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>
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The authors declare no competing financial interests.

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

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

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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...

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

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

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

The overall goal of this procedure is to demonstrate the development of neurospheres from mixed primary hippocampal and cortical neurons isolated from E14-E16 Sprague Dawley rat embryo, in order to offer a robust and low-cost platform for studying the effect of various neurotherapeutic leads. As you know, that brain is a complex network of neurons associated with large number of supporting cells. To understand the brain function, often most of the research is re-initiated from in vitro experiments, which involves commercially available neural lineage derived cell lines.However, this cell lines are transformed cell lines that suffer from genotypic and phenotypic variations. To address this issues, primary neuron culture is the suitable approach. Here, we illustrated a simple and cost-effective protocol to culture primary neurons and built a robust screening platform for various neural therapeutics.The main advantage of this protocol is that just by modulating the cell plating densities, we can showcase two contrasting neuron culture platforms where low-density leads to prolonged primary neuron culture, and high-density generates spontaneous neurospheres. Take two 24-well plates. One for high-density, and another for low-density plating.Transfer 12 mm of sterile glass coverslips in the 24-well plates. Pour 300 microliters of PDL solution in each of the wells in a way that it fully covers the surface of the entire coverslip. Prepare PDL solution at a concentration of 0.1 mg/mL in deionized water.Wrap the plates with aluminum foils to prevent drying, and keep it in the CO2 incubator overnight. Next day, before plating, aspirate the PDL solution. Wash properly with sterile water for two to three times.Then add plating media, and return the plates to the incubator until plating. Sacrifice an E14-E16 timed pregnant Sprague-Dawley rat, Make a V-shaped cut in the abdominal area using sterile forceps and a pair of blunt-end scissors. Remove all the fetuses and carefully place them on the petri plate with cold HBSS solution.Take the embryos out of the embryonic sacs and wash them carefully in a separate petri dish in fresh, cold HBSS. Decapitate the head with the help of sterile scissors. Transfer the heads in the sterile 90 mm petri plates with cold HBSS.Under the stereo microscope, hold the head from the snout region with the sterile serrated forceps, and take out the brain by cutting the skin and the skull open. Remove all the meninges from the hemispheres and the midbrain by holding the brainstem. Carefully remove the intact hemispheres, resembling mushroom caps that contains the hippocampus and the cortex.Collect the hemispheres containing cortex and intact hippocampus in a 15 mL conical tube containing 10 mL of dissociation media. Allow the collected tissues to settle down, and aspirate the dissociation medium, leaving five to ten percent of medium in it. Again, add 10 mL of fresh dissociation medium to it, and repeat this step twice.Add 4.5 mL of dissociation medium and 0.5 mL of trypsin-EDTA solution. Keep it in the incubator at 37 degrees Celsius for 20 minutes for the digestion to proceed. Aspirate the medium and wash with 10 mL of dissociation medium and plating medium, consecutively.Resuspend them in 2.5 mL of plating medium. Keep a 90 mm sterile dish over the inverted cover of the dish and pour it in the base of 90 mm sterile dish. Titrate the digested tissues in the corner base of the dish, using 1000 microliter pipette tip, so as to occupy least volume.Pass the obtained cell suspension through the 70 micrometer cell strainer, excluding any chunks of tissue. Determine the density of viable cells using trypan blue dye exclusion, and count the number of cells in an automated cell counter. Dilute the number of cells obtained in a manner so as to plate 1.5 into 10 to the power of 5 cells per mL for high-density plating, and 20, 000 cells per mL for low-density plating in two separate tubes containing 30 mL of plating medium.Aspirate the previously added plating medium and plate 500 microliters of cells dispersed in plating medium in each well. After that, return the plates to the incubator for four hours. Four hours after plating, examine the cells for adherence under the microscope.After four hours of plating, in both high and low density plates, the neurons show good adherence to the plates. Replace the medium in each well with 500 microliters of fresh maintenance medium, and incubate it in an incubator at 37 degrees Celsius. We have to culture these neurons plated for at high-and low-density.While the low-density plated neurons can be used for prolonged cultures, up to 30 days, by changing the maintenance medium twice a week, the high-density plated neurons starts to spontaneously generate neurospheres, which came day after we maintained in ultra-low attachment plate. After 24 hours of plating, both high-and low-density plated neurons are observed to display healthy morphology. A phase contrast image of the low-density plated neurons, cultured for about seven days, is displayed here.The neurons display healthy morphology, and can be maintained up to 30 days with more vigorous routing and interconnections. The bar diagram here shows around 90 percent viability of the low-density plated neurons even after 30 days of culture, as determined by the MTT assay. The primary neurons here have been characterized by immunostaining with Tuj 1, a marker of differentiated neurons, and tau, a marker of neuronal axons.The purity of the neurons is confirmed by the absence of staining in non-neuronal markers GFAP for astrocytes and O4 for oligodendrocytes. In all the characterization studies, nuclei have been stained with Hoechst 33258. Here, the low-density seeded cells have been stained with astrocytic marker GFAP and neuronal marker Tuj 1 to check the purity of the culture up to seven days.It is observed that this protocol supports the preferential growth of neurons over astrocytes as indicated through the quantitative analysis. Nuclei have been stained with Hoechst 33258. Similarly, in the high-density seeded cells, the astrocytes have been stained with GFAP and neurons by Tuj 1.Here, in the quantitative analysis, around 17 percent astrocytes are observed as compared to 83 percent neurons. The nuclei have been stained with Hoechst 33258. After seven days, spontaneously formed multiple neurospheres in the high-density neurons are observed.Around eighth to tenth day of culture, in the high-density plated neurons, the neurospheres starts to form large projections and bridges consisting of radial glial-like extensions. The black arrows indicate the bridge between two newly formed neurospheres. Alive and dead cell assay have been performed on the neurospheres for 15 days.In dense Calcein AM staining, with absolutely no propidium iodide staining, indicates almost 100 percent viability of the neurospheres in culture. The line graph here indicates an expansion in the size of the neurospheres with the passage of number of days. This overly stained image of neurosphere indicates a rich population of neural progenitor cells through increased expression of NPC markers Nestin and Tuj 1.The neurospheres here show high amounts of astrocytes marked by the strong expression of GFAP, accompanied by an even higher expression of neuronal marker Tuj 1. The nuclei have been stained with Hoechst 33258. So I think our protocol is quite exciting and interesting, considering the fact that from a single strategy we are able to get two platforms:one 2-D and one 3-D.And this will be a great platform for screening different neurotherapeutic tracks. So I guess it's really really useful to all neuroscientists. Also another important aspect is that the neurospheres we opt in are extremely rich in neural progenitor cells, the NPCs.And they can be used to differentiate them into neurons and non-neuronal lineages. So, I feel if, really, people can master this technique by taking help from this video, it will be really really useful for everyone. Thank you.

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Research

JoVE Journal

Neuroscience

8.7K Görüntüleme.  CSIR-Indian Institute of Chemical Biology. Bu prosedürün genel amacı, çeşitli nöroterapötik yol ların etkisini incelemek için sağlam ve düşük maliyetli bir platform sunmak amacıyla, E14-E16 Sprague Dawley sıçan embriyosundan izole edilen karışık primer hipokampal ve kortikal nöronlardan nörosferlerin gelişimini göstermektir. Bildiğiniz gibi, bu beyin çok sayıda destekleyici hücreyle ilişkili karmaşık bir nöron ağıdır. Beyin fonksiyonlarını anlamak için, genellikle araştırmanın çoğu, ticari ola...