Studying the Regeneration of Functional Connections between Spinal Cord Slices Using a Multi-Electrode Array

1. Preparation of MEAs

Note: MEAs are composed of a glass substrate, micro-fabricated metal electrodes and a SU-8 polymer insulation layer. For the purpose of this study, commercially available MEAs were ordered with a customized electrode array layout (Figure 1 A&B). The 68 electrodes are arranged in a rectangular grid that is split into two zones by a 300 µm wide groove free of electrodes, electrical leads and insulation. Each electrode is 40 x 40 µm in size and they are spaced apart by 200 µm from center to center. Four large ground electrodes are positioned around the recording site. They differ from other commercially available standard MEAs in their size (21 mm x 21 mm) and they have no fixed recording chamber.

1. For disinfection rinse MEAs in 100% ethanol (2x), 70% ethanol (1x) and distilled water (2x) for at least 30 sec each. Let dry. Put 10 – 12 MEAs in a clean glass petri dish (150 mm x 25 mm) and close the lid.
2. Autoclave MEAs for 20 min at 120 °C. Store at RT.
3. Prepare the coating solution (see materials list) for the MEAs and store aliquots at -20 °C.
4. Put each MEA in an individual sterile petri dish (35 mm x 10 mm).
5. Use a cooled pipette to put 150 µl chilled coating solution on top of the electrodes and close the lid of the petri dish. After about 10 min, check for air bubbles on top of the electrodes and gently remove them with a rubber-covered spatula if necessary. Let rest for 1 hr at room temperature (RT).
6. Aspirate coating solution residues, wash 1x with medium optimized for prenatal and embryonic neurons and 2x with distilled, sterile water. If MEAs are not used until the next day, keep them in distilled, sterile water at 37 °C in the incubator overnight (O/N). Otherwise, let directly dry at RT.

2. Ingredients Required for the Preparation and Growth of Organotypic Cultures

1. Prepare calcium-free wash solution (see materials list).
2. Reconstitute chicken plasma in wash solution (1:1, shake gently, avoid formation of bubbles), centrifuge at 3,000 x g for about 20 min and decant the clear content into a sterile tube. Aliquot 200 µl into cryotubes and store at -20 °C.
3. Reconstitute thrombin from bovine plasma accordingly (200 U/ml) and sterile-filter (0.2 µm pore filter). Aliquot 200 µl into crytotubes and store at -20 °C.
4. For 30 cultures prepare 100 ml of nutrient medium (see materials list).

3. Spinal Cord Tissue Dissection

Note: Procedure yields, depending on the number of embryos, spinal cord slices for about 25 – 35 co-cultures and is prepared inside a laminar flow hood under sterile conditions.

1. Apply a lethal dose of pentobarbital (0.4 ml) to the mother animal by intramuscular injection. Confirm deep anesthesia by checking the pedal withdrawal reflex. Deliver embryonic day 14 (E14) rat embryos by cesarean section and sacrifice by decapitation.
2. Transfer the bodies of the embryos into a petri dish filled with sterile, chilled, wash solution.
3. Perform one complete transversal cut with a scalpel above the hindlimbs and another one above the forelimbs 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.
4. For slicing, transfer the back pieces containing the spinal cord one at a time on a mounting disk and cut at a thickness of 225 – 250 µm with a tissue chopper. Put a drop of wash solution on the chopped tissue and transfer slices with a spatula in 35 mm x 10 mm petri dishes filled with sterile, chilled, wash solution.
5. For each slice separately, dissect spinal cord away from remaining tissue. Leave dorsal root ganglia attached.
6. Let the slices rest for 1 hr at 4 °C.

4. Mounting Spinal Cord Tissue Slices on MEAs

1. Warm up sterile nutrient medium in the incubator (37 °C, lightly unscrewed lid for oxygenation).
2. Position MEA with sterile tweezers with rubber-covered tips at RT in the petri dish under a stereo microscope with the electrode array in focus. Center a 6 µl droplet of chicken plasma on the clean, dust-free, and sterile electrode array. Using a small spatula, carefully slide two spinal cord sections with ventral sides facing each other into the plasma droplet. Do not touch the electrodes with the spatula.
3. Add 8 µl of thrombin around the chicken plasma droplet. Using the thrombin pipette tip, carefully mix and spread the chicken plasma/thrombin mixture around the two slices. Again, do not touch the brittle electrode array directly. Just before coagulation, aspirate excess chicken plasma/thrombin.
4. Cap the petri dish to retain high humidity while the MEA/culture assembly sits for about 1 hr in a humidified chamber inside the incubator at 37 °C.
5. Carefully add 10 µl of nutrient medium to the culture chamber, cap the petri dish and put back into the incubator for about 30 min.
6. Place each MEA/culture assembly with sterile, rubber-covered tips into a roller tube, add 3 ml of nutrient medium and tightly close the lid. Place the roller tube in the roller drum rotating at 1 – 2 rpm in the incubator in a 5% CO₂-containing atmosphere at 37 °C.
7. Change half of the nutrient medium after 7 days in vitro (DIV) and afterwards 1 – 2 times per week.

5. Mechanical Lesions

1. Take the MEA/culture assembly with sterile, rubber-covered tips out of the roller tube and place in a petri dish without nutrient medium under a stereo microscope with the tissue in focus. Visually verify that the two slices are fused.
2. Hold the MEA/culture assembly steady by placing tweezers with rubber-covered tips on the MEA.
3. Place a scalpel blade in the groove of the MEA close to the tissue slices. Hold the scalpel rather horizontally.
4. Lift the scalpel handle up but let the scalpel blade stay in the groove of the MEA in such a way that the blade "rolls" from base to tip and thereby cuts through the tissue covering the groove.
5. Severe any residual tissue connections with a 25 G needle tip if necessary. Work only in the area within the groove and do not touch the tender edges.
6. Put the MEA/culture assembly back into the roller tube. Provide 3 ml of fresh nutrient medium to the cultures and place them back into the roller drum in the incubator.

6. Electrophysiological Recordings of Spontaneous Activity

1. To investigate functional regeneration among the two spinal cord slices after the chosen number of DIV, mount the MEA/culture assembly in a recording chamber on a microscope and apply about 500 µl extracellular solution (see materials list).
2. Wait 10 min before the first recording to allow the system to stabilize.
3. Record basic spontaneous activity 2x for about 10 min from each activity-detecting electrode of the MEA at RT.
4. To ensure stable extracellular conditions, exchange extracellular solution after every recording session.