The extracellular matrix has been shown to profoundly influence both cell morphology and numerous cellular processes – including adhesion, differentiation, and alignment – through a range of chemical, mechanical, and topographical features. In these studies, we investigate a versatile platform for functionalizing micro-3D-printed (μ-3DP) protein hydrogels via multiphoton excitation of benzophenone-biotin, a photoactivatable ligand capable of reacting with the hydrogel matrix, which is subsequently linked to a biotinylated cell-adhesive peptide through a NeutrAvidin® bridge. This functionalization strategy is potentially applicable to a broad range of hydrogel platforms, enabling biomolecules to be precisely patterned at specified locations within 3D materials. As proof-of-concept of this strategy's utility, we demonstrate that chemical modifications can be made to μ-3DP protein hydrogels that enable Schwann cells to be patterned without altering the mechanical or topographical properties of the hydrogel to an extent that influences SC cell adhesion. The ability to independently control potential cellular cues within in vitro cellular microenvironments is essential to investigating decoupled effects of biomaterial properties on cell-matrix interactions. In addition, we demonstrate feasibility for generating arbitrary immobilized chemical gradient profiles, a result that opens important opportunities for understanding and controlling haptotactic behaviors, such as directed migration, that are key to various tissue regeneration applications.

Due to their short lifespan, rapid division, and ease of genetic manipulation, yeasts are popular model organisms for studying aging in actively dividing cells. To study replicative aging over many cell divisions, individual cells must be continuously separated from their progeny via a laborious manual microdissection procedure. Microfluidics-based soft-lithography devices have recently been used to automate microdissection of the budding yeast Saccharomyces cerevisiae. However, little is known about replicative aging in Schizosaccharomyces pombe, a rod-shaped yeast that divides by binary fission and shares many conserved biological functions with higher eukaryotes. In this report, we develop a versatile multiphoton lithography method that enables rapid fabrication of three-dimensional master structures for polydimethylsiloxane (PDMS)-based microfluidics. We exploit the rapid prototyping capabilities of multiphoton lithography to create and characterize a cell-capture device that is capable of high-resolution microscopic observation of hundreds of individual S. pombe cells. By continuously removing the progeny cells, we demonstrate that cell growth and protein aggregation can be tracked in individual cells for over ∼100 h. Thus, the fission yeast lifespan microdissector (FYLM) provides a powerful on-chip microdissection platform that will enable high-throughput studies of aging in rod-shaped cells.

Living cells reside within anisotropic microenvironments that orchestrate a broad range of polarized responses through physical and chemical cues. To unravel how localized chemical signals influence complex behaviors, tools must be developed for establishing patterns of chemical gradients that vary over subcellular dimensions. Here, we present a strategy for addressing this critical need in which an arbitrary number of chemically distinct, subcellular dosing streams are created in real time within a microfluidic environment. In this approach, cells are cultured on a thin polymer membrane that serves as a barrier between the cell-culture environment and a reagent chamber containing multiple reagent species flowing in parallel under low Reynolds number conditions. Focal ablation of the membrane creates pores that allow solution to flow from desired regions within this reagent pattern into the cell-culture chamber, resulting in narrow, chemically distinct dosing streams. Unlike previous dosing strategies, this system provides the capacity to tailor arbitrary patterns of reagents on the fly to suit the geometry and orientation of specific cells.

The extracellular matrix has been shown to profoundly influence both cell morphology and numerous cellular processes – including adhesion, differentiation, and alignment – through a range of chemical, mechanical, and topographical features. In these studies, we investigate a versatile platform for functionalizing micro-3D-printed (μ-3DP) protein hydrogels via multiphoton excitation of benzophenone-biotin, a photoactivatable ligand capable of reacting with the hydrogel matrix, which is subsequently linked to a biotinylated cell-adhesive peptide through a NeutrAvidin® bridge. This functionalization strategy is potentially applicable to a broad range of hydrogel platforms, enabling biomolecules to be precisely patterned at specified locations within 3D materials. As proof-of-concept of this strategy's utility, we demonstrate that chemical modifications can be made to μ-3DP protein hydrogels that enable Schwann cells to be patterned without altering the mechanical or topographical properties of the hydrogel to an extent that influences SC cell adhesion. The ability to independently control potential cellular cues within in vitro cellular microenvironments is essential to investigating decoupled effects of biomaterial properties on cell-matrix interactions. In addition, we demonstrate feasibility for generating arbitrary immobilized chemical gradient profiles, a result that opens important opportunities for understanding and controlling haptotactic behaviors, such as directed migration, that are key to various tissue regeneration applications.

We report the application of multiphoton microfabrication to prepare conducting polymer (CP)-based biomaterials that were capable of drug delivery and interacting with brain tissue ex vivo, thereby highlighting the potential of multiphoton lithography to prepare electroactive biomaterials which may function as implantable neural biointerfaces (e.g. electrodes).