Culturing Caco-2's on bottom side of Transwells - (Jun/14/2004 )
Hello, I need to culture Caco-2 cells on the underside of Transwell inserts. Does anyone have any suggestions how to do this?
Thanks.
NO
What about flip it the other way around, grow it, and flip it back?
The problem is that the inserts only fit one way in the plate. I've thought about putting the insert upside down in a 100mm dish, trying to put cells on the underside of the membrane, and later transferring it to the proper plate. If I were to do this, how long would the cells need to be on the membrane before inverting it? Thanks so much for your input....Dave
hi!
may I ask why you need them specially at the underside of your transwell insert?
If you want to look at caco layer vs pathogen contact, you can grow them easily on the inside and then add the pathogen to the inside of the transwell.
that would provide the right orientation of the the layer too, since usually pathogens come in for up above (i.e. the lumen) to the cells, and not from behind, so to say.
if you want to avoid contact between caco and the pathogen, why not turn it the other way around. caco inside the transwell, pathogen on the outside. if you want a flow of soluble things and medium from well to transwell, simply add more medium on the outside than on the inside. if you want to generate a flow from the inside out, start the other way around. if you want no flow, just equillibrate the levels of media on the outside and on the inside. All three worked for me.
mike
may I ask why you need them specially at the underside of your transwell insert?
If you want to look at caco layer vs pathogen contact, you can grow them easily on the inside and then add the pathogen to the inside of the transwell.
that would provide the right orientation of the the layer too, since usually pathogens come in for up above (i.e. the lumen) to the cells, and not from behind, so to say.
if you want to avoid contact between caco and the pathogen, why not turn it the other way around. caco inside the transwell, pathogen on the outside. if you want a flow of soluble things and medium from well to transwell, simply add more medium on the outside than on the inside. if you want to generate a flow from the inside out, start the other way around. if you want no flow, just equillibrate the levels of media on the outside and on the inside. All three worked for me.
mike
The model I would like to use involves co-culture with PBMC's, which should be on the serosal side (i.e., the bottom side in a typical Transwell orientation). The problem is, from what I've read, if the PBMC's are added to the bottom compartment, they will simply settle and have minimal contact with the Caco-2 monolayer. To overcome this, I had hoped to try to grow the Caco-2 cells on the underside of the Transwell insert, so that the PBMC's could go in the upper compartment, and gravity would ensure adequate contact with the cell monolayer. Thanks again (and sorry for the long-winded response).
Dave