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

Seong Min Kim,1,2 Masashi Ueki,1 Xueli Ren,3 Jun Akimoto,1 Yasuyuki Sakai,2 Yoshihiro Ito1,3 1Nano Medical Engineering Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama 351-0198, Japan; 2Department of Bioengineering, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan; 3Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, Wako, Saitama 351-0198, JapanCorrespondence: Yoshihiro ItoNano Medical Engineering Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama 351-0198, JapanTel +81 48 467 5809Fax +81 48 467 9300Email y-ito@riken.jpBackground: 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