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

Forfatterne vil gerne vise deres taknemmelighed og respekt for alle dyr, der bidrager med deres celler i denne undersøgelse. Denne undersøgelse blev delvist støttet af tilskud fra National Key R&D Program of China (2019YFA0111200, Yi Liao & Yuan Gao og Grant nr. 2018YFA0107301, Wei Li). Forfatterne takker Jingru Huang og Xiang You fra Central Lab, School of Medicine, Xiamen University for teknisk support i konfokal billeddannelse.

Anvendelsen af forsøgsdyr overholdt reglerne fra Association for Research in Vision and Ophthalmology (ARVO) og blev godkendt af den etiske komité for eksperimentel dyreforvaltning ved Xiamen University.
1. Forberedelse af eksperimentelle kirurgiske anordninger, vævsfordøjelsesenzym og cellekulturbuffer
<ol><li>Forbered det eksperimentelle kirurgiske udstyr ved at rengøre og autoklavere to par oftalmologiske kirurgiske sakse og tang dagen før øjenæbledissektionen og ...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanin+pigment+in+the+pigmented+epithelium+of+the+retina+of+the+embryo+chick+eye+studied+in+vivo+and+in+vino.">Melanin pigment in the pigmented epithelium of the retina of the embryo chick eye studied in vivo and in vino.</a> The Anatomical Record. 18, 260-261 (1920).</li><li>Schnichels, S., 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=Retina+in+a+dish:+Cell+cultures,+retinal+explants+and+animal+models+for+common+diseases+of+the+retina.">Retina in a dish: Cell cultures, retinal explants and animal models for common diseases of the retina.</a> Progress in Retinal and Eye Research. 81, 100880 (2021).</li><li>D'Antonio-Chronowska, A., D'Antonio, M., Frazer, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+human+iPSC-derived+retinal+pigment+epithelium+cells+(iPSC-RPE).">In vitro differentiation of human iPSC-derived retinal pigment epithelium cells (iPSC-RPE).</a> Bio-Protocol. 9 (24), 3469 (2019).</li><li>Hazim, R. A., Volland, S., Yen, A., Burgess, B. L., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+of+the+human+RPE+cell+line,+ARPE-19,+induced+by+nicotinamide.">Rapid differentiation of the human RPE cell line, ARPE-19, induced by nicotinamide.</a> Experimental Eye Research. 179, 18-24 (2019).</li><li>Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R., Hjelmeland, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARPE-19,+a+human+retinal+pigment+epithelial+cell+line+with+differentiated+properties.">ARPE-19, a human retinal pigment epithelial cell line with differentiated properties.</a> Experimental Eye Research. 62 (2), 155-169 (1996).</li><li>. Scientific, T Available from: <a target="_blank" href="https://www.thermofisher.cn/document-connect/document">https://www.thermofisher.cn/document-connect/document</a> (2022)</li><li>. Toyota Available from: <a target="_blank" href="https://www.toyoboglobal.com/seihin/xr/likescience/support/manual/FSQ-201.pdf">https://www.toyoboglobal.com/seihin/xr/likescience/support/manual/FSQ-201.pdf</a> (2022)</li><li>. Abcam Available from: <a target="_blank" href="https://www.abcam.cn/protocols/immunocytochemistry-immunofluorescence-protocol">https://www.abcam.cn/protocols/immunocytochemistry-immunofluorescence-protocol</a> (2022)</li><li>. Zeiss Available from: <a target="_blank" href="https://www.zeiss.com/microscopy/en/products/software/zeiss-zen-lite.html#manuals">https://www.zeiss.com/microscopy/en/products/software/zeiss-zen-lite.html#manuals</a> (2022)</li><li>. 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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=Isolation,+culture+and+characterization+of+primary+mouse+RPE+cells.">Isolation, culture and characterization of primary mouse RPE cells.</a> Nature Protocols. 11 (7), 1206-1218 (2016).</li><li>Langenfeld, A., Julien, S., Schraermeyer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+the+isolation+and+culture+of+retinal+pigment+epithelial+cells+from+adult+rats.">An improved method for the isolation and culture of retinal pigment epithelial cells from adult rats.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 253 (9), 1493-1502 (2015).</li><li>Sonoda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+the+culture+and+differentiation+of+highly+polarized+human+retinal+pigment+epithelial+cells.">A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.</a> Nature Protocols. 4 (5), 662-673 (2009).</li></ol>

Her er beskrevet en detaljeret og optimeret protokol til isolering, dyrkning og karakterisering af RPE-celler fra svineøjeæbler, som genererer en god model til in vitro-karakterisering af RPE-celler og RPE-relaterede lidelsesstudier. Metoder til isolering af RPE fra menneske-, muse- og rotteøjne er tidligere beskrevet23,24,25. Det er dog vanskeligt at få menneskelige øjne i nogle laboratorier, og det rejser normalt...

Retinale pigmentepitel (RPE) er placeret mellem fotoreceptorer og choriocapillaris i det ydre lag af nethinden1 med flere funktioner, herunder dannelse af blod-retinal barriere, transport og udveksling af næringsstoffer og retinale metabolitter, genbrug af vitamin A for at opretholde en normal visuel cyklus og fagocytose og clearance af skurfotoreceptor ydre segmenter (POS'er)2,3 . Da POS'er kræver konstant selvfornyelse for at generere vision, skal RPE-cellerne kontinuerligt opsluge løsrevne POS'er for at opretholde retinal homeostase4. Derfor resulterer RPE-dysfunktion i mange blændende øjensygdomme, såsom aldersrelateret makuladegeneration (AMD)4, retinitis pigmentosa (RP)5, Leber medfødt amaurose6, diabetisk retinopati7 osv. Indtil nu forbliver den nøjagtige patogenese af de fleste af disse sygdomme undvigende. Som et resultat etableres RPE-cellekultur for at studere RPE-cellebiologi, patologiske ændringer og underliggende mekanismer.
Som den enkleste model til at studere cellebiologi blev kulturen af RPE-celler startet allerede i 1920'erne8. Selvom ARPE-19 i vid udstrækning anvendes som RPE-celler, giver tab af pigmentering, brostensmorfologi og især barrierefunktionerne i denne cellelinje anledning til mange bekymringer9. Til sammenligning tilbyder kulturen af primære humane RPE-celler et mere realistisk scenario for fysiologiske og patologiske undersøgelser9. Den relativt begrænsede tilgængelighed begrænser imidlertid deres brug, og etiske problemer eksisterer altid. Derudover brugte flere grupper musemodeller til at dyrke RPE-celler. Imidlertid er museøjets størrelse lille, og en enkelt kultur kræver normalt mange mus, hvilket ikke er praktisk9. For nylig har forskere udviklet nye metoder til at bruge menneskelige embryonale stamceller eller inducerede pluripotente stamceller til at udlede RPE-celler. Selvom denne teknik har særligt potentiale til behandling af arvelige RPE-lidelser, er det tidskrævende og kræver normalt flere måneder at generere modne RPE-celler10. For at overvinde disse problemer introducerer vi her en let at følge protokol til rutinemæssigt at isolere og dyrke RPE-celler med høj renhed i laboratoriet. Under passende dyrkningsforhold kan disse celler vise typiske RPE-funktioner og udvise typiske RPE-morfologier. Derfor kan denne kulturmetode give en god model til at forstå RPE-fysiologi, studere cytotoksicitet, undersøge patologiske mekanismer for relaterede okulære sygdomme og udføre lægemiddelscreeninger.

<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

Det retinale pigmentepitel (RPE) er et monolag af polariserede pigmenterede epitelceller, der ligger mellem choroid og neuroretina i nethinden. Flere funktioner, herunder fagocytose, næringsstof/metabolittransport, vitamin A-metabolisme osv., udføres dagligt af RPE. RPE-celler er terminalt differentierede epitelceller med ringe eller ingen regenerativ kapacitet. Tab af RPE-celler resulterer i flere øjensygdomme, der fører til synshandicap, såsom aldersrelateret makuladegeneration. Derfor er etableringen af en in vitro-kulturmodel af primære RPE-celler, som ligner RPE in vivo mere end cellelinjer, afgørende for de karakteristiske og mekanistiske undersøgelser af RPE-celler. I betragtning af at kilden til menneskelige øjenkugler er begrænset, opretter vi en protokol til dyrkning af primære RPE-celler hos svin. Ved at bruge denne protokol kan RPE-celler let adskilles fra voksne svineøjne. Derefter binder disse dissocierede celler sig til dyrkningsskåle / indsatser, spredes for at danne et sammenflydende monolag og genopretter hurtigt nøglefunktioner i epitelvæv in vivo inden for 2 uger. Ved qRT-PCR er det påvist, at primære RPE-celler hos svin udtrykker flere signaturgener på sammenlignelige niveauer med naturligt RPE-væv, mens ekspressionerne af de fleste af disse gener går tabt/stærkt reduceres i humane RPE-lignende celler, ARPE-19. Desuden viser immunofluorescensfarvningen fordelingen af tæt kryds, vævspolaritet og cytoskeletproteiner samt tilstedeværelsen af RPE65, en isomerase, der er kritisk for vitamin A-metabolisme, i dyrkede primære celler. Alt i alt har vi udviklet en let at følge tilgang til dyrkning af primære svine-RPE-celler med høj renhed og native RPE-funktioner, som kan tjene som en god model til at forstå RPE-fysiologi, studere celletoksiciteter og lette lægemiddelscreeninger.

De primære porcine RPE (pRPE) celler blev dyrket i DMEM / Basic medier med 10% FBS, og cellemorfologi under lysmikroskop blev fotograferet 2 dage (figur 2A), 6 dage (figur 2B) og 10 dage (figur 2C) efter såning. Efter 1 uge blev der observeret et sammenflydende monolag af pigmenterede pRPE-celler med brostensmorfologier.
For bedre at karakterisere de primære pRPE-celler blev primære humane RPE-celler ...

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

Her præsenteres en let at følge metode til dyrkning af primære porcine retinale pigmentepitelceller in vitro .

Primær kultur af porcine retinale pigmentepitelceller

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

RPE-relaterede lidelser er fortsat en stor del af blanding af sygdomme, men uden effektive rester i virtuel kultur af primære RPE-celler til at tjene som en god model for fysiologiske og patologiske undersøgelser af RPE-relaterede lidelser. I denne protokol leverer vi en let at følge protokol til dyrkning af primære IPC'er, som hurtigt kunne gendanne og vedligeholde senere RP-egenskaber, vi til Vi mener, at folk ved at bruge denne metode hurtigt kunne generere RP-celler fra macistate undersøgelser og de beskyttende lægemiddelscreeninger, som kunne lette udviklingen af ny behandling for RPE-lidelser. En trinvis værdidemonstration vil gøre denne metode meget lettere at replikere i forskellige laboratorier, der ønsker at studere RP-celler.For at begynde optø vævsfordøjelsesenzymet malaquad og sterilisere 1X PBS. Suppleret med 2% penicillin og 2% streptomycin ved filtrering af opløsningen gennem en 0,22 mikrometer sprøjtefilterenhed. Vask brøndene på kulturpladen og transwellindsatserne med 1X PBS.PBS fjernes fra brøndene og transwells med en pipette og tilsæt en milliliter frisk 1X PBS til det nedre kammer og 600 mikroliter 10 mikrogram pr. milliliter Laminin-opløsning til det øverste kammer. Inkuber pladerne og transwellindsatserne i denne Heliciculture Incubator natten over ved 37 grader Celsius og 5% kuldioxid. Fjern derefter laminatopløsningen fra det øverste kammer og vask med en milliliter kold 1X PBS to gange, før cellen sættes i plads Rengør laminatflowhætten med 75% ethanol og UV-lys.Forbered tre 50 ml sterile centrifugerør indeholdende omkring 25 ml 75% ethanol, tre 50 ml sterile centrifugerør indeholdende omkring 25 ml 1X PBS og tre 10 centimeter sterile cellekulturskåle indeholdende omkring 15 ml 1X PBS. Sug fire svineøjne i blød i ca. 15 ml 75% ethanol i en 10 centimeter petriskål og brug en saks og tang til at afskære alt det resterende bindevæv og muskler. For at dekontaminere øjenkuglerne skal du blødgøre og vaske fire øjenkugler i tre 50 ml sterile centrifugerør fyldt med 75% ethanol sekventielt.Vask derefter øjenkuglerne i tre 50 ml sterile centrifugerør fyldt med 1X PBS sekventielt. Vend rørene hvert minut for at vaske øjenkuglerne grundigt. Flyt de fire øjenæbler ind i en 10 centimeter steril cellekulturskål indeholdende 1X PBS.Trim den ydre overflade af hvert øjeæble igen for at fjerne synsnerven og små snavs. Brug en saks til at lave et lille snit i krydset mellem limbus og sclera. Og fjern derefter hornhinden, iris, linsen, glaslegemet og neurale nethinden.Overfør RPE choroid sclera-komplekset til en ny 10 centimeter cellekulturskål og lav fire snit for at flade øjenkoppen i form af et firkløver. Udfør ture i EDTA-opløsningsfordøjelsen ved at placere de fire RPE choroid sclera-komplekser i en ny 10 centimeter skål og hælde 20 ml frisk trypsin EDTA-opløsning for at fusionere RPE choroid sclera-komplekserne. Placer skålen i cellekulturinkubatoren ved 37 grader Celsius i næsten 30 minutter.Efter 30 minutter skal du tage skålen ud af inkubatoren og tilsæt 20 ml forvarmte cellekulturmedier med 10% FBS til skålen. For at neutralisere trips i EDTA-opløsning skal du bruge en fem milliliter overførselspipette til at adskille RPE-cellerne ved forsigtigt pipettering flere gange. Saml cellesuspensionerne i 15 ml centrifugerør, og pipette derefter forsigtigt yderligere 10 ml friske kulturmedier med FBS for at vaske RPE Choroids sclera-komplekserne på fadet.RPE-cellerne opsamles ved centrifugering af glassene ved 300 G i fem minutter ved stuetemperatur. Opsug SuperAgent ved hjælp af en pipette og tilsæt yderligere fem milliliter kulturmedier med FBS for at suspendere cellerne igen. Der centrifugeres ved 300 G i yderligere fem minutter.Dekanter SuperAgenten, og ophvirvl cellerne igen med 12 ml kulturmedier. Næste frø 100.000 til 200.000 celler pr. Brønd i 12 Brøndkulturplader eller transwellindsatser. Skift kulturmediet hver anden dag og reducer serumkoncentrationen til 1%når cellerne fuldt ud dækker overfladen af hullerne pr. Indsats.Skift kulturmediet hver anden dag, indtil cellerne høstes. De repræsentative billeder af primære RPE-celler fra svin på dag to, dag seks og dag 10 er vist her. QRTPCR-analysen af mRNA-niveauer af signaturgener i primære RPE-celler til svin, primære humane RPE-celler, ARPE 19-celler og porcint RPE-choroidvæv detekteres af dette grafiske billede.Her blev GAP DH brugt som husholdningsgen for RTPCR. Fire biologiske replikater blev anvendt til primære RPE-celler hos svin og en RPE 19-celle, hvor tre biologiske replikater blev anvendt til primære humane RPE-celler og porcint RPE-choroidvæv. Genekspressionsniveauerne i ARPE 19-celler blev indstillet som kontroller.Den vestlige blodanalyse af RPE 65-proteiner i ARPE 19- og svine-RPE-celler og i primære humane RPE-celler og porcint RPE-choroidvæv er vist i denne figur. Vinculin blev brugt her som lastkontrol. Denne figur viser en to ugers kultur af svin RPE-celler i DMEM Basic-medier med 1% FBS.Celler dyrket med eller uden Lamin blev farvet med RPE 65, natriumkaliumAPTA'er, Z01 og Hex. TR-målinger i celleark, dyrket med eller uden laminin, er afbildet i denne figur. Den vestlige blodanalyse af vaskulær endotelvækstfaktor eller VEGF og pigmentepitelafledt faktor eller PEDF i primære svine-RPE- og ARPE 19-celler er vist i denne figur.De sektorielle funktioner af dyrkede primære svin RPE og ARPE 19 celler er præsenteret i disse billeder. Den store del af denne protokol er at opløse RP-celler fra RPE chide gliale komplekser. Vi anbefaler at bruge friske ture og opløsning hver gang og korrekt kontrollere fordøjelsestiden for at opløse otte 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.

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.

MicroRNA Expression Profiler af menneskelige iPS celler, retinale pigmentepitel arvet fra iPS og Føtal retinale pigmentepitel

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 af humane embryonale stamceller afledt retinal Pigment epitel celler ind i en stor-eyed Model af 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.

Primære cellekulturer fra mus retinale Pigment epitel

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.

Isolation, kultur og genteknologi af pattedyr primære pigment epitelceller til ikke-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 dissektion og kultur af primære mus Retinal Pigment Epitel celler

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 af primære muse retinale pigmenterede 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.

Realtidsanalyse af bioenergetik i primære humane retinale pigmentepitelceller ved hjælp af respirometri med høj opløsning

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 af retinale pigmentepitelceller fra marsvinøjne

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

Protokol til isolering af brystepitelceller fra humant væv

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.

Forfatter Spotlight: Integration af østlig og vestlig medicin til behandling af granulomatøs mastitis

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 og dyrkning af primære humane brystepitelceller

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