Sunny S. Shah

Micropatterning of Proteins and Mammalian Cells on Indium Tin Oxide

This paper describes a novel surface engineering approach that combines oxygen plasma treatment and electrochemical activation to create micropatterned cocultures on indium tin oxide (ITO) substrates. In this approach, photoresist was patterned onto an ITO substrate modified with poly(ethylene) glycol (PEG) silane. The photoresist served as a stencil during exposure of the surface to oxygen plasma. Upon incubation with collagen (I) solution and removal of the photoresist, the ITO substrate contained collagen regions surrounded by nonfouling PEG silane. Chemical analysis carried out with time-of-flight secondary ion mass spectrometry (ToF-SIMS) at different stages in micropatterned construction verified removal of PEG-silane during oxygen plasma and presence of collagen and PEG molecules on the same surface. Imaging ellipsometry and atomic force microscopy (AFM) were employed to further investigate micropatterned ITO surfaces. Biological application of this micropatterning strategy was demonstrated through selective attachment of mammalian cells on the ITO substrate. Importantly, after seeding the first cell type, the ITO surfaces could be activated by applying negative voltage (-1.4 V vs Ag/AgCl). This resulted in removal of nonfouling PEG layer and allowed to attach another cell type onto the same surface and to create micropatterned cocultures. Micropatterned cocultures of primary hepatocytes and fibroblasts created by this strategy remained functional after 9 days as verified by analysis of hepatic albumin. The novel surface engineering strategy described here may be used to pattern multiple cell types on an optically transparent and conductive substrate and is envisioned to have applications in tissue engineering and biosensing.

Micropatterning of bioactive heparin-based hydrogels

This paper describes a UV photopatterning of bioactive heparin-based hydrogels on glass substrates. In this approach, hydrogel micropatterns were formed by UV-initiated thiol–ene reaction between thiolated heparin and diacrylated poly(ethylene) glycol (PEG-DA). Analysis of gelation kinetics showed that photo-crosslinked hydrogels formed faster and were stronger when compared to hydrogels formed by competing Michael addition reaction. To highlight bioactivity of heparin–PEG hybrid gels, hepatocyte growth factor (HGF) was mixed into prepolymer solution prior to hydrogel patterning. Immunostaining showed that HGF was retained after 5 days in the hybrid heparin–PEG hydrogel microstructures but was rapidly released from pure PEG gel microstructures. In a set of experiments further highlighting bioactivity of microfabricated heparin-based hydrogel, primary rat hepatocytes were cultured next to heparin and pure PEG hydrogel disks (∼500 µm in diameter). ELISA analysis revealed that hepatocytes residing next to heparin-based hydrogels were producing ∼4 times more albumin at day 7 compared to cells cultured next to inert PEG hydrogels. In the future, microfabricated heparin-based hydrogels described in this paper will be employed for designing cellular microenvironment in vitro and as vehicles for cell transplantation in vivo.

On-cue detachment of hydrogels and cells from optically transparent electrodes

The goal of the present communication was to develop a strategy for detachment of cells and biomaterial constructs from indium tin oxide (ITO) electrodes.

Quantitative Label-Free Characterization of Avidin-Biotin Assemblies on Silanized Glass

In this study, a time-of-flight secondary ion mass spectrometer TOF-SIMS, operating in the event-by-event bombardment/detection mode was used to characterize avidin-biotin assemblies on silane-modified glass substrates. SIMS was used to analyze several variants of the biointerface, including avidin physically adsorbed on a monofunctional acryl silane surface and covalently attached on monofunctional (amine terminated) and bifunctional (amine and acryl terminated) silanes. The goal of these studies was to determine density of avidin and biotin layers chemically or physically adsorbed on silanized glass substrate. An individual impact of a C(60) projectile used in this study creates a hemispherical crater (∼10 nm in diameter) and emits large numbers of secondary ions from the same nanovolume. Thus, a single impact enables one to unfold distinct secondary ions that span the thickness of the assembled film. This method was used to monitor the presence of glass, silane, and protein ions and to estimate the thickness and density of the avidin layer. In addition, we employed the double coincidence mass spectrometry approach to identify ions coemitted from a specific stratum of the biointerface. This approach was used to determine density of biotin and avidin immobilization while eliminating interferences from isobaric ions that originated from other constituents on the surface. Overall, novel TOF-SIMS quantitative approaches employed here were useful for examining complex biointerfaces and determining both lateral and in depth composition of the film.

Designing Electroactive Biointerface for Spatiotemporal Control of Cell Attachment and Release

In this paper, we demonstrate the use of individually addressable microelectrodes for cell sorting and cell micropatterning applications. Microelectrodes were modified with cell adhesive or non-adhesive molecules and then electrically stimulated to selectively adsorb or desorb proteins and/or mammalian cells. The switching of the surface properties was achieved by the electrochemical desorption of protein-functionalized thiols and poly(ethylene glycol) PEG silane from gold and indium tin oxide (ITO) electrodes respectively. The thiol surfaces were modified with anti-CD4 antibodies and used to capture T-cells. Upon electrical activation of the microelectrodes, both the antibodies and the T-cells were removed from the specific locations on the substrate. In addition, ITO electrodes were modified with cellresistive PEG silane which was later electrochemically desorbed to make the surface adhesive to proteins or cells. This technique was employed to pattern two different cell types on the same substrate.

Patterning cells on optically transparent indium tin oxide electrodes.

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues. In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates. The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive. Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule. Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks. Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns. Micropatterning of several cell types was demonstrated on these substrates. In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.