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In situ monitoring reveals cellular environmental instabilities in human pluripotent stem cell culture.

ABSTRACT: 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.

SUBMITTER: Klein SG

PROVIDER: S-EPMC8826360 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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In situ monitoring reveals cellular environmental instabilities in human pluripotent stem cell culture.

Klein Shannon G SG Alsolami Samhan M SM Arossa Silvia S Ramos-Mandujano Gerardo G Parry Anieka J AJ Steckbauer Alexandra A Duarte Carlos M CM Li Mo M

Communications biology 20220208 1

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 O<sub>2</sub> and CO<sub>2</sub> with optical sensor spots and contemporaneous evaluation of cell proliferation and medium pH in standard batch cul ...[more]

PMID: 35136190

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Project description:Human-induced pluripotent stem cells (hiPSCs) have shown great potential for human health, but their growth and properties have been significantly limited by the traditional monolayer (2D) cell culture method for more than 15 years. Three-dimensional (3D) culture technology has demonstrated tremendous advantages over 2D. In particular, the 3D PGmatrix hiPSC derived from a peptide hydrogel offers a breakthrough pathway for the maintenance and expansion of physiologically relevant hiPSC 3D colonies (spheroids). In this study, the impact of 3D culture conditions in PGmatrix hiPSC on cell performance, integrity, and secretome profiles was determined across two commonly used hiPSC cell lines derived from fibroblast cells (hiPSC-F) and peripheral blood mononuclear cells (hiPSC-P) in the two most popular hiPSC culture media (mTeSR1 and essential eight (E8)). The 3D culture conditions varied in hydrogel strength, 3D embedded matrix, and 3D suspension matrix. The results showed that hiPSCs cultured in 3D PGmatrix hiPSC demonstrated the ability to maintain a consistently high cell viability that was above 95% across all the 3D conditions with cell expansion rates of 10-20-fold, depending on the 3D conditions and cell lines. The RT-qPCR analysis suggested that pluripotent gene markers are stable and not significantly affected by the cell lines or 3D PGmatrix conditions tested in this study. Mass spectrometry-based analysis of secretome from hiPSCs cultured in 3D PGmatrix hiPSC revealed a significantly higher quantity of unique proteins, including extracellular vesicle (EV)-related proteins and growth factors, compared to those in the 2D culture. Moreover, this is the first evidence to identify that hiPSCs in a medium with a rich supplement (i.e., mTeSR1) released more growth-regulating factors, while in a medium with fewer supplements (i.e., E8) hiPSCs secreted more survival growth factors and extracellular proteins. These findings offer insights into how these differences may impact hiPSC behavior, and they deepen our understanding of how hiPSCs respond to 3D culture conditions, aiding the optimization of hiPSC properties in translational biomedical research toward clinical applications.

Dataset Information

In situ monitoring reveals cellular environmental instabilities in human pluripotent stem cell culture.

Publications

In situ monitoring reveals cellular environmental instabilities in human pluripotent stem cell culture.

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