Research in Engineering and Aviation

Development of a Polyacrylamide-Based Stiffness Assay for “High-Throughput” Drug Testing

November 2012

Author(s): Ferguson, D., Zustiak, S.P., Nossal, R., Sackett, D.

Annual Biomedical Research Conference for Minority Students (ABRCMS), San Jose, CA, November 2012.

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National Institute of Health (NIH) Summer Research Program Poster Day, Bethesda, MD, August 2012.


Design of biomaterial-based in-vitro models is an emerging research field that promises to bridge the gap between traditional two-dimensional cell culture and animal models. It is now known that the cell microenvironment, including the properties of the surrounding matrix such as stiffness, affects cell fate. Matrix stiffness regulates signaling, growth, survival, and motility for both normal and cancer cells. Importantly, the matrix stiffness is believed to trigger the onset of tumorigenesis. Our hypothesis is that since matrix stiffness affects cell fate, it will also play a role in drug resistance mechanisms. In order to test this hypothesis we built a high-throughput stiffness assay capable of probing the effect of matrix stiffness on cancer cell responsiveness to anti-cancer drugs. Polyacrylamide (PA) was the biomaterial of choice, since it spans the stiffness ranges of 0.3-300 kPa. The PA gels were coated with collagen to provide adhesion sites necessary for the survival of anchorage-dependent cells. Our assay design criteria involved improving on: preparation time, cost, robustness, ease of use, and high-throughput format. We used two design approaches when casting the PA gels, namely a GelBond and a Stamp assay. For the GelBond assay, PA gels of different stiffness were made on top of a GelBond plastic. These were then dried and cut into shapes appropriate for a 96-well plate. For the Stamp assay PA gels were poured directly into a 96-well plate, and to form a flat gel, we used a “stamp” with the hydrophobic side of a GelBond cover slip pressing down. Overall, the cost and ease of use for the two assays was comparable, but the GelBond assay was more robust and faster to prepare. In order to test the utility of our assay designs, we used three different cancer cell lines – HepG2 (human hepatocellular liver carcinoma), SY5Y (human neuroblastoma), and MDA-MB-231 (human breast adenocarcinoma), to determine how cells responded to anti-cancer drugs with regards to matrix stiffness. All cells were seeded 24 hours before the drug, Taxol - a microtubule stabilizing agent, was administered. MTT, a metabolic activity assay, was used to measure cell viability and proliferation. Our preliminary results indicated that the cells adhered equally well to the gels of different stiffness, but had higher proliferation rates and spreading area on the stiffer gels. In addition, the cells response to Taxol was affected by the matrix stiffness, albeit in a cell dependent manner. In particular, the HepG2 cells responded to Taxol similarly on gels of different stiffness, the MDA-MB-231 cells responded better at higher stiffness, and the SY5Y cells responded better at lower stiffness. In conclusion, we built an assay for the study of stiffness-dependent drug testing and in our preliminary studies, we were able to show that cancer cells respond to drug treatment differently on gels of different stiffness in a cell dependent manner.