The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Abstract

Electrospinning is a technique for producing micro-to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications. Video LinkThe video component of this article can be found at https://www.jove.com/video/2389/ Protocol 1. Poly-L-lactic Acid (PLLA) Spinning Solution 1. Dissolve 0.4 g PLLA in 9 mL chloroform by stirring over low heat. 2. Add 1 mL dimethylformamide to the solution, bringing the final concentration of the solution to 4% PLLA (w/v) in chloroform:DMF 9:1 (v/v). 3. Place the solution in a polypropylene or glass syringe with a blunt 23ga metal tip. Spinning Substrate Preparation 11. Make an 8% (w/v) solution of 85:15 PLGA (poly-lactic-co-glycolic acid) in chloroform by stirring over low heat. 2. Coat clean glass cover slips in PLGA by covering the surface of each cover slip with the PLGA solution. Allow the PLGA to dry to a thin film (approx. 30 min). Electrospinning 21. Secure PLGA coated glass cover slips to collector with conductive carbon tape. For aligned fibers, the collector is a motor-driven wheel. For random fibers, the collector is a stationary plate. 2. Place syringe in pump with tip 20 cm from collector wheel. Set pump to approximately 2 mL/hr and if using a wheel, set the motor to 300-400 RPM. If possible, apply a -2 kV DC bias to the collector and +15 kV to metal tip. In the absence of access to a bipolar power supply, ground the collector. 3. Fibers will jet from the syringe tip and collect on the rotating wheel. Continue spinning until the desired density of fibers is obtained. Swipe the metal tip with a paper towel affixed to a non-conductive rod periodically to prevent clogging at the tip.

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Evaluation of Cisplatin Neurotoxicity in Cultured Rat Dorsal Root Ganglia via Cytosolic Calcium Accumulation

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Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

“…Typically, in primary cultures of mammalian brain neurons, the cells are plated with a relatively high density, on coated coverslips without a glial feeder layer (e.g., [31][32][33][34][35][36][37][38][39] ). When the plating density of neurons is reduced using this method, the initial lack of glial sheet leads to poor neuronal growth and dendritic extension (panels B, C in Figures 8-10), as reported previously [40][41][42] , probably due to poor glial growth 43,44 .…”