We thank CSIR-IICB animal facility. G. D. thanks ICMR, J. K. and V. G. thank DST Inspire, and D. M. thanks DBT, India for their fellowships. S. G. kindly acknowledges SERB (EMR/2015/002230) India for providing financial support.

All experimental procedures involving animal were approved by the Institutional Animal Ethics Committee of CSIR-Indian Institute of Chemical Biology (IICB/AEC/Meeting/Apr/2018/1).
1. Reagent and media preparation
<ol>
	<li>Poly-D-lysine (PDL) solution: prepare PDL solutions at concentrations of 0.1 mg/mL in deionized water and store in 4 &#176;C until use.</li>
	<li>Dissociation medium: To 1 L of sterile, filtered deionized water, combine the following components in the respective concentrations: sodium chloride (8 mg/mL), potassium chloride (0.4 mg/mL), potassium phosphate monobasic (0.06 mg/mL), D-glucose (1 mg/mL), sodium phosphate dibasic (0.479 mg/mL), and 1 M HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 10 mM]. Vortex all the components to aid proper mixing and store in 4 &#176;C until use. 
	NOTE: Use the dissociation medium in ice-cold form during dissociation but at room temperature (RT) for washing and other purposes.</li>
	<li>Plating medium: plating medium consists of the following: minimum essential medium (MEM) Eagle&#39;s with Earle&#39;s Balanced Salt Solution (BSS; 88.4%), D-glucose (0.6%), horse serum (10%), and penicillin/streptomycin (1%). Combine the components in the respective ratios and perform the procedure inside a hood under sterile conditions. 
	NOTE: Always use freshly prepared plating medium to avoid degradation of any component.</li>
	<li>Maintenance medium: prepare the maintenance medium by combining the following in the respective ratios: neurobasal medium (97%), 0.5 mM commercially obtained glutamine sample, B27 serum-free supplement (2%), and penicillin/streptomycin (1%). Combine the components in the respective ratios and perform the procedure inside a hood under sterile conditions. Ensure that all components are freshly prepared.</li>
</ol>
2. Preparation of coverslips
<ol>
	<li>Take a 12 mm diameter round glass coverslip and soak it in 1 M hydrochloric acid (HCl) for 4 h.</li>
	<li>Transfer the coverslips in distilled water using a pair of forceps and swirl it gently to get rid of the acid completely.</li>
	<li>Transfer the washed coverslips for an additional round of cleaning in a beaker containing 100% ethanol.</li>
	<li>Before using the coverslips, dry them well in the laminar hood by keeping them on tissue paper.</li>
</ol>
3. Preparation of poly-D-lysine coated plates for neuron culture
<ol>
	<li>Take two 24 well plates: one for high density plating and another for low density plating. Open the sterile packets only inside the laminar hood.</li>
	<li>Transfer the 12 mm of sterile glass coverslips in the 24 well plates.</li>
	<li>Pour 300 &#181;L of PDL solution (0.1 mg/mL in deionized water) in each well so that it fully covers the surface of the coverslips.</li>
	<li>Wrap the plates with aluminum foil to prevent drying and keep it in the CO2 incubator overnight.</li>
	<li>The next day (before plating), aspirate the PDL solution and wash properly with 300 &#181;L of sterile deionized water two to three times.</li>
	<li>Add 200 &#181;L of freshly prepared plating medium and return the plates to the incubator until plating.</li>
</ol>
4. Removal and decapitation of the fetus
NOTE: Sterilize all surgical instruments packed in aluminum foil in an autoclave at 121 &#176;C (15 psi) for 30 min. This includes a pair of blunt-end scissors, forceps, fine forceps, two fine scissors, and one artery forceps for the entire procedure.
<ol>
	<li>For generating neurons and neurospheres, use a timed-pregnant Sprague Dawley rat and mark the day with vaginal plug detection as E0. 
	&#8203;NOTE: The culture must be performed between E14-E16.</li>
	<li>On the day of culture, place a sterile glass Petri plate on ice and fill it with cold Hank&#39;s Balanced Salt Solution (HBSS).</li>
	<li>Anesthetize an E14-E16 pregnant rat with an intraperitoneal (i.p.) injection of 90 mg ketamine/kg of body weight and 10 mg xylazine/kg, then sacrifice by performing cervical dislocation. 
	NOTE: Rats can also be euthanized by an overdose of pentobarbital or overdose of ketamine with xylazine or diazepam.</li>
	<li>Sterilize the dam&#39;s abdomen by spraying 70% ethanol and make a V-shaped cut in the abdominal area using sterile forceps and a pair of blunt-end scissors.</li>
	<li>Take the embryonic sacs carefully on the Petri plate with cold HBSS solution. 
	NOTE: Do not use the same forceps and scissors that were just used for the skin, as this will contaminate the internal organs. Use a different set of scissors/forceps for the internal organs.</li>
	<li>Take the embryos out of the embryonic sacs in fresh, cold HBSS.</li>
	<li>Decapitate the head with sterile scissors.</li>
</ol>
5. Removal of brain and dissection of the cortex with hippocampus
<ol>
	<li>Before starting, fill 90 mm sterile Petri dishes with cold, sterile HBSS.</li>
	<li>Transfer the heads in the sterile dishes using sterile, blunt-ended dressing forceps.</li>
	<li>Under the stereomicroscope, hold the head from the snout region with sterile, serrated forceps and remove the brain by cutting the skin and skull open.</li>
	<li>Collect all the embryo brains in the same manner in the HBSS solution.</li>
	<li>Remove all meninges from the hemispheres and midbrain by holding the brainstem.</li>
	<li>Carefully remove the intact hemispheres resembling mushroom caps that contain the hippocampus and cortex.</li>
	<li>Collect the hemispheres containing cortex and intact hippocampus in a 15 mL conical tube containing 10 mL of dissociation medium.</li>
</ol>
6. Dissociation of cortical and hippocampal tissue into single neurons
<ol>
	<li>Allow the collected tissues to settle down and aspirate the dissociation medium, leaving 5%-10% of medium in it.</li>
	<li>Add 10 mL of fresh dissociation medium to the tissue, and repeat step 6.1 twice.</li>
	<li>Add 4.5 mL of dissociation medium and 0.5 mL of 0.25% (1x) trypsin EDTA (ethylene diamine tetraacetate) solution.</li>
	<li>Keep the tissue in the incubator at 37 &#176;C for 20 min for the digestion to proceed.</li>
	<li>Aspirate the medium and add 10 mL of dissociation and plating medium consecutively to the digested tissues.</li>
	<li>Allow the digested tissues to settle down and aspirate the dissociation medium. Add 2.5 mL of plating medium and pour into the base of a 90 mm sterile dish.</li>
	<li>Triturate the digested tissues in the corner base of the dish using a 1,000 &#181;L pipette tip to occupy the minimal volume.</li>
	<li>Pass the obtained cell suspension through the 70 &#181;m cell strainer, excluding any chunks of tissue.</li>
	<li>Determine the density of viable cells using the trypan blue dye exclusion method and count the number of cells in an automated cell counter.
	<ol>
		<li>For trypan blue dye exclusion method, take 10 &#181;L of the cell suspension and 10 &#181;L of 0.4% trypan blue stain, mix thoroughly, and add 10 &#181;L of the mixture in one of the two enclosed chambers of the disposable chamber slides.</li>
		<li>Insert the slide containing the mixture into the cell counter and obtain the reading. 
		NOTE: The trypan blue dye exclusion method is based on the principle that live cells (due to their intact membranes) will exclude trypan blue dye and will hence show a clear cytoplasm, compared to a non-viable cell that will easily take up trypan blue and appear blue in color15.</li>
	</ol>
	</li>
	<li>Dilute the number of cells obtained to plate 1.5 x 105 cells/mL for high density and 20,000 cells/mL for low density in two separate tubes containing 30 mL each of the plating medium.</li>
	<li>Aspirate the previously added plating medium from each well and plate 500 &#181;L of cells dispersed in plating medium in each well.</li>
	<li>After that return the plates to the incubator at 37 &#176;C and 5% CO2 for 4 h.</li>
	<li>Examine the cells for adherence under the microscope 4 h after plating.</li>
	<li>If there is proper adherence of the cells in both plates, replace the medium in each well with 500 &#181;L of fresh maintenance medium and incubate at 37 &#176;C.</li>
	<li>Culture these neurons grown at low density for 30 days by changing the maintenance medium 2x per week.</li>
	<li>Culture the neurospheres obtained from the high-density plated neurons in the same maintenance medium by transferring them to the ultra-low attachment plates.</li>
	<li>Characterize the neurons and the neurospheres by immunostaining them with important markers. For immunocytochemistry, first fix the cells/neurospheres using 4% formaldehyde for 30 min on the plate itself, then permeabilize the cells with 0.1% non-ionic detergent for 10 min.</li>
	<li>Add primary antibodies for both neurons (anti-Tuj1, GFAP, O4, tau) and neurospheres (anti-Nestin, GFAP, Tuj1) in phosphate-buffered saline (PBS) at 1:300 concentrations and incubate overnight at 4 &#176;C16. 
	NOTE: The Tuj1 (class III &#946;-tubulin) and tau are positive markers for the primary neurons, while GFAP (glial fibrillary acidic protein) and O4 (oligodendrocyte marker) are negative markers for primary neurons17,18. In the case of neurospheres, Tuj1, GFAP, and Nestin all&#160;serve as positive markers19,20.</li>
	<li>The next day, wash the cells with PBS once or twice and add appropriate secondary antibodies in PBS at 1:600 concentrations at RT for 2 h. 
	NOTE: The anti-Mouse or anti-Rabbit secondary antibodies are selected depending on the host species of the primary antibody added. It should be kept in mind that the secondary antibodies must be conjugated to fluorescence derivatives suitable for fluorescence microscopy purposes.</li>
	<li>Wash the cells again with PBS once or twice.
	<ol>
		<li>Perform nuclear staining of the cells with Hoechst 33258 (1 mg/mL stock solution in deionized water). Prepare 0.1% Hoechst solution in PBS from the stock solution and add it to the cells.</li>
		<li>Incubate the cells with 0.1% Hoechst solution for 30 min, then wash again with PBS.</li>
	</ol>
	</li>
	<li>Add 20 &#181;L PBS (or mounting medium) on the slide and slowly mount the coverslip containing the stained cells over the area of the slide containing PBS. Seal the margins of the coverslip with dibutylphthalate polystyrene xylene (DPX).</li>
	<li>Perform imaging of the fixed cells under a microscope at 10x and 40x magnification.</li>
</ol>

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The authors declare no competing financial interests.

This protocol describes that how by altering the&#160;cell plating densities of primary neurons, two variable neuronal platforms&#160;are obtained. Though this is a simple method, each step must be meticulously performed to achieve the desired results. Other previous methods have either reported long-term primary neuron cultures or neurosphere cultures. Most primary neuron culture protocols have involved the culturing of hippocampal neurons for 3-5 weeks, but most have failed, as the neurons die and wither away due to lo...

The brain is an intricate circuitry of neuronal and non-neuronal cells. For years, scientists have been trying to gain insight into this complex machinery. To do so, neuroscientists initially resorted to various transformed nerve-based cell lines for investigations. However, the inability of these clonal cell lines to form strong synaptic connections and proper axons or dendrites have shifted scientific interest to primary neuron cultures1,2. The most exciting aspect of primary neuron culture is that it creates an opportunity to observe and manipulate living neurons3. Moreover, it is less complex compared to neural tissue, which makes it an ideal candidate for studying the function and transport of various neuronal proteins. Recently, several developments in the fields of microscopy, genomics, and proteomics have generated new opportunities for neuroscientists to exploit neuron cultures4.
Primary cultures have allowed neuroscientists to explore the molecular mechanisms behind neural development, analyze various neural signaling pathways, and develop a more coherent understanding of synapsis. Though a number of methods have reported cultures from primary neurons (mostly from the hippocampal origin5,6,7), a unified protocol with a chemically defined medium that enables long-term culture of neurons is still needed. However, neurons plated at a low density are most often observed, which do not survive long-term, likely due to the lack of trophic support8 that is provided by the adjacent neurons and glial cells. Some methods have even suggested co-culturing of the primary neurons with glial cells, wherein the glial cells are used as a feeder layer9. However, glial cells pose a lot of problems due to their overgrowth, which sometimes override the neuronal growth10. Hence, considering the problems above, a simpler and more cost-effective primary neural culture protocol is required, which can be used by both neurobiologists and neurochemists for investigations.
A primary neuron culture is essentially a form of 2D culture and does not represent the plasticity, spatial integrity, or heterogeneity of the brain. This has given rise to the need for a more believable 3D model called neurospheres11,12.&#160;Neurospheres present a novel platform to neuroscientists, with a closer resemblance to the real, in vivo brain13. Neurospheres are non-adherent 3D clusters of cells that are rich in neural stem cells (NSCs), neural progenitor cells (NPCs), neurons, and astrocytes. They are an excellent source for the isolation of neural stem cells and neural progenitor cells, which can be used to study differentiation into various neuronal and non-neuronal lineages. Again, variability within neurosphere cultures produced using the previously reported protocols presents a barrier to the formulation of a unified neurosphere culture protocol14.
This manuscript presents a protocol in which it is possible to generate both 2D and 3D platforms by alternating cell plating densities from a mixed cortical and hippocampal culture. It is observed that within 7 days free-floating neurospheres are obtained from&#160;high-density plated neurons isolated from E14-E16 Sprague Dawley rat embryo, which upon further culture, form bridges and interconnections through radial glial-like extensions. Similarly, in the low density plated neurons, a primary neuron culture that can be maintained for up to 30 days is obtained by changing the maintenance medium twice per week.

<table><tbody><tr><td>Anti-GFAP</td><td>Abcam</td><td>AB7260</td><td></td></tr><tr><td>Anti- Nestin</td><td>Abcam</td><td>AB92391</td><td></td></tr><tr><td>Anti-O4</td><td>Millipore</td><td>MAB345</td><td></td></tr><tr><td>Anti-Tau</td><td>Abcam</td><td>AB76128</td><td></td></tr><tr><td>Anti-Tuj1</td><td>Millipore</td><td>MAB1637</td><td></td></tr><tr><td>B27 Serum Free Supplement&amp;nbsp;</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>Cell Counter</td><td>Life technologies</td><td>Countess II FL</td><td></td></tr><tr><td>CO2 Incubator</td><td>Eppendorf</td><td>Galaxy 170 R</td><td></td></tr><tr><td>D-glucose&amp;nbsp;</td><td>SDFCL</td><td>38450-K05</td><td></td></tr><tr><td>Ethanol</td><td>Merck Millipore</td><td>100983</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Olympus</td><td>IX83 Model</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma Aldrich</td><td>47608</td><td></td></tr><tr><td>GlutaMax-I Supplement</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>GtXMs IgG Fluor</td><td>Millipore</td><td>AP1814</td><td></td></tr><tr><td>GtXMs IgG (H+L)</td><td>Millipore</td><td>AP124C</td><td></td></tr><tr><td>HEPES</td><td>SRL</td><td>16826</td><td></td></tr><tr><td>Hoechst 33258</td><td>Calbiochem</td><td>382061</td><td></td></tr><tr><td>Horse Serum&amp;nbsp;</td><td>HiMedia</td><td>RM10674</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Rankem</td><td>H0100</td><td></td></tr><tr><td>Laminar Hood</td><td>BioBase</td><td>BBS-V1800</td><td></td></tr><tr><td>MEM Eagle&amp;rsquo;s with Earle&amp;rsquo;s BSS&amp;nbsp;</td><td>Sigma Aldrich</td><td>M-2279</td><td></td></tr><tr><td>Microscope</td><td>Dewinter</td><td>Victory Model</td><td></td></tr><tr><td>Neurobasal Medium&amp;nbsp;</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>Plasticware (24 well plate, cell strainers, and low adherence plates)</td><td>BD Falcon</td><td>353047, 352350 and 3471</td><td></td></tr><tr><td>90 mm Petridishes</td><td>Himedia</td><td>PW001</td><td></td></tr><tr><td>Penicillin/Streptomycin&amp;nbsp;</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Poly-D-Lysine</td><td>Millipore</td><td>A.003.E</td><td></td></tr><tr><td>Potassium Chloride&amp;nbsp;</td><td>Fisher Scientific</td><td>BP366-500</td><td></td></tr><tr><td>Potassium Phosphate Monobasic&amp;nbsp;</td><td>Merck</td><td>MI6M562401</td><td></td></tr><tr><td>Sodium Chloride&amp;nbsp;</td><td>Qualigem</td><td>15918</td><td></td></tr><tr><td>Sodium Phosphate Dibasic&amp;nbsp;</td><td>Merck</td><td>MI6M562328</td><td></td></tr><tr><td>Stereomicrosope</td><td>Dewinter</td><td>Zoomstar Model</td><td></td></tr><tr><td>Triton-X 100</td><td>SRL</td><td>2020130</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>0.25 % Trypsin-EDTA</td><td>Gibco</td><td>25200-072</td><td></td></tr></tbody></table>

generation of neurospheres from mixed primary hippocampal and cortical neurons isolated from e14-e16 sprague dawley rat embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

In this protocol, a simple strategy has been elucidated in which variable cell plating densities from two different neural screening platforms are obtained. Figure 1A,B illustrates the adherence of cells after 4 h of plating the neurons in high and low density plated cells, respectively. On observing the proper adherence of the neurons as shown in Figure 1, the plating medium was replaced by maintenance medium in...

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Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

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Research

JoVE Journal

Neuroscience

8.7K Views.  CSIR-Indian Institute of Chemical Biology. Het algemene doel van deze procedure is om de ontwikkeling van neurosferen aan te tonen van gemengde primaire hippocampal en corticale neuronen geïsoleerd van E14-E16 Sprague Dawley rat embryo, om een robuust en goedkoop platform te bieden voor het bestuderen van het effect van verschillende neurotherapeutische leads. Zoals u weet, dat de hersenen is een complex netwerk van neuronen geassocieerd met een groot aantal ondersteunende cellen. Om de hersenfunctie te begrijpen, wordt het grootste ...

Video: Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

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

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Author Spotlight: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

Source: Lopez-Ramirez, M. A. et al., <a href="https://app.jove.com/v/53617">Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates the generation and differentiation of primary neurospheres. Neural stem cells from zebrafish, in the presence of growth factors, proliferate into free-floating neurospheres with self-renewal capacity, which later differentiate into glial cells and neurons.

1. Preparations

	Prepare 10 ml of dissection medium, and add 200 &#181;l of 100x penicillin-streptomycin into 9.8 ml of Dulbecco&#39;s Modified Eagle ...

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Neuronal Culture Techniques

Advanced Neuronal Models

Generating 3D Co-culture Spheres of Astrocytes and Neurons to Induce Synapse Formation

Source: Cvetkovic, C., et al. <a href="https://app.jove.com/v/58034">Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells.</a> J. Vis. Exp. (2018).
This video demonstrates a procedure for co-culturing the astrocytes and neurons to form 3D spheres. These 3D spheres provide a distinct platform for examining synapse formation and neural network integration, thereby serving as a potential model for research in developmental neuroscience, neurodegenerative diseases, and neuropharmacological interventions.

1. Preparation and Maintenance of 3D Sphere Cocultures

	Dissociate hAstros from 3D aggregates in suspension.&#8203;
	
		Gently dissociate human astrocyte ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

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