Fabrication of Amine-Reactive Polymer Multilayers On Microwell Cell Culture Arrays: Combining Methods for the Topographic Patterning of Cell Substrates with Approaches to Facile Surface Functionalization

Monday, October 17, 2011: 2:00 PM
L100 H (Minneapolis Convention Center)
Adam H. Broderick1, Samira M. Azarin1, Maren E. Buck2, Sean P. Palecek1 and David M. Lynn1, (1)Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, (2)Department of Chemistry, University of Wisconsin-Madison

Numerous past studies have established the important and diverse roles that surface chemistry and substrate topography can play, independently, in directing the functions and fates of cells. The development of methods for the fabrication of topographically patterned surfaces that also display well-defined and tunable surface chemistry could (i) play an important role in understanding the combinatorial effects of these two environmental factors and (ii) provide a platform for the design of new materials and cell culture substrates that provide sophisticated control over a variety of cell behaviors.

While many different lithographic approaches can be used to fabricate topographically patterned surfaces, modification of the surface chemistry of the resulting 3-D substrates for use in biological studies remains a challenge. This problem arises from the fact that many polymers (e.g., polyurethanes) used to create 3-D substrates for cell-based studies are relatively inert and therefore limit opportunities for post-fabrication surface modification. Past studies have demonstrated approaches to spatially resolved functionalization of topographically patterned polymer surfaces by (i) physical adsorption of biological or synthetic macromolecules or (ii) the formation of self-assembled monolayers (SAMs) on thin films of metals (e.g. alkanethiols on gold). While these two approaches are useful for defining surface chemistry in several important contexts, the development of more general methods for the immobilization of chemical and biological functionality on a broader range of topographically patterned surfaces and materials could open the door to new biomedical applications and the development of new research tools.

Here, we present a ‘reactive’ layer-by-layer assembly approach to the fabrication of amine-reactive polymer thin films on the surfaces of topographically patterned polyurethane microwell cell culture arrays used previously for 3-D cell culture. Reactive assembly of branched polyethyleneimine (BPEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) yielded film-coated microwell arrays that could be chemically functionalized post-fabrication by treatment with different amine-functionalized molecules. Characterization using fluorescence microscopy revealed the presence of a uniform and conformal coating on the topographically patterned surface of the arrays. Additional experiments demonstrated that it was possible to functionalize the films post-fabrication by treatment with amine-functionalized small molecules, and that this approach could be used to modify the surface properties of the arrays and their interactions with cells. For example, treatment of film-coated arrays with the small molecule amine D-glucamine resulted in microwell surfaces that resisted the adhesion and proliferation of mammalian fibroblast cells in vitro. The results of other experiments demonstrated that it was possible to functionalize different structural features of these arrays in a spatially resolved manner to create dual-functionalized substrates (e.g., to create arrays having either (i) azlactone-functionalized wells, with regions between the wells functionalized with glucamine, or (ii) substrates with spatially resolved regions of two different cationic polymers). In particular, spatial control over glucamine functionalization yielded 3-D substrates that could be used to confine and control populations of cells within the wells of these arrays for periods of up to four weeks and support the 3-D culture of cubiodal cell clusters.

This approach to the chemical functionalization of topographically patterned 3-D cell culture microarrays could prove useful for the long-term culture and maintenance of cell types (e.g., stem cells) for which the presentation of specific and chemically well-defined 3-D culture environments is believed to be required to control cell growth, differentiation, and other important behaviors. The results of additional studies demonstrating the patterning of arrays with peptide motifs and the potential of this approach to influence the culture of human plutipotent stem cells will be presented. More generally, this approach to the fabrication of reactive thin films provides methods for the straightforward chemical functionalization of otherwise unreactive topographically patterned substrates that could prove useful in a range of other fundamental and applied contexts.

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See more of this Session: Biomaterials I
See more of this Group/Topical: Materials Engineering and Sciences Division