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 ...

<ol>
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E., Vago, R., Baranes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+survival+and+durability+of+neurons+and+astrocytes+on+3-dimensional+aragonite+biomatrices.">Superior survival and durability of neurons and astrocytes on 3-dimensional aragonite biomatrices.</a> Tissue Engineering. 13 (3), 461-472 (2007).</li><li>Shany, B., Vago, R., Baranes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+primary+hippocampal+neuronal+tissue+on+an+aragonite+crystalline+biomatrix.">Growth of primary hippocampal neuronal tissue on an aragonite crystalline biomatrix.</a> Tissue Engineering. 11 (3-4), 585-596 (2005).</li><li>Baranes, D., 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=Interconnected+network+of+ganglion-like+neural+cell+spheres+formed+on+hydrozoan+skeleton.">Interconnected network of ganglion-like neural cell spheres formed on hydrozoan skeleton.</a> Tissue Engineering. 13 (3), 473-482 (2007).</li><li>Morad, T. I., 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=Gliosis+of+astrocytes+cultivated+on+coral+skeleton+is+regulated+by+the+matrix+surface+topography.">Gliosis of astrocytes cultivated on coral skeleton is regulated by the matrix surface topography.</a> Biomedical Materials. 14 (4), 045005 (2019).</li><li>Green, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Therapeutic+Potential+for+Marine+Skeletal+Proteins+in+Bone+Regeneration.">A Therapeutic Potential for Marine Skeletal Proteins in Bone Regeneration.</a> Marine Drugs. 11 (4), 1203-1220 (2013).</li><li>Neto, A. S., Ferreira, J. M. 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Visualization of the ultrastructural interface of cells with the outer and inner-surface of coral skeletons Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/19218486">https://www.ncbi.nlm.nih.gov/pubmed/19218486</a> (2019)</li><li>Drake, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+skeletal+organic+matrix+from+the+stony+coral+Stylophora+pistillata.">Proteomic analysis of skeletal organic matrix from the stony coral Stylophora pistillata.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (10), 3788-3793 (2013).</li><li>Ramos-Silva, P., 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=The+skeletal+proteome+of+the+coral+Acropora+millepora:+the+evolution+of+calcification+by+co-option+and+domain+shuffling.">The skeletal proteome of the coral Acropora millepora: the evolution of calcification by co-option and domain shuffling.</a> Molecular Biology and Evolution. 30 (9), 2099-2112 (2013).</li><li>Peretz, H., Blinder, P., Baranes, D., Vago, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aragonite+crystalline+matrix+as+an+instructive+microenvironment+for+neural+development.">Aragonite crystalline matrix as an instructive microenvironment for neural development.</a> Journal of Tissue Engineering and Regenerative Medicine. 2 (8), 463-471 (2008).</li><li>Morad, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CaCO3 Matrix Dictates Astrocytes Transition to Astrogliosis. , (2019).</li><li>Prada, F., 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=Ocean+warming+and+acidification+synergistically+increase+coral+mortality.">Ocean warming and acidification synergistically increase coral mortality.</a> Scientific Reports. 7, (2017).</li></ol>

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 &#8211; glucose</td>
			<td>Sigma</td>
			<td>#G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#8217;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>H2O2</td>
			<td>Romical</td>
			<td>#007130-72-19</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mixture</td>
			<td>Sigma</td>
			<td>#N4888</td>
			<td></td>
		</tr>
		<tr>
			<td>HANK&#39;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&#181;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&#39;s modified eagle&#39;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 &#8211; Lysine</td>
			<td>Sigma</td>
			<td>#P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>Smart Dentin Grinder</td>
			<td>KometaBio</td>
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			<td>Trypsin</td>
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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...

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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 ...

The significance of this protocol is that it provides a new way to increase the durability and growth of neurons, glia, and stem cells in vitro. It is also applicable for other cells. The main advantage of the technique is the usage of a matrix made of coral skeleton.This matrix nurtures and protects neuro cells and it promotes regrowth. Being supportive for neural cells in vitro, the coral skeleton may function similarly as a regenerative implant in brain injury sites. To begin, use a hammer to break the coral skeleton, and divide it into 0.5 to two centimeter fragments.In order to dissolve organic and non-organic residues, soak the coral skeleton fragments in 10%Sodium hypochlorite solution for 10 minutes. Then wash once with double distilled water. To remove the remaining organic residues, soak the fragments in a one molar sodium hydroxide solution for five minutes, then wash once with double distilled water.Continue with the removal of the organic deposits by soaking the fragments in 30%hydrogen peroxide solution for 10 minutes, and wash the fragments three times with double distilled water. Remove as much excess double distilled water as possible and leave the coral fragments in the hood to dry. Then clean the glass cover slips by soaking them in 10 milliliters of 95%ethanol in a 100 millimeter glass Petri dish.After 15 minutes, remove the ethanol and wash with double distilled water three times, waiting 10 minutes between each wash. Remove the double distilled water and place the dish on an 80 degree Celsius prewarmed heating plate. Then gently stir the cover slips until the double distilled water evaporates.Autoclave the cover slips. Grind the coral skeleton fragments using a mortar and pestle until complete breakdown. A mixture of grains with sizes ranging from 20 to 200 micrometers is obtained.Alternatively, grind the coral skeleton fragments using an electrical grinding machine with a blade length of six centimeters and width of 0.5 to one centimeters, at a velocity of 1000 RPM for 30 seconds. To purify the grains, transfer the grains onto an electrical 40 micron filter mesh strainer. Adjust the electrical strainer settings to shaking at 600 amplitudes per minute, and bouncing at six times per second.Then autoclave the grains. Under sterile conditions, add the coral skeleton grains into a 20 microgram per milliliter PDL, dissolved in Hank's solution to achieve the concentration of five milligrams or 10 milligrams of grains per one milliliter PDL solution. Pour the solution into flasks and dishes at the concentration of approximately two milliliters per 25 square centimeters, and incubate overnight at four degrees Celsius.The grains sink and attach to the bottom. The next day, wash the flasks and dishes once with sterile double distilled water. Let the flasks and dishes dry in the hood.To coat the glass cover slip, first add coral skeleton grains to double distilled water at a concentration of five or 10 milligrams per milliliter. Place the cover slips on a heating plate prewarmed to 80 degrees Celsius. Then drip 40 microliters of the grain solution onto the center of the cover slip, and wait 15 minutes for complete evaporation.This will result in the grains adhering to the cover slip. Autoclave the coated cover slips, and store in sterile conditions. Next, place the cover slips on the lid of a sterile 24 well plate, add 100 microliters of 20 microgram per milliliter PDL solution to each cover slip.Use the tip to ensure that the liquid covers the entire grain region and the rest of the cover slip surface. Cover the lid with the bottom of the plate, and wrap the sides of the place with paraffin film. Incubate overnight at four degrees Celsius.The next day, wash the cover slips once with double distilled water and dry in the hood. After preparing rat pup heads, hold it from the mouth with big tweezers. Cut the skin covering the skull with a small scissor.With clean scissors, 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 small tweezers, peel the skull off from the brain. Separate the brain from the head and put it in a 60 millimeter Petri dish containing two milliliters of cold MEM.Under a stereo microscope, use two small tweezers to dissect the hippocampi. Transfer the hippocampi into the prepared 35 millimeter dish containing MEM. Cut the hippocampi to approximately one millimeter slices using the scalpel.Add 200 microliters of trypsin solution. Mix gently and incubate at 37 degrees Celsius and five to 10%carbon dioxide for 30 minutes. After incubation, add two milliliters of cold MEM to the dish to deactivate the trypsin.Then use a glass Pasteur pipette with the largest diameter to transfer the trypsinized tissue to a 15 milliliter tube containing one milliliter of first day medium, preheated to 37 degrees Celsius. Triturate the tissue by passing it through the largest diameter glass pipette 10 to 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. Let the disassociated tissue pieces sink for two to five minutes.Then transfer the supernatant into another 15 milliliter tube. Count the cells using a hemocytometer. To achieve the preferable cell density between 200, 000 and 400, 000 cells per milliliter.Use first day medium to dilute. Next, seed 100 microliters of cells on each glass cover slip. Make sure to cover the entire cover slip with cells.Incubate at 37 degrees Celsius and five to 10%carbon dioxide. The next day, add 500 microliters of neuronal growth medium to the wells. Using tweezers, remove the first day medium by tilting the cover slip, and gently transferring each cover slip to its appropriate well.Suction the remaining media from the lid. Incubate the plate at 37 degrees Celsius and five to 10%carbon dioxide. Maintain the incubator maximally humidified using a tray of water in the bottom of the incubator.Do not replace the medium. Cultures can be maintained under these conditions up to one month. In this study, manually grinding and electrical grinding machines both yielded similar outcomes, with a mixture of grains ranging between 20 to 200 micrometers in size.The manual or electrical strainers produced two types of grain mixtures. One with grains ranging from 40 to 200 micrometers in size, and the second with the size of 40 micrometers or less. Phase contrast micrographs show that the cultivation of hippocampal cells on cover slips coated with coral skeleton grains formed complex neuronal networks in the absence and presence of the matrix.Cell nuclei staining indicated that 14 days after seeding, cell density was higher on the matrix than on the uncoated cover slips. Staining with microtubule associated protein two showed that a complex neuronal and dendritic network formed on the matrix compared to the control. In addition, glial cells grown on the coral skeleton matrix visualized using the glial fibrillary acidic protein underwent significant morphological changes.They acquired a spikier appearance, with longer processes, than the glial cells grown on the control substrate, where they appeared rounder and flatter. While isolating the cells, It is important to remember to work fast, maintain the pH by adding CO2 if necessary, and work as gently as possible in order to prevent detachment of coral grains.

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1.2K visualizações.  Ariel University. The significance of this protocol is that it provides a new way to increase the durability and growth of neurons, glia, and stem cells in vitro. It is also applicable for other cells. The main advantage of the technique is the usage of a matrix made of coral skeleton.This matrix nurtures and protects neuro cells and it promotes regrowth. Being supportive for neural cells in vitro, the coral skeleton may function similarly as a regenerative implant in brain injury sites. To begin, use a...