I thawed my primary cells and froze them again? serious mistake? - cell culture (Jun/02/2008 )
A question may be silly for me 
We are trying to prepare primary cells from fetal sheep LD muscle, which is very important model for our lab, so My advisor is doing cell culture thing himself. But last week he has to be out for a meeting, then I had to collect two disks of cells and freeze them.
I put 10% DMSO into the cryovials first, then put 1ml suspended cells. I then put them into -80 C freezer. After about 5 min, I suddenly realized I hadn't mixed the DMSO with cells by shaking. But I saw the cells have been frozen. I just thought if DMSO is lying at the bottom of vial, it can't protect cells. Being very upset, I thawed them in warm water, mixed by shaking the vials up and down, then put back to -80C freezer again. 12 hours later, I stored them in liquid N2.
Can anyone tell me happily those cells are still alive? or they are very likely dead, which may make my boss mad.
THANK YOU!!
I would say that the cells are likely to be very unhappy, if they have survived at all. There should have been some cells that were OK in the vials before thawing, but I would suspect that they are no longer alive following the re-freeze. The only real way to tell is to thaw and try growing.
how many cells you estimate to have in each vial. there might be some viable cells. but i'm afraid they won't stay alive for long.
. . had to collect two disks of cells and freeze them.
Hope you have more culture plates.
I hope your advisor will not be angry.
We are trying to prepare primary cells from fetal sheep LD muscle, which is very important model for our lab, so My advisor is doing cell culture thing himself. But last week he has to be out for a meeting, then I had to collect two disks of cells and freeze them.
I put 10% DMSO into the cryovials first, then put 1ml suspended cells. I then put them into -80 C freezer. After about 5 min, I suddenly realized I hadn't mixed the DMSO with cells by shaking. But I saw the cells have been frozen. I just thought if DMSO is lying at the bottom of vial, it can't protect cells. Being very upset, I thawed them in warm water, mixed by shaking the vials up and down, then put back to -80C freezer again. 12 hours later, I stored them in liquid N2.
Can anyone tell me happily those cells are still alive? or they are very likely dead, which may make my boss mad.
THANK YOU!!
First of all IT IS NOT YOUR FAULT!!!!!!!
YOUR SUPERVISOR should have trained you properly in the art of cryogenic freezing.....so it's HIS/HER FAULT.
Secondly cells should be frozen at 1 degree /minute , down to -80, then left overnight in the freezer, then transferred to liquid nitrogen.
As others have said.......the cells will be completely useless.........nothing you can do about that.
Primary cells are more difficult to freeze and resuscitate. You should first practice on cell lines, and commercial ones at that....ones that can be easily replaced........THEN GO ONTO PRIMARIES.............................................again this is YOUR SUPERVISORS FAULT.
Come clean and own up........
Make some more primaries
Good luck
Rhombus