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Cells after thawing - (Aug/16/2006 )

Hi,

I work with primary cells. And I suppose that you know that they divide only for a short period.
Then, I tried to freeze them in order to have cells when it is necessary.

Cells are cultured on collagen. When I put them directly from a biospy (after treatment of course), they expand until they reach 100% confluency. Now, once freezed, and thawed, they still attached to the collagen, they start to divide and grow, but after a short period they completely stop to grow. And I don t understand why they stop since they are well attached and they look nice. Is it due to a mechanism from primary cells or a "simple" problem of serum ?

If anyone has experience in this field....or advices

Thanks

Mif

-MiF-

I don't know why your cells doesn't grow.
I already froze primary cells (fibroblast-like synoviocytes, lymphocytes), and they grow fine after thawing.

what are your cells?

-Missele-

QUOTE (Missele @ Aug 16 2006, 07:56 AM)
I don't know why your cells doesn't grow.
I already froze primary cells (fibroblast-like synoviocytes, lymphocytes), and they grow fine after thawing.

what are your cells?


human nasal respiratory cells....

and I don't understand....

-MiF-

may be they reach the max. point of growth and need passage ..

-strawberry-

Primary cells are not as resistant to the freezing process as cell lines. They also have a Finite number of population doublings that they can achieve before senescence or apoptosis occurs. You mention that the cells at first achieve "100% Confluence". That could be part of the problem. You should never let your cells get to that level of confluence, you should passage/split your cells at between 70 and 80%. Some cells exhibit contact inhibition i.e. when they are in full contact with other cells, the cells become quiescent.
When you freeze your cells you should increase the % FCS in your freezing mixture i.e 10% DMSO, 80% FCS and 10% Media. This can in some cases protect the primary cells from the freezing process.

-Rhombus-

QUOTE (Rhombus @ Aug 17 2006, 06:30 AM)
Primary cells are not as resistant to the freezing process as cell lines. They also have a Finite number of population doublings that they can achieve before senescence or apoptosis occurs. You mention that the cells at first achieve "100% Confluence". That could be part of the problem. You should never let your cells get to that level of confluence, you should passage/split your cells at between 70 and 80%. Some cells exhibit contact inhibition i.e. when they are in full contact with other cells, the cells become quiescent.
When you freeze your cells you should increase the % FCS in your freezing mixture i.e 10% DMSO, 80% FCS and 10% Media. This can in some cases protect the primary cells from the freezing process.



I freeze the cells after treatment of the biospy. Then the cells have not yet been plated.

And I have the same problem when I trypsinize the cells. I already tried with trypsin, trypsin+versene and some other conditions....whithout any success...They have exactly the same problem to grow after spliting.

I found in a book of culture to freeze my cells: Combination of Hepes, FBS (10%), Ham's F12 (<80%) and 10% DMSO...then I wonder whether is it a question of "cells" or a question of "handling".

Thanks

-MiF-

Hi MiF
Are you dropping the cell density too low after subculture (trypsinization) or after thawing your frozen cells? I work with primary cells quite a bit and many of the cell types don't like having their density drop too low after subculture, particularly once they've been passaged a couple of times. They'll often curl up their toes and die if this occurs.

Secondly, I agree with Rhombus, your cells shouldn't be overcrowded prior to freezing (or passaging). Many primary cell lines do not respond well if they're allowed to become overgrown, particularly with the non-specialist (routine) media most research labs use.

-karyotyper-