Establishment of Namalva cell lines which grow continuously in glutamine-free medium

Abstract

Glutamine has been shown to be a preferred energy source for some established cell lines and cancer cells in culture (Kovacevic, 1971; Kovacevic, 1972; Lavietes, 1974). Empirically, glutamine is the most abundant amino acid in most of the culture media developed. The major end product of glutamine metabolism is ammonia. Ammonia build up is one of the limiting factors in the proliferation of mammalian cells in higher density culture and is directly related to the initial glutamine concentration. The susceptibility of glutamine to thermodecomposition prevents the heat sterilization of glutamine-enriched media and this significantly increases the cost of medium preparation at the industrial scale. In an attempt to overcome these drawbacks, a population of Namalva cells capable of growing in glutamine-free media was established. The adapted cells were found to contain a higher level of glutamine synthetase activity which enable them to synthesize sufficient amounts of glutamine for their growth.

Expression of human beta-interferon in Namalwa KJM-1 which was adapted to serum-free medium

“…Namalwa KJM-1 cells which are capable of growing in serum-free medium were established. They were adapted to serum-free RPMI-1640 medium supplemented with insulin (3 yg/ml), transferrin (5 pg/ml), sodium pyruvate (5 mM), sodium selenite (1.25 x 10T7 M), galactose (1 mg/ml), and Pluronic F68 (0.1% w/v) (Hosoi, 1988), and were maintained in the same medium. HEPES (10 mM), penicillin (25 U/ml) and streptomycin (25 pg/ml) were added.…”

Optimization of cell culture conditions for G-CSF (granulocyte-colony stimulating factor) production by genetically engineered namalwa KJM-1 cells

“…They were adapted to serum-and albumin-free RPMI-1640 medium supplemented with 4-(2hydroxyethyl) -1 -piperazineethanesulfonic acid (HEPES) (10 mM), L-glutamine (4 mM), penicillin (25 U/ml), streptomycin (25 gg/ml), insulin (3 gg/ml), transferrin (5 gg/ml), sodium pyruvate (5 mM), sodium selenite (125 nM), galactose (1 mg/mi) and Pluronic F68 (1 mg/ml); we called this ITPSGF medium (Hosoi et al, 1988).…”

“…Biofermenter TM consists of a suspension vessel containing a cone-type cell sedimentation column as cell separator and serving as well for axis of impeller. We previously reported the growth and maintenance of Namalwa KJM-1 cells, a serumindependent subline of Namalwa cells (B lymphoblastoid cells), in suspension culture at high density in chemically defined serum-and albumin-free ITPSGF medium (Hosoi et aL, 1988).…”

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An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al, 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized.

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Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium

“…Namalwa KJM-1, which can grow in serum-free medium, was established as described previously (Hosoi, 1988). It was cultured in RPMI-1640 medium supplemented with HEPES (10 mM), L-glutamine (4 mM), penicillin (25 U/ml), streptomycin (25 gg/ml), insulin (3 gg/ml), transferrin (5 gg/ml), sodium pymvate (5 raM), sodium selenite (1.25 x 10-7M), galactose (1 mg/ml) and Pluronic F68 (0.1% w/v) (Sato, 1983).…”