Download Conditioning Cells to the Compliance of the Soft Underlying

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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
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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).