DOI: 10.5061/dryad.41ns1rnd9
title: In situ monitoring reveals cellular environmental instabilities in human pluripotent stem cell culture

description: 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.
description: A publication search was conducted in February 2020 to obtain
 data of pH, dO<sub>2</sub>, and dCO<sub>2</sub> 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<sup>®</sup> and Google Scholar search engines, which
 included “cell culture” AND “pH”, “O<sub>2</sub>”,
 “CO<sub>2</sub>”, “buffering capacity”, “dissolved oxygen”,
 “dissolved carbon dioxide”, “dissolved gases”. We extracted mean pH,
 O<sub>2</sub> and CO<sub>2</sub> 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, dO<sub>2</sub> and
 dCO<sub>2</sub> 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, O<sub>2</sub>, and
 CO<sub>2</sub> values, we extracted data for incubation times
 that were common across the publications to facilitate
 comparison.
description: 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
 CO<sub>2</sub> and O<sub>2</sub> 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
 %.