Supplementary Materials

3D bioprinting of collagen to rebuild components of the human heart

A. Lee, A. R. Hudson, D. J. Shiwarski, J. W. Tashman, T. J. Hinton, S. Yerneni, J. M. Bliley, P. G. Campbell, A. W. Feinberg

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Figs. S1 to S15
  • Table S1
  • Captions for Movies S1 to S10
  • References

Images, Video, and Other Media

Movie S1
FRESH v2.0 Printing CMU. Time-lapse sequence of 3D bioprinting the letters "CMU" from an Alcian blue-stained 4% (w/v) alginate ink using FRESH v2.0. Printing is performed at room temperature and raised to > 37°C after completion, melting the gelatin support bath and releasing the print. Alginate was used instead of collagen as it provided higher contrast within the support bath, making it easier to visualize the printing method.
Movie S2
Perfusion of 10 kDa fluorescent dextran through the FRESH v2.0 printed collagen perfusion tube. Video shows the peristaltic flow of a 10 kDa fluorescein dextran through a 1.4 mm inner diameter collagen tube during the initial 2 minutes, sped up 6x. Fluorescence intensity has been pseudo-colored with the fire LUT.
Movie S3
Flythrough of a FRESH v2.0 printed collagen construct with 1 mm diameter printed pores and ~30 μ diameter micropores due to removal of the gelatin microparticle support bath. Confocal reflectance microscopy of a printed collagen construct reveals an extensive and uniform porous network purposely generated by the incorporation of the gelatin microparticles into the construct during the printing process and removal during the release process.
Movie S4
3D rendering of vascularization in the FRESH v2.0 printed VEGF doped collagen construct after 10 days in vivo. 3D visualization and flythrough of a printed collagen construct showing a multiscale vascularized network (lectin, red) and nuclei (DAPI, blue) down to vessels as small as 5 μ in diameter.
Movie S5
In vivo vascularized network within the FRESH v2.0 printed collagen construct contains red blood cells. Multiphoton imaging at 870 nm excitation of a printed collagen construct following implantation into mice. Vessels are seen at depths of up to 200 μ and contain red blood cells.
Movie S6
Calcium imaging of FRESH v2.0 printed human ventricle during spontaneous and paced contractions. Ventricles were FRESH v2.0 printed using human stem cell-derived cardiomyocytes and adult human cardiac fibroblasts. Shown in sequence are (i) side views of a ventricle during spontaneous and 1 Hz and 2 Hz field stimulated contractions. (ii) Top-down view of a ventricle during spontaneous and 1 Hz and 2 Hz field stimulated contractions. (iii) Side view of blebbistatin treated ventricle during 1 Hz point stimulation with a bipolar electrode followed by an overlaid time delay map of conduction. (iv) Top-down view of ventricle during spontaneous contractions showing two simultaneous wave fronts followed by an overlaid time delay map of conduction. (v) Side view of ventricle during spontaneous contractions demonstrating pinned rotor-like electrical activity followed by an overlaid time delay map of conduction.
Movie S7
Handling and pulsatile flow testing of FRESH v2.0 printed collagen trileaflet heart valve. A trileaflet valve 28 mm in diameter is handled after fixation with glutaraldehyde. The valve is capable of sustaining its weight in air and is printed with high enough fidelity to prevent the fusion of the leaflets, allowing for individual actuation. The valve is then placed in an in vitro pulsatile flow loop and imaged using an endoscope. The pressure in the system was lowered to 25/15 mmHg by adjusting the stroke rate and volume to 30 BPM and 30CC, respectively, to approximate the physiologic pressures experienced by the pulmonary valve in vivo.
Movie S8
FRESH v2.0 printed collagen trileaflet heart valve imaged by μCT. A 3D reconstruction of the trileaflet heart valve printed from collagen with 2% (w/v) barium sulfate. Incorporating barium sulfate into the ink increases X-ray contrast and allows for the internal geometry of the valve to be visualized. The entire valve structure is panned through first in the xz plane followed by top-down through the xy plane.
Movie S9
Computational generation, 3D printing and perfusion of MRI-templated multiscale vasculature. Movie depicts the design and fabrication process of the generative vasculature followed by perfusion of a glycerol blood substitute through the printed vascular network, with vessels ranging from 5 mm down to ~100 μ in diameter.
Movie S10
3D rendering and digital sectioning of μCT of the FRESH v2.0 printed collagen heart. An adult human heart from MRI was scaled to down to neonatal size 37 mm in diameter, FRESH v2.0 3D printed using collagen and imaged by μCT. Imaris image analysis software was used to import and visualize the μCT data (grey). Surface renderings of the heart were used to enhance visualization of the external printed features (tan). Digital sectioning of the printed heart revealed the internal architecture and detail contained within the print including the valves and trabeculae.