Figure 1

Schematic representation of the experimental setup. (A) Timeline of the performed procedures. (B) Two days before culturing (DIV-2), fish are subjected to ONC to prime RGCs and improve their outgrowth in culture. In addition, MFDs are assembled by adhering the PDMS top part on a cleaned (acid treated) glass cover slip using plasma treatment. Next, MFDs are coated overnight with consecutively PLL (DIV-2) and Laminin (DIV-1). For both coatings, a volumetric gradient is used to ensure coating of the grooves. Additionally, a Laminin concentration gradient (from the AC to the SDC) is used to promote axonal outgrowth toward the AC. The next morning, adult zebrafish retinas are isolated, followed by an enzymatic and mechanical dissociation to obtain a single cell suspension. To remove dead cells and debris, the suspension is centrifuged and resuspended in fresh fish medium, and retinal cells are seeded in the SDC of an open compartment MFD (SOC450). To stimulate outgrowth of RGC axons toward the AC, a volumetric gradient from the SDC to the AC is created and conditioned retinal cells are also seeded in the AC. Cells are cultured at 30°C and 5% CO₂ and monitored daily. At DIV 2, RGC axonal outgrowth can be examined. At DIV 3, a vacuum-assisted axotomy is performed, whereafter axonal regeneration can be evaluated. To visualize the regenerative process and characterize mitochondrial dynamics or dendritic remodeling, respectively, Tg(Tru.gap43:GFP)^mil (gap43), Tg(Tru.gap43:mitoEGFP-2A-TagRFP-CAAX)^ulb17 (gapmito) or a sparsely-labeled, mixed culture of wild type (WT) and (gap43) retinal neurons are seeded in the SDC. AC, axonal compartment; DIV, days in vitro; MFD, microfluidic device; ONC, optic nerve crush; PLL, Poly-L-lysine; RGC, retinal ganglion cell; SDC, somatodendritic compartment.