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3D Neurite Outgrowth Model

REPROCELL’s physiologically relevant 3D neurite outgrowth model provides a means of studying the effects of inhibitory or neurotoxic compounds on neurite outgrowth, and of molecules that enhance neurite outgrowth. The model was co-developed with the laboratory of REPROCELL’s CSO, Professor Stefan Przyborski* at Durham University, UK and is based on StemRNA™ Neuro neurospheres grown in Alvetex® Scaffold.

[* Stefan Przyborski is Chief Scientific Officer (CSO) of REPROCELL Europe Ltd and Professor of Cell Technology at Durham University, UK.]

  • iPSC-derived StemRNA™ Neuro cells (other neuronal or pluripotent stem cell-derived neuronal starting points can also be used)
  • Measure inhibition and recovery of neurite outgrowth
  • Explore pathological changes in functional human cells in a physiologically relevant in vitro 3D model
  • Assess many physiological and pathological ECM coatings (laminin, CSPGs), known pathway inhibitors, and test therapeutic lead compounds

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The loss of neuronal connectivity can occur due to spinal cord injury (SCI), Alzheimer’s Disease (AD), and other causes. The release of amyloid-β species from damaged cells builds up in glial scars formed in SCI or deposited in senile plaques in AD, causing the collapse of apical cones and the perturbation of actin dynamics within neurite outgrowths, leading to loss of connectivity and retreat. 

Various small molecules have been demonstrated to restore and enhance neurite outgrowth density and length in vitro. However, the various mechanisms for many of these processes are not well known and 2D cell culture methods for testing of new therapies often do not accurately reflect complex 3D neuronal structures. REPROCELL’s 3D neurite outgrowth model displays greater sensitivity to drug effects and more accurately reflects human biology.

Co-culture models can also be established, e.g. glial cells with neurons (e.g. Clarke et al. (2016).1

alvetex-science-neurite-outgrowth

Above: Immunocytochemistry analysis showing neurites penetration though Alvetex Scaffold in our 3D outgrowth model. Neurites were visualized by staining with the pan neuronal marker TUJ-1 (green). Nucleii in the neurosphere are stained blue.

 

A

2D Model

B

Alvetex 3D model 

Above: The presence of exogenous amyloid-β40 (Aβ40) causes a retreat of neurite outgrowth in both the 2D and 3D models; however, drug compound effects are more sensitively measured in 3D. Test compounds were NEP1-40 (competitive NgR (Nogo receptor) inhibitor), ibuprofen (Rho A inhibitor), and Y-27632 (ROCK inhibitor). While the introduction of any of these three compounds restores neurite outgrowth (i.e. increase in neurite density, not shown), in the 2D model none were observed to restore outgrowth length to that of the vehicle control (A.). In the 2D model, neurite outgrowth length is measured via the neurites radiating from neurospheres. But in the Alvetex Scaffold 3D model, outgrowth length can by better estimated by counting the number of neurites that have penetrated all the way through the scaffold (B.). In the more physiologically relevant 3D model, ibuprofen restored neurite penetration levels while Y-27632 significantly enhanced neurite penetration levels. From: Goncalves and Przyborski (Sept. 2024).2

References

  1. A robust and reproducible human pluripotent stem cell derived model of neurite outgrowth in a three-dimensional culture system and its application to study neurite inhibition
    Kirsty E. Clarke, Daniel M. Tams, Andrew P. Henderson, Mathilde F. Roger, Andrew Whiting, Stefan A. Przyborski (June 2016). Neurochemistry International. DOI: 10.1016/j.neuint.2016.12.009
  2. Modulation of the Nogo signaling pathway to overcome amyloid-β-mediated neurite inhibition in human pluripotent stem cell–derived neurites
    Kirsty Goncalves, Stefan Przyborski (September 2024). Neural Regeneration Research. DOI: 10.4103/NRR.NRR-D-23-01628

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