All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of materials
- Sterilize all instruments and ddH2O using an autoclave before performing the experiment. Instruments include but are not limited to surgical scissors, ophthalmic scissors, forceps, spoons nucleus divider, glass dishes (60–100 mm diameter), and glass beakers.
- Prepare the Krebs solution (Table 1): Dissolve all the chemicals with ddH2O before use. Dissolve CaCI2 separately and add slowly into the mixed solution while stirring to avoid sediment.
- Prepare the Rinse media, which consists of F12 media with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (100x). Add 5 mL of FBS and 0.5 mL of antibiotic/antimycotic to 44.5 mL of F12 media.
- Prepare neuron media A, which comprises neurobasal A media with 2% B-27, 1% FBS, 1% L-glutamine, 1% antibiotic/antimycotic (100x), and 0.1% glia-derived neurotrophic factor (GDNF). Add 200 μL of B-27, 100 μL of FBS, 100 μL of L-glutamine, 100 μL of antibiotic/antimycotic (100x), and 10 μL of GDNF (10 μg/mL) to 9.5 mL of neurobasal A media.
NOTE: Prepare neuron media A within one week of usage to ensure freshness of B-27, L-glutamine, and GDNF.
- Prepare neuron media B using the same procedure as the preparation of neuron media A but without adding FBS.
- Prepare digestion solution 1 by dissolving 20 mg of collagenase type II, 6 mg of bovine serum albumin, and 200 μL of antibiotic/antimycotic (100x) in 10 mL of oxygen-stable Krebs solution.
- Prepare digestion solution 2 by diluting 1 mL of 0.25% trypsin with 4 mL of Hank's balanced salt solution.
- Prepare a plate with coated coverslips.
- Use forceps in a laminar flow bench when placing glass coverslips into a 48-well culture plate.
- Dilute poly-D-lysine with ddH2O to a concentration of 0.1 mg/mL as poly-D-lysine stock. Store the stock at –20 °C, and thaw before
- Add 40 μL of poly-D-lysine stock on top of each coverslip and incubate the solution at room temperature for 10 min.
NOTE: Adjust the concentration to 4 μg/cm2 for different plates with different basal areas. For example, if the basal area of one well from a 24-well plate is 2 cm2, we should transfer 80 μL of poly-D-lysine stock in a well. Each cm2 would contain 4 μg of poly-D-lysine.
- Remove poly-D-lysine in the 48-well plate, and rinse coverslips with ddH2O thrice.
- Dry the plate in a laminar flow hood for at least 30 min to ensure that the plate is anhydrous.
- Store the plate at 4 °C before laminin coating for 1 day at most. Storing the plate at −20 °C is preferable for long-term storage.
- Thaw laminin at 4 °C. Dilute laminin with ddH2O to a 50 μg/mL concentration as laminin stock. Store the stock at –20 °C and thaw at 4 °C before use.
- Use a pipette to transfer 100 μL of diluted laminin to the top of each coverslip and incubate laminin at 4 °C for 1 h.
NOTE: Adjust the concentration to 5 μg/cm2 for different plates with different basal areas. For example, if the basal area of one well from a 24-well plate is 2 cm2, then transfer 200 μL of diluted laminin into a well. Each cm2 would contain 5 μg of laminin.
- Remove the laminin solution, and rinse coverslips with ddH2O once. Perform this operation on the edge of the coverslip to avoid scraping.
NOTE: Coated coverslips could be stored at 4 °C for 2 weeks at most.
2. Bladder harvest
- Obtain five-week-old Sprague–Dawley rats.
- Infuse carbogen (95% oxygen, 5% CO2) into the Krebs solution for at least 30 min in an ice bath to reach a stable oxygen status and pH level.
- After euthanasia via cervical dislocation, soak the rats in 75% ethanol for 30 s for sterilization.
- Place rats on a sterilized surgical towel and expose their abdomen. Open the abdominal cavity and reveal the bladder with a set of scissors and forceps.
- Lift the bladder gently and cut the bladder from the bladder neck with another set of scissors and forceps to avoid cross-contamination. Place the bladder rapidly in a cold, oxygen-stable Krebs solution to improve cell survival.
NOTE: Once the bladder is removed, perform the following operations rapidly to improve the prospect of neuron survival.
- Add Krebs solution into three glass dishes and glass beakers.
- Prepare glass dishes and beakers with Krebs solution in an ice bath for pre-cooling.
- Mark these containers with numbers 1–3 correspondingly to prevent confusion.
- Pair each glass dish with forceps and a spoons nucleus divider.
- In glass dish 1, cut open the bladder with ophthalmic scissors and unfold it with forceps and a spoons nucleus divider.
- Rinse the bladder in glass beaker 1 and place it in glass dish 2.
- Eliminate adherent fat on the tissue surface using forceps and ophthalmic scissors in a glass dish 2.
- Rinse the bladder in glass beaker 2, and place it in glass dish 3.
- Gently scrape the bladder using the forceps and the spoons nucleus divider onto glass dish 3 to remove exogenous attachments.
- Rinse the bladder in glass beaker 3, transfer the bladder to a 15 mL centrifuge tube with 14 mL of cold Krebs solution, and spin the sample for 1 min at 356 x g and 4 °C.
- Repeat the previous step twice in two other tubes with Krebs solution to reduce contamination.
3. Two-step bladder digestion
- Transfer the bladder from the centrifuge tube to a 2 mL vial containing 1 mL of digestion solution 1. Use ophthalmic scissors to cut the bladder into small pieces (smaller than 1 mm) in solution.
- Mix the bladder solution with 9 mL of digestion solution 1 in a sterile cell culture dish (100 mm diameter). Perform step 1 digestion in a shaking incubator for 1 h under 5% CO2, 37 °C, and 200 rpm.
- After step 1 digestion, centrifuge the solution at 356 x g at 4 °C for 8 min.
- Place digestion solution 2 in a 37 °C water bath for preheating.
- After centrifugation, remove the supernatant that contains digestion solution 1 and harvest the cell sediment. Some remaining liquid is allowed. Complete removal of the solution may promote cell loss.
- Mix cell sediment with warm digestion solution 2 in a 15 mL centrifuge tube and shake the mixture while digesting in a 37 °C water bath for 5 min. Do not exceed 7 min of step 2 digestion, or neurons will perish.
- After digestion, immediately deactivate trypsin in the mixture with 10 mL of cold rinse media.
NOTE: Perform the following steps at 0 °C–4 °C. An ice bath could provide such conditions.
- Harvest the cell sediment after centrifugation at 356 x g at 4 °C for 8 min. Remove as much media as possible because the remaining trypsin harms cell growth.
- Resuspend sediment with 3 mL of neuron media gently. Ensure that air bubbles are not generated in the solution containing cells for a high survival rate.
- Filter the mixture media through a 70 µm cell strainer into a 50 mL centrifuge tube.
- Keep the filtrate on a shaker at 30 rpm in an ice bath for 30 min. This step is not necessary but recommended.
- Collect cells by centrifugation at 356 x g at 4 °C for 8 min, and gently resuspend the cell pellets in 1 mL of neuron media A.
- Add 500 µL of cell mixture into each well of the prepared 48-well plate.
- Culture cells in an incubator at 37 °C and 5% CO2.
- Replace all the media with neuron media B in 1 h to provide a serum-free culture.
- Change half of the neuron media B every 3 days.
NOTE: Neurons are ready for immunocytochemical experiments after 5–7 days of culture.
Table 1. Krebs solution composition
| Ingredients | Molarity (mM) |
| NaCI | 120 |
| KCI | 5.9 |
| NaHCO3 | 25 |
| Na2HPO4·12H2O | 1.2 |
| MgCI2·6H2O | 1.2 |
| CaCI2 | 2.5 |
| Glucose | 11.5 |
