Guiding neuronal development with in situ microfabrication

Abstract

We report the ability to modify microscopic 3D topographies within dissociated cultures, providing a means to alter the development of neurons as they extend neurites and establish interconnections. In this approach, multiphoton excitation is used to focally excite noncytotoxic photosensitizers that promote protein crosslinking, such as BSA, into matrices having feature sizes >250 nm. Barriers, growth lanes, and pinning structures comprised of crosslinked proteins are fabricated under conditions that do not compromise the viability of neurons both on short time scales and over periods of days. In addition, the ability to fabricate functional microstructures from crosslinked avidin enables submicrometer localization of controllable quantities of biotinylated ligands, such as indicators and biological effectors. Feasibility is demonstrated for using in situ microfabrication to guide the contact position of cortical neurons with micrometer accuracy, opening the possibility for engineering well defined sets of synaptic interactions.biofabrication ͉ multiphoton cell patterning ͉ growth cone S tudies of neuronal function increasingly rely on methods for precisely manipulating cellular properties. Innovations in electrophysiology, photolytic release of effectors, and inducible knockout technologies (1-3) have made it possible to explore cellular phenomena at levels of reduction few anticipated a quarter-century ago. Despite this technological revolution, approaches for influencing neuronal morphology, motility, and interconnectivity remain relatively primitive, a limitation of considerable importance to fundamental and applied neuroscience. An ability to prescribe the exact location at which an extending neurite makes contact with a target cell, or to constrain neuronal migration at a specific time point in development, would be of great value to studies of signal transduction and integration within individual cells and neural networks.Neurite orientation and growth can be modified in real time by various stimuli, including diffusible neurotrophin gradients (4), electric fields (5), and near-IR light (6), but these approaches exert relatively coarse influences over neurite pathfinding and have not been used to accurately guide cellular interactions. Finer delimitation of neurite development can be achieved by using patterned surfaces and topologies microfabricated in silicon and other materials (7-9); such structures, however, must be prepared before cells are introduced for culture when the detailed features of neurite arborization cannot be known.Multiphoton excitation provides an alternative approach for constructing 3D defined microscopic materials that, in principle, could be fabricated within cellular environments. Used extensively in 3D fluorescence imaging, multiphoton excitation also has proved useful for promoting photochemical reactions with high spatial and temporal control (10-13). Application of this strategy to ''direct-write'' material fabrication can be achieved by focusing light from a pulsed femtosec...

Multiphoton fabrication of chemically responsive protein hydrogels for microactuation

“…Taken together, these results imply that a broad range of hydrogel swelling profiles will be possible by judicious selection [or engineering (37)] of protein building blocks. Further, protein matrices composed of avidin, which we previously have demonstrated retain biotin-binding functionality (23)(24)(25), showed attenuated swelling in acid and denaturant solutions in the biotinbound state compared with the unbound state ( Fig. 2d and Fig.…”

“…We and others have shown that direct-write protein matrices can retain ligand-binding and catalytic functionality similar to that of their component proteins (23)(24)(25)(26)(27), implying that native electrostatic and hydrophobic interactions governing protein conformation remain significantly intact within microstructures. However, at pH extremes, unfavorable electrostatic interactions can disrupt the native state and result in protein unfolding (31,32).…”

“…Protein microstructures are fabricated by using an aqueous direct-write procedure in which a pulsed laser beam is focused to submicrometer dimensions to promote formation of crosslinks between oxidizable residues (28-30) via multiphoton excitation of a photosensitizer (23). This concept is illustrated in Fig.…”

“…In addition to having the potential to undergo shape and hydration changes in response to chemical triggers, proteins contain a large number of weak acids and bases, similar to polymers used in pH-responsive hydrogels (5). Importantly, matrices composed of functional photocrosslinked proteins can be localized in 3D microenvironments by using multiphoton fabrication (23)(24)(25)(26)(27). Thus, multiphoton-fabricated protein structures could provide high-resolution 3D control over the topography of a stimuliresponsive material-facilitating greater mechanical functionality and incorporation of responsive hydrogels into more complex 3D devices-while potentially providing specificity (e.g., through ligand binding) over volumetric responses.…”

Mask-Directed Multiphoton Lithography

“…By scanning the fabrication voxel through reagent solution, complex three-dimensional microscale objects 1,5 have been produced with lateral feature sizes as small as ∼120 nm using synthetic materials 1 and ∼250 nm using proteins. 4 Figure 1A demonstrates our approach for mask-directed multiphoton lithography. Here, light from a femtosecond titanium: sapphire (76 kHz) laser is sent through an unmodified confocal scan box to raster the beam in a rectangular pattern at a focal plane positioned between the scan box and an inverted microscope.…”