Primary epithelial cell line - Isolation of epithelial cells (Aug/24/2004 )

Hello!

Can anyone help me?

I have to establish a primary cell line consisting of epithelial cells. Does somebody know a protocol for removing fibroblast cells from mucous epithelial tissue? I won't have much starting material and I would like to be able to keep them as a line for as much as 15 passages.

Thanks a bunch
Caro

Cell culture 101, "trypsinize" the whole population (fibroblast and epithelial) - the first to come off will be fibroblast and epithelial cells are normally resistant to trypsin.

Furthermore, do regular "cell seeding", epithelial cells will be faster to attach to the bottom of dish than fibroblasts.

By repeating these procedures, you can get a fairly pure epithelial population.

Hi

I have a specific tissue (nose epithelial) I want to make the cultures from, and they have a limited life span, so I think by this method I'd just get the isolated and then they'd die.
Can I make use of serum free media that enhances growth of epithelial cells and then check them with anti vimentin antibodies, or is there a better method. I have found a method where they place the minced tissue in media + serum for a while, then remove the tissue, trypsinize, and place it in the special media (DMEM/Ham's F12, Nasal epithelial cell growth medium(oligene) or Airway epithelial cell growth medium(promocell)). It sounds a bit too easy?
Are there anybody who knows anything about these media and protocols for establishin primary epithelial cells.

Caro

Try out the following method gien below. This method has been sucessfully used recently to isolate rat Alveolar epithelial cells (Am J Pathol. 2005 May;166(5):1321-32.). Good luck.


Protocol Reference - Kao WW-Y, Prockop DJ: Proline analogue removes fibroblasts from cultured mixed cell populations. Nature 1977, 266:63-64