Name: primary culture of neurons isolated from embryonic mouse cerebellum
Uploaded: 2019-10-26T14:00:00.000+00:00
Duration: 8 min 9 s
Description: Watch this Scientific Journal Video about Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum at JoVE.com

These studies were supported by grants from the Natural Sciences and Engineering Research Council (HM: NSERC Discovery Grant # RGPIN-2018-06040), and Children Hospital Research Institute of Manitoba (HM: Grant # 320035), and the ALS Canada-Brain Canada Arthur J. Hudson Translational Team Grant (JK, HM).

All animal procedures were performed in accordance with institutional regulations and the Guide to the Care and Use of Experimental Animals from the Canadian Council for Animal Care and has been approved by local authorities (&ldquo;the Bannatyne Campus Animal Care Committee&rdquo;). All efforts were made to minimize the number and suffering of animals used. Adequate depth of anesthesia was confirmed by observing that there was no change in respiratory rate associated with manipulation and toe pinch or corneal reflex.
1. Preparation
NOTE: Schedule the provision of timed pregnant mice, based on the research plan, for post-conception E12&ndash;E18. Choice of the timing depends on the desired cell characteristics (see below). Prepare the cover slips and plates at least 2 days before the experiment. Poly-L-ornithine is used as a coating material to enhance cell attachment to the cover slips.
<ol>
<li>Coat the cover slips 2 days before cell isolation.
<ol>
<li>Place the round cover slips in a 24 well plate, under sterile conditions in a biosafety cabinet. Leave a gap between each cover slip to avoid any contamination.</li>
<li>Add 90 &micro;L of poly-L-ornithine (PLO, 500 &micro;g/mL) to the center of each cover slip. Close the cap and gently place the plate in a 37 &deg;C/5% CO2 incubator for 2 days. NOTE: A greater volume may spill off the cover slips during incubation. The cover slip does not need to be completely covered with PLO after placing a drop. During overnight incubation, PLO distributes equally to the edge of the cover slips.</li>
</ol>
</li>
<li>One day before cell isolation, prepare the culture medium I (DMEM/F-12 containing putrescine 100 &micro;M, sodium selenite 30 nM, L-glutamine 3.9 mM, gentamicin 3.5 &micro;g/mL, tri-iodothyronine (T3) 0.5 ng/mL, and N3 supplements [progesterone 4 &micro;M, insulin 20 &mu;g/mL, transferrin 20 mg/mL]) and seeding medium (culture medium I [without N3 and T3] containing 10% fetal bovine serum [FBS]) and store them in 4 &deg;C. Prepare the trypsin working solution (0.25%) in DMEM/F12 and keep it at 4 &deg;C. NOTE: The medium compositions are provided in Table 1.</li>
<li>On the day of cell isolation, place culture medium I and seeding medium in the incubator at 37 &deg;C. NOTE: Before starting the cerebellum isolation, make sure that all the tools and working surfaces are sterile.</li>
<li>Wash cover slips.
<ol>
<li>Take the 24 well plate out of the incubator.</li>
<li>Wash the cover slips in the 24 well plate 3x with double distilled water (DDW) in a biosafety cabinet under sterile conditions. Each time let the cover slips soak in DDW for 5 min before starting aspiration.</li>
<li>Leave the cover slips in a biosafety cabinet for at least 2 h to completely dry.</li>
</ol>
</li>
</ol>
2. Cerebellum collection
<ol>
<li>Prepare three 10 cm sterile plastic Petri dishes filled with ice-cold 1x phosphate-buffered saline (PBS), 3 Petri dishes filled with ice-cold 1x Hank&rsquo;s balanced salt solution (HBSS), and approximately 5 Petri dishes filled with ice-cold dissection medium (1x HBSS containing gentamicin 10 &mu;g/mL). Keep them on ice.</li>
<li>Anesthetize the E18 CD1 pregnant mouse with 40% isoflurane. Perform cervical dislocation on the mouse. Sterilize the abdomen with 70% ethanol solution.</li>
<li>Use a pair of scissors to make a skin incision from the pubic symphysis to the xiphoid process. Then hold the skin with forceps and open the abdominal cavity.</li>
<li>Excise the uterine horns with forceps and wash them 3x in the ice-cold 1x PBS on ice. NOTE: The following steps should all be conducted on ice to minimize the metabolic rate and prevent tissue and cell damage.</li>
</ol>
3. Dissecting the cerebellum
<ol>
<li>After the last washing step, using a pair of fine forceps separate the embryos from the uterus in 1x HBSS and transfer them to the ice-cold dissection medium. In the dissection medium, decapitate the embryos with scissors. Place the tissue in a clean dissection medium. NOTE: Using a stereomicroscope for microdissection is optional.</li>
<li>Hold the skull with fine forceps and cut through the calvarium with a pair of small scissors from the lateral aspect of the skull in a line from the foramen magnum to the external acoustic meatus and inferior border of the orbital cavity. NOTE: Taking this step exposes the cranial cavity at the level of the skull base and makes it easier to remove the brain.</li>
<li>Using a pair of fine forceps, remove the skull base and peel the skull away from the brain.</li>
<li>Carefully remove the meninges on the cerebellum, starting from the lateral surface of the middle cerebellar peduncle and pons.</li>
<li>Cut both cerebellar peduncles and separate the cerebellum from the rest of the brain (Figure 1).</li>
<li>Immediately place the collected cerebella in a sterile 15 mL conical tube filled with 14 mL of DMEM/F12 on ice.</li>
<li>Centrifuge the tube at 1,000 x g, 4 &deg;C for 1 min, 3x. Each time gently remove the supernatant with a pipette and resuspend the pellet in fresh, ice-cold DMEM/F12. NOTE: To avoid losing the samples, use the cabinet suction as little as possible.</li>
</ol>
4. Cerebellum dissociation
<ol>
<li>Add 2 mL of prewarmed (37 &deg;C) trypsin to the pellet from step 3.7 and gently pipet for adequate mixing.</li>
<li>Place the tube in a 37 &deg;C water bath for 12 min.</li>
<li>After incubation, bring the tube to a biosafety cabinet and add 10 mL of DMEM/F12 to inactivate the trypsin.</li>
<li>Centrifuge the mixture at 1,200 x g for 5 min. Discard the supernatant and resuspend the pellet in fresh DMEM/F12. Repeat 3x.</li>
<li>Prewet a sterile plastic transfer pipet with DMEM/F12.</li>
<li>After washing and the final centrifugation, add 3.5 mL of DNase working solution (1 mL of DNase I stock solution [0.05% DNase + 12 mM MgSO4 + 1x HBSS] in 500 &micro;L of heat-inactivated FBS and 2 mL of DMEM/F12) to the pellet in the same tube.</li>
<li>Triturate the tissue with a pipet at least 30x until the mixture attains a homogenous milky color.</li>
</ol>
5. Cell collection
<ol>
<li>Add 10 mL of ice-cold DMEM/F12 to the mixture.</li>
<li>Centrifuge the sample at 1,200 x g, 4 &deg;C for 5 min.</li>
<li>Carefully remove the supernatant without disturbing the pellet.</li>
<li>Remove the seeding medium from the incubator.</li>
<li>Add 500 &micro;L of prewarmed seeding medium to the pellet and mix it very well using a pipet to resuspend the cells.</li>
<li>Count the cells using a hemocytometer.</li>
<li>Dilute the cell suspension with the seeding medium to a density of 5 x 105 cells/mL.</li>
<li>Under a biosafety cabinet, add 90 &micro;L of the diluted mixture to each well at the center of the cover slip. NOTE: Do not load the particles that did not suspend in the DNase.</li>
<li>Place the plate in the incubator at 37 &deg;C for 3&minus;4 h.</li>
<li>After incubation, add 500 &micro;L of prewarmed culture medium I to each well and place the plate back into the incubator (37 &deg;C).</li>
</ol>
6. Treatment of the recovered cells
<ol>
<li>After 7 days, replace the old medium with fresh culture medium II (culture medium I supplemented with cytosine arabinoside [Ara-C, 4 &mu;M] and 100 &mu;g/mL bovine serum albumin [BSA]; see Table 1). NOTE: This step is critical to avoid growth of nonneuronal cells.</li>
<li>Monitor the culture medium once a day. If the pH changes (indicated by a marked change in color, usually yellower), remove half (almost 250 &micro;L) of the old medium from all wells and add 300 &micro;L of prewarmed culture medium I to each of them to avoid nutrient loss. NOTE: Phenol red in culture medium is a good indicator of the pH of the medium and activity of the cells. Try to minimize the exposure time of the cells to out of incubator conditions. This prevents any stress that might affect their viability.</li>
</ol>
7. Cell collection and fixation
NOTE: Depending on the experimental design, the cells can be collected at any day, at any timepoint.
<ol>
<li>Prepare a separate 24 well plate with the corresponding numeric organization and add 100 &micro;L of 4% paraformaldehyde (PFA) to each well.</li>
<li>To harvest cells on the desired days (depending on the experimental protocol), gently remove the cover slips from the wells of the original culture plate and place them in the corresponding wells of the PFA-filled plate.</li>
<li>Add PFA to the wells to completely immerse the cover slips. NOTE: To avoid cell detachment from the cover slip, do not add the PFA directly on the cover slip.</li>
<li>Keep the PFA plate at 4 &deg;C for 30&minus;120 min.</li>
<li>After incubation, restore the plate to room temperature.</li>
<li>Gently wash the cover slips 3x for 5 min with 1x PBS.</li>
<li>Proceed to the immunostaining process. NOTE: In this study, a fluorescence microscope equipped with a camera was used to capture the images and assembled into montages using an image editing software application.</li>
</ol>

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The authors have nothing to disclose.

The use of primary cultures is a well-known method applicable for all types of neurons17,18,19. In the presented protocol, we explain how to isolate cerebellar neurons and maintain their viability with optimum survival in vitro for a maximum of 3 weeks. Primary culture of cerebellar cells, which were isolated at E15&minus;E18, confirms the collection of three classes of large neurons: PCs, Golgi cells, and CNs. The cell bodies and projections of Golgi cells (some of which emerge at E19&minus;P5) and soma of granule cells can be detected by neurogranin (NRGN) and SMI32 antibodies, respectively, within 21 days of culture19,21.
A critical factor for the survival and maintenance of cerebellar neurons is the type of culture medium used. FBS supplemented medium has been a standard condition for cell lines and primary culture for decades. However, ethical concerns, variability in serum composition, and its potential to be a source of contamination, have led to some caution22. Recently, supplement-enriched DMEM/F-12 medium was found to reduce the gap between physiological conditions and in vitro neuronal models. The medium suggested by Furuya et al. enhanced survival rate of PCs and improved their dendrite differentiation compared with the widely used basal medium eagle (BME)-based serum-free medium17,18,23. This medium has become the basis of chemically defined media implemented in cerebellum cell primary culture.
Primary cell culturing of neurons has limitations. As in any in vitro culture, cells can survive for a limited period of time. Without exception, primary neuronal cells cultured in vitro can only be passaged a limited number of times. Excessive passaging will affect cellular health, function, and phenotype, and as such render variable experimental results. Therefore, neuronal/nonneuronal primary culture-based experiments usually take place within the first 3 weeks of starting the culture24. The in vitro conditions have not yet been sufficiently optimized to serve all cells of the nervous system simultaneously, in a way similar to their natural environment. For example, using Ara-C to inhibit proliferation of nonneuronal cells is quite common in studies where achieving a higher survival rate of neuronal cells is a priority. Thus, the quality of primary cultures relies on methods that artificially rebalance the in vitro cell population in favor of the cell types of interest25,26. Despite its limitations, primary cell culture is an excellent approach to study cellular mechanisms, signaling pathways, and pheno- and genotypic changes under conditions where optimized cellular growth (subject to the specific research aims) is carefully characterized and compared to the in vivo situation. In addition, primary cerebellar culture as a simplified model system provides a controlled microenvironment to investigate the plethora of morphological and physiological effects mediated by the numerous molecules affecting neuronal cells27,28. However, to what extent the physiological properties of these cells are preserved outside of the in vivo setting remains to be fully elucidated.
The presented method is a cost-effective general protocol for culturing primary cells. It provides broader options for manipulating cells and testing drugs without changing the nature of the cells, which is crucial to (pre-)clinical trials.

For several decades, cell lines have been widely used as a high throughput tool in preclinical studies and biological research. Cost-effectiveness, fast growth, and reduction of live animal use are some benefits of using these cells. However, genetic alterations and phenotypical changes accumulate after several passages in vitro1. Misidentification of cell lines and genetic dissimilarity from primary cells can lead to irreproducible experiments and false conclusions2,3,4,5. Therefore, in spite of some similarities to differentiated cells such as neurons (e.g., neurotransmitters, ion channels, receptors, and other neuron-specific proteins), neuronal cell lines cannot replicate the full phenotype of neurons. Using mature neurons is another option; however, these cells are non-dividing postmitotic cells that are difficult to propagate in culture. Moreover, re-entry into the cell cycle may precipitate apoptosis6.
Three-dimensional (3D) cell culture, organotypic slice cultures, and organoid cultures have been developed to provide an environment in which cells can arrange into a 3D form that mimics the in vivo setting. Thus, cell-to-cell communication, migration, invasion of tumor cells into surrounding tissues, and angiogenesis can be studied7. However, additional costs of using extra cellular matrix (ECM) proteins or synthetic hydrogels as a bedding, difficulty in imaging, and compatibility with high-throughput screening instruments are considerable drawbacks of 3D cell culturing. A major disadvantage of organotypic tissue slice culture is the use of a large number of animals and the adverse effects of axotomy, which leads to inaccessibility of targets and growth factors for axons, and consequently neuronal death8.
Therefore, an alternate approach, which avoids the problems with cell lines, the difficulty of growing mature cells, and complexity of tissues, is in vitro maturation of immature primary cells. Primary cells are derived directly from human or animal tissue and dissociated using enzymatic and/or mechanical methods9. The main principles of isolation, seeding, and maintenance in culture medium are similar regardless of the tissue source. However, the trophic factors necessary to promote proliferation and maturation are highly cell specific6.
Knowing the &lsquo;birthdate&rsquo; of each cerebellar cell type is a prerequisite for designing a primary culture experiment. In general, Purkinje cells (PCs) and the neurons of the cerebellar nuclei (CN), are born before the smaller cells, including interneurons (e.g., basket, stellate cells) and granule cells. In mice, PCs emerge between embryonic day (E)10&ndash;E13, whereas CN neurons at approximately E9&ndash;E1210.
Other cerebellar neurons are born much later. For example, in mice, the Golgi subpopulation of interneurons are generated from VZ at (~E14&minus;E18) and the remaining interneurons (basket cell and stellate cells) located in the molecular layer emerge from dividing progenitor cells in the white matter between early postnatal (P)0&ndash;P711. Granule cells are generated from the external germinal zone (EGZ), a secondary germinal zone that is derived from the rostral rhombic lip and goes through terminal division after birth. But before their precursors arise from the rhombic lip from E13&ndash;E16, the cells have already migrated rostrally along the pia surface to make a thin layer of cells on the dorsal surface of the cerebellum anlage. Nonneuronal macroglial cells such as astrocytes and oligodendrocytes, which originate from the ventricular neuroepithelium, are born at E13.5&minus;P0 and P0&minus;P7 respectively11,12,13,14,15. Microglia are derived from yolk-sac primitive myeloid progenitor cells between E8&ndash;E10 and after invasion into the central nervous system can be detected in the mouse brain by E916.
The method presented in this article is based on the one originally developed by Furuya et al. and Tabata et al.17,18, which was optimized for primary culturing of Purkinje cells derived from Wistar rat cerebella. We have now adapted this method and carefully modified it to study the growth of mouse cerebellar neurons19. Unlike in our new protocol, cold dissection medium is the main washing buffer used during dissection and dissociation steps before adding seeding medium in Furuya&rsquo;s protocol17. This buffer lacks the nutrition, growth factors, and hormones (all in Dulbecco&rsquo;s modified Eagle medium:nutrient mixture F-12 [DMEM/F12]) that are necessary to support cell growth and survival during the aforementioned steps. In addition, based on our extensive experience with murine primary cerebellar cultures, we have used 500 &mu;L of culture medium in each well (instead of 1 mL) and increased the tri-iodothyronine concentration to 0.5 ng/mL, which improves growth of neuronal cells, in particular those with a Purkinje cell phenotype, and promotes the outgrowth of dendritic branches in culture. The principal method featured in this article can be broadly applied to other small rodents (e.g., squirrels and hamsters) during embryonic development and can be used to study cerebellar neurogenesis and differentiation in the various embryonic stages, which differ between species.

<table><tbody><tr><td>Adobe Photoshop CS5 Version 12</td><td>Adobe Inc</td><td></td><td></td></tr><tr><td>Anti-Sodium Channel (SCN)PN4 (Nav1.6)</td><td>Sigma</td><td>S0438</td><td>3 &amp;mu;g/mL</td></tr><tr><td>Bovine Serum Albumin</td><td>Millipore Sigma</td><td>A3608</td><td></td></tr><tr><td>CALB1</td><td>Swant Swiss Antibodies (Polyclonal)</td><td>CB38</td><td>1/5000 dilution</td></tr><tr><td>CALB1</td><td>Swant Swiss Antibodies (Monoclonal)</td><td>300</td><td>1/1000 dilution</td></tr><tr><td>Cytosine &amp;beta;-D-arabinofuranoside or Cytosine Arabinoside (Ara-C)</td><td>Millipore Sigma</td><td>C1768</td><td></td></tr><tr><td>DNase I from bovine pancreas</td><td>Roche</td><td>11284932001</td><td></td></tr><tr><td>Dressing Forceps</td><td>Delasco</td><td>DF-45</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM-F12)</td><td>Lonza</td><td>12-719F</td><td></td></tr><tr><td>Fisherbrand Cover Slips: Circles</td><td>Fisher Scientific</td><td>12-545-81</td><td></td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15710-064</td><td></td></tr><tr><td>Hanks&amp;rsquo; Balanced Salt Solution (HBSS)</td><td>Gibco</td><td>14185-052</td><td></td></tr><tr><td>Insulin from bovine pancreas</td><td>Millipore sigma</td><td>I5500, I6634, I1882, and I4011</td><td></td></tr><tr><td>Large Scissor</td><td>Stoelting</td><td>52134-38</td><td></td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Metallized Hemacytometer</td><td>Hausser Bright-Line</td><td>3100</td><td></td></tr><tr><td>Microdissection Forceps</td><td>Merlan</td><td>624734</td><td></td></tr><tr><td>Pattern 5 Tweezer</td><td>Dixon</td><td>291-9454</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Fisher BioReagents</td><td>BP399-26</td><td></td></tr><tr><td>Poly-L-Ornithine</td><td>Millipore Sigma</td><td>P4638</td><td></td></tr><tr><td>Progesteron (P4)</td><td>Millipore sigma</td><td>P8783</td><td></td></tr><tr><td>PVALB</td><td>Swant Swiss Antibodies</td><td>235</td><td>1/1500 dilution</td></tr><tr><td>Samll Scissor</td><td>WPI Swiss Scissors, 9cm</td><td>504519</td><td></td></tr><tr><td>Sodium Selenite</td><td>Millipore Sigma</td><td>S9133</td><td></td></tr><tr><td>Transferrin</td><td>Millipore Sigma</td><td>T8158</td><td></td></tr><tr><td>Tri-iodothyronine (T3)</td><td>Millipore Sigma</td><td>T2877</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>Zeiss Fluorescence microscope</td><td>Zeiss</td><td>Z2 Imager</td><td></td></tr></tbody></table>

primary culture of neurons isolated from embryonic mouse cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Based on the different birthdates of neuronal subtypes in the cerebellum, cultures from E12−E18 mouse embryos yielded different cell types. Large projection neurons, such as CN neurons (E9−E12) and PCs (E10−E13), emerged early during cerebellar development. In mice, granule and Golgi cells arose between ~E13–E18 and underwent terminal divisions up to postnatal week 4.
Replacing old medium I with fresh culture medium II at days in vitro (DIV) 7 will eventually prevent glial cell proliferation. The interneurons of the molecular layer, such as basket and stellate cells, differentiate after birth. Therefore, most of the cells in the culture medium at E18 were expected to be a combination of PCs, granule cells, CN, and some Golgi cells. Calbindin 1 (CALB1), a specific marker of PCs, was used to track the morphological changes during the 21-day time course. On DIV 0, the cell bodies of PCs were detectable (e.g., 10−11 h of incubation), but the outgrowth of neurite had not yet started (Figure 2A). By DIV 3, the axonal extension showed progress, while dendritic processes had just started. This status remained almost unchanged until the second week in vitro (WIV) (Figure 2B–D). On DIV 10, a few sparse branches and primary dendrites with spines started growing (Figure 2E). Sprouting of the new primary dendrites occurred continuously after DIV 14. Therefore, after DIV 14, the number of secondary and tertiary dendrites increased and developed wide branches at DIV 21 (Figure 2F,G).
It is important to know that the timing of the morphological changes in vitro may not follow the same pattern as in vivo. Following these results, in another experiment the dissociated cerebellar primordia from E12, E13, E14, and E15 were cultured for 3 weeks. The cultured PCs from E12 and E13 did not develop any dendritic outgrowth on DIV 18 except axonal extensions (Figure 3A,B). However, after the same period of time, the dissociated cerebellar primordium cultures from E14 and E15 showed dendritic outgrowth and arborization (Figure 3C,D). This growth rhythm variation among neural cells in vitro sheds light on a big change that happened in their natural environment between the early and late stage of cerebellar development, which needs to be applied in vitro for medium optimization.
Along with PCs, there are other neuronal cell types in the cerebellum that develop during dissociated primary cerebellar cultures that can be detected using specific neuronal markers. Double immunofluorescence labeling for CALB1 and a calcium-binding albumin protein, parvalbumin (PVALB), showed that CALB1 expression was exclusively restricted to PCs in primary cerebellar cultures, while PVALB was expressed in CALB1+ neurons (i.e., PCs) and PVALB+/CALB1– neurons, which are molecular layer interneurons (basket/stellate cells) (Figure 4A–C). The alpha subunit (Nav1.6) of the voltage-gated sodium channel (SCN) is a marker for granule cells and PCs in the cerebellum20. Double labeling with anti-SCN and anti-CALB1 shows the colocalization of granule cell bodies and PCs in culture medium on DIV 21 (Figure 4D–F). For other specific cerebellar cell types from dissociated primary cerebellar cultures, please see Marzban and Hawkes19.
<img alt="Anatomy diagram; mouse brain lobes labeled: cerebellum, midbrain, forebrain, medulla oblongata." class="xfigimg" src="/files/ftp_upload/60168/60168fig01.jpg" data-altupdatedat="1747736610000" data-alt="Figure 1"> 
Figure 1: Dorsal aspect of the mouse brain outlining the cerebellum at E18. The meninges are indicated by yellow squares. The location of the cerebellum is outlined by the yellow line, which is limited rostrally by the midbrain and caudally by the medulla oblongata (outlined by brown dashed lines). The cerebellum vermis and hemisphere are shown by brown dashed lines. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60168/60168fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Neuronal development over days; fluorescence microscopy; CALB marker; axonal and dendritic growth." class="xfigimg" src="/files/ftp_upload/60168/60168fig02.jpg" data-altupdatedat="1747736610000" data-alt="Figure 2"> 
Figure 2: Development of Purkinje cells (PCs) derived from the mouse cerebellum at E18 in primary culture for days in vitro (DIV) 0–21. (A) PC somata (arrowheads) were labeled by immunofluorescence using anti-calbindin 1 (CALB1) at DIV 0 (after 10 h). (B) The first axonal extension (arrow) and early dendritic outgrowth (arrowhead) appear on DIV 3. The PCs’ dendritic outgrowth and development (arrowhead) continue on DIV 5 (C) and DIV 7 (D). The PCs’ dendritic branches are clearly distinguishable on DIV 10 (E) and DIV 14 (F) (arrowheads) and elaborate dendritic trees are detectable at DIV 21 (arrowhead) (G). Scale bars: A = 100 μm (applies to panels B, C, D, E and F); G = 50 μm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60168/60168fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Neuronal development stages, CALB1 expression, fluorescence microscopy, embryonic mouse neurons." class="xfigimg" src="/files/ftp_upload/60168/60168fig03.jpg" data-altupdatedat="1747736610000" data-alt="Figure 3"> 
Figure 3: Development of Purkinje cells (PCs) derived from the mouse cerebellar primordium at E12, E13, E14, and E15 after 18 days in primary culture (DIV 18). The axons are the only extensions (arrow) from the PC somata in primary culture of the E12 and E13 cerebella primordium after 18 days in vitro (DIV 18) (A,B). The PCs’ dendrite outgrowth and extension develop by DIV 18 (arrowhead) only from E14 (C) and E15 (D) cerebellar primary cultures. Scale bar = 20 μm (applies to panels A−D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60168/60168fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Confocal microscopy images of CALB1, PVALB, SCN neuron interactions; fluorescence labeling." class="xfigimg" src="/files/ftp_upload/60168/60168fig04.jpg" data-altupdatedat="1747736610000" data-alt="Figure 4"> 
Figure 4: Immunofluorescence labeling of PCs, GABAergic interneurons (basket and stellate cells), and granule cells from E18 mouse cerebellar primary culture at DIV 21. (A−C) Double labeling with anti-CALB1 (green) and anti-PVALB (red) shows PCs and a few interneurons (arrow) in contact with PC dendritic arbors. (D−F) Voltage-gated sodium channels (SCNs) in PC bodies with elaborated dendrites and numerous small granule cell bodies (arrowhead) are labeled with anti-SCN (red) and double-labeled with anti-CALB1 (green). Abbreviations: Purkinje cells = PCs; CALB1 = calbindin 1; PVALB = parvalbumin; SCN = voltage-gated sodium channel. Scale bar = 100 μm (applies to A−F). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60168/60168fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Basic medium</td>
			<td>Putrescine</td>
			<td>Sodium selenite</td>
			<td>L-glutamine</td>
			<td>Gentamicin</td>
			<td>T3</td>
			<td>N3</td>
			<td>BSA</td>
			<td>Ara-C</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Culture medium I</td>
			<td>DMEM/F12</td>
			<td>100 µM</td>
			<td>30 nM</td>
			<td>3.9 mM</td>
			<td>3.5 µg/mL</td>
			<td>0.5 ng/mL</td>
			<td>Progesterone 4 µM, Insulin 20 μg/mL, Transferrin 20 mg/mL</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Seeding medium</td>
			<td>DMEM/F12</td>
			<td>100 µM</td>
			<td>30 nM</td>
			<td>3.9 mM</td>
			<td>3.5 µg/mL</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>10% with culture medium I</td>
		</tr>
		<tr>
			<td>Culture medium II</td>
			<td colspan="7">Culture medium I</td>
			<td>100 μg/mL</td>
			<td>4 μM</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 1: Media compositions.

Watch this Scientific Journal Video about Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum at JoVE.com

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

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19.8K Views.  University of Manitoba. Primaire neuronale celcultuur is een ideaal modelsysteem om gescheiden neuronen in vitrocultuur te bestuderen. Het voordeel van het gebruik van deze methode is de mogelijkheid om de cellulaire kenmerken van gescheiden cellen, zoals mechanismen, functies en morfologie in vitro voorwaarden te behouden voor een paar weken zo dicht mogelijk bij in vivo conditie. Zet de ronde afdek slips in een 24-put plaat onder de biosafety kast.Voeg 90 microliter poly-L-ornithine toe aan elke afdeksel. S...

Video: Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Biology

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Embryonic Mouse Brain Mixed Cell Cultures to Study Infection and Innate Immunity

Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of neurons, astrocytes, oligodendrocytes, and microglia. Due to increasing efforts to replace and reduce animal use, a variety of in vitro cell culture systems have been developed to explore innate cell properties, which allow the development of therapeutics for CNS infections and pathologies. Whilst certain research questions can be addressed by human-based cell culture systems, such as (induced) pluripotent stem cells, working with human cells has its own limitations with regard to availability, costs, and ethics. Here, we describe a unique protocol for isolating and culturing cells from embryonic mouse brains. The resulting mixed neural cell cultures mimic several cell populations and interactions found in the brain in vivo. Compared to current equivalent methods, this protocol more closely mimics the characteristics of the brain and also garners more cells, thus allowing for more experimental conditions to be investigated from one pregnant mouse. Further, the protocol is relatively easy and highly reproducible. These cultures have been optimized for use at various scales, including 96-well based high throughput screens, 24-well microscopy analysis, and 6-well cultures for flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. This culture method is a powerful tool to investigate infection and immunity within the context of some of the complexity of the CNS with the convenience of in vitro methods.

Author Spotlight: Establishing Mixed Neuronal and Glial Cell Cultures from Embryonic Mouse Brains to Study Infection and Innate Immunity  

This protocol presents a unique way of generating central nervous system cell cultures from embryonic day 17 mouse brains for neuro(immuno)logy research. This model can be analyzed using various experimental techniques, including RT-qPCR, microscopy, ELISA, and flow cytometry.

Models of the central nervous system (CNS) must recapitulate the complex network of interconnected cells found in vivo. The CNS consists primarily of ...

 Primary Microglia and Neuron Co-Culture Protocol for CNS Interaction Studies

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Author Spotlight: In Vitro Co-Culture Model for Studying Microglia-Neuronal Interactions in Disease Conditions

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

Modeling Neuronal Injury in Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video demonstrates the generation of a neuronal injury model in mouse primary cerebellar granule neurons. It outlines the steps involved in inducing neuronal injury using NMDA, an excitatory neurotransmitter receptor agonist, and glycine, a co-agonist. NMDA and glycine bind to specific neuronal receptors, causing their overstimulation and resulting in a massive intracellular calcium influx, which leads to neuronal cell damage and death.

1. Experimental Preparation
NOTE: The following stock solutions can be prepared and maintained until use.

	Dissection Solution
	
		Dissolve 3.62 g of ...

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Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a> J. Vis. Exp. (2017)
This video demonstrates the technique for isolating and culturing primary mouse cerebellar granule neurons. Cells are treated with an antimetabolite to eliminate proliferating glial cells and produce a pure culture of cerebellar granule neurons.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Preparation
NOTE: ...

Neuronal Culture Techniques

Primary Neuronal Culture

Isolating and Culturing Murine Cerebellar Granular Neuron Progenitors

Source: Bovio, P., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56631">Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark.</a> J. Vis. Exp. (2018)
This video demonstrates the isolation and cultivation of cerebellar neural progenitor cells from a mouse pup&#39;s cerebellum. Cerebellar granule neuron progenitors are isolated and purified through differential adhesion, effectively removing astrocytes. These cultured progenitors subsequently differentiate into cerebellar granule neurons.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1.&#160;Preparations

	Preparations ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

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