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>

<ol>
	<li>Giffard, R. G., Ouyang, Y. B., Squire, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture:+primary+neural+cells.">Cell culture: primary neural cells.</a> Encyclopedia of Neuroscience. , 633-637 (2009).</li><li>Huang, Y., Liu, Y., Zheng, C., Shen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+Cross-Contamination+and+Misidentification+of+278+Widely+Used+Tumor+Cell+Lines.">Investigation of Cross-Contamination and Misidentification of 278 Widely Used Tumor Cell Lines.</a> PLoS One. 12 (1), 0170384 (2017).</li><li>Lorsch, J. R., Collins, F. S., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixing+problems+with+cell+lines.">Fixing problems with cell lines.</a> Science. 346 (6216), 1452-1453 (2014).</li><li>Kaur, G., Dufour, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines:+Valuable+tools+or+useless+artifacts.">Cell lines: Valuable tools or useless artifacts.</a> Spermatogenesis. 2 (1), 1-5 (2012).</li><li>Capes-Davis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Check+your+cultures!+A+list+of+cross-contaminated+or+misidentified+cell+lines.">Check your cultures! A list of cross-contaminated or misidentified cell lines.</a> International Journal of Cancer. 127 (1), 1-8 (2010).</li><li>Frade, J. M., Ovejero-Benito, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cell+cycle:+the+neuron+itself+and+its+circumstances.">Neuronal cell cycle: the neuron itself and its circumstances.</a> Cell Cycle. 14 (5), 712-720 (2015).</li><li>Antoni, D., Burckel, H., Josset, E., Noel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+a+breakthrough+in+vivo.">Three-dimensional cell culture: a breakthrough in vivo.</a> International Journal of Molecular Science. 16 (3), 5517-5527 (2015).</li><li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Voloboueva, L., Sun, X., Ouyang, Y. B., Giffard, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture:+primary+neural+cells.">Cell culture: primary neural cells.</a> Reference Module in Neuroscience and Biobehavioral Psychology. , (2017).</li><li>Marzban, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+commitment+in+the+developing+cerebellum.">Cellular commitment in the developing cerebellum.</a> Frontiers in Cellular Neuroscience. 8, 450 (2014).</li><li>Sudarov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascl1+genetics+reveals+insights+into+cerebellum+local+circuit+assembly.">Ascl1 genetics reveals insights into cerebellum local circuit assembly.</a> Journal of Neuroscience. 31 (30), 11055-11069 (2011).</li><li>Rahimi-Balaei, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrin+II+Is+Ectopically+Expressed+in+Microglia+in+the+Cerebellum+of+Neurogenin+2+Null+Mice.">Zebrin II Is Ectopically Expressed in Microglia in the Cerebellum of Neurogenin 2 Null Mice.</a> Cerebellum. 18 (1), 56-66 (2019).</li><li>Rahimi-Balaei, M., Bergen, H., Kong, J., Marzban, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Migration+During+Development+of+the+Cerebellum.">Neuronal Migration During Development of the Cerebellum.</a> Frontiers in Cellular Neuroscience. 12, 484 (2018).</li><li>Buffo, A., Rossi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin,+lineage+and+function+of+cerebellar+glia.">Origin, lineage and function of cerebellar glia.</a> Progress in Neurobiology. 109, 42-63 (2013).</li><li>Alder, J., Cho, N. K., Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Precursor+Cells+from+the+Rhombic+Lip+Are+Specified+to+a+Cerebellar+Granule+Neuron+Identity.">Embryonic Precursor Cells from the Rhombic Lip Are Specified to a Cerebellar Granule Neuron Identity.</a> Neuron. 17 (3), 389-399 (1996).</li><li>Reemst, K., Noctor, S. C., Lucassen, P. J., Hol, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Indispensable+Roles+of+Microglia+and+Astrocytes+during+Brain+Development.">The Indispensable Roles of Microglia and Astrocytes during Brain Development.</a> Frontiers in Human Neuroscience. 10, 566 (2016).</li><li>Furuya, S., Makino, A., Hirabayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+culturing+cerebellar+Purkinje+cells+with+differentiated+dendrites+under+a+mixed+monolayer+setting.+Brain+research.">An improved method for culturing cerebellar Purkinje cells with differentiated dendrites under a mixed monolayer setting. Brain research.</a> Brain Research Protocols. 3 (2), 192-198 (1998).</li><li>Tabata, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reliable+method+for+culture+of+dissociated+mouse+cerebellar+cells+enriched+for+Purkinje+neurons.">A reliable method for culture of dissociated mouse cerebellar cells enriched for Purkinje neurons.</a> Journal of Neuroscience Methods. 104 (1), 45-53 (2000).</li><li>Marzban, H., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+promotes+the+development+of+deep+cerebellar+nuclear+neurons+in+dissociated+mouse+cerebellar+cultures.">Fibroblast growth factor promotes the development of deep cerebellar nuclear neurons in dissociated mouse cerebellar cultures.</a> Brain Research. 1141, 25-36 (2007).</li><li>Schaller, K. L., Caldwell, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+distribution+of+voltage-gated+sodium+channels+in+the+cerebellum.">Expression and distribution of voltage-gated sodium channels in the cerebellum.</a> Cerebellum. 2 (1), 2-9 (2003).</li><li>Alder, J., Cho, N. K., Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+precursor+cells+from+the+rhombic+lip+are+specified+to+a+cerebellar+granule+neuron+identity.">Embryonic precursor cells from the rhombic lip are specified to a cerebellar granule neuron identity.</a> Neuron. 17 (3), 389-399 (1996).</li><li>van der Valk, J., Gstraunthaler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fetal+Bovine+Serum+(FBS)+-+A+pain+in+the+dish.">Fetal Bovine Serum (FBS) - A pain in the dish.</a> Alternatives to Laboratory Animals. 45 (6), 329-332 (2017).</li><li>Froud, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development,+benefits+and+disadvantages+of+serum-free+media.">The development, benefits and disadvantages of serum-free media.</a> Developments in Biological Standardization. 99, 157-166 (1999).</li><li>Kwist, K., Bridges, W. C., Burg, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cell+passage+number+on+osteogenic+and+adipogenic+characteristics+of+D1+cells.">The effect of cell passage number on osteogenic and adipogenic characteristics of D1 cells.</a> Cytotechnology. 68 (4), 1661-1667 (2016).</li><li>Uysal, O., SevimLi, T., SevimLi, M., Gunes, S., Eker Sariboyaci, A., Barh, D., Azevedo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+tissue+culture:+the+base+of+biotechnology.">Cell and tissue culture: the base of biotechnology.</a> Omics Technologies and Bio-Engineering. , 391-429 (2018).</li><li>Seibenhener, M. L., Wooten, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+hippocampal+neurons+from+prenatal+mice.">Isolation and culture of hippocampal neurons from prenatal mice.</a> Journal of Visualized Experiments. (65), e3634 (2012).</li><li>Nelson, K. B., Bauman, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thimerosal+and+autism.">Thimerosal and autism.</a> Pediatrics. 111 (3), 674-679 (2003).</li><li>Marzban, H., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+promotes+the+development+of+deep+cerebellar+nuclear+neurons+in+dissociated+mouse+cerebellar+cultures.">Fibroblast growth factor promotes the development of deep cerebellar nuclear neurons in dissociated mouse cerebellar cultures.</a> Brain Research. 1141, 25-36 (2007).</li></ol>

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 a...

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.

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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&#8722;E18 mouse embryos yielded different cell types. Large projection neurons, such as CN neurons (E9&#8722;E12) and PCs (E10&#8722;E13), emerged early during cerebellar development. In mice, granule and Golgi cells arose between ~E13&#8211;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 gl...

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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19.7K Views.  University of Manitoba. 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.

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/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.

Modelowanie uszkodzenia neuronów w pierwotnych neuronach ziarnistych móżdżku myszy

1. Experimental Preparation
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Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

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

Izolacja i hodowla pierwotnych neuronów ziarnistych móżdżku myszy

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/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.

Izolowanie i hodowla mysich progenitorów neuronów ziarnistych móżdżku

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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Rozdziały w tym wideo

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

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary Culture of Mouse Dopaminergic Neurons

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

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

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Modelowanie uszkodzenia neuronów w pierwotnych neuronach ziarnistych móżdżku myszy

Izolacja i hodowla pierwotnych neuronów ziarnistych móżdżku myszy

Izolowanie i hodowla mysich progenitorów neuronów ziarnistych móżdżku