Outline‐etching image segmentation reveals enhanced cell chirality through intercellular alignment

Abstract

Cells cultured on micropatterns exhibit a chiral orientation, which may underlie the development of left–right asymmetry in tissue microarchitectures. To investigate this phenomenon, fluorescence staining of nuclei has been used to reveal such orientation. However, for images with high cell density, analysis is difficult because of the overlapping nuclei. Here, we report an image processing method that can acquire cell orientations within dense cell populations. After initial separation based on Boolean addition of binarized images using global and adaptive thresholds, the overlapping nucleus contours in the binarized images were segmented by iteratively etching the outlines of nuclei, which allowed the orientations of each cell to be extracted from densely packed cell clusters. In applying this technique to cultured C2C12 myoblasts in micropatterned stripes on different substrates, we found an enhanced chiral orientation on glass substrate. More important, this enhanced chirality was consistently observed with increased intercellular alignment and independent of cell–cell distance or cell density, suggesting that intercellular alignment plays a role in determining the chiral orientation. By segmenting single cells with intact orientation, this technique offers an automated method for quantitative analysis with improved accuracy, providing an essential tool for studying left–right asymmetry and other morphogenic dynamics in tissue formation.

Remnant Effects of Culture Density on Cell Chirality After Reseeding

“…Therefore, cells with higher aspect ratio, i.e., more elongated cells, may demonstrate enhanced intercellular alignment to amplify the expression of chiral orientation. We used absolute, acute angle difference |Δθ| between one cell and its neighbors as a quantitative measure of intercellular alignment, as reported previously . After averaging the |Δθ| of pairs of cells with different cell–cell distance, we found that |Δθ| was reduced with shorter cell–cell distance, suggesting a better intercellular alignment between cells and their nearest neighbors (Figure c).…”

“…We used absolute, acute angle difference |Δθ| between one cell and its neighbors as a quantitative measure of intercellular alignment, as reported previously. 25 After averaging the |Δθ| of pairs of cells with different cell−cell distance, we found that |Δθ| was reduced with shorter cell−cell distance, suggesting a better intercellular alignment between cells and their nearest neighbors (Figure 3c). More importantly, the |Δθ| of cells after low-density culture was always greater than the |Δθ| of cells after high-density culture, and was even greater on PDMS substrate compared to that on glass substrate.…”

An Image-Guided Intrascaffold Cell Assembly Technique for Accurate Printing of Heterogeneous Tissue Constructs

“…A global image threshold value G is selected, and the gray value of each image point is compared to said value. If the gray value ( x ) is lower than the threshold value G , it is given the set value 0, and if it is over the threshold value, it is given the value 255: ,, The threshold value is used to convert the image to binary form. Additional operations, if required, are performed to remove image imperfections such as incorrectly produced connected components.…”

Cell chirality in cardiovascular development and disease

“… 13,14,16,19,59 While the specific mechanisms that regulate cell chirality are only beginning to be understood, it is widely accepted that the actin-based cytoskeleton, which is heavily involved in determining cell morphology and motility, plays a crucial role in cell chirality. 60–64 The microtubule network is shown to be mostly disposable, which is consistent with its more prominent role in molecular transport. 16 How the molecular chirality of actin and related structures relates to the cellular phenotype is not very well understood.…”

“… 64,65 Similar nuclear alignment was also observed by Huang et al using an outline etching image segmentation strategy. 60 Interestingly, inhibition of actin polymerization led to the reversal of the CCW chirality of C2C12 mouse myoblasts, supporting the role of the actin cytoskeleton in chirality development. 16 Similar biased alignment and migration were also observed with the vascular mesenchymal cells.…”