Mammalian Cell Growth on Microcarriers
Spitzer, John Joseph
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The initial purpose of this project was to study the development of resistance of V79 cells to antitumor drugs. Since the frequency of resistant cells is very low (l0 -6 - 10 -7), amicrocarrier culture technique was developed using beads to carry a-large number of cells while reducing the. amount of medium, labour, and chances of contamination. This report defines the critical parameters for optimizing the microcarrier culture technique. The condition for maximum cell attachment, i,e., infrequent stirring of cell/bead suspension, was opposite that required for even distribution of cells on beads. Since we considered cell attachment to be the more important parameter, we optimized that factor while obtaining reasonably even distribution. We found that a ratio of 8-10 cells/bead and a static attachment period with occasional swirling enhanced the probabilities of cells attaching to beads and a high percentage of beads bearing cells. In most early experiments, cell growth stopped at about 10 6 cells/mI. This could have been due to depletion of nutrients, accumulation of toxic factors, low pH, etc. We found that supplementation of the medium with pyruvate and non-essential amino acids did not improve cell attachment or result in a more even cell distribution on beads. However, it significantly increased (by 25%) the final cell yield. A four-fold increase in cell yield was obtained when 50% of the culture medium was replaced daily compared to every two or three days. Even under these conditions, however, beads were not confluent with cells when plateau was reached indicating that the technique can be further optimized. The addition of 10 nM HEPES buffer to the medium did not offset the lowering of pH in the culture. The following factors did· not affect. the final cell yield: overtrypsinization of the starting culture, the use of confluent cells to seed the culture, and the use of medium without antibiotics.