Coarse-graining analysis for non-invasive, label-free, and quantitative evaluation of undulating motion in cultured oral mucosa epithelial cell sheets

Abstract

Background Cultured oral mucosa epithelial cell sheets (COMECS) represent a promising therapeutic approach for epithelial repair, such as corneal reconstruction or esophageal mucosa defects; however, current quality assessment methods are largely invasive and destructive. Although optical flow (OF)-based analysis has shown promise in evaluating keratinocyte motility, its sensitivity decreases under confluent conditions. A novel coarse-graining (CG) analysis combined with OF is introduced as a candidate non-invasive, label-free imaging parameter to quantitatively evaluate the characteristic undulating motion patterns (local directional synchrony of cell motion) of mature COMECS and to provide a potential basis for future quality assessment. Methods COMECS were manufactured from passage 2 (p2) and passage 5 (p5) oral keratinocytes using standard (cultured in serum-containing medium) and sub-standard (cultured in serum-free medium) protocols. Time-lapse microscopy was conducted at multiple time points (48-, 96-, and 192-h post manufacture). The OF analysis-derived microscopic velocity fields were subjected to CG analysis to generate macroscopic velocity fields. The undulating index (UI) and mean undulating index (MUI) were calculated to quantify the directional synchrony of cellular motion. Western blotting was used to evaluate p63 and PITX1 protein expression levels to correlate the undulating motion patterns with biological markers of keratinocyte undifferentiation and oral epithelial identity. Results OF-based CG analysis successfully differentiated undulating motion patterns between the standard and sub-standard COMECS, with average MUI values higher in the standard COMECS ( p < 0.05). Time-course analysis of p2 COMECS indicated a substantially lower average MUI at 48 h than at 96 h and 192 h, suggesting time-dependent changes in collective motion behavior. A passage-dependent comparison showed apparent temporal patterns between p2 and p5 COMECS, with p5 sheets exhibiting earlier peak undulation but reduced sustainability. The p63 and PITX1 expression levels were elevated in standard COMECS, and were associated with enhanced undulating motion patterns. Conclusion This novel OF-based, CG analysis enabled non-invasive, label-free, and quantitative assessment of COMECS quality using undulating motion pattern evaluation, demonstrating both applicability and feasibility for regenerative medicine quality control applications.