293SF Metabolic Flux Analysis during Cell Growth and Infection with an Adenoviral Vector

Abstract

Metabolic flux quantification of cell culture is becoming a crucial means to improve cell growth as well as protein and vector productions. The technique allows rapid determination of cell culture status, thus providing a tool for further feeding improvements. Herein, we report on key results of a metabolic investigation using 293 cells adapted to suspension and serum-free medium (293SF) during growth and infection with an adenoviral vector encoding the green fluorescence protein (GFP). The model developed contains 35 fluxes, which include the main fluxes of glycolysis, glutaminolysis, and amino acids pathways. It requires specific consumption and production rate measurements of amino acids, glucose, lactate, NH(3), and O(2), as well as DNA and total proteins biosynthesis rate measurements. Also, it was found that extracellular protein concentration measurement is important for flux calculation accuracy. With this model, we are able to describe the 293SF cell metabolism, grown under different culture conditions in a 3-L controlled bioreactor for batch and fed-batch with low glucose. The metabolism is also investigated during infection under two different feeding strategies: a fed-batch starting at the end of the growth phase and extending during infection without medium change and a fed-batch after infection following medium renewal. Differences in metabolism are observed between growth and infection, as well as between the different feeding strategies, thus providing a better understanding of the general metabolism.

Development and optimization of an adenovirus production process

“…A predominant set of data, used in this paper to exemplify the approaches, has been generated in the authors' laboratory [11][12][13][14][15][16][17][18][19][20] using derived clones from HEK-293 cells [21]. However, whenever possible, these data are discussed in light of conclusions from published papers in the field.…”

293 cell cycle synchronisation adenovirus vector production

“…To achieve cell cycle arrest in G1/G0 phase, 293 cells were incubated at 29 C, 31 C, 33 , and 35 C and compared with a control at 37 C. As shown in Table 3, the only incubation temperature where cell death occurred was 29 C; for all other temperatures tested specific growth rates ranged from 2.2 (at 35 C) to 12.6 (at 31 C) times less than the ones verified in the control (37 C). This is expected since the optimal growth temperature for most mammalian cell lines is between 35 C and 37 C. 27 Concerning cell cycle synchronisation, the results ( Figure 5) show that the highest percentage of cells at the G1/G0 phase occurred at 31 C and 67 h of incubation, with a maximal synchronisation of 77%, representing a 1.3-fold increase compared with the control for the corresponding period of time. To check whether a longer incubation period at 31 C could increase the proportion of cells arrested in G1/G0 phase, cells were incubated for 100 h at 31 C; however, no further increase was observed (data not shown).…”

“…The cytometric parameters were set to provide accurate discrimination between nonfluorescent negative cells and positive GFP-fluorescence cells on a FL1 vs. FSC density plot to estimate the proportion of infected cells: One fluorescent cell corresponds to one infectious particle (ip) when less than 30% of the cells are fluorescent. 31 Data analysis was performed using FlowMax V V R software. Titer variations were always in the order of AE10%.…”

Reduction of Proteinuria through Podocyte Alkalinization

“…It is based on podocyte mitochondrial proteome expression analysis (20) and podocyte mRNA expression profiles (supplemental Table S2). This information was combined with a metabolic network information previously employed for hybridomas (21-24), CHO cells (25), HEK 293 cells (26,27), and hepatocytes (28) because all eukaryotic cells share considerable similarities in their respective operative metabolic enzyme compositions.…”