Protein SIX4 (SIX4) Myosin heavy chain 1 (MYH1) Myosin heavy chain eight (MYH8) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Forward Primer five -GCGCCATCCAGTACATTGAGC-3 5 CGGAGTGCCATCAGCTACATTG-3 five -TTC AAG GAG AAG TCG CGC AAC-3 five -GGCACTGTGGACTACAACATCG-3 5 –MNI137 site CTACCAAAGGCAAGGCCGAG-3 Reverse Primer five -ACGATGGACGTAAGGGAGTGC-3 five -TCCACGTTTGCTCCTCCTTCC-3 five -ACT GGG GTT GCC ATC CGA TTC-3 five -TTT CTT TCC ACC ACC GCC ACC-3 five -ATCTGCTTCAGCACTAGCGTATG-5 -ACAACTTTGGCATTGTGGAAGGG-5 -TACTTGGCAGGTTTCTCCAGGC-Gels 2021, 7,15 of5.12. Scanning Electron Microscopy (SEM) and Scanning Electron Cryomicroscopy (cryoSEM) Samples had been ready for SEM as follows: bioprinted constructs have been fixed in two.five paraformaldehyde for 30 min at 37 C, then rinsed three times using a sodium cacodylate buffer for five minutes every. Samples had been passed via successive dehydration measures in ethanol (50 , 70 , 90 , and 95 ethanol for 10 min, and 100 ethanol for 15 min). The samples have been then dried by soaking within a hexamethyldisilazane (HMDS) resolution overnight and attached around the SEM stubs. Ultimately, the samples have been sputter coated with 10 nm gold. Pictures were observed utilizing FEI Verios 460L FEGSEM under high vacuum circumstances. Samples have been prepared for cryoSEM as follows: bioprinted constructs were positioned onto a cryoSEM sample holder and plunged into liquid nitrogen (LN2) slush to snap freeze samples and stay away from ice crystal formation. Frozen samples had been placed inside a sample preparation chamber maintained at -180 C under high vacuum situations. Next, samples had been sublimated at -90 C for two minutes. Lastly, samples were gold-sputter coated for 120 s. Photos have been observed applying FEI Quantra 200 in cryoSEM mode, at -180 C beneath higher vacuum. Photos had been taken at 15 kV. five.13. Calcium Imaging Intracellular calcium transients were assessed by loading bioprinted grids with five Fluo-4 AM dye in extracellular recording remedy (145 mM NaCl, five mM KCl, two.six mM CaCl2 , 1 mM MgCl2 , ten mM Na-HEPES, and 5.six mM D-glucose at pH 7.4) [44]. After 20 min of incubation at 37 C, the dye was removed, then the cells have been incubated for an additional 20 min in fresh extracellular resolution. Activity was observed beneath a fluorescent microscope (Eclipse FN1, Nikon) and recorded with Visiview imaging software (Visitron Systems GmbH). Fluorescence images have been recorded with an iXon Ultra camera at six.67 Hz for 90 s (Oxford Instruments). Videos have been analyzed with Fmoc-leucine-d10 PPAR ImageJ software program (National Institute of Overall health). Calcium transients had been expressed as F/F ((Fmax – Frest)/Frest). 5.14. In Vivo Study A computer-aided design and style (CAD) file was developed applying Tinkercad (Autodesk Inc.) for the 3D printing of chambers to residence the bioprinted muscle and AV loop. The chamber design and style was based on equivalent devices previously described [22,45], with modifications to accommodate for the bioprinted muscle. Structures have been printed in good quality, glossyfinish settings with MED610 (Stratasys) on an Objet30 3D printer (Stratasys). The chambers were cleaned and sterilized following a previously described protocol [46]. The chambers were soaked in sterile PBS overnight just before use. Bioprinted grids of muscle have been differentiated in vitro for 1 week, prior to transfer into chambers for in vivo implantation. Two chambers were prepared with bioprinted muscle, when two additional chambers had been prepared with GelMA-only (acellular) grids to serve as controls. This study was approved by the St. Vincent’s Hospital Animal Ethics Committee (Melbourne, AEC.
