1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatric Research, Education and Clinical Center, Veterans Affairs Ann Arbor Health System
Leach, M. K., Feng, Z., Gertz, C. C., Tuck, S. J., Regan, T. M., Naim, Y., et al. The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds. J. Vis. Exp. (48), e2389, doi:10.3791/2389 (2011).
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.
1. Poly-L-lactic Acid (PLLA) Spinning Solution
2. Spinning Substrate Preparation1
5. Protein Coating
6. Obtain E15 Rat Embryos3
*All experiments were done in accordance with the NIH Guide for Care and Use of Laboratory Animals as approved by the University of Michigan Committee on Use and Care of Animals.
8. Motor Neuron (MN) Processing5,6,7
9. Sensory Neuron (SN) Processing7
10. Plating and Culture
12. Representative Results:
The typical morphology of aligned and random electrospun fibers are shown in Figure 1. We typically obtain MN purity of >90% neurons7,8 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. Immunostained DRG cultured for three days on glass and fibers are depicted in Figure 3.
Figure 1. Fiber alignment is manipulated by choice of collector. A.) Aligned fibers spun onto a rotating wheel collector and B.) random fibers spun onto a stationary plate collector. Scale bars = 20 μm.
Figure 2. Representative motor neurons stained for TuJ1 (green) and DAPI (blue) after 24 hrs of culture on PLL coated A.) fibers and B.) glass. Scale bars = 20 μm.
Figure 3. Representative DRG stained for neurofilament (green) after 3 days of culture on PLL coated A.) glass and B.) fibers. Scale bars = 200 μm.
|Stock solution:||MN media:||DRG/SN media:|
|10 mg mL-1 albumin||12.5 μl||--|
|10 mg mL-1 apo-transferrin||50 μl||40 μl|
|50 μg mL-1 β-estradiol||2.7 μl||2.2 μl|
|0.1 mg mL-1 biotin||50 μl||--|
|16 mg mL-1 catalase||8 μl||--|
|15 mg mL-1 D- galactose||50 μl||--|
|50 μg mL-1 hydrocortisone||3.7 μl||2.9 μl|
|0.63 mg mL-1 progesterone||0.5 μl||--|
|16 mg mL-1 putrescine||50 μl||--|
|50 μg mL-1 selenium||3.4 μl||4.1 μl|
|2.5 mg mL-1 superoxide dismutase||50 μl||--|
|* 500 ng mL-1 nerve growth factor||--||1 mL|
|* 100X PSN||0.5 mL||0.5 mL|
|* B27||1 mL||1 mL|
|* 2 mM L-glutamine||35 μl||35 μl|
|* Neurobasal||to 50 mL||to 50 mL|
Table 1. Add starred (*) components to media just prior to use. The remaining components can be prepared as a stock solution and kept at -20°C until needed6,7.
|anti-β-tubulin (TuJ1) (1:1000)||✓||X||X|
|anti-NGFR p75 (1:500)||✓||✓||X|
Table 2. The selection of primary antibody is dependent on the goals of the investigator. The above are several antibodies and the concentrations we have used with success in our laboratory. S-100 is particularly useful to identify Schwann cells and/or check for contaminating fibroblasts, but note that it must be used in combination with NGFR p75 to distinguish between glia and fibroblasts4. We have also noticed that NGFR p75 stains neurites very lightly while neurofilament or TuJ1 are excellent choices if the goal is to visualize individual neurites.
This protocol has several critical steps. The first involves the proper production of the electrospun fiber substrates. In liquid media, the PLLA fibers, PLGA film and PLGA moat will separate from the glass cover slip as a single unit. If the PLGA is omitted, the PLLA fibers will not remain a flat sheet - they will curl up into an unusable tangle. Thus, the PLGA film is included as a suitable substrate to maintain fiber alignment during culture. The moat is added after spinning both to ensure that the fibers are securely fixed to the PLGA film and to add structural rigidity to the film. The moat also serves as a useful handle when the substrates are manipulated during fixing, staining and mounting. Trituration is the second critical step. Every effort should be made to avoid the formation of bubbles. Additionally, we have obtained much higher yields when a fire-polished pipette is used for this step. To fire polish, light a Bunsen burner and attach a bulb to the pipette. Pass the tip of the pipette quickly through the flame 2-3 times while continuously squeezing and releasing the bulb - air flow through the pipette will help prevent the tip from closing completely. Examine the tip of the pipette to ensure the hole is still open and that the edges appear slightly rounded before use.
Electrospinning is a very versatile process; the electrospinning parameters can be modified to produce fibers with a variety of morphologies. Tan et al. (2005) details a systematic study of parameters affecting the diameter of PLLA electrospun fibers. Wang et al. (2009) presents a detailed study of the parameters influencing the alignment of electrospun PLLA fibers.
No conflicts of interest declared.
NIH K08 EB003996
|PLLA||Boehringer Ingeheim||Resomer L210|
|L15||GIBCO, by Life Technologies||11415|
|Biotin||Fisher Scientific||AC 23009-0010|
|Neurobasal||GIBCO, by Life Technologies||21103-049|
|PSN||GIBCO, by Life Technologies||15640|
|L-glutamine||Nalge Nunc international||1680149|
|Trypsin||Nalge Nunc international||1689149|
|Fetal bovine serum||GIBCO, by Life Technologies||26140|
|Optiprep||Accurate Chemical & Scientific Corporation||2011|
|B27||GIBCO, by Life Technologies||17504-044|
|Normal goat serum||Sigma-Aldrich||G6767|
|Carbon tape||Ted Pella, Inc.||13073-1|
|Prolong Gold +DAPI||Invitrogen||P36931|