Cell Engraftment into BMOs

[Karolin Stumpf]

Dear TooT,

When engrafting primary cells or cell lines into the BMOs, do you just add labeled cells to the BMO wells or also spin them down together afterwards? And how do you deal with non-engrafted (potentially overgrowing) cells in the culture medium that might compete for growth factors etc. with the organoids?
On that note, did you usually add lower numbers of cell lines when compared to primary cells?

Best,

Karo

[aude-anais.olijnik]

Dear Karo,

Thanks for this. We usually mix the cells to engraft in media and directly add them to the wells without needing to spin them. 

You might need to test different numbers of cells to engraft if you are worried the cells might be overgrowing. The engraftment outcome will be cell-type dependent, because different cells will have different engraftment potential and will proliferate at different rates too. We usually engraft 5000 to 10000 cells depending on the experiment. Cells which have not engrafted but that are still present in the well will be washed and discarded on the day of analysis when we collect the organoids for analysis.

Thanks,

Aude.

[Karolin Stumpf]

Thanks for the reply!

Did you ever test spinning them down together at low speed and if so, were there any differences compared to just adding them to the media?

Best,

Karo

[Abdullah Khan]

Yep we usually do a slow spin (3 min at 200G) to facilitate the engraftment. You can do a couple of minutes anywhere between 100-300G, but be gentle as they will have been through a lot already! 

[Salim Atakhanov]

Hi everyone,

when engrafting cells with fluorescent labels (CellVue or Calcein) - after how many days does the labeling disaper? Do you know how different is CellVue  from calcein for labeling donor cells?

Cheers,

Salim

[Abdullah Khan]

Hi Salim, 

We haven't really used CellVue for viability/proliferation tracking long term. Mostly just for imaging after engraftments to confirm the cell's presence. CellVue seems to be better for fixation. 

We've used CellTrace on a lot of samples now and the uper limit is around 2 weeks, but that does seem to hugely depend on what sample you are engrafting. For good healthy donor and MF samples for example, at 2 weeks there is a lot of loss of the dye and I would feel at this stage we've already lost the ability to robustly track patient cells. For other samples which are less proliferative, e.g. myeloma - we have sample which is brightly CellTrace labeled at 3 weeks +. 

It's possible you can get some longer term labelling with something ultra bright like Quantum Dots or similar technologies. I think we have some but haven't had a chance to test them. I think ultimately it is mostly about how many times cells divide/proliferate after you label them (and this of course can be very condition dependent).

Hope this helps!

Abs

[Salim Atakhanov]

Hi Abs,

thanks a lot! Yeah, Calcein really quickly goes away, BrdU the same. maybe I can try QDots, thanks for the hint! 

I have another question: form day 14 onward when I engrafted BMOs with exogenous cells, do I still need to out VEGF-A and C? Or would you recommend just adding the cytokines for my cells to differentiate into a certain lineage? I.e. do the BMOs still need to constantly receive VEGFs ? 

Cheers,

Salim

[Abdullah Khan]

Although in the protocol we ween off the VEGFs and they will be stable without VEGF/FGF for a while, I suspect supplementing with low level VEGFs will help in the longer term cultures (depending how far out you want to go) + not have any deleterious effects on maintaining the vessel network. You don't need a high concentration past day 16 (10ng/mL at 72 hours is plenty). We tend not to supplement with these as per Nat Protocols paper, but moving into longer time courses it is probably a good idea. 

Does that make sense? 

Abs

[Salim Atakhanov]

Dear all,

I wonder if you updated the protocol for the engrafting the exogenous cells into BMOs or not (VEGF 20ng/ml or better 10ng/ml?), but wanted to ask why the media for engraftment slightly varies from the Phase IV, e.g. no Knockout serum, no CD Lipids? Do cells better engraft if there is no K.O. Serum and CD Lipids? And if we want long term culture with exogenous cells, would you recommend adding just 10ng/ml of VEGF-A+C and FGF2 or not add them at all?

In general, your expertise and tips and tricks on how to improve the engraftment would be highly appreciated!

Best,

Salim

[Abdullah Khan]

Hi Salim, 

Are you having trouble with your engraftments? It would help to understand what the issue is if you don't mind explaining the problem. 

Generally there is a degree of 'disease dependence' on what the engraftment cocktail is e.g. for Myeloma we supplement IL7. My general rule of thumb is to try and keep the media minimal for engraftments so you don't accidentally swamp any signal coming from your donor cells, though of course the balance is keeping them alive ! 

To clarify your points: 

I wonder if you updated the protocol for the engrafting the exogenous cells into BMOs or not (VEGF 20ng/ml or better 10ng/ml?), 

We've not tested if 20ng vs 10ng/mL VEGF makes a difference - to be honest I don't think it will have a particular effect on your engraftment efficiency. 

but wanted to ask why the media for engraftment slightly varies from the Phase IV, e.g. no Knockout serum, no CD Lipids? Do cells better engraft if there is no K.O. Serum and CD Lipids? 

The engraftment media does include KO serum + CD lipids as per Phase IV medium - sorry if this is not clear. It is essentially phase IV media + a few different supplements. 

And if we want long term culture with exogenous cells, would you recommend adding just 10ng/ml of VEGF-A+C and FGF2 or not add them at all?

I think if you wanted to culture them for longer than we do, it would be helpful to maintain the 10ng/mL VEGFAC/FGF2 (they do this in the Klein paper and that implies they can maintain the vessels for longer). I'd be curious to see if this works. 

As for updated protocols - we have some improvements that I will share in the next couple of months hopefully I have been a bit swamped with grant application related stuff. 

If you're happy to talk through your problems I can give more general troubleshooting. Some tips would be: 

- When you add the donor cells do a gentle spin (3 minutes at 100G) just to encourage them into the organoid 

- If possible wait for at least 72 hours before your first media change - avoids the risk of accidentally aspirating some cells out. 

Yuqi and Zoe in particular might have some other suggestions! WE tend to sort CD34s and chuck them straight in. We have done some 'dilution' engraftments and get good results with a few different donor cell types down to sub 1000 cells/organoid (I haven't dared to go lower than this yet but Zoe has I think). 

Best and as always, thanks for tooting ;) 

Abs

[Karolin Stumpf]

Hi Abs and Salim, 

just thought I still share my experience with Cell Trace for proliferation tracking: When it comes to quite proliferative primary AML samples, there will be strong dye loss even after only 5 days. For other samples, one can robustly distinguish patient cells for over a week.

In case any of you has used CellTrace so far: What was your experience when using CellTrace and then fixing for imaging, regarding dye stability until imaging?

Which specific microscope did you use for confocal z-stacks of CT+ engrafted cells into the BMOs? Where there any other steps of the whole mount imaging protocol that you had to adjust for this?

Best

Karo

Abdullah Khan schrieb am Sonntag, 10. März 2024 um 18:18:24 UTC+1:

[Salim Atakhanov]

Dear Karolin,

I just received the CellTrace to test in the upcoming weeks. Thanks for providing your info about the primary cells. I took this paper https://pmc.ncbi.nlm.nih.gov/articles/PMC11732577/ as reference for the planning of primary cell engraftment with CellTrace kit. Here they fixed it with 4%PFA for the imaging. Did you have any trouble after fixation or you didn't test yet? 

Best,

Salim

[Karolin Stumpf]

Hi Salim,

so far, I have not performed imaging after engraftment with CTV-labeled primary cells. Will keep you updated!

Best

Karo

[Salim Atakhanov]

Hi all,

which cell tracer / cell proliferation kit is best to use to track the primary donor cells in the BMOs? I tried CellTracer C34564 on primary cells and put them in suspension culture to see for how long they can preserve the dye, but 7 days after there was almost nothing... I will highly appreciate if people can share their experience. 

Many thanks in advance !

BW,

Salim