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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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.
Elleman GL, Courtney KD, Anders V and Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88–95.
Fleischaker RJ and Sinskey AJ (1981) Oxygen demand and supply in cell culture. Eur. J. Appl. Microbiol. Biotechnol. 12: 193–197.
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.
Goetghebeur S and Hu W-S (1991) Cultivation of anchoragedependent animal cells in microsphere-induced aggregate culture. Appl. Microbiol. Biotechnol. 34: 735–741.
Johansson A and Nielsen V (1980). Biosilon a new microcarrier. Develop. Biol. Standard. 46: 125–129.
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.
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.
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.
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.
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.
Lazar A (1991) Immobilization of animal cells in fixed bed bioreactors. Biotech. Adv. 9: 411–424.
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.
Litwin J (1984) The growth of human diploid fibroblasts as aggregates with cellulose fibers in suspension. Develop. Biol. Standard. 60: 237–242.
Looby D and Griffiths JB (1988) Fixed bed glass sphere (porosphere) bioreactors for animal cells. Cytotechnology 1: 339–346.
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.
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.
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.
Paterson Jr MK (1979) Measurement of cell growth and viability of cells in culture. Methods Enzymol. 58: 141–143.
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.
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.
Robert J, Cote J and Archambault J (1991) Surface immobilization of anchorage-dependent mammalian cells. Biotechnol. Bioeng. 39: 697–706.
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.
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.
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.
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.
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.
Taylor P (1985) In: Gilman AG, Goodman LS, Rall T and Murad F (eds) Pharmacological Basis of Therapeutics (pp 110–129), MacMillan, New York.
Tolbert WR, Hitt MM and Feder J (1980) Cell aggregate suspension culture.In Vitro 16: 486–490.
Van Wezel AL (1982) Cultivation of anchorage-dependent cells and their application. J. Chem. Technol. Biotechnol. 32: 318–323.
van Wezel AL (1984) Microcarrier technology present status and prospects. Develop. Biol. Standard. 55: 3–9.
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.
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.
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.
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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