Simple methodology to score micropattern quality and effectiveness.

Abstract

Micropatterns (MPs) are widely used as a powerful tool to control cell morphology and phenotype. However, methods for determining the effectiveness of how well cells are controlled by the shape of MPs have been inconsistently used and studies rarely report on this topic, indicating lack of standardization. We introduce an evaluation score that quantitatively assesses the MP fabrication quality and effectiveness, which can be broadly used in conjunction with all currently available MP design types. This score uses four simple and quick steps: (i) scoring MP and (ii) background fabrication quality, (iii) defining the type(s) of MP of interest, and (iv) assigning so-called efficiency descriptors describing cell behavior. These steps are based on visual inspection and quick categorization of various aspects of MP fabrication quality and cell behaviour, presented in illustrations and microscopy image examples intended to serve as a reference "atlas". To illustrate the advantage of using this score, we determined differences in cell morphology and F-actin intensity between scored vs. non-scored cells. These measurements, which could be different in other studies, were chosen because both are understood as markers of cell phenotype and function. We combined intensity-calibrated immunofluorescence microscopy and image-based single cell protein analysis. Importantly, significant differences in cell morphology and cytoskeletal protein content between scored vs. non-scored cells were noted: the unconditional inclusion of all experimental read-outs (i.e., all MP data regardless of MP quality and effectiveness) into the final results significantly misjudged the experimental readouts vs. only including experimental read-outs of quality-controlled and effective MPs, identified by scoring. Specifically, non-scoring underestimated the F-actin intensity per cell and quantitative cellular morphometric descriptors circularity and solidity and overestimated aspect ratio. Scoring improved the precision of cellular readouts, advocating the use of a MP quality and efficiency score as a quantitative decision-supporting tool in deciding whether or not particular MPs should be used for experiments, saving time and money. This simple scoring methodology can be used for improving MP fabrication, comparing results across studies, benefiting basic science studies and potential future clinical use of MPs by introducing standardization.