Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Summary

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

Abstract

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague–Dawley rats. In this method, the neurons and glia were incubated in a 37 °C, 5% CO₂ incubator for 5–7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by β-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

Introduction

The lower urinary tract has two main functions: periodic urine storage and micturition¹. The lower urinary tract nervous system (LUTNS) controls these functions and is delicate and susceptible to many neuropathies, which can be innate (porphyria), acquired (Lyme disease), secondary to disease states (diabetic cystopathy), drug induced (hemorrhagic cystitis), surgery caused (abdominoperineal resection), or injury caused (traumatic spinal cord injury)^2,3,4,5,6,7. In physiological/pathological studies, in vivo and in vitro experiments are equally important. While in vivo research on LUTNS has been conducted at organ, cellular, and molecular levels for some time, in vitro research on primary neurons from the urinary bladder is almost nonexistent^8,9. Although the present study is limited, we hope to pioneer research in this area so that other researchers could improve it. In this manner, this co-culture may lead to a cellular understanding of physiological dysfunction in phenotypes, such as bladder neuron dysfunction.

In contrast to enteric muscles with a clear directionality of the muscle cells into discrete layers, the muscles of the bladder are unorganized¹⁰. Therefore, instead of peeling off the outer layer of the bladder, this method proposes digesting the entire bladder to reduce the difficulty of operation and shorten the preprocessing time for a high cell survival rate.

Following this method, we can obtain a mixed culture of neurons and other cells. The other cells are indispensable because their presence mimics an in vivo environment¹¹. In addition, such cells provide the substances that are unavailable in the medium.

This method involves two steps for digestion. First, collagenase type II is used to hydrolyze collagen, followed by trypsin, to dissociate the tissue into cells¹⁰. In this manner, bladder tissues are dispersed into single cells and then grow relatively independent. When the culture of neurons matures, the neurons can be used for imaging or functional assays.

Protocol

All experimental protocols and animal procedures complied with the ethical principle guidelines of the National Research Council.

1. Preparation of materials

1. 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 in diameter), and glass breakers.
2. Prepare the Krebs solution as follows (Table 1): Dissolve all the chemicals together with ddH₂O prior to use. Dissolve CaCI₂ separately and add slowly into the mixed solution while stirring to avoid sediment.
3. 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.
4. 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.
5. Prepare neuron media B using the same procedure as the preparation of neuron media A but without adding FBS.
6. 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.
7. Prepare digestion solution 2 by diluting 1 mL of 0.25% trypsin with 4 mL of Hank’s balanced salt solution.
8. Prepare a plate with coated coverslips.
   1. Use forceps in a laminar flow bench when placing glass coverslips into a 48-well culture plate.
   2. 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 use.
   3. Add 40 µL of poly-D-lysine stock on top of each coverslip, and incubate the solution at room temperature for 10 min.
      NOTE: For different plates with different basal areas, adjust the concentration to 4 µg/cm². For example, if the basal area of one well from a 24-well plate is 2 cm², then we should transfer 80 µL of poly-D-lysine stock in a well. Each cm² would contain 4 µg of poly-D-lysine.
   4. Remove poly-D-lysine in the 48-well plate, and rinse coverslips with ddH₂O thrice.
   5. Dry the plate in a laminar flow hood for at least 30 min to ensure that the plate is anhydrous.
   6. 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.
   7. Thaw laminin at 4 °C. Dilute laminin with ddH₂O to a concentration of 50 µg/mL as laminin stock. Store the stock at –20 °C, and thaw at 4 °C before use.
   8. 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: For different plates with different basal areas, adjust the concentration to 5 µg/cm². For example, if the basal area of one well from a 24-well plate is 2 cm², then transfer 200 µL of diluted laminin in a well. Each cm² would contain 5 µg of laminin.
   9. 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

1. Obtain five-week-old Sprague–Dawley rats.
2. 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.
3. After euthanasia via cervical dislocation, soak the rats in 75% ethanol for 30 s for sterilization.
4. 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.
5. 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 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.
6. Add Krebs solution into three glass dishes and glass breakers.
7. Prepare glass dishes and glass breakers with Krebs solution in an ice bath for precooling.
8. Mark these containers with numbers 1–3 correspondingly to prevent confusion.
9. Pair each glass dish with forceps and a spoons nucleus divider.
10. In glass dish 1, cut open the bladder with ophthalmic scissors, and unfold it with forceps and a spoons nucleus divider.
11. Rinse the bladder in glass breaker 1, and place it in glass dish 2.
12. Eliminate adherent fat on the tissue surface using the forceps and ophthalmic scissors in glass dish 2.
13. Rinse the bladder in glass breaker 2, and place it in glass dish 3.
14. Gently scrape the bladder using the forceps and the spoons nucleus divider onto glass dish 3 to remove exogenous attachments.
15. Rinse the bladder in glass breaker 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.
16. Repeat the previous step twice in two other tubes with Krebs solution to reduce contamination.

3. Two-step bladder digestion

1. 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.
2. Mix the bladder solution with 9 mL of digestion solution 1 in a sterile cell culture dish (100 mm in diameter). Perform step 1 digestion in a shaking incubator for 1 h under 5% CO₂, 37 °C, and 200 rpm.
3. After step 1 digestion, centrifuge the solution at 356 x g at 4 °C for 8 min.
4. Place digestion solution 2 in a 37 °C water bath for preheating.
5. 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.
6. 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.
7. 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 condition.
8. 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 is harmful to cell growth.
9. 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.
10. Filter the mixture media through a 70 µm cell strainer into a 50 mL centrifuge tube.
11. Keep the filtrate on a shaker at 30 rpm in an ice bath for 30 min. This step is not necessary but recommended.
12. 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.
13. Add 500 µL of cell mixture into each well of the prepared 48-well plate.
14. Culture cells in an incubator at 37 °C and 5% CO₂.
15. Replace all the media with neuron media B in 1 h to provide a serum-free culture.
16. Change half of the neuron media B every 3 days.
    NOTE: Neurons are ready for immunocytochemical experiments after 5–7 days of culture.

Results

In the process of primary cell culture, the cells acquired were round with bright and clear boundaries before the attached state. As the neurons grew, dendrites and axons started to be distinct. After 5–7 days of culture, the neurons reached a mature form with long projections, which were ideal for imaging or function studies. Although most of the impurities and cell debris could be removed due to changing media, certain residuals attached to poly-D-lysine and laminin coating were visible (Figure 1

Discussion

Plate Preparation
The use of glass coverslips in 6-, 12-, or 48-well culture plates for immunofluorescent or calcium imaging experiments is an economical and sample-sparing operation. Cells grow well in plates without coverslips during the preparation of primary cell cultures. Therefore, coverslips are dispensable in experiments, such as Western blot or polymerase chain reaction. Furthermore, coating is a necessary step before plating cells, with or without coverslips. Laminin and poly-D-lysine are...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin	Gibico	15050065	Enzyme digestion
48-well culture plate	Corning	3548	Coating dish
antibiotic/antimycotic	Gibico	15240062	Culture media/Rinse media
Anti-Glial Fibrillary Acidic Protein Antibody	Santa Cruz	sc-33673	ICC
B-27	Gibico	17504044	Culture media
BSA Fraction V	Gibico	332	Enzyme digestion
Choline Acetyltransferase Antibody	Abcam	ab18736	ICC
CO2 Incubator	Heraeus	B16UU	Cells culture
Collagenase type II	Sigma	2593923	Enzyme digestion
DMEM/F-12	Gibico	11330032	Rinse media
DYNLL2 Antibody	Santa Cruz	sc-13969	ICC
Fetal Bovine Serum	Gibico	10100147	Culture media/Rinse media
Forceps	Shanghai Jin Zhong Medical Devices	1383	10 cm; Sterile operation
Glass breakers	Huan Qiu Medical Devices	1101	50 ml; Sterile operation
Glass coverslips	WHB Scientific	WHB-48-CS	Coating dish
Glass dishes	Huan Qiu Medical Devices	1177	100 mm; Sterile operation
Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488	Southernbiotech	3050-30	ICC
Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555	Southernbiotech	3050-30	ICC
Hoechst 33342	BD	561908	ICC
Laminar flow bench	Su Jie Medical Devices	CB 1400V	Sterile operation
Laminin	Sigma	L2020	Coating dish
L-glutamine	Gibico	25030081	Culture media
MAP-2 Antibody	Affinity	AF5156	ICC
Murine GDNF	Peprotech	AF45044	Culture media
Neurobasal-A Medium	Gibico	10888022	Culture media
Ophthalmic scissors	Shanghai Jin Zhong Medical Devices	J21010	12.5 cm; Sterile operation
Pipettes	Eppendorf	3120000240	100-1000 ul; Reagent and sample pipetting
Pipettes	Eppendorf	3120000267	10-100 ul; Reagent and sample pipetting
Poly-D-lysine	Sigma	P7280	Coating dish
Refrigerated centrifuge	Ping Fan Instrument	TGL-16A	Enzyme digestion
Shaking incubator	Haimen Kylin-Bell Lab Instruments	T8-1	Enzyme digestion
SLC17A9 Antibody	MBL International	BMP079	ICC
Spoons nucleus divider	Shanghai Jin Zhong Medical Devices	YZR030	12 cm; Sterile operation
Substance P Antibody	Santa Cruz	sc-58591	ICC
Surgical scissors	Shanghai Jin Zhong Medical Devices	J21130	16 cm; Sterile operation
Surgical towel	Fu Kang Medical Devices	5002	40 x 50 cm; Sterile operation
Synapsin-1 Antibody	CST	5297T	ICC
Tubulin beta Antibody(β-III-tubulin)	Affinity	AF7011	ICC