I am isolating crypts from the jejunum of black-6 mice and growing them into 'spherical' apical-in intestinal organoids. I have had lots of success growing these 'spherical' organoids in a 40 uL bubble of matrigel on a 24-well plate and supplementing advanced DMEM with R-spondin-1 (1000 ng/uL), Noggin (50 ng/uL), and EGF (100 ng/uL).
When plating the organoid monolayer I take those 'spherical' organoids, pellet them, resuspend in TrypLE Digestion Enzyme and pipette up and down intermittently throughout a 10 minute period. I then pellet those single cells and plate them on a thin layer of matrigel in a 96-well plate. I use 1 24-well of spherical organoids for each 96-well of monolayers. After 24 hours the cells have always attached but given additional time they never appear to spread or divide.
I have tried growing in half conditioned media (conditioned by the spherical organoids) and half new, I have tried growing them in a 24-well plate thinking crowding might be an issue - to no effect. I have tried varying the concentration of cells I am plating, varying the matrigel thickness, I have also tried plating crypt fragments (organoids broken by 23G needle) instead of single cells, but I see no spread or growth. I am supplementing with the same concentrations of R-Spondin-1, noggin, and EGF that I use for the spherical organoids.
Is there a trick to this I am not seeing?
Any and all brainstorming is deeply encouraged and appreciated.
Hi! Hav you considered these?
1. Add ~10uM CHIR99012 and ~2mM valproic acid to maintain stemness before seeding
2. Add Wnt3a in medium when passaged to monolayer, it grows faster (Wnt-3a afamin is best). in this case, it needs differentiation after confluent using ENR(low R) medium or EN medium. you can differentiate to certain cells
(ex. IWP-2 and valproic acid differentiate to enterocytes)
in my experience, bacterial contaminations will slow cell growth, making it difficult to expand in monolayers.
Passage at least once before monolayer culture, always keep ROCK inhibitor during monolayer culture, consider coating the plate with higher % matrigel (~5%)