Table 3.

Troubleshooting table.

Problem	Possible Causes	Differential Diagnosis	Solution
Cells insufficiently attach	Insufficient cell density	Check flow cell under microscope during attachment. Are cells less than confluent?	Use more cells or resuspend cells into a smaller volume.
	Cell detachment secondary to attachment	Check flow cell under microscope during attachment. Search for attached cells. Apply flow. Do attached cells subsequently detach?	Decrease flow rate.
	Insufficient DNA on cells	Use Support Protocol 2. Is MFI below 10?	Increase concentration of DNA, decrease number of cells, use alternative lifting, or use alternative DNA.
	Cells are clumping	Spot 5uL cell suspension on glass slide and inspect under microscope. Are cell clumps evident?	Increase trypsinization time. Increase pipetting when resuspending cells. Increase % BSA and/or EDTA in flow buffer. Handle cells more gently.
	Insufficient accessible DNA on surface	Use Support Protocol 3. Are fluorescent spots visible under microscope?	Use new aldehyde slides and new sodium borohydride. Increase hybridization time. Prepare new activation buffer.
	Surface occluded by cell debris	Use Support Protocol 4, then try again. Did the cell attachment improve?	Use Support Protocol 4 before starting synthesis. If this is a multistep synthesis, decrease attachment time for previous steps.
Cells attach to incorrect DNA spots	Insufficient flow rate	Check flow cell under microscope after attachment. Apply forceful flow. Did some of the inappropriate cells detach?	Increase flow rate. Alternatively, wash with PBS as warm as 37 °C to increase wash stringency.
	DNA strands improperly designed	Use DINAmelt and/or melting curve analysis to assess cross-hybridization between DNA strands.	Redesign DNA strands. Alternatively, wash with PBS as warm as 37 °C to increase wash stringency.
Cells attach non-specifically to surface	Surface insufficiently passivated	Measure sessile drop contact angle of water on freshly dried surface. Is contact angle < 90 degrees?	Use fresh silanization reagents. Increase silanization time.
	Flow cell buckling	Flow food coloring into flow cell. Is the intensity of color fainter in the center of the flow cell?	Decrease clamp pressure. Increase thickness of PDMS flow cells.
	Cells are clumping	Spot 5uL cell suspension on glass slide and inspect under microscope. Are cell clumps evident?	Increase trypsinization time. Increase pipetting when resuspending cells. Increase % BSA and/or EDTA in flow buffer. Handle cells more gently.
	Flow rate insufficient	Check flow cell under microscope after attachment. Apply forceful flow. Did some of the inappropriate cells detach?	Increase flow rate.
Cells die	Temperature too high	Use infrared thermometer to measure temperature.	Adjust amount of/distance from ice to reach target 1–4C range.
	Manipulations taking too long	Label cells with DNA and set them aside, on ice, for duration of manipulations. Compare viability (e.g., using Trypan blue) before/after.	Decrease number of synthetic steps, decrease hybridization time per step, optimize your workflow.
	Flow rate too high	Repeat experiment, reducing the flow rate as much as possible. Does viability improve?	Decrease flow rate.
Assembles fall apart during synthesis	Flow rate too high	Check flow cell under microscope after assembly. Apply flow. Do assemblies disassociate?	Decrease flow rate.
	Organoids too tall for the flow cell	Does the synthetic scheme contain any 5-step-or-greater assemblies?	Redesign synthetic scheme.
	Organoids anchored to surface at too few points	Are any >=3-step assemblies anchored by a single cell? Are any >=4-step assemblies anchored by <=4 cells?	Redesign synthetic scheme and/or pattern.
	Insufficient DNA on cells	Use Support Protocol 2. Is MFI below 10?	Increase concentration of DNA, decrease number of cells, use alternative lifting, or use alternative DNA.
	Temperature too low	Use infrared thermometer to measure temperature.	Adjust amount of/distance from ice to reach target 1–4C range.
Assemblies grow at inappropriate rate	Cells are clumping	Spot 5uL cell suspension on glass slide and inspect under microscope. Are cell clumps evident?	Increase trypsinization time. Increase pipetting when resuspending cells. Increase % BSA and/or EDTA in flow buffer. Handle cells more gently.
	Insufficient DNA on cells	Use Support Protocol 2. Is MFI below 10?	Increase concentration of DNA, decrease number of cells, use alternative lifting, or use alternative DNA.
Gel does not flow into flow cell.	Viscosity too high.	Prepare a solution of 85% glycerol at 20 °C. Is your matrix more viscous than this?	Reformulate matrix. Consider lowering bulk protein concentration.
	Matrix prematurely gelling.	Once gel ceases flowing, pry the flow cell off the surface. Is gelled material left behind? (this is a destructive assay)	Reformulate matrix and/or adjust temperature/light/speed to prevent premature gelation.
Gel distorts/fractures	Poor manual manipulation	Was the blade flush with the flow cell during 3D transfer? Were your movements smooth and sure?	Practice on empty flow cells. Drink less coffee.
	Failure to remove gel outside flow cell	Before 3D transfer, are there traces of gel outside the flow cell but still connected to the gel within the flow cell?	Use razor blade to cut off excess gel.
	Gel shrinkage/swelling	Are air bubbles evident within the flow cell after 3D transfer? Has gel pulled away from the inlet and outlet?	Reformulate matrix. Consider adding surfactants.
	Inadequate gel toughness	If the gel is gently poked with a capillary tube, does it fracture?	Reformulate matrix. Consider increasing bulk protein concentration.
	Gel curls on itself	During 3D transfer, once the gel is released from the surface and inverted, does the gel ball up?	Add surfactants such as 1% BSA to gel, or increase gel density.
Gel remains on surface during 3D transfer	Surface insufficiently passivated	Measure sessile drop contact angle of water on freshly dried surface. Is contact angle < 90 degrees?	Use fresh silanization reagents. Increase silanization time.
Gel detaches from bottom of culture vessel	Insufficient adhesion between gel layers	Add a distinct dye, such as food coloring, to each gel layer. Do the dyes indicate that only certain gel layers detached?	Reformulate matrix. Consider increasing bulk protein concentration or adding adhesive proteins.