Unhappy cells on H&E - ?infection

[Ellen Nuttall Musson]

Hi TooT

We just finished up out first run of BM organoids in the lab following your excellent protocol with some degree of success.

We did some initial validation tests with H&E staining, IHC and flow.

A high proportion of the cells in the H&E sections at the organoid centres look stressed/pyknotic. I wondered if we might have picked up an infection at some point, although the media always looked clear and we were careful about maintaining sterile conditions.

The other thing I wondered was whether they were using up the media resulting in stress. I changed the media every 48 hours (50/50) once they were in the 96 well plate and used higher volumes generally (approx 200ul per well).

My questions are:

- Have you ever seen similar appearances in the absence of infection?

- If not, have you ever tried introducing antibiotics later in the protocol once the cells have differentiated somewhat e.g. after mesodermal and haematopoietic induction?

Many thanks for your help through this group!

Best wishes

Ellen

[Abdullah Khan]

Hi Ellen - thanks for Tooting! 

To be able to understand this a bit better, I would need to see the images. Would you be happy to share? I can't think of an experiment where we saw notably pyknotic cells in H&E I'm afraid. 

Ultimately your flow should also be telling (if there is a notable granularity on your scatter gates or odd live/dead staining). 

We have added P/S after patient cell engraftments on d12/d14 onwards and not seen any issues so that's ok to do if you're worried about infection. 

Abs 

[Ellen Nuttall Musson]

Hi Abs

Thanks a lot. Attached are an example of the H&E and the initial flow gates - doesn't look like there is overrepresented granularity but did have quite a lot of dead cells - before seeing the H&E I put this down to pretty vigarous digestion (used collagenase D but had to do several rounds of extra pipetting to get rid of all visible clumps).

BW

E

[Abdullah Khan]

Hmm that's an odd one I don't think I've come across it before. Let me discuss with the group in the week.

Viability post Col D even with vigorous pipetting should be in the order of 80%+. Could be down to a few different things e.g. infection. Your media volume might be quite high (200uL can deprive 3D cultures of Oxygen apparently...). Either way I'd suggest repeating anyway to see if there was an issue with this culture! 

Sorry I can't be more definitive this one is a bit of a head scratcher... hopefully come up with something and get back to you! 

Abs