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

Yazarlar, bu çalışmada hücrelerine katkıda bulunan tüm hayvanlara şükranlarını ve saygılarını göstermek istemektedir. Bu çalışma kısmen Çin Ulusal Anahtar Ar-Ge Programı'ndan (2019YFA0111200, Yi Liao & Yuan Gao ve Hibe no. 2018YFA0107301, Wei Li) gelen hibelerle desteklenmiştir. Yazarlar, konfokal görüntülemede teknik destek için Xiamen Üniversitesi Central Lab, Tıp Fakültesi'nden Jingru Huang ve Xiang You'ya teşekkür ediyor.

Deney hayvanlarının kullanımı, Görme ve Oftalmoloji Araştırmaları Derneği'nin (ARVO) düzenlemelerine uygun ve Xiamen Üniversitesi Deneysel Hayvan Yönetimi Etik Kurulu tarafından onaylandı.
1. Deneysel cerrahi cihazların, doku sindirim enziminin ve hücre kültürü tamponunun hazırlanması
<ol><li>Göz küresi diseksiyonundan bir gün önce iki çift oftalmik cerrahi makas ve forseps temizleyerek ve otoklavlayarak ve daha sonra kutuyu gece boyunca 65 ° C'de ...

<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. 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Burada, RPE hücrelerinin domuz gözbebeklerinden izolasyonu, kültürü ve karakterizasyonu için ayrıntılı ve optimize edilmiş bir protokol, RPE hücrelerinin in vitro karakterizasyonu ve RPE ile ilişkili bozukluk çalışmaları için iyi bir model oluşturmaktadır. RPE'nin insan, fare ve sıçan gözlerinden izole edilmesi için yöntemler daha önce 23,24,25 olarak tanımlanmıştır. Bununla birlikte, bazı ...

Retinal pigment epiteli (RPE), retina1'in dış tabakasındaki fotoreseptörler ve koryokapillaris arasında bulunur ve kan-retinal bariyerin oluşturulması, besin maddelerinin ve retinal metabolitlerin taşınması ve değişimi, normal bir görme döngüsünü sürdürmek için A vitamininin geri dönüşümü ve fagositoz ve dökülen fotoreseptör dış segmentlerinin (POS'lar) temizlenmesi gibi birçok fonksiyona sahiptir2,3 . POS'lar görme üretmek için sürekli kendini yenilemeyi gerektirdiğinden, RPE hücrelerinin retinal homeostazı korumak için sürekli olarak ayrılmış POS'ları yutması gerekir4. Bu nedenle, RPE disfonksiyonu, yaşa bağlı makula dejenerasyonu (AMD)4, retinitis pigmentosa (RP)5, Leber konjenital amaurozus6, diyabetik retinopati7 gibi birçok kör edici göz hastalığına neden olur. Şimdiye kadar, bu hastalıkların çoğunun kesin patogenezi belirsizliğini korumaktadır. Sonuç olarak, RPE hücre biyolojisini, patolojik değişiklikleri ve altta yatan mekanizmaları incelemek için RPE hücre kültürü kurulmuştur.
Hücre biyolojisini incelemek için en basit model olarak, RPE hücrelerinin kültürü 1920'lerin8'i kadar erken bir tarihte başlatıldı. ARPE-19, RPE hücreleri olarak yaygın olarak kullanılmasına rağmen, pigmentasyon kaybı, parke taşı morfolojisi ve özellikle bu hücre hattındaki bariyer fonksiyonları birçok endişeyi gündeme getirmektedir9. Buna karşılık, birincil insan RPE hücrelerinin kültürü, fizyolojik ve patolojik çalışmalar için daha gerçekçi bir senaryo sunmaktadır9. Bununla birlikte, nispeten sınırlı kullanılabilirlik kullanımlarını kısıtlar ve etik sorunlar her zaman mevcuttur. Ek olarak, birkaç grup RPE hücrelerini kültürlemek için fare modellerini kullandı. Bununla birlikte, fare gözünün boyutu küçüktür ve tek bir kültür genellikle uygun olmayan birçok fare gerektirir9. Son zamanlarda, bilim adamları insan embriyonik kök hücrelerini kullanmak için yeni yöntemler geliştirdiler veya RPE hücrelerini türetmek için pluripotent kök hücreleri indüklediler. Bu teknik kalıtsal RPE bozukluklarının tedavisi için özel bir potansiyele sahip olmasına rağmen, zaman alıcıdır ve olgun RPE hücrelerinin üretilmesi genellikle birkaç ay gerektirir10. Bu sorunların üstesinden gelmek için, burada laboratuvarda rutin olarak yüksek saflıkta RPE hücrelerini izole etmek ve kültürlemek için takip edilmesi kolay bir protokol sunuyoruz. Uygun kültür koşulları altında, bu hücreler tipik RPE fonksiyonları gösterebilir ve tipik RPE morfolojileri sergileyebilir. Bu nedenle, bu kültür yöntemi RPE fizyolojisini anlamak, sitotoksisiteyi incelemek, ilgili oküler hastalıkların patolojik mekanizmalarını araştırmak ve ilaç taramaları yapmak için iyi bir model sağlayabilir.

<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

Retina pigment epiteli (RPE), retinadaki koroid ve nöroretina arasında yer alan polarize pigmentli epitel hücrelerinin tek katmanıdır. Fagositoz, besin / metabolit taşınması, A vitamini metabolizması vb. dahil olmak üzere birçok fonksiyon RPE tarafından günlük olarak gerçekleştirilir. RPE hücreleri, rejeneratif kapasitesi çok az olan veya hiç olmayan terminal olarak farklılaşmış epitel hücreleridir. RPE hücrelerinin kaybı, yaşa bağlı makula dejenerasyonu gibi görme bozukluğuna yol açan çoklu göz hastalıklarına neden olur. Bu nedenle, RPE'ye hücre hatlarından daha çok benzeyen primer RPE hücrelerinin in vitro kültür modelinin oluşturulması, RPE hücrelerinin karakteristik ve mekanik çalışmaları için kritik öneme sahiptir. İnsan gözbebeklerinin kaynağının sınırlı olduğu gerçeğini göz önünde bulundurarak, birincil domuz RPE hücrelerini kültürlemek için bir protokol oluşturuyoruz. Bu protokolü kullanarak, RPE hücreleri yetişkin domuz gözbebeklerinden kolayca ayrılabilir. Daha sonra, bu ayrışmış hücreler kültür tabaklarına / eklerine bağlanır, akıcı bir tek katman oluşturmak için çoğalır ve epitel dokusunun temel özelliklerini in vivo olarak 2 wks içinde hızla yeniden oluşturur. qRT-PCR ile, birincil domuz RPE hücrelerinin doğal RPE dokusu ile karşılaştırılabilir seviyelerde çoklu imza genleri eksprese ettiği, bu genlerin çoğunun ekspresyonlarının insan RPE benzeri hücrelerde, ARPE-19'da kaybolduğu / yüksek oranda azaldığı gösterilmiştir. Ayrıca, immünofloresan boyama, sıkı birleşme, doku polaritesi ve sitoiskelet proteinlerinin dağılımını ve ayrıca kültürlenmiş primer hücrelerde A vitamini metabolizması için kritik bir izomeraz olan RPE65'in varlığını gösterir. Toplamda, RPE fizyolojisini anlamak, hücre toksisitelerini incelemek ve ilaç taramalarını kolaylaştırmak için iyi bir model olarak hizmet edebilecek yüksek saflıkta ve doğal RPE özelliklerine sahip birincil domuz RPE hücrelerinin kültürüne takip edilmesi kolay bir yaklaşım geliştirdik.

Primer porsin RPE (pRPE) hücreleri DMEM/Basic ortamında %10 FBS ile kültürlendi ve ışık mikroskobu altında hücre morfolojisi tohumlamadan sonra 2 gün (Şekil 2A), 6 gün (Şekil 2B) ve 10 günde (Şekil 2C) fotoğraflandı. 1 wk'dan sonra, parke taşı morfolojileri olan pigmentli pRPE hücrelerinin birleşik bir tek katmanı gözlendi.
Primer pRPE hücrelerini daha iyi karakterize etmek için, ...

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

Burada, primer domuz retinal pigment epitel hücrelerinin in vitro olarak kültürlenmesi için takip edilmesi kolay bir yöntem sunulmaktadır.

Domuz Retinal Pigment Epitel Hücrelerinin Primer Kültürü

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

RPE ile ilişkili bozukluklar, hastalıkların harmanlanmasının önemli bir parçası olmaya devam etmektedir, ancak primer RPE hücrelerinin sanal kültüründe, RPE ile ilişkili bozuklukların fizyolojik ve patolojik çalışmaları için iyi bir model olarak hizmet edecek etkili kalıntılar yoktur. Bu protokolde, daha sonraki RP özelliklerini hızlı bir şekilde geri yükleyebilen ve koruyabilen birincil IPC'leri kültüre takip etmesi kolay bir protokol sağlıyoruz, bu yöntemi kullanarak insanların masistat çalışmalarından ve RPE bozuklukları için yeni tedavinin geliştirilmesini kolaylaştırabilecek koruyucu ilaç taramalarından hızlı bir şekilde RP hücreleri üretebileceklerine inanıyoruz. Adım adım değer gösterimi, bu yöntemin RP hücrelerini incelemek isteyen farklı laboratuvarlarda çoğaltılmasını çok daha kolay hale getirecektir.Doku sindirim enzimi malaquad'ı eritmeye başlamak ve 1X PBS'yi sterilize etmek. Çözeltiyi 0.22 mikrometrelik bir şırınga filtre ünitesinden filtreleyerek% 2 penisilin ve% 2 streptomisin ile desteklenir. Kültür plakasının kuyucuklarını ve transwell eklerini 1X PBS ile yıkayın.PBS'yi kuyucuklardan ve transwelllerden bir pipetle çıkarın ve alt odaya bir mililitre taze 1X PBS ve üst odaya mililitre başına 600 mikrolitre 10 mikrogram Laminin çözeltisi ekleyin. Bu Helisykültür İnkübatöründe plakaları ve transwell eklerini gece boyunca 37 santigrat derece ve% 5 karbondioksitte inkübe edin. Bundan sonra, laminat çözeltisini üst odadan çıkarın ve hücreyi yerleştirmeden önce iki kez bir mililitre soğuk 1X PBS ile yıkayın Laminat akış başlığını% 75 etanol ve UV ışığı ile temizleyin.Yaklaşık 25 mililitre %75 Etanol içeren üç adet 50 mililitrelik steril santrifüj tüpü, yaklaşık 25 mililitre 1X PBS içeren üç adet 50 mililitre steril santrifüj tüpü ve yaklaşık 15 mililitre 1X PBS içeren üç adet 10 santimetre steril hücre kültürü kabı hazırlayın. Dört domuz gözbebeğini 10 santimetrelik bir Petri kabında yaklaşık 15 mililitre% 75 Etanol içine batırın ve tüm artık bağ dokularını ve kaslarını kesmek için bir çift makas ve forseps kullanın. Göz kürelerini dekontamine etmek için, sırasıyla% 75 etanol ile doldurulmuş üç adet 50 mililitre steril santrifüj tüpünde dört göz küresini ıslatın ve yıkayın.Daha sonra göz kürelerini sırayla 1X PBS ile doldurulmuş üç adet 50 mililitrelik steril santrifüj tüpünde yıkayın. Göz kürelerini iyice yıkamak için tüpleri her dakika ters çevirin. Dört göz küresini 1X PBS içeren 10 santimetrelik steril bir hücre kültürü kabına taşıyın.Optik siniri ve küçük kalıntıları çıkarmak için her göz küresinin dış yüzeyini tekrar kesin. Limbus ve skleranın kesiştiği noktada küçük bir kesim yapmak için makas kullanın. Ve sonra kornea, iris, lens, vitreus gövdesi ve nöral retinayı çıkarın.RPE koroid sklera kompleksini yeni bir 10 santimetre hücre kültürü kabına aktarın ve göz kapağını dört yapraklı yonca şeklinde düzleştirmek için dört kesim yapın. Dört RPE koroid sklera kompleksini yeni bir 10 santimetrelik kaba yerleştirerek ve RPE koroid sklera komplekslerini birleştirmek için 20 mililitre taze tripsin EDTA çözeltisi dökerek EDTA çözeltisi sindiriminde geziler gerçekleştirin. Çanağı hücre kültürü inkübatörüne yaklaşık 30 dakika boyunca 37 santigrat derecede yerleştirin.30 dakika sonra, çanağı inkübatörden çıkarın ve,% 10 FBS ile 20 mililitre ön sıcak hücre kültürü ortamı ekleyin. EDTA çözeltisindeki yolculukları nötralize etmek için, RPE hücrelerini birkaç kez hafifçe pipetleyerek ayırmak için beş mililitrelik bir transfer pipeti kullanın. Hücre süspansiyonlarını 15 mililitrelik santrifüj tüplerinde toplayın ve ardından RPE Choroids sklera komplekslerini çanak üzerinde yıkamak için FBS ile 10 mililitre taze kültür ortamını nazikçe pipetleyin.RPE hücrelerini, tüpleri oda sıcaklığında beş dakika boyunca 300 G'de santrifüj ederek toplayın. SuperAgent'ı bir pipet kullanarak aspire edin ve hücreleri yeniden askıya almak için FBS ile beş mililitre kültür ortamı daha ekleyin. Beş dakika daha 300 G'de santrifüj.SuperAgent'ı boşaltın ve hücreleri 12 mililitre kültür ortamı ile yeniden askıya alın. Sonraki tohum kuyu başına 100.000 ila 200.000 hücreyi 12 Kuyu kültür plakasına veya transwell eklere yerleştirin. Kültür ortamını her iki günde bir değiştirin ve hücreler ekleme başına kuyucukların yüzeyini tamamen kapladığında serum konsantrasyonunu% 1'e düşürün.Hücreler toplanana kadar kültür ortamını her iki günde bir değiştirin. Primer domuz RPE hücrelerinin ikinci gün, altıncı gün ve 10. gündeki temsili görüntüleri burada gösterilmiştir. Primer domuz RPE hücreleri, primer insan RPE hücreleri, ARPE 19 hücreleri ve domuz RPE koroid dokusundaki imza genlerinin mRNA düzeylerinin QRTPCR analizi bu grafik görüntü ile tespit edilmiştir.Burada GAP DH, RTPCR için temizlik geni olarak kullanılmıştır. Birincil domuz RPE hücreleri için dört biyolojik replikasyon ve birincil insan RPE hücreleri ve domuz RPE koroid dokusu için üç biyolojik replikasyonun kullanıldığı bir RPE 19 hücresi kullanılmıştır. ARPE 19 hücrelerindeki gen ekspresyon seviyeleri kontrol olarak belirlendi.ARPE 19 ve domuz RPE hücrelerinde ve primer insan RPE hücrelerinde ve domuz RPE koroid dokularında RPE 65 proteinlerinin Batı kan analizi bu şekilde gösterilmiştir. Vinculin burada yükleme kontrolü olarak kullanılmıştır. Bu şekil, DMEM Basic ortamında% 1FBS ile iki haftalık bir domuz RPE hücresi kültürünü göstermektedir.Lamin ile veya Lamin olmadan kültürlenen hücreler RPE 65, sodyum potasyum APTA'lar, Z01 ve Hex ile boyandı. Lamininli veya lamininsiz kültürlenmiş hücre tabakalarındaki TR ölçümleri bu şekilde tasvir edilmiştir. Primer domuz RPE ve ARPE 19 hücrelerinde vasküler endotel büyüme faktörü veya VEGF ve pigment epitel kaynaklı faktör veya PEDF'nin batı kan analizi bu şekilde gösterilmiştir.Bu görüntülerde kültürlenmiş primer domuz RPE ve ARPE 19 hücrelerinin sektörel fonksiyonları sunulmuştur. Bu protokolün büyük kısmı, RPE chide glial komplekslerinden RP hücrelerini çözmektir. Sekiz RP hücresini çözmek için her seferinde taze geziler ve çözeltiler kullanmanızı ve sindirim süresini uygun şekilde kontrol etmenizi öneririz.

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 Kültür İlköğretim İnsan Fallop Tüp Epitel Hücreleri

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 ...

Biyoloji

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.

İnsan iPS Hücreleri MicroRNA İfade Profilleri, iPS Kaynaklanan Retina pigment epiteli ve Fetal Retina pigment epiteli

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.

Büyük gözlü manken coğrafi Atrofi, içine insan embriyonik kök hücre elde edilen retina Pigment epitel hücrelerinin subretinal nakli

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.

Fare retina Pigment epiteli birincil hücre kültürleri

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.

Viral Olmayan Gen Tedavisi için Memeli Primer Pigment Epitel Hücrelerinin İzolasyonu, Kültürü ve Genetik Mühendisliği

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.

Birincil Fare Retina Pigment Epitel Hücrelerinin Verimli Diseksiyonu ve Kültürü

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.

Primer Fare Retinal Pigmentli Epitel Hücrelerinin İzolasyonu

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.

Primer İnsan Retinal Pigment Epitel Hücrelerinde Yüksek Çözünürlüklü Respirometri Kullanılarak Biyoenerjetik Biyoenerjetik Analizinin Gerçek Zamanlı Analizi

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.

Retinal Pigment Epitel Hücrelerinin Guinea Pig Gözlerinden İzolasyonu

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

Meme Epitel Hücrelerini İnsan Dokusundan İzole Etme Protokolü

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.

Yazar Spotlight: Granülomatöz Mastit Tedavisi için Doğu ve Batı Tıbbının Entegrasyonu

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.

Primer İnsan Meme Epitel Hücrelerinin İzolasyonu ve Kültürü

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