Somehow this question ended up in the "Penicillin" category? So trying again.
I am working with primary murine jejunal enteroids/spheroids, but I am having difficulty keeping them growing robustly past Passage #3. I would love some help! Details below.
My lab, based on how collaborators do things, has been using rat tail collagen type I as the basement membrane matrix for years now to grow human and mouse enteroids and colonoids (or spheroids, as well as in 2D culture). We previously used 1.0% collagen (in a neutralized buffer solution) to coat tissue culture plates in a thick patty (~1 mm) for 2D growth, but this was quite delicate and so I have started using 1.5% collagen. I also use 3.2 mg/mL or higher neutralized collagen as a 3D matrix to embed the enteroids which works pretty well. I have experimented with thin coating with collagen I and other products more similar to Matrigel (which is in a severe shortage right now), but I haven't found that one works better than the others so far. Embedding crypts in Matrigel-like products also didn't look much different from those embedded in collagen I.
We use 50% L-WRN conditioned medium, which is checked for efficacy with the TOPFlash assay for each large batch. The 50% Conditioned Medium contains Advanced DMEM/F12 medium with 20% FBS, 1X Penicillin/Streptomycin, and 1X GlutaMAX. The other 50% is the same medium without the WRN part: Advanced DMEM/F12 medium with 20% FBS, 1X Penicillin/Streptomycin, and 1X GlutaMAX. This is stored at -80 degrees Celsius, and thawed overnight at 4 degrees Celsius for use the next day. Medium is never freeze-thawed more than once. To the thawed medium, we add 50 ng/mL murine recombinant EGF, 1X B-27 Supplement (without Vitamin A), 10 mM Nicotinamide, 10 mM HEPES, 1.25 mM N-acetylcysteine, 50 ug/mL Primocin, 3 uM SB202190, 10 nM Gastrin, and 10 nM PGE2. ROCK inhibitor (Y-27632, 10 uM) is used for the first 2 days of culture. Medium is changed every 48 hours and cells passaged every 5-8 days (depending on confluency).
I have done an extensive literature review, and there is very little consensus out there on how to grow murine enteroids. Most labs use Matrigel (or similar), but some use Collagen I. Many use thin coating of tissue culture plates. There are additional components that can be added to the medium, including B-27 Supplement (which HAS vitamin A), A-83-01 (ALK5 inhibitor), N2 Supplement, Thiazovivin (ROCK inhibitor), CHIR99021 (GSK3 inhibitor), LDN-193189 (ALK inhibitor), SB431542 (TGF-βR Inhibitor), Blebbistatin (myosin inhibitor), and Valproic Acid (HDAC inhibitor/NOTCH signaling activator).
Additionally, some labs don't use 10% serum (or even less, when purified growth factors are added instead of conditioned medium), whereas I am using 20% FBS. I know serum contains many differentiating factors, so perhaps this is my problem? I am using the recommended FBS from Sigma (#F2442 is recommended from one publication). The FBS is heat-inactivated (although I'm not sure that is necessary). I do not freeze-thaw the FBS or the Conditioned Medium more than once.
Another twist (which I have not yet attempted) is the "inside-out" organoids grown in suspension that I am interested in trying as an alternative to the typical 2D monolayer TEER and FITC-dextran, etc., methods of measuring permeability.
My question is mostly how to propagate the enteroids (no differentiation) for longer than 3 passages, when my yield of proliferating ISCs seems to fall off. I am new to the epithelial world and this seems like a very daunting task to sort this out!! ....and I have been working on it for quite some time. Please help!
Thanks, Erin
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