Live Imaging Axon Stretch Growth Bioreactor

Live Imaging Axon Stretch Growth Bioreactor Joseph R. Loverde 1,2, Vivian C. Ozoka 1, Robert Aquino,1 Ling Lin 1, Bryan J. Pfister PhD 1 1Departments of Biomedical Engineering, New Jersey Institute of Technology and 2University of Medicine and Dentistry of New Jersey, Newark, New Jersey. Address for all authors: New Jersey Institute of Technology CHEN Building, Rm 301 111 Lock Street Newark, NJ 07103 973-596-3401 Joseph R. Loverde, MS Graduate Student, Biomedical Engineering Jrl3@ Vivian C. Ozoka, MS Graduate Student, Biomedical Engineering vo3@ Robert Aquino, BS Graduate Student, Biomedical Engineering rja5@ Ling Lin Undergraduate Student, Biology Department ll75@ *Corresponding Author Bryan J. Pfister, PhD Assistant Professor, Biomedical Engineering New Jersey Institute of Technology University Heights Newark, NJ 07102 pfister@ J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .Abstract Strategies for nervous system repair arise from knowledge of growth mechanisms via a growth cone. The distinctive process of axon stretch growth is a robust, long-term growth that may reveal new pathways to accelerate nerve repair. Here, a live imaging bioreactor was engineered to closely explore cellular events initiated by applied tension. The stretch growth potential between adult and embryonic DRG neurons was investigated; an important difference in nerve repair. Embryonic axons were capable of unidirectional stretch growth rates of 4mm/day and reliably reached 4cm in length within 2 weeks. Adult axons could only reach 2mm/day and took over 3 weeks to reach 4cm. Utilizing time-lapse imaging, we observed growth cone motility in coordination with stretch growth. Upon initiation of stretching, growth cones retracted. However, within 10 hours of continuous stretching, growth cones extended at a rate of 0.2mm/day opposite the direction of applied tension; contributing to overall axon elongation. We analyzed fast mitochondrial transport under increasing levels of strain to determine the effect of stretch on axonal transport. Transport began to diminish at 24% strain, and almost completely diminished at 39% strain. Surprisingly, axons recovered and were capable of subsequent stretch growth. When tension was completely released (-5% strain), stretch grown axons retracted at rates up to 6.1µm/s and slowed as resting tension was restored. This ability to assess the process of axon stretch growth in real time will allow detailed study of how tension can be used to drive axonal growth and retraction. Keywords: nerve growth; axon stretch; live imaging; bioreactor; development J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .Introduction A common goal for nervous system repair is to regenerate new axonal pathways to restore function (Schmidt and Leach 2003; Bareyre, Kerschensteiner et al. 2004). Regenerating axons appear to recapitulate a developing axon, extending via a growth cone. Accordingly, the development of repair strategies has extensively examined axon extension and guidance during development (Tessier-Lavigne and Goodman 1996; Hall 2001; Yu and Bargmann 2001; Dickson 2002). These are the principles that researchers and clinicians apply in efforts to regenerate and repair the adult nervous system (Hudson, Evans et al. 1999; Lee and Wolfe 2000; Bunge 2001; Evans 2001; Fry 2001; Hall 2001; McKerracher 2001; Fawcett 2002; Geller and Fawcett 2002; David and Lacroix 2003; Blight 2004; Pfister, Gordon et al. 2011). Research has explored the physical and cellular environment to enhance axon regeneration in animal models including: implanting peripheral nerve (David and Aguayo 1981; Houle and Tessler 2003; Houle, Tom et al. 2006; Tom and Houle 2008) or biomaterials as physical guides (Jain, Kim et al. 2006; Dodla and Bellamkonda 2008; Pfister, Gordon et al. 2011), support cell transplantation such as schwann or olfactory ensheathing cells, and therapies to counteract inhibitors of axon growth or enhance growth; for review (Anderson, Howland et al. 1995; Zompa, Cain et al. 1997; Stichel and Muller 1998; McDonald 1999; Bunge 2001; McKerracher 2001; Fawcett 2002; Hulsebosch 2002; Bunge and Pearse 2003; Pfister, Gordon et al. 2011); (Miya, Giszter et al. 1997; McDonald, Liu et al. 1999; Park, Liu et al. 1999; Imaizumi, Lankford et al. 2000; Ramon-Cueto, Cordero et al. 2000; Bareyre, Kerschensteiner et al. 2004). While efforts to enhance regeneration have made great strides, repairing peripheral and spinal cord injuries remain a formidable challenge. J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .Conventional knowledge on axon growth typically considers only the navigation of growth cones and formation of synaptic connections during early development and regeneration (Yu and Bargmann 2001; Dickson 2002). Indeed, developing animals continue to undergo substantial growth and nerves must grow in length to accommodate the increasing separation between target and the neuronal soma (Bray 1984; Heidemann, Lamoureux et al. 1995; Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004). For example, the giraffe’s neck increases by ~2cm/day at peak growth (Dagg and Foster 1982), suggesting that axons in the neck are forced to rapidly expand as well. This recently recognized process of Axon Stretch Growth (ASG) likely represents a secondary phase of rapid and long-term axon growth that drives the formation of long nerves. In adults, it is conceivable that the nervous system is forced to grow when tissue expands such as during pregnancy, extreme fluctuations in weight, or use of tissue expanders for plastic surgery (Swanson and Argenta 1988; Wood and McMahon 1989; Battiston, Buffoli et al. 1992; Iwahira and Maruyama 1993; Johnson, Lowe et al. 1993; Malis, MacMillan et al. 1995; Takei, Mills et al. 1998; Vekris, Bates et al. 1999; Elwood, Ingram et al. 2000; Hu, Tang et al. 2001; Kroeber, Diao et al. 2001; De Filippo and Atala 2002; Riordan, Budny et al. 2003; Zeng, Xu et al. 2003; Del Frari, Pulzl et al. 2004; Tang, Hu et al. 2004; Whitesides and Meyer 2004; Park, Kim et al. 2006). Our goal is to identify and explore this unique process in attempt to discover new mechanisms to enhance regeneration of axons following injury. Previously, we explored the application of stretch growth as a tissue engineering method to produce large living nerve constructs. Far exceeding the rate of growth cone extension, we found that stretch growth could sustain growth rates of 1cm/day for several days (Pfister, Iwata et al. 2004; Pfister, Iwata et al. 2006). These extreme stretch growth conditions stimulated expansion of the central portion of axon cylinders in caliber as well as length, while maintaining J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .a normal cytoskeletal ultrastructure and the ability to generate and convey action potentials (Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006). Indeed, stretch growth likely induces unknown cellular mechanisms to alter protein synthesis and transport to rapidly assemble the axon. The robust growth rates and lengths accommodated by ASG present an opportunity to discover new mechanisms to stimulate regeneration. Research from other groups also show that axons can grow under the application of mechanical force highlighting this is a distinctively different process from growth-cone growth (Heidemann, Lamoureux et al. 1995; O'Toole, Lamoureux et al. 2008; Lamoureux, Heidemann et al. 2010). As Biomedical Engineers, we dedicated our research efforts to develop the tools needed to investigate the processes that accommodate ASG. The original ASG bioreactor was developed to create copious amounts of nervous tissue for transplantation in animal injury models (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Iwata et al. 2006). This system only allowed examination of fixed time points, limiting analysis to absolute comparisons between differing preparations. To effectively study the biomechanical and biological aspects of stretch growth, the system was reengineered to accommodate real-time imaging and quantification techniques. Here we report new findings from long-term live microscopic monitoring of axons as they are stretch grown into long axon tracts. J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .Materials and Methods Principles of Operation The ASG bioreactor was engineered to gradually apply tension to axon bundles spanning two separate culture substrates (Pfister, Iwata et al. 2004; Pfister, Iwata et al. 2006; Loverde, Tolentino et al. 2011). To accomplish this, a flexible strip of fluoropolymer film (Aclar 33C film, Structure Probe Inc., West Chester, PA) was positioned over the bottom of the culture chamber. This ‘towing substrate’ underlaid the moving population of cells. The 'stationary substrate' was the bottom of the culture chamber. First, plated neurons were given time to extend axons from the towing substrate onto the stationary substrate via growth cone extension. The towing substrate was then pulled using a micromotion system, applying a gradual stretch to the spanning axons. Starting as a short distance between the soma and growth cones, axons were stretch-grown into long fasciculated unidirectional axon tracts several centimeters in length. Bioreactor Culture Chamber The bioreactor culture chamber was designed using Pro/ENGINEER software (PTC, Needham, MA, USA) to produce a 3D schematic for fabrication (supplemental figure 1). The major features were a compact size for use within the confines of a microscope stage, a coverslip stationary substrate that accommodated oil immersion objectives, an improved towing mechanism, and incubation controls, Figure 1. Structural components, (enclosure walls, stretching frame w/lanes, towing block, towing rod supports, luer lock mounts) were machined out of polyetheretherketone (PEEK) on a vertical milling machine (Bridgeport, Elmira, NY). Lids were machined out of transparent polycarbonate for viewing of cultures and microscopy. Fasteners and hardware (towing rods, guide rods, luer lock gas ports, screws & washers) were J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .purchased in 316 stainless steel (McMaster-Carr, Elmhurst, IL). These materials have proven both highly biocompatible and corrosion resistant, while autoclavable for sterilization, see materials Table I. The culture chamber stretching frame consisted of three independent lanes (elongated wells) each measuring 1 x 8cm that allowed for parallel experiments with a controlled variable. A glass coverslip measuring 4.8 x 6.5cm with #1 thickness (#4865-1, Brain Research Labs, Newton, MA) was glued to the bottom of the stretching frame for each experiment. The coverslip served as the stationary substrate for the attachment of growth cones and as a window to permit oil immersion microscopy, Figure 2. Here, the design of the towing apparatus has been improved over previous systems. The Aclar towing substrates were glued to arched legs, which extended down and into each culture lane; rigidly holding the substrates. This design eliminated slack in the towing substrates, and ensured that each manipulation of the towing block positively engaged the axons, Figure 2A, part vii. Adjustment screws allowed the legs to be independently lowered to contact the coverslip bottom. Conducting sustained, live imaging during stretch growth required the bioreactor to double as an incubator. Temperature was maintained using a closed system of heating elements, thermistors and a temperature controller, as listed in Table II. A heated lid was fabricated from a polycarbonate frame with an inset Indium Tin Oxide (ITO) coated glass slide, Figure 2A. The base of the culture chamber consisted of a stainless steel heat sink with a flexible silicone heating element to deliver uniform heating across the culture lanes, Figure 2B. Both heating elements were controlled by a two-channel temperature controller with thermistors for temperature feedback. J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .Efficient pH buffering of culture media was maintained by continuous perfusion of premixed air from a tissue culture incubator. Using an aquarium air pump, air was passed from the incubator through 1/8" ID (3.175mm) PVC tubing to the culture chamber. An inline aquarium regulator valve was used to control flow rate, while a 0.2µm syringe filter was used to maintain air sterility. Luer lock fittings were used for quick attachment of tubing to the culture chamber. For humidification, air was routed from the inlet through liquid reservoirs built into the hollowed walls of the culture chamber. Air was returned to the incubator through an outlet located opposite the inlet, Figure 1 & 3C. Bioreactor Preparation The towing culture substrates were made from 0.002” (50.8µm) Aclar film (Table I) cut into thin strips measuring roughly 25mm x 5mm using a scalpel. To minimize thickness further, the 5mm edge of the substrates was gently sanded with 1200-grit sandpaper. The coverslip and Aclar were washed with laboratory detergent, rinsed excessively with deionized water and sterilized in 70% ethanol. The bioreactor culture chamber was autoclave sterilized separately and dried inside a tissue culture hood. All subsequent assembly was performed inside the hood using aseptic technique. First, the towing substrates were attached starting with adjustment of the legs fully retracted away from the bottom of the chamber. The back of the towing legs were coated with Silicone RTV (Dow Corning #732, McMaster-Carr) using sterile cotton-tipped swabs. The Aclar strips were applied oriented with the sanded edges facing the lid, Figure 2B. Next, the bottom of the chamber was lightly coated with silicone glue and the coverslip was attached, Figure 2C. Using a swab, excess glue and air pockets were removed by pressing against the J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .bottom of the coverslip. To allow for complete curing of the glue, the culture chamber was dried in the hood for 2 days. Prior to cell plating, the towing substrates were lowered into the chamber to make contact with the coverslip. Optimal overlap was determined to be approximately 2-3mm as measured from the tip of the Aclar. Each lane was coated at the substrate interface with 1mL of 10µg/mL high molecular weight Poly-D-Lysine (PDL) (#354210, BD Biosciences, Bedford, MA) in serum free media for 1 hour. The lanes were drained and rinsed gently with deionized water and a final rinse with culture media before plating. Neuronal Culture Experiments were performed using dorsal root ganglia (DRG) neurons isolated from embryonic day 15 rat pups. Alternatively, adult DRGs were obtained from rats of 16 to 20 weeks age. 5-10 embryonic DRG explants or 25,000 dissociated adult DRGs were plated directly onto the towing substrates within ~500µm of the edge using a stereo microscope, Figure 2D. A ‘puddle’ of approximately 100-500µl media was formed during plating in which cells were allowed to adhere for 1-2 hours before filling the lanes with growth medium (Neuralbasal w/B-27, 0.5mM L-Glut., 1% heat inactivated FBS, 20% glucose, 20ng/ml NGF, 20uM FdU + 20 uM Uridine). Media changes were done prior to stretch growth in order to avoid disturbance of actively stretch-growing axons. All animal protocols have been approved by the Rutgers University IACUC. J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .Axon Stretch Growth The bioreactor culture chamber was docked to a computer-controlled micromotion system consisting of a linear motion table and microstepper motor controlled by a programmable motor indexer (table II). A delrin chassis was fabricated to position the culture chamber and linear motion table on the stage of the microscope such that they do not move during experimentation, Figure 1. The linear motion table was attached to the chassis using an Acrylonitrile Butadiene Styrene (ABS) plastic mount printed on a rapid prototype, 3-Dimensional printer (SST 1200es, Dimension, Inc., Eden Prairie, MN). The bioreactor was seated within a square cutout section of the chassis, allowing the microscope objective to contact the viewing window. The towing rods of the bioreactor were fastened to the linear motion table by an ABS adapter printed on the 3D printer. When axons extended onto the coverslip by at least 1mm, typically 5 days, stretch was applied by towing neuronal soma away from the growth cones by taking a series of short 2µm steps. The stretching motion was programmed with motion control software (Si Programmer, Applied Motion Products, Watsonville, CA) that sets the following parameters: 1) number of motor steps (size of stretch steps), 2) interval between steps (stretch frequency), and 3) number of iterations (total stretch length). Stretch growth of embryonic axons was initiated at a net 1mm/day by taking 2µm steps every 172 seconds over 500 iterations, Table III. Since stretch induced growth is strain limited, the stretch rate begun slowly and gradually accelerated to the desired rate (Pfister, Iwata et al. 2004; Pfister, Iwata et al. 2006). J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .Live Imaging A major obstacle to imaging was the floatation of stretch grown axons above the working distance of high magnification objectives. Stretch grown axons were only adhered to substrates at the proximal and distal ends while the central portions did not form substrate adhesions during stretch growth. Accordingly, we developed a depressor tool to constrain floating axons within working distance of high magnification objectives during microscopy, supplemental figure 2. The depressor base was machined from PEEK with a height adjustable perch to which an Aclar "finger" was glued. The Aclar was folded to extend from the base and acts as a mild spring. The tip of the Aclar finger was placed on top of the imaged axon segment and the height of the Aclar was adjusted vertically by a thumbscrew to gently bring floating axons to within focal distance. For phase contrast imaging, the bioreactor was seated to the stretch growth dock on a Nikon TE2000-S inverted microscope. To collect long duration time-lapse sequences, a 4x objective was used with a 0.63x camera lens to increase the field of view to ~3.5mm. Time-lapse software (Q-Imaging, Q-Capture Pro, Surrey, BC, CA) was used to create time-lapse sequences and was set to acquire one image every 7.5 minutes over 48-hour periods. For fluorescent imaging, high magnification images were taken to observe intra-axonal transport during and following stretch growth. Mitochondria were stained with MitoTracker Red (M7512, Invitrogen, Carlsbad, CA) and visualized within stretch grown axons using confocal microscopy. Briefly, cultures were stained for 1 minute in 100 nM MitoTracker Red and rinsed three times with Phosphate Buffered Saline. After incubation for 60 minutes, mitochondria were visualized on a Nikon TE2000-E confocal microscope using a 60x oil immersion objective with 561nm laser excitation. Select planes were imaged for 10-20 minute time-lapse sequences at a rate of 0.5Hz to analyze mitochondrial movement. Laser power was set to just 0.5% in order to J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .avoid photo bleaching and heating damage to axons over time. Pixel dwell was further reduced to ~6µs in order to limit exposure of the axons to the laser. It was found that 1.5 seconds of scanning with a delay of 0.5 seconds produced the best results, enabling time-lapse of select axon segments at 0.5Hz for at least 1 hour. While these settings did not provide detailed morphology of mitochondria, they were a necessary trade-off in order to record sustained axonal transport (Miller and Sheetz 2006). Time-lapse sequences (stacks) were imported into ImageJ (National Institutes of Health, Bethesda, MD) where bundles were cropped and rotated to span horizontally (transform/rotate, no interpolation). Kymographs were created by reslicing the axon bundle (stacks/reslice, avoid interpolation, 1.0 spacing) to produce a stack of graphs to measure fast axonal transport, Figure 9B. Fast transport was identified by faint diagonal lines, while docked or slow transport was identified by vertical lines. To measure fast transport, diagonal lines were traced in order to find the slope (velocity & direction) and length (distance) of moving mitochondria. All data was exported to Excel (Microsoft, Redmond, WA) for subsequent analysis. J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m t h i s p r o o f .Results Optimization of Incubation for Live Imaging Conducting prolonged live imaging of ASG required accurate incubation control for optimal results. To control temperature outside the incubator and prevent condensation on the lid (which distorted transmitted light), the device was heated from the top and bottom. The media temperature was maintained within 35-37°C by placing the heat block and heated lid on separate controller channels with independent feedback control. To prevent large fluctuations in temperature, the position of the temperature probes for feedback was important. For the heat block, a temperature probe (thermistor #1, table II) was optimally positioned within the culture media at the plating area of the culture lanes (within any of the 3 lanes). For the heated lid, a temperature probe (thermistor #2, table II) was positioned on top of the insulating lid, adjacent to the heating element and leads, Figure 1. Final temperature controller settings for the heat block/lid were 33°/30°C, bandwidth 11/13, and gain of 50/60, respectively. To maintain culture media at a physiological pH of 7.4, mixed air containing 6.5% CO 2 was pumped into the bioreactor from a tissue culture incubator. Airflow was adjusted until faint bubbling could be seen within the liquid reservoirs. pH was maintained throughout all experiments by inspection of Phenol Red and occasional measurement. If, however, airflow was set incorrectly, the culture media either evaporated or turned basic due to excessive or insufficient flow, respectively. Liquid reservoirs typically lasted 1-2 weeks before drying when airflow was set correctly. J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o mUnidirectional Axon Stretch Growth Previous ASG experiments separated two populations of interconnected neurons located on the stationary and towing substrates, resulting in bi-directional axon polarity, Figure 4A. Here, axons were stretch grown unidirectionally with active growth cones at one end, Figures 4B. Although neuronal soma could be plated on either the towing or stationary substrates, optimal imaging of the distal axon and growth cones was achieved by plating soma on the towing substrates, Figure 5. However, despite precise plating, a small number of cells frequently migrated onto the stationary substrate, (supplemental figure 3). While some cells migrated along the substrates directly, others eventually migrated along the axons spanning the substrates. To achieve unidirectional polarity of dissociated cells, an extra step was required during plating to temporarily contain suspended cells on the towing substrates. For each lane, a 100µL pipet tip was cut in half approximately 2-3mm from the widest end, and placed on the towing substrate to serve as a well in which to plate cells. Axons from embryonic DRG explants were stretch grown unidirectionally for up to 2 weeks at rates up to 6mm/day. Stretch growth was initiated at 1mm/day and increased by 1mm/day every 24 hours according to the rate escalation schedule in table III. It was found that axons could routinely grow without disconnection at stretch rates up to 4mm/day over a 5 day escalation schedule (pre-stretch, 1, 2, 3, 4mm/day). Consistently, stretch growth could be sustained at 4mm/day until the system ran out of travel (over 1 week producing >4cm long axons). If, however, the stretch rate was increased on day 6 to 5mm/day, axons would start to disconnect within 24-48 hours. Since stretch rates above 4mm/day could not be sustained, 4mm/day was considered to be the maximum growth rate. J o u r n a l o f N e u r o t r a u m aL i v e I m a g i n g A x o n S t r e t c h -G r o w t h B i o r e a c t o r (d o i : 10.1089/n e u .2010.1598)l e h a s b e e n p e e r -r e v i e w e d a n d a c c e p t e d f o r p u b l i c a t i o n , b u t h a s y e t t o u n d e r g o c o p y e d i t i n g a n d p r o o f c o r r e c t i o n . T h e f i n a l p u b l i s h e d v e r s i o n m a y d i f f e r f r o m。