Some queries regarding A549 cell culture - (Jul/10/2014 )

Respected Sirs,

          I have been culturing A549 cells for some time and have encountered certain problems. I am my guide's first student and thus have no seniors to turn to. I'll be most grateful if you can answer some of my questions as given below:

 1) My objective is to study certain signalling pathways in A549 cells, hence I have to continuously culture A549 cells. Upto what passage no. may I culture A549 cells so that my primary objective is not affected adversely?

 2) One problem I have repeatedly encountered is that A549 cells suddenly have a change in their morphology. They increase in size, loses its characteristic shape accompanied by the appearance of numerous vacuoles. It seems the cells are stressed somehow but the question is what might have caused such stress? (I culture the cells in DMEM high glucose, cells are maintained at 37degrees and 5% CO2.)

        Thanking you for your patience and eagerly waiting for your reply.

A general rule for cell culture is that you can keep a cell line under continuous culture for about 10 passages before you start to see changes in morphology and behaviour.  This will vary a bit depending on the cell line and how thorough you are with your cell culture (e.g. not letting them get over confluent before passaging etc.)

Cells that get large and flat with many vacuoles are senescent and are no longer part of the dividing cell population (and never will be again), they can survive passaging to some extent, but won't divide.  There are usually a few senescent cells in any given population, but an increase is a sign that your cells are getting old and you should get up more.