I'm trying to isolate primary epithelial cells from colons of rats that have been exposed to a carcinogen and are suspected to have aberrant crypt foci. I have tried with trypsin but the number of cells obtained was very low. Does anyone know of an efficient procedure to obtain primary cells? Can collagenase be used? If so, which type?
I would use the protocol described in the paper I have listed below. They use EDTA solutions to efficiently and quickly release the intestinal epithelium. I have only done this in mice, but suspect they will work well with rat colons as well. Keep in mind that the protocols, as written, are for the isolation of intestinal crypt stem cells. Depending on which cell population you want, you may need to make modifications to the protocol (or save samples that you would otherwise discard if following the protocol exactly). Also remember that once these cells are dissociated, they are very prone to apoptosis because they lose the key signals from the WNT pathway. If you are trying to keep them alive, you will want to follow the precise culture conditions listed in their paper.
http://www.ncbi.nlm.nih.gov/pubmed/22610555
A couple of other very useful papers are the following:
http://www.ncbi.nlm.nih.gov/pubmed/23097115
--- This is another method for isolating intestinal cells. I prefer the method above, but they are similar.
http://www.ncbi.nlm.nih.gov/pubmed/21889923
--- A nice overview of the growth conditions required for small intestine v. colon v. tumors from mice which may be relevant to your work, depending on your downstream applications.
Good luck! Feel free to post again if other questions arise.
I would use the protocol described in the paper I have listed below. They use EDTA solutions to efficiently and quickly release the intestinal epithelium. I have only done this in mice, but suspect they will work well with rat colons as well. Keep in mind that the protocols, as written, are for the isolation of intestinal crypt stem cells. Depending on which cell population you want, you may need to make modifications to the protocol (or save samples that you would otherwise discard if following the protocol exactly). Also remember that once these cells are dissociated, they are very prone to apoptosis because they lose the key signals from the WNT pathway. If you are trying to keep them alive, you will want to follow the precise culture conditions listed in their paper.
http://www.ncbi.nlm.nih.gov/pubmed/22610555
A couple of other very useful papers are the following:
http://www.ncbi.nlm.nih.gov/pubmed/23097115
--- This is another method for isolating intestinal cells. I prefer the method above, but they are similar.
http://www.ncbi.nlm.nih.gov/pubmed/21889923
--- A nice overview of the growth conditions required for small intestine v. colon v. tumors from mice which may be relevant to your work, depending on your downstream applications.
Good luck! Feel free to post again if other questions arise.
I have been using several enzymes for the separation of oral epithelium. I found dispase is very good to do the jop. Epithelial cells can be dissociated with trypsin in ca and mg free buffer.
You may try cocktel enzymes as separation/disosociation may be different from one tissue to the other and in different animals.
This paper is specific for epithelial cells of colon of rats
Cancer Res. 1981 Jan;41(1):168-75.
Isolation and characterization of epithelial cell types from the normal rat colon.
Chopra DP, Yeh K, Brockman RW.
I am attaching it here.
Good luck!
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