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

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

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 1, 2, and 3 to prevent confusion.
In glass dish 1, 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 the forceps and ophthalmic scissors. Then, rinse the bladder in beaker 2 and place it in dish 3.
Gently scrape the bladder using the forceps and the spoons nucleus divider to remove exogenous attachments. Rinse the bladder in glass beaker 3 and transfer it to a 15-milliliter centrifuge tube with 14 milliliters of cold Krebs solution. Centrifuge the sample for 1 minute at 360 times g and 4 degrees Celsius.
Transfer the bladder from the centrifuge tube to a 2-milliliter vial containing 1 milliliter of digestion solution. Use ophthalmic scissors to cut the bladder into small pieces. Mix the bladder solution with 9 milliliters of digestion solution 1 in a sterile cell culture dish. Perform the first digestion in a shaking incubator for 1 hour at 37 degrees Celsius and 5% carbon dioxide at 200 RPM. After the first digestion, centrifuge the solution at 300 times g and 4 degrees Celsius for 8 minutes.
Meanwhile, place digestion solution 2 in a 37-degree Celsius water bath for preheating. After centrifugation, remove most of the supernatant that contains digestion solution 1, leaving some supernatant to avoid cell loss.
Resuspend the cells in warm digestion solution 2 and shake the mixture while digesting in a 37-degree Celsius water bath for 5 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 4 degrees Celsius for 8 minutes. Then, remove as much supernatant as possible to eliminate all trypsin.
Gently resuspend sediment with 3 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 4 degrees Celsius for 8 minutes. Then, gently resuspend the cell pellets in 1 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.

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Neuronal Culture Techniques

Primary Neuronal Culture

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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
<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 diameter), and glass beakers.</li>
	<li>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.</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 &#956;L of B-27, 100 &#956;L of FBS, 100 &#956;L of L-glutamine, 100 &#956;L of antibiotic/antimycotic (100x), and 10 &#956;L of GDNF (10 &#956;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 &#956;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&#39;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</li>
		<li>Add 40 &#956;L 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 &#956;g/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 &#956;L of poly-D-lysine stock in a well. Each cm2 would contain 4 &#956;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 50 &#956;g/mL concentration 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 &#956;L of diluted laminin to the top of each coverslip and incubate laminin at 4 &#176;C for 1 h. 
		NOTE: Adjust the concentration to 5 &#956;g/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 &#956;L of diluted laminin into a well. Each cm2 would contain 5 &#956;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 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.</li>
	<li>Add Krebs solution into three glass dishes and glass beakers.</li>
	<li>Prepare glass dishes and beakers with Krebs solution in an ice bath for pre-cooling.</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 beaker 1 and place it in glass dish 2.</li>
	<li>Eliminate adherent fat on the tissue surface using forceps and ophthalmic scissors in a glass dish 2.</li>
	<li>Rinse the bladder in glass beaker 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 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&#160;g&#160;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 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&#160;g&#160;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 conditions.</li>
	<li>Harvest the cell sediment after centrifugation at 356 x&#160;g&#160;at 4 &#176;C for 8 min. Remove as much media as possible because the remaining trypsin harms 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&#160;g&#160;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>
Table 1. Krebs solution composition
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>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>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>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 beakers</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>Laminar flow bench</td>
			<td>Su Jie Medical Devices</td>
			<td>CB 1400V</td>
			<td>Sterile operation</td>
		</tr>
		<tr>
			<td>Murine GDNF&#160;</td>
			<td>Peprotech&#160;</td>
			<td>AF45044&#160;</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>Spoons nucleus divider</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>YZR030</td>
			<td>12 cm; Sterile operation</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>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>
	</tbody>
</table>

Source: Wang, R. et al., <a href="https://app.jove.com/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.

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

Take a euthanized rat.&#160;
Open the abdomen, excise the bladder, and immerse it in buffer.
Cut open the bladder and remove adherent fat and tissue debris.
Transfer the bladder to a tube containing buffer, centrifuge, and remove the debris.
Place the tissue in a solution containing collagenase and mince it. Add more collagenase to degrade the extracellular matrix, loosening the cells.
Centrifuge and discard the supernatant. Add trypsin to disrupt the cell-cell junctions, releasing the cells.
Add rinse media containing proteins to inactivate trypsin, then centrifuge, and remove the supernatant.
Resuspend the cells in media; filter them to remove aggregates, and obtain a single-cell suspension.
Centrifuge and remove the supernatant. Resuspend the cells in media.
Plate the cells on coated coverslips to facilitate neuronal and glial cell attachment. Remove the non-attached cells.
Add neuron media containing growth factors to promote cellular maturation, establishing a primary neuronal and glial culture.

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Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

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During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

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JoVE Journal

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Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

낮은 밀도 차 해마 신경 세포 문화

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

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Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

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

내레이션 대본

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