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

Dette arbeidet ble finansiert av Kamin-programmet til det israelske handels- og arbeidsdepartementet og av Qrons Inc., 777 Brickell Avenue Miami, FL 33131, USA.

Bruken av dyr i denne protokollen ble godkjent av National Animal Care and Use Committee.
MERK: Kalsiumkarbonerte korallskjeletter bør brukes i krystallinsk form av aragonitt. Koralltypene som hittil er testet for nevrale kulturer er Porites Lutea, Stylophora Pistillata og Trachyphyllia Geoffroyi. Skjelettene kan kjøpes hele eller malt.
1. Rengjøring av korallskjelettbitene
FORSIKTIG: Følgende trinn skal utføres i en ...

<ol>
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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+physical+structure+and+charge+on+neurite+extension+in+a+3D+hydrogel+scaffold.">The influence of physical structure and charge on neurite extension in a 3D hydrogel scaffold.</a> Journal of Biomaterials Science, Polymer Edition. 9 (10), 1049-1069 (1998).</li><li>Carballo-Molina, O. A., Velasco, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+scaffolds+and+delivery+systems+to+enhance+axonal+regeneration+after+injuries.">Hydrogels as scaffolds and delivery systems to enhance axonal regeneration after injuries.</a> Frontiers in Cellular Neuroscience. 9, (2015).</li><li>George, J., Hsu, C. C., Nguyen, L. T. 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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. 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Teknikken som presenteres her beskriver en måte å forbedre vedlikehold og funksjonalitet av nevrale celler i kultur. Dette oppnås ved å feste cellene til en matrise laget av korallskjelettkorn som nærer cellene og fremmer deres vekst og aktivitet. Ved hjelp av denne teknikken øker kapasiteten til nevralkulturmodellen for å etterligne cellenes miljø i hjernen.
Innføringen av matrisen som et kultursubstrat har flere fordeler i forhold til andre substrater som brukes i klassiske nevrale ...

Kulturer av dissosierte nevrale celler, i dette tilfellet hippocampusceller, er en verdifull eksperimentell modell for å studere nevral vekst og funksjon ved å gi høy celleisolasjon og tilgjengelighet 1,2,3. Denne typen kultur brukes ofte i nevrovitenskap, stoffutvikling og vevsteknikk på grunn av den store mengden informasjon som kan samles inn, for eksempel veksthastighet og levedyktighet, nevrotoksisitet, nevrittutvekst og nettverk, synaptisk tilkobling og plastisitet, morfologiske modifikasjoner, nevrittorganisasjon og ledninger, etc.1,4,5,6,7.
Til tross for kulturens betydning, blir de kultiverte cellene vanligvis tvunget til å vokse på glassdeksel i et todimensjonalt monolag. Disse strenge miljømodifikasjonene reduserer signifikant nevrale cellers evne til å overleve over tid, fordi glassdeksler er ikke-nærende substrater med lav vedheftstyrke, og viser en lavere kapasitet til å støtte cellevekst 8,9,10,11.
Fordi dyrkede nevrale celler blir tvunget til å vokse under utfordrende forhold, vil en viktig tilnærming for å forbedre overlevelsen være å etterligne sitt naturlige miljø så mye som mulig12,13. Dette kan oppnås ved å bruke biomaterialer som vil fungere som matriser og etterligne den ekstracellulære matrisen til cellene, slik at de kan danne en vevlignende struktur og bistå i næring14.
Bruken av biomaterialer er en lovende tilnærming til å forbedre cellekulturer, fordi de fungerer som biokompatible stillas, gir mekanisk stabilitet og forbedrer en rekke celleegenskaper, inkludert vedheft, overlevelse, spredning, migrasjon, morfogenese og differensiering15,16,17. Flere typer biomaterialer brukes til å forbedre forholdene til cellene in vitro. Blant dem er biopolymerer, eller biologiske komponenter som vanligvis er en del av den ekstracellulære matrisen av cellene. Disse biomaterialene brukes mest som en form for polymeriserte beleggmidler eller hydrogeler18,19,20. På den ene siden gir matrisene nevnt ovenfor cellene et kjent 3D-miljø å vokse i, oppmuntre deres vedheft til parabolen og gi dem mekanisk støtte21,22. På den annen side forstyrrer deres polymeriserte form og innesperring av cellene i hydrogeler cellens tilgang til pleiende komponenter som er tilstede i vekstmediet, og gjør også oppfølgingen av cellene ved mikroskopiske metoder vanskeligere23.
Koralleksoskjeletter er biologiske marine-opprinnelsesmatriser. De er laget av kalsiumkarbonat, har mekanisk stabilitet og er biologisk nedbrytbare. Tidligere studier som bruker korallskjelettet som matrise for dyrking av nevrale celler i kultur har vist mye større vedheft, sammenlignet med glassdeksler24,25. I tillegg demonstrerte nevrale celler dyrket på korallskjelett sin evne til å innta kalsiumet skjelettet består av, som beskytter nevrale celler under forhold med næringsmangel26. Videre er korallskjelettet en støttende og nærende matrise som øker overlevelsen av nevrale celler, oppfordrer dannelsen av nevrale nettverk, øker graden av synaptisk tilkobling og muliggjør dannelsen av vevlignende strukturer27,28. Nylige studier har også vist at overflatetopografien til korallskjelettmatrisen spiller en avgjørende rolle i distribusjonen og aktiveringen av gliaceller 8,29. Korallskjelett er også effektivt som matrise for dyrking av andre celletyper, for eksempel osteocytter30,31, hepatocytter og kardiomyocytter i kultur (upubliserte data).
Derfor er korallskjelett en lovende matrise for dyrking av celler in vitro. Dermed beskriver protokollen beskrevet nedenfor teknikken for å dyrke nevrale celler på korallskjelett for å produsere mer stabile og velstående nevrale kulturer enn de som oppnås ved eksisterende metoder. Denne protokollen kan også være nyttig for dyrking av kardiomyocytter, hepatocytter og andre celletyper.

<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>
			<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

Kulturer av dissosierte hippocampus-, nevron- og gliaceller er en verdifull eksperimentell modell for å studere nevral vekst og funksjon ved å gi høy celleisolasjon og et kontrollert miljø. Imidlertid er overlevelsen av hippocampusceller in vitro kompromittert: de fleste celler dør i løpet av den første uken av kulturen. Det er derfor av stor betydning å identifisere måter å øke holdbarheten til nevrale celler i kultur.
Kalsiumkarbonat i form av krystallinsk aragonitt avledet fra skjelettet av koraller kan brukes som en overlegen, aktiv matrise for nevrale kulturer. Ved å pleie, beskytte og aktivere gliaceller forbedrer korallskjelettet overlevelsen og veksten av disse cellene in vitro bedre enn andre matriser.
Denne protokollen beskriver en metode for å dyrke hippocampusceller på en korallmatrise. Denne matrisen genereres ved å feste korn av korallskjeletter til kulturretter, kolber og glassdeksler. Kornene hjelper til med å forbedre miljøet i cellene ved å introdusere dem til et fint tredimensjonalt (3D) miljø for å vokse på og danne vevlignende strukturer. 3D-miljøet introdusert av korallskjelettet kan optimaliseres for cellene ved sliping, noe som muliggjør kontroll over kornets størrelse og tetthet (dvs. matriksruheten), en egenskap som har vist seg å påvirke glialcelleaktiviteten. Videre gjør bruken av korn observasjon og analyse av kulturen enklere, spesielt ved bruk av lysmikroskopi. Derfor inneholder protokollen prosedyrer for generering og optimalisering av korallinmatrisen som et verktøy for å forbedre vedlikehold og funksjonalitet av nevrale celler in vitro.

For å forberede korallskjelettmatrisen ble hele korallskjelettet (figur 1A) brutt i 0,5-2 cm fragmenter ved hjelp av en hammer (figur 1B) og grundig rengjort fra organiske rester gjennom tre trinn (trinn 1 i protokollen) ved bruk av 10% hypoklorittløsning, 1M NaOH-løsning og 30%H2O2-løsning(figur 1C). Korallfragmentene ble godt rengjort da skjelettfargen endret seg fra brun (figur 1D</s...

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

Dissociert hippocampus cellekultur er et sentralt eksperimentelt verktøy i nevrovitenskap. Nevral celleoverlevelse og funksjon i kultur forbedres når korallskjeletter brukes som matriser, på grunn av deres nevrobeskyttende og nevromodulative roller. Derfor viser nevrale celler dyrket på korallmatriks høyere holdbarhet, og er dermed mer tilstrekkelige for dyrking.

Økende holdbarhet av dissosierte nevrale cellekulturer ved bruk av biologisk aktiv korallinmatriks

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.

Research

JoVE Journal

Bioengineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Ikke-kontakt, Label-fri Overvåking av celler og ekstracellulær matriks med Raman spektroskopi

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Shock Wave Application to Cell Cultures

Shock Wave Søknad til cellekulturer

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Analyse av celle migrasjon innenfor en tre-dimensjonal Kollagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Forbedret 3D Hydrogel kulturer av primære gliacellene for In Vitro modellering av Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

En ny Tenorrhaphy sutur teknikk med vev konstruert kollagen graft å reparere store sene defekter

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Fabrikere en nyre Cortex ekstracellulær Matrix-avledet Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Produksjon av ekstracellulær Matrix fiber via oppofrende hul Fiber membran cellekultur

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...