Extraction and culture as individual organoids

[Karolin Stumpf]

Dear TooT members,

did you ever face problems extracting organoids from the hydrogel on day 13, meaning too strong adherence of aggregates to hydrogel and thus no separation after centrifugation even after multiple rounds of resuspension with Pasteur pipettes and subsequent centrifugation even at 500xg?
If so, what would you expect as likely causes and would you recommend trying to separate gel from aggregates by addition of TrypLE for example or will this be too harsh on the aggregates?

Best,

Karo

 

[Abdullah Khan]

It happens sometimes! Usually because of too much protein in there - it's hard to pin down specifically the why. You can centrigure a bit harder (I've gone up to 700G when this as happened). 

We've not really done an enzyme wash to clear excess material but it is an option. 

If you end up with difficulties separating, I would suggest spinning at the higher speed and doing the best you can - but when you replate do a spin into the plate to compress your excess ECM on to the organoid. That way it gets incorporated into the structure and will be less of an issue. I wouldn't worry over much - perhaps you can share some images and data at end point so we can see your structure and cell composition?

Good luck! 

[aude-anais.olijnik]

It can be an issue indeed. My hypothesis is that it comes from the Matrigel composition since you will have batch to batch variability and some Matrigel vials are more protein concentrated and could be "heavier" and therefore settle down with the organoids during a spin.

If you really struggle and find that you still have a lot of leftover hydrogel around the organoids, I have found that doing a couple of extra washes with PBS helps a little bit. In a nutshell:

1) Try and extract as per protocol (remove conditioned media and put aside).

2) Remove as much hydrogel on top of the organoid pellet by pipetting it out with a P1000 pipette. Leave about 2-3mm hydrogel layer on top of the organoids to be sure not to pipette them out (the organoids will break if taken out with a P1000 tip).

3) Transfer the organoids + leftover hydrogel mixture to a 1.5mL Eppendorf tube with a Pasteur pipette.

4) Top up to 1mL with PBS.

5) Very gently mix the organoids and PBS together (~10 times) with a Pasteur pipette. It will help to break the hydrogel layer.

6) Spin 5min/RT/400g

7) Remove supernatant carefully

8) Repeat steps 4-7 one more time. I wouldn't do more than one time in case this is too disruptive for the organoids: no real data here, just a feeling ;)

I hope this helps!

[Karolin Stumpf]

Thanks for the advice!
I have attached some brightfield images of aggregates from the first run of the differentiation protocol at different timepoints before and after hydrogel extraction
(day7, 10, 13 and day 16 and 19 of the protocol, taken at 4x magnification: https://drive.google.com/drive/folders/1lYywXKya2TQL9yw5XRRa06cc8_Hnv713?usp=drive_link).
Aggregate size varies quite a bit, generally being smaller than what you end up with (maximum of 1000 to 1500 µM at day 19). Smaller ones seem to produce more vascular sprouts, also minorly lower levels of endothelial cells assessed by flow on day 14.

Regarding their composition, I will get back to you soon with plotted data. Shortly, CD45+ fraction increases from day 14-19 (assessed on day 14, 16 and 19) with good stromal cell variety (MSCs and endothelial cells at roughly 20% and 10% of live cells respectively on day 14 already) and quite some erythroid priming (30% of live cells being CD71+CD235a- on day 14).

I was thinking of adding TPO and EPO at a later timepoint or lower concentration for less erythroid priming.

For your hematopoietic and stromal flow panel: Do you still include a Lin cocktail for the hemato. panel or just go by CD45 positivity and ery exclusion? And do you always gate for erythrocytes before gating for CD45 or actually only consider CD45-CD71+CD235a+ cells as erys?

Looking forward to your feedback!

[Abdullah Khan]

Hi Karo, 

I've requested access and will check out your images soon. 

All sounds about right. The Ery expansion makes sense too and dropping your EPO concentration will fix that (I would suggest doing that at an early time point mind) 

We don't have a Lin in the stromal panel and just do an Ery exclusion CD71/235 then CD45- cells are what we treat as our stromal compartment. 

Hope this helps, 

Abs

[Karolin Stumpf]

First of all: Happy New Year to all Toot members (off to another great year of BMOs and BMO-related questions)!

Secondly, do you perform media replacements or additions besides just volume adjustments after day 13?

Best,

Karo

[Abdullah Khan]

Hey Karo - HNY! 

I don't follow the question sorry - media changes are performed throughout (50/50 in and out).

Thanks for TooTing

Abs

[Karolin Stumpf]

Hi Abs, 

I meant whether you add APEL2 or StemPro34 to single organoids in culture in a ULA 96 well plate from day 13 onwards until day 18 or 20ish or until any downstream analysis and if so, at which time points would you add additional media (e.g. every other day or less frequently?)? I hope this makes it a little clearer.

Best,

Karo

[Yuqi]

Hi Karo,

We normally do a 50/50 media change every 2-3 days, simply just taking 50 ul out carefully and add 50 ul of fresh media in. Depending on your experiment timeline, you could also adjust the refeeding frequency based on that, for instance, I've left the media in for up to 4 days at most when I was doing a 96-hour drug treatment. Hope this helps.

Best wishes,

Yuqi

[Salim Atakhanov]

Hi everyone,

did someone try to do TGFb stimulation for more than 3 days? Does it influence somehow the viability of cells? 

Today is day16 for my BM organoids and they have those cells produced. Do you guys observe the same? hematopoietic cells will be released from the organoid ? Attached is the representative image.

Best,

Salim

[Abdullah Khan]

Hi Salim, 

Yes we have done a few different time courses and doses of TGFb - to be honest I would suggest doing this for your experiments after you've set up (we can discuss in detail if you like) because it can impact results as it's a pleiotropic factor. A very high dose for more than 3 days will impact viability, but you can get good fibrosis with low doses over longer times. 

So we do see release of haematopoietic cells some times - not always. We have stripped G-CSF from the cocktail from day 10 onwards which mitigates this and now it's something we only really see on occasion. Did you have G-CSF in the media throughout? It is a potent chemotactic agent so my theory is that is the main culprit. 

Abs

[Salim Atakhanov]

Hi Abs,

yeah, I had G-CSF, for my future diffs I will try to remove it as you said. Let's see what happens :) thanks!

Best,

Salim