An MRC-5 cell based high-throughput, high-content imaging assay to identify hits against Trypanosoma cruzi intracellular parasites.

The discovery of effective therapies for Trypanosoma cruzi (T. cruzi), the causative agent of Chagas disease, remains one of the most pressing challenges in parasitology and global health. Despite the significant burden posed by Chagas disease, especially in Latin America, only two drugs, benznidazole and nifurtimox, are currently available for treatment. These drugs are often limited by side effects and long treatment durations. There is an urgent need for effective new therapies, requiring innovative and physiologically relevant assay platforms for high-throughput identification of compounds active against T. cruzi. Here we discuss the development, optimization, evaluation and validation of a robust and highly reproducible high-throughput, high-content imaging assay in a 384-well microplate format to quantitatively assess the effects of compounds on intracellular T. cruzi amastigotes infecting MRC-5 human lung fibroblasts. The multiplexed assay design enables concurrent evaluation of compound-induced cytotoxicity on host cells within the same well, serving as an early indicator of host cell viability and compound selectivity.