How can we isolate a specific cell type from the others in a tissue?

The mammalian retina is made of several different cell types, each of them having a specific role for a correct functioning of the retina itself.

To understand the complexity of the contribution of each cell to the retinal physiology, it is important to study both the way the different cells interact with each others, and the peculiar features of each different cell type.

How can we isolate a single cell type amongst the others cell types in the retina in order to study them?

Over the years scientists came up with different ways to do that.

One of the most recent and broadly used, due to its compatibility with many different assays afterwards, is the immunopanning technique.

What is immunopanning?

Immunopanning allows to isolate the cells by using an antibody that recognizes an antigen, meaning a protein expressed only on the cell type that we want to analyze.

For instance, what can we do to isolate retinal ganglion cells (RGCs) from the mouse retina?

RGCs are the cells responsible for transmitting the visual information from the eye to the brain. Their axons gather together making up the optic nerve. These cells express on their plasma membranes the protein Thy1. In the retina, Thy1 is expressed only in RGCs (outside the retina is expressed for instance in some population of blood cells).

After isolation, the mammalian retina is treated with a solution containing a specific enzyme (in this case the enzyme used is called papain) that disrupts the protein interaction between cells. Protein interactions contribute to keep the retinal cells all together. After this treatment, by a simple mechanical dissociation, we can obtain a cell suspension: all the retinal cells are now floating in a solution, including RGCs.

At this point an antibody directed against Thy1 is added to the cell suspension. The antibody will bind only to the cell population expressing Thy1, which means RGCs. This antibody is conjugated to a very small metal bead.

The cell suspension is added to a little column placed in a magnetic field. Following the gravity law, the cells will go through the column and will fall down in a container placed under the column. However, as the RGCs expressing Thy1 are labeled by an antibody with a metal bead, they will be retained in the column by the magnetic field. At this point we will have all the other retinal cell types in the container, while in the column, retained by the magnetic field, there will be only RGCs.

By removing the column from the magnetic field and washing with a solution, the cells will now be ready to go through it. We will collect them in another container.

A pure population of RGCs can now be plated on a special substrate and a special medium that will support the growth of axons.

In the figure are shown few examples of cultures of mouse retinal ganglion cells isolated by immunopanning.

(A detailed protocol in the "Protocols" section of this website)