Micropatterned nanolayers immobilized with nerve growth factor for neurite formation of PC12 cells

Abstract

Background

Nerve regeneration is important for the treatment of degenerative diseases and neurons injured by accidents. Nerve growth factor (NGF) has been previously conjugated to materials for promotion of neurogenesis.

Materials and methods

Photoreactive gelatin was prepared by chemical coupling of gelatin with azidobenzoic acid (P-gel), and then NGF was immobilized on substrates in the presence or absence of micropatterned photomasks. UV irradiation induced crosslinking reactions of P-gel with itself, NGF, and the plate for immobilization.

Results

By adjustment of the P-gel concentration, the nanometer-order height of micropatterns was controlled. NGF was quantitatively immobilized with increasing amounts of P-gel. Immobilized NGF induced neurite outgrowth of PC12 cells, a cell line derived from a pheochromocytoma of the rat adrenal medulla, at the same level as soluble NGF. The immobilized NGF showed higher thermal stability than the soluble NGF and was repeatedly used without loss of biological activity. The 3D structure (height of the formed micropattern) regulated the behavior of neurite guidance. As a result, the orientation of neurites was regulated by the stripe pattern width.

Conclusion

The micropattern-immobilized NGF nanolayer biochemically and topologically regulated neurite formation.

Keywords:

• nerve growth factor
• photoreactive gelatin
• micropatterned immobilization
• PC12 cell
• neurite outgrowth

Acknowledgment

This work was financially supported by KAKENNHI from JSPS (15H01810). S.M.K. was financially supported by the Junior Research Associate (JRA) program from RIKEN. We thank Dr. Hiroyuki Kamiguchi at the RIKEN Center for Brain Science for useful discussions. We also thank Mitchell Arico from Edanz Group for editing a draft of this manuscript.

Disclosure

The authors report no conflicts of interest in this work.