Propagation of Strain L (Earle) Cells in Agitated Fluid Suspension Cultures.

Abstract

Strain L mouse fibroblasts (Earle) were grown in a medium composed of horse serum, synthetic mixture 199 (Morgan, Morton and Parker) and Hanks balanced salt solution. Cells, suspended in 80 ml of this medium contained in a rubber stoppered 250 ml Erlenmeyer flask, were shaken on a New Brunswick rotary shaker at 100 rpm. When cultures were started at pH 7.4 with cells taken from the stationary growth phase, a lag of 24-48 hours was seen during which time the cells adjusted the pH to approximately 7.0. After this interval the cells entered a phase of logarithmic growth which lasted for 4-5 days and during which time the pH remained almost constant. A generation time of approximately 40 hours and a maximum cell density of 1.5-2.0 x 106 cells/ml was noted. When cultures were started with cells from the logarithmic growth phase and in a medium having a pH of 7.0, no lag was seen. Changes in the concentration of serum in the medium over the range of 5-40% had no significant effect on the generation time though serum levels below 20% gave a corresponding decrease in maximum cell density. Inoculum size over the range of 5 x 104 - 5 x 105 had no effect on growth rate. The level at which the pH stabilized during logarithmic growth was directly proportional to the serum control of the medium.