My latest experiments have involved measuring the release of MMP cleavable peptides from hydrogels. A short, simplified breakdown:
The MMP cleavable peptide with a fluroescent molecule is conjugated to the hydrogel material monomer, PEGDA (polyethylene glycol diacrylate). The PEGDA is polymerized. Hydrogel samples are put in the presence of cells producing activated MMP-2. The MMP should enter the hydrogel, cleave the peptide, and release part of the peptide (including the fluorescent molecule) into the cell media. By measuring the fluorescence in the cell media, the amount of peptide released can be determined. A control group is done where hydrogels are only in the presence of media (no cells), to verify that release is taking place.
I did the experiment above, measuring release at different time points up to 96 hours, but my results showed no significant difference in release between the cell group and the control group. My initial thought was that there were differences in conjugation. Because I used a separate group of hydrogels for each time point, perhaps one group had more conjugated peptide than another. Tomorrow I’m going to try measuring release from the same group of hydrogels at different time points.
Another suggestion made by my PI is that the cells don’t produce active MMP when they become c0nfluent (covering the entire area of the growing surface). I’m using HT-1080s (a human fibrosarcoma cell line), which are known to produce active MMP-2. However, they divide very quickly. I did my experiment with a cell density of 500,00 cells/mL, and they became confluent within 24 hours of seeding.
I’m reviewing the thesis and lab notebooks of a previous student who did a lot of work on MMP-2 expression from cells. I haven’t finished reviewing her experiments, but I noticed she used much lower cell densities than I am using. A lower cell density is what I’ll attempt if the experiment I try tomorrow doesn’t show a significant release difference between experimental and control groups.