Mj. Keen et Tw. Steward, ADAPTATION OF CHOLESTEROL-REQUIRING NS0 MOUSE MYELOMA CELLS TO HIGH-DENSITY GROWTH IN A FULLY DEFINED PROTEIN-FREE AND CHOLESTEROL-FREE CULTURE-MEDIUM, Cytotechnology, 17(3), 1995, pp. 203-211
NS0 has been used as a fusion partner for the production of hybridomas
and has more recently been engineered to produce recombinant protein.
A protein-free culture medium, designated W38 medium, has previously
been developed which supported high density growth of rat myeloma and
hybridoma cell lines. NS0 cells failed to grow in W38 medium and in a
number of protein-free culture media which support the growth of other
myeloma cell lines. NS0 cells are derived from the NS-1 cell line, wh
ich is known to require exogenous cholesterol. It was found that NS0 c
ells grew in W38 medium supplemented with phosphatidylcholine, cholest
erol, and albumin and that NS0 were auxotrophic for cholesterol. Prote
in-free growth of NS0 cells was achieved by using beta-cyclodextrin to
replace albumin as a lipid carrier. The maximal cell density reached
in this protein-free medium was in excess of 1.5 x 10(6) cell ml(-1).
The lipid supplements in the medium precipitated after a few days stor
age at +4 degrees C. In order to overcome this problem a protocol was
developed which allowed NS0 cells to be adapted to cholesterol-indepen
dent growth in W38 medium. NS0.CF (cholesterol-independent NS0 cells)
were cultured continuously in W38 medium for several months. In shake
flask culture a cell density of 2.4 x 10(6) cells ml(-1) was achieved
in W38 medium compared with 1.41 x 10(6) cells ml(-1) in RPMI 1640 med
ium containing 10% foetal bovine serum. NS0.CF cells readily grew in a
1 live stirred bioreactor using W38 medium supplemented with Pluronic
F68 reaching a density of 3.24 x 10(6) cells ml(-1). NS0.CF were clon
ed protein-free by limiting dilution in W38 medium, giving colonies in
wells that were seeded at an average density of 0.32 cells per 200 mu
l. This study has demonstrated for the first time the growth of a cho
lesterol-requiring mouse myeloma cell line in a completely defined pro
tein-free medium and its subsequent adaptation to cholesterol-independ
ence.