Hello!
I am growing mouse intestinal organoids. After growing them for a while I started noticing some single cells that originate from the organoids (it looks like they are coming out of the organoid) and grow in 2D (take a look at image attached). We are using growth factor reduced matrigel ( the matrigel is brand new and we keep it in the freezer) and WENR culture medium that includes: advanced DMEM/f12, b27, N2, acetylcysteine, EGF, noggin, rspondin and wnt3a. Do you have any idea what could be the problem? Or what would you recommend to solve it?
Hi Ioanna,
one possibility could be that your organoids are at the bottom of the well and it is therefore not very unusual that some of the cells adhere to the plate and grow in a 2-D structure. Have you tried to first plate a layer of your matrigel/media mix without your cells into the wells? Incubate just this layer for ~30min and then add your matrigel/media/cell mix into the well. This might prevent your organoids from floating to the bottom and attaching to the plastic of the plate.
Hope that helps
Patrick
Hi Patrick,
Thank you very much for your suggestion! I will give it a try
Ioanna
Hi Patrick,
I am curious that what is the matrigel/media ratio in the mix? will the mix gel?
Hi He Kangxin
a final concentration of 5mg/ml should work well. However, depending on the application you might want to change the concentration.
Hope that helps!
Patrick
Hello Lokesh!
Thanks a lot for the suggestion! Let's see if it works out