I routinely culture MOLT-4 and a range of T-ALL cell lines, and these do grow reasonably well. Not knowing all the steps you have taken, I have taken the liberty to write what I do with my MOLT-4 cells for freezing and thawing. Perhaps you can contrast and compare with your method, bearing in mind that there are many ways to freeze and thaw cells of course!

FREEZING DOWN:I freeze down my cells as follows:

1) Make freeze mixture of 5 mL 10% Media, 2 mL DMSO, 2 mL 100% FBS. (does get warm upon addition of DMSO - so make sure you cool it down to RT prior to adding cells at step 3 below - or you run the risk of killing your cells)

2) Spin down cells, resuspend in small volume of complete media (i.e. 10% FBS RPMI) - keeping in mind the number of vials you wish to make.

3) Place an equal volume of cell suspension with equal volume of freeze mix (i.e. 500 uL cell suspension + 500 uL Freeze mix)

4) Place in Cryobox and then in -80. Transfer to Liq Nitrogen the next day or at the earliest moment (as the longer they stay at -80, viability does decrease)

THAWING:

Method 1 (slow and sure): I take vial out of liquid nitrogen and warm in 37 degree water bath, or in incubator to thaw. Then CAREFULLY pipette cell suspension in 15 mL tube and top up with pre warmed complete 10% FCS RPMI to 5 mL, and then spin for 5 min at 800 rpm (slower than usual). Then remove supernatant and resuspend in media and place in T25 or T75 depending on cell density and cell number. This will remove all the DMSO.

Method 2 (if you are in a hurry).: Take vial thaw out, add thawed cell suspension direct to flask with media. But - make sure the next day, you collect all the cells, spin down, remove supernatant to get rid of DMSO, and then resuspend in fresh media and culture as per usual. Residual DMSO I find can slow the cells down a bit - not always - but best to remove it as soon as possible in my view.

Anyway, hope this helps a bit, you may be doing all this already and so it could all just be a case of bad freeze down vial which happens from time to time. Hence why it is important to freeze a few vials at a time and in different batches.

Good luck with the trouble shooting.

Hi Sridharan,

the most trivial question: did you check for mycoplasma? In my experience, especially the suspension cell lines have to be checked often and carefully.

Other possible reason: did you change your serum (supplier, charge or similar)?

Another possible solution for the problem: thaw one of your earliest back-ups and check if they behave the same.

Good luck

Michael

Hi Sridharan,

I may give you an opinion. Your cell line grows as suspension culture. So when you take them out from nitrogen, cell suspension could contain too many dead cells with healthy ones. I suggest you when you take frozen cells, put them into horizontal 75T flask for 1 or 2 weeks. In this way you can separate dead cells from alive ones. If they stay together, dead cells will cause dead signal which kills your healthy cells. Put cells into flask very diluted and horizontally you will see your healthy cells grows much more faster.

Good luck

Cigdem

I routinely culture MOLT-4 and a range of T-ALL cell lines, and these do grow reasonably well. Not knowing all the steps you have taken, I have taken the liberty to write what I do with my MOLT-4 cells for freezing and thawing. Perhaps you can contrast and compare with your method, bearing in mind that there are many ways to freeze and thaw cells of course!

FREEZING DOWN:I freeze down my cells as follows:

1) Make freeze mixture of 5 mL 10% Media, 2 mL DMSO, 2 mL 100% FBS. (does get warm upon addition of DMSO - so make sure you cool it down to RT prior to adding cells at step 3 below - or you run the risk of killing your cells)

2) Spin down cells, resuspend in small volume of complete media (i.e. 10% FBS RPMI) - keeping in mind the number of vials you wish to make.

3) Place an equal volume of cell suspension with equal volume of freeze mix (i.e. 500 uL cell suspension + 500 uL Freeze mix)

4) Place in Cryobox and then in -80. Transfer to Liq Nitrogen the next day or at the earliest moment (as the longer they stay at -80, viability does decrease)

THAWING:

Method 1 (slow and sure): I take vial out of liquid nitrogen and warm in 37 degree water bath, or in incubator to thaw. Then CAREFULLY pipette cell suspension in 15 mL tube and top up with pre warmed complete 10% FCS RPMI to 5 mL, and then spin for 5 min at 800 rpm (slower than usual). Then remove supernatant and resuspend in media and place in T25 or T75 depending on cell density and cell number. This will remove all the DMSO.

Method 2 (if you are in a hurry).: Take vial thaw out, add thawed cell suspension direct to flask with media. But - make sure the next day, you collect all the cells, spin down, remove supernatant to get rid of DMSO, and then resuspend in fresh media and culture as per usual. Residual DMSO I find can slow the cells down a bit - not always - but best to remove it as soon as possible in my view.

Anyway, hope this helps a bit, you may be doing all this already and so it could all just be a case of bad freeze down vial which happens from time to time. Hence why it is important to freeze a few vials at a time and in different batches.

Good luck with the trouble shooting.