Name: two methods for establishing primary human endometrial stromal cells from hysterectomy specimens
Uploaded: 2014-05-23T14:00:00
Description: Watch this Scientific Journal Video about Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens at JoVE.com

Wij danken de gezamenlijke inspanningen van dr. Thao Dang en de leden van haar lab voor het gebruik van hun beeldvorming en microscoop apparatuur. We danken ook de biorepository en Tissue Research Facility (BTRF) kern, Jeff Harper, en de bewoners aan de Universiteit van Virginia voor het verstrekken van ons met baarmoeder weefsel. Wij danken Karol Szlachta voor de hulp met schematisch overzicht.

Hysterectomie exemplaren gebruikt in dit manuscript werden verzameld in overeenstemming met een universiteit IRB goedgekeurde ethiek protocol genummerde IRB-HSR # 14424. 1. Sample Overname van Clinical Bron <ol><li> Verkrijgen overheid en het institutioneel bepaalde ethische richtlijnen en documentatie goedkeuring voordat u begint. </li><li> Voeren alle stappen in steriele omstandigheden. </li><li> Domein-patiënt afgeleide weefsel in medium (RPMI of DMEM / High Glucose) in een 50 ml buis ...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+production+by+cultured+human+endometrial+stromal+cells:+identification+of+interstitial+collagenase,+gelatinase-A,+gelatinase-B,+and+stromelysin-1+and+their+differential+regulation+by+interleukin-1+alpha+and+tumor+necrosis+factor-alpha.">Matrix metalloproteinase production by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase-A, gelatinase-B, and stromelysin-1 and their differential regulation by interleukin-1 alpha and tumor necrosis factor-alpha.</a> J Clin Endocrinol Metab. 79, 530-536 (1994).</li><li>Dimitriadis, E., Robb, L., Salamonsen, L. 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Andere groepen hebben beschreven en aangepaste methodologie voor de bereiding van endometriale stromale culturen, waarvan de meeste gebruik collagenase 4,12,13,15-18. In dit manuscript hebben we methodologie en bewijsmateriaal voor twee vereenvoudigde primaire endometriale stromale kweekmethoden, die beide worden gebruikt door ons laboratorium om economische redenen en de gemakkelijke verkrijgbaarheid van trypsine en / of een scheermesje. Wanneer onze twee methoden vergelijken, zo...

De menselijke baarmoeder corpus bestaat uit drie lagen, de perimetrium (of serosa), het myometrium en het endometrium. Onderscheidende elk van deze lagen een belangrijke stap endometrium cellijnen vast. De perimetrium is de buitenste laag van de baarmoeder en samengesteld uit dunne, sereuze cellen. De myometrium is de dikke, middelste laag van de baarmoeder en bestaat uit gladde spiercellen. Het endometrium is geïdentificeerd als de binnenste laag van de baarmoeder en omvat epitheliale en stromale cel populaties. Het endometrium is verder onderverdeeld in de basalis laag waarvan stamcelpopulatie wordt verondersteld dat de functionalis laag herbevolking ongeveer elke 28 dagen 1. De functionalis laag van het menselijke endometrium ondergaat significante biochemische en morfologische veranderingen in respons op circulerende hormonen. Deze hormonen zijn afgeleid van de hypofyse en de eierstokken. Degecoördineerde productie en afgifte van hormonen resulteert in een voortplantingscyclus. De voortplantingscyclus is ontworpen om het endometrium te bereiden op mogelijke embryo implantatie gebeurtenissen. Bij mensen wordt de voortplantingscyclus bekend als &quot;de menstruele cyclus&quot; en onderverdeeld in drie fasen - proliferatieve, secretoire en menstruatie. De proliferatieve fase betreft de verspreiding van de functionalis endometrium laag terwijl de secretoire fase wordt gekenmerkt door functionalis rijping. Specifiek extracellulaire veranderingen, afscheidingen en celdifferentiatie signaal een mogelijke inplanting. Als implantatie niet geschieden vóór het einde van de secretoire fase wordt de functionalis baarmoederslijmvlies laag vergoten tijdens de menstruele fase. Het belang van de menstruatie en de gebeurtenissen die het vergieten van de functionalis laag triggeren zijn nog gedebatteerd. In mensen, is gesteld dat de menstruatie is het gevolg van een specifieke mid-secretoire fase differentiatie gebeurtenis bekendals &quot;spontane decidualization&quot; 2. In dit manuscript hebben we gedetailleerde methodologie voor zowel endometrium stromale celisolatie methoden, en gebruik een combinatie van immunofluorescentie en digitale beelden naar de werkzaamheid van deze benaderingen te tonen. Daarnaast we een algemeen in vitro model van spontane decidualization endometrium stromale cel isolatie bevestigen.

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			<td dir="LTR" height="24" style="height:24px;width:235px;">0.25 Trypsin or 0.05% Trypsin&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">SH3023602 or SH30004202&#160;&#160;</td>
			<td style="width:63px;"></td>
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		<tr height="27">
			<td dir="LTR" height="27" style="height:27px;width:235px;">1.7 micro Centrifuge Tube&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Genesee Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">22-272A&#160;</td>
			<td></td>
		</tr>
		<tr height="25">
			<td dir="LTR" height="25" style="height:25px;width:235px;">1&#181;l,20&#181;l, 200ml and 1000&#181;l Pipette&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Genesee Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">24-401,24-402, 24-412, 24-430&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td dir="LTR" height="22" style="height:22px;width:235px;">15ml Conical Tube&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">339650</td>
			<td></td>
		</tr>
		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">50ml Conical Tube&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">339652</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">6cm Cell Culture Dish&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo scientific&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">12-556-002&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td dir="LTR" height="22" style="height:22px;width:235px;">8 well Chambers&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">AB-4162&#160;</td>
			<td></td>
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		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">Acetate&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher scientific&#160;</td>
			<td dir="LTR" style="width:216px;">C4-100&#160;</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">AMV RT Enzyme/Buffer&#160;</td>
			<td dir="LTR" style="width:201px;">Bio Labs&#160;</td>
			<td dir="LTR" style="width:216px;">M077L&#160;</td>
			<td></td>
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		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Bovine Serum Albumin (BSA)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP-1605-100&#160;</td>
			<td></td>
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		<tr height="23">
			<td dir="LTR" height="23" style="height:23px;width:235px;">Buffered Zinc Formalin&#160;</td>
			<td dir="LTR" style="width:201px;">Thermo&#160;</td>
			<td dir="LTR" style="width:216px;">59201ZF&#160;</td>
			<td></td>
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		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Charcoal strip FBS&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher&#160;</td>
			<td dir="LTR" style="width:216px;">NC9019735&#160;&#160;</td>
			<td></td>
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		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Chloroform&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP1145-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Cover slip&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Brand&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">12-544D&#160;</td>
			<td></td>
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		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">Cyclic AMP (cAMP)&#160;</td>
			<td dir="LTR" style="width:201px;">Sigma&#160;</td>
			<td dir="LTR" style="width:216px;">B7880</td>
			<td></td>
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		<tr height="24">
			<td dir="LTR" height="24" style="height:24px;width:235px;">DMEM/High Glucose&#160;</td>
			<td dir="LTR" style="width:201px;">Hyclone&#160;</td>
			<td dir="LTR" style="width:216px;">SH30243FS&#160;</td>
			<td></td>
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		<tr height="25">
			<td dir="LTR" height="25" style="height:25px;width:235px;">dNTP&#160;</td>
			<td dir="LTR" style="width:201px;">Bioline&#160;</td>
			<td dir="LTR" style="width:216px;">BIO-39025&#160;</td>
			<td></td>
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		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Donkey Anti Goat -TRITC&#160;</td>
			<td dir="LTR" style="width:201px;">Santa Cruz&#160;</td>
			<td dir="LTR" style="width:216px;">SC-3855&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Donkey Serum&#160;</td>
			<td dir="LTR" style="width:201px;">Jackson&#8217;s lab&#160;</td>
			<td dir="LTR" style="width:216px;">&#160;017-000-002&#160;</td>
			<td></td>
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		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">E Cadherin Antibody&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Epitomics&#160;</td>
			<td dir="LTR" style="width:216px;">1702-1</td>
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		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Ethanol&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;&#160;</td>
			<td dir="LTR" style="width:216px;">BP2818-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Fetal Bovine Serum (FBS)&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific</td>
			<td dir="LTR" style="width:216px;">03-600-511&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Fungizone Amphotericin B&#160;</td>
			<td dir="LTR" style="width:201px;">Gibco&#160;</td>
			<td dir="LTR" style="width:216px;">15290-018&#160;</td>
			<td></td>
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		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">GAPDH Probe&#160;</td>
			<td dir="LTR" style="width:201px;">Life Technologies&#160;</td>
			<td dir="LTR" style="width:216px;">HS99999905&#160;</td>
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		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Glycogen&#160;</td>
			<td dir="LTR" style="width:201px;">5Prime&#160;</td>
			<td dir="LTR" style="width:216px;">2301440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Goat Anti Mouse -FITC&#160;</td>
			<td dir="LTR" style="width:201px;">Jackson&#8217;s Lab&#160;</td>
			<td dir="LTR" style="width:216px;">115-096-003&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Isopropanol&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP2618-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:235px;">Kanamycin&#160;&#160;</td>
			<td dir="LTR" style="width:201px;">Fisher Scientific&#160;</td>
			<td dir="LTR" style="width:216px;">BP906-5&#160;</td>
			<td></td>
		</tr>
		<tr height="41">
			<td dir="LTR" height="41" style="height:41px;width:235px;">Medroxyprogesterone acetate (MPA)&#160;</td>
			<td dir="LTR" style="width:201px;">Sigma&#160;</td>
			<td dir="LTR" style="width:216px;">M1629</td>
			<td></td>
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two methods for establishing primary human endometrial stromal cells from hysterectomy specimens

Vele inspanningen gedaan om te bepalen in vitro celkweek systemen. Deze systemen zijn ontworpen om een model van een groot aantal in vivo processen. Celkweek systemen die voortkomen uit menselijke endometrium monsters zijn geen uitzondering. Toepassingen variëren van normale cyclische fysiologische processen endometrium pathologieën zoals gynaecologische kankers, infectieziekten en voortplantingsfunctie. Hier bieden we twee methoden voor het vaststellen van primaire endometrium stromale cellen van chirurgisch weggesneden endometrium hysterectomie exemplaren. De eerste methode wordt aangeduid als &quot;het schrapen methode&quot; en omvat mechanische schrapen met chirurgische of scheermesjes, terwijl de tweede methode wordt aangeduid als &quot;de trypsine-methode. &#39;Deze laatste methode wordt de enzymatische activiteit van trypsine om de scheiding van de elementen en bevorderen cel uitgroei. We illustreren stap-voor-stap methode door middel van digitale beelden en microscopie. We hebben ook provide voorbeelden voor het valideren van endometrium stromale cellijnen via kwantitatieve real time polymerase kettingreactie (qPCR) en immunofluorescentie (IF).

Zoals benadrukt in de sectie Protocol, moet u alle methoden onder overheid, institutionele en ethische richtlijnen uit te voeren bij het ​​hanteren en het voorbereiden van menselijk weefsel. Inbegrepen in dit manuscript is een illustratie van de algemene werkschema van &quot;het schrapen methode&quot; (figuur 1A) en &quot;de trypsine-methode&quot; (figuur 1B) gebruikt om primaire kweken endometrial stellen. Deze werkwijzen worden in deta...

Watch this Scientific Journal Video about Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens at JoVE.com

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Oprichting primaire endometrium stromale celkweek systemen van hysterectomie exemplaren is een waardevolle biologische techniek en een cruciale stap voorafgaand aan het nastreven van een breed scala van onderzoek wil. We beschrijven hier twee methoden om stroma culturen chirurgisch weggesneden endometriale weefsel van menselijke patiënten vastgesteld.

Twee methoden voor het vaststellen van primaire humane Endometriale Stromal Cellen uit hysterectomie Exemplaren

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

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