CHO Suspension Research Articles

Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction

In order to preserve the in vivo metabolite levels of cells, a quenching protocol must be quickly executed to avoid degradation of labile metabolites either chemically or biologically. In the case of mammalian cell cultures cultivated in complex media, a wash step previous to quenching is necessary to avoid contamination of the cell pellet with extracellular metabolites, which could distort the real intracellular concentration of metabolites. This is typically achieved either by one or multiple centrifugation/wash steps which delay the time until quenching (even harsh centrifugation requires several minutes for processing until the cells are quenched) or filtration.In this article, we describe and evaluate a two-step optimized protocol based on fast filtration by use of a vacuum pump for quenching and subsequent extraction of intracellular metabolites from CHO (Chinese hamster ovary) suspension cells, which uses commercially available components. The method allows transfer of washed cells into liquid nitrogen within 10–15s of sampling and recovers the entire extraction solution volume. It also has the advantage to remove residual filter filaments in the final sample, thus preventing damage to separation columns during subsequent MS analysis. Relative to other methods currently used in the literature, the resulting energy charge of intracellular adenosine nucleotides was increased to 0.94 compared to 0.90 with cold PBS quenching or 0.82 with cold methanol/AMBIC quenching.

Analysis of microRNA transcription and post-transcriptional processing by Dicer in the context of CHO cell proliferation

CHO cells are the mammalian cell line of choice for recombinant production of therapeutic proteins. However, their low rate of proliferation limits obtainable space-time yields due to inefficient biomass accumulation. We set out to correlate microRNA transcription to cell-specific growth-rate by microarray analysis of 5 CHO suspension cell lines with low to high specific growth rates. Global microRNA expression analysis and Pearson correlation studies showed that mature microRNA transcript levels are predominately up-regulated in a state of fast proliferation (46 positively correlated, 17 negatively correlated). To further validate this observation, the expression of three genes that are central to microRNA biogenesis (Dicer, Drosha and Dgcr8) was analyzed. The expression of Dicer, which mediates the final step in microRNA maturation, was found to be strongly correlated to growth rate. Accordingly, knockdown of Dicer impaired cell growth by reducing growth-correlating microRNA transcripts. Moderate ectopic overexpression of Dicer positively affected cell growth, while strong overexpression impaired growth, presumably due to the concomitant increase of microRNAs that inhibit cell growth. Our data therefore suggest that Dicer dependent microRNAs regulate CHO cell proliferation and that Dicer could serve as a potential surrogate marker for cellular proliferation.

13C labeling dynamics of intra- and extracellular metabolites in CHO suspension cells

13C labeling dynamics of intra- and extracellular metabolites in CHO suspension cells

Sampling and quenching of CHO suspension cells for the analysis of intracellular metabolites

Sampling and quenching of CHO suspension cells for the analysis of intracellular metabolites

Generation of a Potent Recombinant Homophilic Chimeric Anti-CD20 Antibody

Previously we increased the potency of therapeutic antibodies in targeting, induction of apoptosis, and growth inhibition in vitro and in vivo by chemically conjugating a homophilic peptide to the antibody. Here, we describe the construction of a chimeric fusion gene derived from the murine anti-CD20 antibody (1F5) variable region, with an engineered homophilic domain at the C-terminus of the human IgG1 sequence. The construct was expressed in CHO suspension cells and purified. The potency of the homophilic anti-CD20 antibody was compared to a chimeric antibody without the engineered homophilic domain. In this comparison, the homophilic anti-CD20 antibody showed increased binding to a human CD20 cell line, and significantly more ADCC, CDC, and induction of apoptosis in three cell lines. In addition, the homophilic anti-CD20 antibody demonstrated increased inhibition of proliferation of two cell lines. These data show that homophilic fusion protein antibodies with enhanced therapeutic potency can be produced with industry-standard fermentation protocols.

Investigations on Mechanical Stress Caused to CHO Suspension Cells by Standard and Single‐Use Pumps

AbstractMechanical stress caused to transfected Chinese hamster ovary (CHO) suspension cells by reusable and single‐use magnetically levitated, bearingless centrifugal pumps was investigated. Cell death rates were determined for different pump speeds and compared with data from a peristaltic and a 4‐piston diaphragm pump. Furthermore, the fluid distribution inside the PuraLev® 200 pump was modeled using computational fluid dynamics. The results reveal considerably lower mechanical stress to CHO cells caused by the magnetically levitated bearingless centrifugal pumps than by the peristaltic and diaphragm pump.

Evaluation of sampling and quenching procedures for the analysis of intracellular metabolites in CHO suspension cells

Background Metabolomics, aiming at the quantification of all extracellular and intracellular metabolites, is a valuable tool for characterizing, understanding and manipulating the physiology of mammalian cells. While extracellular metabolite analysis is well established, required quenching and extraction procedures for intracellular metabolite analysis in mammalian suspension cells are not yet routinely available. In this study a simple sampling and quenching protocol using ice-cold 0.9% saline as quenching solution [1] was tested on CHO cells. Quenching efficiency, preservation of cell integrity as well as cell separation and the necessity of washing steps were evaluated and possible sources of error are discussed.

Characterization of the Batl (Bacteroides aerotolerance) operon in Bacteroides fragilis: isolation of a B. fragilis mutant with reduced aerotolerance and impaired growth in in vivo model systems.

YT135.2.8, a Tn4400' insertion mutant of Bacteroides fragilis strain TM4000, grows poorly when used to infect Monika or Chinese hamster ovary (CHO) cell monolayers and is outcompeted by wild-type strains in mixed infections. YT135.2.8 also shows defects in the rat granuloma pouch model system in monoculture and is completely outcompeted by the wild-type strain in a mixed infection. In addition, this mutant shows defects in a new model system consisting of CHO suspension cell columns. All of these defects may be explained by the finding that YT135.2.8 shows decreased tolerance to exposure to atmospheric oxygen (less aerotolerant). The monolayer growth defect (MGD) of YT135.2.8 can be influenced significantly by the presence of sulphur-containing reducing agents (cysteine, dithiothreitol, thiodiglycol) or the non-sulphur reducing agent Tris-(2-carboxylethyl)phosphine (TCEP). The defects in YT135.2.8 can be complemented by a 6.6 kb fragment of the B. fragilis chromosome. DNA sequencing of this fragment and of the regions flanking the Tn4400' insertion in the B. fragilis chromosome revealed the presence of five open reading frames, corresponding to genes bat (Bacteroides aerotolerance) A, B, C, D, E, which form the Batl operon; Tn4400' inserted within batD. All of the hypothetical proteins possess one or more membrane-spanning domains. BatA and BatB show high similarity to each other but, like BatD, they show no match to sequences of known function in the databases. BatC and BatE contain 2-4 repeated sequences similar to the tetratricopeptide repeats (TPRs) seen in many eukaryotic proteins. The function of TPR sequences in protein interactions in other systems leads to the suggestion that the Bat proteins form a complex. The Batl complex may be involved in the generation or export of reducing power equivalents to the periplasm of the B. fragilis cell.

Sialic acid of mammalian cell lines.

AbstractApproximately two‐thirds of the total sialic acid (S.A.) per cell of a number of cell lines (L‐929, L5178Y, HeLa, C13, P183, and CHO) was located at the cell surface but was inaccessible to the action of trypsin, pronase, lysozyme, β‐glucuronidase, or hyaluronidase. The mean surface density of S.A. ranged from 5.4 × 105 molecules/μ2 surface area for the L5178Y cell to 16.1 × 105 molecules/μ2 for the P183 cell. The P183 cell line, which is a polyoma virus‐transformed derivative of Stoker's C13 line, consistently contained more S.A. per cell than the latter under a variety of growth conditions, although the two lines did not differ in mean cell volume. When mean cell volume of C13, P183, or CHO cells was experimentally manipulated by thymidine or colcemide blockade, S.A. content per cell followed size changes closely. No evidence could be found for a shift in total S.A. per unit cell volume accompanying the period of maximum mitotic activity of partially synchronized CHO suspension cultures. Comparisons between cells grown on glass and the same cells grown in suspension, or between cells grown to different densities on glass, indicated no differences in the characteristic S.A. content per cell.