Divergent responses of human intestinal organoid monolayers using commercial in vitro cytotoxicity assays

• Published in: PloS one Vol. 19; no. 6; p. e0304526
• Main Authors: Lewis, Miranda A, Patil, Ketki, Ettayebi, Khalil, Estes, Mary K, Atmar, Robert L, Ramani, Sasirekha
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10-06-2024
• Subjects: Analysis; Assaying; Auranofin; Biology and Life Sciences; Cell culture; Cell lines; Cell Survival - drug effects; Cells; Cytotoxicity; Dehydrogenases; Drug development; Drug interactions; Drug testing; Gastrointestinal surgery; Genetic modification; Genetically modified organisms; Humans; Infectious diseases; Intestinal Mucosa - cytology; Intestinal Mucosa - drug effects; Intestinal Mucosa - metabolism; Intestine; Intestines - cytology; L-Lactate dehydrogenase; L-Lactate Dehydrogenase - metabolism; Lactate dehydrogenase; Medicine and Health Sciences; Metabolism; Organoids; Organoids - cytology; Organoids - drug effects; Organoids - metabolism; Regression analysis; Research and Analysis Methods; Stem cells; Toxic diseases; Toxicity
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0304526

In vitro models, such as primary cells and continuous cell lines routinely used for evaluating drug candidates, have limitations in their translational relevance to human diseases. Organotypic cultures are increasingly being used to assess therapeutics for various cancers and infectious diseases. Monitoring drug cytotoxicity in cell cultures is crucial in drug development, and several commercially available kits for cytotoxicity assessment offer distinct advantages and limitations. Given the complexity of organoid cultures, including donor-driven variability, we investigated drug-treated, tissue stem cell-derived human intestinal organoid responses with commonly used cell cytotoxicity assay kits. Using seven different compounds, we compared the cytotoxicity assay performance of two different leaky membrane-based and two metabolism-based assays. Significant variability was seen in reported viability outcomes across assays and organoid lines. High baseline activity of lactate dehydrogenase (LDH) in four human intestinal organoid lines required modification of the standard LDH assay protocol. Additionally, the LDH assay reported unique resilience to damage in a genetically-modified line contrasting results compared to other assays. This study highlights factors that can impact the measurement of cell cytotoxicity in intestinal organoid models, which are emerging as valuable new tools for research and pre-clinical drug testing and suggest the need for using multiple assay types to ensure reliable cytotoxicity assessment.