An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

All animal care and experimental procedures were in accordance with and approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University.

1. Preparation of Sterile Poly-D-Lysine- and Laminin-Coated Glass Coverslips in 24-Well Plates

1. All procedures for step 1 are performed in sterile conditions; under a hood, and with sterile reagents. Glass coverslips and double deionized water (ddH₂O) should be sterilized in advance. Preparation of plates can be done up to two weeks before neuron isolation.
2. Under the hood, use sterile forceps to place autoclaved glass coverslips in 12 wells of a 24-well plate.
3. Prepare poly-D-lysine stock ahead of time. Add 50 ml of sterile ddH2O to 5 mg poly-D-lysine. Vortex and store in 3 ml aliquots at -20 °C. Thaw aliquots before use.
4. For a final concentration of 1 ml poly-D-lysine stock per 25 cm²: pipette 80 μl of poly-D-lysine stock on top of each glass coverslip (2 cm²). Let solution settle for 10 min.
5. Remove poly-D-lysine using vacuum and rinse coverslips 3x with sterile ddH₂O.
6. Allow plates to dry for at least 2 hr under the hood.
7. Plates may be stored at 4 °C or – 20 °C before proceeding to laminin coating.
8. Prepare laminin stock ahead of time. Thaw laminin on ice and keep on ice during use. Dilute to a concentration of 50 μg/ml; pay attention to lot concentration; each lot will be different. Depending on lot concentration, approximately 20 ml ddH₂O will be added to each vial of laminin. Aliquot (5 ml) and store at -80 °C. Thaw aliquots on ice before use.
9. Coat coverslips with 5 μg/cm² laminin; pipette 200 μl of laminin stock on each coverslip.
10. Incubate laminin solution on coverslips for 1 hr.
11. Aspirate remaining laminin solution and rinse coverslips once with ddH₂O. Avoid scraping surface of coverslips.
12. Store plates at 4 °C for up to two weeks.

2. Advance Preparation of Neuron Isolation Solutions

1. Prepare Krebs solution (in mM: 118 NaCl, 4.6 KCl, 1.3 NaH₂PO₄, 1.2 MgSO₄, 25 NaHCO₃, 11 glucose, and 2.5 CaCl₂). Place 13.79 g NaCl (FW 58.44), 0.686 g KCl (FW 74.55), 0.312 g NaH₂PO₄ (FW 120), .289 g MgSO₄ (FW 120.4), 4.20 g NaHCO₃ (FW 84.01), 3.96 g glucose (FW 180.2), and 0.555 g CaCl₂ (FW 111) in 2 L ddH₂0. Chill to 4 °C.
2. Prepare rinse media (F12 media with 10% fetal bovine serum (FBS) and antibiotic/antimycotic): To a 500 ml bottle of F12 media, add 50 ml FBS and 5 ml of Antibiotic/Antimycotic 100x Liquid (Gibco).
3. Prepare enteric neuron media (Neurobasal A media with B-27, 2 mM L-glutamine, 1% FBS, 10 ng/ml, Glial Derived Neurotrophic Factor (GDNF), and Antibiotic/Antimycotic 100x Liquid). To make GDNF stock, add 1 ml ddH2O to 10 μg GDNF and freeze back in 50 μl aliquots, store at -80 °C. For a 50 ml vial of complete neuron media, combine 47.5 ml Neurobasal A media, 1 ml B-27 (50x), 500 μl FBS, 500 μl 200 mM L-glutamine (Gibco) 50 μl GDNF stock, and 500 μl Antibiotic/Antimycotic 100x Liquid. Media is made in small 50 ml batches to ensure freshness of GDNF and to avoid contamination of large amounts of this expensive cell media mixture.

3. Harvest Longitudinal Muscle/Myenteric Plexus (LMMP) Preparation from Mice

1. Appropriate national and institutional ethics should be in place before performing animal experiments. Adult Swiss Webster mice weighing over 25 g (typically over 8 weeks of age) are housed in groups of 6 prior to experiments. Tissues from two mice are used for the isolation of ileal enteric neurons and glia and three mice are used for isolation of colonic cells.
2. Prepare surgical area. Gather small surgical scissors, angled forceps, cotton swabs, three 200 ml glass beakers, and a glass/plastic rod (paintbrush). Ensure all supplies are thoroughly washed and rinsed before use to minimize contamination. This step can be performed the day before the experiment.
3. Place Krebs solution on ice and bubble with carbogen (95% oxygen, 5% CO₂) for at least 30 min to stabilize pH.
4. Turn on various equipment and warm chemicals to stabilize at desired temperatures; heat water bath(s) to 37 °C, cool centrifuge to 4 °C, place Hank’s balanced salt solution (HBSS) and 0.5% trypsin in water bath (37 °C). Complete neuron media and rinse media should remain refrigerated.
5. Place 150 ml ice cold bubbled Krebs in three 200 ml glass beakers labeled ‘dirty’, ‘clean’, and ‘LMMP’. Bubble beaker labeled ‘LMMP’ with carbogen.
6. Following approval from ethics committee, euthanize mouse by cervical dislocation or CO₂ asphyxiation.
7. Place mouse in dorsal recumbence on surgical surface, clean skin with 70% EtOH, and lift abdominal skin using forceps. Using scissors, open abdominal cavity and reveal internal digestive organs.
8. Remove the length of the gastrointestinal tract by lifting a section of ileum and revealing its mesentery. Snip through mesentery with scissors to gently remove and unravel ileum and colon, being careful not to pull mesentery from the ileum/colon.
9. After the full length of intestine is unraveled, remove the ileum by cutting through the intestine distal to the stomach and proximal to the cecum. Note: this procedure can also be performed with colonic tissue by removing the colon from distal to the cecum to proximal to the anus.
10. Tissues can be further divided between proximal and distal colon, and jejenum, and ileum. The rest of this procedure will refer to isolation of the ileum; the procedure is the same for the isolation of colon LMMP. Place the entire ileum in a glass container with ice cold Krebs marked ‘dirty’.
11. Create a tool to clean the ileum; blunt a large 20 G needle using a filing stone and attach it to a large syringe (10 ml) containing ice cold Krebs.
12. Clean fecal matter from the ileum by sectioning the ileum into three or more large pieces. Remove an ileal section from the beaker and place the blunted needle into the end of an ileal piece.
13. Gently run Krebs through the gut section until all fecal matter is removed into a separate waste container. Place the cleaned section into the container of Krebs marked ‘clean’. Repeat until entire ileum is cleaned.
14. To remove the LMMP, cut the ileum into small segments, approximately 2 – 4 cm. Place a segment of ileum on a plastic or glass rod; ileum should fit snuggly but not be loose or taut (~2 mm). Begin removal of the LMMP by gently removing bits of mesentery still attached to the gastrointestinal (GI) tract using a forceps. Prevent the GI tract from rotating around rod by gently pinning the tube to the rod using the thumb.
15. Separate the LMMP from the underlying circular muscle; first gently rub the edge of the forceps along the entire line where the mesentery was attached, from top to bottom of the segment, gently creating a gap in the longitudinal muscle. Then gently tease away the longitudinal muscle using a cotton swab wetted with Krebs.
16. Begin at the top of the gap in the longitudinal muscle and tease away using the lightest horizontal stroke while applying very light pressure until the longitudinal muscle just begins to separate from the circular muscle; do this down the entire strip along the mesentery attachment point.
17. Then gently begin to work around the GI tube; moving from top to bottom and back as the longitudinal muscle is slowly separated from the circular muscle all the way around the tube. When complete, the LMMP will naturally come off the remainder of the GI tube.
18. Place the resulting thin strip of longitudinal muscle in the beaker marked ‘LMMP’. Repeat for all the segments.
19. After all the strips of LMMP have been gathered from one mouse, repeat procedure for any other mice being used. Thoroughly rinse the ‘dirty’ and ‘clean’ beakers before re-use.
20. Rinse the LMMP 3x to remove biological contamination. Fill three 2 ml Eppendorf tubes with ice cold Krebs. Place the LMMP strips in the first tube and spin for 30 sec at 356 x g in a centrifuge cooled to 4 °C. Carefully remove the supernatant with a pipette and move the LMMP strips to next clean Krebs filled tube. Repeat this procedure for each remaining tube.

4. Digest LMMP

1. Prepare digestion solution by placing 13 mg collagenase type 2 and 3 mg BSA in 10 ml carbogen-bubbled Krebs solution.
2. Place segments of rinsed LMMP into digestion solution and use scissors to snip LMMP into tiny pieces.
3. Digest LMMP for 60 min at 37 °C in water bath with shaker while being gently bubbled with carbogen.
4. After digestion is complete, gather cells by centrifugation for 8 min at 356 x g in centrifuge cooled to 4 ° C.
5. From this point forward all procedures should be done under sterile conditions in a cell culture hood! All reagents and containers should be sterilized before use.
6. During centrifugation, prepare 0.05% trypsin solution by placing 1 ml warmed 0.25% trypsin and 4 ml warmed HBSS into a sterile 50 ml cell culture tube.
7. After centrifugation, remove and discard supernatant. Do not use a vacuum; this will likely suck up the cell pellet due to low centrifugation speeds. Carefully remove the cell pellet and place into clean tube with 5 ml of 0.05% trypsin solution.
8. Digest cells in 0.05% trypsin solution in 37 °C water bath with shaking for 7 min. Do not exceed 7 min of total trypsin treatment or neurons will perish.
9. Neutralize trypsin with 10 ml of cold rinse media after 7 min of trypsin treatment.
10. Centrifuge cells for 8 min at 356 x g. Remove and discard media.
11. During centrifugation, balance a section of sterilized Nitex mesh on top of a sterile 15 ml cell culture tube.
12. After centrifugation, remove and discard supernatant. Gently resuspend cell mixture by triturating in 3 ml complete neuron media. Avoid generating air bubbles during this step and all future steps in which cell mixture is triturated. All triturations should be done very gently. Filter cell solution through Nitex mesh into clean 15 ml cell culture tube.
13. Optional: Cap cell culture tube and place in refrigerated Hula mixer (or any mixer that provides rotation) with gentle rolling and tilting for 30 min. This step is optional but recommended.
14. Optional: After Hula mixer, add 2 ml cold rinse media to wash cells.
15. Gather cells by centrifugation (8 min, 356 x g). Remove and discard supernatant.
16. Resuspend cells in 1,200 μl complete neuron media. Triturate cells gently using 1 ml plastic pipette tip. Do not generate air bubbles. Pipette slowly and gently until most chunks are broken up and cells are suspended into liquid.
17. Add 750 μl of complete media to each of the 12 wells containing a pre-coated glass coverslip and add 100 μl of the triturated cell solution.
18. Incubate neurons in cell culture incubator at 37 °C with 5% CO₂.
19. Change half of the cell media every 2 days.
20. Neurons are ready for electrophysiological recordings after 1 – 2 days in culture.