Exogenous cell engraftment issues in BMOs

[Aparajitha Vaidyanathan]

Hi Abs,

Many congratulations on the comBO paper, it’s a fantastic piece of work and well deserved!

I am hoping you might be able to share some insight into exogenous cell engraftment into BMOs, as I been struggling to get this to work reliably. I tested engraftment using GFP-labelled leukemia lines (SEM, KOPN-8, and RS4;11) as well as CellTrace-labelled MOLM-14 cells, at both Day 14 and Day 21 timepoints, without success so far.

My approach has been to count and resuspend 5,000–10,000 cells in Phase IV media and add them directly on top of the BMOs in 96-well ULA plates. I have also tried centrifugation (100×g for 3 min) to encourage contact, but neither condition has worked. When I wash and image the BMOs after 1-3 days by fluorescence and confocal microscopy, the exogenous cells appear to be adhering to the surface rather than engrafting into the organoid.

I would really welcome any suggestions on where I might be going wrong or how to optimize the protocol, whether it's the timing, cell numbers, media conditions, or something else entirely.

Thanks so much for your time!

Kind regards, 

Aparajitha

[Abdullah Khan]

Hi Aprajitha, 

Thank you and sorry to hear you are having trouble. We've done SEM and MOLM13s, not sure about MOLM14s. 

I'll chat to Yuqi and Eleanor - but I'll ask a few questions first. 

- This is the OG protocol? Cancer Discovery + Nature Protocols? 

- Do you have any images from a bench top microscope + confocal? 

- How are you imaging (Whole mount? Clearing? Objective?) 

Thanks! We will chat and get back to you. -give us a poke if you don't hear in a week. 

Abs