Dark side of the moon: us and them
The aim of the project is to create a microenvironment in the form of 3D matrix droplet that could be used to observe cell migration applied in cancer research. What we study is the invasion assay of tumor organoids in collagen droplet matrix. This way specific cellular behaviour in regard to collagen matrix can be observed in real time using both light and two photon microscopy.
First step in a thousand mile journey
What we do first is to prepare the organoids. Organoid can be easily defined as a sphere of cells that spontaneously forms in standard media. After a few days of growth it is time to transfer the organoid from liquid media and embed it into 3D collagen matrix.
Nature of the collagen droplet is the tricky part
In order to establish just right micro-environment we made two kinds of Petri dishes : fluorodish – in which the droplet will be on a flat structure with no other support and “home made” dishes with holes- in which we created the holes in a shape that would support the droplet.
3D matrix is another tricky part
Here, we choose to examine different matrix composition to see how the cells behave according to these variations. First we want to see the difference in various collagen concentrations, because higher concentrations will form a denser fiber network and that will prevent cells from migrating . Second, we will compare plain collagen matrix with the collagen matrix enriched with fibronectin, structural protein molecule shown to have impact on cell migration. Last, but not least, we will examine whether the presence of cancer associated fibroblasts(CAFs) in the collagen matrix has any impact on cancer migration and invasion since there is a controversy whether they stimulate or inhibit cancer invasion.
Picture is worth a thousand words
To finish, we take pictures of each organoid under the light microscope (one picture per day) so that we can follow the evolution and behaviour of the organoids in the drops (and so, their invasion of the matrix), in different conditions.
Look from inside the group
The preparation of the drops is quite long, it took us maybe 4h30 to have everything prepared and one hour to take the pictures. We only did the experiment once and Ralitza explained everything we weren’t familiar so we are sure that we will be much faster next time. Once the organoid preparations are done, we just have to come every day and take pictures over the next few days, it takes about 30 min.