Hello everyone! I'm trying to grow porcine intestinal organoids from small intestinal crypts. I used 4-5 wks piglet for crypts isolation and organoids cultivation succeeds. However, when organoids were subcultured, some of them formed in different shapes compared with the primary organoid. I attached organoid images (Up: Images of primary organoids Day 1 to Day 5 / Down: Images of subcultured organoid on Day 4). It was similar to primary cultured organoid when many were cultured, but they spheroid shapes when few were cultured. I used organoid culture media (1xN2 supplement, 1xB27 supplement, 1mM N-acetylcysteine, 50ng/ml EGF, 100ng/ml Noggin, 2mM glutamax, 10mM HEPES, 100ug/ml Primocin, 10mM nicotinamide, 10uM Y-27632, 10uM SB202190, 500nM A 83-01, 2.5uM CHIR99021). In our lab condition, we used conditioned media (10% R-spondin media and 70% Wnt3A media) and Y-27632 inhibitor used first 2 Day. Give me any suggestion plz.
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have you checked this?
Hi, I have experience with human and mouse intestinal organoids. If I am understanding well your question, you want to know why you have cyst organoids (bubble-like) and organoids with more invaginations, isn't it? Normally, they would start with cyst form and as the days pass the organoids will create invaginations. How did you passage them? Did you break them into single cells or organoid pieces?
Thank you for your answer, Mar Margalef-Català
When I passage organoids, I break organoid into organoid fragments.
Briefly,
1. Remove the matrigel using cell recovery solution (Corning)
2. Centrifuge organoids and suspension small volume media (~200 ul volume)
3. Break organoids using pipetting mechanically (40~50 times)
4. To remove the debris and obtain pure organoid fragments, pass them a 40um cell strainer
5. After centrifugation, the organoid fragments are suspended in a matrigel and seeding to a culture plate
Is there any problem with my subculture method?
I am not sure still what is your question, sorry. I don;t think there is anything wrong with your subculture method. I normally was using TrypleExpress to break the organoids into single cells, but what you are doing is fine too. I was asking how you were passaging your organoids to try to know how you were ending up with different morphologies. If that is what you want to know, you may be having single cells that form full organoids (cyst form) and pieces of organoids from the previous passage more differentiated that end up forming the more invaginated organoids. How do your wells look right after passage?