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Evaluation of anchorage-dependent cell propagation systems for production of human acetylcholinesterase by recombinant 293 cells

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Abstract

Production of recombinant human acetylcholinesterase (AChE) by a high producer human embryonic kidney cell line (293) was evaluated by three main cell propagation systems; surface propagator, fixed-bed reactor and stirred microcarrier cultures. The recombinant cell line expresses AChE levels as high as 10–20 mg/l/day. System productivities in either the surface propagator (multitray system), or in the fixed-bed reactor (polyurethane macroporous sponges) were 4–8 mg AChE/l/day during a production period of 8 days. Similar productive rates, yet longer production periods (up to 22 days), were obtained in microcarrier (MC) cultures using either polystyrene beads (Biosilon); collagen-coated dextran beads (Cytodex-3); or gelatin macroporous beads (Cultispher-G). Best results were obtained in an aggregate cculture using cellulose beads charged with diethylaminoethyl (DEAE) groups, (Servacel), as carriers. In this culture, a system productivity of 6–10 mg/l/day was maintained for 28 days.

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References

  • Almgren J, Nilsson C, Petersson AC and Nilsson K (1991) Cultispher-microporous gelatin microcarriers-new applications. In: Spier RE, Griffiths JB and Meignier B (eds). Production of Biologicals from Animal Cells in Culture (pp. 434–438). Butterworth-Heinemann, Oxford.

    Google Scholar 

  • Elleman GL, Courtney KD, Anders V and Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88–95.

    Google Scholar 

  • Fleischaker RJ and Sinskey AJ (1981) Oxygen demand and supply in cell culture. Eur. J. Appl. Microbiol. Biotechnol. 12: 193–197.

    Google Scholar 

  • Gebb C, Clark JM, Hirtenstein MD, Lindgren G, Lindskog U, Lundgren B and Vretblad P (1982) Alternative surfaces for microcarrier culture of animal cells. Develop. Biol. Standard. 50: 93–102.

    Google Scholar 

  • Goetghebeur S and Hu W-S (1991) Cultivation of anchoragedependent animal cells in microsphere-induced aggregate culture. Appl. Microbiol. Biotechnol. 34: 735–741.

    Google Scholar 

  • Johansson A and Nielsen V (1980). Biosilon a new microcarrier. Develop. Biol. Standard. 46: 125–129.

    Google Scholar 

  • Kerem A, Kronman C, Bar-Nun S, Shafferman A and Velan B. (1993) Interrelation between assembly and secretion of recombinant human acetylcholinesterase. J. Biol. Chem. 268: 180–184.

    Google Scholar 

  • Kotler M, Reuveny S, Mizrahi A and Shahar A (1985) Ion exchange capacity of DEAE microcarriers determined the growth pattern of cells in culture. Develop. Biol. Standard. 60: 255–261.

    Google Scholar 

  • Kronman C, Velan B, Gozes Y, Leitner M, Flashner Y, Lazar A, Marcus D, Sery T, Papier Y, Grosfeld H, Cohen S and Shafferman A (1992) Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit. Gene 121: 295–304.

    Google Scholar 

  • Lazar A, Reuveny S, Mizrahi A, Whiteside JP and Spier RE (1987) Production of biologicals by animal cells immobilized in polyurethane foam matrix. In: Spier RE and Griffiths JB (eds). Modern Approaches to Animal Cell Technology. (pp. 437–448). Butterworths, Seven Oaks.

    Google Scholar 

  • Lazar A, Silberstein L, Mizrahi A and Reuveny S (1988) An immobilized hybridoma culture perfusion system for production of monoclonal antibodies. Cytotechriology 1: 331–337.

    Google Scholar 

  • Lazar A (1991) Immobilization of animal cells in fixed bed bioreactors. Biotech. Adv. 9: 411–424.

    Google Scholar 

  • Lind W, Jager V, Lucki-Lange M and Wagner R (1991) Characterization of protease activity in serum-free culture supernatants of hybridomas and recombinant mammalian cells. In: Spier RE, Griffiths JB and Meignier B (eds). Production of Biologicals from Animal Cells in Culture. (pp. 196–202), Butterworths Seven Oaks.

    Google Scholar 

  • Litwin J (1984) The growth of human diploid fibroblasts as aggregates with cellulose fibers in suspension. Develop. Biol. Standard. 60: 237–242.

    Google Scholar 

  • Looby D and Griffiths JB (1988) Fixed bed glass sphere (porosphere) bioreactors for animal cells. Cytotechnology 1: 339–346.

    Google Scholar 

  • Massouli J, Pezzementi L, Bon S, Krejci E and Valette F-M (1993) Molecular and cellular biology of cholinesterases. Progress in Neurobiol. 41: 31–91.

    Google Scholar 

  • Nilsson L, Nilsson L, Petterson AC and Almgren J (1990) Growth of cells on macroporous microcarriers. In: Murakami H (ed). Trends in Animal Cell Culture Technology. (pp. 35–39). Kodansha, Tokyo.

    Google Scholar 

  • Ordentlich A, Barak D, Kronman C, Flashner Y, Leitner M, Segall Y, Ariel N, Cohen S, Velan B and Shafferman A (1993) Dissection of the human acetylcholinesterase active center determinants of substrate specificity. J. Biol. Chem. 268: 17083–17095.

    Google Scholar 

  • Paterson Jr MK (1979) Measurement of cell growth and viability of cells in culture. Methods Enzymol. 58: 141–143.

    Google Scholar 

  • Peshwa MV, Kyung YS, McClure PB and Hu WS (1993) Cultivation of mammalian cells as aggregates in bioreactors: Effect of calcium concentration on spatial distribution of viability. Biotechnol. Bioeng. 41: 179–187.

    Google Scholar 

  • Reuveny S, Zheng ZB and Epstein L (1986) Evaluation of a cell culture fermenter. Am. Biotechnol. Lab. Jan–Feb 28–39.

  • Reuveny S (1990). Microcarrier culture systems. In Lubiniecki AS (ed). Large-Scale Mammalian Cell Culture Technology. (pp. 271–341) Marcel Dekker, New York.

    Google Scholar 

  • Robert J, Cote J and Archambault J (1991) Surface immobilization of anchorage-dependent mammalian cells. Biotechnol. Bioeng. 39: 697–706.

    Google Scholar 

  • Sanders PG (1990). Protein production by genetically engineered mammalian cell lines. In: Spier RE and Griffiths JB (eds). Animal Cell Biotechnology, vol. 4 (pp 15–70), Academic Press, London.

    Google Scholar 

  • Sanford KK, Earle WR, Evans VS, Walltz JK and Shanon JE (1951) The measurement of proliferation in tissue cultures by enumeration of cell nuclei. J. Natl. Cancer Inst. 11: 773–795.

    Google Scholar 

  • Shafferman A, Kronman C, Flashner Y, Leitner M, Grosfeld H, Ordentlich A, Gozes Y, Cohen S, Ariel N, Barak D, Harel M, Silman I, Sussman JL and Velan B (1992a) Mutagenesis of human acetylcholinesterase; identification of residues involved in catalytic activity and in polypeptide folding. J. Biol. Chem. 267: 17640–17648.

    Google Scholar 

  • Shafferman A, Velan B, Ordentlich A, Kronman C, Grosfeld H, Leitner M, Flashner Y, Cohen S, Barak D and Ariel N (1992b) Substrate inhibition of acetylcholinesterase: residues involved in signal transduction from the surface to the catalytic center. EMBO J. 11: 3561–3568.

    Google Scholar 

  • Shafferman A, Velan B, Ordentlich A, Kronman C, Grosfeld H, Leitner M, Flashner Y, Cohen S, Barak D and Ariel N (1992c) Acetylcholinesterase catalysis protein engineering studies. In: Shafferman A and Velan B (eds) Multidiciplinary Approaches to Cholinesterase Functions (pp. 165–175). Plenum Pub., London.

    Google Scholar 

  • Taylor P (1985) In: Gilman AG, Goodman LS, Rall T and Murad F (eds) Pharmacological Basis of Therapeutics (pp 110–129), MacMillan, New York.

    Google Scholar 

  • Tolbert WR, Hitt MM and Feder J (1980) Cell aggregate suspension culture.In Vitro 16: 486–490.

    Google Scholar 

  • Van Wezel AL (1982) Cultivation of anchorage-dependent cells and their application. J. Chem. Technol. Biotechnol. 32: 318–323.

    Google Scholar 

  • van Wezel AL (1984) Microcarrier technology present status and prospects. Develop. Biol. Standard. 55: 3–9.

    Google Scholar 

  • Velan B, Kronman C, Grosfeld H, Leitner M, Gozes Y, Flashner Y, Sery T, Cohen S, Ben-Aziz R, Seidman S, Shafferman A and Soreq H (1991a) Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme. Cell. Mol. Neurobiol. 11: 143–156.

    Google Scholar 

  • Velan B, Grosfeld H, Kronman C, Leitner M, Gozes Y, Lazar A, Flashner Y, Marcus D, Cohen S and Shafferman A (1991b) The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. J. Biol. Chem. 266: 23977–23984.

    Google Scholar 

  • Velan B, Kronman C, Leitner M, Grosfeld H, Flashner Y, Marcus D, Lazar A, Kerem A, Bar-Nun S, Cohen S and Shafferman A (1992) Molecular organization of recombinant human acetylcholinesterase. In: Shafferman A and Velan B (eds) Multidiciplinary Approaches to Cholinesterase Functions. (pp. 39–47). Plenum Pub., London.

    Google Scholar 

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Lazar, A., Reuveny, S., Kronman, C. et al. Evaluation of anchorage-dependent cell propagation systems for production of human acetylcholinesterase by recombinant 293 cells. Cytotechnology 13, 115–123 (1993). https://doi.org/10.1007/BF00749938

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  • DOI: https://doi.org/10.1007/BF00749938

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