Name: isolation of sertoli cells and peritubular cells from rat testes
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Forfatterne vil gerne takke Guido Verhoeven og Ludo Deboel, Leuven, som var yderst hjælpsomme i oprettelse isolering af primære Sertolicellerne i Meinhardt laboratorium i Giessen. Monika Fijak, Gießen, er anerkendt for hendes hjælp med figur 1A og rådgivning. Undersøgelser blev støttet af Deutsche Forschungsgemeinschaft gennem tilskud BH 93 / 1-1 (SB) og finansiering af International Research Graduate College JLU Gießen (Tyskland) / Monash University (Melbourne, Australien) GRK 1871 (SB). Der blev også opnået fra et tilskud af staten Hessen i programmet &quot;Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz&quot; (LOEWE) kaldet &quot;Männliche Infertilität bei Infektion &amp; Entzündung&quot; (MIBIE).

1. Dyreetiske Statement Eksperimenter er beskrevet her, blev udført i overensstemmelse med retningslinjerne i Regierungspräsidium Giessen, Tyskland, som den lokale myndighed og bekræft til den tyske adfærdskodeks for pasning og anvendelse af dyr til forsøg (Tilladelse nr.: GI 20/23 Nr A 31 / 2012). 2. Udarbejdelse af Medier, enzymløsninger og Dyr <ol><li> Forberede 1% jod alkohol ved at opløse 1 g iod i 100 ml ethanol. </li><li> Forbered 2x 500 ml Dulbecco phosphatbufret saltv...

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The seminiferous tubules are bounded by a circular layer of peritubular cells and a basal lamina on the luminal side. Sertoli cells are resting on the basal lamina, establish the blood testis barrier through formation of occluding junctions between adjacent cells and provide the structural framework for the organisation of the seminiferous epithelium. Sertoli cells and adjacent spermatogenic cells maintain intimate contacts throughout germ cell development providing physical contact and communication alike. Therefore, wh...

Testiklerne producerer mandlige kønsceller og kønshormoner, dvs, androgener. Organet består af to rum. I det interstitielle rum, som repræsenterer omkring 10-12% af den samlede testikelkræft bind 1, steroidgenese foregår inden for Leydig-celler. Det rørformede kammer udgør ca. 60-80% af den testikel bind 1 og indeholder kimceller og to typer af somatiske celler - peritubulære celler og Sertoliceller. Testiklerne er delt af bindevæv septa i 250-300 lobules, hver indeholdende 1-3 stærkt snoede sædkanalerne. Disse tubuli er omgivet af en basalmembran, et ark af collagen, og et rundtgående lag af peritubulære celler (figur 1A). Den germinale epitel er placeret på den luminale side af basalmembranen. Sertoliceller er store langstrakte celler, der spænder over hele germinale epitel fra basalmembranen til hulrummet. De er stærkt attgjorde ondt til basalmembranen og udgøre et sammenhængende cellulær ark gennem basolaterale organiserede tight junctions, der okkluderer germinale epitel fra interstitium og repræsenterer blod-testis-barrieren. Sertolicellerne har prominente cytoplasmatiske fremskrivninger og forgreninger, der sætter dem i stand til at komme ind i stramme morfologiske og funktionelle kontakt med en artsspecifik, men konstant antal kønsceller. Diploide germinale stamceller formering og differentiering til spermatogonier. Under meiose I kortlivede tetraploide spermatocytter genereres der udvikler videre i fire haploide spermatider under meiose II. Alle kimceller er indbyrdes forbundet ved cytoplasmatiske broer således at de danner en cellulær net. Den væsentligste begivenhed under modning af spermatider er ekstrudering af store dele af cytoplasmaet, danner resterende organer, i en proces kaldet sædcelledannelse. Resterende organer fagocyteres af Sertoli celler. Sene spermatider derefter frigives tilde rørformede hulrum og transporteret ind bitestiklen for yderligere modning. Sertoliceller og kønsceller synes at gensidigt koordinere spermatogenesen topografisk og funktionelt. Udarbejdelse af individuelle testikelkræft celletyper startede næsten et århundrede siden, da små testikel stykker blev dyrket og celletyper identificeres ved mikroskopi 2. Ved omhyggelig dissektion af tubuli efter åbning tunica albuginea med fint-tippes pincet var det senere muligt at adskille rørformet og interstitielle rum 3. I 1975 Welsh og Wiebe indført en kollagenase behandling for at befri tubuli i at hæfte interstitiel væv og en pancreatin behandling til fjernelse af det ydre peritubular cellelag 4. Fra tidligt på unge umodne rotter (ca. 20 dage gamle) var blevet anvendt, hvor Sertoli celler omfatter en stor del af den rørformede cellepopulation fordi spermatogenesen ikke er begyndt endnu. I denne alder rotte SertOLI celler ophører med at dele sig, og tætte forbindelser mellem naboceller danner så blod-testis-barrieren er etableret 5. Uafhængigt af Welsh og Wiebe Dorrington et al. Indført en kombination af trypsin og deoxyribonuclease efterfulgt af soybeen trypsin inhibitor og collagenase behandling, der blev offentliggjort i samme år 6. Begge grupper anvendes også mekanisk kraft (tegning de spaltede rørformede fragmenter gentagne gange gennem en sprøjtenål ​​eller en Pasteur pipette henholdsvis) for at frembringe en homogen cellesuspension til galvanisering, der indeholder ca. 70% Sertoliceller. Efter 3 dage i kultur, ved hjælp af et medium, der er serumfrit, procentdelen af ​​Sertoli celler stiger til ca. 90%. Dette kunne især tilskrives en død kontaminerende kimceller. Resterende peritubulære celler (PTC), dog holde solidt fastgjort via deres ekstracellulære matrix. PTC&#39;er, men ikke Sertoli celler, vides at producerefibronectin, der kan tjene som markør-protein til estimering kontaminering med PTC&#39;er. Derfor Tung et al. ræsonnerede, at en yderligere hyaluronidase behandling kan forbedre renheden af Sertolicellen fraktion 7. Faktisk kunne de vise, at en yderligere behandling kunne reducere forurening med PTC&#39;er ca. 20-fold, som bliver særlig tydeligt, når et serum-holdigt medium i sammenligning anvendes til dyrkning af oprensede Sertoliceller. Fra da af den forbedrede procedure for Tung et al. blev den fremherskende protokol og blev flittigt brugt af andre større grupper på området 8 (figur 1B). Under collagenasebehandling flertallet af PTC&#39;er frigives og kan isoleres parallelt med Sertoliceller. Ud fra følgende betragtninger PTC&#39;er kraftigt formere og reagerer ikke på follikelstimulerende hormon (FSH), behøver Sertoliceller ikke undergår mitose længere, og reagere på FSH ved karakteristiske morfologiske ændringerog en stigning i cyklisk adenosinmonophosphat (cAMP) koncentration 9. Meget lignende enzymatisk nedbrydning protokoller kan anvendes til isolering af primære Sertoliceller fra andre dyr som mennesket 10, muse 11,12, sibirisk hamster 13 eller yak 14. Til fjernelse af store mængder forurenende kønsceller en hypotonisk chok kan anvendes ved afslutningen af den isolation procedure 15. Dette muliggør også en effektiv isolering af Sertoliceller fra voksen rotte testikler 16. En beriget Sertolicellen suspension kan også separeres fra kimceller ved udpladning af suspensionen på lectin retter overtrukket med Datura stramonium agglutinin 17. Kun Sertoliceller og et par resterende PTC&#39;er overholde lectin plader. Primær Sertoli cellekulturer, hovedsageligt fra rotten, er oprindeligt brugt til undersøgelse lydhørhed over for hormoner eller oprettelse cellelinjer som muse Sertolicellen line TM4 18. Denne cellelinie er blevet undersøgt i mere end hundrede studier frem til i dag. I en translationel tilgang Sertoliceller er blevet brugt til immun-beskyttelse af co-dyrkede celler og væv som i co-transplantation af fremmedfjendske eller allogene pancreas-øer for langsigtet graftoverlevelse uden systemisk immunosuppression 19. Isolerede Sertoli-celler er også blevet anvendt i co-kultur eksperimenter til undersøgelse epitel-mesenkymale (Sertoliceller - PTCc) og somatisk-kønsceller (Sertoliceller - kimceller) interaktioner 20, 21. Senest primære Sertoliceller er blevet anvendt til undersøgelse af ekspression af Toll-lignende receptorer og sekretionen af proinflammatoriske cytokiner samt signaltransduktionsegenskaberne kaskader fører til cytokinekspression efter infektion med patogene og uropatogen E. coli 22. Andre nylige undersøgelser brugte Sertoliceller for at studere testikelkræft immun privilege 23 og viste, at testosteron-forbehandling undertrykker LPS-inducerede inflammatoriske respons 24.

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	<tbody>
		<tr height="80">
			<td height="80" style="height:80px;width:289px;">Actin (smooth muscle) antibody clone 1A4</td>
			<td style="width:111px;">Dako</td>
			<td style="width:124px;">M0851</td>
			<td style="width:289px;">Monoclonal mouse anti human antibody. 
			Use 1:100 dilution for immunofluorescence.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Albumin bovine fraction V 
			Standard grade, lyophilized</td>
			<td style="width:111px;">Serva</td>
			<td style="width:124px;">11930.04</td>
			<td style="width:289px;">Filter (0.45 &#181;m) BSA solutions used for immunofluorescence.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Corning&#8482; Cell Strainers 70&#160;&#181;m</td>
			<td style="width:111px;">Corning</td>
			<td style="width:124px;">431751</td>
			<td>White color</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Collagenase A from Clostridium histolyticum</td>
			<td style="width:111px;">Roche</td>
			<td style="width:124px;">10103586001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">DAPI mountant ProLong&#174; Gold Antifade</td>
			<td style="width:111px;">Life technologies</td>
			<td style="width:124px;">P-36931</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">D-glucose</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">G8644</td>
			<td>100 g/l</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Dulbecco&#180;s PBS without Ca2+/Mg2+</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">DNase I</td>
			<td style="width:111px;">Roche</td>
			<td style="width:124px;">10104159001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Electron microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td>EM 109S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Fluorescence microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td style="width:124px;">Axioplan 2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Hyaluronidase from bovine testis</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">H3506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Inverted light microscope</td>
			<td style="width:111px;">Olympus</td>
			<td style="width:124px;">CKX41</td>
			<td>Routine cell culture microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Mouse IgG / IgM (H+L) polyclonal 
			secondary Antibody&#160;</td>
			<td style="width:111px;">Life technologies</td>
			<td style="width:124px;">A-10684</td>
			<td>Alexa Fluor&#174; 488 conjugate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Oil Red O</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">O0625</td>
			<td style="width:289px;">Has replaced Sudan III and Sudan IV 
			because of brighter color.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Paraformaldehyde</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">P6148</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Penicillin (5,000&#160;U/ml)/ 
			Streptomycin (5,000 &#181;g/ml)</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">15070-063</td>
			<td>100x solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">RPMI-1640</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">21875-034</td>
			<td>Contains 300 mg/L L-glutamine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Trypsin from porcine pancreas</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">T5266</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:289px;">Trypsin inhibitor from soybean</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">T6522</td>
			<td style="width:289px;">The Sigma product is considerably cheaper than the previously used 
			BPTI (Aprotinin) from Roche.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:289px;">Vimentin antibody (V9)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">V6630</td>
			<td style="width:289px;">Monoclonal mouse anti pig vimentin antibody.&#160; Use 1:100 dilution for immunofluorescence.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Wistar WU rats</td>
			<td style="width:111px;">Charles River</td>
			<td style="width:124px;">N/A</td>
			<td>Should be 19 days old on day of experiment.</td>
		</tr>
	</tbody>
</table>

isolation of sertoli cells and peritubular cells from rat testes

Testiklerne, og især den mandlige gamet, udfordrer immunsystemet på en unik måde, fordi differentieret sperm først vises på tidspunktet for puberteten - mere end ti år efter etableringen af ​​systemiske immun tolerance. Spermatogene celler udtrykker en række proteiner, der kan ses som ikke-selv af immunsystemet. Testiklerne skal derefter kunne etablere tolerance over for disse neo-antigener på den ene side, men stadig være i stand til at beskytte sig mod infektioner og tumorudvikling på den anden side. Derfor testiklerne er en af ​​et par immune privilegerede steder i kroppen, der tåler fremmede antigener uden at fremkalde en skadelig inflammatorisk immunrespons. Sertoli celler spiller en central rolle for vedligeholdelse af denne immune privilegeret miljø af testiklerne og også forlænge overlevelsen af ​​cotransplanted celler i et fremmed miljø. Derfor primære Sertolicellerne er et vigtigt redskab til at studere immun privilegium testiklerne, der ikke kan være easily erstattet af etablerede cellelinjer eller andre cellulære modeller. Her præsenterer vi en detaljeret og omfattende protokol til isolering af Sertoliceller - og peritubulære celler hvis det ønskes - fra rotte testikler inden for en enkelt dag.

Den beskrevne procedure tillader isolering af det ca. 12 x 10 7 Sertoli celler fra 10 rotte testikler. 3 x 10 6 celler udplades per brønd på en 6-brønds plade, således at seks til syv plader med 6 brønde er tilgængelige for eksperimenter på dag 7. Den trypsin-DNase I fordøjelsen er den mest kritiske trin under Sertolicellen isolation. Hvis fordøjelse på dette tidspunkt skrider for langt, er forholdet mellem kønsceller / Sertoliceller vil uforholdsmæssigt...

Watch this Scientific Journal Video about Isolation of Sertoli Cells and Peritubular Cells from Rat Testes at JoVE.com

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

Primære Sertoliceller er nødvendige for at studere testis-immune privilegium, signaltransduktion under betændelse eller infektion, og udnyttelse af deres immunbeskyttende egenskaber. Her beskriver vi en enzym-baseret protokol til isolering af højtoprensede primære Sertoliceller og peritubulære celler fra rotte testikler.

Isolering af Sertoliceller og peritubulære Celler fra rotte Testikler

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Research

JoVE Journal

Immunology and Infection

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The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

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Isolering af Funktionel Cardiac immunceller

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

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Isolering af Mouse Lung dendritiske celler

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Primære Mikroglia Isolering af blandede gliacelle Kulturer af neonatal rottehjerne Tissue

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Isolering og karakterisering af dendritiske celler og makrofager fra mus Tarmen

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Adipose Tissue Immune Cells

Isolering af fedtvæv immunceller

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolering af myeloide dendritiske celler og epitelceller fra human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

Isolation of Double Negative ÃƒÅ½Ã‚Â±ÃƒÅ½Ã‚Â² T Cells from the Kidney

Isolering af Double Negative αβ T-celler fra nyren

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Isolation of Murine Lymph Node Stromal Cells

Isolering af murine lymfeknuder stromacellers

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...