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

Vi takker de felles anstrengelser av Dr. Thao Dang og medlemmer av hennes lab for bruk av deres bildebehandling og mikroskop utstyr. Vi takker også Biorepository og Tissue Research Facility (BTRF) kjerne, Jeff Harper, og beboerne ved Universitetet i Virginia for å gi oss med livmor vev. Vi takker Karol Szlachta for hjelp med Skjematisk oversikt.

Hysterektomi prøver som brukes i dette manuskriptet ble samlet i samstemmighet med et universitet IRB-godkjent etikk protokoll nummerert IRB-HSR # 14424. En. Sample Oppkjøp fra Clinical Source <ol><li> Skaff regjeringen og institusjonsbaserte etiske retningslinjer og dokumentasjon godkjenning før du begynner. </li><li> Gjennomføre alle trinn i sterile forhold. </li><li> Bevar pasient-avledet vev i mediet (RPMI-eller DMEM / High Glucose) i et 50 ml rør ved 4 ° C dersom prøven ikke ka...

<ol>
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M., Hampton, A. L., Riley, S. C., Findlay, J. K., Salamonsen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+endothelin+released+by+human+endometrial+epithelial+cells+in+culture.">Production and characterization of endothelin released by human endometrial epithelial cells in culture.</a> J Clin Endocrinol Metab. 79, 1625-1631 (1994).</li><li>Siegfried, J. M., Nelson, K. G., Martin, J. L., Kaufman, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+identification+of+cultured+cells+from+human+endometrium.">Histochemical identification of cultured cells from human endometrium.</a> In Vitro. 20, 25-32 (1984).</li><li>Rawdanowicz, T. J., Hampton, A. L., Nagase, H., Woolley, D. E., Salamonsen, L. 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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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cyclic+AMP+and+estrogen/progesterone+on+the+transcription+of+DNA+methyltransferases+during+the+decidualization+of+human+endometrial+stromal+cells.">Effect of cyclic AMP and estrogen/progesterone on the transcription of DNA methyltransferases during the decidualization of human endometrial stromal cells.</a> Mol Hum Reprod. 19, 302-312 (2013).</li><li>Tamura, 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=Induction+of+IGFBP-1+expression+by+cAMP+is+associated+with+histone+acetylation+status+of+the+promoter+region+in+human+endometrial+stromal+cells.">Induction of IGFBP-1 expression by cAMP is associated with histone acetylation status of the promoter region in human endometrial stromal cells.</a> Endocrinology. 153, 5612-5621 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American Society for Reproductive Medicine: Quick facts about infertility. , (2013).</li><li>Salker, M., 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=Natural+selection+of+human+embryos:+impaired+decidualization+of+endometrium+disables+embryo-maternal+interactions+and+causes+recurrent+pregnancy+loss.">Natural selection of human embryos: impaired decidualization of endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss.</a> PLoS One. 5, (2010).</li></ol>

Andre grupper har beskrevet og tilpasset metodikk for utarbeidelse av endometrial stromal kulturer, de fleste som benytter collagenase 4,12,13,15-18. I dette manuskriptet, har vi gitt metodikk og dokumentasjon for to forenklede primære endometrial stromal kultur metoder, som begge benyttes av vår lab for økonomiske årsaker og den praktiske tilgjengeligheten av trypsin og / eller et barberblad. Når man sammenligner våre to metoder, både med hell generere levedyktige primærk...

Den menneskelige uterus korpus består av tre lag, det perimetrium (eller serosa), myometrium, og endometrium. Skille hver av disse lagene er et viktig skritt for å etablere endometrial cellelinjer. Den perimetrium er det ytterste lag av uterus og består av tynne, serøse celler. Den myometrium er tykk, midtre lag av uterus og består av glatte muskelceller. Endometrium er identifisert som det indre laget av livmoren og omfatter epiteliale og stromal cellepopulasjoner. Endometriet er videre delt inn i basalis lag som stamcelle befolkningen er en hypotese å befolke functionalis lag ca hver 28 dager en. Den functionalis lag av human endometrium gjennomgår betydelige biokjemiske og morfologiske endringer i respons til sirkulerende hormoner. Disse hormonene er avledet fra hypofysen og eggstokkene. Denkoordinert produksjon og frigjøring av hormoner resultater i en reproduksjonssyklusen. Den reproduktive syklus er designet for å forberede endometriet for potensielle embryo implantasjon hendelser. Hos mennesker er den reproduktive syklus kjent som &quot;menstruasjonssyklus&quot;, og deles inn i tre faser - proliferativ, sekretoriske, og menstruasjons. Proliferasjonsfasen innebærer spredning av functionalis livmor lag mens sekretoriske fasen er preget av functionalis modning. Spesielt ekstracellulære forandringer, sekreter, og cellulær differensiering signalisere en potensiell implantasjon. Ved implantasjonen ikke finner sted før slutten av den sekretoriske fase, er functionalis endometrial sjikt felle i løpet av menstruasjons fase. Betydningen av menstruasjon og de hendelser som utløser den utstøtingen av functionalis lag er fortsatt blir debattert. I mennesker er det blitt stilt at menstruasjonen er resultatet av en bestemt mid-sekretoriske fase differensiering hendelse kjentsom &quot;spontan decidualization&quot; to. I dette manuskriptet, gir vi detaljert metodikk for både endometrial stromal celle isolasjon metoder, og bruker en kombinasjon av immunfluorescens og digitale bilder for å demonstrere effekten av disse tilnærmingene. I tillegg har vi anvende en vanlig brukt in vitro modell av spontan decidualization å bekrefte endometrial stromal celleisolasjon.

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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>
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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>
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		<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>
		</tr>
		<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>
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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>
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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>
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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>
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		<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>
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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>
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		<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>
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		<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>
		</tr>
		<tr height="21">
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two methods for establishing primary human endometrial stromal cells from hysterectomy specimens

Mange forsøk har vært viet til å etablere in vitro cellekultursystemer. Disse systemene er designet for å modellere et stort antall in vivo prosesser. Cellekultursystemer som følge av menneskelige endometrial prøvene er ikke noe unntak. Applikasjoner varierer fra normale cykliske fysiologiske prosesser i endometriale patologier slik som gynekologisk kreft, infeksjonssykdommer, og forplantnings mangler. Her gir vi to metoder for å etablere primær endometrial stromale celler fra kirurgisk resekterte endometrial hysterektomi prøver. Den første metode er referert til som &quot;skrape-metoden&quot;, og innbefatter mekanisk skraping ved bruk av kirurgisk eller barberblader, mens den andre metoden er betegnet &quot;den trypsin-metoden&quot;. Denne sistnevnte metode anvender den enzymatiske aktiviteten av trypsin for å fremme separasjon av celler og primære celleutvekst. Vi illustrere trinn-for-trinn metode gjennom digitale bilder og mikroskopi. Vi også kundenee eksempler for å validere endometrial stromal cellelinjer via kvantitative sanntid polymerase kjedereaksjoner (qPCR) og immunfluorescens (IF).

Som understreket i protokollen delen, pass på å utføre alle metoder under offentlig, institusjonelle og etiske retningslinjer ved håndtering og tilberedning menneskelig vev. Inkludert i dette manuskriptet er en illustrasjon av den generelle arbeidsflyten av &quot;skrapemetode&quot; (figur 1A), og &quot;den trypsin-metoden&quot; (figur 1B) som brukes til å etablere primær endometrial kulturer. Disse metoder er beskrevet i detalj i <stro...

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

Etablering av primær endometrial stromal cellekultursystemer fra hysterektomi prøver er et verdifullt biologisk teknikk og et avgjørende skritt før forfølge en lang rekke forsknings mål. Her beskriver vi to metoder som brukes for å etablere stromal kulturer fra kirurgisk resekterte endometrial vev av menneskelige pasienter.

To metoder for å etablere primær Menneskelig Livmor stromale celler fra Hysterektomi Prøver

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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Medicine

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Retinal detachment (RD) describes a separation of the neurosensory retina  from the retinal pigmented epithelium (RPE). The RPE is essential for normal  ...

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Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

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Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

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Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...