This study was supported by the National Natural Science Foundation of China (Grant no. 81673676) and Dongguan Science and Technology Bureau (Grant no. 2019622101002). The authors thank Dr. Maryrose Sullivan (Assistant Professor in Surgery, Harvard Medical School) for technical consulting.

All experimental protocols and animal procedures complied with the ethical principle guidelines of the National Research Council.
1. Preparation of materials
<ol>
	<li>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&#8211;100 mm in diameter), and glass breakers.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;L of B-27, 100 &#181;L of FBS, 100 &#181;L of L-glutamine, 100 &#181;L of antibiotic/antimycotic (100x), and 10 &#181;L of GDNF (10 &#181;g/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.</li>
	<li>Prepare neuron media B using the same procedure as the preparation of neuron media A but without adding FBS.</li>
	<li>Prepare digestion solution 1 by dissolving 20 mg of collagenase type II, 6 mg of bovine serum albumin, and 200 &#181;L of antibiotic/antimycotic (100x) in 10 mL of oxygen-stable Krebs solution.</li>
	<li>Prepare digestion solution 2 by diluting 1 mL of 0.25% trypsin with 4 mL of Hank&#8217;s balanced salt solution.</li>
	<li>Prepare a plate with coated coverslips.
	<ol>
		<li>Use forceps in a laminar flow bench when placing glass coverslips into a 48-well culture plate.</li>
		<li>Dilute poly-D-lysine with ddH2O to a concentration of 0.1 mg/mL as poly-D-lysine stock. Store the stock at &#8211;20 &#176;C, and thaw before use.</li>
		<li>Add 40 &#181;L 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 &#181;g/cm2. For example, if the basal area of one well from a 24-well plate is 2 cm2, then we should transfer 80 &#181;L of poly-D-lysine stock in a well. Each cm2 would contain 4 &#181;g of poly-D-lysine.</li>
		<li>Remove poly-D-lysine in the 48-well plate, and rinse coverslips with ddH2O thrice.</li>
		<li>Dry the plate in a laminar flow hood for at least 30 min to ensure that the plate is anhydrous.</li>
		<li>Store the plate at 4 &#176;C before laminin coating for 1 day at most. Storing the plate at &#8722;20 &#176;C is preferable for long-term storage.</li>
		<li>Thaw laminin at 4 &#176;C. Dilute laminin with ddH2O to a concentration of 50 &#181;g/mL as laminin stock. Store the stock at &#8211;20 &#176;C, and thaw at 4 &#176;C before use.</li>
		<li>Use a pipette to transfer 100 &#181;L of diluted laminin to the top of each coverslip and incubate laminin at 4 &#176;C for 1 h. 
		NOTE: For different plates with different basal areas, adjust the concentration to 5 &#181;g/cm2. For example, if the basal area of one well from a 24-well plate is 2 cm2, then transfer 200 &#181;L of diluted laminin in a well. Each cm2 would contain 5 &#181;g of laminin.</li>
		<li>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 &#176;C for 2 weeks at most.</li>
	</ol>
	</li>
</ol>
2. Bladder harvest
<ol>
	<li>Obtain five-week-old Sprague&#8211;Dawley rats.</li>
	<li>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.</li>
	<li>After euthanasia via cervical dislocation, soak the rats in 75% ethanol for 30 s for sterilization.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Add Krebs solution into three glass dishes and glass breakers.</li>
	<li>Prepare glass dishes and glass breakers with Krebs solution in an ice bath for precooling.</li>
	<li>Mark these containers with numbers 1&#8211;3 correspondingly to prevent confusion.</li>
	<li>Pair each glass dish with forceps and a spoons nucleus divider.</li>
	<li>In glass dish 1, cut open the bladder with ophthalmic scissors, and unfold it with forceps and a spoons nucleus divider.</li>
	<li>Rinse the bladder in glass breaker 1, and place it in glass dish 2.</li>
	<li>Eliminate adherent fat on the tissue surface using the forceps and ophthalmic scissors in glass dish 2.</li>
	<li>Rinse the bladder in glass breaker 2, and place it in glass dish 3.</li>
	<li>Gently scrape the bladder using the forceps and the spoons nucleus divider onto glass dish 3 to remove exogenous attachments.</li>
	<li>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 &#176;C.</li>
	<li>Repeat the previous step twice in two other tubes with Krebs solution to reduce contamination.</li>
</ol>
3. Two-step bladder digestion
<ol>
	<li>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.</li>
	<li>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 &#176;C, and 200 rpm.</li>
	<li>After step 1 digestion, centrifuge the solution at 356 x g at 4 &#176;C for 8 min.</li>
	<li>Place digestion solution 2 in a 37 &#176;C water bath for preheating.</li>
	<li>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.</li>
	<li>Mix cell sediment with warm digestion solution 2 in a 15 mL centrifuge tube, and shake the mixture while digesting in a 37 &#176;C water bath for 5 min. Do not exceed 7 min of step 2 digestion or neurons will perish.</li>
	<li>After digestion, immediately deactivate trypsin in the mixture with 10 mL of cold rinse media. 
	NOTE: Perform the following steps at 0 &#176;C&#8211;4 &#176;C. An ice bath could provide such condition.</li>
	<li>Harvest the cell sediment after centrifugation at 356 x g at 4 &#176;C for 8 min. Remove as much media as possible because the remaining trypsin is harmful to cell growth.</li>
	<li>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.</li>
	<li>Filter the mixture media through a 70 &#181;m cell strainer into a 50 mL centrifuge tube.</li>
	<li>Keep the filtrate on a shaker at 30 rpm in an ice bath for 30 min. This step is not necessary but recommended.</li>
	<li>Collect cells by centrifugation at 356 x g at 4 &#176;C for 8 min, and gently resuspend the cell pellets in 1 mL of neuron media A.</li>
	<li>Add 500 &#181;L of cell mixture into each well of the prepared 48-well plate.</li>
	<li>Culture cells in an incubator at 37 &#176;C and 5% CO2.</li>
	<li>Replace all the media with neuron media B in 1 h to provide a serum-free culture.</li>
	<li>Change half of the neuron media B every 3 days. 
	NOTE: Neurons are ready for immunocytochemical experiments after 5&#8211;7 days of culture.</li>
</ol>

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The authors declare no major conflict of interest.

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 common choices in coating neurons, particularly laminin, which is essential for neuron growth17.
Media Preparation 
After first medium replacement, cell isolation requires media without serum because serum stimulates cell division and leads to a limited neuron-growing space18. Thus, neuron growth factors are crucial. The quality of B27 and GDNF can vary largely from different batches and cause major effects on neuron growth19. Therefore, checking the lot number of the media is recommended when neuron yield is poor. Meanwhile, fresh media stock is crucial; the required amount should be calculated and prepared in advance each time before media are replaced.
Animals 
Sprague&#8211;Dawley rats are used in this method. C57BL/6 mice are also acceptable in this experiment. Therefore, other strains of rats or mice may also be adopted for this method despite a few variations in morphology and neuronal circuitry. In terms of different animal models, researchers should develop an optimized and targeted protocol. Furthermore, young animals should always be considered prior to the application of this method.
Tissue Treatment 
During the experiments, except for the digestive process, keeping tissues at a low temperature is essential to increase cell viability, which can reduce cell metabolism and avoid energy deficit. Oxygen levels, nutrition, and pH can also affect cell yield11. Moreover, for other tissues, we suggest that researchers perform this method with adjusted digestion condition.
Cell Culture 
One remarkable characteristic of neurons when inoculated is their quick adherence to coated plates20. In this case, changing media after 1 h of culture is recommended to gain a high proportion of neurons. Moreover, when most of the cells start to grow pseudopodium, the frequency of changing media can be reduced appropriately depending on the color of the media and the cellular state. Primary cell culture in a good state displays black soma with a bright border.
Most nerve cells isolated from the bladder are bladder intramural ganglia, which consist of afferent and autonomic efferent innervations of the bladder13. Moreover, no major pelvic ganglia are present in the harvested tissue. It distributes below the bladder neck21.
Limitation 
This is a preliminary research to isolate and culture neurons and glia. Many attempts had been done, like cytarabine treatment or density gradient centrifugation. However, the proportion of desired cells was still not ideal, and even more cell loss appeared. Moreover, traditional digestive conditions in this protocol, such as 37 &#176;C, are likely to kill off some sensitive neuron types, and cause potential gene expression artifacts22.
In conclusion, this protocol offers a method to culture neurons and glia from rat bladder. The isolation is easy to repeat, time efficient, and involves minimal microbial contamination. Although improvement evoking the purity of neurons is necessary, we hope this method contributes to LUTNS research.

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 nonexistent8,9. 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.

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

isolation and culture of primary neurons and glia from adult rat urinary bladder

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&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;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 &#946;-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.

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&#8211;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).
After proper culture, neurons could be identified via typical &#946;-III-tubulin and MAP-2 immunostaining10,12. In addition, glia was specifically identified via GFAP immunostaining10. Mature neurons developed synaptic spines, which were close to the presynaptic specializations identified by the immunostaining of the synaptic protein maker, synapsin-1 (Figure 2)12. These results indicated that mature cells with well-developed synapses were obtained through this method. This result suggests its important role in future function studies.
Meanwhile, several neuron subtypes were recognized through immunocytochemistry experiments (Figure 3). Peptidergic neurons, which contain various neuropeptides, were immunostained with substance P13. Purinergic neurons with expressed vesicular nucleotide transporters were identified via SLC17A9 staining14. Nitrergic neurons were visualized with DYNLL-2, which connects nNOS with motor proteins in neurons15. Cholinergic neurons were immunoreactive with choline acetyltransferase16.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61177/61177fig1.jpg" /> 
Figure 1. Phase-contrast images of primary cells isolated from the rat bladder culture taken at 1, 3, and 7 days after plating (A, B, C, respectively). Scale bar: 50 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61177/61177fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61177/61177fig2.jpg" /> 
Figure 2. Immunofluorescence images of primary cells isolated from the rat bladder. Confocal microscopy analysis showed neuron cytoskeleton protein staining (&#946;-III-tubulin, RRID: AB_2827688, 1:200) in primary culture neurons (A) and in whole mount bladder preparation (B). In primary culture neurons, neuronal phosphoprotein immunostaining (MAP 2, RRID:AB_2827689, 1:200) was also visualized (C). Glia were identified via glial fibrillary acidic protein staining (D; RRID: AB_627673, 1:50). Synapsins were visualized via synapsin protein staining (Synapsin-1, RRID: AB_2798146, 1:200) in cellular (E) and tissue (F) levels. The secondary antibodies used were as follows: Alexa Fluor 488 (green, goat anti-rabbit lgG, 1:200), Alexa Fluor 555 (red, goat anti-mouse lgG, 1:200). The nucleus was visualized using Hoechst 33342 (A, C, D, E; blue, 1 &#956;g/mL). Scale bar: 50 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61177/61177fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61177/61177fig3.jpg" /> 
Figure 3. Immunofluorescence images of several neuron subtypes of primary neurons. Peptidergic neurons were immunostained with substance P (A; RRID: AB_785913, 1:50). Purinergic neurons were identified via SLC17A9 staining (B; RRID: AB_10597575, 1:200). Nitrergic neurons were visualized via DYNLL-2 staining (C; RRID: AB_654147, 1:50). Cholinergic neurons were immunoreactive with choline acetyltransferase (D; RRID: AB_2244867, 1:100). The secondary antibodies used were as follows: Alexa Fluor 488 (green, goat anti-rabbit lgG, 1:200), Alexa Fluor 555 (red, goat anti-mouse lgG, 1:200). The nucleus was visualized using Hoechst 33342 (A, B, C, D, blue, 1 &#956;g/mL). Scale bar: 50 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61177/61177fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Ingredients</td>
			<td>Molarity (mM)</td>
		</tr>
		<tr>
			<td>NaCI</td>
			<td>120</td>
		</tr>
		<tr>
			<td>KCI</td>
			<td>5.9</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;12H2O</td>
			<td>1.2</td>
		</tr>
		<tr>
			<td>MgCI2&#183;6H2O</td>
			<td>1.2</td>
		</tr>
		<tr>
			<td>CaCI2</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>11.5</td>
		</tr>
	</tbody>
</table>
Table 1. Krebs solution composition

Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

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

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

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

isolation-culture-primary-neurons-glia-from-adult-rat-urinary

Nanyang Technological University

Research

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Neuroscience

3.8K Views.  Guangzhou University of Chinese Medicine. This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

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

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Developmental Biology

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Immunology and Infection

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information regarding glial activities at the cellular and molecular levels has been obtained through in vitro cell culture systems. The in vitro systems utilized, mainly include primary glia derived from neonatal brain cortical tissue and immortalized cell lines. However, these systems may not reflect the characteristics of spinal cord glial cells in vivo. In order to further investigate the roles of spinal cord glial cells in the development of peripheral nerve injury-induced neuropathic pain using a culture system that better reflects the in vivo condition, our laboratory has developed a method to establish primary spinal cord mixed glial cultures from adult mice. Briefly, spinal cords are collected from adult mice and processed through papain digestion followed by myelin removal with a density-gradient medium. Single cell suspensions are cultured in complete Dulbecco&#39;s modified Eagle media (cDMEM) supplemented with 2-mercaptoethanol (2-ME) at 35.9 oC. These culture conditions were optimized specifically for the growth of mixed glial cells. Under these conditions, cells are ready to be used for experimentation between 12 - 14 d&#160;(cells are usually in log phase during this time) after the establishment of the culture (D 0) and can be kept in culture conditions up to D 21. This culture system can be used to investigate the responses of spinal cord glial cells upon stimulation with various substances and agents. Besides neuropathic pain, this system can be used to study glial responses in other diseases that involve pathological changes of spinal cord glial cells.

The development of neuropathic pain involves pathological changes of spinal cord glial cells. A reliable glial culture system derived from adult spinal cord tissue and designed for studying these cells in vitro is lacking. Therefore, we show here how&#160;to establish primary mixed glial cultures from adult mouse spinal cord tissue.

It has been well-accepted that spinal cord glial responses contribute significantly to the development of neuropathic pain. Tremendous information ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

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

Source: Wang, R. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61177">Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder.</a> J. Vis. Exp. (2020).
This video demonstrates a technique for generating a primary culture of neurons and glia from a rat&#39;s urinary bladder. It outlines the steps involved in tissue preparation, enzymatic digestion to obtain single cells, and culturing the cells in a neuron-specific medium to establish a primary neuronal and glial culture.

Izolacja i hodowla pierwotnych neuronów i gleju z pęcherza moczowego szczura

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat

Source: Mothe, A., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52732">Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord.</a> J. Vis. Exp. (2015).
This video presents a method for isolating neural stem/progenitor cells from the periventricular tissue of the spinal cord in adult rats. The technique includes incubating the tissue fragments in a solution containing proteolytic enzymes to disrupt the extracellular matrix and release the cells. Subsequently, the released cells are plated in a medium containing specific growth factors that enhance neural stem/progenitor proliferation and promote aggregation into three-dimensional neurospheres.

Izolacja nerwowych komórek progenitorowych łodygi z obszaru okołokomorowego rdzenia kręgowego dorosłego szczura

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of Dissection ...