I am currently growing organoids from the small intestine and the colon plated in matrigel. For experiments, I will be using the monolayers. What is a better approach for conducting the experiments: to seed the cells on collagen or on matrigel? What are the advantages/disadvantages? The matrigel already contains a couple of growth factors, which I will also have in the cell culture medium.
Thank you for sharing your experience.
I agree with the previous comment - besides containing a cocktail of varying growth-factors (differing also in concentration quite a lot from batch-to-batch) it is difficult to get an even coating with Matrigel. Collagen would be easier in this respect.
If you need Matrigel for your experiments I suggest you dilute the stock-solution substantially and maybe even incubate the plates simply with PBSafter coating to reduce growth-factor content - that could make your experiments more comparable over time when using different Matrigel-batches.
Best regards,
Christian
collagen is the best for monolayer experiment.
Matrigel is used for 3D culture model experiment. its difficult to use for monolayer experiment.
regards
Murali
Hi guys - thank you for your comments.
I have seen that some researchers use matrigel to coat the transwells and some use collagen. I was wondering why. My pilot experiments using collagen worked quite well and I will continue using collagen for growing monolayers. I measured TER and got good values. In a next setting, I will also perform stainings in order to confirm polarization of the epithelial cells. Any advice on that (staining of cells grown on transwells)?
@Neil: we are going to test a bunch of immunological settings (co-cultures).
In that case Collagen will work fine. As long as you are getting good resistances you are good to go. Depending on the size of your filters you may want to stain in-filter. If they are small filters they tend to curl up when you cut them out. Fortunately there are many antibodies you can use to look for polarity. Good luck
Thank you Neil.
Can I do double-stainings on the monolayers on the filters or is this not recommended? Any experience on that?
Sure double staining is fine. Your best bet is to do confocal microscopy so you dont see the filter, but we did it and its fine. The other way is to embed the filter in gelatin and section it, but confocal should work for you.
Great - thank you so much. Really appreciate.
My plan actually was to do confocal.
Hello Sandra,
Im also going organoids in monolayer but honestly couldn't manage to make it confluent. they first look "like confluent" with about 70-80% cell, but after washing or changing media, they just turn from 40 to 10% remaining cells. may i know what is the collagen concentration and how much collagen do you use to coat your well? thank you(~.~)
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