Table 2.

Guideline for the development of topographic cell culture devices with controlled or removed confounding factors.

Stage	Requirements	Strategies	Notes
Structure generation related assessments	“One structural parameter” difference	1. Select a fabrication method with high accuracy, high throughput in accordance with the desired surface geometry	Replica molding may be a feasible method, others can also be selected if meeting these requirements
		2. Select an appropriate substrate material that compatible with the chosen method	Material selection should meet other requirements
		3. Optimize the technique details and material parameters	The details need to be optimized vary with methods
	Minimum and consistent chemical properties	1. Choose a biologically inert material	Because the chemical molecules-induced cellular effects will interfere with investigating the sole topographic structures-mediated cell responses
		2. Avoid bioactive coatings
		3. Thoroughly remove the chemical residues
	Consistent structure-independent differences in physical properties	1. Unify the fabrication conditions to control the surface stiffness	Its main affecting factors may be various in different methods
		2. Other types of confounding physical properties (wettability, roughness, and charge, etc.) should also be managed by referring to the way we control stiffness.
Cell culture related assessments	Geometrically and physicochemically stable under sterilization	Choose a material that is stable under sterilization condition, and select its compatible sterilization method	Stability in high temperature, irradiation, and ethanol, etc. should be considerate
	Geometrically and physicochemically stable during immersion	Choose an un-swellable and un-degradable material	Deionized water is suggested to be the assessment medium
	Immobile during immersion	Choose a material with high density or fixable property	Material selection should meet other requirements
	No structure-independent toxicity	1. Thoroughly remove the chemical residues
		2. Choose an un-degradable material
Cell response analysis related assessments	For morphological assay: Not disturb signal transmission and collection	Choose a material that is transparent, non-magnetic, non-autofluorescence	Material selection should meet other requirements
	For hydrodynamic flow assay: Immobile during shear stress	Choose a material with high density or fixable property	Material selection should meet other requirements
	For wound-healing assay: Geometrically stable after wound preparation	Prepare wounds by healing inserts rather than physical scratching	Commercial or self-designed healing inserts both can meet the requirement
	For CCK-8, MTT assays: Not to bring absorbance difference	Detect the absorbance without the substrate materials	Ensure the same volume reaction reagent being transferred to the new plate
	For RT-qPCR, WB assays: Not affect sample quality and internal reference	1. Choose a material with low solubility in the cell lysis reagents.	Material selection should meet other requirements
		2. Carefully choose internal reference	β-actin is not stably expressed on different topographies
	There are a large number of methods for analyzing cell responses. The management of confounding factors should be carried out following our guideline, when other cell response detecting methods are performed.