Table 1: A troubleshooting guide to identify and resolve potential failures that can arise from this protocol.

In particular, poor adhesion of cells to the pattern can have many root causes and this guide should help with the identification and resolution of those issues.

Result	Possible Cause(s)	Suggested Fixes
Photolithography – features are cracked	Inconsistent or inadequate soft-bake	Increase time of soft-bake up to 3 minutes; verify actual temperature of hotplate and increase temperature as necessary
Photolithography – features are not sharp or have photoresist remaining within them	Under-development	Increase time that slide spends in developer solution; incorporate gentle agitation
Photolithography – features inconsistent across slide	UV light may not be centered or not focused properly	Adjust UV light setup to ensure collimated light of uniform intensity
Cells don’t adhere to patterned spots with high efficiency	Not enough DNA on surface	Confirm that DNA is present on surface by hybridizing the slide with fluorescent complementary oligos and then imaging under microscope
	Cells are inadequately labeled with CMO	Add fluorescent complementary oligos to cell suspension and confirm fluorescence via flow cytometry
	Not enough cells over pattern	Collect cells by washing out from PDMS flow cell, centrifuge, and re-suspend in lower volume to concentrate the cells
	Too much remaining CMO in cell suspension, hybridizing with DNA on slide	Add another wash step. Be sure to remove as much supernatant as possible with each wash.
	Too much internalization of CMO due to time and temperature	Work quickly after labeling the cells with CMO; keep cells and slide on ice and use ice-cold reagents
Cells clump	Cells were not adequately separated during trypsinization	Use PBS + 0.04% EDTA during cell washes; pass cell suspension through 35 µm filter before the final wash
Cells adhere non-specifically	If in one specific area – could be due to scratches on slide, misalignment of PDMS flow cells, or spillage of DNA outside the pattern region	Avoid scratches, be careful to align the PDMS flow cells to the pattern region
	If cells are adhering everywhere – inadequate blocking or washing	Add in more washes after patterning the cells; pipet more vigorously during washes; block with 1% BSA for longer before starting cell patterning; silanize slide (optional step 3) or confirm silanization was successful by measuring contact angle of water droplet
Bubbles form within flow cell	Pipetting errors, uneven hydrophilic surface created during plasma oxidation	If bubbles are small, add PBS to the inlet of the flow cell and they may be washed out. If bubbles are larger, apply gentle pressure to the PDMS flow cell, nudging the bubbles towards the inlet or outlet.
Cells initially adhere to pattern but are removed during washes, patterning of other cell types, or adding the hydrogel precursor	The shear forces from pipetting too vigorously can cause the cells to detach from the surface	Pipet more gently during subsequent washes, rounds of cell patterning, or adding hydrogel precursors. Because the hydrogel precursors are viscous, they are more likely to cause the pattern to dislodge, so take extra caution. Multilayered structures tend to be top-heavy and are more susceptible to being dislodged.
Tissue deforms during 3D transfer	Hydrogel sticks to slide	Confirm hydrophobicity of slide using contact angle measurements
		Use razor blade to lift PDMS fully on both edges, allowing PBS to float under the tissue
		This can happen with pure collagen hydrogels – consider adjusting the protein concentration or composition of hydrogel
	Cells don’t transfer with the hydrogel and remain on the slide	Increase Turbo DNAse concentration or increase incubation time
	Hydrogel is not solid enough	Increase incubation time and/or the gelation mechanism for the hydrogel in question (e.g. for collagen, make sure pH is correct)
	Hydrogel tears upon removing PDMS	Make PDMS flow cells hydrophilic using plasma oxidation before beginning experiment so that they detach easily upon adding media. Use forceps very gently to detach the PDMS.