The following protocols describe general procedures for subculturing mammalian cells in suspension culture. Note that the procedure for passaging insect cells differs from that for mammalian cells on several crucial steps. For more information, refer to Notes on Subculturing Insect Cells.
For passaging your own cell line, we recommend that you closely follow the instructions provided with each product you are using in your experiments. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant phenotypes to a complete failure of the cell culture.
Passaging Suspension Cultures
Subculturing suspension cells is somewhat less complicated than passaging adherent cells. Because the cells are already suspended in growth medium, there is no need to treat them enzymatically to detach them from the surface of the culture vessel, and the whole process is faster and less traumatic for the cells. Replacement of growth medium is not carried out in suspension cultures; instead, the cells are maintained by feeding them every 2 to 3 days until they reach confluency. This can be done by directly diluting the cells in the culture flask and continue expanding them, or by withdrawing a portion of the cells from the culture flask and diluting the remaining cells down to a seeding density appropriate for the cell line. Usually, the lag period following the passaging is shorter than that observed with adherent cultures.
Suspension Culture Vessels
Suspension cultures can be maintained in sterile culture flasks (e.g., shaker flasks without baffles) that are not tissue-culture treated; however, spinner flasks (i.e., stirrer bottles) specifically designed for suspension cell culture allow for superior gas exchange and permit higher volumes of cells to be cultured.
Spinner flasks have two basic designs; the medium is agitated (i.e., stirred) by a hanging stir-bar assembly or with a vertical impeller. The vertical impeller provides better aeration. The total culture volume in a spinner flask should not exceed half of the indicated volume of the spinner for proper aeration (e.g., a 500 mL spinner should never contain more than 250 mL of culture).