Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers

Gertz, Caitlyn C.; Leach, Michelle K.; Birrell, Lisa K.; Martin, David C.; Feldman, Eva L.; Corey, Joseph M.

doi:10.1002/dneu.20792

Cited by 79 publications

(82 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We typically obtain MN purity of >90% neurons 7,8 with this protocol and we have maintained DRG cultures for as long as one week without any significant fibroblast growth. Figure 2 shows typical MN appearance on glass and fibers after 24 hrs of culture and immunostaining.…”

Section: Representative Resultsmentioning

confidence: 99%

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

Leach¹,

Feng²,

Gertz³

et al. 2011

JoVE

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Representative Resultsmentioning

confidence: 99%

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

Leach¹,

Feng²,

Gertz³

et al. 2011

JoVE

Self Cite

View full text Add to dashboard Cite

show abstract

“…In other studies, aligned PLLA nanofibrous scaffold also supported the growth and extension of dorsal root ganglion (DRGs) [20,21] and primary motor neurons [20]. Primary motor neurons developed neurites earlier on PLLA nanofibers when compared to PLLA films [22]. However, no differences in neurite number and length were detected between aligned and random PLLA fibers [22].…”

Section: Effect Of Fiber Orientation and Sizementioning

confidence: 92%

“…Primary motor neurons developed neurites earlier on PLLA nanofibers when compared to PLLA films [22]. However, no differences in neurite number and length were detected between aligned and random PLLA fibers [22]. For human Schwann cells on PCL scaffolds, aligned fibers promoted cytoskeleton and nuclei alignment along the fiber.…”

Section: Effect Of Fiber Orientation and Sizementioning

confidence: 93%

Electrospun Nanofibrous Materials for Neural Tissue Engineering

2011

View full text Add to dashboard Cite

Abstract:The use of biomaterials processed by the electrospinning technique has gained considerable interest for neural tissue engineering applications. The tissue engineering strategy is to facilitate the regrowth of nerves by combining an appropriate cell type with the electrospun scaffold. Electrospinning can generate fibrous meshes having fiber diameter dimensions at the nanoscale and these fibers can be nonwoven or oriented to facilitate neurite extension via contact guidance. This article reviews studies evaluating the effect of the scaffold's architectural features such as fiber diameter and orientation on neural cell function and neurite extension. Electrospun meshes made of natural polymers, proteins and compositions having electrical activity in order to enhance neural cell function are also discussed.

show abstract

“…Gertz et al showed that electrospun submicron fibers can significantly enhance neuritogenesis, maturation and the polarity formation of neurons compared to the two dimensional substrates, such as glass [12]. On the other hand, natural bio-polymers such as gelatin, which is produced by hydrolyzing collagen, should be more relevant than synthetic polymers in terms of biocompatibility [13].…”

Section: Introductionmentioning

confidence: 99%

Patch method for culture of primary hippocampal neurons

Tang

Severino

Iseppon

et al. 2017

Microelectronic Engineering

View full text Add to dashboard Cite

In vitro culture of primary neurons, especially hippocampal neurons, is important for understanding cellular mechanisms in neurobiology. Actually, this is achieved by using culture dish or glass slide with surface coated proteins. Here, we proposed a patch method to culture primary neurons on a monolayer of gelatin nanofibers, electrospun and crosslinked on a microfabricated honeycomb frame of poly (ethylene glycol) diacrylate (PEGDA). By using such a patch method, neural networks could be formed with a minimal cell-exogenous materials contact and a maximal exposure of the cells to the medium. Interestingly, hippocampal cells, especially astrocytes, showed in-vivo like morphology and most of neurons were found in the porous areas inside the honeycomb compartments although the nanofibers were deposited everywhere of the frame. Finally, calcium imaging showed that primary neurons have a higher degree of neural activity on the patch than on glass.

show abstract

Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers

Cited by 79 publications

References 55 publications

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

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

Electrospun Nanofibrous Materials for Neural Tissue Engineering

Patch method for culture of primary hippocampal neurons

Contact Info

Product

Resources

About