The maximum cell population density of mouse fibroblast (strain LS) cells growing in static suspension culture was found to be directly proportional to the dialysed calf serum concentration. This was due to choline limitation and the fact that serum protein was a major source of choline. The growth yield (Y) was 3.2 x 105 cells/µg choline chloride.Studies on the role of serum in the presence of excess choline showed the following. When protein was omitted from the medium, cell death occurred. Whole serum protein could be replaced by either (1) bovine serum albumin fraction V, or (2) crystalline bovine serum albumin + sodium pyruvate and α-ketoglutarate, or (3) polyvinylpyrrolidone + methylcellulose + pyruvate and α-ketoglutarate.The population doubling time was 24 h in the presence of whole serum protein and increased considerably with the substitutes (1-3). The increase in maximum cell population density (without medium changes) exceeded 2.9 x 106 cells/ml with either whole serum protein or substitutes (1) and (2). With serum substitute (3) the maximum increase in population density was reduced to 1.6 x 106 cells/ml.
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