Bioprinting Vascularized Cardiac Patches

iPSCs Culture: iPSCs were generated from omental stromal cells and were a kind gift from Dr. Rivka Ofir, Ben Gurion University. The undifferentiated cells were cultivated on culture plates, pre coated with Matrigel (BD, Franklin Lakes, New Jersey), diluted to 250 µg mL⁻¹ in Dulbecco's modified Eagle medium (DMEM)/F12 (Biological Industries, Beit HaEmek, Israel). Cells were maintained in NutriStem (Biological Industries) medium containing 1% penicillin/streptomycin (Biological Industries) and cultured under a humidified atmosphere at 37 °C with 5% CO₂. Medium was refreshed daily and cells were passaged weekly by treatment with 1 U mL⁻¹ dispase (Stemcell Technologies, Vancouver, Canada) followed by mechanical trituration.
Cardiomyocyte Differentiation from iPSCs: Cells were differentiated as previously described.9, 31 Briefly, growth media (NutriStem) was refreshed daily until iPSCs reached 100% confluence. At this point (day 0) medium was changed to RPMI (Biological Industries) supplemented with 0.5% glutamine (Biological Industries), B27 minus Insulin (X50, Invitrogen, Carlsbad, California) and 10 × 10⁻⁶m CHIR‐99021 (Tocris, Bristol, UK). Medium was refreshed every other day. At day 2, CHIR‐99021 was removed from media. At day 4, 5 × 10⁻⁶m IWP‐2 (Tocris) was added to media and was removed on day 6. At day 8, contracting implants were observed and medium was changed to medium supplemented with 0.5% glutamine, B27 minus retinoic acid (×50, Invitrogen), and 1 × 10⁻⁶m retinoic acid (Sigma‐Aldrich). After day 10, medium was changed to M‐199 (Biological Industries), supplemented with 0.1% penicillin/streptomycin, 5% fetal bovine serum (FBS, Biological Industries), 0.6 × 10⁻³m CuSO4 · 5H2O, 0.5 × 10⁻³m ZnSO4 · 7H2O, 1.5 × 10⁻⁶m vitamin B12 (Sigma‐Aldrich), this media was refreshed every other day.
Endothelial Cell Differentiation from iPSCs: Cells were differentiated as previously described with modifications.9, 32 Briefly, After iPSCs reached ≈90% confluence (day 0), medium was changed to 50% (v/v) Neurobasal (Invitrogen) 50% (v/v) DMEM/F12 (Biological Industries), supplemented with 1% penicillin/streptomycin, 1% glutamine, B27 minus retinoic acid, N2 supplement (×100, Invitrogen), 1% nonessential amino acids (Invitrogen), 10 × 10⁻⁶m β‐mercaptoethanol (Gibco, Welltham, Massachusetts), 8 × 10⁻⁶m CHIR‐99021 (Tocris), and BMP4 20 ng mL⁻¹ (R&D, Minneapolis, Minnesota). On day 3, medium was changed to EGM‐2 (Lonza, Basel, Switzerland), supplemented as according to the manufacturer instructions, and was refreshed every other day.
Cell Dissociation from Matrigel‐Coated Plates: Cells grown on Matrigel‐coated plates were dissociated by enzyme digestion with collagenase type II (95 U mL⁻¹, Worthington, Lakewood, New Jersey) and pancreatin (0.6 mg mL⁻¹, Sigma‐Aldrich) in DMEM (37 °C, 30 min), followed by TrypLE express (STEMCELL) treatment.
Mathematical Modeling: Anonymous CT Images of a human heart have been contributed by the courtesy of Tel Aviv Sourasky Medical Center, Israel. The digital data file was then analyzed using RadiAnt DICOM viewer (Medixant). The left ventricle major blood vessels were segmented and measured. Based on these measurements, a 3D model of the cardiac patch was generated using COMSOL Multiphysics software. Oxygen concentration profile was calculated based on Fick's second law, Michaelis–Menten equations and the following data: Maximum cellular O² consumption rate of 5.44 × 10⁻² nmol s⁻¹ cm⁻³, Michaelis–Menten constant for oxygen consumption of 3.79 nmol cm⁻³, diffusion coefficient (oxygen in hydrogel) of 1 × 10⁻⁹ m² s⁻¹ (Figure S1c,d). The model was then supplemented with blood vessels, ensuring that no region reach critical oxygen concentration (2.64 × 10⁻³ mol m⁻³).28, 33Fluorescent Endothelial and Fibroblast Cell Culture: Red fluorescent protein‐expressing human neonatal dermal fibroblast (RFP‐HNDF) cells (Angio‐Proteomie, Boston, Massachusetts) were grown in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1% glutamine, 1% nonessential amino acids, and 0.2% β‐mercaptoethanol. Green/red fluorescent protein‐expressing primary human umbilical vein endothelial cells (GFP/RFP‐HUVECs, Angio‐Proteomie) were maintained in EGM‐2.
Neonatal Cardiac Cell Isolation: Neonatal cardiac cells were isolated according to Tel Aviv University ethical use protocols from intact ventricles of 1‐ to 3‐day‐old neonatal Sprague‐Dawley rats as previously reported.34 Cells were isolated using 6 cycles (37 °C, 30 min each) of enzyme digestion with collagenase type II (95 U mL⁻¹) and pancreatin (0.6 mg mL⁻¹) in DMEM. After each round of digestion, cells were centrifuged (600 g, 5 min) and resuspended in M‐199 culture medium supplemented with 0.6 × 10⁻³m CuSO4 · 5H2O, 0.5 × 10⁻³m ZnSO4 · 7H2O, 1.5 × 10⁻³m vitamin B12, 500 U mL⁻¹ penicillin, and 100 mg mL⁻¹ streptomycin, and 0.5% FBS. To enrich the cardiomyocyte population, cells were suspended in culture medium with 5% FBS and were pre‐plated twice for 45 min. Cell number and viability were determined by a hemocytometer and trypan blue exclusion assay.
Bioinks Preparation: Omenta were decellularized as previously described.9 Briefly, human omenta (Helsinky #0237‐16‐ASF, Assaf Harofeh Medical Center, Israel; a consent was obtained from all subjects), or omenta from the remains of healthy pigs (Kibutz Lahav – designated for the food industry), were washed with phosphate buffered saline (PBS) (at least three human and ten pig omenta were used). Then, transferred to hypotonic buffer (10 × 10⁻³m Tris, 5 × 10⁻³m ethylenediaminete‐traacetic acid (EDTA), and 1 × 10⁻⁶m phenylmethanesulfonyl‐fluoride, pH 8.0) for 1 h. Next, tissues were frozen and thawed three times in the hypotonic buffer. The tissues were washed gradually with 70% (v/v) ethanol and 100% ethanol for 30 min each. Lipids were extracted by three, 30 min washes of 100% acetone, followed by 24 h incubation in a 60/40 (v/v) hexane: acetone solution (solution was exchanged three times in 24 h). The defatted tissue was washed in 100% ethanol for 30 min and incubated over‐night (O.N.) at 4 °C in 70% ethanol. Then, the tissue was washed four times with PBS (pH 7.4) and incubated in 0.25% Trypsin‐EDTA solution (Biological Industries) O.N. The tissue was washed thoroughly with PBS and incubated in 1.5 m NaCl (solution was exchanged three times in 24 h), followed by washing in 50 × 10⁻³m Tris (pH 8.0), 1% triton‐X100 (Sigma‐Aldrich) solution for 1 h. The decellularized tissue was washed in PBS followed by double distilled water and then frozen (–20 °C) and lyophilized. The dry, decellularized omentum was ground into powder (Wiley Mini‐Mill, Thomas Scientific, Swedesboro, NJ). The milled omentum was then enzymatically digested for 96 h at RT with stirring, in a 1 mg mL⁻¹ solution of pepsin (Sigma‐Aldrich, 4000 U mg‐1) in 0.1 M HCl. Subsequently, pH was adjusted to 7.4 using 5 m NaOH and either DMEM/F12 × 10 or PBS ×10 (Biological industries). The final concentration of decellularized omentum in the titrated solution was 1% (w/v). For the personalized bioink preparation, omentum gel 1% (w/v) was homogenized at 15 000 rpm for 2 min (Silent Crusher‐M with 8F generator probe, Heidolph Brinkmann, Schwabach, Germany) and then weighted. Subsequently, while constantly stirred, the gel was allowed to reduce under a jet of sterile air until reached 1/3 of its initial weight. The concentrated gel (2.5% w/v) was then centrifuged at 300 g for 2 min to remove air bubbles and stored at 4 °C until use. Dissociated iPSC derived CMs or neonatal rat cardiac cells were then dispersed in M‐199 medium and mixed with the omentum gel, reaching a final hydrogel concentration of 1% w/v with cells concentration of 1 × 10⁸ mL⁻¹. The cell‐laden ink was loaded into a syringe and kept at 4 °C. Sacrificial ink: Gelatin hydrogel was prepared by dissolving 15% w/v gelatin (from porcine skin, type A, Sigma‐Aldrich) in 40 °C warmed EBM‐2 (Lonza). The solution was then filtered by 0.22 µm syringe filter and kept at 4 °C until further use. Cell‐laden gelatin ink was generated by dispersing ECs in warm EGM‐2 medium, mixed with prewarmed gelatin ink at a 1:2 v/v ratio, reaching a final concentration of 10% w/v gelatin and 1.5 × 10⁷ cells mL⁻¹. HNDF cells were added to the bioink to a final concentration of 3 × 10⁶ cells mL⁻¹. The cell‐laden ink was then loaded into a syringe and allowed to cool to room temperature (22 °C).
In the support bath method, in order to form the blood vessel perimeters, the personalized hydrogel bioink was used, encapsulating ECs at a concentration of 2 × 10⁷ cell mL⁻¹.
Support Medium Preparation: For the generation of the printing support medium, an aqueous solution containing 0.32% (w/v) sodium alginate (PROTANAL LF 200 FTS, a generous gift from FMC BioPolymer), 0.25% (w/v) Xanthan gum (XANTURAL 180, kindly provided by CP Kelco), and 9.56 × 10⁻³m calcium carbonate (as suspension, Sigma‐Aldrich) was prepared. While constantly stirred, the mixture was supplemented with freshly prepared, predissolved d‐(+)‐gluconic acid δ‐lacton (Sigma‐Aldrich) to reach a final concentration of 19.15 × 10⁻³m. This results in a slow decrease in the pH and solubilization of the calcium carbonate and liberation of the calcium ion that crosslinks the alginate. When the solution's viscosity is increased to a level that prevents precipitation of the calcium carbonate, the stirring was stopped and the mixture was incubated at RT for 24 h. Double distilled water at four times the volume of the resulted hydrogel were then added, followed by homogenization at 25 000 rpm (HOG‐020 homogenizer with GEN‐2000 generator probe, MRC ltd, Israel). The homogenate was centrifuged at 15 800 g for 20 min. The pellet was resuspended in DMEM/F12 (HAM) 1:1 culture media (Biological Industries) and centrifuged again, after which the supernatant was discarded. The pellet was then supplemented with 1% (w/v) xanthan gum in DMEM/F12 (HAM) 1:1 media (reaching a final concentration of 0.1%) followed by vigorous vortexing to homogenize the mixture.
Cardiac Patches Printing Process: Cardiac patches were printed using 3DDiscovery printer (regenHU, Villaz‐Saint‐Pierre, Switzerland). The bioinks were extruded through 30G needles onto glass slides. First, the CMs cell laden omentum gel was extruded in a crisscross geometry, creating the two lower layers of the patch. The third layer was composed of omentum gel, creating the supporting walls between which ECs laden gelatin ink was deposited to generate the vascular network. On top, two layers of crisscross CMs cell laden omentum gel were extruded, encapsulating the printed vessels. The printed patches were then incubated at 37 °C for 30 min to crosslink the omentum gel and to liquefy the gelatin, followed by submerging in EGM‐2 media for further culturing.
Printing in a Support Bath: Support medium was transferred into a transparent, open sterile plastic box immediately prior to printing. The a‐cellularized or cellularized constructs were then printed (3D Discovery printer) by extrusion (through 30G needles) according to designs generated by BioCAD drawing software (regenHU) or according to data from STL files (sliced and processed by BioCAM software (regenHU)), which were downloaded from Thingiverse (www.thingiverse.com) (“Spheres in sphere” by Syzguru11 (modified), under the Creative Commons – Attribution license‐ CC BY 3.0 – https://creativecommons.org/licenses/by/3.0/; “Hand” by Teak (unmodified), under the Creative Commons – Attribution license – CC BY 3.0 https://creativecommons.org/licenses/by/3.0/; “Anatomical Human Heart” by 517860 (modified), under the Creative Commons – Attribution – Share Alike license – CC BY‐SA 3.0 https://creativecommons.org/licenses/by‐sa/3.0/). The cellularized constructs were printed using two omentum bioinks containing CMs and ECs. To improve visualization, constructs could be printed with bioinks supplemented with 1 µm blue or red polystyrene microparticles (Sigma‐Aldrich) or with lipid nanoparticles encapsulating cy3 or cy5 molecules, which were a kind gift from Prof. Dan Peer, Tel Aviv University. Upon completion of the printing process, the box was incubated at 37 °C for 45 min to crosslink the personalized hydrogel. Then, support medium was gradually aspirated and replaced with EGM‐2 medium containing alginate lyase 1 U mL⁻¹ (Sigma‐Aldrich). The printed construct was then cultured O.N., allowing final, complete degradation of the alginate particles. Finally, the medium was changed to fresh EGM‐2 medium for further culturing.
Rheological Properties: Rheological measurements (n = 3) were taken using Discovery HR‐3 hybrid Rheometer (TA Instruments, DE) with 8 mm diameter parallel plate geometry with a Peltier plate to maintain the sample temperature. The samples were loaded at a temperature of 4 °C, which was then raised to 37 °C to induce gelation; during which the oscillatory moduli of samples were monitored at a fixed frequency of 0.8 rad s⁻¹ and a strain of 1%. Compression tests on the printed or decellularized35 hearts were performed with 20 mm diameter parallel plate geometry which compressed the samples at a rate of 5 µm s⁻¹.
Immunostaining, Confocal Imaging, and Calcium Imaging: Cells/tissues were fixed in 4% formaldehyde, permeabilized with 0.05% (v/v) triton X‐100 and blocked with PBS, 1% bovine serum albumin, 10% FBS, and stained with primary antibodies followed by secondary antibodies (as indicated in the antibody list). Cells/tissues were imaged using an upright confocal microscope (Nikon ECLIPSE NI‐E) and inverted fluorescence microscope (Nikon ECLIPSE TI‐E). Images were processed and analyzed using NIS elements software (Nikon Instruments). Representative images from at least three different experiments were chosen. For calcium imaging, the cardiac patches were incubated with 10 × 10⁻⁶m fluo‐4 AM (Invitrogen) and 0.1% Pluronic F‐127 (Sigma‐Aldrich) for 45 min at 37 °C. Cardiac patches were then washed in culture medium and imaged using an inverted fluorescence microscope. Videos were acquired with an ORCA‐Flash 4.0 digital complementary metal‐oxide semiconductor camera (Hamamatsu Photonics) at 100 frames s⁻¹.
Antibody and Dyes List: Antibodies for NKX2‐5 (ab91196, 1:500), Troponin (ab47003, 1:100), CD31 (ab32457, 1:100), OCT4 (ab27985, 1:100), Ki67 (ab16667, 1:250), and Cytopainter deep red (ab138894) were acquired from Abcam (Cambridge, MA). Antibodies for actinin (A7811, 1:500) were acquired from Sigma‐Aldrich. Antibodies for Vimentin (1117481A, 1:100) were acquired from Invitrogen. Secondary antibodies: FITC‐conjugated goat anti‐mouse (ab6785, 1:800) and Alexa Flour 555‐conjugated goat anti‐mouse (ab150118, 1:500) have been acquired from Abcam. Alexa 647‐conjugated goat anti‐mouse (115‐605‐003, 1:500) and Alexa Fluor 488‐conjugated goat anti‐rabbit (111‐545‐144, 1:500) have been acquired from Jackson (Pennsylvania). For nuclei detection, the cells were incubated for 3 min with Hoechst 33258 (1:100) (Sigma‐Aldrich).
Viability Assay: Cell viability was determined using a Live/Dead fluorescent staining with fluorescein diacetate (Sigma‐Aldrich, 7 µg mL⁻¹) and propidium Iodide (Sigma‐Aldrich, 5 µg mL⁻¹) for 10 min at 37 °C. The number of live and dead cells was determined by manual counting using NIS Elements software (Nikon) from at least three different microscopic field (n ≥ 3 in each experiment), visualized by inverted fluorescence microscope.
Scanning Electron Microscopy (SEM): Human omentum hydrogel samples were fixed with 2.5% glutaraldehyde (24 h at 4 °C), followed by graded incubation series in ethanol–water solutions (25–100% (v/v)). All samples (n ≥ 3) were critical point dried, sputter‐coated with gold in a Polaron E 5100 coating apparatus (Quorum technologies, Lewis, UK) and observed under JSM‐840A SEM (JEOL, Tokyo, Japan).
Statistical Analysis: Statistical analysis data are presented as means ± s.d. Differences between samples were assessed by student's t‐test. p < 0.05 was considered significant. ns denotes not significant. Analyses were performed using GraphPad prism version 6.00 for windows (GraphPad Software).