TABLE 2.

Troubleshooting table.

Step	Problem	Possible reason	Solution
14	Photoresist thickness is inconsistent	The hotplate is inadequately leveled	Level the hotplate before soft baking the SU-8
	Photoresist is too soft	The soft bake time is insufficient	Increase the soft bake time by 2–5 h
21	Features lift off silicon substrate on development	The total exposure time is insufficient	Increase the total exposure time by 20–50%
	Features appear malformed—usually elliptical in shape instead of rectangular	The soft bake time is insufficient, leading to retention of solvent in the photoresist and diffusion of the cross-linker	Use a longer soft bake time
		The wafer is heated excessively during UV exposure or postexposure bake	Reduce the duration of each exposure, increase the cool-off interval between exposures and ensure that the wafer is baked at the appropriate temperature
46	Cell–hydrogel solution does not readily fill the PDMS mold during injection	Plasma treatment is insufficient to render the PDMS mold fully hydrophilic	Increase the time of plasma treatment
		Cell–hydrogel mixture is partially polymerized as the time between adding thrombin and injection is too long	Decrease the number of molds to be filled by the same cell–hydrogel mixture
48	Cell–hydrogel mixture in the mold disintegrates on the addition of culture medium	Thrombin is not mixed well within the cell–hydrogel solution to produce uniform cross-linking of fibrinogen. Incomplete mixing may also result in an uneven distribution of calcium ions, potentially creating points of weakness in the gel	Pipette up and down an increased number of times after adding thrombin into the cell–hydrogel solution to ensure adequate mixing
49	The formed NRSKM bundles disintegrate 1 or 2 days after the onset of spontaneous twitching	The working concentration of fibrinogen (2 mg ml−1) is too low to withstand the contractions of the differentiated myotubes	Increase the concentration of fibrinogen up to 4 mg ml−1. Higher concentrations inhibit cell spreading and alignment
	The particular design of the post array does not result in the desired cell alignment	The layout of the post array does not give rise to the adequate strain field required to induce the desired cell alignment	Either increase post length or decrease post spacing to increase the degree of cell alignment
		Alternatively, the fibrinogen concentration is too high to permit cell spreading and alignment	Alternatively, lower the concentration of fibrinogen in the gel
	The formed constructs detach from the Velcro frame	The volume of cell–gel solution adsorbed onto the Velcro frame is insufficient to yield strong anchoring	Use pipette tip to evenly spread cell–gel solution onto the entire Velcro frame after injection
	Fibrin gel adheres to the PDMS molds	Coating with pluronic solution is insufficient to prevent gel adhesion	Make sure that pluronic solution has wetted the entire PDMS surface
			Increase pluronic coating time by 1 h and pluronic concentration to 1% (wt/vol)
	Cells settle to the bottom of the gel during hydrogel polymerization	Hydrogel polymerization takes too long, which permits the cells to precipitate at the bottom	Increase relative thrombin concentration (per mg fibrinogen)
			Gelation at 37 °C can also accelerate the polymerization
50A(x) and B(xv)	Intensity of immunostaining decreases with depth below the tissue surface	Penetration of antibodies may not be sufficient to reach cell layers below the tissue surface	Apply antibodies while stirring on an orbital shaker and/or increase the incubation time by 1–2 h

NRSKM, Neonatal rat skeletal myoblasts; PDMS, polydimethylsiloxane.