Abigail Pulsipher

Synthetic chemoselective rewiring of cell surfaces: generation of three-dimensional tissue structures.

Proper cell-cell 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 cell-cell 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.

Engineering cell surfaces via liposome fusion.

In this study, we have rewired cell surfaces with ketone and oxyamine molecules based on liposome fusion for applications in cell-surface engineering. Lipid vesicles, functionalized with ketone and oxyamine molecules, display complementary chemistry and undergo recognition, docking, and subsequent fusion upon covalent oxime bond formation. Liposome fusion was characterized by several techniques including matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS), light scattering, fluorescence resonance energy transfer (FRET), and transmission electron microscopy (TEM). When cultured with cells, ketone- and oxyamine-containing liposomes undergo spontaneous membrane fusion to present the respective molecules from cell surfaces. Ketone-functionalized cell surfaces serve as sites for chemoselective ligation with oxyamine-conjugated molecules. We tailored and fluorescently labeled cell surfaces with an oxyamine-conjugated rhodamine dye. As an application of this cell-surface engineering strategy, ketone- and oxyamine-functionalized cells were patterned on oxyamine- and ketone-presenting surfaces, respectively. Cells adhered, spread, and proliferated in the patterned regions via interfacial oxime linkage. The number of ketone molecules on the cell surface was also quantified by flow cytometry.

Directing Neuronal Signaling through Cell-Surface Glycan Engineering

The ability to tailor plasma membranes with specific glycans may enable the control of signaling events that are critical for proper development and function. We report a method to modify cell surfaces with specific sulfated chondroitin sulfate (CS) glycosaminoglycans using chemically modified liposomes. Neurons engineered to display CS-E-enriched polysaccharides exhibited increased activation of neurotrophin-mediated signaling pathways and enhanced axonal growth. This approach provides a facile, general route to tailor cell membranes with biologically active glycans and demonstrates the potential to direct important cellular events through cell-surface glycan engineering.

Surface chemistry and cell biological tools for the analysis of cell adhesion and migration.

Cells exist in a complex, dynamic, and highly evolving environment, a key component of which is the extracellular matrix (ECM). The ECM provides structural support for the cell and also contains a host of supramolecular assemblies of proteins and glycosaminoglycans, which play a vital role in the cell’s behavior. In order to undergo fundamental biological processes, cells must adhere to the underlying ECM. Upon receiving and responding to complex molecular signals, cells migrate from the various epithelial layers to target locations, where they differentiate to form specialized cells that make up various organs and tissues. As a result, cell adhesion and migration are critical to processes such as embryogenesis, normal tissue repair, the immune response, wound healing, and angiogenesis. Moreover, improper cell adhesion and migration have been implicated in disease states such as tumor invasion in cancer cell metastasis. Therefore, understanding the mechanisms of cell adhesion and migration will impact a broad range of research communities including medicine and cell-based biotechnologies. In the past few years, the surface-chemistry community has actively pursued the development and integration of strategies to control the interface between cells and a solid support. In doing so, tailored substrates that aim to mimic the ECM and induce cellular behavior have been generated. Different materials have been exploited for studies in cell biology, however, self-assembled monolayers (SAMs) of alkanethiolates on gold remain the ideal model platform due to a number of factors. The organic coupling strategies amenable to thiol chemistry permit a vast variety of biomolecules to be tethered to the surface, as well as to be confined to selective regions so that a specific biointeraction can be observed. In addition to this system’s compatibly with cell culture, the nonfouling properties of oligo(ethylene glycol)-alkanethiols enable the creation of complexly patterned and mixed SAMs. Another advantage to this model platform is the wide range of analytical and patterning techniques available to simulate and characterize cell behavior. Therefore, SAMs have been used in many biological studies including the interrogation of protein–protein, protein– cell, carbohydrate–protein, lipid–carbohydrate, and cell–cell interactions, as well as employed in the investigation of cell adhesion, polarization, and migration. Furthermore, technologies to create dynamic SAM gradients with immobilized adhesion molecules and other chemoattractants have been developed to aid in the elucidation of the mechanism of directed migration. In this Minireview, the design and utility of dynamic surfaces for biological analysis will be discussed. The structure, physical properties, and advantages of SAMs of alkanethiolates on gold for use as model substrates are first summarized. Specific applications of SAMs as dynamic surfaces for the analysis of cell adhesion and migration are then reported.

Long-lived engineering of glycans to direct stem cell fate.

Glycans mediate many critical, long-term biological processes, such as stem cell differentiation. However, few methods are available for the sustained remodeling of cells with specific glycan structures. A new strategy that enables the long-lived presentation of defined glycosaminoglycans on cell surfaces using HaloTag proteins (HTPs) as anchors is reported. By controlling the sulfation patterns of heparan sulfate (HS) on pluripotent embryonic stem cell (ESC) membranes, it is demonstrated that specific glycans cause ESCs to undergo accelerated exit from self-renewal and differentiation into neuronal cell types. Thus, the stable display of glycans on HTP scaffolds provides a powerful, versatile means to direct key signaling events and biological outcomes such as stem cell fate.

Cell‐Surface Engineering by a Conjugation‐and‐Release Approach Based on the Formation and Cleavage of Oxime Linkages upon Mild Electrochemical Oxidation and Reduction

We report a strategy to rewire cell surfaces for the dynamic control of ligand composition on cell membranes and the modulation of cell-cell interactions to generate three-dimensional (3D) tissue structures applied to stem-cell differentiation, cell-surface tailoring, and tissue engineering. We tailored cell surfaces with bioorthogonal chemical groups on the basis of a liposome-fusion and -delivery method to create dynamic, electroactive, and switchable cell-tissue assemblies through chemistry involving chemoselective conjugation and release. Each step to modify the cell surface: activation, conjugation, release, and regeneration, can be monitored and modulated by noninvasive, label-free analytical techniques. We demonstrate the utility of this methodology by the conjugation and release of small molecules to and from cell surfaces and by the generation of 3D coculture spheroids and multilayered cell tissues that can be programmed to undergo assembly and disassembly on demand.

Rapid in Situ Generation of Two Patterned Chemoselective Surface Chemistries from a Single Hydroxy-Terminated Surface Using Controlled Microfluidic Oxidation

In this work, we develop a new, rapid and inexpensive method to generate spatially controlled aldehyde and carboxylic acid surface groups by microfluidic oxidation of 11-hydroxyundecylphosphonic acid self-assembled monolayers (SAMs) on indium tin oxide (ITO) surfaces. SAMs are activated and patterned using a reversibly sealable, elastomeric polydimethylsiloxane cassette, fabricated with preformed micropatterns by soft lithography. By flowing the mild oxidant pyridinium chlorochromate through the microchannels, only selected areas of the SAM are chemically altered. This microfluidic oxidation strategy allows for ligand immobilization by two chemistries originating from a single SAM composition. ITO is robust, conductive, and transparent, making it an ideal platform for studying interfacial interactions. We display spatial control over the immobilization of a variety of ligands on ITO and characterize the resulting oxime and amide linkages by electrochemistry, X-ray photoelectron spectroscopy, contact angle, fluorescence microscopy, and atomic force microscopy. This general method may be used with many other materials to rapidly generate patterned and tailored surfaces for studies ranging from molecular electronics to biospecific cell-based assays and biomolecular microarrays.

Renewable and Optically Transparent Electroactive Indium Tin Oxide Surfaces for Chemoselective Ligand Immobilization and Biospecific Cell Adhesion

In this report, we show the successful transfer of a sophisticated electroactive immobilization and release strategy to an indium tin oxide (ITO) surface to generate (1) optically transparent, robust, and renewable surfaces, (2) inert surfaces that resist nonspecific protein adsorption and cell attachment, and (3) tailored biospecific surfaces for live-cell high-resolution fluorescence microscopy of cell culture. By comparing the surface chemistry properties on both ITO and gold surfaces, we demonstrate the ITO surfaces are superior to gold as a renewable surface, in robustness (durability), and as an optically transparent material for live-cell fluorescence microscopy studies of cell behavior. These advantages will make ITO surfaces a desired platform for numerous biosensor and microarray applications and as model substrates for various cell biological studies.

Expedient generation of patterned surface aldehydes by microfluidic oxidation for chemoselective immobilization of ligands and cells.

An expedient and inexpensive method to generate patterned aldehydes on self-assembled monolayers (SAMs) of alkanethiolates on gold with control of density for subsequent chemoselective immobilization from commercially available starting materials has been developed. Utilizing microfluidic cassettes, primary alcohol oxidation of tetra(ethylene glycol) undecane thiol and 11-mercapto-1-undecanol SAMs was performed directly on the surface generating patterned aldehyde groups with pyridinium chlorochromate. The precise density of surface aldehydes generated can be controlled and characterized by electrochemistry. For biological applications, fibroblast cells were seeded on patterned surfaces presenting biospecifc cell adhesive (Arg-Glyc-Asp) RGD peptides.

Remote Control of Tissue Interactions via Engineered Photo-switchable Cell Surfaces

We report a general cell surface molecular engineering strategy via liposome fusion delivery to create a dual photo-active and bio-orthogonal cell surface for remote controlled spatial and temporal manipulation of microtissue assembly and disassembly. Cell surface tailoring of chemoselective functional groups was achieved by a liposome fusion delivery method and quantified by flow cytometry and characterized by a new cell surface lipid pull down mass spectrometry strategy. Dynamic co-culture spheroid tissue assembly in solution and co-culture tissue multilayer assembly on materials was demonstrated by an intercellular photo-oxime ligation that could be remotely cleaved and disassembled on demand. Spatial and temporal control of microtissue structures containing multiple cell types was demonstrated by the generation of patterned multilayers for controlling stem cell differentiation. Remote control of cell interactions via cell surface engineering that allows for real-time manipulation of tissue dynamics may provide tools with the scope to answer fundamental questions of cell communication and initiate new biotechnologies ranging from imaging probes to drug delivery vehicles to regenerative medicine, inexpensive bioreactor technology and tissue engineering therapies.