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How to design a coculture system with transwell insert for influenza virus studi


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#1 Mohamed 1984

Mohamed 1984

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Posted 11 January 2014 - 02:37 AM

Hi all,

 

Is there is any one who can help me getting some information about a protocol for coculturing A549 and THP-1 cell using transwell insert to study miRNAs in Influenza virus infection ? Important is the size of the pores ( I need it to be less than 0.3 µm) because I wanted to have both cell cultured separatelly by the membrane that aloow passing of cytokine but not the virus

 

Thanks



#2 rowlingr

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Posted 13 January 2014 - 03:11 AM

Hi Mohamed - here are some ideas.

If you are prepared to try out 3D cell culture, you could use separated co-culture in the following ways:

1) Use a scaffold (like the Alvetex well inserts) where you culture one of the cell types in the scaffold (say the monocyte line), add a layer of ECM (like collagen) on the top of the scaffold and then culture the epithelial cell type on the top surface. This will allow you to keep the cell lines separated and study paracrine effects. it also has the benefit of allowing the cells to grow in 3D 

2) You can also try simply growing one of the cells in 2D in the bottom of a 6 well or 12 well plate and grow the other cell type in 3D in a hanging Alvetex well insert

3) You coudl grow one type of cell in 3D in an Alvetex plate and the second cell also in 3D in an Alvetex well insert. 

 

Both 2 and 3 above will allow you to use co-culture but study paracrine effects.

 

Here is an application  note that describes some of this - 

http://reinnervate.c...rtal-co-culture

 

 

Kind regards and let me know if you have any questions.

 

Richard



#3 Mohamed 1984

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Posted 16 January 2014 - 04:50 AM

Thanks. I never tried to make this 3D culture. Could you define the difference between it and the normal co-culture ?

I heared some suggestions regarding coculture and it stated that if you want to study the effect of cytokines secreated from one cell to the other ( without showing virus release effect). you can take the first cell, infect them, take supernantent, inactivate the virus in supernatent , the remaining would be the cytokines , add this supernatent to the other cells

 

Other suggestions is to take the supernatent, define the cytokines inside and order artificial medium containg them only with no virus.

 

What is your opinion and how can i attenuate virus or even filtrate it from the supernatent ?

 

 

Thanks


Edited by Mohamed 1984, 16 January 2014 - 04:51 AM.





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