Name: increasing durability of dissociated neural cell cultures using biologically active coralline matrix
Uploaded: 2020-06-03T13:30:00.000+00:00
Duration: 9 min 22 s
Description: Watch this Scientific Journal Video about Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix at JoVE.com

This work was funded by the KAMIN program of the Israeli Trade and Labor Ministry and by Qrons Inc., 777 Brickell Avenue Miami, FL 33131, US.

The use of animals in this protocol was approved by the National Animal Care and Use Committee.
NOTE: Calcium carbonated coral skeletons should be used in the crystalline form of aragonite. The coral types tested so far for neural cultures are Porites Lutea, Stylophora Pistillata, and Trachyphyllia Geoffroyi. The skeletons can be purchased whole or ground.
1. Cleaning the coral skeleton pieces
CAUTION: The following steps should be performed in a chemical hood at room temperature, because the solutions described below are hazardous and may cause burns and irritations.
<ol>
	<li>Use a hammer to break the coral skeleton and divide it into 0.5&#8211;2 cm fragments. In order to dissolve organic and non-organic residues, soak the coral skeleton fragments in 10% sodium hypochlorite solution for 10 min, then wash once with double distilled water (DDW).</li>
	<li>To remove the remaining organic residues, soak the fragments in a 1M NaOH solution for 5 min, then wash once with DDW. Continue with the removal of the organic deposits by soaking the fragments in 30% H2O2 solution for 10 min, then wash the fragments 3x with DDW.</li>
	<li>Remove as much excess DDW as possible and leave the coral fragments in the hood to dry (1&#8211;8 h).</li>
</ol>
2. Cleaning the glass coverslips
<ol>
	<li>Transfer the glass coverslips into a 100 mm glass Petri dish. Add 10 mL of 95% ethanol for 15 min.</li>
	<li>Remove the ethanol and wash with DDW 3x, waiting 10 min between each wash. Place the dish on an 80 &#176;C pre-warmed heating plate until the DDW evaporates. Gently stir the coverslips within the plate several times while drying to keep them from sticking to each other.</li>
	<li>Autoclave the coverslips.</li>
</ol>
3. Preparation of coral skeleton grains
<ol>
	<li>Grind the coral skeleton fragments using a mortar and pestle (manual grinding) until complete breakdown. The outcome is a mixture of grains with sizes ranging from 20 &#181;m&#8211;200 &#181;m.</li>
	<li>Alternatively, grind the coral skeleton fragments using an electrical grinding machine at a velocity of 1,000 rpm for 30 s (blade length = 6 cm; width = 0.5 cm&#8211;1.0 cm). The resulting grain size is similar to the size range produced by the manual grinding.</li>
</ol>
4. Purification of grains of a specific size range
NOTE: If control over the size of the grains in a matrix is desired, use the following filtration-based grain purification procedure.
<ol>
	<li>Transfer the grains onto a manual or electrical 40 &#181;m filter mesh strainer.</li>
	<li>Divide the grains into two specific ranges by sieving the grains through the strainer (if using an electrical strainer, the following conditions are recommended: shaking = 600 amplitudes/min, bouncing = 6/s). This procedure produces two groups of grain sizes, one &#60;40 &#181;m, and the second &#62;40 &#181;m. 
	NOTE: The two sizes can be determined by using strainers of varying meshes. If a more restricted range is desired, then re-sieve each group from step 4.2 through strainers with different meshes.</li>
	<li>Autoclave the grains.</li>
</ol>
5. Preparation of coral grain-coated dishes or coverslips
<ol>
	<li>For coating flasks, plates, or Petri dishes 
	NOTE: The following steps should be performed under sterile conditions.
	<ol>
		<li>Add the coral skeleton grains into a 20 &#181;g/mL poly-D-lysine (PDL) solution dissolved in Hanks&#39; solution. The concentration recommended is 5 mg/10 mg of grains per 1 mL PDL solution.</li>
		<li>Pour the solution into flasks and dishes (approximately 2 mL/25 cm2) and incubate overnight at 4 &#176;C. The grains sink and attach to the bottom.</li>
		<li>The next day, wash the flasks and dishes once with sterile DDW. Let the flasks and dishes dry in the hood. 
		NOTE: It is preferable to use freshly coated flasks and dishes. The coated flasks and dishes can be used up to a week after coating if preserved at 4 &#176;C. However, the effectiveness of the matrix may be compromised.</li>
	</ol>
	</li>
	<li>Coating glass coverslips (12 mm diameter, 0.17 mm thickness)
	<ol>
		<li>Add coral skeleton grains to DDW at any desired concentration. The common densities used for neural cells are 5 mg/mL and 10 mg/mL. Pour 40 &#181;L of the grain solution onto the center of the coverslip (Figure 4).</li>
		<li>Place the coverslips on a heating plate prewarmed to 80 &#176;C and wait for complete evaporation (usually 15 min). Under these conditions, the grains adhere to the coverslip. Autoclave the coated coverslips. Store in sterile conditions.</li>
		<li>A day before culture, place the coverslips on the lid of a sterile 24 well plate. Add 100 &#181;L of 20 &#181;g/mL PDL solution to each coverslip. Use the tip to ensure that the liquid covers the entire grain region and the rest of the coverslip surface.</li>
		<li>Cover the lid with the bottom of the plate. Wrap the sides of the plates with paraffin film. Incubate overnight at 4 &#176;C.</li>
		<li>The next day, wash once with DDW and dry in the hood. 
		NOTE: It is preferable to use freshly coated coverslips.</li>
	</ol>
	</li>
</ol>
6. Cultivation of hippocampal dissociated cells on coral skeleton grain-coated glass coverslips
NOTE: The method for hippocampal dissociated cell culture was modified from previously published procedures24,27. The preparation of the culture is described for four rat pups. The expected yield from each hippocampus is 1&#8211;1.5 x 106 cells.
<ol>
	<li>Setup
	<ol>
		<li>Prepare an empty 60 mm Petri dish. Pour 2 mL of minimal essential medium (MEM) into a 60 mm Petri dish and put on ice.</li>
		<li>Pour 2 mL of MEM into a 35 mm dish and put it in an incubator at 37 &#176;C, 5&#8211;10% CO2. Pour 2 mL of MEM into a 15 mL tube and keep it at 4 &#176;C.</li>
		<li>Pour 1 mL of First Day Medium into a 15 mL tube and put it in an incubator at 37 &#176;C, 5-10% CO2. 
		NOTE: The composition of the First Day Medium is described in the Table of Materials.</li>
		<li>Thaw 200 &#181;L of a 2.5% trypsin solution and incubate at 37 &#176;C.</li>
		<li>Prepare three glass Pasteur pipettes with 0.5 mm, 0.75 mm, and 1.0 mm diameter heads using a flame.</li>
		<li>Prepare adequate surgical tools: one big scissor, two small scissors, one big tweezer, four small tweezers, and one scalpel.</li>
		<li>Prepare a stereomicroscope in the hood.</li>
	</ol>
	</li>
	<li>Using a big scissor, sacrifice 0&#8211;3 day old Sprague Dawley rat pups by separating their heads from their bodies into an empty 60 mm Petri dish (step 6.1.1). Dispose of the bodies.</li>
	<li>Pick up the head by holding it from the mouth with a big tweezer. Cut the skin covering the skull with a small scissor.</li>
	<li>With a clean scissor, enter beneath the skull (on the side of the cerebellum) and cut the skull to the right and to the left to enable its removal. Using a small tweezer, peel the skull off from the brain.</li>
	<li>With a clean tweezer, separate the brain from the head and put it in a 60 mm Petri dish containing 2 mL of cold MEM (see step 6.1.2).</li>
	<li>Using two small tweezers, dissect the hippocampi under a stereomicroscope. Transfer the hippocampi into the prepared 35 mm dish (from step 6.1.2) containing MEM. 
	NOTE: Steps 6.3&#8211;6.6&#160;should be repeated for each pup.</li>
	<li>Cut the hippocampi to approximately 1 mm slices using the scalpel. Add 200 &#181;L of the trypsin solution (diluted to a final concentration of 0.25%). Mix gently and incubate at 37 &#176;C, 5&#8211;10% CO2 for 30 min.</li>
	<li>After incubation, add 2 mL of cold MEM (step 6.1.2) to the dish to deactivate the trypsin. Transfer the trypsinized tissue to a 15 mL tube containing 1 mL First Day Medium preheated to 37 &#176;C (step 6.1.3) using the glass Pasteur pipette with the largest diameter (step 6.1.5). 
	NOTE: The best medium to tissue (v:v) ratio for further processing of the tissue is 1 mL/8 hippocampi. Avoid transferring the medium with the tissue as much as possible.</li>
	<li>Triturate the tissue by passing it through the largest diameter glass pipette 10&#8211;15 times. Then, repeat this process using the glass pipette with the medium diameter. Continue triturating with the smallest diameter pipette. Avoid bubbling to reduce cell death.</li>
	<li>Let the remaining&#160;tissue pieces sink for 2&#8211;5 min, then transfer the supernatant into another 15 mL tube. Count the cells using a hemocytometer. 
	NOTE: The preferable cell density is 200,000&#8211;400,000 cells/mL. Use First Day Medium to dilute or centrifugation at 470 x g to concentrate the cells.</li>
	<li>Seed 100 &#181;L of cells on each glass coverslip. While seeding, make sure to cover the entire coverslip with cells. Incubate at 37 &#176;C, 5&#8211;10% CO2.</li>
	<li>The next day, add 500 &#181;L of Neuronal Growth Medium to the wells. 
	NOTE: The composition of the Neuronal Growth Medium is described in the Table of Materials.</li>
	<li>Gently transfer each coverslip to its appropriate well using a tweezer while removing the First Day Medium by tilting the coverslip.&#160;Suction the remaining media from the lid.</li>
	<li>Incubate at 37 &#176;C, 5&#8211;10% CO2. Avoid replacing the medium throughout incubation.&#160;Cultures can be maintained under these conditions up to 1 month. The major concern is humidity. Therefore, make sure to maintain the incubator maximally humidified.</li>
</ol>

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The authors declare that they have no competing financial interests.

The technique presented here describes a way to improve the maintenance and functionality of neural cells in culture. This is achieved by adhering the cells to a matrix made of coral skeleton grains that nurtures the cells and promotes their growth and activity. Using this technique increases the capacity of the neural culture model to mimic the cells&#39; environment in the brain.
The introduction of the matrix as a culture substrate has several advantages over other substrates used in classi...

Cultures of dissociated neural cells, in this case hippocampal cells, are a valuable experimental model for studying neural growth and function by providing high cell isolation and accessibility1,2,3. This type of culture is frequently used in neuroscience, drug development, and tissue engineering due to the large amount of information that can be collected, such as rates of growth and viability, neurotoxicity, neurite outgrowth and networking, synaptic connectivity and plasticity, morphological modifications, neurites organization and wiring, etc.1,4,5,6,7.
Despite the significance of the cultures, the cultivated cells are usually forced to grow on glass coverslips in a two-dimensional monolayer. These strict environmental modifications significantly decrease the ability of neural cells to survive over time, because glass coverslips are non-nurturing substrates with a low adhesion strength, exhibiting a lower capacity to support cell growth8,9,10,11.
Because cultivated neural cells are forced to grow in challenging conditions, an essential approach to enhance their survival would be to imitate their natural environment as much as possible12,13. This could be achieved by using biomaterials that will act as matrices and mimic the extracellular matrix of the cells, enabling them to form a tissue-like structure and assist in their nourishment14.
The use of biomaterials is a promising approach in improving cell cultures, because they act as biocompatible scaffolds, providing mechanical stability and enhancing a variety of cell properties, including adhesion, survival, proliferation, migration, morphogenesis, and differentiation15,16,17. Several types of biomaterials are used to improve the conditions of the cells in vitro. Among them are biopolymers, or biological components that are usually part of the extracellular matrix of the cells. These biomaterials are mostly used as a form of polymerized coating agents or hydrogels18,19,20. On the one hand, the matrices mentioned above give the cells a familiar 3D environment to grow in, encourage their adhesion to the dish, and give them mechanical support21,22. On the other hand, their polymerized form and the confinement of the cells within hydrogels disturbs the access of the cells to nurturing components present in the growth media and also makes the follow up of the cells by microscopic methods more difficult23.
Coral exoskeletons are biological marine-originated matrices. They are made of calcium carbonate, have mechanical stability, and are biodegradable. Previous studies using the coral skeleton as a matrix for growing neural cells in culture have shown much greater adhesion, compared to glass coverslips24,25. In addition, neural cells grown on coral skeleton demonstrated their capability to intake the calcium the skeleton is composed of, which protects the neural cells in conditions of nutrient deprivation26. Moreover, the coral skeleton is a supportive and nurturing matrix that increases the survival of neural cells, encourages the formation of neural networks, elevates the rate of synaptic connectivity, and enables the formation of tissue-like structures27,28. Recent studies have also shown that the surface topography of the coral skeleton matrix plays a crucial role in the distribution and activation of glial cells8,29. Also, coral skeleton is effective as a matrix for cultivation of other cell types, such as osteocytes30,31, hepatocytes, and cardiomyocytes in culture (unpublished data).
Hence, coral skeleton is a promising matrix for cultivation of cells in vitro. Thus, the protocol detailed below describes the technique of cultivating neural cells on coral skeleton for producing more stable and prosperous neural cultures than those achieved by existing methods. This protocol may also be useful for cultivation of cardiomyocytes, hepatocytes, and other cell types.

<table><tbody><tr><td>24-well plates</td><td>Greiner</td><td>#60-662160</td><td></td></tr><tr><td>B-27</td><td>Gibco</td><td>#17504-044</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>#A4503</td><td></td></tr><tr><td>D &amp;ndash; glucose</td><td>Sigma</td><td>#G8769</td><td></td></tr><tr><td>Dulbecco&#039;s Minimal Essential Eagle (DMEM)</td><td>Sigma</td><td>#D5796</td><td></td></tr><tr><td>Electrical sieve</td><td>Ari Levy</td><td>#3700</td><td></td></tr><tr><td>Fetal Bovine Serun (FBS)</td><td>Biological Industries</td><td>#04-007-1A</td><td></td></tr><tr><td>First Day Medium</td><td></td><td></td><td>85.1% Minimum Essential Eagle&amp;rsquo;s medium (MEM), 11.5% heat-inactivated fetal bovine serum, 1.2% L-Glutamine and 2.2% D-Glucose.</td></tr><tr><td>Flasks</td><td>Greiner</td><td>#60-690160</td><td>25cm^2, Tissue culture treated</td></tr><tr><td>Fluoro-deoxy-uridine</td><td>Sigma</td><td>#F0503</td><td></td></tr><tr><td>Glass Coverslips</td><td>Menzel-Glaser</td><td>#BNCB00120RA1</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Romical</td><td>#007130-72-19</td><td>Hazardous</td></tr><tr><td>Ham&#039;s F-12 Nutrient Mixture</td><td>Sigma</td><td>#N4888</td><td></td></tr><tr><td>HANK&#039;S solution</td><td>Sigma</td><td>#H6648</td><td></td></tr><tr><td>Kynurenic acid</td><td>Sigma</td><td>#K3375</td><td></td></tr><tr><td>L - glutamine</td><td>Sigma</td><td>#G7513</td><td></td></tr><tr><td>Manual strainer (40&amp;micro;m)</td><td>VWR</td><td>#10199-654</td><td></td></tr><tr><td>Minimun Essential Eagle (MEM)</td><td>Sigma</td><td>#M2279</td><td></td></tr><tr><td>Mortar and pestle</td><td>De-Groot</td><td>4-P090</td><td></td></tr><tr><td>NaClO (Sodium Hypochlorite)</td><td>Sigma</td><td>#425044</td><td>Hazardous</td></tr><tr><td>NaOH</td><td>Sigma</td><td>#S8045</td><td>Hazardous</td></tr><tr><td>Neuronal Growth Medium</td><td></td><td></td><td>45% MEM, 40% Dulbecco&#039;s modified eagle&#039;s medium (DMEM), 10% Nutrient mixture F-12 Ham, 0.25% (w/v) bovine serum albumin (BSA), 0.75% D-glucose, 0.25% L-Glutamine, 0.5% B-27 supplement, 0.1% kynurenic acid, 0.01% of 70 % uridine and 30% fluoro-deoxy-uridine.</td></tr><tr><td>Petri dish</td><td>Greiner</td><td>#60-628160, #60-627160</td><td>60mm, 35mm, respectively.</td></tr><tr><td>Poly D &amp;ndash; Lysine</td><td>Sigma</td><td>#P7280</td><td></td></tr><tr><td>Smart Dentin Grinder</td><td>KometaBio</td><td>#GR101</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>#15-090-046</td><td></td></tr><tr><td>Uridine</td><td>Sigma</td><td>#U3750</td><td></td></tr></tbody></table>

increasing durability of dissociated neural cell cultures using biologically active coralline matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.
Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.
This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

In order to prepare the coral skeleton matrix, the entire coral skeleton (Figure 1A) was broken into 0.5&#8211;2 cm fragments using a hammer (Figure 1B) and thoroughly cleaned from organic residues through three steps (step 1 in the protocol) using 10% hypochlorite solution, 1M NaOH solution, and 30% H2O2 solution (Figure 1C). Coral fragments were well-cleaned when the skeleton color changed from brown (<strong...

Watch this Scientific Journal Video about Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix at JoVE.com

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

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1.2K Views.  Ariel University. Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

Video: Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Neuroscience

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

Developmental Biology

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular matrix (osteoid) express the Pdpn gene to form cellular dendrites and transform into preosteocytes. Later, preosteocytes express the Dmp1 gene to promote matrix mineralization and thereby transform into mature osteocytes.This process is called osteocytogenesis. IDG-SW3 is a well-known cell line for in vitro studies of osteocytogenesis. Many previous methods have used collagen I as the main or the only component of the culturing matrix. However, in addition to collagen I, the osteoid also contains a ground substance, which is an important component in promoting cellular growth, adhesion, and migration. In addition, the matrix substance is transparent, which increases the transparency of the collagen I-formed gel and, thus, aids the exploration of dendrite formation through imaging techniques. Thus, this paper details a protocol to establish a 3D gel using an extracellular matrix along with collagen I for IDG-SW3 survival. In this work, dendrite formation and gene expression were analyzed during osteocytogenesis. After 7 days of osteogenic culture, an extensive dendrite network was clearly observed under a fluorescence confocal microscope. Real-time PCR showed that the mRNA levels of Pdpn and Dmp1 continually increased for 3 weeks. At week 4, the stereomicroscope revealed an opaque gel filled with mineral particles, consistent with the X-ray fluorescence (XRF) assay. These results indicate that this culture matrix successfully facilitates the transition from osteoblasts to mature osteocytes.

Here, we present a protocol for culturing IDG-SW3 cells in a three-dimensional (3D) extracellular matrix.

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular ...

Co-Culture of Schwann Cells with DRG Neurons on a Pre-Stretched Membrane

Source: Liu, C., et al. <a href="https://app.jove.com/v/54085">An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons.</a> J. Vis. Exp. (2016).
The video demonstrates the co-culturing of dorsal root ganglion (DRG) neurons with Schwann cells. DRG neurons are first grown on a pre-stretched membrane for aligned axon growth. Schwann cells are then introduced, which attach to the axons, insulating them and promoting neural cell survival and function.

1. Culture of dorsal root ganglion (DRG) on Pre-stretched Surface

	Preparation of Pre-stretched Anisotropic Surface
	
		Mix a 10:1 solution of base and ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons

Source: Sharif, R., et al. <a href="https://app.jove.com/v/56308">Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea.</a> J. Vis. Exp. (2018)
This video demonstrates a technique to generate a three-dimensional co-culture model system of primary human corneal fibroblasts and differentiated neuronal cells. This co-culture system can be used to study the stromal-nerve interactions.

1. Primary HCFs (human corneal fibroblasts) Culture and 3D Constructs Assembly

	To assemble 3D constructs from explants previously grown in EMEM 10% FBS ...

Preparation of Collagen-Coated Compartmented Culture Dishes for Neuronal Cell Culture

Source: Zepecki, J. P., et al. <a href="https://app.jove.com/v/59744">Real-Time monitoring of human glioma cell migration on dorsal root ganglion axon-oligodendrocyte co-cultures.</a> J. Vis. Exp. (2019)
This video demonstrates constructing a three-compartment cell culture system using collagen and non-reactive grease. This system is used to observe interactions between glioma cells and axons in real-time.


	Compartmented culture of Rat Dorsal Root Ganglia (DRGs), Oligodendrocytes (OPCs), and hGCs (human glioma cells)

1. Preparation of compartmented culture ...