11 replies to this topic

[DSLJ]

Anyone share their protocol for Freezing down and thawing THP-1 monocytes, have just got a new batch but in the past have found them very difficult to grow from frozen.

[SuMi]

I freeze my THP-1s in Recovery Cell Culture Freezing Media from Gibco at a concentration of 2x10(6)/mL. But I have used 10% DMSO in cell media as well and this works fine. I freeze them at -80oC using a Mr Frosty and transfer them to liquid nitrogen after 2 days.

To take out THP-1 stocks, I prewarm two 5 mL aliquots of media and then take the vial out of liquid nitrogen, thaw as quickly as possible in the water bath and transfer the contents of the vial to one 5 mL tube of media as soon as it starts to defrost. I centrifuge them at 800xg for 3 minutes and then resuspend the pellet in the second 5 mL of media and transfer them to a T25 flask.

This works for me. After 2 days I have enough cells to transfer them to a T75 and then they're flying!

[rhombus]

SuMi, on Oct 1 2009, 11:43 AM, said:

I freeze my THP-1s in Recovery Cell Culture Freezing Media from Gibco at a concentration of 2x10(6)/mL. But I have used 10% DMSO in cell media as well and this works fine. I freeze them at -80oC using a Mr Frosty and transfer them to liquid nitrogen after 2 days.

To take out THP-1 stocks, I prewarm two 5 mL aliquots of media and then take the vial out of liquid nitrogen, thaw as quickly as possible in the water bath and transfer the contents of the vial to one 5 mL tube of media as soon as it starts to defrost. I centrifuge them at 800xg for 3 minutes and then resuspend the pellet in the second 5 mL of media and transfer them to a T25 flask.

This works for me. After 2 days I have enough cells to transfer them to a T75 and then they're flying!

BIG MISTAKE,

THP-1 need to be frozen in 10-20 Glycerol as the cryogenic preservative.....the reason being:-

Researchers use DMSO to DIFFERENTIATE these cells into macrophages.

Thus if you use DMSO in the freezing mixture, you will differentiate cells in the process.

Hope this advice is not to late.

Rhombus

[SuMi]

I've never had a problem with cells differentiating in the Cell Recovery freezing medium. The only time my cells have differentiated when I didn't want them to was when I let the seeding density get too high (>1.5e6/ml) and they spontaneously differentiated.
I have tried making stocks with glycerol and the recovery was very poor when I thawed them

[DSLJ]

Thanks for the replies, my recipe for freeze media is Complete media 45%, FCS 50%, DMSO 5%.  Centrifuge cells, remove excess media and add cold freeze media drop wise on the cells, the freeze in an insulated box at -80 and then 24 hours later transfer to liquid nitrogen.  Is 2 x 106 your normal final concentration in the freeze media, I have been using much higher concentrations maybe thats where I have been going wrong!

[miBunny]

When first plating after thawing, upping the FBS to 20%  can help them bounce back.  A lot of the protocols will use glycerol for the cropreservant because DMSO can induce differentiation but glycerol does not protect the cells as well as DMSO (it does not penetrate into the membranes as well).  Adding the extra protein by doubling the FBS helps to "cushion" and protect the fragile cell membranes until they can recover.

[DSLJ]

Thanks for that, I have another problem now it looks like 50% of my THP-1s are sticking to the bottom of the flask - is this spontaneous differentiation and what are the causes other than overcrowding which I dont think was the problem unless my cell counts are wrong.

[SuMi]

Sounds like they have differentiated into macrophages. Have they become more oval in shape rather than round? I don't know what else would cause it besides the seeding density being too high. I have seen them starting to change shape when I left them for 2 days without feeding even though the density didn't look very high.

[hoshi]

rhombus, on Oct 1 2009, 09:09 AM, said:

BIG MISTAKE,

THP-1 need to be frozen in 10-20 Glycerol as the cryogenic preservative.....the reason being:-

Researchers use DMSO to DIFFERENTIATE these cells into macrophages.

Thus if you use DMSO in the freezing mixture, you will differentiate cells in the process.

Hope this advice is not to late.

Rhombus

I want to use THP-1 in an undifferentiated state as a monocytic model cell line. I am currently having difficulty replicating some prior publication data and I was wondering if these issues are due to THP-1 becoming differentiated while freezing the cells.

I found some frozen THP-1 cells which were frozen with 10% DMSO, 50% FBS and 40% RPMI by a previous lab member.
Is it normal for THP-1 cells to not be all round and symmetrical like U937 or HL-60 cells?

Also what surface markers do your THP-1 express? The only reference I could find, reported THP-1 to be CD14+/-, CD33+, CD13+, CD11c+, CD4+ but my cells are CD14-, CD33+, CD13+, CD11c+/- and CD4+/-.

How much differentiation would one expect if THP-1 were frozen with 10% DMSO?
If DMSO induces differentiation and ATCC uses 5% DMSO to freeze their cells, how "good" is it to use cells bought from ATCC?

Thank you

Edited by hoshi, 19 November 2009 - 12:14 PM.

[DSLJ]

Have started back with a fresh batch of THP-1s, just wondering about cell concentrations you freeze down at 2x 106 cells/ ml seems very low?
What concentrations do you use?

[SuMi]

I always freeze 2x10(6)/ml. If you freeze down too high a concentration of cells they will not all be protected by the cryoprotectant and there will be a high percentage of dead cells when you thaw them anwyay

[DSLJ]

SuMi, on Jan 25 2010, 08:15 PM, said:

I always freeze 2x10(6)/ml. If you freeze down too high a concentration of cells they will not all be protected by the cryoprotectant and there will be a high percentage of dead cells when you thaw them anwyay

Thanks for that, its just with other cell lines I always freeze down at  a much higher (107) type concentration.  Will try the 2 x 106 and see how it goes