A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.
During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.
1. Overview of The Axon Stretch Growth Bioreactor System
2. Preparation of Bioreactor Chamber
3. Neuronal Cultures
4. Axon Stretch Growth
5. Representative Results:
Axonal processes can undergo remarkably rapid and robust stretch growth. Initially, the process begins with a period of slow stretching (≤ 1mm/day) that consists of small, infrequent displacements. Within the first 24 hours of stretching, mechanotransduction of neuronal growth pathways occurs, whereby neurons begin addition to the axon cylinder. Within 24 hours of continuous ASG, axons show an increasing tolerance to greater and more frequent displacements. In general, axons can withstand an increase in the stretch rate of 1mm/day every 12-24 hours (Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006). Increasing the stretch rate too soon, however, may lead to more rapid growth of select axons but will also lead to pathological occlusion that causes disconnection.
Stretch-growing axons have the tendency to form bundles, resembling the architecture of fascicles. Utilizing current protocols, the central, stretch grown portion of axon bundles have no adhesions to the culture substrate. Only the initially adhered, proximal and distal segments of stretch-growing axons remain attached to the culture substrates. Accordingly, the central portion of stretch grown axons float freely, and are sensitive to disruption due to handling.
For a variety of reasons, some axons cannot grow at the applied stretch rate. For example, a DRG neuron with two axonal processes, both of which are undergoing stretch, may not be able to translate sufficient protein and grow at the applied stretch rate. Axons that cannot accommodate the applied stretch will thin following Poisson’s effect. Subsequent stretch will lead to occlusion of the axons, inhibiting assembly, leading to pathological disconnection. The majority of axons, however, are able to undergo ASG successfully and only a small percentage of axons undergo this pruning-like process.
Figure 1. Axon Stretch Growth Bioreactor System. (A) Bioreactor culture chamber & Automated linear motion table, (B) Step motor drive controller and Si Programming software.
Figure 2. Bioreactor Culture Chamber. This cartoon depicts the bioreactor chamber from the top with the lid removed. The position of the towing block reflects the end stage of axon stretch growth. Stretch grown axons can be seen in bundles within the culture lanes.
Figure 3. Culture Substrate Plating Interface. This cartoon depicts the components of the towing mechanism within each lane of the bioreactor from side view. (Top) Correct overlap of the towing and stationary culture substrates. (Bottom) Excessive overlap of the towing and stationary substrates causes the tip of the towing substrate to curl.
Movie 1. Attachment of Culture Substrates to Bioreactor Chamber. Click here to watch video
Movie 2. Plating of DRG Explants onto Towing Substrates. Click here to watch video
Movie 3. SiProgrammer Usage. Click here to watch video
Movie 4. Growth Cone Extension onto Stationary Substrate. Click here to watch video
Movie 5. Axon Stretch Growth. Click here to watch video
Day | Step |
1 | Sterilization & Drying |
2 | Gluing & Assembly |
4 | Coatings & Neuronal Culture Plating |
9 | Stretch Growth Start |
Table 1. Experiment Schedule.
Time [hr] | Stretch Rate [mm/day] | Dwell Time [s] | Total Length [mm] | Total Stretch Time [days] | |
Pretension | 24 | 1 | 172.8 | 0 | 1 |
Stretch | 24 | 1 | 172.8 | 1 | 2 |
Stretch | 24 | 2 | 86.4 | 3 | 3 |
Stretch | 24 | 3 | 57.6 | 6 | 4 |
Stretch | 24 | 4 | 43.2 | 10 | 5 |
Stretch | 24 | 5 | 34.6 | 15 | 6 |
Table 2. Stretch Rate Schedule. All stretch steps are 2 μm in displacement (10 step motor steps = 2 μm stretch).
Two critical steps should be observed during preparation of bioreactors. First, an optimal overlap at the substrate interface is needed to ensure that axons can cross onto the stationary substrate. Aclar that is excessively curled or otherwise imperfect should not be used (figure 3). To optimize the overlap, confirm that the towing substrate is sanded evenly and contacts the stationary substrate uniformly over a 2-3mm long contact patch. The overlap should be optimized prior to each experiment by carefully adjusting the height of the towing legs.
Second, while providing for attachment of neurons, substrate coatings sustain considerable sheering forces caused by displacement of the bioreactor and contractile tension of the axons (Heidemann and Buxbaum 1990; Pfister, Iwata et al. 2004; Loverde, Ozoka et al. 2011). Substrates should be rinsed thoroughly with sterilized water both prior to and after lysine coating. Coatings should be applied from freshly thawed aliquots and spread as evenly as possible. Importantly, the substrates should not be moved or otherwise disturbed during the adhesion period. In subsequent steps, avoid contact with the substrates during plating, and pipet all solutions from the far end of the lanes away from the substrate interface.
Tolerances in the connections of each bioreactor component can manifest in the form of slack. During the initial period of ASG, slack in the system is evident as movement of the automated linear motion table occurs without movement of the towing block. Slack can vary per experiment, but is typically <1mm in our experience. For this reason, a “pre-tension” slack elimination phase of 1mm/day, for one day, precedes the ASG schedule during which the bioreactor parts engage and begin movement of the towing substrates.
Troubleshooting may be required if the displacement step of the automated linear motion table does not match the displacement of the towing block after the pre-tension phase is complete. Asynchronous, inaccurate displacements of the towing block are associated with ‘stiction’ or static friction within the towing hardware and flexing of the adaptor. To prevent these issues from occurring, movement of the towing block should be checked by hand, after assembly, for smooth near-effortless movement. If binding occurs, the towing assembly should be freed prior to experimentation. Sufficient stiffness of the adaptor is also necessary to assure accurate, synchronous displacements are not overcome by stiction of the towing hardware.
The authors have nothing to disclose.
This work was funded by NSF CAREER CBET-0747615. The authors would like to thank Drs. Douglas H. Smith and David F. Meaney for their mentorship and support.
Name of component | Company | Catalogue number | Comments |
PolyEtherEtherKetone (PEEK) | McMaster-Carr, Elmhurst, IL | 8504K69 | Custom made bioreactor chamber |
Polycarbonate | McMaster-Carr, Elmhurst, IL | 8574K28 | Custom made bioreactor chamber lid |
Stepper motor | Applied Motion Products, Watsonville, CA | HT23-397 | |
Linear motion table | Servo Systems, Montville, NJ | MIPS-2-10-1.0mm | |
Step motor drive controller | Applied Motion Products, Watsonville, CA | Si2035 | |
Aclar | Structure Probe Inc., West Chester, PA | 1859 | Towing culture substrates |
No. 1 coverslip | Brain Research Labs, Newton, MA | 4865-1 | Stationary culture substrate |
Silicon RTV | McMaster-Carr, Elmhurst, IL | 7587A37 | |
Cotton-tipped swabs | McMaster-Carr, Elmhurst, IL | 7074T62 | |
Poly-D-Lysine | BD, Bedford, MA | 354210 |