a lot of cell death/debris between day 0 and 3

[Maria Semiannikova]

Hi Everyone!

I was wondering if anyone has experienced a lot of cell death/debris between days 0 and 3-5? On day 0 my aggregates look really great, round regular shapes, clear non-blebby edges, minimal debris in the wells, but after hypoxia incubation (and I have also tried to leave them in a normal incubator in parallel in case it is an incubator issue) the aggregates look very poorly: a lot of debris, very blebby edges, etc. By day 5 they mostly recover but don't look amazing. And after being put into the gel they really start to take off and grow. They seem to not like the transition into the StemDiff media maybe?

Has anyone experienced anything similar?

I have attached a pic of the aggregates on D0 and then the same aggregates on D3. Can attach more pics if necessary. 

Thank you for your help!

Best wishes, 

Maria 

[Abdullah Khan]

Hi Maria, 

Sorry for the delay in getting back to you - missed the original method. 

Looking at the day 0 images, I think your starting material is a bit too big. The unusual thing is they look like they are folding into two layers in the first image, which is quite unusual (refer to the protocols paper for size and morphology). 

My feeling is your starting material is too large - so extend your EDTA incubation by a minute and pipette a touch more vigorously to to break up aggregates. Remember to filter using at least the 150um cell filter (if you can't get a hold of these 100um will also do). If the initial aggregates are too large, you will have a necrotic core and lots of cell death. 

Hope that helps!

BW
Abs

[Maria Semiannikova]

Hi Abs, 

Thanks for getting back to me! The aggregates in that image are all mostly 80-100um in diameter, some are 70-80 and just a few are 100-120. Which I believe is the range you had in your paper?

I have tried filtering through 150um, 100um, and even a 70um filter and I have already played around with the EDTA incubation times and I originally actually thought I was pipetting too much so when the starting aggregates were smaller (like 50-80um) they looked even worse or completely fell apart by day 3 so when I reduced pipetting and they became a bit larger like in this image they survive better but their condition is quite bad by day 3 and then by day 5 they start to get better and once embedded they grow ok. That's why I decided to write to you because I feel like I have already tried a few of the most obvious things because I also thought about starting size. So if you have any other ideas let me know because I believe this step is what causes inconsistencies between experiments because obviously the aggregates are undergoing some kind of stress which most definitely affects the differentiation of different populations and thus affects proportions of the populations. 

Best wishes, 

Maria 

[Abdullah Khan]

That's the right range - looking at the image, it is pretty variable with some much larger aggregates in there. I think where you have a lot of aggregates in a well, with some larger ones you are likely to deplete media quickly and have more cell death. In any case, my main worry with that image is the layered look of the aggregates which is a bit unusual. 

Sounds like you trouble shooted the first stage in any case - can you let me know what your end points looked like? What proportions of populations do you get? Is there good vascularisation? 

Also what is the source of your cells? Am I remembering correctly in saying they are KOLFs? Has their potency/karyotype been confirmed? 

Thanks!

Abs