   6. Prepare primary antibody solutions in PBS plus 1% BSA to block non-specific bindings and incubate them overnight at 4 °C.
      1. For 2D hiPSC-CMs, use 1/400 dilution of Anti-cardiac α-ACTN (sarcomeric actinin) mouse antibody. For 2D hiPSC-CFs, use 1/500 dilution of Anti-DDR2 mouse antibody. For 3D tissues, combine the two CM- and CF-specific antibodies.
   7. On the next day, aspirate the antibody solution and perform 3 washes with PBS at RT, 5 min each.
   8. Dilute the secondary antibodies (Alexa-Fluor 488 and Alexa-Fluor 594) at 1/200 and incubate for 1 h at RT in the dark to identify mouse and rabbit primary antibodies, respectively. Repeat the PBS wash three times.
   9. To counterstain the nuclei, incubate samples with 1/500 dilution of 4',6-diamidino-2-phenylindole (DAPI) for 20 min at RT in the dark. Repeat the PBS wash three times and store the samples at 4 °C in PBS.
   10. Examine the samples with a confocal microscope, acquire images, and process them with suitable software such as Fiji.
   11. For 3D mini tissues, transfer them carefully with PBS between two glass coverslips to allow good imaging.
       NOTE: A 40x oil immersion or higher objective is recommended to obtain Z-stack images at high resolution.
3. Cell viability analysis
   NOTE: Alamar Blue assay (AB) is used to measure cell metabolic activity, which is directly related to cell viability in 3D cardiac mini tissues. Perform the entire process protecting samples from light and under sterile conditions.
   1. Prepare 10% (v/v) AB solution in RPMI 1640 medium without phenol red (as it could interfere with colorimetric measurement).
   2. Carefully transfer cardiac mini tissues to a 48-well plate with sterile tweezers. Incubate constructs with 300 µL of AB solution per tissue for 1 h 30 min (adjust time experimentally) at 37 °C.
      NOTE: As cells reduce resazurin through the action of metabolic enzymes, a color change in the culture medium will be generated, which will be measured by spectrophotometry at 570 nm and 600 nm.
   3. For measurement, collect 100 µL per tissue of the metabolized AB solution in a 96-well plate and read absorbances.
      NOTE: Samples can be cultured after this analysis by changing the medium to Tissue Maintenance Medium once more.
4. Contraction analysis
   NOTE: Cardiac mini tissues start spontaneously to beat, usually 1-2 days after tissue generation.
   1. Manually measure the beating frequency by observing the tissues under the optical microscope (beats/min). If many tissues are measured at the same time, keep the plates warm before each reading.
      NOTE: As temperature decreases, the beating rate also starts to decrease.
   2. For extended cardiac contractility assessment, obtain optical microscopy videos of the beating tissues. At 4x to capture a larger plane or at 10x from different tissue areas. Record around 30 s per video (at least 3 beats).
   3. Use a frame rate of at least 30 frames/s and save the video in .AVI format.
      NOTE: Here, videos have been analyzed with a custom-made tracking point algorithm developed for MATLAB¹⁰. With this method, the contraction speed, contraction maximum displacement (amplitude), and contraction direction can be obtained for each video.
   4. Directly run the algorithm in MATLAB for the videos contained in the analysis folder. It will automatically provide a Results Excel document with the values of contraction speed per frame, contraction amplitude per frame, contraction angle (direction), and its respective standard deviations¹⁰.
   5. Multiply 'Contraction speed per frame' by the camera resolution (µm/pixel) and by the camera frame rate (frames/s). This will give the contraction speed final value (µm/s).
   6. Multiply 'Contraction amplitude per frame' by the camera resolution (µm/pixel), and this will give the contraction amplitude final value (µm).
      NOTE: It would be possible to analyze contraction kinetics in more depth using software such as MUSCLEMOTION, as discussed elsewhere^19,20. The imaging setup must capture at least 60 frames/s for analysis with the software.

Results

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 eliminates non-hiPSC-CMs, resulting in cultures composed of more than 90% of cells expressing cardiac troponin protein (cTNT⁺) at day 21 (Figure 1B). These robust beating monolayers show expression of the contractile sarcomeric actinin protein (ACTN) by IF staining (Figure 1C, D).

On the other side, the success of this protocol relies on generating cardiac-specific fibroblasts through an intermediate epicardial state induction. Here, after Wnt signaling reactivation by CHIR, epicardial cells are obtained at day 8 from cardiac mesoderm progenitors (Figure 2A). These hiPSC-EpiCs are large cells grouped into single colonies that, when replating with fibroblast medium, give rise to cardiac fibroblast cells with spindle-shaped morphology (Figure 2C). Thus, after 18 total days of differentiation, hiPSC-CFs present over 90% expression of DDR2 by FACS analysis (Figure 2B). This collagen receptor, characterized as a CF marker, is also observable by IF staining (Figure 2D).

Figure 1: hiPSC-CMs differentiation timeline and immunofluorescence and flow cytometry characterization. (A) The overall scheme of the differentiation of CMs derived from hiPSCs and purification steps. (B) Flow cytometry quantification of hiPSC-CMs purity (percentage of cTNT⁺ cells) at day 21. (C) Representative phase contrast image of cells after purification steps (day 21). Scale bar = 100 µm. (D) Confocal image of fully differentiated hiPSC-CMs stained with ACTN (green) and nuclei stained with DAPI (blue). Scale bar = 25 µm. Please click here to view a larger version of this figure.

Figure 2: hiPSC-CFs differentiation timeline and immunofluorescence and flow cytometry characterization. (A) The overall scheme of the differentiation of CFs derived from hiPSC-epicardial cells. (B) Flow cytometry quantification of hiPSC-CFs purity (percent of DDR2⁺ cells) at day 18 and representative phase contrast image of the culture (C); scale bar = 100 µm. (D) Confocal image of hiPSC-CFs stained with DDR2 (green) and nuclei stained with DAPI (blue). Scale bar = 25 µm. Please click here to view a larger version of this figure.

Generation of PCL scaffolds by MEW
The mechanical properties of the scaffolds are determined by the total number of layers, fiber density, diameter, distribution, and pore geometry. These are fundamental aspects that can strongly influence cell behavior. Once established, the equipment set up (Figure 3A) and following the printing settings described in this protocol (7 kV, 10 mm collector distance, 2 bar, 80/65 °C head/nozzle, 1080 mm/s collector speed and 23 G syringe tip), 15-layer square scaffolds were generated with square pore geometry of 500 µm x 500 µm (Figure 3B). PCL fibers were deposited layer-by-layer correctly, presenting a fiber diameter of around 15 µm.

Figure 3: MEW equipment and fibrillar scaffold fabrication. (A) MEW setup (i) and Mach3 software (ii); view of the printer head and the collector plate (iii), and Taylor cone formation during printing (iv). (B) Phase contrast images of the fibrillar square scaffolds (i) and amplification of the precise fiber deposition (ii). Scale bar = 250 µm. Please click here to view a larger version of this figure.

Characterization of 3D cardiac mini tissue functionality
Following single-cell dissociation, the Cell Mix composed of 8:2 hiPSC-CMs:hiPSC-CFs was seeded within fibrin hydrogels onto the MEW PCL scaffolds (Figure 4A). After 1 h of incubation at 37 °C, fibrin is stably formed, with cells uniformly distributed throughout the gel, covering the entire pores of the mesh (Figure 4B). This is important since hiPSC-CMs are mostly non-mitotic, and it is not possible to rely on cell growth to fill in the pores as with other cell types. The cells evidenced rapid remodeling of the surrounding matrix by cell elongation, with a localized spontaneous beating as early as 2 days after plating. IF staining analysis showed mixed cell distribution of the cells throughout the gel, interacting with PCL mesh, with a majority of CMs (ACTN⁺ VIM^-) interspaced with VIM⁺ ACTN^- hiPSC-CFs. hiPSC-CMs exhibit a well-organized sarcomere structure marked by regularly spaced ACTN protein staining (Figure 4C). Cardiac tissues of 1 million cells showed stabilized contraction throughout the entire mesh, with a beating frequency of around 30 bpm at day 7 (28.93 ± 11.2, mean ± SD). This functional capacity was sustained throughout all days in culture, with a small decline until day 14 (17.18 ± 12.6, mean ± SD) (Figure 4D). When analyzing metabolic activity, tissues have shown to maintain cell viability throughout culture, with no significant changes, here up analyzed as day 7 vs. day 14 (Figure 4E). Lastly, track point analysis of the tissues at 1 week showed contraction speed of 38.53 µm/s ± 19.8 (mean ± SD, n=16) and contraction amplitude of 28.91 µm ± 15 (mean ± SD, n=16) (Figure 4F).

Figure 4: Generation of human 3D cardiac mini tissues and characterization of tissue structure, functionality, and cell viability. (A) The overall scheme of tissue generation combining hiPSC-derived cardiac cells, MEW printed scaffolds, and fibrin hydrogel encapsulation. (B) Representative phase contrast images of beating mini tissues. Scale bar = 250 µm. (C) Confocal Z-stack image of 3D tissue organization; hiPSC-CMs are stained with ACTN (green), hiPSC-CFs are stained with VIM (red), and nuclei are stained with DAPI (blue). The MEW mesh is indicated by arrowheads. Scale bar = 250 µm. (D) Evolution of beating rate of the tissues from generation to day 14. Data are represented as mean with SD of N = 3, n = 2-4. (E) Cell viability (analyzed by metabolic activity) of tissues between days 7 and 14. Data are represented as mean with SD of N = 3, n = 3-6. (F) Contraction analysis of spontaneously beating cardiac mini tissues at 1 week. Contraction speed (µm/s) and contraction amplitude (µm) are represented as mean with SD (n = 16). Please click here to view a larger version of this figure.

Discussion

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 include maintaining the pluripotency characteristics of the hiPSCs by preventing confluency from exceeding 90%, optimizing the dilution of hiPSC at passaging to achieve adequate confluency at the start of CM or CF differentiation, and fine-tuning the concentration of CHIR for each cell line to ensure efficient mesoderm induction^4,15. It is essential to ensure that cultures achieve over 90% purity and that the different cardiac cell types are generated separately. This allows for the formation of functional tissues where the specific roles of each cell type can be properly studied, especially when using them for disease modeling or pharmacological testing. To maintain this high level of cell purity, it is crucial to rigorously carry out the metabolic selection of cardiomyocytes in the glucose-restrictive medium. On the other hand, when obtaining healthy cardiac fibroblasts, the ability to modulate their phenotype and transition into myofibroblasts via the TGF-β pathway offers an opportunity to model conditions like cardiac fibrosis, described elsewhere²¹. In this protocol, the focus is on generating non-activated, healthy fibroblasts. To achieve this, it is essential to maintain the TGF-β inhibitor (SB) in the culture medium after the differentiation process is completed. Additionally, hiPSC-CFs culture should not exceed six passages, as this could trigger fibroblast activation and transdifferentiation due to the supra-physiological stiffness of tissue culture plastic²².

Moving on to the process of generating artificial 3D scaffolds, MEW stands out as a highly promising technology due to its ability to produce highly ordered micro- to nanoscale fiber architectures using biocompatible materials. Compared to other additive manufacturing technologies, MEW provides superior control over fiber diameter through the adjustment of system parameters such as temperature, collection speed, and applied voltage^23,24. However, consistent and precise fiber production requires careful fine-tuning of these parameters to maintain fiber stability. Environmental factors such as room temperature and humidity can introduce minor but significant deviations in scaffold production, underscoring the need for controlled fabrication conditions. Any imbalance can result in alterations in fiber diameter or inaccurate deposition, affecting scaffold morphology and mechanical properties²⁴. Therefore, the optimization of all the above-mentioned parameters is one of the critical steps of this protocol that must be optimized in each laboratory. Here, to fabricate the fibrillar scaffolds integrated into the 3D constructs, PCL medical grade has been selected as the polymer of choice for MEW printing, as it is not only the gold standard in this field but also offers several advantageous material properties. PCL has a low melting point (~60°C) and is semi-conductive, which simplifies the processing requirements and enhances the consistency of fiber formation during the printing. From a biomedical perspective, PCL is highly biocompatible, supporting cell attachment and growth, making it ideal for tissue engineering scaffolds²⁵. Recent innovations also emphasize the use of materials like PCL due to their ability to balance biodegradability with mechanical stability, ensuring that scaffolds provide adequate support for tissue regeneration while gradually degrading without producing harmful byproducts^25,26. This is essential for the potential applicability of current constructs for in vivo cardiac regeneration studies. Also, the tunability of MEW scaffold geometries offers further potential for investigating both cell behavior and mechanical properties in tissue-engineered constructs. By modifying pore size and scaffold stiffness, MEW enables the creation of environments that simulate the extracellular matrix influencing cellular responses. This provides a versatile platform for studying cell-scaffold interactions and enhances MEW's potential for advancing the fields of Tissue Engineering and Regenerative medicine^26,27,28. In comparison to other techniques like 3D bioprinting, which offers lower precision and resolution, MEW's ability to produce ordered architectures with controlled precision adds tremendous value to its application.

Despite these advantages, MEW presents certain limitations compared to other 3D printing technologies, particularly regarding the maximum height of printable structures. The accumulation of electrostatic charges within the deposited fibers becomes problematic when scaffold heights exceed approximately 4 mm, leading to distortions in the top layers due to charge repulsion²⁹. In this case, the challenge in generating thicker tissues is less about PCL fiber repulsion, as these tissues typically do not exceed 200 µm in thickness, and more about the reduced oxygen availability in thicker constructs (beyond 200-300 µm)³⁰. This reduction in oxygen leads to hypoxia and subsequent cell death. To overcome this issue and make 3D constructs suitable for in vivo regenerative studies, future improvements in tissue thickness will require the integration of vascularization and perfusion. This is crucial, as incorporating vascular networks into 3D constructs will ensure adequate oxygen and nutrient availability throughout the tissue, which is a current focus in the field of cardiac tissue engineering^30,31,32.

It is well known that the native myocardium consists of various cardiovascular cells, including cardiomyocytes, fibroblasts, smooth muscle cells, and endothelial cells, all organized in a highly structured and aligned manner⁷. Although current tissue models incorporate only CMs and CFs, the precise arrangement of these cells, obtained from hiPSCs, within the 3D structure marks a significant advancement. The use of MEW scaffolds plays a crucial role in achieving this organization, as the scaffolds provide a well-defined microstructure that promotes proper alignment and cell organization. In this model, the cardiomyocytes exhibit well-organized sarcomeres and demonstrate strong contraction, which are key characteristics of functional heart tissue. This level of cellular alignment and organization is crucial for mimicking native heart tissue and represents a significant improvement over other models, such as cardiac organoids, where cells often exhibit random alignment³³. This model, therefore, provides a more physiologically relevant system for studying heart function and diseases, offering better control over cell organization, which is essential for the accurate modeling of cardiac tissues.

However, to develop truly representative models of heart disease, one significant challenge is overcoming the immaturity of the generated tissues. This issue largely stems from the immature nature of hiPSC-CMs obtained in vitro³⁴. These cells do not fully replicate the functional properties of adult cardiomyocytes, so enhancing their maturation is essential. This can be achieved through two primary approaches: first, by promoting the maturation of the hiPSC-CMs themselves, and second, by improving the maturity of the overall multi-phenotypic tissue. Strategies such as mechanical and electrical stimulation have shown promise in driving both functional and structural maturation, helping to create tissue models that more closely mimic native adult heart tissue^35,36. These are applicable to the present model with small changes, given the self-standing nature of the MEW-based tissues.

Regarding the technical steps of the tissue fabrication protocol, it is important to note that the CM: CF ratio embedded in the fibrin hydrogel can vary. It has been reported that for engineered cardiac constructs, a fibroblast proportion of 5%-20% is necessary to mimic the natural beating behavior of the tissues as it could increase the action potential propagation rate³⁷. In this study, a 20% hiPSC-CFs level was selected to better assess fibroblast responses in subsequent toxicology studies. The total number of cells seeded can also be adjusted, as tissues can be regularly generated with 0, 5, 10, or 20% hiPSC-CFs. Always calculate this proportion relative to the total volume of the tissue, not its diameter. Before the process of encapsulation, good cell viability must be ensured. For that, try to discard the TrypLE solution correctly when harvesting cells and avoid keeping the cells disaggregated and pelleted into single cells for too long before seeding. Additionally, as cardiomyocytes are very sensitive to mechanical stress, make sure not to centrifuge at higher g than specified, or pipetting too strongly.

For potential therapeutic applications, it is essential to encapsulate cells within a material that supports both viability and functionality. In this context, fibrin has been chosen due to its high biocompatibility, biodegradability, and ability to mimic components of the extracellular matrix³⁸. The limitations of this material arise from its significant batch-to-batch variability and relatively low mechanical strength. However, these issues are addressed through the reinforcement provided by the MEW fibrillar scaffolds. To ensure the remodeling capacity of the cells within this porous matrix, obtaining a homogeneous solution of the Hydrogel Mix is important. For that, ensure the Cell Mix pellet is sufficiently dry, discard any remaining volume that could dilute the final hydrogel solution, and mix thoroughly with thrombin. For bigger constructs, the Fibrinogen/Thrombin ratio should also be adjusted relative to the total volume of the tissue. Avoiding air bubbles is crucial for achieving a well-defined and homogeneous 3D structure, allowing cells to spread properly. Shortly after the addition of thrombin, an increase in the gel's viscosity should be noticeable, along with a slight color change from pink (Tissue Generation Medium) to yellow.

Lastly, one of the most important steps to ensure the success of the process is adding aprotinin to the medium where the tissues are transferred after the fibrin gel has polymerized for 1 h at 37 °C. Aprotinin, a serine protease inhibitor, prevents fibrinolysis (fibrin degradation) by inhibiting proteolytic enzymes such as plasmin, which are released in the body or culture^17,39. Without inhibition, these enzymes would break down the fibrin into its degradation products. By preserving the integrity of the fibrin scaffold, aprotinin allows the matrix to remain functional for extended periods, maintaining the 3D structure essential for supporting long-term cell viability and functionality in the engineered tissues.

Materials

List of materials used in this article

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