Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

The overall goal of the following experiment is to study functional regeneration of intraspinal connections in organotypic spinal cord co cultures using multi electrode arrays, so-called meas. This is achieved by culturing two transversal spinal cord slices next to each other on meas to mimic intraspinal connections between different spinal cord segments. In vitro the slices grow and within a few days in vitro fuse along the sides facing each other.

As a second step. Lesions are performed after different timeframes in culture, which leads to a complete separation of the slices. Next, the cultures are left in the incubator for at least two weeks.

In order to give the spinal networks time to regenerate the activity burst between the two slices show that cultures lesioned at an early age show a high potential for functional regeneration, whereas this ability is clearly reduced in cultures lesioned at later ages. The main advantage of this technique over existing methods like slices, cultured on membranes, dissociated cultures, or in BSAs, is that the combination of Ergon typic cultures with multi electrode arrays allows us to assess functional aspects of regeneration in the microenvironment of the spinal cord with direct experimental access To begin fabricate or purchase pre-made multi electrode arrays like the one shown here, sterilize the multi electrode arrays by rinsing them for 30 seconds, twice in 100%ethanol, once in 70%ethanol, and then twice in distilled water. Once dry, put 10 to 12 arrays in a glass Petri dish and close the lid after you have autoclave the arrays for 20 minutes at 120 degrees Celsius.

Place each multi electrode array into a separate sterile 35 millimeter Petri dish. Take a cooled pipette out of the freezer. Use it to put 150 microliters of chilled extracellular matrix coating solution on top of the electrode.

Close the lid of the Petri dish and incubate the array for one hour at room temperature. After about 10 minutes, check for air bubbles on top of the electrodes and gently remove them with a rubber covered spatula. If necessary, check for the disappearance of all bubbles.

Next, aspirate the coating solution. Wash the erase with medium, optimized for prenatal and embryonic neurons, and then rinse them twice with distilled sterile water. Once rinsed, let them sit at room temperature until dry.

Immediately following extraction from the mother, transfer the decapitated E 14 rat embryos into a Petri dish filled with sterile chilled wash solution. Perform one complete transversal cut with a scalpel above the hind limbs and another one above the four limbs, and remove the limbs from the body. Next, make a cut in the frontal plane to separate viscera from the back piece containing the spinal cord.

Next, transfer the back pieces containing the spinal cord, one at a time onto a mounting disc. Cut the cord at a thickness of 225 to 250 microns using a tissue chopper. Then put a drop of wash solution on the chopped tissue and transfer the slices into 35 millimeter by 10 millimeter Petri dishes filled with sterile chilled wash solution.

Dissect the spinal cord away from any remaining tissue on each of the slices. Taking care to leave the dorsal root ganglia attached. Transfer the slices into a 35 millimeter by 10 millimeter Petri dish filled with sterile chilled wash solution.

Then let the slices rest for one hour at four degrees Celsius. Put a Petri dish with a coated micro electrode array inside under a stereo microscope. Bring the array into focus and center.

A six microliter droplet of chicken plasma on the electrode array. Using a small spatula carefully slide two spinal cord sections with their ventral size facing each other into the plasma droplet. Next at eight microliters of thrombin around the chicken plasma droplet.

Then use the pipette tip to carefully mix and spread the chicken, plasma and thrombin mixture. Just before the chicken plasma and thrombin mixture becomes too stringy and starts to coagulate, aspirate the excess liquid, then cap the Petri dish and place it in a humidified chamber. Place the chamber inside an incubator at 37 degrees Celsius for about an hour After incubation, carefully add 10 microliters of nutrient medium to the sample.

Cap the Petri dish and place it back into the incubator for an additional 45 minutes. Next, place each of the multi electrode array culture assemblies into a roller tube and add three milliliters of nutrient medium. Close the lid tightly and place the roller tube into the roller drum.

Rotate the drum at one to two RRP M in the incubator at 37 degrees Celsius using sterile rubber tip forceps. Remove the multi electrode array culture assemblies from the roller tube and place them into a Petri dish under a stereo microscope. Bring the tissue into focus and verify that the two slices are fused.

Next, hold the assembly steady and place a scalpel blade in the groove of the multi electrode array. Close to the tissue slices. Hold the scalpel rather horizontally and then lift the scalpel handle up.

But let the scalpel blade stay in the groove of the array in such a way that the blade rolls from base to tip cutting through the tissue, covering the groove. Sever any residual tissue connections with a 25 gauge needle tip if necessary, work only in the area within the groove and do not touch the tender edges. Put the multi electrode array culture assemblies back into the roller tube and add three milliliters of fresh nutrient medium to the cultures.

Then place the roller tube back onto the roller drum in the incubator at 37 degrees Celsius. Mount a multi electrode array culture assembly into a recording chamber and apply about 500 microliters of extracellular solution to the array. Then mount the assembly on the microscope.

Wait 10 minutes for the system to stabilize, and then record basic spontaneous activity from each of the activity detecting electrodes for 10 minutes. Repeat the recordings a total of two times to ensure stable extracellular conditions. Exchange the extracellular solution after every recording session if desired.

Disinhibit the network by applying extracellular solution containing one micromolar of strict nine and 10 micromolar of gazin, and wait for at least two minutes before recording the electrical activity to investigate the potential for functional recovery of the co cultures derived from the spinal cord of E 14 rat embryos. Lesions were performed in a time window of eight to 28 days in vitro. Two to three weeks later, the spontaneous neuronal activity was recorded.

Using the multi electrode array, the raw data is then transformed into raster plots, followed by network activity plots to visualize the total activity separately per side in each slice. The spontaneous activity is usually organized in bursts shown here under disinhibition slices that were separated at a young age appear here as functionally connected. However, those separated at an older age appear asynchronous.

Using this information, the amount of synchronized bursts between slices at various ages can be determined and visualized as shown here Following this procedure. Other methods like immunohistochemical staining can be performed in order to answer additional questions like what cell types contribute to the functional connection between the two spinal quart slices.