A method to rapidly analyze the simultaneous release of multiple pharmaceuticals from electrospun fibers.

Electrospun fibers are a commonly used cell scaffold and have also been used as pharmaceutical delivery devices. In this study, we developed a method to analyze the release of multiple pharmaceuticals from a single electrospun fiber scaffold and determine how each pharmaceutical's loading concentration affects the release rate of each pharmaceutical. Our analysis methods were tested on electrospun fibers loaded with two pharmaceuticals: 6-aminonicotinamide (6AN) and ibuprofen. Pharmaceutical concentration in electrospun fibers ranged from 1.5% to 8.5% by weight. We found that 6AN release was dependent on the concentration of 6AN and ibuprofen loaded into the fibers, while ibuprofen release was only dependent on the loading concentration of ibuprofen but not 6AN. Unexpectedly, ibuprofen release became dependent on both 6AN and ibuprofen loading concentrations when fibers were aged for 1-month post-fabrication at room temperature in the laboratory followed by a 4-hour incubation inside the cell culture incubator at 37 °C and 5% CO2. One additional discovery was an unknown signal that was attributed to the medical grade syringes used for electrospinning, which was easily removed using our method. These results demonstrate the utility of the methods developed here and indicate multiple agents can be released concomitantly from electrospun fibers to meet the demands of more complex tissue engineering approaches. Future work will focus on analysis of pharmaceutical release profiles to exploit the dependencies on pharmaceutical loading concentrations.

Combining electrospun nanofibers with cell-encapsulating hydrogel fibers for neural tissue engineering.

A promising component of biomaterial constructs for neural tissue engineering are electrospun fibers, which differentiate stem cells and neurons as well as direct neurite growth. However, means of protecting neurons, glia, and stem cells seeded on electrospun fibers between lab and surgical suite have yet to be developed. Here we report an effort to accomplish this using cell-encapsulating hydrogel fibers made by interfacial polyelectrolyte complexation (IPC). IPC-hydrogel fibers were created by interfacing acid-soluble chitosan (AsC) and cell-containing alginate and spinning them on bundles of aligned electrospun fibers. Primary spinal astrocytes, cortical neurons, or L929 fibroblasts were mixed into alginate hydrogels prior to IPC-fiber spinning. The viability of each cell type was assessed at 30 min, 4 h, 1 d, and 7 d after encapsulation in IPC hydrogels. Some neurons were encapsulated in IPC-hydrogel fibers made from water-soluble chitosan (WsC). Neurons were also stained with Tuj1 and assessed for neurite extension. Neuron survival in AsC-fibers was worse than astrocytes in AsC-fibers (p < 0.05) and neurons in WsC-fibers (p < 0.05). As expected, neuron and glia survival was worse than L929 fibroblasts (p < 0.05). Neurons in IPC-hydrogel fibers fabricated with WsC extended neurites robustly, while none in AsC fibers did. Neurons remaining inside IPC-hydrogel fibers extended neurites inside them, while others de-encapsulated, extending neurites on electrospun fibers, which did not fully integrate with IPC-hydrogel fibers. This study demonstrates that primary neurons and astrocytes can be encapsulated in IPC-hydrogel fibers at good percentages of survival. IPC hydrogel technology may be a useful tool for encapsulating neural and other cells on electrospun fiber scaffolds.

Nanofibrous scaffolds for the guidance of stem cell-derived neurons for auditory nerve regeneration.

Impairment of spiral ganglion neurons (SGNs) of the auditory nerve is a major cause for hearing loss occurring independently or in addition to sensory hair cell damage. Unfortunately, mammalian SGNs lack the potential for autonomous regeneration. Stem cell based therapy is a promising approach for auditory nerve regeneration, but proper integration of exogenous cells into the auditory circuit remains a fundamental challenge. Here, we present novel nanofibrous scaffolds designed to guide the integration of human stem cell-derived neurons in the internal auditory meatus (IAM), the foramen allowing passage of the spiral ganglion to the auditory brainstem. Human embryonic stem cells (hESC) were differentiated into neural precursor cells (NPCs) and seeded onto aligned nanofiber mats. The NPCs terminally differentiated into glutamatergic neurons with high efficiency, and neurite projections aligned with nanofibers in vitro. Scaffolds were assembled by seeding GFP-labeled NPCs on nanofibers integrated in a polymer sheath. Biocompatibility and functionality of the NPC-seeded scaffolds were evaluated in vivo in deafened guinea pigs (Cavia porcellus). To this end, we established an ouabain-based deafening procedure that depleted an average 72% of SGNs from apex to base of the cochleae and caused profound hearing loss. Further, we developed a surgical procedure to implant seeded scaffolds directly into the guinea pig IAM. No evidence of an inflammatory response was observed, but post-surgery tissue repair appeared to be facilitated by infiltrating Schwann cells. While NPC survival was found to be poor, both subjects implanted with NPC-seeded and cell-free control scaffolds showed partial recovery of electrically-evoked auditory brainstem thresholds. Thus, while future studies must address cell survival, nanofibrous scaffolds pose a promising strategy for auditory nerve regeneration.

Mechanical tension applied to substrate films specifies location of neuritogenesis and promotes major neurite growth at the expense of minor neurite development.

One obstacle in neural repair is facilitating axon growth long enough to reach denervated targets. Recent studies show that axonal growth is accelerated by applying tension to bundles of neurites, and additional studies show that mechanical tension is critical to all neurite growth. However, no studies yet describe how individual neurons respond to tensile forces applied to cell bodies and neurites simultaneously; neither do any test motor neurons, a phenotype critical to neural repair. Here we examine the growth of dissociated motor neurons on stretchable substrates. E15 spinal motor neurons were cultured on poly-lactide-co-glycolide films stretched at 4.8, 9.6, or 14.3 mm day(-1). Morphological analysis revealed that substrate stretching has profound effects on developing motor neurons. Stretching increases major neurite length; it also forces neuritogenesis to occur nearest poles of the cell closest to the sources of tension. Stretching also reduces the number of neurites per neuron. These data show that substrate stretching affects neuronal morphology by specifying locations on the cell where neuritogenesis occurs and favoring major neurite growth at the expense of minor neurites. These results serve as a building block for development of new techniques to control and improve the growth of neurons for nerve repair purposes.

Dimethyl Fumarate Protects Neural Stem/Progenitor Cells and Neurons from Oxidative Damage through Nrf2-ERK1/2 MAPK Pathway.

Multiple sclerosis (MS) is the most common multifocal inflammatory demyelinating disease of the central nervous system (CNS). Due to the progressive neurodegenerative nature of MS, developing treatments that exhibit direct neuroprotective effects are needed. Tecfidera™ (BG-12) is an oral formulation of the fumaric acid esters (FAE), containing the active metabolite dimethyl fumarate (DMF). Although BG-12 showed remarkable efficacy in lowering relapse rates in clinical trials, its mechanism of action in MS is not yet well understood. In this study, we reported the potential neuroprotective effects of dimethyl fumarate (DMF) on mouse and rat neural stem/progenitor cells (NPCs) and neurons. We found that DMF increased the frequency of the multipotent neurospheres and the survival of NPCs following oxidative stress with hydrogen peroxide (H2O2) treatment. In addition, utilizing the reactive oxygen species (ROS) assay, we showed that DMF reduced ROS production induced by H2O2. DMF also decreased oxidative stress-induced apoptosis. Using motor neuron survival assay, DMF significantly promoted survival of motor neurons under oxidative stress. We further analyzed the expression of oxidative stress-induced genes in the NPC cultures and showed that DMF increased the expression of transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) at both levels of RNA and protein. Furthermore, we demonstrated the involvement of Nrf2-ERK1/2 MAPK pathway in DMF-mediated neuroprotection. Finally, we utilized SuperArray gene screen technology to identify additional anti-oxidative stress genes (Gstp1, Sod2, Nqo1, Srxn1, Fth1). Our data suggests that analysis of anti-oxidative stress mechanisms may yield further insights into new targets for treatment of multiple sclerosis (MS).

Growth of primary motor neurons on horizontally aligned carbon nanotube thin films and striped patterns.

OBJECTIVE: Carbon nanotubes (CNTs) are attractive for use in peripheral nerve interfaces because of their unique combination of strength, flexibility, electrical conductivity and nanoscale surface texture. Here we investigated the growth of motor neurons on thin films of horizontally aligned CNTs (HACNTs). APPROACH: We cultured primary embryonic rat motor neurons on HACNTs and performed statistical analysis of the length and orientation of neurites. We next presented motor neurons with substrates of alternating stripes of HACNTs and SiO2. MAIN RESULTS: The neurons survived on HACNT substrates for up to eight days, which was the full duration of our experiments. Statistical analysis of the length and orientation of neurites indicated that the longest neurites on HACNTs tended to align with the CNT direction, although the average neurite length was similar between HACNTs and glass control substrates. We observed that when motor neurons were presented with alternating stripes of HACNTs and SiO2, the proportion of neurons on HACNTs increases over time, suggesting that neurons selectively migrate toward and adhere to the HACNT surface. SIGNIFICANCE: The behavior of motor neurons on CNTs has not been previously investigated, and we show that aligned CNTs could provide a viable interface material to motor neurons. Combined with emerging techniques to build complex hierarchical structures of CNTs, our results suggest that organised CNTs could be incorporated into nerve grafts that use physical and electrical cues to guide regenerating axons.

A rapid and reproducible assay for modeling myelination by oligodendrocytes using engineered nanofibers.

Current methods for studying oligodendrocyte myelination using primary neurons are limited by the time, cost and reproducibility of myelination in vitro. Nanofibers with diameters of >0.4 µm fabricated from electrospinning of liquid polystyrene are suitable scaffolds for concentric membrane wrapping by oligodendrocytes. With the advent of aligned electrospinning technology, nanofibers can be rapidly fabricated, standardized, and configured into various densities and patterns as desired. Notably, the minimally permissive culture environment of fibers provides investigators with an opportunity to explore the autonomous oligodendrocyte cellular processes underlying differentiation and myelination. The simplicity of the system is conducive to monitoring oligodendrocyte proliferation, migration, differentiation and membrane wrapping in the absence of neuronal signals. Here we describe protocols for the fabrication and preparation of nanofibers aligned on glass coverslips for the study of membrane wrapping by rodent oligodendrocytes. The entire protocol can be completed within 2 weeks.

Synthesis, copolymerization and peptide-modification of carboxylic acid-functionalized 3,4-ethylenedioxythiophene (EDOTacid) for neural electrode interfaces.

BACKGROUND: Conjugated polymers have been developed as effective materials for interfacing prosthetic device electrodes with neural tissue. Recent focus has been on the development of conjugated polymers that contain biological components in order to improve the tissue response upon implantation of these electrodes. METHODS: Carboxylic acid-functionalized 3,4-ethylenedioxythiophene (EDOTacid) monomer was synthesized in order to covalently bind peptides to the surface of conjugated polymer films. EDOTacid was copolymerized with EDOT monomer to form stable, electrically conductive copolymer films referred to as PEDOT-PEDOTacid. The peptide GGGGRGDS was bound to PEDOT-PEDOTacid to create peptide functionalized PEDOT films. RESULTS: The PEDOT-PEDOTacid-peptide films increased the adhesion of primary rat motor neurons between 3 and 9 times higher than controls, thus demonstrating that the peptide maintained its biological activity. CONCLUSIONS: The EDOT-acid monomer can be used to create functionalized PEDOT-PEDOTacid copolymer films that can have controlled bioactivity. GENERAL SIGNIFICANCE: PEDOT-PEDOTacid-peptide films have the potential to control the behavior of neurons and vastly improve the performance of implanted electrodes. This article is part of a Special Issue entitled Organic Bioelectronics-Novel Applications in Biomedicine.

Highly Aligned Poly(3,4-ethylene dioxythiophene) (PEDOT) Nano- and Microscale Fibers and Tubes.

This study reports a facile method for the fabrication of aligned Poly(3,4-ethylene dioxythiophene) (PEDOT) fibers and tubes based on electrospinning and oxidative chemical polymerization. Discrete PEDOT nano- and microfibers and nano- and microtubes are difficult to fabricate quickly and reproducibly. We employed poly(lactide-co-glycolide) (PLGA) polymers that were loaded with polymerizable 3,4-ethylene dioxythiophene (EDOT) monomer to create aligned nanofiber assemblies using a rotating glass mandrel during electrospinning. The EDOT monomer/PLGA polymer blends were then polymerized by exposure to an oxidative catalyst (FeCl3). PEDOT was polymerized by continuously dripping a FeCl3 solution onto the glass rod during electrospinning. The resulting PEDOT fibers were conductive, aligned and discrete. Fiber bundles could be easily produced in lengths of several centimeters. The PEDOT sheath/PLGA core fibers were immersed in chloroform to remove the PLGA and any residual EDOT resulting in hollow PEDOT tubes. This approach made it possible to easily generate large areas of aligned PEDOT fibers/tubes. The structure and properties of the aligned assemblies were measured using optical microscopy, electron microscopy, Raman spectroscopy, thermal gravimetric analysis, and DC conductivity measurements. We also demonstrated that the aligned PEDOT sheath/PLGA core fiber assemblies could be used in supporting and directing the extension of dorsal root ganglia (DRG) neurons in vitro.

Combining topographical and genetic cues to promote neuronal fate specification in stem cells.

There is little remedy for the devastating effects resulting from neuronal loss caused by neural injury or neurodegenerative disease. Reconstruction of damaged neural circuitry with stem cell-derived neurons is a promising approach to repair these defects, but controlling differentiation and guiding synaptic integration with existing neurons remain significant unmet challenges. Biomaterial surfaces can present nanoscale topographical cues that influence neuronal differentiation and process outgrowth. By combining these scaffolds with additional molecular biology strategies, synergistic control over cell fate can be achieved. Here, we review recent progress in promoting neuronal fate using techniques at the interface of biomaterial science and genetic engineering. New data demonstrates that combining nanofiber topography with an induced genetic program enhances neuritogenesis in a synergistic fashion. We propose combining patterned biomaterial surface cues with prescribed genetic programs to achieve neuronal cell fates with the desired sublineage specification, neurochemical profile, targeted integration, and electrophysiological properties.

A culture system to study oligodendrocyte myelination processes using engineered nanofibers.

Current methods for studying central nervous system myelination necessitate permissive axonal substrates conducive to myelin wrapping by oligodendrocytes. We have developed a neuron-free culture system in which electron-spun nanofibers of varying sizes substitute for axons as a substrate for oligodendrocyte myelination, thereby allowing manipulation of the biophysical elements of axonal-oligodendroglial interactions. To investigate axonal regulation of myelination, this system effectively uncouples the role of molecular (inductive) cues from that of biophysical properties of the axon. We use this method to uncover the causation and sufficiency of fiber diameter in the initiation of concentric wrapping by rat oligodendrocytes. We also show that oligodendrocyte precursor cells display sensitivity to the biophysical properties of fiber diameter and initiate membrane ensheathment before differentiation. The use of nanofiber scaffolds will enable screening for potential therapeutic agents that promote oligodendrocyte differentiation and myelination and will also provide valuable insight into the processes involved in remyelination.

Electrospun fibers and tissue engineering.

Electrospinning is an exciting technique attracting more and more attention as a potential solution to the current challenges in the field of tissue engineering. This technique can be used to produce fibrous scaffolds with excellent biocompatibility and biodegradability, as well as suitable micro-/nanostructure to induce desired cellular activities and to guide tissue regeneration. In order to develop electrospun fibrous scaffolds for these applications, different biocompatible materials including natural polymers, synthetic polymers and inorganic substances and preparations have been used to fabricate electrospun fibers with different structures and morphologies. This review briefly describes the development of the technique, focusing on several typically electrospun materials, surface modification of electrospun fibers, and current applications in tissue engineering.

Critical variables in the alignment of electrospun PLLA nanofibers.

Electrospun polymer nanofibers show promise as components of scaffolds for tissue engineering because of their ability to orient regenerating cells. Our research focuses on aligned electrospun fiber scaffolds for nerve regeneration. Critical to this are highly aligned fibers, which are frequently difficult to manufacture reproducibly. Here we show that three variables: the distance between the spinneret tip and collector, the addition of DMF to the solvent, and placement of an aluminum sheet on the spinneret together greatly improve the alignment of electrospun poly-L-lactide (PLLA) nanofibers. We identified the most important variable as tip-to-collector distance. Nanofiber alignment was maximal at 30cm compared to shorter distances. DMF:chloroform (1:9) improved nanofiber uniformity and was integral to maintaining a uniform stream over the 30cm tip-to-collector distance. Other ratios caused splattering of the solution or flattening or beading of the fibers and non-uniform fiber diameter. The aluminum sheet helped to stabilize the electric field and improve fiber alignment provided that it was placed at 1cm behind the tip, while other distances destabilized the stream and worsened alignment. This study demonstrates that control of these variables produces dramatic improvement in reproducibly obtaining high alignment and uniform morphology of electrospun PLLA nanofibers.

Stages of neuronal morphological development in vitro--an automated assay.

Following plating in vitro, neurons pass through a series of morphological stages as they adhere and mature. These morphological stage transitions can be monitored as a function of time to evaluate the relative health and development of neuronal cultures under different conditions. While morphological development is usually quite obvious to the experienced eye, it can often be difficult to quantify in a meaningful way. Morphology quantification typically relies on manual image measurement and can therefore be tedious, time consuming and prone to human error. Here we report the successful development of an automated process using the commercially available image analysis program MetaMorph(®) to analyze the morphology and quantify the growth of embryonic spinal motor neurons in vitro. Our process relied on the Neurite Outgrowth and Cell Scoring modules included in MetaMorph(®) and on analyzing the exported data with an algorithm written in MATLAB(®). We first adopted a series of stages of motor neuron development in vitro. Neurons were classified into these stages directly from the available output of MetaMorph(®) using the algorithm written in MATLAB(®). We validated the results of the automated analysis against a manual analysis of the same images and found no statistically significant difference between the two methods. When properly configured, automated image analysis with MetaMorph(®) is a rapid and reliable alternative to manual measurement and has the potential to accelerate the research process.

The culture of primary motor and sensory neurons in defined media on electrospun poly-L-lactide nanofiber scaffolds.

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.

Electrospinning fundamentals: optimizing solution and apparatus parameters.

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

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

Neuritogenesis, neuronal polarity formation, and maturation of axons and dendrites are strongly influenced by both biochemical and topographical extracellular components. The aim of this study was to elucidate the effects of polylactic acid electrospun fiber topography on primary motor neuron development, because regeneration of motor axons is extremely limited in the central nervous system and could potentially benefit from the implementation of a synthetic scaffold to encourage regrowth. In this analysis, we found that both aligned and randomly oriented submicron fibers significantly accelerated the processes of neuritogenesis and polarity formation of individual cultured motor neurons compared to flat polymer films and glass controls, likely due to restricted lamellipodia formation observed on fibers. In contrast, dendritic maturation and soma spreading were inhibited on fiber substrates after 2 days in vitro. This study is the first to examine the effects of electrospun fiber topography on motor neuron neuritogenesis and polarity formation. Aligned nanofibers were shown to affect the directionality and timing of motor neuron development, providing further evidence for the effective use of electrospun scaffolds in neural regeneration applications.

Conducting-polymer nanotubes improve electrical properties, mechanical adhesion, neural attachment, and neurite outgrowth of neural electrodes.

An in vitro comparison of conducting-polymer nanotubes of poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(pyrrole) (PPy) and to their film counterparts is reported. Impedance, charge-capacity density (CCD), tendency towards delamination, and neurite outgrowth are compared. For the same deposition charge density, PPy films and nanotubes grow relatively faster vertically, while PEDOT films and nanotubes grow more laterally. For the same deposition charge density (1.44 C cm(-2)), PPy nanotubes and PEDOT nanotubes have lower impedance (19.5 +/- 2.1 kOmega for PPy nanotubes and 2.5 +/- 1.4 kOmega for PEDOT nanotubes at 1 kHz) and higher CCD (184 +/- 5.3 mC cm(-2) for PPy nanotubes and 392 +/- 6.2 mC cm(-2) for PEDOT nanotubes) compared to their film counterparts. However, PEDOT nanotubes decrease the impedance of neural-electrode sites by about two orders of magnitude (bare iridium 468.8 +/- 13.3 kOmega at 1 kHz) and increase capacity of charge density by about three orders of magnitude (bare iridium 0.1 +/- 0.5 mC cm(-2)). During cyclic voltammetry measurements, both PPy and PEDOT nanotubes remain adherent on the surface of the silicon dioxide while PPy and PEDOT films delaminate. In experiments of primary neurons with conducting-polymer nanotubes, cultured dorsal root ganglion explants remain more intact and exhibit longer neurites (1400 +/- 95 microm for PPy nanotubes and 2100 +/- 150 microm for PEDOT nanotubes) than their film counterparts. These findings suggest that conducting-polymer nanotubes may improve the long-term function of neural microelectrodes.

Patterning N-type and S-type neuroblastoma cells with Pluronic F108 and ECM proteins.

Influencing cell shape using micropatterned substrates affects cell behaviors, such as proliferation and apoptosis. Cell shape may also affect these behaviors in human neuroblastoma (NBL) cancer, but to date, no substrate design has effectively patterned multiple clinically important human NBL lines. In this study, we investigated whether Pluronic F108 was an effective antiadhesive coating for human NBL cells and whether it would localize three NBL lines to adhesive regions of tissue culture plastic or collagen I on substrate patterns. The adhesion and patterning of an S-type line, SH-EP, and two N-type lines, SH-SY5Y and IMR-32, were tested. In adhesion assays, F108 deterred NBL adhesion equally as well as two antiadhesive organofunctional silanes and far better than bovine serum albumin. Patterned stripes of F108 restricted all three human NBL lines to adhesive stripes of tissue culture plastic. We then investigated four schemes of applying collagen and F108 to different regions of a substrate. Contact with collagen obliterates the ability of F108 to deter NBL adhesion, limiting how both materials can be applied to substrates to produce high fidelity NBL patterning. This patterned substrate design should facilitate investigations of the role of cell shape in NBL cell behavior.

The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons.

Aligned electrospun nanofibers direct neurite growth and may prove effective for repair throughout the nervous system. Applying nanofiber scaffolds to different nervous system regions will require prior in vitro testing of scaffold designs with specific neuronal and glial cell types. This would be best accomplished using primary neurons in serum-free media; however, such growth on nanofiber substrates has not yet been achieved. Here we report the development of poly(L-lactic acid) (PLLA) nanofiber substrates that support serum-free growth of primary motor and sensory neurons at low plating densities. In our study, we first compared materials used to anchor fibers to glass to keep cells submerged and maintain fiber alignment. We found that poly(lactic-co-glycolic acid) (PLGA) anchors fibers to glass and is less toxic to primary neurons than bandage and glue used in other studies. We then designed a substrate produced by electrospinning PLLA nanofibers directly on cover slips pre-coated with PLGA. This substrate retains fiber alignment even when the fiber bundle detaches from the cover slip and keeps cells in the same focal plane. To see if increasing wettability improves motor neuron survival, some fibers were plasma etched before cell plating. Survival on etched fibers was reduced at the lower plating density. Finally, the alignment of neurons grown on this substrate was equal to nanofiber alignment and surpassed the alignment of neurites from explants tested in a previous study. This substrate should facilitate investigating the behavior of many neuronal types on electrospun fibers in serum-free conditions.