Micropatterns of double-layered nanofiber scaffolds with dual functions of cell patterning and metabolite detection

This paper describes the development of multi-functional nanofiber scaffolds consisting of multiple layers of nanofiber scaffolds and nanofiber-incorporated poly(ethylene glycol) (PEG) hydrogels. As a proof-of-concept demonstration, we fabricated micropatterned polymeric nanofiber scaffolds that were capable of simultaneously generating cellular micropatterns within a biomimetic environment and detecting cellular metabolic products within well-defined microdomains. To achieve this goal, we designed nanofiber scaffolds with both vertical and lateral microdomains. Vertically heterogeneous structures that were responsible for multi-functionality were realized by preparing double-layered nanofiber scaffolds consisting of an antibody-immobilized bottom layer of nanofibers and an upper layer of bare polystyrene (PS) nanofibers by a two-step sequential electrospinning process. Photopatterning of poly(ethylene glycol) (PEG) hydrogel on the electrospun nanofibers produced laterally heterogeneous micropatterned nanofiber scaffolds made of hydrogel microwells filled with a nanofibrous region, which is capable of generating cell and protein micropatterns due to the different interactions that cells and proteins have with PEG hydrogels and nanofibers. When HepG2 cells were seeded into resultant nanofiber scaffolds, cells selectively adhered within the 200 μm × 200 μm PS fiber microdomain and formed 180.2 ± 6.7 μm spheroids after 5 days of culture in the upper layer. Furthermore, immobilized anti-albumin in the bottom layer detected albumin secreted by micropatterned HepG2 cells with higher sensitivity than flat PS substrates, demonstrating successful accomplishment of dual functions using micropatterned double-layered nanofiber scaffolds.