The importance of scoring recognition fitness in spheroid morphological analysis for robust label-free quality evaluation.

Because of the growing demand for human cell spheroids as functional cellular components for both drug development and regenerative therapy, the technology to non-invasively evaluate their quality has emerged. Image-based morphology analysis of spheroids enables high-throughput screening of their quality. However, since spheroids are three-dimensional, their images can have poor contrast in their surface area, and therefore the total spheroid recognition by image processing is greatly dependent on human who design the filter-set to fit for their own definition of spheroid outline. As a result, the reproducibility of morphology measurement is critically affected by the performance of filter-set, and its fluctuation can disrupt the subsequent morphology-based analysis. Although the unexpected failure derived from the inconsistency of image processing result is a critical issue for analyzing large image data for quality screening, it has been tackled rarely. To achieve robust analysis performances using morphological features, we investigated the influence of filter-set's reproducibility for various types of spheroid data. We propose a new scoring index, the "recognition fitness deviation (RFD)," as a measure to quantitatively and comprehensively evaluate how reproductively a designed filter-set can work with data variations, such as the variations in replicate samples, in time-course samples, and in different types of cells (a total of six normal or cancer cell types). Our result shows that RFD scoring from 5000 images can automatically rank the best robust filter-set for obtaining the best 6-cell type classification model (94% accuracy). Moreover, the RFD score reflected the differences between the worst and the best classification models for morphologically similar spheroids, 60% and 89% accuracy respectively. In addition to RFD scoring, we found that using the time-course of morphological features can augment the fluctuations in spheroid recognitions leading to robust morphological analysis.