Hydrogel Lifting

[Alyssa Monique Monterroso]

Hi all,

I hope this email finds everyone well! 

I'm reaching out today to see if anyone else has ran into the issue I'm currently having during the hydrogel embedding (Nature protocols 2024 step 35). 

I've been adding phase IIIA media very carefully after polymerization but have seen consistently in my past attempts a section of the hydrogel lift from the plate (image attached). 

Has anyone else had this happen and if yes, would you be willing to share tips on how to avoid the lifting? Also since only a section of the gel lifted, are the organoids still submerged in media salvageable? 

Thank you so much everyone for reading through my questions and looking forward to hearing your thoughts!

Kind regards, 

Aly 

[Abdullah Khan]

Hi Aly, 

Firstly embedded organoids are salvageable from this so definitely not the end of the world! 

We found this seems to be because of batch to batch variation in protein content in matrigel (and sometimes how well other gel factors polymerise). There are a couple of solutions: 

(1) spiking the gel with low level fibrin helps improve stability (but low ammounts as too much makes for an overly stiff gel that doesn't induce sprouting). 

(2) switching to the granular method in the comBO paper has proven to solve this (so far... touch wood). 

Hope this helps! Thanks for tooting, 

Abs