Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins

Abstract

Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth. Key points • Less and more efficient glucose utilization for suspension cell growth • Concerted alteration of metabolic enzyme activity and protein expression • Protein candidates to interfere glycolytic activity in MDCK cells

The impact of serum-free culture on HEK293 cells: From the establishment of suspension and adherent serum-free adaptation cultures to the investigation of growth and metabolic profiles

“…A similar observation was recently reported that, compared to their adherent progenitors, suspension HEK293 cell lineages have downregulated gene expression related to oxidative phosphorylation, nucleotide, and amino acids metabolism except for cholesterol metabolism by genomic, transcriptomic, and metabolic gene analysis ( Malm et al, 2020 ). Moreover, proteomics analysis showed down-regulated enzyme activity or decreased peptide intensity related to glycolysis, PPP, and partially TCA cycle pathway in the exponential growth phase of suspension compared to adherent culture in MDCK cells ( Pech et al, 2021 ).…”

“…In addition, a recent publication detected a 4-fold increase in the ratio of AMP/ATP in MDCK cells when transitioned from adherent to suspension culture, which is in accordance with our culture models. They further postulated that it could implicate the involvement of the AMPK pathway ( Pech et al, 2021 ). Therefore, the link between EMT mechanisms mediated by IA, SDH, and AMPK pathways could be further investigated to understand the more detailed metabolism of a transition from adherent to suspension cells.…”

Optimizing VLP production in gene therapy: Opportunities and challenges for in silico modeling

“…[ 153 ] Alterations between adherent and suspension cultures were investigated based on label‐free proteomes and metabolomes in combination with enzyme activity assays. [ 154 ] The generated data were integrated into a MFA model, which was generated based on different media compositions. [ 155 ] Besides differentially expressed intracellular proteins, alterations in catalytic activities were observed for enzymes, including Pirin, CAV1, or AMPK, which are known to influence cellular metabolism directly.…”

“…Consequently, growth rates in suspension culture were reduced by up to 30%. [ 154 ] A proteome‐based comparative study of adherent and suspension cell lines reveiled differently expressed proteins involved in intercellular adhesion, junction, and adhesion to the extracellular matrix. [ 156 ] These data may form the basis for enzyme‐constrained MDCK cell modeling on genome scale as soon as a reference model is available.…”