Good day, I am a fairly new researcher having problems in culturing the THP-1 Blue monocytic cell line. I am trying to make a bank of these cells for future use; however, whenever i culture these cells, they always show a very low viability percent (around 30% to 40%)
These cells are from the suspension type and this is what i am doing:
1) I thaw a vial and culture them in T-75cm flask in 10ml of Rosswell Park Memorial Institute (RPMI) 1640 culture media. I leave them overnight in the media.
2) I then proceed to centrifuge the cells at 1,300 - 1,400 rpm x 5 minutes to remove the dimethyl sulfoxide (DMSO).
3) The cells are then cultured in T-75cm flask with 10ml of RPMI media for 2-3 days.
4) After 2 or 3 days, i obseve that there appears to be a great number of cells. I get excited and proceed to centifuge them at 1,300-1,400rpm x 5 minutes. The pellet appears great in size, i remove the supernatant and proceed to count the cells.
5) I count the cells with the trypan blue procedure (white cells are alive while dead cells are blue) and i see that almost all of them are in blue. I count them anyways and proceed to validate my results with the TC20 Automated Cell Counter by BIO-RAD and confirm that they show similar/identical percentage of live vs dead cells. The cells are useless so i have discard them.
IMPORTANT NOTES:
DATA SHEET CATALOGUE # thp-cd14sp
-The cells are in an early passage (passage +6)
-The incubator parameters are 37C and 5% CO2.
- I really do not like the date sheet of these cells. They recommend to centrifuge the cells at 3,000 - 5,000rpm x 5 minutes. I did this once and they all died (around 15% viability).
- The cells were provided as a gift from my committee member. The first time i subcultured these cells and count them, it resulted in a 95% viability. I started with 3 million cells and they double to 6million to almost 7 million cells. I froze these cells and after a while, i took a vial and thaw them and they started dying since then.
-We had suspicions that it was our incubator which was improperly cleaned. However, i sub-cultured the cells in two different incubator and they resulted with almost exactly the same viability (33% of primary incubator and 35% in secondary incubator).
-I was given a free culture media of RPMI from my co-workers. However, i am still very new and i was told this media works even though it is expired. I culture them with this media the first time and it resulted in 95% viability. The media expired on AUG 2013 and i fear it was maybe this? If it was this then why did they proliferate so fast with such a good viability?
-We have a fairly new RPMI culture media. However, this expired in AUG 2017. I prepared the media and culture another vial and they died with a 45% viability.
-My laboratory partners think they may have died during the freezing procedure. The freezing media recommended by this data sheet is RPMI 1640 with 20% FBS and 10% DMSO. We have suspicions that the 10% more of FBS may have killed them during the freezing procedure.
- I even count the cells with the RPMI media exp of AUG 2017 and count the cells the next day after removing the DMSO and they were at 45% viability.
Please, i would like suggestions for what to do.
If I'm reading your initial message correctly, you thaw the vial, put in flask and immediately put in incubator for overnight culture. This is wrong.
Thaw the vial and as soon as possible spin down the cells and remove the medium containing the DMSO from cryopreservation, then resuspend in fresh medium and put in culture overnight.
I don't like that they say 3000-5000 rpm on the datasheet because RPM will have variable RCF depending on the diameter of the centrifuge that you use. Thaw the cells (1 mL) and pull up the cells in 9 mL of pre-warmed RPMI, then you should spin the cells at 125x g (rcf) for 5-7 minutes to pellet the cells from the vial, remove the entire supernatant, and resuspend in fresh medium.
Normal THP-1 cells require a small amount of beta mercaptoethanol in the RPMI to grow properly, but I see that these particular cells from Invivogen don't. I'm not sure why.
I agree with Jordan, you should remove DMSO by centrifugation as soon as possible after thawing. It is probably the reason why you have low viability. I just wanted to add few points which should make THP1 culturing a bit easier. They should be kept in a concentration between 0.2 and 0.8 million cells per ml. I usually count them daily as they like to be disagregated. When they reach 0.8M/ml i split them in half. On Fridays i seed them around 0.3M/ml to keep them happy over weekend. Also we usually freeze cells in 10% DMSO in FBS as it improves survival. I usually grow them without beta mercaptoethanol and they are ok but you can add it and see if it makes a difference for you. Hope that helps
Good day, Jordan and Sylwia and thank you for your responses. At one time, i did thawed the cells and then quickly centrifuged the cells to remove the supernatant + DMSO. However, they resulted in a very low viability. I later then culture another vial by thawing the cells and culturing them overnight to remove the DMSO later which also resulted in death.
Is it possible that the expired culture media is affecting the viability? A culture media expired in AUG 2013 vs AUG 2017 may kill the cells? From what i know, the media was stored in the dark all this time.
It's hard to say exactly what would have degraded in that time. Always best to use non-expired materials so that you can be sure what the variables are. Try finding a neighboring lab to borrow some RPMI from. It's quite common.
As a side note. My laboratory partners may think it could have been the freezing media. The freezing media contains RPMI 1640 media, 20% FBS and 10% DMSO. We usually use the standard 10% FBS for freezing media; however, the data sheet suggested 20% FBS. Could a 10% more FBS affect cellular viability?
Highly unlikely in my opinion. I know people who freeze their THP-1 cells in 90% FBS with 10% DMSO (and no RPMI at all) and have good success.
Actually, having 90% FBS usually improves cells recovery after freezing. People use lower concentration in the freezing mix because FBS is more expensive than media. Maybe your cells are kept too long in the freezing mix before you freeze them down? The freezing mix should be cold as cold DMSO is less toxic than warm one. And cells should be frozen in Mr frosty as soon as possible after adding the mix. I've worked with expired RPMI before (about 1 year after expiry date) and i didn't notice any difference, although i wouldn't use 5 years old media. Do the cells finally recover after thawing or do they also struggle to grow?
Thank you, Jordan and Sylwia, i feel now more relief with the FBS part. In response to your question, Sylwia, the cells struggle to grow after thawing. I thawed the cells, left them overnight and removed the DMSO the next day and counted them. They had a 45% viability from one day to another. You might think the RPMI expired in 2013 killed them?
I'm not sure if expired RPMI killed the cells, but probably it didn't help them. try to get fresh RPMI at least you will have one less thing to worry about. However, i wanted to know what happens if you grow your cells longer, a week after thawing, do they recover or do they still have poor viability. It may help us to troubleshoot your problem
Well, i did tried to grow them when they were at passage +6 for around 1 week and a half by changing the culture media every 2- 3 days. When i first received the cells, the passage was at +5 which i successfully made a bank of cells in passage +6 with a 95% healthy cell viabiltiy with the expired RPMI media. I froze all the cells at +6 at -80C for around 1 month or so using freezing media of (expired RPMI, 20% FBS and 10% DMSO) with each vial containign 3 million cells for around 1 month or so.
I subcultured passage +6 to +7 for around 1 week and a half which at passage +7, it went down to 83% viabiltiy and at passage + 8 it went down to 40% viability during the 1 week and a half of recovery.
I completely agree with Jordan. I would like to add couple of things to it.
1. Instead of T75, try T25 for first overnight culture. THP1 tend to be happy when the concentration is above 5X10^5/ml.
2. For freezing, I resuspend my cell pellet in FBS, and then aliquot it in the cryovials. Then to each cryovials, I add equal volumes of 10% DMSO/FBS.
Thank you all for your answers, a committee member will provide me a frozen vial of THP-1 Blue cells and a new RPMI media was ordered from ATCC. Hope everything goes well this time.
Hi,
Your main mistake is made in the beginning when you thaw the cells.
To preserve cells and avoid intracellular ice formation the freezing medium is enhanced with glycerol/DMSO etc. to pull out the water from intracellular space. Therefore when you leave the cells after thawing in freezing solution without washing it out you are simply intoxifying the cells.
Therefore to preserve the cells viability you have to change your protocol. Before you launch thawing at 37C prepare in advance the medium with 10%FCS/FBS and and 1% of antibiotics (50.5 units/ml penicillin, 50.5 μg/ml streptomycin and 101 μg/ml neomycin, Sigma). Fill the 15mL falcon with 10mL of your full medium (FBS+ antibiotics) and add the thawed cells suspension. Mix it delicately in hands and immediately centrifuge to remove all toxic agents. Remove the supernatant without disturbing the cells pellet. Then re-suspend the cells pellet in full medium and seed the cells in TC flask.
Usually after two days cells should be already well attached and you should change the medium. Then after leave the cells for about three days - observe the medium color change as an indicator of a pH. Add fresh media and wait till the required cells number will be achieved and then divide them into new TC flasks.
Good luck!
Katarzyna
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