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Protein-free culture medium improvement: testing additives and their interactive effects in 96-well plates (1993)

Côte, Johanne ; Kamen, Amine A. ; André, Gérald

Springer

Applied microbiology and biotechnology 39 (1993), S. 577-584

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ISSN: 1432-0614

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract In order to achieve a rational screening of additives suspected to improve antibody production over basal Dulbecco's Modified Eagle Medium (DMEM), we have developed a 96-well plate method for simultaneously testing individual and interactive effects of multiple additives. Cell viabilities were determined directly in each well by a colorimetric assay (MTT assay) and antibody production was measured by an enzyme-linked immunosorbent assay (ELISA) performed on well supernatants. Such as supplemented culture medium might considerably reduce production costs since commercial serum- or protein-free media are sold at serum-enriched basal medium costs. The CBM-P22 mouse cell line used in this study was shown to be sensitive to key amino acids, oxalacetic acid, ethanolamine and selenium at low cell density (〈1 × 105 cells·ml−1). When these cells were inoculated in 96-well plates their antibody productivity was improved (sevenfold) by adding these additives to the basal DMEM as evidenced by ELISA absorbance readings. This improved productivity, obtained with the supplemented DMEM, named DOWSENs, is comparable to the one obtained with the commercial, but costly, protein-free hybridoma medium (PFHM II), taken here as the positive control. Results were confirmed by growing these cells in different media in standard (25 cm2) T-flask static culture. In addition, specific amino acid consumption, as analysed by HPLC, showed that asparagine and tryptophan when added to DMEM may improve antibody production, even if these amino acids are not limiting or highly consumed in PFHM II. Using this 96-well plate assay allows the assessment of a large number of different assays with a few milligrams of the product(s) tested. This is a rapid technique that gives results for additives that are not easily quantified by analytical techniques or for the study of interactive effects, which is time consuming and labour-intensive when done in individual T-flasks.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00205055

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Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells (1994)

Garnier, Alain ; Côté, Johanne ; Nadeau, Isabelle ; [et al.]

Springer

Cytotechnology 15 (1994), S. 145-155

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ISSN: 1573-0778

Keywords: Adenovirus ; human 293S cells ; recombinant protein ; scale-up ; metabolism

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract Human 293S cells, a cell line adapted to suspension culture, were grown to 5×106 cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×105 cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×106 cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00762389

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Surface immobilization of anchorage-dependent mammalian cells (1992)

Robert, Joëlle ; Côté, Johanne ; Archambault, Jean

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 39 (1992), S. 697-706

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ISSN: 0006-3592

Keywords: anchorage-dependent mammalian cells ; immobilization ; fibers ; bioreactor ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Anchorage-dependent HeLa cells were successfully cultured on two fibrous materials (A07 and R100) with porosities of 75-125 and 40 μm, void fractions of 92% and 81%, and fiber diameters of 7.6 and 10.2 μm, respectively, in 100-mL spinner flasks and 2-L stirred tank bioreactors. The matrix was formed into a fixed vertical spiral configuration. All cultures displayed rapid (≤2-3 h) attachment of inoculated cells (≥95%) to the matrix, uniform coverage of the immobilizing area with viable cells, and no significant amount of cell debris in the medium. Spinner flask cultures indicated that the denser material R100 showed better results in terms of final cell density. The growth of HeLa cells on material R100 in both culture systems was similar to that observed in tissue culture dishes (specific growth rate ∼0.03-0.04 h-1, maximum cell density of 8 × 106-9 × 106 cells · mL-1, and yields of 0.4 × 108 cells · mM-1 on glucose and 2 × 108-3 × 108 cells · mM-1 on glutamine). Scale-up of this culture technique in a 2-L bioreactor under perfusion with pH and dissolved oxygen (DO) control yielded cell densities of up to 1.6 × 106 cells · mL-1. Two other anchorage-dependent mammalian cells (ADC) known to be cultured with difficulty in roller bottles or with micro carriers were easily grown on material R100 in spinner flasks. The performance of this culture technique was compared to other ADC culture systems.

Additional Material: 8 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/bit.260390702

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Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells (1998)

Côté, Johanne ; Garnier, Alain ; Massie, Bernard ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 59 (1998), S. 567-575

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ISSN: 0006-3592

Keywords: adenovirus ; adenoviral vectors ; 293 cells ; recombinant proteins ; serum-free medium ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing β-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both β-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 567-575, 1998.

Additional Material: 5 Ill.

Type of Medium: Electronic Resource

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