On Friday I observed Dr. Keese while he performed an experiment using an array with filters. By growing cells on a porated filter, we area able to better simulate an in vivo environment, and to measure the barrier function between cell junctions. There are many different types of cell junctions,  each with a different strength. We used MDCK II (canine kidney cells) in our experiment which form tight junctions. These cells are very hard to separate, which makes it difficult for us to passage them. We added 3 rinses of a solution called EDTA which takes away the magnesium and calcium ions of the cells. Then, we added trypsin, which is an enzyme that essentially eats away at the cell walls, weakening their junctions. Once the cells became rounded and were no longer attached to one another or the surface they were grown on, we were ready to put them into our array!

The electrode array we used today has a very deep well which allows for our filters and a suspended electrode to sit in the well. Here is what our electrode array looks like:

Here is a diagram of a single well in our electrode:

As you can see, medium sits above and below the cells, therefore creating an environment that would be similar to that of the body. 

So what, exactly are we measuring in this type of experiment? Using the two electrodes, we can pass a current through the layer of cells growing on top of the filter, therefore seeing how tight the cells junctions are. This measurement is called barrier function, which is much higher for MDCK II cells than the BSC-1 cells I have been working with. The higher measure of barrier function, the tighter cell junctions are. For example, endothelial cells that comprise the blood-brain barrier would have an extremely high barrier function because they must have a low permeability in order to keep out microorganisms and other large or hydrophilic molecules. 

I also worked on passaging my BSC-1 cells. Here some candid shots of me working with my cells:
(Photo credits to my awesome mentor, Dr. Keese!)

Holding a flask of BSC-1 cells

Working in the hood