application note: how to culture cortical neural stem cells and neurons in 3D environment

3D cell culture techniques for culturing neural stem cells and neurons provide highly useful tools for studying neurogenesis, neuropathology, toxicology and effects of new drug targets. They also allow the potential for side by side comparisons of disease state cells vs healthy controls for the study of neurological disorders and diseases. The use of such systems overcomes some of the limitations of standard 2D culture systems in which neural cells are cultured as a monolayer. The recapitulation of the complex microenvironment in which neural cells exists allows 3D culture systems to bridge the gap between traditional cell culture approaches and in vivo models such as transgenic mice. By mimicking the "stem cell niche" and physiological environments both mechanically and spatially, the crosstalk between the cells and environment leads to a closer scenario to what can be seen in the brain. There are number of different approaches to 3D culture of neuronal cells including generation of organoids/spheroids or scaffolding systems such as collagen hydrogels and nanofiber scaffolds.

To investigate the suitability of Axol Neural Cells for use in a 3D culture system we used TAP Biosystems RAFT technology, a collagen based hydrogel system. The resulting gels were stained using immunocytochemistry and analysed using confocal microscopy.


Axol Cortical Neural Stem Cells

Foxg1 - Nestin 

On culturing Axol Neural Stem Cells (NSCs) on top of the gel, cell migration into the matrix was observed. The cells went on to form the 3D structure of commonly seen neural rosettes. The rosette structure viewed in this way shows the cell bodies of the NSCs forming outwards into a spherical pattern alongside the nestin, which becomes a meshing. Outside of the rosette, there is a matrix of cells in a non-uniform conformity.

axol 3d cortical neural stem cells

Axol Human Cerebral Cortical Neurons

Tbr1- Tuj1

On culturing the human Cerebral Cortical Neurons (hCCNs) on top of the RAFT gel, they formed a uniform static layer of cell bodies. Neurites projected out of these cells and grew downwards, creating a network of interconnected neurites. The results demonstrate that the RAFT collagen matrix was suitable for culturing hCCNs to achieve neurite outgrowth, although little neuronal migration was observed.

Image: Left Panel: Top View, Top Right Panel: Bottom View, Bottom Panel: Side View

axol 3d cortical neural stem cells