Nonpolar and Polar Lipids Composition in Recombinant Chinese Hamster Ovary Cells by Nile Red Staining Flow Cytometry

Chinese hamster ovary (CHO) cells are the preferred system for producing recombinant proteins (RPs), and many have been used effectively in therapies. Specific productivity is related to the intrinsic genetics, biochemical, and metabolic characteristics of each producing clone. Through cell growth, lipids play a crucial role in biology. They serve as energy sources, form the bilayer of organelles and cell membranes, and are involved in the regulation of metabolic stress. To understand lipid requirements during culture, the cellular lipid composition must be considered. However, lipid studies involve complex analytical techniques, such as nuclear magnetic resonance (NMR) and mass spectrometry (MS), which require lengthy processing. Here, we explored the nonpolar and polar lipid composition in CHO cells stained with Nile red (NR), a fluorescent dye with solvatochromic properties, using flow cytometry. We describe a form to determine nonpolar and polar lipids using four different configurations of excitation wavelength laser lines and 10 different wavelength bandpass filters to acquire the fluorescence emission by flow cytometry. We show that the nonpolar and polar lipid composition changes throughout the kinetic growth of three clones of the stably transfected CHO cell line DG44 (CHO‐DG44). Nonpolar lipids tend to increase in the stationary phase while polar lipids progressively decrease throughout the kinetic growth. This study provides insight into changes in lipid composition during the growth phases of CHO cells, which can be used to improve bioprocesses for CHO and other cell lines used for RP production. Practical applications : An increasing number of recombinant proteins are produced in mammalian cells. They are amply used not only in the biomedical field but also in the food industry. A necessary approach to improving the bioprocesses involved in recombinant protein production is to understand and control needed physical parameters such as temperature and oxygen consumption, as well as the required metabolites, by maintaining a continuous record of culture conditions throughout the process. This work describes a procedure for rapidly assessing changes in nonpolar and polar lipids throughout cell culture. This technique could be used to estimate lipid requirements in cell lines used in recombinant protein production and improve culture medium conditions.