Neurite Outgrowth Guidance using micropatterns

Protocol and images generously provided by Dr. Sandrine Vitry who ran the experiments.

Retrovirus and Genetic Transfer Unit lead by Dr. Jean-Michel Heard,

Neuroscience Department, Institut Pasteur, Paris, France.

Background Neurite outgrowth quantification can be challenging when neurons are grown on a homogeneous adhesive surface in the absence of any cell guidance. Here you can visualize how neurites can be observed in cultures of primary cortical neurons on gridded micropatterns. Dendrite and axon growth are guided by the geometry of the micropatterns, and follow PDL/laminine coated lines. Primary neurons can be maintained in culture for several days on micropatterns: in this example cells were fixed at Day4 post-seeding.   Figure 1. Cortical neurons on gridded micropatterns stained for: nuclei (Hoechst, blue), axonal marker SMI132 (green), somatodendritic marker MAP2 (red) and early neuronal marker ßIII-Tubulin (magenta). Images were acquired on a Carl Zeiss Apotome (20X Magnification)

Experimental Description*

*Please contact us for a full protocol description

Briefly,  dissociated cortical neurons were seeded on micropatterns -previously coated with poly-D-Lysine and laminin- and maintained in culture for 4 days (Figure 2).

Figure 2: Contrast-phase images of cortical primary neurons on gridded micropatterns. Light yellow: representation of micropattern grid

After 4 days of culture, cells were fixed and stained for nuclei (Hoechst, blue, Fig3A), axonal marker SMI132 (green, Fig3A) somatodendritic marker MAP2 (red, Fig3B) and early neuronal marker ßIII-Tubulin (magenta, Fig3C). Images were acquired on a Carl Zeiss Apotome (20X Magnification) (Figure 3). Fig 3D: Merge.

In area 1: neurites and neurons on non-patterned surfaces

In area 2: grid-patterned neurons

Figure 3: Immuno-staining of cortical primary neurons on gridded micropatterns.