Figure 2.

Micro-fabricated “Nichoid” scaffold for 3D cell culture. A) Nichoid architecture. From left to right: scanning electron microscopy (SEM) image of a Nichoid single-module; SEM image of up-scaled repetitive matrixes of Nichoids; picture of 50 mm² micro patterned glass coverslip with Nichoids. Nichoid culture 3D-substrates are produced via two-photon laser polymerization of SZ2080 negative photoresist with near-infrared exposure following a CAD geometry. B) Possible microscopy configurations to use on Nichoid culture substrates. Thanks to the device's versatility, this scaffold enables optical accessibility both in transmission and in reflection modality. C) Immunofluorescence images obtained via confocal fluorescence microscopy of GFAP (red), β-ACTIN (green) and DNA (blue), in human Adipose derived Stem Cells (hADSCs) after a 7-day expansion inside the Nichoid and in standard conditions (2D-Control). The images demonstrate that nuclear morphology and protein organization and localization (both β-ACTIN and GFAP) differed between the two culture systems. Cytoskeletal markers merge (yellow signal) into cellular protrusions inside Nichoids, while a poor signal appears in flat conditions. D) Real-time PCR analysis of specific gene targets shows a significant gene expression difference (up-regulated in green, and down-regulated in red) between Nichoid and Control conditions, both using a standard culture medium and also using a culture medium that induces adipogenic or neural differentiation.