Name: isolation and culture of primary neurons and glia from adult rat urinary bladder
Uploaded: 2020-05-23T14:45:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

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 ...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+function+and+dysfunction+in+acute+intermittent+porphyria.">Nerve function and dysfunction in acute intermittent porphyria.</a> Brain. 131, 2510-2519 (2008).</li><li>Rantell, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+utility+of+urodynamics,+including+ambulatory,+and+24+h+monitoring,+in+predicting+upper+urinary+tract+damage+in+neuro-urological+patients+and+other+lower+urinary+tract+dysfunction?+ICI-RS+2017.">What is the utility of urodynamics, including ambulatory, and 24 h monitoring, in predicting upper urinary tract damage in neuro-urological patients and other lower urinary tract dysfunction? ICI-RS 2017.</a> Neurourology and Urodynamics. 37, 25-31 (2018).</li><li>Halperin, J. 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(202), 395-423 (2011).</li><li>Moriyama, Y., Hiasa, M., Sakamoto, S., Omote, H., Nomura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+nucleotide+transporter+(VNUT):+appearance+of+an+actress+on+the+stage+of+purinergic+signaling.">Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling.</a> Purinergic Signal. 13 (3), 387-404 (2017).</li><li>Chaudhury, A., He, X. D., Goyal, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+Va+plays+a+key+role+in+nitrergic+neurotransmission+by+transporting+nNOS&#945;+to+enteric+varicosity+membrane.">Myosin Va plays a key role in nitrergic neurotransmission by transporting nNOS&#945; to enteric varicosity membrane.</a> American journal of physiology. Gastrointestinal and Liver Physiology. 301 (3), 498-507 (2011).</li><li>Le Berre-Scoul, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+enteric+neuron-glia+coculture+system+reveals+the+role+of+glia+in+neuronal+development.">A novel enteric neuron-glia coculture system reveals the role of glia in neuronal development.</a> The Journal of Physiology. 595 (2), 583-598 (2017).</li><li>Hayashi, H., Yamada, M., Kumai, J., Takagi, N., Nomizu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+activities+of+laminin-111-derived+peptide-chitosan+matrices+in+a+primary+culture+of+rat+cortical+neurons.">Biological activities of laminin-111-derived peptide-chitosan matrices in a primary culture of rat cortical neurons.</a> Archives of Biochemistry and Biophysics. 648, 53-59 (2018).</li><li>Bottenstein, J. 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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...

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 border="0" cellpadding="0" cellspacing="0" style="width:1061px;" width="1061">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">0.25% trypsin</td>
			<td style="width:279px;">Gibico</td>
			<td style="width:119px;">15050065</td>
			<td style="width:296px;">Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">48-well culture plate</td>
			<td>Corning</td>
			<td style="width:119px;">3548</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">antibiotic/antimycotic</td>
			<td>Gibico</td>
			<td>15240062</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Glial Fibrillary Acidic Protein Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-33673</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27</td>
			<td>Gibico</td>
			<td>17504044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA Fraction V</td>
			<td>Gibico</td>
			<td>332</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Choline Acetyltransferase Antibody</td>
			<td>Abcam</td>
			<td>ab18736</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 Incubator</td>
			<td>Heraeus</td>
			<td>B16UU</td>
			<td>Cells culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type II</td>
			<td>Sigma</td>
			<td>2593923</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">DMEM/F-12</td>
			<td>Gibico</td>
			<td>11330032</td>
			<td>Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DYNLL2 Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-13969</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Gibico</td>
			<td>10100147</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>1383</td>
			<td>10 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass breakers</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1101</td>
			<td>50 ml; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips</td>
			<td>WHB Scientific</td>
			<td>WHB-48-CS</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass dishes</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1177</td>
			<td>100 mm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>BD</td>
			<td>561908</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminar flow bench</td>
			<td>Su Jie Medical Devices</td>
			<td>CB 1400V</td>
			<td>Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>Gibico</td>
			<td>25030081</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAP-2 Antibody</td>
			<td>Affinity</td>
			<td>AF5156</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Murine GDNF</td>
			<td>Peprotech</td>
			<td>AF45044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal-A Medium</td>
			<td>Gibico</td>
			<td>10888022</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ophthalmic scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21010</td>
			<td>12.5 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000240</td>
			<td>100-1000 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000267</td>
			<td>10-100 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Refrigerated centrifuge</td>
			<td>Ping Fan Instrument</td>
			<td>TGL-16A</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>Haimen Kylin-Bell Lab Instruments</td>
			<td style="width:119px;">T8-1</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SLC17A9 Antibody</td>
			<td>MBL International</td>
			<td>BMP079</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spoons nucleus divider</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>YZR030</td>
			<td>12 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Substance P Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-58591</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21130</td>
			<td>16 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical towel</td>
			<td>Fu Kang Medical Devices</td>
			<td>5002</td>
			<td>40 x 50 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synapsin-1 Antibody</td>
			<td>CST</td>
			<td>5297T</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubulin beta Antibody(&#946;-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 (Figu...

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 ...

This co-culture method may lead towards cellular understanding of physiological dysfunction in phenotypes, such as bladder neuron dysfunctions. The main advantage of this protocol is high efficiency. The entire isolation can be completed in six hours.Demonstrating the procedure will be Jiao Zhang, a postgraduate student from my laboratory. Begin by placing rats on a sterilized surgical towel and exposing the abdomen. Use scissors and forceps to open the abdominal cavity and reveal the bladder.Use another set of scissors and forceps to gently lift the bladder and cut it from the bladder neck. Then quickly place the bladder in cold oxygen stable Krebs solution to improve cell survival. Add Krebs solution into three glass dishes and three glass beakers and place them in an ice bath for pre-cooling.Number the dishes and beakers one, two, and three to prevent confusion. In glass dish one, open the bladder with ophthalmic scissors and unfold it with forceps and a Spoons nucleus divider. Rinse the bladder in glass beaker one and place it in glass dish two.Eliminate adherent fat on the tissue surface using the forceps and ophthalmic scissors, then rinse the bladder in beaker two and place it in dish three. Gently scrape the bladder using the forceps and the Spoons nucleus divider to remove exogenous attachments. Rinse the bladder in glass beaker three and transfer it to a 15 milliliter centrifuge tube with 14 milliliters of cold Krebs solution.Centrifuge the sample for one minute at 360 times G and four degrees Celsius. Transfer the bladder from the centrifuge tube to a two milliliter vial containing one milliliter of digestion solution. Use ophthalmic scissors to cut the bladder into small pieces.Mix the bladder solution with nine milliliters of digestion solution one in a sterile cell culture dish. Perform the first digestion in a shaking incubator for one hour at 37 degrees Celsius and 5%carbon dioxide at 200 RPM. After the first digestion, centrifuge the solution at 300 times G and four degrees Celsius for eight minutes.Meanwhile, place digestion solution two in a 37 degree Celsius water bath for preheating. After centrifugation, remove most of the supernatant that contains digestion solution one, leaving some supernatant to avoid cell loss. Resuspend the cells in warm digestion solution two and shake the mixture while digesting in a 37 degree Celsius water bath for five minutes.Once digestion is complete, immediately deactivate the trypsin by adding 10 milliliters of cold rinse media. Centrifuge the cells at 360 times G and four degrees Celsius for eight minutes, then remove as much supernatant as possible to eliminate all trypsin. Gently resuspend sediment with three milliliters of neuron media, making sure to not generate air bubbles in the solution.Filter the suspension through a 70 micrometer cell strainer into a 50 milliliter centrifuge tube. Collect cells by centrifugation at 360 times G at four degrees Celsius for eight minutes, then gently resuspend the cell pellets in one milliliter of neuron media A and add 500 microliters of cell suspension to each well of a 48-well plate. Culture cells at 37 degrees Celsius and 5%carbon dioxide.The cells isolated from the rat bladder culture were imaged at one, three, and seven days after plating. As the neurons grew, dendrites and axons became distinct. After five to seven days of culture, the neurons reached a mature form with long projections which were ideal for imaging or function studies.After proper culture, neurons were identified with beta-three tubulin and MAP2 immunostaining and glia were identified with GFAP immunostaining. Mature neurons developed synaptic spines, which were close to the presynaptic specializations identified by immunostaining of Synapsin-1. Meanwhile, several neuron subtypes were recognized through immunohistochemistry experiments.Peptidergic neurons which contain various neuropeptides were immunostained with substance-P. Purinergic neurons with expressed vascular nucleotide transporters were identified via SLC17A9 staining. Nitrogenic neurons were visualized with DOIN-LL2 which connects neural NOS with motor proteins in neurons.And cholinergic neurons were immunoreactive with choline acetyltransferase. When performing the second digestion, remember to have the cold rinse medium beside you and ready to use. In this way, you can immediately deactivate the trypsin.When the cell culture matures, immunocytochemistry can be performed. This will reveal expression and distribution of target proteins in the cells. This method provides researchers with an in vitro model to explore diseases like bladder neuron dysfunction.

Research

JoVE Journal

Neuroscience

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

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

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Spinal Cord Neurons Isolation and Culture from Neonatal Mice

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Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

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