Fig. 4.

Controlling or removing the confounding factors derived from micro/nano topographic structural cell response detection. (A-F) Ensuring precise cell morphology evaluation: (A) Translucency assessment. (B) Observe cell density and status without confounding factors through optical microscopy. (C) Fluorescence interference assessment. (D) Observe subcellular structures without confounding factors. (E) Magnetic properties assessment. (F) Observe the interaction of cell and topographic structure without confounding factors. (G-H) Ensuring precise cell adhesion affinity evaluation: (G) Pictures of the generated cell culture devices after undergoing shear stress. (H) Evaluating cell adhesion affinity without confounding factors. (I-J) Ensuring precise cell migration evaluation: (I) Assessing the integrity of topographic structures after preparing wound by pipette tip scratching or healing inserts through SEM. (J) Evaluating cell migration without confounding factors. (K-M) Ensuring precise cell proliferation evaluation: (K–L) The influence of topographic structures on absorbance with or without the detection reagents of CCK-8 assay, and removing the confounding absorbance by detecting without substrate materials. (M) Evaluating cell proliferation without confounding factors by the optimized protocol. (N–S) Ensuring precise gene expression and protein synthesis evaluation: Assessing the concentration (N, O) and the quality (P, Q) of the mRNA or the protein extracted from the cells cultured on the substrate materials. (R, S) Evaluating the expression of housekeeping molecules in RT-qPCR and WB assays.