After a few passages I am getting change in morphology. When I had changed the serum it had also shown some changes in cell proliferation and growth pattern.
By definition a "cell line" is immortal and has potential to proliferate indefinitely. However, it is likely that if these "cell lines" are derived from patients, they are heterogeneous and exhibit heterogeneity (and associated morphological changes) in culture. Therefore, one may want to develop a clonal population and characterize it and then select a desired clonal population. Notably, culture conditions (glucose levels and the levels of serum growth factors in serum) do influence the characteristics. Therefore, to maintain reproducibility from an experiment to next, one needs to control the culture conditions.
It is usually recommended to change cultivation vessel every time you subculture cells. If you see changes in cell line characteristics when grown for longer time in the same vessel, I would stick to this recommendation. With robust cell lines we sometime use the same vessel for upto four subcultivations but it seems this is not the case of you cell lines.
Maybe you can clarify if you are using standard cell lines or those that you have isolated from a tumor.
Irrespective, using the same dish or flask has been shown to improve cell adherence, since the flask contains fragments of adhesion proteins (like collagen, fibronectin, etc.) put down by the previous culture of cells, and this promotes the binding of the next batch of cells. It is like "pre-coating" the flask with these proteins. Some cells are sensitive to the presence of these fragments, and some are not. This is why, the pre-coating of plates with collagen etc., can improve the growth of many cells, like neurons, and also change their morpology etc. Thus, using the same flask for 1-2 cultures may be good, but may change things if this is stretched out.
The attached paper is an old, but very elegantly informative paper on how this can happen. (Effect of substrate on cell growth.pdf)
Changing the serum, or even the batch of serum can have effects. BUT, generally lot-to-lot variation from the same manufacturer can be considered as small. But, changing manufacturers for the serum can make a difference due the very complex nature of the composition of serum itself.
Even changing the % serum, can affect the replicative life span of a cell type. see this attached paper. (Replicative life-span....pdf). Thus, increasing the medium from say 35 mls to 50 mls in a T-150 flask can keep cells happier over the long term! Dont be too stingy with your medium added.
Although you can find this in many cell culture books, have a look at these links also:
https://www.researchgate.net/post/Could_change_of_FBS_Fetal_Bovine_Serum_affect_the_quality_of_the_cell
http://www.labome.com/method/Cell-Culture-Media-A-Review.html
Good luck !
It is usually recommended to perform the assays after the third passage.
I would suggest never ever use the same vessles, contamination will be a problem- I would always change vessels.
I would also suggest to change flask each passage, or, at least, after 1-2 passages. Extracellular matrix fragments change adhesion, so that proliferation etc.
the cells will change a little in each passage, and as the cells are heterogeneous there is a continuous process of selection of the best fitted cells in the population. Therefore, it is recommended to use parallel cultures to validate your experimental results.
Hi Chandra,
I worked on CACO-2, HT29 and T84 colon cancer cell lines.
For all cells lines, 60-80% confluence was attained in 3-4 days.
Don't work after more 10 passages because of cells derivation.
Don't dilute too much your cells when you make your passages avoid to clone cells (this type of cells line are heterogen).
Did you prepare other type of cell line in your laboratory?
You can have changes in morphology if you contaminate your preparation with other cell type (i.e. fibroblast).
Virginie
I am still wondering what passage means for old cell lines ...hela cells is the prototype!
I am always susupicious when I see written on a plate "hela passage 5"...only 5 passage from the date hela were eestablised in cell culture dishes....nearly 50years ago????
any hints..
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