Freezing and thawing of cells - (Sep/09/2004 )

Hi everyone!
I posted earlier too about not being able to get viable cells. To refresh memory, .....I am working with Hybridoma cells. They are kept frozen in vapor N2 as liquid N2 temperature is too cold for them. And I am mentioning how I thawed the last vial...

First day, I thawed a vial(in about 2 minutes at 37 degrees) and split it into two by resuspending it in two 20 cm2 flasks that hold around 4-5ml at a time. So, in short I thawed a vial into 10 ml medium and 2 fasks. VIABLE Cell concentration was 0.6
Second day-VIABLE Cell concentration was 0.91 and 0.78 for the two flasks from day1. I seeded the cells at seed concentration of 0.4, and what was remaining (approximately half of the culture was left over), I centrifuged it at 900 rpm for 10 minutes. I tried lower speeds with previous dead cells and found that no less than 900 rpm was any good).
The third day, the VIABLE Cell concentration was 0.1025 and 0.075 and 0.1175
for seeded flask-1, seeded flask-2 and centrifuged flask-3. So, I had not much choice but to centrifuge the two seeded flasks and resuspend the combined cell culture into 4 ml fresh medium. I wanted the cells to be enough to be able to condition the medium.
The culture that had centrifuged cells also did not have high viable cell conc., so I just transferred it into a new flask and added 0.3 ml of fresh medium. I now think I should not have transferred to another flask.
Do you think there is something the matter with my ways? The medium has added glutamine to it, now.
I have seen that with all the vials that I thawed, te cells seem to grow till a day after thawing, but after that they just go downhill. What could cause this? One thing for sure, I do not centrifuge immediately, I keep for a day and the centrifuge.
I hope this is detailed enough to be abe to judge.

Penny

Hi,
I have been thinking about the problems with my hybridoma cell line that could be hampering growth. It would be helpful if I could get a second opinion from people around who have some experience with hybridoma cell line/ eukaryotic cells.
So far, I have tried 4 vials and a common trend amongst all is that the cells survive till 2 days(incl. the day of thawing) and then there is a sharp decline in the viable cell count, untill the cells are completely gone in say 4-5 days. So far, I have tried replenishing the media with Glutamine, centrifuging, maintaning a high viable cell concentration of 4x10^05 cells but the cells don't seem to proliferate.
1) When the cells were frozen, they were kept overnight in a -80 degrees freezer and transferred to liquid nitrogen the next day; I went through number of cell freezing protocols and they seem to suggest that the vials need to be cooled/frozen gradually. Does it seem that freezing method was improper?
2)Could be a problem with the medium... it expired on 08/05 but i replenished it with Glutamine again.
3) DMSO needs to removed immediately? I am not aware if it is a critical factor for my cell line. I have not found any information that confirms so.I remove it a day after thawing by centrifuging at 800-1100 rpm for 5 to 10 min.
4) Centrifugation of culture is done a day after thawing to get rid of DMSO, maybe the cells die of necrosis due to shear.
5) Subculturing errors...I am not sure what ways would that be going wrong?
6) Is dilution of a vial into around 20 ml of media a concern as it might induce ostomic shock? the person before me seems to have used this dilution ratio and had no problems, really. I guess stepwise addition of medium could help!
7) Any other reasons you could attribute?
These are the only possible reasons I can think of?
If anyone can give any suggestions or feedback..it could help me figure out the flaw in my case.
Waiting to hear from you people
Penny

Hi Penny,

First of all, don't worry, I've had problems with cell culture too! I don't culture hybridomas, but I do culture some fussy mammalian cells.
First of all i would check that you have the correct medium for your cell type and correct conditions in the incubator - it could be a problem with CO2 that causes cell death. Make sure the medium is in date and has the correct supplement of glutamine if it's not already in it, and fetal calf serum (FCS).

When freezing your cells, you should freeze slowly. I use a freezing medium made up of FCS, 10% medium and 10% DMSO. Trypsinise or bang off the cells as normal and centrifuge at 1200rpm for 5mins. resuspend in 1ml of freezing medium and as you do - -80 overnight sounds fine.

When thawing, I thaw the vial in some 70% IMS in distilled water at 37 degrees, then transfer the cells to 5ml of fresh warmed medium in a 15ml tube and centrifuge at 1200rpm for 5mins, to wash out the DMSO straight away. discard the supernatant and resuspend the cells in the desired amount of fresh medium and put up into one flask straight away. Leave the cells to settle overnight and have a look at them the next day.

I don't know if this will help, this is just the method I use and it works just fine for me. I know someone who cultures hybridomas, so if you still have problems after this I will ask for their methods!

Good luck
HannahBelle

Hi Hannah,
Thanks a lot for the suggestions!
I have already suuplemented my medium with L Glutamine, and I use FBS.
I will try it this way and lets hope for the best!
Penny

I'm trying to expand some new working stocks of neural progenitors, but I am having problems with their attachment, the first passge after thawing.

The cells grow very fast directly after thawing, and I have to passage on day 3. After that, the cells start looking funky. The cells are clumping, attachment is poor, even a gentle media change can rinse a lot of them off. The clumps proliferate in suspension. I've even seen sheets of what look like spread cells with processes, floating in the flasks.

It seems like it has to be something with the trypsin, or the water quality... I don't see any contamination, and we've been testing regularly for mycoplasma.

Has anyone else had similar problems?

Hi there!
I have a few general questions for all the animal cell culture people.
1. Can a medium containing 3.7 g/L sodium bicarbonate be grown in a 5% co2 incubator? for hybridomas?
2. how critical is the presence of sodium pyruvate in the growth medium? And to what concentration? I think it is just a energy source and the cells should functin fine even without it
Waitin for the feedbacks!!
Penny

1. Can a medium containing 3.7 g/L sodium bicarbonate be grown in a 5% co2 incubator? for hybridomas?

I know of some medium that is buffered with sodium bicarbonate, generally requiring around 5% CO2 settings for the incubator. However, the people who work on it have found that actively growing cultures, particularly as they approach confluence, tend to overwhelm the acidic buffering capacity. This can be seen by the phenol red indicator - culture medium becomes yellow. Therefore, they often kept a separate incubator set at lower CO2 concentration (around 3%) to which they removed cells as they became more confluent. Frequent refeeding could also alleviate the acidity. The cells will lose viability if left acid for too long.



2. how critical is the presence of sodium pyruvate in the growth medium? And to what concentration? I think it is just a energy source and the cells should functin fine even without it

All ready discussed in this forum.

Vetticus

Thanks a lot!
Penny

Hi Everyone!
I have yet another question.. what is cause of heat inactivation of FBS..... before thawing or subculturing..I place the culture medium(incl. FBS) in the incubator(5% c02 at 37 degrees) for an hour. Could it lead to FBS inactivation?
Penny

Hi ALL!!
Well, I found the answers to my previous questions...thanks (a lot) to the helpful souls we have at the forum....
There is yet another question, though.
I am to freeze my very own Hybridoma- cell bank this week and how it was done was that the vials were kept in a -80 degrees freezer overnight...but I think that causes the cells to cool down too fast causing problems with recovery later on.
I read somewhere that the next best option is to put a styrofoam box into the freezer for a day and the next day centrifuge the cells into vials and put them in the styrofoam box for a day.
However, another popular opinion is that this is not such a good practice after all, as the cooling rate is below the recommended 1 degree per minute.
SO, I am confused....(obviously)......Previously, we used to freeze the cells directly in a -80 freezer, but the recovery is 30-40% with that. So, upon thawing it is hard to revive the cells..
Pl. help me choose lesser of the 2 evils...
Thanks,
Penny