generation and differentiation of primary neurospheres from zebrafish neural stem cells

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

To prepare for neurosphere generation, fill each well of a 24-well plate with 300 microliters of fresh Z-condition medium. Then, add 200 microliters of the cell suspension to each well, seeding them at a density of approximately 500 cells per microliter. Afterwards, incubate the plate at 30 degrees Celsius in 5% carbon dioxide.
After one day of culture, view the cells in the 24-well plate under a microscope. Expect to observe single-cell suspensions at this stage. If any debris is seen to have collected in the center of a well, remove it by pipetting out approximately 100 microliters of medium, and replace this liquid by adding 100 microliters of fresh Z-condition medium. Once all debris has been removed, incubate the plate under the conditions described previously.
After an additional day of culture, transfer 250 microliters of cell suspension from a single well into a new, empty well. Repeat this step for all 24 wells. Then, add 250 microliters of fresh Z-condition medium to each well, and homogenize the suspensions by gently pipetting up and down. Incubate the plates again under the same conditions and repeat this expansion procedure after an additional day of culture. Expect to observe a progressive increase in the size of neurospheres on the third and fourth days of this period.
To begin passaging, after four days, remove 250 microliters of medium, but no neurospheres, from each well. Then use a 1-milliliter pipette to mechanically dissociate the neurospheres in the remaining medium volume. Proceed to pool the cell suspension from each dissociated well. Then, count the cells with a hemocytometer, and distribute 250 microliters of primary culture supernatant containing 800 cells per microliter into each well of a new 24-well plate. Continue by adding 250 microliters of Z-condition medium to each well and then incubating the plate as previously described.

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1. Preparations
<ol>
	<li>Prepare 10 ml of dissection medium, and add 200 &#181;l of 100x penicillin-streptomycin into 9.8 ml of Dulbecco&#39;s Modified Eagle Medium F12 (DMEM/F12).</li>
	<li>Prepare L-Cysteine solution: to 10 ml of water for tissue culture, and add 120 mg of L-cysteine. Store the L-cysteine solution at 4 &#176;C for up to 2 weeks or in aliquots at -20 &#176;C for up to 1 year.</li>
	<li>Prepare DNase I solution: to 1 ml of water for tissue culture, add 10 mg of DNase I. Store the DNase I solution at 4 &#176;C for up to 2 months.</li>
	<li>Prepare papain solution: to prepare papain solution, add 100 &#181;l papain (approximately 140 units), 100 &#181;l DNase I (1%), and 200 &#181;l L-cysteine (12 mg/ml) into 5 mL of DMEM/F12. Freshly prepare the papain solution every time and sterilize with a 0.22 &#181;m pore size filter before use.</li>
	<li>Prepare washing solution: to prepare 100 ml of washing solution, add 650 &#181;l glucose 45%, 500 &#181;L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES 1 M and 5 ml fetal bovine serum (FBS) into 93.85 ml Dulbecco&#39;s phosphate buffered saline (DPBS) 1x. Sterilize the washing solution with a 0.22 &#181;m pore size filter before use. Store the washing solution at 4 &#176;C for up to 2 months.</li>
	<li>Prepare insulin solution: to prepare 2 ml of insulin solution, add a 25 &#181;l drop of 10 N sodium hydroxide (NaOH) and 100 mg insulin into 2 ml of water for tissue culture.</li>
	<li>Prepare epidermal growth factor (EGF) and fibroblast growth factor (FGF) solutions: Dissolve both mitogens in DMEM/F12 at 100 &#181;g/ml concentration. Store as 10 &#181;l aliquots at -20 &#176;C.</li>
	<li>Prepare B-27 and N-2 media: store B-27 and N-2 supplements at -20 &#176;C as 500 &#181;l and 1 ml aliquots, respectively.</li>
	<li>Prepare Z-differentiation condition medium: to prepare 100 ml of Z-differentiation condition medium, add 40 &#181;l insulin (50 mg/ml), 500 &#181;l B-27, 1 ml N-2, 650 &#181;l glucose 45% and 1 ml of 100 x penicillin-streptomycin into 97.81 ml of DMEM/F12. Sterilize the Z-differentiation condition medium with a 0.22 &#181;m pore size filter before use. Store the Z-differentiation condition medium at 4 &#176;C for up to 1 week.</li>
	<li>Prepare Z-condition medium: to prepare 50 ml of Z-condition media, add 10 &#181;l of EGF and 10 &#181;l of FGF into 50 ml of sterile Z-differentiation condition medium (20 ng/ml). Store the Z-condition medium at 4 &#176;C for up to 1 week.</li>
	<li>Prepare coating solution for differentiation culture: for 5 ml extracellular coating solution, add 100 &#181;l of the extracellular matrix solution (e.g., Matrigel) into 4.9 ml of DMEM/F12. Thaw extracellular matrix solution at 4 &#176;C on ice. Once defrosted, keep at 4 &#176;C for up to 2 weeks.</li>
</ol>
2. Dissection of the Adult Zebrafish Brain
<ol>
	<li>Prepare a dissection bed by filling a 100 mm x 15 mm Petri dish with gel ice packs. Then place the lid on the petri dish and incubate at -20 &#176;C until the gel freezes. On top of the lid place a square of clean filter paper and wrap both the filter paper and petri dish with plastic film.</li>
	<li>Clean and sterilize all the microdissection instruments by 70% ethanol or heat before each use. Place all sterilized dissection instruments near the dissecting microscope and, right before euthanasia, place the dissection bed under the microscope with optical fiber illumination.</li>
	<li>Collect 2 adult zebrafish for a whole brain neurosphere preparation; and 3 to 4 zebrafish to generate neurospheres from dissected brain regions.</li>
	<li>Euthanize adult zebrafish (8-12 months old) using a protocol approved by the Institutional Animal Care and Use Committee. Next, immerse the fish in 75% ethanol for 5-10 sec and quickly place in the dissection bed followed by decapitation at the level of the gills using a surgical blade.
	<ol>
		<li>To euthanize animals, administer an overdose (300 mg/L) of tricaine methane sulfonate until the animal&#39;s heartbeat gradually slows down and circulation stops, then immerse in iced water.</li>
	</ol>
	</li>
	<li>Turn the head dorsal side down, and using the scissors make a longitudinal cut from the cut side to the mouth. Using the forceps expose the base of the skull and remove all the adjacent tissue. Cut the lateral walls of the skull from the beginning of the spinal cord towards the tectum.</li>
	<li>Using the scissors, cut and remove the optic nerves and then remove both sides of the most lateral part of the skull at the level of the tectum. Turn the head ventral side up. Using forceps, peel off the rest of the most apical part of the skull.</li>
	<li>Transfer the brain along with the remaining part of the skull into a new dish with the dissection medium (1.1). Clean the brain tissue in the dissection medium using the micro knife&#39;s plastic handle, keeping all brain structures intact.</li>
	<li>From this point, continue the protocol using the whole zebrafish brain.
	<ol>
		<li>Alternatively adapt this protocol to specific brain regions to generate neurospheres from the whole zebrafish brain or from the telencephalon, tectum/diencephalon or cerebellum dissected with a fresh scalpel. Use a neural specific fluorescent zebrafish neural transgenic line to dissect the brain region of interest according to&#160;Figure 1.</li>
	</ol>
	</li>
</ol>
3. Single Cell Dissociation of Adult Brain
<ol>
	<li>Perform all subsequent work in a biological safety cabinet. Transfer the brain tissue into a 1.5 ml tube containing 800 &#181;l of dissection medium (prepared in step 1.1). Remove the dissection medium by pipetting, without touching the brain tissue.</li>
	<li>Add 500 &#181;l of papain solution (step 1.4) and digest the brain tissue at 37 &#176;C for 10 min. Do not incubate longer than 15 min as it could decrease cell viability.</li>
	<li>After incubation, transfer the brain tissue with 500 &#181;l of the papain solution into a 15 ml conical tube using a 1,000 &#181;l pipet tip cut at ~0.25 inches from the bottom. Gently dissociate the brain tissue by slowly pipetting up and down 10 times with the same tip. Do not pipet up and down more than 15 times and do not generate air bubbles during this step, as it could alter cell viability.</li>
	<li>Incubate the brain tissue again at 37 &#176;C for 10 min followed by pipetting up and down 10-13 times using an uncut 1,000 &#181;l regular tip. Do not pipet up and down more than 15 times, to avoid cell death.</li>
	<li>Stop the enzymatic digestion by adding 2 ml of washing solution (step 1.5) and centrifuge the cell suspension at 800 x g for 5 min at room temperature&#160;(RT). Carefully decant the supernatant and resuspend the pellet in the remaining solution by tapping the tube vigorously with a finger and then by pipetting up and down with a 1,000 &#181;l regular tip. Next, add 2 ml of washing solution.</li>
	<li>At this stage, check under the microscope that a single cell suspension has been obtained. Centrifuge the cell suspension again at 800 x g for 5 min at RT. Remove the supernatant and re-suspend the pellet using 1 ml of fresh Z-condition medium (step 1.10).</li>
	<li>Stain cells by using trypan blue&#160;and count the living cells using a hemocytometer by excluding blue dead cells. Prepare a 24-well plate with 200 &#181;l of cell suspension and 300 &#181;l of fresh Z-condition medium in each well. Seed cells at a density of ~500 cells/&#181;l. Maintain cultured cells in an incubator at 30 &#176;C in 5% CO2, since zebrafish brain-derived neurospheres do not grow well at 37 &#176;C.</li>
</ol>
4. Generation of Primary Neurospheres
<ol>
	<li>After 1 day&#160;in vitro&#160;(DiV1), observe the single cell suspension obtained from the adult whole brain under the microscope and note whether debris has accumulated in the center of the well. Carefully remove any debris by pipetting approximately 100 &#181;l of medium (less if possible). Next add 100 &#181;l of fresh Z-condition medium. Single cell suspensions should be observed at this time (Figure 2A&#160;DiV1).</li>
	<li>After 2 days&#160;in vitro&#160;(DiV2), expand the cultured cells. Using a 1,000 &#181;l pipette with the tip cut, collect and transfer 250 &#181;l of cell suspension from each well into a new well. Add 250 &#181;l of fresh Z-condition medium into all wells. Before expanding the cultured cells, homogenize the suspension very gently by pipetting up and down throughout the well.</li>
	<li>Repeat steps 4.1 and 4.2 for the following 4 days&#160;in vitro&#160;(DiV4). A progressive increase in size of neurospheres should be visible at the center and edges of the well at DiV3 and DiV4 (Figure 2B&#160;and&#160;C).&#160; 
	NOTE: Primary neurospheres can be processed for passage or differentiation to assess multipotency. Alternatively, they can be processed for nucleofection or lipo-transfection&#160;to characterize the role of specific genes during the differentiation process.</li>
</ol>
5. Passaging of Primary Neurospheres
<ol>
	<li>Remove 250 &#956;l of tissue culture from each well and mechanically dissociate DiV4 neurospheres with a 1 ml pipette. Do not pipet up and down too rapidly as air bubble formation may increase cell death.</li>
	<li>Count the cells with a hemocytometer and distribute 800 cells/&#181;l in 250 &#181;l of primary culture supernatant into each well of a 24-well plate.</li>
	<li>Add 250 &#181;l of Z-condition medium to each well.</li>
	<li>After 2 days&#160;in vitro&#160;(DiV2), expand the cultured cells as reported in step 4.2 and 4.3.</li>
</ol>
6. Differentiation of Primary Neurospheres
<ol>
	<li>Sterilize cover glasses by immersion in 70% ethanol for 10 min, then dry cover glasses at RT by placing them into each well of a 12-well plate.</li>
	<li>Add 300 &#181;l of extracellular coating solution (step 1.11) at the center of the cover glass in such a way that it can expand widely and cover the entire cover glass. Then, incubate at 37 &#176;C for 1 h (using the humidified cell culture incubator).</li>
	<li>Remove the extracellular coating solution after incubation, and dry the cover glass for 2 hr at 37 &#176;C.</li>
	<li>Remove 250 &#181;l of tissue culture from each well. Mechanically dissociate all the DiV4 primary neurospheres per well with a 1 ml pipette (with wider-end tip) by pipetting up and down.</li>
	<li>Seed dissociated neurosphere cell suspensions at a cell density of 4 x 103&#160;cells/ml on previously prepared extracellular matrix-coated cover glasses (step 6.1-6.3) and keep for at least 30 min, at 30 &#176;C in 5% CO2,&#160;until attached to the substrate. Then, remove the culture medium and rapidly add 1 ml of pre-warmed fresh Z-differentiation condition medium (step 1.9) before culturing the cells for 24 hr&#160;at 30 &#176;C in 5% CO2.</li>
	<li>Every two days, replace half of the Z-differentiation condition medium during the time of cell differentiation.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DPBS 1X</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 1X</td>
			<td>Life Technologies</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine hydrochloride monohydrate&#160;</td>
			<td>Sigma</td>
			<td>C6852-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td>N-2 supplement (100x) liquid&#160;</td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>Life Technologies</td>
			<td>15630</td>
			<td>1M</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose 45%&#160;</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin&#160;</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Life Technologies</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF-basic&#160;</td>
			<td>Peprotech</td>
			<td>&#160;100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Human EGF&#160;</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Sigma</td>
			<td>I5500-50 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma</td>
			<td>DN25-10mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain&#160;</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>Becton Dickinson</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA&#160;</td>
			<td>TCI</td>
			<td>P0018</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>AmericanBio</td>
			<td>AB11072-04000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine MS-222</td>
			<td>Sigma</td>
			<td>A5040</td>
			<td>Stock solution of 4 mg/ml.&#160;</td>
		</tr>
		<tr>
			<td>Trycold gel&#160;</td>
			<td>Sigma</td>
			<td>TGP8</td>
			<td>gel pack</td>
		</tr>
		<tr>
			<td>Trypan blue stain&#160;</td>
			<td>Life Technologies</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

<img alt="Figure 1" src="/files/ftp_upload/22347/22347_Figure1.jpg" /> 
Figure 1: Differentiation of Zebrafish Brain-derived Neurospheres.&#160;(A-C,&#160;E) Phase contrast images of whole brain-derived neurospheres cultured in the Z-differentiation medium during 1 (A, DiVd1), 3 (B, DiVd3) and 4 (C&#160;and&#160;D, DiVd4) days. (E) D...

1. Preparations

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

Begin with a multiwell plate containing a growth medium enriched with growth factors.
Seed a single-cell suspension of zebrafish neural stem cells and incubate.&#160;
Initially, the stem cells utilize the growth factors and proliferate.
Remove debris and add the fresh medium to promote continuous cell proliferation and spontaneous aggregation to form free-floating primary neurospheres.
Next, transfer the neurospheres to a fresh well containing the growth medium.
Incubate under the same conditions to induce a progressive expansion of the neurospheres.
Pipette repeatedly to mechanically dissociate the neurospheres into small clusters.
Next, transfer them onto an extracellular matrix coated well, facilitating their adhesion.&#160;
Replace the growth medium with a growth factor-free differentiation medium containing insulin.
The absence of growth factors and the presence of insulin stimulate cell differentiation into glial cells and neurons with neuronal projections.

24 Görüntüleme. Zebra Balığı Nöral Kök Hücrelerinden Primer Nörosferlerin Üretilmesi ve Farklılaşması

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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.

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.

E14-E16 Sprague Dawley Sıçan Embriyosu İzole Karışık Primer Hipokampal ve Kortikal Nöronlardan Nörosfer Lerin Üretimi

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

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Nörobilim

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

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

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

Postnatal (P1−3) Fare Nörojenik Nişlerden Nörosferlerin İzolasyon ve Genişlemesi

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

Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the differentiation and proliferation potential of neural stem cells, as well as to generate cell lines than can be assayed over time. Also, neurospheres can create a niche (in vitro) that allows the modeling of the dynamic changing environment, such as varying growth factors, hormones, neurotransmitters, among others. Microtus ochrogaster (prairie vole) is a unique model for understanding the neurobiological basis of socio-sexual behaviors and social cognition. However, the cellular mechanisms involved in these behaviors are not well known. The protocol aims to obtain neural progenitor cells from the neurogenic niches of the adult prairie vole, which are cultured under non-adherent conditions, to generate neurospheres. The size and number of neurospheres depend on the region (subventricular zone or dentate gyrus) and sex of the prairie vole. This method is a remarkable tool to study sex-dependent differences in neurogenic niches in vitro and the neuroplasticity changes associated with social behaviors such as pair bonding and biparental care. Also, cognitive conditions that entail deficits in social interactions (autism spectrum disorders and schizophrenia) could be examined.

We established the conditions to culture neural progenitor cells from the subventricular zone and dentate gyrus of the adult brain of prairie voles, as a complementary in vitro study, to analyze the sex-dependent differences between neurogenic niches that could be part of functional plastic changes associated with social behaviors.

Yetişkin Çayır Voles Nörojenik Nişler Türetilmiş Nörosferkültürü

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the ...

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

TRANSKRIPT

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells