Published: May 11, 2011 r2011 American Chemical Society 8704 dx.doi.org/10.1021/ja2022569 | J. Am. Chem. Soc. 2011, 133, 8704–8713 ARTICLE pubs.acs.org/JACS Synthetic Chemoselective Rewiring of Cell Surfaces: Generation of Three-Dimensional Tissue Structures Debjit Dutta, Abigail Pulsipher, Wei Luo, and Muhammad N. Yousaf* Department of Chemistry and Carolina Center for Genome Science, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States b S Supporting Information ’ INTRODUCTION Cells that make up tissues and organs exist and communicate within a complex, three-dimensional (3D) environment. The spatial orientation and distribution of extracellular matrix (ECM) components directly influences the manner in which cells receive, integrate, and respond to a range of input signals. 1 As such, cellular interactions with ECM molecules and/or other cells have been extensively investigated for fundamental studies in development, cell motility, differentiation, apoptosis, paracrine signaling, and applications in tissue engineering. 2,3 There has been tremendous effort toward the design and fabrication of 3D scaffolds that mimic ECM properties and induce tissue formation in vitro, utilizing various biomaterials, biodegradable polymers, 4 collagen, 5 and hydrogels. 6,7 Among the major challenges facing the use of these technologies for tissue engineering are the abilities to force contact between multiple cell types in 3D to control the spatial and temporal arrangement of cellular interac- tions and tailor and mold the biomaterial to recapitulate the 3D, in vivo environment under laboratory constraints. Without the use of engineered scaffolds in culture, most cells are unable to form the necessary higher-order 3D structure required for the anatomical mimicry of tissue and are limited to random migra- tion, generating two-dimensional (2D) monolayers. As a result, several approaches, including the use of dielectrophoretic forces, 8,9 laser-guided writing, 10À12 surface manipulation, 13 and a number of lithographic printing techniques 14À17 have been integrated with 3D scaffold designs to produce multitype cellular arrays 9,11,17,18 or 3D cell clusters or spheroids. 7,8,13 In a recent study, 3D aggregates consisting of multiple cell types were formed within a hydrogel matrix through DNA hybridization after cell surfaces were engineered with complementary short oligonucleotides via a metabolic labeling approach. 7 However, for some applications, the presentation of cell-surface DNA may not be stable for extended time periods in cell culture or in vivo. Cell-surface engineering methodologies have primarily been of interest in molecular biology. As such, biosynthetic approaches have been employed to introduce different functional groups on cell surfaces. In a pioneering study, an unnatural derivative of N- acetyl-mannosamine, which bears a ketone group, was converted to the corresponding sialic acid and metabolically incorporated onto cell-surface oligosaccharides, resulting in the cell surface display of ketone groups. 19 However, metabolic or genetic methods may alter many of the biochemical pathways required for normal cell function and not all cell lines possess this metabolic machinery. Thus, there is a growing demand for general tools that can provide simple alternatives to the complex genetic and biosynthetic methods. Other approaches to cell-surface Received: March 11, 2011 ABSTRACT: Proper cellÀcell communication through physical contact is crucial for a range of fundamental biological processes including, cell proliferation, migration, differentiation, and apoptosis and for the correct function of organs and other multicellular tissues. The spatial and temporal arrangements of these cellular interactions in vivo are dynamic and lead to higher-order function that is extremely difficult to recapitulate in vitro. The development of three-dimensional (3D), in vitro model systems to investigate these complex, in vivo interconnectivities would generate novel methods to study the biochemical signaling of these processes, as well as provide platforms for tissue engineering technologies. Herein, we develop and employ a strategy to induce specific and stable cellÀcell contacts in 3D through chemoselective cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. This strategy uses liposome fusion for the delivery of ketone or oxyamine groups to different populations of cells for subsequent cell assembly via oxime ligation. We demonstrate how this method can be used for several applications including, the delivery of reagents to cells for fluorescent labeling and cell-surface engineering, the formation of small, 3D spheroid cell assemblies, and the generation of large and dense, 3D multilayered tissue-like structures for tissue engineering applications.