Livingston, Sarah

Cell Attachment to Alginate Hydrogel Structures

Faculty Mentor: Dr. Alonzo Cook, Chemical Engineering

Introduction

One of the most pressing needs within the medical community is the demand for
transplantable organs. On average, 22 people per day on average die waiting for a
transplant. Even if a patient receives an organ, the risks associated with the transplant
are high, from the immunosuppressant drugs required to the risk of Graft-versus-host
disease (GvHD). If organs could be engineered using a patient’s own cellular material,
transplants could take place without the risk of GvHD or the need for
immunosuppressants. One of the keys to creating any organ is first building a vascular
network that could provide vital nutrients to any surrounding tissue. Our group is
working to create fully functional and patient-specific blood vessels in vitro using a 3D
printer. Our specialized 3D printer would print a scaffold that would then be seeded with
a patient’s own cells to create a viable vascular tissue.

In order to allow both printing and subsequent cellular growth, the scaffold needs to be
of an optimal density for printing, maintain its shape after printing, and then facilitate
good cellular attachment and invasion. Our team has chosen to work with alginate, a
hydrogel, in order to meet these needs. Alginate is typically purchased in a powder form
and can then be dissolved in water to form a gel. Our team has experimented with
different concentrations of the gel to find one best for printing, and we’ve used
methacrylic anhydride to crosslink the gel and maintain its structure. Next, we needed to
find a method that would make the gel a better candidate for cellular attachment and
invasion. For this, we had several ideas. One was to lyophilize (or freeze-dry) the
alginate structure to make it more porous, to allow the cells to infiltrate further into the
gel. Another was to coat the gel with fibronectin, a glycoprotein that functions in cell
adhesion in vivo. These ideas were the subject of this project.

Methodology

In preparation for the experiments, cells from a mouse endothelial line (MS1) were
cultured in a DMEM growth media containing 10% FBS (fetal bovine serum) and 1%
penicillin/streptomycin. HUVECs (human umbilical vein endothelial cells) were also
cultured with the same media.

To test the effects of lyophilization and fibronectin coating on cellular attachment to
alginate, we made 15 balls of 6% alginate, some of which were then lyophilized, coated
with fibronectin, or both, while a few were left as controls. These balls were then placed
in a multi-well plate and allowed to absorb the cell growth media, after which the MS1
cells were added. These were left to incubate to allow the cells to grow and infiltrate
throughout the gel. After incubation, each well was observed under a light microscope.

Results

Under the light microscope, it appeared that fibronectin coating did indeed increase
growth, and it was unclear whether lyophilization aided cellular infiltration or not. Further
experiments and expanded analysis methods are needed to confirm these results.

Discussion

During the experiment and after its completion, several problems arose. First among
these was that light microscopy alone was an inadequate analysis method. Given that
the alginate balls were in a small multi-well plate, it was hard to distinguish between
growth of cells on the alginate and growth of cells on the plate wall with the microscope
we were using. In future studies, we plan on analyzing solely the alginate using
histological visualization methods. Another problem was maintaining a
contamination-free growth environment. Because of the processes through which the
gel is prepared and crosslinked, it can be difficult to ensure it is sterile. While the gel is
exposed to UV-light during the crosslinking process, this alone has not been completely
efficacious in sterilizing the gel. Since the gel is water based, autoclaving poses a
problem as well. Some of our latest efforts have been focused on inventing a method to
maintain gel sterility throughout the gel’s preparation. The third problem was in the
continued culture of both the MS1 cells and HUVECs. Both of our cultures fell prey to a
bacterial contamination, and back-up stores of the cells were apparently lost in a move
between liquid nitrogen containers earlier this year. We plan on using iPS (induced
pluripotent stem) cells in the long run, which we will differentiation into the different cell
types needed to make a human blood vessel. This differentiation process is already
underway in the lab as well, so we will likely use these cells in our continued
experiments with fibronectin instead of the MS1s or HUVECs we had previously
experimented with.

Conclusion

In conclusion, fibronectin coating seems to increase cellular attachment to an alginate
hydrogel structure, but further research is needed to confirm and expand upon our
results.