Protein SIX4 (SIX4) Myosin heavy chain 1 (MYH1) Myosin heavy chain eight (MYH8) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Forward Primer 5 -GCGCCATCCAGTACATTGAGC-3 5 CGGAGTGCCATCAGCTACATTG-3 five -TTC AAG GAG AAG TCG CGC AAC-3 5 -GGCACTGTGGACTACAACATCG-3 5 -CTACCAAAGGCAAGGCCGAG-3 Reverse Primer five -ACGATGGACGTAAGGGAGTGC-3 5 -TCCACGTTTGCTCCTCCTTCC-3 five -ACT GGG GTT GCC ATC CGA TTC-3 5 -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 were ready for SEM as follows: bioprinted constructs have been fixed in two.5 paraformaldehyde for 30 min at 37 C, then rinsed 3 times using a sodium Adaphostin supplier cacodylate buffer for five minutes each. Samples were passed via successive dehydration measures in ethanol (50 , 70 , 90 , and 95 ethanol for ten min, and 100 ethanol for 15 min). The samples were then dried by soaking in a hexamethyldisilazane (HMDS) resolution overnight and attached around the SEM stubs. Finally, the samples were sputter coated with ten nm gold. Photos had been observed employing FEI Verios 460L FEGSEM under higher vacuum situations. Samples have been ready for cryoSEM as follows: bioprinted constructs have been positioned onto a cryoSEM sample holder and plunged into liquid nitrogen (LN2) slush to snap freeze samples and keep away from ice crystal formation. Frozen samples have been placed within a sample preparation chamber maintained at -180 C under higher vacuum situations. Next, samples have been sublimated at -90 C for two minutes. Ultimately, samples have been gold-sputter coated for 120 s. Images have been observed utilizing FEI Quantra 200 in cryoSEM mode, at -180 C under higher vacuum. Photos were taken at 15 kV. five.13. Calcium Imaging Intracellular calcium transients have been assessed by loading bioprinted grids with 5 Fluo-4 AM dye in extracellular recording answer (145 mM NaCl, five mM KCl, two.six mM CaCl2 , 1 mM MgCl2 , ten mM Na-HEPES, and 5.6 mM D-glucose at pH 7.four) [44]. After 20 min of incubation at 37 C, the dye was removed, and after that the cells were incubated for one more 20 min in fresh extracellular option. Activity was observed under a fluorescent microscope (Eclipse FN1, Nikon) and recorded with Visiview imaging application (Visitron Systems GmbH). Fluorescence photos have been recorded with an iXon Ultra camera at 6.67 Hz for 90 s (Oxford Instruments). Videos were analyzed with ImageJ software program (National Institute of Health). Calcium transients had been expressed as F/F ((Fmax – Frest)/Frest). 5.14. In Vivo Study A computer-aided style (CAD) file was designed applying Tinkercad (Autodesk Inc.) for the 3D printing of chambers to property the bioprinted muscle and AV loop. The chamber style was according to equivalent devices Quin C1 Data Sheet previously described [22,45], with modifications to accommodate for the bioprinted muscle. Structures were printed in premium 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 had been soaked in sterile PBS overnight before use. Bioprinted grids of muscle were differentiated in vitro for a single week, before transfer into chambers for in vivo implantation. Two chambers had been prepared with bioprinted muscle, while two additional chambers were prepared with GelMA-only (acellular) grids to serve as controls. This study was authorized by the St. Vincent’s Hospital Animal Ethics Committee (Melbourne, AEC.
