The overall goal of the following experiment is to apply tensile force to the axonal process of a neuron in order to stimulate and sustain its growth. This is achieved by plating neurons within a computer, controlled by a reactor upon a micro manipulated towing substrate. After plating, neurons are cultured up to one week during which axons extend onto a stationary substrate via growth cone extension.
Next Axon stretch growth is performed by towing the plated cell bodies away from the axon terminals adhere to the stationary substrate. The tissue engineering technique of Axon stretch growth is capable of sustained growth rates up to 10 millimeters per day, resulting in long vesicular bundles of enlarged axon tracts based on microscopic analysis. The main advantage of this technique over other methods like growth current extension, is that axon stretch growth allows to experiment to grow a axon tracks to any desirable length in shorter time periods.
The implications of axon stretch growth or that allows us to recreate the biomechanical signals that stimulate nerve growth. These signals can be studied in order to exploit them and develop new strategies to treat diseased or injured nerves. So the Axon stretch growth method can help answer key fundamental questions about the development and maintenance of the nervous system.
Importantly, we can explore the mechanisms that allow axons to grow at rates faster than we thought possible. The Axon stretch growth bioreactor system has three main components. First, a bioreactor chamber when neurons are cultured and stretched.
Second, an automated linear motion table, which applies stretching forces. And lastly, a step motor drive controller with software to control stretch growth. Here the protocol is demonstrated using a miniaturized system that has the capability to operate under a microscope.
Fabrication of bioreactor prototypes was done using a vertical milling machine for biocompatibility and ease of sterilization culture. Chamber components were machined out of Polyether ether ketone corrosion resistant three 16 stainless steel fasteners were used to complete the assembly. The bioreactor culture chamber contains a stretching frame, an adjustable towing block, towing rods for external manipulation and disposable towing and stationary culture substrates.
The automated linear motion table consists of a linear motion table with a stepper motor mounted to a delrin table. A software program called P si programmer is used to program and monitor the drive controller that interfaces with the stepper motor. Make towing culture substrates by cutting LAR film into thin strips, roughly 0.5 by 2.5 centimeters.
Lightly sand the lower one third of the towing culture substrates on both sides, using fine 1, 200 grit sand paper to facilitate the growth of axons from the towing substrates onto the stationary substrate. Use of a divider tab helps to sand more evenly. Cut a five by seven centimeter piece of LAR or use a number one glass cover slip to serve as the stationary substrate.
Clean the culture substrates and the bioreactor chamber with a dilute Alcan solution and rinse thoroughly with deionized water. Sterilize the culture substrates by immersion in 70%ethanol for 30 minutes. Autoclave sterilize the bioreactor chamber within an autoclavable container.
After sterilization, transfer the culture substrates and bioreactor chamber to a tissue culture hood and allow to air dry. A box of pipette tips can be used for drying of the culture substrates. Insert the towing culture substrates using sterile cotton tip swabs.
Glue the towing culture substrates to the towing block legs. With a thin layer of silicon RTV, the substrate should be glued at the non sanded portion while contact with the sanded culture surface should be avoided. Next, glue the stationary culture substrate to the bottom of the bioreactor chamber.
Remove excess glue and air pockets by gently depressing a dry swab against the bottom of the stationary substrate. Let the chamber dry for two full days inside the tissue culture hood. After two days, adjust the towing block legs to achieve a two to three millimeter overlap between the tips of the standard towing substrates and the stationary substrate.
Crucial attention must be paid to the size of the overlap. If it is too large, the tips of the towing substrates will deflect off of the stationary substrate and reduce the number of axons that span the overlap. Position the towing block at the starting position with the towing rods retracted inside the bioreactor.
Tighten the immobilization screws to prevent movement of the towing rods. Prior to stretch growth working in a tissue culture hood pulled one milliliter of high molecular weight poly de lycine in serum free media at the substrate interface of each lane. Allow the solution to sit for one hour at room temperature after an hour rinse three times with deionized water, followed by a final rinse with culture media plate dorsal root ganglion explan from E 16 rat pups onto the sanded towing culture substrates.
Using a stereo microscope first dilute the explan in drops using petri dishes, and then collect three to four eggplants using a 100 microliter pipette plate. X plants at the edge of the towing substrate in a small puddle of media up to one milliliter of culture. Media is sufficient to prevent evaporation for several hours while limiting movement of the eggplants.
Fasten the lid of the bioreactor and transfer to an incubator for one hour to allow eggplants to a adhere. After one hour, fill the bioreactor with culture media at the point farthest from the eggplants to avoid dislodging them. Incubate the bioreactor for five days to allow DRG neurons to extend axonal processes onto the stationary substrate.
Within the tissue culture hood, fill the reservoirs of the bioreactor with phosphate buffered saline to humidify the chamber and limited aberration of media. Perform a final media change filling the lanes of the bioreactor to capacity seat. The bioreactor within the automated linear motion table in a non humidified incubator.
Fasten the towing rod adapter to the towing rods. Using Cy programmer software. Jog the stage at the linear motion table to align with the towing rod adapter and fasten it to the stage for convenience.
Prepare s programmer sequences in advance in order to manipulate the stage in specific increments. Prior to starting the Axon Stretch growth program, loosen the immobilization screws on the bioreactor chamber. Stretches applied in the stepwise fashion by taking a series of small displacement steps separated by resting periods.
This paradigm starts by taking two micron steps every 172 seconds resulting in a net one millimeter stretch over the first 24 hours following pretension. After one day, the stretch rate can be accelerated by increasing the displacement step size, or decreasing the resting time. Representative results of the axon stretch growth process are shown here.
First cells within the plated implants migrate onto the towing substrate. Over the next five days, growth cones extend axonal processes onto the stationary substrate. Once growth cones have extended by at least one millimeter across the stationary substrate, axon stretch growth is begun.
The towing substrate is manipulated away from the AED growth cones In a series of steps, the elasticity of the spanning axons is immediately apparent as they are stretched. Typically, axons initially thin, but within hours enter a growth phase in which they recover and maintain their diameters. Within 24 hours of continuous stretch growth, axons show increased tolerance to stretch and are able to sustain increments in the stretch rate.
As the axons grow even longer and FAS circulate, they demonstrate increasing tolerance to stretching. Here the flotation of stretch grown axons is apparent as the bioreactor is intentionally bumped by hand. Occasionally, axons may disconnect from the stationary substrate due to insufficient adhesion.
This is the most common failure and is most likely to occur if the initial step size is too large or the stationary substrate was inadequately coated. Here a portion of axons disconnect from the stationary substrate upon initiation of stretch growth due to too large of a step size. While carrying out this procedure, it is important to take your time preparing the substrate interface.
The toe and substrate should overlap the stationary substrate by two to three millimeters, and DRG should be plated as close to the interface as possible While stretch growth is occurring. You'll have a unique opportunity to study Axon growth using live fluorescent probes or by lysing the cells as they are stretching in order to study gene expression. After watching this video, you should have a good understanding of how to use applied tension to drive Axon growth.
Using this technique, we are discovering that axons have an amazing capacity to grow faster than we thought possible, and to links that cannot be achieved by any other means.
