Effect of Inter-individual Variability on a Phenotypic Endpoint Battery in an In Vitro Co-culture Model of the Human Respiratory Tract after Exposure to Model Reactive Gas Acrolein
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Many untested, ubiquitous inhalable toxicants may drive adverse human health effects. The “Tox21” program seeks to increase chemical testing throughput by integrating in vitro new approach methodologies (NAMs) to address challenges including developing tiered-testing frameworks and replacing costly and lengthy in vivo models. The use of non-immortalized cells from native human tissue (i.e. primary cells) in NAMs incorporates inter-individual variability into in vitro models that is not recapitulated by inbred rodent strains or immortalized cell lines. The use of primary cell-based NAMs for the evaluation of inhaled chemicals on the respiratory tract also poses challenges, including evaluating the impact of inter-individual variability on in vitro system assessment. To this end, we characterized inter-individual variability across 14 donors using an in vitro co-culture model of the human tracheobronchial respiratory tract and using acrolein as a model reactive gas, a ubiquitous indoor and environmental combustion byproduct. The co-culture model was characterized by a battery of 16 physiologically-relevant endpoints assessing viability, epithelium physiology and function, and intracellular communication. We found that 1) differentiated donor epithelium exhibited highly variable barrier height and goblet cell density prior to insult. 2) 6 of 16 endpoints exhibited sensitivity (i.e. statistical significance after insult) and detected variability at baseline or after insult. For example, all donors exhibited high viability at baseline; however, exposure to acrolein revealed 4 resilient individuals (cell death < 20%) and 3 highly responsive individuals (cell death > 80%) with several intermediate donor responses. Alternatively, barrier integrity (TEER) exhibited high variability at baseline and a significant decrease in barrier integrity for all donors after acrolein exposure. The extensive response range prompted us to assess the relative sensitivity of each endpoint as many published NAMs have used only 3-5 primary cell donors. 3) We calculated that sub-samplings of an n = 3 donor group from the 14 donor population will fall within the 14 donor confidence interval in ~70% of sub-samplings for all endpoints. 4) Furthermore, we assessed the minimum number of donors required to detect a significant response to acrolein exposure. For example, TEER requires 7 donors to detect a robust significant response between acrolein and control populations; however, viability requires 9 donors to reach the same robust conclusion. These observations are among the first to define the impact of inter-individual variability on NAM performance, revealing variability in baseline donor cell culture, differential sensitivity within the endpoint battery, and variability in donor response. While it is clear that 14 donors fail to represent the human population, this work is necessary to inform more complex NAM infrastructure to evaluate risk to known susceptible populations and address populations that are marginalized in the existing published research. Does not reflect EPA policy.