Research Program
Cellular Microdynamics

Objectives:

• To understand the mechanism of enzymatic surface-initiated polymerization (ESIP) of polyhydroxybutyrate (PHB) and to enhance the growth of PHB on the solid surfaces
• To investigate the interaction between various mammalian cell lines and the ESIP PHB derivatized surfaces and evaluate their performance for 2D patterning of mammalian cells.
• To evaluate the use of ESIP PHB as a surface modification for 2D patterning of mammalian cells.

Methods
Growth and functionalization of ESIP PHB on the solid surfaces. PHB is a biodegradable and biocompatible aliphatic polyester produced by a variety of microorganisms as a reserve energy source. Here, site-specific attachment of the key catalytic enzyme, PHA synthase, on lithographic-fabricated surfaces and subsequent addition of 3-hydroxybutyryl-CoA substrates (3HB-CoA), allowed us to create spatially ordered PHB polymeric micro-/nano-structures on the patterned surfaces via in situ ESIP. Bovine serum albumin (BSA) proteins were added during the immobilization as well as the polymerization steps in order to enhance PHB surface polymerization forming thick PHB-BSA films up to 1 μm in height with full surface coverage (Figure 1A). By using biotinylated BSA conjugates, we were able to incorporate biotin groups into the PHB polymer matrix, thus generating a bioactive surface that can be used for displaying other functional biomolecules through streptavidin-biotin interaction on the PHB structures (Figure 1B).

In vitro biocompatibility studies of mouse embryonic fibroblasts and embryonic stem cells on ESIP PHB and ESIP PHB-BSA fabricated surfaces. Various gold patterned substrates used for our surface polymerization and cells studies were fabricated using standard lithography techniques housed within NBTC & CNF at Duffield Hall. We also collaborated with Professor Jun-Lin Guan ( College of Veterinary Medicine , Cornell University ) to study the interaction between mouse embryonic fibroblasts (MEFs) and the fabricated ESIP PHB patterned surfaces and with Professor Tom Sato’s lab (Weill Medical College of Cornell University) to study mouse embryonic stem (ES) cells.

Summary
Cell studies performed with MEFs showed a substantially increased cell attachment and proliferation on the fabricated PHB surface as compared to the unmodified surface. Moreover, we demonstrated that micropatterning of PHB structures created through a combination of parylene lift-off and in situ biofabrication of PHB could be used for mammalian cell patterning (Figure 2). Besides MEF cells, we also evaluated the biocompatibility assessments of the fabricated PHB surface with  mouse ES cells. Our preliminary results suggested that the in situ fabricated PHB polymer on a planar gold surface could provide a favorable 2D matrix for ES cells maintenance and propagation. More embryonic bodies (EBs) were formed after two days of cultivation on this PHB surface in comparison to the unmodified gold surface. In addition, these resulting EBs were found to remain in an undifferentiated state for over a week without the need for fibroblast feeder cells (Figure 3A-B). Besides the PHB planar surface, we also evaluated the effect of a simple 2D geometrical shape of the ESIP-PHB and found that the mES cells localized onto the PHB stripes, especially during and after EB formation (Figure 3C-D). Our current goal is to use the fabricated PHB surfaces as a promising biomaterial for culturing and differentiating ES cells into other cells, such as epithelial cells, nerve cells, and bone cells. In addition, we will evaluate the effects of different geometric patterned surfaces fabricated with PHB, which we hope to find out information that will offer us new levels of control over ES cell behavior.

Accomplishments

• Obtained a better understanding about the mechanism of ESIP PHB on the solid surfaces and could further functionalize the ESIP PHB matrix via biotinyated BSA
• Demonstrated that the fabricated ESIP PHB surfaces could be used as favorable scaffolds for 2D patterning of mammalian cells
• Proved that the fabricated ESIP PHB surfaces are biocompatible for maintaining the culture of mouse embryonic stem cells in an undifferentiating state