Automated high-throughput high-content autophagy and mitophagy analysis platform

Abstract

Autophagy and mitophagy play a central role in cellular homeostasis. In pathological conditions, the flow of autophagy and mitophagy can be affected at multiple and distinct steps of the pathways. Unfortunately, the level of detail of current state of the art analyses does not allow detection or dissection of pathway intermediates.Moreover, is conducted in low-throughput manner on bulk cell populations. Defining autophagy and mitophagy pathway intermediates in a high-throughput manner is technologically challenging, and has not been addressed so far. Here, we overcome those limitations and developed a novel high-throughput phenotyping platform with automated high-content image analysis to assess autophagy and mitophagy pathway intermediates. R

Assessment of Ethanol-Induced Toxicity on iPSC-Derived Human Neurons Using a Novel High-Throughput Mitochondrial Neuronal Health (MNH) Assay

“…Recent studies used MEA to investigate the effect of compounds on spontaneous neuronal activity and network activity ( Zwartsen et al, 2018 ). Various HT screening methods have also been established ( Saunders et al, 2014 ; Iannetti et al, 2015 ; Gelles and Chipuk, 2016 ; Daniele et al, 2017 ; Kepiro et al, 2018 ; Arias-Fuenzalida et al, 2019 ; Varkuti et al, 2020 ). Nonetheless, there is a need for additional in vitro systems centered on human iPSC derivatives in order to capture different aspects of human neurotoxicity ( Schmidt et al, 2017 ).…”

Assessment of ethanol-induced toxicity on iPSC-derived human dopaminergic neurons using a novel high-throughput mitochondrial neuronal health (MNH) assay

“…Recent studies used MEA to investigate the effect of compounds on spontaneous neuronal activity and network activity (34). Various HT screening methods have also been established (35)(36)(37)(38)(39)(40)(41). Nonetheless, there is a need for additional in vitro systems centered on human iPSC derivatives in order to capture different aspects of human neurotoxicity (42).…”

Generalising from Conventional Pipelines: A Case Study in Deep Learning-Based for High-Throughput Screening

“…However, "autophagy flux", in which the different autophagy phases can be measured, has been established as one of the best approaches to study autophagy in pathological conditions. Our dataset uses the Rosella pH-sensitive biosensor 21 that allows for the identification of the different autophagy phases 13 . The gold standard in segmentation is the manual creation of label masks by experts.…”

“…Notwithstanding, such an approach requires a large amount of work from highly trained researchers. An existing solution to the segmentation problem of autophagy events is based on CIP techniques specifically optimised for fluorescent microscopy analysis of cells with the Rosella biosensor to report autophagy events as suggested in 13 . In this study, we demonstrate how to effectively leverage this approach developing two alternative pipelines (CDL and MDL) to improve the final segmentation quality.…”

“…This renders CIP algorithms still relevant since images automatically generated (e.g. using an HTS system), can be processed with techniques that require simple thresholding and basic corrections yielding an acceptable quality of segmentation ( 13 ).…”