Hi,
I am using normal Human Epidermal Melanocytes neonatal (HEMn) cells and they used to adhere and grow normally in ready-to-use Melanocyte Growth Medium. However, after freezing them for about 3 months, when I culture them now they adhere properly for about 24 hours then they start flouting and dying.
Does anyone have any idea why are they dying? or perhaps any suggestion apart from changing media.
Thanks in advance!
It sounds to me that the problem probably lies in your freezing-thawing protocol. Which medium do you use for freezing? If that contains DMSO, you should thaw the cells rapidly and get rid of the DMSO asap once they are thawed (either by centrifuging the cells before plating or by changing the medium directly after adherence of the cells).
Hi Anthal,
Thanks for your answer.
I used the same ready-to-use Melanocyte Growth Medium+10% DMSO for freezing and they used to work fine after freezing for a short time but this time the only difference is that the freezing time was a bit longer.
Regarding DMSO, I always change the first medium after thawing as soon as the cells adhere but I haven't actually tried centrifugation before plating for this cell line which I might give it ago.
Usually it is best to spin down once it is thawed, directly mixed with fresh warm media and spin down probably at low rpm. I spin down at 800rpm about 200g. However if the cells still do not recover then I would suggest steps as follow. I have revived cells kept in LN for more than 3 years using this method.
- Resuspend gently as the cells might be very fragile and I put them in smaller flask or plate from their original flask (ie 1 vial originated from one 6cm plate, I will plate onto 35mm or 6 well plate which is almost half of the surface area) as probability of cell survival after long period of cryo is quite low.
- The next day (24 hours) usually almost half will float but just leave it for a few days to make sure viable cells attached. After 3-4 days, it might not be confluent so collect the media from the plate, spin down and mix fresh media (3:2 old:fresh) put back into plate.
- Collected pellet is up to you if you want to resuspend onto another flask and just leave it longer. Old media contains growth factor secreted when the cells are recovering.
I don't know if there is any serum in your ready-to-go freezing medium, but otherwise you could also consider to add 20% serum to your freezing medium. Also the way you freeze the cells can be important. The way we do it for most of our cells is:
Resuspend your cells in freezing medium A (medium + 20% serum) at the appropriate concentration. Distribute over your cryovials (500-750 ul per vial). Then drip in the same volume of freezing medium B (medium + 20% serum + 20% DMSO) and immediately freeze using a Mr Frosty. In this way, your cells are not exposed long to the DMSO before freezing. By using A and B at 1:1 ratio, your final concentrations will be 20% serum and 10% DMSO.
Good luck!
After checking the viability of the cells and confirmed that they are in a good state moments after thawing, I got suspicious about the media because it was the first time to buy it from that particular company and it turned out that the media was surprisingly killing my cells which is unfortunate.
I have prepared a new media by myself and fortunately solved the problem and the cells are growing perfectly now.
Many thanks for your answers, well appreciated
- Badges
- Science method