Isolated node engineering of neuronal systems using laser direct write

Abstract

Current limitations to the engineering of ex vivo and in vitro neural environments are hampering the ability to understand underlying neurophysiology. High levels of spatial specificity, reproducibility and viability have been previously reported using laser direct write (LDW) to print cells. However, despite the significant need no one has yet reported laser assisted printing of primary mammalian neuronal cells, an inherently sensitive but critically important population. Herein, we describe the use of LDW to reproducibly and accurately pattern viable dorsal root ganglion (DRG) neurons and supportive cells capable of neural outgrowth and network formation. Our demonstrated ability to engineer and control distinct micro-environmental components unlocks the potential for high throughput experiments to both understand underlying physiology and investigate therapeutic interventions.

Laser‐Induced Forward Transfer: Fundamentals and Applications

“…Due to the limited healing capacity of this tissue, this application remains particularly challenging. Results obtained after printing neurons and glial cells through LIFT pave the way for the future generation of complex cellular models with unprecedented cellular organization …”

Bioprinting Neural Systems to Model Central Nervous System Diseases

“…≈90% (day 6) [36] Optic-based Laser-induced forward transfer (LIFT) ≈200 µm [39] E15 rat primary dorsal root ganglia (DRG) [39] Live-Dye /PI: ≈85% (24 h post printing) [39] • Successful prints performed with hyaluronic acid and Matrigel [40] • Neurite growths reported [39] • Proven in situ differentiation of bioprinted cells [34] • Low cell density: ≈80 cells per drop [39] • Limited manufacturer diversity might affect device accessibility [41] Not assessed [40] hiPSC [40] Trypan Blue: ≈82% (2-3 h post printing) [40] Stereolithography (SLA) ≈190 µm [42] Mouse NSCs (NE-4C) [42] Calcein AM/PI: ≈100-70%, 40-120 mW laser power) [42] • ≈5 µm micrometer-scale resolution a chievable [43] • Custom devices a vailable [44] • Combined with 3D printing using PCL fibers [42] • Used with conductive graphene-loaded bioinks [45] • Potential cell damage due to UV light exposure…”

Neural Engineering

“…By day 7 in culture, neurite extensions connected these islands and the expression of synaptic marker, VGLUT2, was visible. However, the cells were not constrained to the area of their printed islands, so the nodal organization induced by printing has begun to degrade by day 7 (Curley et al, 2016 ).…”