High Content System for Quantifying Mitochondrial Morphology in Patient-Derived Human Cells

Abstract Mitochondrial function is critical for cellular health, with dysfunction contributing to human diseases. Structural changes in mitochondria, such as size and shape, reflect alterations in bioenergetics, fission-fusion dynamics, and metabolic homeostasis. Existing morphological quantification is outdated, can be biased, technologically limited, or overly complex. This study presents a high content system for quantifying morphology using open-access resources and widely available equipment. Fibroblasts were stained with PKmito TM Dye Deep Red, imaged via automated confocal microscopy, and analyzed with CellProfiler™ and KNIME ® . We tested different imaging conditions and found live-cell confocal imaging at 60x magnification provided the most precise measurements. Using this system, we found that human Huntington Disease fibroblast mitochondria were significantly smaller and more circular, suggesting increased fission. To confirm our results, we employed other mitochondrial assays and found elevated expression of the fission protein Drp1, reduced respiration, impaired iron uptake, and increased membrane potential. This system offers a robust, unbiased high content approach to studying mitochondrial morphology in disease.