Hi Rafael ,
I am culturing the instinal cell lines but not worked on primary cultures but might be soon doing that. Any queries??
Hello Nikhil,
I'm starting to cultivate epithelial organoids from small intestine based on the protocol below:
"The development of a method for the preparation of rat intestinal epithelial
cell primary cultures"
(G. S. EVANS1*, N. FLINT1, A. S. SOMERS1, B. EYDEN2 and C. S. POTTEN1)
however its an old paper and I'd like to know what protocol is used nowadays...or if its still used the same technique.
Best regards from Brazil
Rafael
I culture quite often Intesinal stem cells as organoid on marigel. Which protocol are you using and what is the aim of your experiments? If you write about this then perhaps I can be of help to you.
I'm trying to cultivate intestinal cell primary cultures from adult rats in a 3D scaffold for some studies, and I´m using the protocol from the paper :
""The development of a method for the preparation of rat intestinal epithelial
cell primary cultures"
(G. S. EVANS1*, N. FLINT1, A. S. SOMERS1, B. EYDEN2 and C. S. POTTEN1)"
(that author used fetal rats and I´m using adult rats. He used whole small intestine and Im trying to use just 5 cm of it)
I think I just need to change the dispase concentration, however I´d like to know if researchers are using a different protocol or if im going to the right way.
Thank You all for the answers!
PS: Thomas , thank You very much!!! It will help me a lot!!
That is a very old and difficult protocol. You would do much better by following Sato's method of culturing on Matrigel. The only problem is that the method uses lots of R-spondin. Try to find someone who has cloned it and get enough to try it. The published method was for mouse but will work with rat. Good luck.
Hi Rafael,
As Robert suggested, Sato's method is nice. Along with this I have tried atleast two methods described in the old paper. All of them worked well. Well, you use fatal intestine to avoid contamination of gut microbiota, easy to digest in short time and few contiminant cells. When you use adult intestine you have to wash 5-6 times in HBSS +P/S after cutting into 5 mm pieces. In my experience the younger the mice the better is cell viability after final purification. If you have younger mice you need to use whole intestine but from adult mice you can take part of it, infact less starting material is better as you have less undesired cells after enzyme digestion/EDTA dissociation. Once you have enriched crypt cell fraction, best way is to use Matrigel (as lots of publication suggest) and you will get organoids in a week. You can also culture them on collagen matrix.
Send me your email address, I will send you my step-by-step details protocol but off course for mice. I am sure you can use the same method for rat.
You can also try this method PLoS ONE 6(11): e26898. doi:10.1371/journal.pone.0026898, here they isolation SEMF and crypt cells in the same protocol.
Cheers!
Shahzad
Hi Shahzad,
Thank you so much!!
my email is: [email protected] or [email protected]
Best regards
Rafael
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