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Primary cell line - (Apr/19/2013 )

Hi,

Is there any body to advice me how I handle the primary cell I bought from company. when I cryopreserve it and again thaw it cell become different morphological and slow in growth. I need really help. Thank you

-virusproteomics-

Did the company tell you in the product sheet how many passages the cells can be passed? Primary cells are not immortalized and have a limited number of cell division, then they become senescent giving a different morphology and stop to grow.

-pcrman-

pcrman on Sat Apr 20 07:24:58 2013 said:


Did the company tell you in the product sheet how many passages the cells can be passed? Primary cells are not immortalized and have a limited number of cell division, then they become senescent giving a different morphology and stop to grow.
It is about 15. I cropreserved it at passage no 1. After thawing the cell the morphology and growing rate decreased substantially.

-virusproteomics-

What cell type is it? Did you have a look on the ATCC (atcc.org) website for similar lines and protocols?

-bob1-

It is a primary human epithelial airway cells from Lonza

-virusproteomics-

Hi VP,

 

These cells require collagen coated surfaces when they're fresh out of nitrogen, so check that you have done this. I use Collagen I rat tail, but collagen IV is also fine. If you're using the Lonza bulletkit for medium, try also culturing without the gentamicin that they include. My cells are sensitive to this and go into growth arrest if I persist with GA. What you describe does sound like growth arrest. Are you getting any membrane blebbing at all? This looks like little ball like structures extending on stalks from the cells. The first time I saw them I thought, "Oh goody, they're spreading out!", but alas, no - they were producing apoptotic bodies!

 

I would start again with fresh cells (you say you froze at p1, but is that YOUR p1? If so, what passage did they come in at? Don't forget to add that on).

-roscopcat-