Mammalian cell cultures are a keystone resource in biomedical research, but the results of published experiments often suffer from reproducibility challenges. This has led to a focus on the influence of cell culture conditions on cellular responses and reproducibility of experimental findings. Here, we perform frequent in situ monitoring of dissolved O₂ and CO₂ with optical sensor spots and contemporaneous evaluation of cell proliferation and medium pH in standard batch cultures of three widely used human somatic and pluripotent stem cell lines. We collate data from the literature to demonstrate that standard cell cultures consistently exhibit environmental instability, indicating that this may be a pervasive issue affecting experimental findings. Our results show that in vitro cell cultures consistently undergo large departures of environmental parameters during standard batch culture. These findings should catalyze further efforts to increase the relevance of experimental results to the in vivo physiology and enhance reproducibility.

A publication search was conducted in February 2020 to obtain data of pH, dO₂, and dCO₂ in the medium of mammalian cell line cultures. The resulting dataset delivered 203 measurements of 12 different cell lines from seven published studies, examining primary cells, cancer cells, and stem cells that were published between 1971 -2019 and included our own data. In the publication search, we used a combination of keywords in PubMed NCBI^® and Google Scholar search engines, which included “cell culture” AND “pH”, “O₂”, “CO₂”, “buffering capacity”, “dissolved oxygen”, “dissolved carbon dioxide”, “dissolved gases”. We extracted mean pH, O₂ and CO₂ values, as well as the time at which the values were recorded throughout the incubation (reported in hours since inoculation). We used these data to produce ∆ values that represented the difference between measurements taken at time zero minus the levels measured at intervals during the cultures. We collected mean pH, dO₂ and dCO₂ values for all cell cultures within each publication, including those of the same cell type with slight differences in media formulations (e.g., higher or lower concentrations of serum or differing concentrations of buffers) to represent the diversity of changes expected in mammalian cell cultures. Where publications reported a time-series of pH, O₂, and CO₂ values, we extracted data for incubation times that were common across the publications to facilitate comparison.

Environmental parameter data of in situ measurements in the medium of 12 different cell lines. Missing values represent unavailable data. For columns that contain quantitative data (e.g. working volume = 40mL, time of measurements = 24 hours), the "unit" column to the right contains the unit of measurement. Publications reported CO₂ and O₂ concentrations in different units (mmHg, % air saturation, and absolute %) and were converted into units of an absolute percent (%) under the assumption that experiments were conducted at ambient atmospheric pressure (101.325 kPa). Columns labeled "units reported" represent the values reported in the original publication that were converted into units of an absolute %.