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1 1 Conditioning Cells to the Compliance of the Soft Underlying Substrate Sana Syed M.S., 1Alexandra Blanco, 2Joseph Schober PhD, *1Silviya Zustiak PhD Department of Biomedical Engineering, Saint Louis University, Saint Louis, Missouri, USA 2 Department of Pharmacy, Southern Illinois University Edwardsville, Illinois, USA Statement of Purpose: Current research has demonstrated that cells can adapt to their microenvironment by altering their phenotype, and in some cases, even their genotype. Yet, since the development of cell culture techniques, cells have been continuously propagated on tissue culture polystyrene (TCP), which is mostly inert and orders of magnitudes stiffer than the stiffest tissue in our body. Thus, it is expected that continuous use of TCP can cause phenotypic and genotypic changes in the cells. We suggest that alternate in vitro cell culture techniques, which incorporate substrates of physiology-relevant properties, will be a more representative platform for studying cell behaviors than the current TCP standard. In lieu of recent discoveries, which pinpoint the importance of substrate compliance on cell fate, we examined the effect of long-term culturing of cells on substrates of varying rigidities on cell behaviors, such as morphology, cytoskeletal protein expression, cell spreading rate, and drug responsiveness. Methods: MDA-MB-231 (human breast carcinoma) and MCF-7 (human breast carcinoma) cells were used in this research study. Cells were seeded onto collagen coated 1 kPa and 100 kPa polyacrylamide (PA) gels, as well as collagen coated TCP (control). PA gels were created on GelBond PAG Film (GE Healthcare). Cells were seeding onto the gels and supplemented with RPMI media (10% fetal bovine serum and 1 % penicillin streptomycin); they were then re-passaged onto new soft and stiff PA gels after 72 hours, and this process was repeated consecutively for 3 passages. Cell images were taken at 24 h, 48 h, and 72 h after seeding for a total of 9 days (3 consecutive passages) and were analyzed on ImageJ for changes in morphology, proliferation rate, and attachment efficiency. Live imaging was conducted on postconditioned and pre-conditioned cells to evaluate the spreading rate. Cytoskeletal protein expression was performed by staining for nucleus, actin, and vinculin to evaluate changes between conditioned and unconditioned samples. Different doses of paclitaxel were used to screen for changes is cell viability between conditioned cells and unconditioned cells. These methods helped in identifying the variance between cells that have been cultured longterm and those that have not. Results: When cultured continuously for 9 days (3 passages) on soft 1 kPa PA gels, cells exhibited decreased circularity and increased spreading area compared to cells that had only been seeded on the soft gels for 1 day; it was also noticed that spreading area and circularity increased for the stiff gel as well. No significant changes were noticed in cell attachment efficiency, however cells on 1 kPa gels did show a slightly smaller efficiency compared to cells on 100 kPa and TCP. During conditioning to the soft substrate, our observations Figure 1. Spreading area of MDA-MB-231 cells on 1 kPa and 100 kPa PA gels over a 9 day period consisting of consecutive re-passaging back onto gels. indicated an increased proliferation rate of cells during the last passage when compared to the first passage. Although not able to quantify accurately, cells also displayed increased actin and vinculin expression after being conditioned on the soft 1 kPa PA gels, compared to cells that that were not conditioned. Through live imaging, we were able to visualize the increased spreading rate of conditioned cells, indicating adaptation to the soft substrate when compared to TCP and stiff substrates. Lastly, conditioning of cells to the soft substrates also seemed to increase their sensitivity to paclitaxel as compared to non-conditioned cells or cells seeded on stiff substrates and TCP. Conclusions: Our results indicate that an adaptation period might be necessary for cells that have been continuously propagated on stiff TCP substrates prior to assessing cell-behaviors since cells could acquire phenotypic changes upon conditioning. This is the first study to show the difference in cell behavior between cells that have only had one day to adapt to their underlying substrate versus cells that have had a longer time to adapt (i.e. 9 days). Future studies include quantifying the expression of vinculin and actin and conducting a durotaxis study with the conditioned cells. Another potential future experiment would be to assess the micro-RNA to look for genotypic changes within the cells. References: Syed, SM. Sim PA mwell stiff assay for stiff dep cell resp. 2015; 97(10).