(776h) STEP Enabled Long Time Culture of Primary Hepatocytes in Multiple Layers

Introduction:
Chronic
diseases of the liver including hepatitis and cirrhosis account for a large
portion of the 16,000 ? 17,000 person-long waiting list
for a liver transplantation.  Only
5,000-6,000 liver donations are available each year, meaning only 63% of people
needing a transplant are unable to receive one. 
To alleviate this deficit, bioartifical liver
assist devices (BLADs) have been developed which perform the functions of a
normal liver outside of the body.  The
design and implementation of more successful assist devices is driven by the
need to maintain hepatocyte functionality over longer periods of time.  In this work, hepatocytes are seeded onto an
ECM-mimicking, highly aligned nanofibrous network.  As single cells attach and spread, they pull
against these fibers and cause deflections which allow migratory forces to be
probed.  We believe that an understanding
of the magnitude and nature of these forces will allow improvements to be made
to BLAD construction to create more viable assist devices for the many in need
of them.

Materials
and Methods: Using the previously-reported
Spinneret-based Tunable Engineered Parameters (STEP) technique, nanofibrous
scaffolds were created with fused intersections.  Using xylene as a solvent,
suspended polystyrene and polyurethane fibers of 500 nm and 300 nm diameter,
respectively, were spun in criss-cross layers.  Primary rat hepatocytes were seeded onto
these constructs, which were then imaged with an incubating microscope.  Migration behavior was analyzed via timelapse videos and fluorescence imaging.  Cells were fixed and stained for nucleus
(DAPI), actin (phalloidin) and focal adhesion (paxillin) locations.

Results
and Discussion: After examining over
20 cells who had deflected nearby fibers, it was found
that the average hepatocyte generated a force of approximately 1.5 nN.

Conclusions:  We have shown that nanofibrous scaffolds carefully
arranged for fiber spacing and layouts can cause cells to preferentially attach
in specific shapes.  According to focal
adhesion immunohistochemistry, these configurations inherently display
different attachment and migration forces which may prove useful in directed
differentiation in the future.