Name: primary culture of porcine retinal pigment epithelial cells
Uploaded: 2022-09-23T14:00:00
Description: Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

Författarna vill visa sin tacksamhet och respekt för alla djur som bidrar med sina celler i denna studie. Denna studie stöddes delvis av bidrag från National Key R&D Program of China (2019YFA0111200, Yi Liao & Yuan Gao och Grant nr 2018YFA0107301, Wei Li). Författarna tackar Jingru Huang och Xiang You från Central Lab, School of Medicine, Xiamen University för tekniskt stöd inom konfokal avbildning.

Användningen av försöksdjur följde bestämmelserna från Association for Research in Vision and Ophthalmology (ARVO) och godkändes av etikkommittén för experimentell djurhantering vid Xiamen University.
1. Beredning av experimentella kirurgiska anordningar, vävnadsuppslutningsenzym och cellodlingsbuffert
<ol><li>Förbered de experimentella kirurgiska apparaterna genom att rengöra och autoklavera två par oftalmiska kirurgiska saxar och pincett dagen före ögonglobs...

<ol>
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Här har ett detaljerat och optimerat protokoll för isolering, odling och karakterisering av RPE-celler från svinögonbollar, vilket genererar en bra modell för in vitro-karakterisering av RPE-celler och RPE-relaterade sjukdomsstudier beskrivits. Metoder för isolering av RPE från mänskliga, mus- och råttögon har beskrivits tidigare23,24,25. Det är dock svårt att få mänskliga ögonbollar i vissa laboratorier,...

Retinalpigmentepitelet (RPE) är beläget mellan fotoreceptorer och choriokapillaris i det yttre skiktet av näthinnan1 med flera funktioner, inklusive att bilda blod-retinalbarriären, transportera och utbyta näringsämnen och retinala metaboliter, återvinna vitamin A för att upprätthålla en normal visuell cykel och fagocytos och clearance av shed-fotoreceptor yttre segment (POS)2,3 . Eftersom POS kräver konstant självförnyelse för att generera syn, måste RPE-cellerna kontinuerligt uppsluka fristående POS för att upprätthålla retinal homeostas4. Därför resulterar RPE-dysfunktion i många bländande ögonsjukdomar, såsom åldersrelaterad makuladegeneration (AMD)4, retinitis pigmentosa (RP)5, Leber medfödd amauros6, diabetisk retinopati7, etc. Hittills är den exakta patogenesen av de flesta av dessa sjukdomar fortfarande svårfångad. Som ett resultat etableras RPE-cellodling för att studera RPE-cellbiologi, patologiska förändringar och underliggande mekanismer.
Som den enklaste modellen för att studera cellbiologi startades odlingen av RPE-celler redan på 1920-talet8. Även om ARPE-19 används i stor utsträckning som RPE-celler, ger förlust av pigmentering, kullerstensmorfologi och särskilt barriärfunktionerna i denna cellinje upphov till många problem9. I jämförelse erbjuder kulturen av primära mänskliga RPE-celler ett mer realistiskt scenario för fysiologiska och patologiska studier9. Den relativt begränsade tillgängligheten begränsar dock deras användning och etiska problem finns alltid. Dessutom använde flera grupper musmodeller för att odla RPE-celler. Musögats storlek är dock liten, och en enda kultur kräver vanligtvis många möss, vilket inte är bekvämt9. Nyligen har forskare utvecklat nya metoder för att använda mänskliga embryonala stamceller eller inducerade pluripotenta stamceller för att härleda RPE-celler. Även om denna teknik har särskild potential för behandling av ärftliga RPE-störningar, är det tidskrävande och kräver vanligtvis flera månader för att generera mogna RPE-celler10. För att övervinna dessa problem introducerar vi här ett lätt att följa protokoll för att rutinmässigt isolera och odla RPE-celler med hög renhet i laboratoriet. Under lämpliga odlingsförhållanden kan dessa celler visa typiska RPE-funktioner och uppvisa typiska RPE-morfologier. Därför kan denna odlingsmetod ge en bra modell för att förstå RPE-fysiologi, studera cytotoxicitet, undersöka patologiska mekanismer för relaterade ögonsjukdomar och genomföra läkemedelsundersökningar.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ARPE-19 cells</td>
			<td>CCTCC</td>
			<td>GDC0323</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Yeasen</td>
			<td>36101ES60</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscopy</td>
			<td>Zeiss</td>
			<td>LSM 880 with Airyscan</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Touch</td>
			<td>Bio-Rad</td>
			<td>1708370</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Sangon</td>
			<td>F619301</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish</td>
			<td>NEST</td>
			<td>121621EH01</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well culture plate</td>
			<td>NEST</td>
			<td>29821075P</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F12 Medium</td>
			<td>Gibco</td>
			<td>C11330500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM basic Medium</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM2</td>
			<td>World Precision Instruments</td>
			<td>EVOM2</td>
			<td>For TER measurement</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ExCell Bio</td>
			<td>FSP500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>A-11034</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Mouse IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>0300-0108P</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti Rabbit IgG (H/L):HRP</td>
			<td>Bio-Rad</td>
			<td>5196-2504</td>
			<td></td>
		</tr>
		<tr>
			<td>hydrocortisone</td>
			<td>MCE</td>
			<td>HY-N0583/CS-2226</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342 solution (20 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 96 Instrument</td>
			<td>Roche</td>
			<td>5815916001</td>
			<td></td>
		</tr>
		<tr>
			<td>Liothyronine</td>
			<td>MCE</td>
			<td>HY-A0070A/CS-4141</td>
			<td></td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM(1X)+GlutaMAX Medium</td>
			<td>Gibco</td>
			<td>10566-016</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM NEAA(100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP syringe filter unit</td>
			<td>Millipore</td>
			<td>SLGPR33RB</td>
			<td></td>
		</tr>
		<tr>
			<td>N1</td>
			<td>Sigma-Aldrich</td>
			<td>SLCF4683</td>
			<td></td>
		</tr>
		<tr>
			<td>NcmECL Ultra</td>
			<td>New Cell&#38;Molecular Biotech</td>
			<td>P10300</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat Powdered Milk</td>
			<td>Solarbio</td>
			<td>D8340</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>SparkJade</td>
			<td>SJ-MV0061</td>
			<td></td>
		</tr>
		<tr>
			<td>Na+-K+ ATPase antibody</td>
			<td>Abcam</td>
			<td>ab76020</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PAGE Gel Fast Preparation Kit(10%)</td>
			<td>Epizyme</td>
			<td>PG112</td>
			<td></td>
		</tr>
		<tr>
			<td>primary Human RPE cells&#160;</td>
			<td>-</td>
			<td>-</td>
			<td>Generous gift from Shoubi Wang lab&#160;</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad by Dotmatics</td>
			<td>version 8.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktails</td>
			<td>APExBIO</td>
			<td>K1024</td>
			<td></td>
		</tr>
		<tr>
			<td>PRE65 antibody</td>
			<td>Proteintech</td>
			<td>17939-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>PEDF antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-390172</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>100 x penicillin/streptomycin&#160;</td>
			<td>Biological Industries</td>
			<td>03-031-1BCS</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>RARBIO</td>
			<td>RA-9005</td>
			<td></td>
		</tr>
		<tr>
			<td>ReverTra Ace qPCR RT Master Mix</td>
			<td>Toyobo</td>
			<td>FSQ-201</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>601052</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile centrifuge tubes</td>
			<td>NEST</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Damas-beta</td>
			<td>107-35-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo-Fisher&#160;</td>
			<td>15596026</td>
			<td>RNA extraction solution</td>
		</tr>
		<tr>
			<td>TB Green Fast qPCR Mix</td>
			<td>Takara</td>
			<td>RR430A</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well transwell inserts</td>
			<td>Labselect</td>
			<td>14212</td>
			<td></td>
		</tr>
		<tr>
			<td>VEGF antibody</td>
			<td>Proteintech</td>
			<td>19003-1-AP</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
		<tr>
			<td>VEGF ELISA kit</td>
			<td>Novusbio</td>
			<td>VAL106</td>
			<td></td>
		</tr>
		<tr>
			<td>ZO-1 antibody</td>
			<td>ABclonal</td>
			<td>A0659</td>
			<td>Recognize both human and porcine proteins</td>
		</tr>
	</tbody>
</table>

primary culture of porcine retinal pigment epithelial cells

Retinal pigmentepitel (RPE) är ett monolager av polariserade pigmenterade epitelceller, belägna mellan choroid och neuroretina i näthinnan. Flera funktioner, inklusive fagocytos, närings- / metabolittransport, vitamin A-metabolism, etc., utförs av RPE dagligen. RPE-celler är terminalt differentierade epitelceller med liten eller ingen regenerativ kapacitet. Förlust av RPE-celler resulterar i flera ögonsjukdomar som leder till synnedsättning, såsom åldersrelaterad makuladegeneration. Därför är upprättandet av en in vitro-odlingsmodell av primära RPE-celler, som mer liknar RPE in vivo än cellinjer, avgörande för de karakteristiska och mekanistiska studierna av RPE-celler. Med tanke på att källan till mänskliga ögonbollar är begränsad skapar vi ett protokoll för att odla primära RPE-celler från svin. Genom att använda detta protokoll kan RPE-celler enkelt dissocieras från vuxna ögonbollar från svin. Därefter fäster dessa dissocierade celler till odlingsskålar / insatser, sprider sig för att bilda ett sammanflytande monolager och återupprättar snabbt viktiga egenskaper hos epitelvävnad in vivo inom 2 wks. Genom qRT-PCR visas det att primära RPE-celler hos svin uttrycker flera signaturgener på jämförbara nivåer med inbyggd RPE-vävnad, medan uttrycken för de flesta av dessa gener förloras/reduceras kraftigt i humana RPE-liknande celler, ARPE-19. Dessutom visar immunofluorescensfärgningen fördelningen av tät korsning, vävnadspolaritet och cytoskelettproteiner, liksom närvaron av RPE65, ett isomeras som är kritiskt för vitamin A-metabolism, i odlade primära celler. Sammantaget har vi utvecklat en lätt att följa metod för att odla primära RPE-celler hos svin med hög renhet och inbyggda RPE-funktioner, vilket kan fungera som en bra modell för att förstå RPE-fysiologi, studera celltoxiciteter och underlätta läkemedelsundersökningar.

De primära RPE-cellerna (pRPE) odlades i DMEM/Basic media med 10% FBS, och cellmorfologi under ljusmikroskop fotograferades vid 2 dagar (figur 2A), 6 dagar (figur 2B) och 10 dagar (figur 2C) efter sådd. Efter 1 wk observerades ett sammanflytande monolager av pigmenterade pRPE-celler med kullerstensmorfologier.
För att bättre karakterisera de primära pRPE-cellerna odlades primära humana RPE-celler (h...

Watch this Scientific Journal Video about Primary Culture of Porcine Retinal Pigment Epithelial Cells at JoVE.com

Primary Culture of Porcine Retinal Pigment Epithelial Cells

Här presenteras en lätt att följa metod för att odla primära svinretinala pigmentepitelceller in vitro .

Primär odling av svin retinala pigmentepitelceller

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

RPE-relaterade störningar är fortfarande en stor del av blandningssjukdomar, men utan effektiva rester i virtuell odling av primära RPE-celler för att fungera som en bra modell för fysiologiska och patologiska studier av RPE-relaterade störningar. I detta protokoll tillhandahåller vi ett lätt att följa protokoll för att odla primära IPC: er, som snabbt kan återställa och behålla senare RP-egenskaper, vi tror att genom att använda denna metod kan människor snabbt generera RP-celler från macistate-studier och skyddande läkemedelsundersökningar som kan underlätta utvecklingen av ny behandling för RPE-störningar. En stegvis värdedemonstration kommer att göra denna metod mycket lättare att replikera i olika laboratorier som vill studera RP-celler.För att börja tina vävnadsuppslutningsenzymet malaquad och sterilisera 1X PBS. Kompletteras med 2% penicillin och 2% streptomycin genom att filtrera lösningen genom en 0,22 mikrometer sprutfilterenhet. Tvätta brunnarna på odlingsplattan och transwellinsatserna med 1X PBS.Ta bort PBS från brunnarna och transwells med en pipett och tillsätt en milliliter färsk 1X PBS till den nedre kammaren och 600 mikroliter 10 mikrogram per milliliter Laminin-lösning till den övre kammaren. Inkubera plattorna och transwellinsatserna i denna helicikulturinkubator över natten vid 37 grader Celsius och 5% koldioxid. Ta sedan bort laminatlösningen från den övre kammaren och tvätta med en milliliter kall 1X PBS två gånger innan du sätter cellen Rengör laminatflödeshuven med 75% etanol och UV-ljus.Förbered tre 50 ml sterila centrifugrör som innehåller cirka 25 ml 75% etanol, tre 50 ml sterila centrifugrör som innehåller cirka 25 ml 1X PBS och tre 10 centimeter sterila cellodlingsskålar som innehåller cirka 15 ml 1X PBS. Blötlägg fyra ögonbollar från svin i cirka 15 ml 75% etanol i en 10 centimeter petriskål och använd en sax och pincett för att skära av alla kvarvarande bindväv och muskler. För att dekontaminera ögonbollarna, blötlägg och tvätta fyra ögonbollar i tre 50 ml sterila centrifugrör fyllda med 75% etanol sekventiellt.Tvätta sedan ögonbollarna i tre 50 ml sterila centrifugrör fyllda med 1X PBS sekventiellt. Vänd rören varje minut för att tvätta ögonbollarna noggrant. Flytta de fyra ögonbollarna till en 10 centimeter steril cellodlingsskål som innehåller 1X PBS.Trimma den yttre ytan av varje ögonglob igen, för att ta bort optisk nerv och små skräp. Använd sax för att göra ett litet snitt i skärningspunkten mellan limbus och sclera. Och ta sedan bort hornhinnan, iris, linsen, glaskroppen och neural näthinnan.Överför RPE choroid sclera-komplexet till en ny 10 centimeter cellodlingsskål och gör fyra snitt för att platta ögonkoppen i form av en fyrklöver. Utför resor i EDTA-lösningssmältning genom att placera de fyra RPE-choroid sclera-komplexen i en ny 10 centimeter skål och hälla 20 ml färsk trypsin EDTA-lösning för att slå samman RPE-choroid sclera-komplexen. Placera skålen i cellodlingsinkubatorn vid 37 grader Celsius i nästan 30 minuter.Efter 30 minuter, ta skålen ur inkubatorn och tillsätt 20 ml förvärmda cellodlingsmedier med 10% FBS till skålen. För att neutralisera resorna i EDTA-lösning använd en fem milliliters överföringspipett för att dissociera RPE-cellerna genom att försiktigt pipettera flera gånger. Samla cellsuspensionerna i 15 ml centrifugrör och pipettera sedan försiktigt ytterligare 10 ml färskt odlingsmedium med FBS för att tvätta RPE Choroids sclera-komplexen på skålen.Samla upp RPE-cellerna genom att centrifugera rören vid 300 G i fem minuter vid rumstemperatur. Aspirera SuperAgent med en pipett och tillsätt ytterligare fem milliliter odlingsmedia med FBS för att åter suspendera cellerna. Centrifugera vid 300 G i ytterligare fem minuter.Dekantera SuperAgent och suspendera cellerna igen med 12 ml odlingsmedier. Nästa frö 100 000 till 200 000 celler per brunn i 12 brunnsodlingsplattor eller transwellinsatser. Byt odlingsmediet varannan dag och minska serumkoncentrationen till 1% när cellerna helt täcker ytan på brunnarna per insats.Byt odlingsmediet varannan dag tills cellerna skördas. De representativa bilderna av primära rpe-celler för svin dag två, dag sex och dag 10 visas här. QRTPCR-analysen av mRNA-nivåer av signaturgener i primära RPE-celler hos svin, primära humana RPE-celler, ARPE 19-celler och RPE-koroidvävnad hos svin detekteras av denna grafiska bild.Här användes GAP DH som hushållningsgen för RTPCR. Fyra biologiska replikat användes för primära RPE-celler för svin och en RPE 19-celler där tre biologiska replikat användes för primära humana RPE-celler och rpe-koroidvävnad hos svin. Genuttrycksnivåerna i ARPE 19-celler sattes som kontroller.Den västerländska blodanalysen av RPE 65-proteiner i ARPE 19- och svin-RPE-celler och i primära humana RPE-celler och porcin-RPE-koroidvävnader visas i denna figur. Vinculin användes här som lastkontroll. Denna figur visar en två veckors odling av rpe-celler från svin i DMEM Basic-media med 1% FBS.Celler odlade med eller utan Lamin färgades med RPE 65, natriumkalium-APTA, Z01 och Hex. TR-mätningar i cellark, odlade med eller utan laminin visas i denna figur. Den västra blodanalysen av vaskulär endoteltillväxtfaktor eller VEGF och pigmentepitel härledd faktor eller PEDF i primära svin RPE- och ARPE 19-celler visas i denna figur.De sektoriella funktionerna hos odlade primära rpe- och ARPE 19-celler presenteras i dessa bilder. Den stora delen av detta protokoll är att lösa upp RP-celler från RPE-chide glialkomplex. Vi rekommenderar att du använder färska resor och lösning varje gång och korrekt kontroll av matsmältningstiden för att lösa upp åtta RP-celler.

primary-culture-of-porcine-retinal-pigment-epithelial-cells

Research

JoVE Journal

Biology

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex vivo Kultur av primära humana äggledaren epitelceller

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

MikroRNA Expression Profiler av Human iPS-celler, pigmentepitelet härledas från iPS, och Fetal pigmentepitelet

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Subretinal Transplantation av mänskliga embryonala stamceller härstammar-retinal Pigment epitelial celler till en stor-eyed modell av geografisk atrofi

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

Primära cellkulturer från mus Retinal pigmentepitel

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

Isolering, kultur och genteknik av däggdjurs primära pigment epitelceller för icke-viral genterapi

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Effektiv dissekering och kultur av primära mus retinal pigment epitelceller

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

Isolering av primära musretinala pigmenterade epitelceller

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Realtidsanalys av bioenergetik i primära humana retinala pigmentepitelceller med hjälp av högupplöst respirometri

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

Isolering av retinala pigmentepitelceller från marsvinsögon

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

Protokoll för isolering av bröstepitelceller från mänsklig vävnad

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

Författare i fokus: Integrering av österländsk och västerländsk medicin för behandling av granulomatös mastit

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

Isolering och odling av primära humana bröstepitelceller

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...