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.

Figure 1. Hepatic arrangement around STEP Scaffolds

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.