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. 2008 Jul;4(4):863-75.
doi: 10.1016/j.actbio.2008.02.020. Epub 2008 Mar 12.

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

Joseph M Corey  1 , Caitlyn C Gertz, Bor-Shuen Wang, Lisa K Birrell, Sara L Johnson, David C Martin, Eva L Feldman
Affiliations

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

Joseph M Corey et al. Acta Biomater. 2008 Jul.

Abstract

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.

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Figures

Fig. 1
Fig. 1
Photograph of electrospinning apparatus. All components are pictured in A, including a DC power supply (1), syringe pump (2), syringe filled with PLLA solution and connected to an electrode (3), wheel with substrates attached (4) and a motor that rotates the wheel (5). A close-up of the wheel is pictured in B showing the grounding electrode (1), glass cover slips attached to the wheel with masking tape (2) and a PLGA stripe painted on to each cover slip (3).
Fig. 2
Fig. 2
Identification of E15 rat spinal motor and sensory neurons by immunostaining. In order to confirm the identity of cultured cells, motor neurons were labeled for ChAT (A) and sensory neurons for CGRP (B). Cells in the motor neuron culture stained positively for ChAT, which showed punctate expression in both the cell body and neurites. Neurons from dissociated DRG stained positively for CGRP, expressed in both cell body and neurites, confirming their identity as sensory neurons. All cells were stained with DAPI (blue). Scale bar = 25 μm.
Fig. 3
Fig. 3
Survival of E15 motor neurons and sensory neurons on candidate materials used to fasten aligned nanofibers to cover slips. Cells were plated at 50 mm−2. Live neurons were stained with fluorescein diacetate (FD) and dead neurons with propidium iodide (PI). Survival was calculated as the number of live cells divided by total cells in a field. Each field was then normalized to the mean percent survival on glass controls. (A) After 4 DIV, motor neuron survival on bandage was decreased by 39% compared to glass controls. Survival on PLGA was similar to glass. (B) Example of FD–PI staining of motor neurons on PLGA 75:25. (C) After 5 DIV, sensory neuron survival was decreased by 93% on silicone glue and 28% on bandage compared to glass controls. Survival on PLGA was similar to glass. (D) Example of FD–PI staining of sensory neurons on PLGA 85:15. Data shown are averages of four trials for motor neurons and three trials for sensory neurons. *p < 0.05 and ***p < 0.001; error bars = standard error of the mean. Scale bar in (B) and (D) = 50 μm.
Fig. 4
Fig. 4
Candidate substrate designs for aligned electrospun PLLA nanofibers. Photos (A, C, E) and SEM images (B, D, F) of nanofibers spun onto glass cover slips. (A, B) Fibers were directly electrospun on cover slips attached to the rotating target wheel. No glue was used to fasten them to the cover slip. (C, D) Fibers were electrospun on the rotating target wheel, removed, and glued to the cover slip at the ends of the fiber bundle with PLGA (85:15). (E, F) Cover slips were painted with a stripe of PLGA down the center and taped to the wheel, and PLLA fibers were electrospun while the PLGA was still wet. Additionally, a “moat” was created in this preparation by painting a wall of PLGA on the perimeter of the cover slip to hold the cell suspension at the time of plating. SEM images reveal that fiber alignment is equal among each substrate design. Cover slips are 22 × 22 mm2, scale bar = 20 μm.
Fig. 5
Fig. 5
Preservation of fiber alignment after submersion. Substrates were immersed in PBS until fiber bundles floated off the underlying cover slips. (A) A fiber bundle that had been electrospun directly onto a cover slip without a fastener detached and floated in the media. The flat shape of the bundle was lost and the bundle twisted onto itself. (B) SEM photo of the same sample shows a small portion of the extensive loss of fiber alignment. (C) Fibers that are electrospun on wet PLGA retain their macroscopic architecture even after detachment, allowing handling throughout cell culture, fixation and immunostaining. (D) Alignment of the fibers is retained on the microscopic level, as seen in the SEM image. Scale bar = 20 μm.
Fig. 6
Fig. 6
Primary motor and sensory neurons (E15) grown on electrospun PLLA nanofibers in serum-free media. Nanofibers were coated with polylysine for motor neurons and collagen I for sensory neurons. In these images, fibers are aligned from left to right. Neurons are stained for neurofilament at 4 DIV (green). (A) Motor neuron processes are elongated and oriented parallel to the fiber bundle. Neurites are not perfectly aligned, as can be seen at the axonal branching point (arrow). (B) A higher magnification of a single motor neuron shows the alignment of the major neurite (arrow) and dendrites (arrowhead). (C) Similar to motor neurons, the long processes of sensory neurons are highly aligned and oriented parallel to the fibers. (D) Other cell types in the culture reveal similar alignment. Schwann cells labeled with S-100 (red) were seen closely associated with sensory neurites (arrowhead) and between sensory neuron processes (arrow). All cells were stained with DAPI (blue). Scale bar = 50 μm.
Fig. 7
Fig. 7
Motor neuron survival as a function of surface wettability. Some samples were plasma treated to increase surface wettability. On polylysine-coated fibers and films cells were plated at a density of 50 or 100 mm−2 and stained with fluorescein diacetate and propidium iodide at 4 DIV. Survival was calculated as the number of live cells divided by total cells in a field. Each field was then normalized to the mean percent survival on glass controls. Motor neuron survival rates were similar on glass, PLLA films and untreated PLLA fibers. Motor neuron survival was significantly decreased on plasma treated fibers at a cell density of 50 mm−2. This decrease was not observed at a density of 100 cells mm−2. Values are the average of three trials. *p < 0.05 and ***p < 0.001; error bars = standard error of the mean.
Fig. 8
Fig. 8
Quantification of fiber and neurite alignment. Representative images of fibers (A), motor neurons on fibers (B), motor neurons on flat surfaces (C), sensory neurons on fibers (D) and sensory neurons on flat surfaces (E) are shown alongside their corresponding FFT images. FFT images were produced by selecting representative squares 256 pixels on a side in SEM images and 512 pixels on a side in photomicrographs. FFTs were generated from the outlined regions as shown. Yellow to orange depicts high intensity of signal and magenta to blue depicts low intensity. The FWHM, a measure of the shape of the FFT image, was calculated using a MATLAB script for each sample. (F) Graph of the FWHM of fibers and both types of neurons grown on both fiber and planar substrates. Smaller FWHM values result from narrower FFT images and correspond to more highly aligned features in the spatial image. Fiber alignment and neurite alignment of both cell types on fibers were not statistically different from one another. Both motor and sensory neurons grown on fibers were more highly aligned compared to when grown on planar surfaces. Data shown as mean + standard error of the mean (n ranges from 19 to 41). ***p < 0.001, scale bar in A = 20 μm; scale bars in B–E = 100 μm.
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