High cell density attenuates reactive oxygen species: Implications for in vitro assays

Abstract

In vitro cell-based assays are an essential and universally used step in elucidation of biological processes as well as in drug development. However, results obtained depend on the validity of protocols used. This statement certainly pertains to in vitro assays of oxidative stress. The holy grail of in vitro models is reliability and predictability of outcomes that relate to a single variable like addition of hydrogen peroxide or xanthine oxidase. Without such validated outcomes, comparison of results among different laboratories is not possible. Achieving this goal requires a thorough understanding of the complex interplay between the cells, their environment, and the experimental assays. Furthermore, as this knowledge is attained, it must be disseminated and used to update and standardize existing protocols. Here, we confirm and extend the effect of pyruvate and cell density on in vitro oxidative stress assays. Cell viability was assessed using a colorimetric assay measuring the reduction of a tetrazolium salt (XTT) into a colored formazan dye. Extracellular hydrogen peroxide concentrations were measured using the foxp3 assay. We confirmed a previously reported finding that pyruvate, a common ingredient in cell culture media, acts as an extracellular scavenger of reactive oxygen species. We also demonstrated that cell density directly correlates with resistance to oxidative stress in tissue culture. It is theorized that the protective effect due to cell density predominantly relates to intracellular factors such as reduced glutathione and extracellular factors such as catalase.

On Plasma Fractionation Treatment and Its Implications in Cells

“…quiescence) in confluent cultures and also of quiescent cells being less sensitive to DNA damage-induced cell death are both well-established concepts in cell biology. An additional mechanism has also been proposed for the reduced sensitivity of high-density cultures to oxidative stress based on their having a greater antioxidant capacity per well, which results in greater quenching of extracellular reactive oxygen species [15]. Consistent with these phenomena and our observations, it has been previously reported that confluent HaCaT cells are more resistant to oxidative stress than their subconfluent counterparts [16].…”

High-throughput three-dimensional characterization of morphogenetic signals during the formation of the vertebrate retina

“…Previous research suggests that cell density is pivotal in regulating neural precursor cell function, influencing the expression levels of various proteins and reactive oxygen species (ROS) [56]. Additionally, cell density is directly associated with resistance to oxidative stress [57], as well as cell differentiation, survival, migration, and proliferation [58, 59].…”

Methods Applied to the In Vitro Primary Toxicology Testing of Natural Products: State of the Art, Strengths, and Limits

“…In an in vitro study using dermal fibroblasts, Kim et al demonstrated that high cell densities led to improved ROS resistance by increasing the total antioxidant capacity of the cell culture [157]. The authors suggested that cell density should be considered as a critical point when studying oxidative stress.…”