Freezing and thawing of cells - (Sep/09/2004 )
Hi all,
I had read all the suggestions given in the page, thought maybe someone can give me some suggestions to my protocol as well.
I am relatively new in ACC field. Recently i just got 3 vials of Pig bone marrow cells (primary cells). As far as i know, those vials were frozen for about... 5 months in - 80'C. Will that affect the cells viability since they are stored in -80'C for so long and also they were taken in & out of the -80'C occasionally?
When i thawed the cells which i was given and observed them, they were like..... DEAD! omg... i'm not sure whether is the protocol or if is just ME.
i thawed the cells according to the protocol:
take vials out from -80'C
thaw quickly in 37'C water bath
transfer content to 15ml centrifuge tube
add 10ml complete media dropwise to content
spin @1500rpm, 5mins
discard media (with DMSO as cryopreservative)
resuspend pellet with 10ml media
cell count & viability check
seed suspension to T25 and incubate.
When i did a viability check and cell count... i have got something like 45% viability for my cells, but after the incubation overnight... they did not attach to flask and were still in suspension. Its my first time dealing with primary cells and i'm not too sure why they always die after the incubation? Never had such problems with cell lines.
HELP!
Sg_ACC
while i think it might be difficult to agree on a procedure that will work best for most, please make sure NOT to
most cryovials can - under certain circumstances - let some of the liquid nitrogen leak into them. any cryovial coming from liquid nitrogen storage is therefore to be considered dangerous and explosive for the first few minutes. take it out of the tank using usual safety precautions, place it into a special container, and let it sit for a few minutes. THEN take it to the water bath.
This is not hearsay, it has happened to me more then once!
take care.m
yup i agree.... it happened to my frd, the vial exploded and injured her face.
http://www.protocol-online.org/prot/Cell_B...tion/index.html
For bacteria it's important to freeze quickly.
That's because when freezig slowly, ice cristals may form.
Strange that it's opposite for other cells.
Can someone explain why?
hi
for general and detailed informations on using cryopreservatives, badcell gave a very good link :
fundamentals of cryobiology
[quote=shilpi13,Sep 9 2004, 11:32 PM]
hi
I am facing problems in thawing of cells. The procedure which we follow is- we take stock from liq. N2 then though it in water bath at 37 degree. Then we take medium in falcon (5ml) then add thawed cells dropwise into it. We then centrifuge it at 1500rpm for 5 mins.
but the problem is that after thawing i am unable to get viable cells.
Almost all cells are dead. So what all modification shall i make ? Its really hard to revive cell line from frozen stock?
well, as for me...if you have thawed cells add to them PBS sloooooowly drop PBS...I use 5ml PBS to 1 ml of cells...subsequently spin down them 1500 rpm 5 min....discard supernatant and add normal medium
When i thawed the cells which i was given and observed them, they were like..... DEAD! omg... i'm not sure whether is the protocol or if is just ME.
When i did a viability check and cell count... i have got something like 45% viability for my cells, but after the incubation overnight... they did not attach to flask and were still in suspension. Its my first time dealing with primary cells and i'm not too sure why they always die after the incubation? Never had such problems with cell lines.
HELP!
First of all... don't panic. Primary cell lines can be touchy little things.
In your case, you were working with primary marrow cells. I haven't done that particular tissue preparation, but it has to come out crammed with red blood cells no matter how much they try to wash and separate tissue types during preparation. So I'll bet that your initial count was full of erythrocytes, as well as a fair amount of debris.
So: the debris is going to stain as dead, giving you low viability. The erythrocytes, on the other hand, are going to stain as live cells - but they aren't going to attach and grow into a viable culture. If you've been counting erythrocytes as live cells and basing your inoculation density on that count, it's likely that you're inoculating at a MUCH lower density than you planned on.
This is mostly a matter of getting used to what a primary cell line looks like out of the amp. The erythrocytes will be round and smaller than the cells that you're looking to culture; but most notably they will have none of the internal detail that you see in normal, nucleated cells.
Primary cell lines vary. I've seen some that are very forgiving of being at low density, and other cells that simply die off when it's too low. The next time you thaw one of these amps, count them very conservatively - the only thing you should count as a live cell is one that looks like it has the potential to divide and grow. And inoculate heavier. The worst that happens is that you have to split the cells sooner.
Good luck... and keep us posted.
Katherine
"For bacteria it's important to freeze quickly.
That's because when freezig slowly, ice cristals may form.
Strange that it's opposite for other cells.
Can someone explain why?"
Bacteria have cell walls, and that makes them more rugged. My area is mammalian cell culture, and they only have cell membranes, which are much more fragile. Addition of DMSO to the freeze medium for mammalian cells is to prevent the formation of ice crystals that would puncture those membranes. My understanding is that a slow freeze actually lessens the occurrence of ice crystals.
Bacteria are tough. I don't know if it's actually important to freeze them quickly, or if you freeze them quickly because it's simpler and you know the bacteria will survive.
Katherine
hi!
well, i don't know for bacteria.
i work with 4 different multiple myeloma cell lines, which i thawed after they were frozen for 8 months. i got poor viability in two lines, but they recovered pretty well. generally, i follow the rule "slow freezing, quick thawing", but with some modifications.
thawing:
1) put cells in 37 degrees bath till thawed
2) ASAP transfer them into snap-caps and dilute with RPMI dropwise (1:10 at least, to dilute DMSO; washing DMSO is not necessary since 3)
3) transfer them into a flask and add 20-30 ml RPMI (i think it is enough to dilute DMSO since my cells are ok after that)
freezing:
i freeze cells with 50% FCS, 40% RPMI and 10% DMSO, 4-5 x 10e6 per tube
1) resuspend palleted cells with FCS
2) transfer them in cryotubes
3) add DMSO+RPMI (20% DMSO, 80% RPMI) DROPWISE
4) ASAP transfer them in cotton-embedded box and put in freezer -80 degrees
course, it is much easier to handle malignant cells than normal ones, 
.
Hey,
Dont try to thaw fully at 37 degree water bath completely, let the cells remain frozen partially, keep ur tubes and everything ready in the mean time. Instead of adding all of ur media in the 5ml flacon tube , add0.5 ml in the the partially thawed vial. This will help decrease the DMSO concentration also when the cells are fully thawed. Keep rinsing the thawwing vial with ur media till u get everything out.
Let meknow if you see any improvements.
-PAN