Carboxylate modified polystyrene latex beads

The integrated microfluidic device was filled with PBS and operated at different rpm, from 1200 to 4000. The device inlet was connected to a tube of PBS solution and the outlet was connected to a 15 mL empty tube. By measuring the time for the pump to fill up the 15 mL outlet tube, we can drive the pumping flow rate at different rpm. To calculate the flow velocity, 10 μL fluorescent carboxylate-modified polystyrene latex beads (Sigma, Saint Louis, MO, USA) were dispersed in 2 mL distilled water; the beads have an average size of 1 μm. Diluted beads solution was injected into the integrated pump for the experiments. The pump was then run at 1500 rpm, which resulted in a flow rate around 50 μL/min. Fluorescent pictures were taken at 100 ms exposure time inside the cell differentiation chamber. The flow velocity was calculated by measuring the travel distance divided by the 100 ms exposure time.

GelMA-myoblast constructs were fixed with 10% formalin for 30 min, then blocked and permeabilized for an hour with 10% normal donkey serum made up with a PBS of 0.1% TritonX-100. Immunofluorescent staining was performed for sarcomeric myosin (mouse anti-MF20, Developmental Studies Hybridoma Bank). Cells were incubated in the primary antibody (1:400) overnight at 4 °C. Cells were then incubated with the secondary antibody Alexa Fluor 594-conjugated donkey anti-mouse IgG (1:2000, Molecular Probes) and Alexa Fluor 488 Phalloidin (1:100, Thermo Fisher Scientific) for 60 min at 37 °C. Nuclei were stained with 1 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 15 min at room temperature. Samples were washed in PBS and imaged with an inverted fluorescence microscope (Olympus IX70). The 3D rendered z-stack images were taken with confocal microscopy. A total of 0.5 µm red fluorescent beads at a concentration of 25 µL/mL were added to the bioink (aqueous suspension of carboxylate–modified polystyrene latex beads, Sigma-Aldrich). After printing, the cells were then stained with Alexa Fluor 488 Phalloidin, as described above. Confocal imaging was performed with a NikonA1Plus confocal microscope using a Nikon Plan Fluor 20× DIC L N1 N.A. 0.75 objective lens, and the images were processed using NIS-Elements software (Nikon).