Name: fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro
Uploaded: 2018-06-20T12:30:00
Description: Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

De auteurs erkennen de steun van onderzoekssubsidies van nationale Natural Science Foundation van China (31371390), het programma van de staat High-Tech ontwikkelingsproject (2014AA020702) en het programma van Guangdong wetenschap en technologie (2016B030231001).

Alle dierlijke werk werd verricht overeenkomstig de Akte van dierenwelzijn, institutioneel richtsnoeren en goedgekeurd door institutionele Animal Care en gebruik Comité, zon Yat-sen-universiteit.
1. voorbereiding van de TEM
<ol><li>Uitvoeren van muizen door cervicale dislocatie en verwijder de tongen met behulp van steriele chirurgische schaar en pincet.</li>
	<li>De tongen in 75% ethanol voor 3 min. onderdompelen en elke tong gestoken met een 1,5 mL Eppendorf (EP) buis met 1 mL 10 mM steri...

<ol>
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Oncol. 29, 1488-1494 (2011).</li><li>Zhao, L., Huang, L., Yu, S., Zheng, J., Wang, H., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+tongue+tissue+as+an+in+vitro.+model+for+studying+tongue+cancer+and+tongue+regeneration.">Decellularized tongue tissue as an in vitro. model for studying tongue cancer and tongue regeneration.</a> Acta Biomaterialia. 58, 122-135 (2017).</li><li>Ng, S. L., Narayanan, K., Gao, S., Wan, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restricted+progenitors+for+the+repopulation+of+decellularized+heart.">Lineage restricted progenitors for the repopulation of decellularized heart.</a> Biomaterials. 32, 7571-7580 (2011).</li><li>Ott, H. 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Een goed gangbaar protocol voor de fabricage van decellularized ECM moet behouden de oorspronkelijke samenstelling van de ECM tijdens het verwijderen van cellulaire componenten in weefsels bijna volledig21. Ondanks momenteel gerapporteerde decellularization protocollen waarvoor perfusie door middel van de therapieën materialen te verwijderen cellulaire door convectie vervoer, mechanische agitatie goedgekeurd hier, bekend als een traditionele, eenvoudige en goedkope methode22</su...

De tong heeft verschillende belangrijke functies, zoals deglutition, articulatie en proeverij. Dus, de verslechtering van de tong functie heeft grote impact op patiënten levenskwaliteit1. De meest voorkomende maligniteit in de mondholte is tong plaveiselcelcarcinoom (TSCC), die meestal voorkomt bij mensen die drinken van alcohol of roken van tabak2.
In de afgelopen jaren, is weinig vooruitgang geboekt in fundamenteel onderzoek op TSCC. Het gebrek aan efficiënte in vitro onderzoek modellen blijft als een van de grootste problemen. Dus, de extracellulaire matrix (ECM) blijkt te zijn van een mogelijke oplossing. Aangezien ECM een complex netwerk frame samengesteld uit onderdelen van de hoogst georganiseerde matrix is, zou Steiger materiaal met een ECM-achtige structuur en samenstelling bevoegd voor kankeronderzoek zijn. Decellularized ECM kan perfect de niche bieden voor de cellen van dezelfde oorsprong in vitro, die blijkt te zijn van het belangrijkste voordeel van ECM.
ECM kan worden bewaard met cellulaire componenten wordt verwijderd uit de weefsels door middel van de decellularization met behulp van detergentia en enzymen. Verschillende ECM componenten, met inbegrip van collageen, fibronectine en laminin in de decellularized matrix bieden een native-weefsel-achtige communicatie voor gekweekte cellen, bevordering van de overleving, proliferatie en differentiatie van de cellen3. Bovendien kan de immunogeniciteit voor transplantatie worden gereduceerd tot een minimaal niveau aan de afwezigheid van cellulaire componenten in ECM.
Tot nu toe hebben fabricage methoden voor decellularized ECM geprobeerd in verschillende weefsels en organen, zoals hart4,5,6,7, lever8,9,10 ,11, Long12,13,14,15,16,17, en nier18,19 , 20. echter geen relevant onderzoek is te vinden op soortgelijke werkzaamheden in de tong om het beste van onze kennis.
In deze studie werd decellularized tong extracellulaire matrix (TEM) zowel efficiënt en goedkoop vervaardigd door een reeks van fysieke, chemische en enzymatische behandeling. Vervolgens werd de TEM gebruikt om te recapituleren TSCC in vitro, tonen een passende simulatie voor TSCC gedrag en ontwikkeling. TEM heeft goede biocompatibiliteit, evenals de mogelijkheid om te begeleiden van de cellen naar de weefsel-specifieke niche, waarmee wordt aangegeven dat de TEM groot potentieel in TSCC onderzoek3 wellicht. Het hier getoonde protocol biedt u de keuze voor onderzoekers bestuderen op pathogenese of klinische therapieën voor TSCC.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C57-BL/6J mice</td>
			<td style="width:292px;">Sun Yat-sen University Laboratory Animal Center</td>
			<td style="width:119px;"></td>
			<td style="width:264px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">HB15-GR-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium chloride</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A100395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dibasic Sodium Phosphate</td>
			<td style="width:292px;">Guangzhou Chemical Reagent Factory</td>
			<td style="width:119px;">BE14-GR-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Phosphate Monobasic&#160;</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A501211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL EP tube</td>
			<td style="width:292px;">Axygen</td>
			<td style="width:119px;">MCT-150-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-low temperature freezer&#160;</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3.5 cm cell culture dish</td>
			<td style="width:292px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">153066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 cm cell culture dish</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">V900502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium chloride</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">449164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNase</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium sulphate</td>
			<td style="width:292px;">Sangon Biotech</td>
			<td style="width:119px;">A601988</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">158968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium bicarbonate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kanamycin</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">PHR1487</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical suture</td>
			<td style="width:292px;">Shanghai Jinhuan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">250 mL wide-mouth bottle</td>
			<td style="width:292px;">SHUNIU</td>
			<td style="width:119px;">1407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic stirrer</td>
			<td style="width:292px;">AS ONE</td>
			<td style="width:119px;">1-4602-32</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">CO2 incubator</td>
			<td style="width:292px;">SHEL LAB</td>
			<td style="width:119px;">SCO5A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL syringe</td>
			<td style="width:292px;">Hunan Pingan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 mL centrifuge tube</td>
			<td style="width:292px;">Greiner</td>
			<td style="width:119px;">227270</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cal27 cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Tongue squamous cell carcinoma cell line</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">U2OS cell</td>
			<td style="width:292px;">Chinese Academy of Science, Shanghai Cell Bank</td>
			<td style="width:119px;"></td>
			<td>Human osteosarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:292px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes free acid</td>
			<td style="width:292px;">BBI</td>
			<td style="width:119px;">A600264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:292px;">Hyclone</td>
			<td style="width:119px;">SH30084.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4 &#176;C fridge</td>
			<td style="width:292px;">Haier</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water purifier</td>
			<td style="width:292px;">ELGA</td>
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fabrication of tongue extracellular matrix and reconstitution of tongue squamous cell carcinoma in vitro

De methoden zijn om te bouwen van een model voor effectieve en realistische voor tong squamous cell carcinoma (TSCC) in vitro, gemaakt naar producten decellularized tong extracellulaire matrix (TEM) waarmee functionele steigers voor TSCC bouw. TEM voorziet een niche in vitro celgroei, differentiatie en cel migratie. De microstructuren inheemse extracellulaire matrix (ECM) en biochemische composities bewaard in de decellularized matrix wordt voorzien dat weefsel-specifieke niches verankering van cellen. De fabricage van TEM kan worden gerealiseerd door deoxyribonuclease (DNase) spijsvertering gepaard met een ernstige van organische of anorganische voorbehandeling. Dit protocol is eenvoudig te bedienen en zorgt voor een hoog rendement voor de decellularization. De TEM toonde gunstige cytocompatibility voor TSCC cellen onder statische of stirred kweekomstandigheden, waardoor de bouw van de TSCC model. Een zelf-gemaakte bioreactor werd ook gebruikt voor de aanhoudende stirred voorwaarde voor celkweek. Gereconstrueerde TSCC met behulp van TEM toonde de kenmerken en eigenschappen die lijken op klinisch TSCC Histopathologie, het potentieel in TSCC onderzoek suggereert.

Dit protocol voor de bereiding van TEM ontpopt zich tot efficiënte en geschikte. De TEM toonde perfecte decellularization vergeleken met de moedertaal weefsels. De werkzaamheid van decellularization werd bevestigd door de Haematoxyline-eosine (HE) kleuring (Figuur 1A-B). De HE kleuring resultaten bleek volledige verdwijning van nucleaire kleuring in TEM (Figuur 1B). DNA inhoud kwantificering van...

Watch this Scientific Journal Video about Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro at JoVE.com

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

Fabricage van tong extracellulaire Matrix en reconstructie van tong plaveiselcelcarcinoom In Vitro

Een methode is hier weergegeven voor de voorbereiding van de tong extracellulaire matrix (TEM) met efficiënte decellularization. De TEM kan worden gebruikt als functionele steigers voor de wederopbouw van een tong plaveiselcelcarcinoom (TSCC) model onder statische of stirred cultuuromstandigheden.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Research

JoVE Journal

Cancer Research

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Een Model voor perineurale invasie in hoofd en nek plaveiselcelcarcinoom

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

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Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

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Vier-kleur Fluorescentie Immunohistochemie van T-cel Subpopulations in Archival Formalin-Fixed, Paraffine-Embedded Human Oropharyngeal Squamous Cell Carcinoma Monsters

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Verplaatsen naar boven: Een eenvoudig en flexibel In Vitro driedimensionale invasie Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Analyseren van de communicatie tussen monocyten en primaire borstkankercellen in een extracellulaire Matrix extraheren (ECME)-gebaseerde driedimensionaal systeem

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Therapie testen in een sferoïde gebaseerde 3D cel cultuur Model voor hoofd en nek plaveiselcelcarcinoom

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Intramucosal enten van plaveiselcelcarcinoom cellen in muizen voor Tumor immuun profileren en evaluatie van de reactie van de behandeling

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Driedimensionale bot extracellulaire Matrix Model voor Osteosarcoom

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Het bestuderen van normale weefsel straling effecten met behulp van extracellulaire matrix hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Time-lapse 2D-beeldvorming van fagocytische activiteit in M1 macrophage-4T1 muis borstkanker cellen in co-culturen

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...