Adventures in Cell Seeding
For the past month or so, I’ve been working on cell seeding of hydrogels. In order to demonstrate penetration of my hydrogel system by MMP-2, I have to place cells on the top layer of my hydrogel. But cells do not like to grow on hydrogels. When placed in a tissue culture plate, cells will do fine growing on the bottom of the well, but not on the hydrogel surface.
To improve cell attachment, I’ve added a short peptide sequence, RGD, to the top (polyacrylamide) layer of my hydrogels. The letters RGD stand for the one letter initials of the pertinent amino acids: aRginine, Glycine, and aspartic aciD. Basically, it works like this:
We all know the human body is made up of cells. In addition to cells, there ares other proteins in the spaces between the cells known as the extracellular matrix (ECM). Collagen is the best-known example of an ECM protein, but there are many others. Cell membranes have proteins called integrins that attach to the ECM. The RGD peptide is the attachment site for cell integrins. The cells will bind to the RGD and attach to the hydrogel surface.
That’s the idea, at least.
I first tried seeding my hydrogels with U-87 MG cells, a line of human glioblastoma cells. I put cells on three groups of hydrogels: 1) Hydrogels with RGD, 2) Hydrogels with the peptide DGR (same amino acids, different sequence), 3) Hydrogels with no peptide. The purpose of the DGR group is to ensure it’s the specific RGD sequence that has the effect, not just the presence of a peptide. I assessed cell viability using an MTS assay. I won’t go into all the details of this, but the pertinent facts are that it’s commonly used for to test cell viability, and it doesn’t give an exact count of living cells but is useful for providing comparisons between groups.
My results were not promising. Either there was no difference between hydrogels with RGD and without RGD, or in some cases, the hydrogels without RGD had greater viability. I repeated this a couple times to make sure it wasn’t a problem with my technique, but saw no improvement in results. So, I wondered, what was the problem?
It was suggested that I look at published scientific papers to see if anyone else had grown these type of cells on polyacrylamide hydrogels. A search of PubMed showed no results for U-87 MG cells. So now the question was: were my cells not growing on the hydrogels because there was a problem with the hydrogels (or my technique), or was there something unfavorable about this type of cell growing with polyacrylamide?
To rule out a problem with the hydrogels, I tried seeding them with a different type of cell: NIH/3T3. The 3T3s are human fibroblast cells. They’re not cancer, but there are several published papers successfully demonstrating 3T3s on polyacrylamide hydrogels with RGD. I tried this last week, and the results were much better. I still need to make a few modifications to this system, but I expect this portion of my project will be done soon, and I’ll be able to progress. It’s a good feeling when your creation works!