Hi everybody. I've bought human CD34+ derived iPSC line from Life Technologies. In the initial recovery as soon as I got the cells, it worked great. However when I cryopreserved the cells with E8 medium with 10% DMSO and thaw them, I found massive cell death.
Here are the details:
Day 1. Thaw cells as fast as possible. Culture them with E8 medium + ROCK inhibitor.
Day 2. Most of the cells are viable. Change the medium, with ROCK inhibitor.
Day 3. Start to see modest cell death. There're still some decent looking colonies. Change the medium, with ROCK inhibitor.
Day 4. Massive cell death. Although there're still some cells left (but not quite look like colonies) adherent in the dish (vitronectin coated), I am worry that eventually all the cell will die.
So is that normal or I did something wrong with the culture? I will definitely try longer culture.
Usually ROCK inhibitor is only used for short time (max 24 hour). Plus, you may want to use other freezing medieum (i.e. Synth-a-freeze) to improve the viability.
Hi Sihan,
I went through a similar ordeal. I was using in house derived hES cells lines for years and then suddenly whenever I used to thaw these cells, they would attach appear healthy but after first media change they would start dying. I changed every variable and for me it came down to lot to lot variations in the media from life technologies. In your case have the cells that you cryopreserved successfully revived earlier? if not you may have to standardize your cryopreservation
1. Ensure the percentage of DMSO is 10% (hope Life technologies also recommend 10%)
2. Gently resuspend cells in the cold mix (10%DMSO chilled) before being placed in cryovial
3. Also what method of cooling you use has a an affect, hope you ensure the cryovials are quickly transferred from the cooling system to liquid nitrogen canister.
4. Also ensure that the media lot is being tested my the company.
Many pluripotent cells lines can be revived without ROCK inhibitor, so that does not seem a problem to me. With pluripotent cells, revival can be poor, but the cells that remain viable can quickly make up, also assess the usual percent viability of your cell line.
These are general suggestions. Hope this is of some help to you.
Prasad.
As Yanuar mentioned, I would shorten the incubation with ROCK medium. Maybe you can add at day1, replace the medium without ROCK at the end of day3. The temperature of your thawing medium can be 37ºC.
Also, as Prasad mentioned, it is very important the way you freeze them. Use pre-chilled at freezing medium 4ºC and check for the cell viability at that point using trypan blue. As well, shorten the time when they are in contact with freezing medium at liquid state.
Cheers.
As some folks already told you, I don't think is useful anymore to add Y-27 after 72 hours. You could start from removing it after 24 hours and see if you have some improvements. In my experience, usually it takes between 7 and 10/12 days for iPS cells to recover from unthawing, in a MEF conditioned medium context.
Another issue could be.......are your cells actively growing, and NOT confluent, at the time you are freezing them down. If cells are not in log phase of growth when they are fozen down this can cause them to thaw poorly.
Have a look in expression of CD49d before and after cryopreservation and thawing!
I usually thaw frozen cells asap, and let them to get use to the new temperature and osmolarity of the culture media. With this protocol, I enhanced cell survival up to ±80%.
Here you have my protocol: 1) submerge the vial in a 37C degree bath until you could see that it thawed. 2) Transfer carefully the thawed cells into a tube containing the exact same amount of slight warm media (1:1, i.e. 1000 ul frozen media, you will add 1000 ul of fresh media). 3) wait 5 minutes and add more media (1:1, add 2 ml of fresh media) and wait again for 5 minutes. 4) finally add 4 ml of fresh media, and centrifuge the tube. 5) transfer cells to culture plate 5) continue the culture as normal. 6) the day after, chance the culture media.
Good luck.
Dear Sihan Wu,
I believe that your Protocol was correct. Maybe you need to pay attention in your trypsine-time that you use in your IpSc colonies and damage you adhesion molecules protein like, then after seeded they died.
I'm having the same problem here with my cells. I follow your same protocol. Differences are that I'm detaching cells with non-enzimatic solution and plating them in matrigel hesc qualified. If you found what is going on, please send me an answer. Thanks!
Hi,
The iPSC cells grown in the E8 medium on vitronectin should be detached as colonies. Try to use EDTA to detach the cells in the aggregates. This method of iPS cell detached didn't need centrifugation before cells freezing. The iPS cells viability is better when we use EDTA to detached iPS cells in comparison to trypsin before cells freezing,
Good luck
Dear Sihan,
I can understand your situation, this does happen with pluripotent stem cells.
While cryopreserving try to have atleast 1 million to 10 million cells per vial.
While this scenario which you mentioned is not normal but it does happen regularly, i would also advice make sure the cryocontainer is upto the required levels and make sure that your cryovials are not kept out when someone else is trying to take his or her cryovial out.
All the best.
Regards,
Prasad.
Dear Sihan Wu,
it seems that some factors are not optimized. Both, the freezing AND the thawing protocol need to be optimized for best cell recovery. Feel free to have a look at one of our recent publications (attached you will find a graphical abstract and the corresponding review). The graphical abstract comprises the most important points, which all should be optimized for best results. In Table 1 and 2 of the review critical steps are listed and how to improve them. As a dissociation solution I can recommend the Gentle Cell Dissociation Reagent (STEM CELL Technologies) or EDTA. An often overlooked factor is that the cells must be in the exponential (logarithmic) growth phase before freezing them, which is approx. 2-4 days after the last passaging.
Best regards,
Markus
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