Released cells

[Cecilia Reyneri]

Hi Toot!

First of all, congratulation on the ComBO publication, truly impressive validation!

I have two questions regarding the cells released from the organoids. 

1. I was wondering if you routinely observe great amount of released cells in the media, even without the usage of the 3D-printed fluidic device. We observe it already at DIV21 in a line-dependent way (some lines released a lot of cells, others none) and they are highly enriched in hematopoietic cells, but we do not use any fluidic device or shaking, so I was wondering your experience with this.

2. I was wondering if you could share the protocol you used for the Cytospin (speed and centrifugation time), because in my experience with Cytospin cells smash and loose their morphology once on the slide.

Thank you in advance!

Cecilia

[Abdullah Khan]

Thanks Cecilia - glad you think so! 

1. I was wondering if you routinely observe great amount of released cells in the media, even without the usage of the 3D-printed fluidic device. We observe it already at DIV21 in a line-dependent way (some lines released a lot of cells, others none) and they are highly enriched in hematopoietic cells, but we do not use any fluidic device or shaking, so I was wondering your experience with this.

We see this too! Working assumption is that they follow an oxygen gradient. Magnitude varies from line to line, but so far (others feel free to disagree) when there's a good haem fraction you get cells coming out. 

2. I was wondering if you could share the protocol you used for the Cytospin (speed and centrifugation time), because in my experience with Cytospin cells smash and loose their morphology once on the slide.

I'll ask Yuqi to reply to this - but we do a 'longer' slow spin (300G for 8 minutes) seems to give nice cytospins! But we had the same problem for ages. 

Abs 

[Yuqi Shen]

Hi Cecilia,

Just to add a comment on the cytospin conditions - we typically use 400rpm for 5min with low acceleration as our standard setting. The optimal setting could depend on your cell types and the machine configuration. If you’re after more fragile cell types like megakaryocytes, I'd reduce the speed to 300rpm and extend the spin time to 6-8min. Hope this is helpful!

BW,

Yuqi

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