Aligned Electros bun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. Here, electros BUN fiber scaffolds are generated and primary rat sensory and motor neurons are grown on them. First, fibrous scaffolds are electro spun onto glass cover slips.
Primary neurons are then obtained from embryonic day 15 rats and cultured on the electros spun fiber scaffolds. The neurons are allowed to adhere and grow then they are fixed and stained using immuno cyto chemical techniques. Imaging results reveal changes in the morphology of neurons cultured on electros spun fibers, indicating that the nano topography of the electros spun fibers can direct neurite outgrowth.
The main advantage of this technique over existing methods, such as the use of transformed cell lines, is that primary neurons and vitro more closely mimic the behavior of neurons and vivo then do transformed cells. These techniques offer viewers the ability to use electro fun fibers with other cell types. Alternatively, viewers may wish to use cultured motor and sensory neurons for other investigative purposes.
Generally, individuals new to this method will struggle because electro spinning is a finicky process, which we explain in more detail in our accompanying video article. To prepare electros fun fiber substrates begin by making an 8%solution of 85 to 15 polylactic co glycolic acid or PLGA in chloroform and a 4%poly L lactide or PLLA solution in chloroform by stirring over a low heat next coat clean glass cover slips in PLGA by covering the surface of each cover slip. With the PLGA solution, allow the PLGA to dry to a thin film, which takes approximately 30 minutes.
Once the PLGA is dry, use conductive carbon tape to secure A PLG. A coated cover slip to either a motor driven wheel for generating aligned fibers or a stationary plate for random fibers. Place a syringe containing the 4%PLLA solution in a pump with a tip, position 20 centimeters from the collector.
Set the pump to approximately 0.22 milliliters per hour. Set the wheel motor to 300 to 400 rotations per minute. Apply a minus two kilovolt DC bias to the collector and plus 15 kilovolts to the conductor plate, which is in contact with the metal tip.
In the absence of access to a bipolar power supply ground, the collector start the syringe pump fibers will jet from the syringe tip and collect on the rotating wheel. Continue spinning until the desired density of fibers is obtained to prevent clogging of the metal tip periodically swipe it with a paper towel affixed to a non-conductive rod following spinning pipetter line of PLGA around the perimeter of the cover slip. Essentially placing a moat around the electros.
Fun samples allow the PLGA to dry. This will maintain the alignment of the fibers during culture. The electro spun fibers and glass controls need to be coated in protein prior to cell culture.
To do this, cover the substrates in polyol, lysine, laminin, or fibronectin for at least one hour. Then aspirate the excess solution until the substrates are dry. If polyol lysine is used, the substrate should be rinsed in sterile water and dried twice following the initial coating.
Once they are dry, place the substrates in six well tissue culture plates for cell culture to prepare the cultured cells that will be grown on electros bun fibers. Obtain E 15 embryos from a euthanized pregnant rat. Begin by sterilizing the area with 70%ethanol and then tenting the skin of the abdomen.
Cut through the skin and muscle layers to expose the abdominal cavity. Locate the uterus and detach it at the cervix and ovaries. Transfer the uterus to a sterile dish.
Then remove the embryos from the uterus with scissors and place them immediately into warm L 15. After decapitating the embryos, place an embryo on its side while protecting the spinal cord with one set of forceps. Use the other set to strip away the limbs and abdominal organs.
Next, turn the embryo and hold it steady with one set of forceps. Sniff along the length of the embryo from the neck to the tail until the entire spinal cord is exposed. Grasp the end of the spinal cord carefully, then pull it over itself towards the tail.
To remove it, the cord will pull free with membrane and dorsal root ganglia attached. If a dorsal root ganglia x explan, or dissociated sensory neuron culture is to be performed, snip the dorsal root ganglia off the cord and transfer them to a fresh dish of warm L 15. For motor neuron culture.
Grasp the membrane from the spinal cord at the anterior end of the cord and peel it down. Similar to removing a long sock, repeat this process for all of the embryos and then using forceps. Chop the cords into small pieces to obtain motor neuron cultures.
Pour the chopped spinal cords and L 15 into a conical tube and spin it at 180 times gravity for two to three minutes. To pellet the pieces, remove the supinate and add three milliliters of trips into the pelleted cords. Place the tube in a 37 degrees Celsius water bath and incubate it for 20 minutes.
Following the incubation, add three milliliters of FBS to the conical tube. Then spin at 180 times gravity for two to three minutes to repel it. Meanwhile, COTA fire polished pasta pipette with FBS by drawing the FBS into the pipette and then expelling it.
This prevents bits of tissue from sticking to the glass during tri ation. After the spin, remove the supinate. Then add one pasta pipette full of L 15 to the conical tube triturate gently using the FBS coated pipette until the solution is homogenous with no visible clumps.
Avoid bubbles. Prepare one tube with three milliliters of 9%opre in L 15 for every two embryos. If there is an odd number of embryos, add one drip.
The homogenized solution on top of the opre solution dividing the homogenate evenly among all the tubes. The homogenate will form a visible cloudy layer on top of the Opti prep solution. Spin the tubes at 700 times gravity for 15 minutes.
Carefully collect the top two milliliters from each tube and pull in a 50 milliliter conical. Fill the conical with L 15 and spin at 180 times gravity for five minutes to rinse the cells of opre. Remove the supinate.
The pellet is rarely visible to the eye. At this stage, Reese, suspend the cells in a small amount of plating media. Count the cells using triam blue exclusion to identify live cells.
The cells are now ready for plating. A purity of greater than 90%neurons is expected to obtain sensory neuron cultures. Begin by pouring the dorsal root ganglia removed from the spinal cords and L 15 into a conical tube.
Spin up 180 times gravity for two to three minutes to pellet the pieces following the centrifugation. Pour off the supinate and add three milliliters of trips into the pelleted DRG incubate in a 37 degree Celsius water bath. After 10 minutes, remove the tube from the water bath and add three milliliters of FBS to the tube centrifuge at 180 times gravity for two to three minutes to repel it.
In the meantime, co a fire polished pasta pipette with FBS. As before, use the FBS coated pipette to add one pipette full of L 15 to the pellet and tri rate gently until the solution is homogenous with no visible clumps. Avoid bubbles after spinning the homogenate at 180 times gravity for five minutes.
Remove the supena and re suspend the cells in a small amount of sensory neuron plaing media. Count the cells using trian blue exclusion to identify live cells. The cells are now ready for plating to culture sensory, or motor neurons.
Begin by covering the protein coated electro bun fiber substrates with a few drops of medium. Then add an appropriate volume of cell suspension. Dissociated cells should be plated at a low density.
Allow the cells to adhere for at least one hour in the incubator before flooding the wells with medium and returning them to the incubator to culture. Dorsal root ganglia eggplants cover the protein coated electro spun fiber substrates with a few drops of media. Then add dorsal root ganglia to the medium.
Arrange the dorsal root ganglia so that they are evenly distributed across the substrate. Approximately four dorsal root ganglia will fit on a 22 by 22 millimeter cover slip. Place the dorsal root ganglia cultures in the incubator at 37 degrees Celsius and allow them to adhere for at least four hours following the incubation.
Flood the wells with media and return them to the incubator. Cultures can be maintained for up to four days without media change. For longer cultures, half media changes should be done with feed media.
Feed medium is the same composition as plating medium minus the L-glutamine. Once the cells have reached the desired time point, immuno cyto chemistry can be performed according to the instructions in the accompanying written protocol. This SEM image illustrates the typical morphology of aligned and random electros spun fibers.
The fiber alignment is manipulated by the choice of the collector. Note that fiber spun onto a rotating wheel collector are uniformly aligned. In contrast, fiber spun onto a stationary collector have a random morphology.
These images show motor neuron stain for beta tubulin shown in green and DPI nucleus staining in blue. After 24 hours of culture on PLL coated fibers and glass on fibers, motor neurons typically possess few processes which project from the cell body parallel to the direction of fiber alignment, which is left to right in this image. The major processes of cells cultured on fibers are much longer than the major processes of cells cultured on glass at equivalent time.
Points on glass motor neurons typically possess more processes at 24 hours, which extend radially from the cell body. The neurons cultured on fibers. The major process is also shorter on glass than those seen on fibers in these images.
Dorsal root ganglia are stained for neuro filament shown in green. After three days of culture on PLL coated glass and fibers on glass neurites emanate radially from the dorsal root ganglion. There is also significant overlap of adjacent neurites on fibers.
The neurites also emanate radially from the ganglion. However, they subsequently turn and follow the direction of the aligned fibers. There is significantly less overlap of adjacent neurites Once these procedures are mastered.
Electro spinning can be accomplished in approximately 30 minutes, and motor and sensory neurons can be obtained in approximately two hours. While attempting this procedure, it's important to remember to use sterile technique. The use of a fire polished pipette for the iteration step is necessary to maximize neuronal survival.
Additionally, it is important to incubate the adhesion molecule and the electro spun fibers for the appropriate amount of time. For example, if the fibers are coated with polylysine for less than one hour, there will be insufficient coating to support cell adhesion. However, if polylysine is left on too long, such as overnight, the fibers can degrade.
It is also important to remember that most microscopes will not allow the visualization of neurons on fibers during culture. This is because the fibers and neuronal processes look so much alike. After watching this video, you should have a good understanding of how to grow primary cultures of motor and sensory neurons from embryonic day 15 rats on electro spun fiber substrates.
Don't forget that working with high voltages can be extremely hazardous and appropriate. Precautions should always be taken to isolate the operator from the electric spinning setup.
