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

Sabine Pech,Markus Rehberg,Dirk Benndorf,Albert Sickmann,Udo Reichl,Yvonne Genzel,Robert Janke,Thilo Muth,Erdmann Rapp

doi:10.1007/s00253-021-11150-z

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

Highlights

Adaptation of mammalian cell lines to growth in suspension in a chemically defined medium has significant advantages for the design and optimization of manufacturing processes for biologicals
The segregated model was in good agreement with the measured data points for cell number, Glc, Gln, and Glu concentrations (Fig. 1)
Maximum cell specific growth rates of 0.013–0.014 h−1 were observed and represent a reduction to 25–30% compared to MDCKADH (Table 1)

Summary

Introduction

Adaptation of mammalian cell lines to growth in suspension in a chemically defined medium has significant advantages for the design and optimization of manufacturing processes for biologicals. In combination with biotechnological tools, which are widely used to study the influence of the medium on cellular metabolism and growth rate (Cruz et al 1999; Genzel et al 2005; Sussman et al 1980), the behavior of cellular systems can be studied in more detail It is barely understood how concerted changes on the proteome, metabolome, and growth enzyme level compose a system response, which enables suspension growth. This is partly because of the highly complex mechanism of cell line adaptation and partly because of the immense number of samples to be analyzed and evaluated

Methods

Results

Conclusion