Name: generation of neurospheres from mixed primary hippocampal and cortical neurons isolated from e14-e16 sprague dawley rat embryo
Uploaded: 2019-08-31T13:00:00
Description: Watch this Scientific Journal Video about Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo at JoVE.com

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

All experimental procedures involving animal were approved by the Institutional Animal Ethics Committee of CSIR-Indian Institute of Chemical Biology (IICB/AEC/Meeting/Apr/2018/1).
1. Reagent and media preparation
<ol>
	<li>Poly-D-lysine (PDL) solution: prepare PDL solutions at concentrations of 0.1 mg/mL in deionized water and store in 4 &#176;C until use.</li>
	<li>Dissociation medium: To 1 L of sterile, filtered deionized water, combine the following components in the respective concentrat...

<ol>
	<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>Masters, J. R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+line+misidentification:+the+beginning+of+the+end.">Cell line misidentification: the beginning of the end.</a> Nature Reviews Cancer. 10 (6), 441-448 (2010).</li><li>Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells, 2nd edition. , 339-370 (1998).</li><li>Geschwind, D. H., Konopka, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience+in+the+era+of+functional+genomics+and+systems+biology.">Neuroscience in the era of functional genomics and systems biology.</a> Nature. 461 (7266), 908-915 (2009).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nature Protocol. 1, 2406-2415 (2006).</li><li>Lu, Z. M., Piechowicz, M., Qiu, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simplified+Method+for+Ultra-Low+Density,+Long-Term+Primary+Hippocampal+Neuron+Culture.">A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture.</a> Journal of Visual Experiments. 109, e53797 (2016).</li><li>Kaneko, A., Sankai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Culture+of+Rat+Hippocampal+Neurons+at+Low+Density+in+Serum-Free+Medium:+Combination+of+the+Sandwich+Culture+Technique+with+the+Three-Dimensional+Nanofibrous+Hydrogel+PuraMatrix.">Long-Term Culture of Rat Hippocampal Neurons at Low Density in Serum-Free Medium: Combination of the Sandwich Culture Technique with the Three-Dimensional Nanofibrous Hydrogel PuraMatrix.</a> PLoS ONE. 9 (7), e102703 (2014).</li><li>Banker, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+interactions+between+astroglial+cells+and+hippocampal+neurons+in+culture.">Trophic interactions between astroglial cells and hippocampal neurons in culture.</a> Science. 209 (4458), 809-810 (1980).</li><li>Dotti, C. G., Sullivan, C. A., Banker, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+establishment+of+polarity+by+hippocampal+neurons+in+culture.">The establishment of polarity by hippocampal neurons in culture.</a> Journal of Neuroscience. 8 (4), 1454-1468 (1988).</li><li>Piret, G., Perez, M. T., Prinz, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Support+of+Neuronal+Growth+Over+Glial+Growth+and+Guidance+of+Optic+Nerve+Axons+by+Vertical+Nanowire+Arrays.">Support of Neuronal Growth Over Glial Growth and Guidance of Optic Nerve Axons by Vertical Nanowire Arrays.</a> ACS Applied Materials &amp; Interfaces. 7 (34), 18944-18948 (2015).</li><li>Campos, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurospheres:+Insights+biology+into+neural+stem+cell+biology.">Neurospheres: Insights biology into neural stem cell biology.</a> Journal of Neuroscience Research. 78 (6), 761-769 (2004).</li><li>Ahmed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Culture+of+Neural+Stem+Cells.">The Culture of Neural Stem Cells.</a> Journal of Cellular Biochemistry. 106, 1-6 (2009).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Molecular Neurobiology. 34 (3), 153-161 (2006).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Current Protocols in Immunology. 111, A3.B.1-A3.B.3 (2015).</li><li>Pradhan, K., 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=Neuro-Regenerative+Choline-Functionalized+Injectable+Graphene+Oxide+Hydrogel+Repairs+Focal+Brain+Injury.">Neuro-Regenerative Choline-Functionalized Injectable Graphene Oxide Hydrogel Repairs Focal Brain Injury.</a> ACS Chemical Neuroscience. 10 (3), 1535-1543 (2019).</li><li>Ray, B., Bailey, J. A., Sarkar, S., Lahiri, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+immunocytochemical+characterization+of+primary+neuronal+cultures+from+adult+rat+brain:+Differential+expression+of+neuronal+and+glial+protein+markers.">Molecular and immunocytochemical characterization of primary neuronal cultures from adult rat brain: Differential expression of neuronal and glial protein markers.</a> Journal of Neuroscience Methods. 184 (2), 294-302 (2009).</li><li>Robinson, A. P., Rodgers, J. M., Goings, G. E., Miller, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Oligodendroglial+Populations+in+Mouse+Demyelinating+Disease+Using+Flow+Cytometry:+Clues+for+MS+Pathogenesis.">Characterization of Oligodendroglial Populations in Mouse Demyelinating Disease Using Flow Cytometry: Clues for MS Pathogenesis.</a> PLoS ONE. 9 (9), (2014).</li><li>Osterberg, N., Roussa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+primary+neurospheres+generated+from+mouse+ventral+rostral+hindbrain.">Characterization of primary neurospheres generated from mouse ventral rostral hindbrain.</a> Cell and Tissue Research. 336 (1), 11-20 (2009).</li><li>Bernal, A., Arranz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nestin-expressing+progenitor+cells:+function,+identity+and+therapeutic+implications.">Nestin-expressing progenitor cells: function, identity and therapeutic implications.</a> Cellular and Molecular Life Sciences. 75 (12), 2177-2195 (2018).</li><li>Qu, Q. 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=High-efficiency+motor+neuron+differentiation+from+human+pluripotent+stem+cells+and+the+function+of+Islet-1.">High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1.</a> Nature Communications. 5, 3449 (2014).</li><li>Bradke, F., Dotti, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+neurons+retain+the+capacity+to+generate+axons+from+dendrites.">Differentiated neurons retain the capacity to generate axons from dendrites.</a> Current Biology. 10 (22), 1467-1470 (2000).</li><li>Theocharatos, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Progenitor+Cell+Proliferation+and+Neuronal+Differentiation+in+Enteric+Nervous+System+Neurospheres.">Regulation of Progenitor Cell Proliferation and Neuronal Differentiation in Enteric Nervous System Neurospheres.</a> PLoS ONE. 8 (1), (2013).</li><li>Binder, E., 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=Enteric+Neurospheres+Are+Not+Specific+to+Neural+Crest+Cultures:+Implications+for+Neural+Stem+Cell+Therapies.">Enteric Neurospheres Are Not Specific to Neural Crest Cultures: Implications for Neural Stem Cell Therapies.</a> PLoS ONE. 10 (3), e0119467 (2015).</li><li>Cordey, M., Limacher, M., Kobel, S., Taylor, V., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+the+Reliability+and+Throughput+of+Neurosphere+Culture+on+Hydrogel+Microwell+Arrays.">Enhancing the Reliability and Throughput of Neurosphere Culture on Hydrogel Microwell Arrays.</a> Stem Cells. 26 (10), 2586-2594 (2008).</li><li>Ladiwala, U., Basu, H., Mathur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembling+Neurospheres:+Dynamics+of+Neural+Progenitor/Stem+Cell+Aggregation+Probed+Using+an+Optical+Trap.">Assembling Neurospheres: Dynamics of Neural Progenitor/Stem Cell Aggregation Probed Using an Optical Trap.</a> PLoS ONE. 7 (6), (2012).</li></ol>

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

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

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

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

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

Watch this Scientific Journal Video about Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo at JoVE.com

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

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

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

Research

JoVE Journal

Neuroscience

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

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Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

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Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...