Bu çalışma PAT için bir Ulusal Sağlık Enstitüsü, hibe Ey10540 tarafından finanse edildi.

1. İris Hücre Kültürü <ol><li> 7 SBD (anestezi, PBS içinde hazırlanan 3-aminobenzoate metan sülfonik asit etil% 0.1) ve kalsiyum magnezyum ücretsiz Hanks solüsyonu (CMF) yer gözbebekleri toplayın. </li><li> Eldivenleri değiştirin ve doku kültürü kaputu çalışır. </li><li> Lugol&#39;s-EtOH göz topları 3 saniye süreyle sterilize edin. </li><li> CMF 2 kez yıkayın. </li><li> Hanks gözleri aktarın ve diseksiyon başlayın. </li><li> Iris kornea kompleks inceleyin ve nöral retina kaldırmak. </li><li> Kompleksleri Hanks yeni bir çanak içine aktarın. </li><li> # 15 bistüri, ayrı kompleksleri, sırt ve karın yarısı içine kullanma. </li><li> 1 ml L-15 içeren bir tabak içinde iris parçaları (ilk ventral) ve yer çıkarın. </li><li> % 15 (150 ul) dispase hacmi (7.5 adet / ml konsantrasyon) ekleyin. </li><li> 27 ° C (parafilm sarın yemek) 2 saat süreyle inkübe edin. </li><li> Stroma (27 Place tripsin çözüm ° C) IPE hücrelerin izole. </li><li> 1.5 ml tüp içine IPE hücreleri toplayın. </li><li> RT 1000 rpm (oda sıcaklığında) 2 dakika süreyle santrifüj, supernatant çıkarın. </li><li> RT 1000 devirde 2 dakika boyunca 1 ml Hanks ve santrifüj ile yıkayınız süpernatant kaldırmak. </li><li> 2 saat boyunca inkübe 27 ° C, 1 ml tripsin çözüm ekle </li><li> 2 dakika boyunca oda sıcaklığında 1000 rpm&#39;de santrifüj, tripsin çıkarın. </li><li> 2 dakika 1000 rpm&#39;de santrifüj, yıkamak için 1 ml L-15 ekleyin. </li><li> Yavaş yavaş yukarı ve aşağı 800 ul L-15, pipetlemeyin ~ 10 kat (12 kat fazla bağlılık azaltır) ekleyin. </li><li> Plaka 27 24 iyi kollajen kaplı plaka ve inkübe IPE hücreleri ° C 2 hafta (Genellikle, 4 dorsal ve ventral 4 kuyulardan 7 SBD elde edilen). </li><li> Bu aşamada hücre, gen transfeksiyon veya indükleyen veya lens rejenerasyon ile müdahale rollerini belirlemek için faktörler ile tedavi için uygundur. </li></ol> 2. İris Hücreler Birleştirilmesi <ol><li> Plakalar içeren iris hücreleri hafifçe 400 inoküle, girdap dispase çözüm 20μl ekleyin. </li><li> Plakalar 27 ° C&#39;de gece boyunca inkübe edin. </li><li> Pipetleyin yavaşça yukarı ve aşağı hücreleri çıkarmak için </li><li> Eppendorf tüp ve RT az 1000 rpm&#39;de 2 dakika spin icine koyun hücreleri. </li><li> 1 ml tam L-15 ekleyerek orta ve yıkama çıkarın, oda sıcaklığında 1000 rpm&#39;de 2 dakika döndürmek orta kaldırmak. </li><li> Toplam ortalama 2000-7000 hücreleri kullanın. Her bir tüp içine agrega başına 200 ul L-15 ekleyin. </li><li> Yeni Eppendorf tüpleri böler hücreleri (200 ul). </li><li> 2 dakika süreyle 1000 rpm&#39;de RT dönerler. </li><li> 48 saat boyunca inkübe 27 ° C. </li></ol> 3. Aggregrated Hücreler implantasyonu <ol><li> Newt gözün kornea bir yarık olun ve objektif çıkarın. </li><li> Lens çıkarıldıktan sonra, ventral tarafında gözün kornea dokusunun hemen altında IPE hücre agrega yerleştirin. </li><li> SBD onlara lens yeniden sağlamak için bir ay süre ile muhafaza edilir. </li></ol> 4. Newt Göz gömülmesi <ol><li> Agrega ile implante newt gözleri kaldırın ve fosfat tamponlu salin (PBS). </li><li> En az 4 saat veya gece boyunca 4 ° C&#39;de% 4 paraformaldehid sabitleyin. </li><li> PBS, 4 ° C de 30 dakika yıkayın. </li><li> 4 ° C, 30 dakika: 0.85% serum fizyolojik ile şımartın. </li><li> RT: Tuzlu / Ethanol (1:1) 15 dakika boyunca yıkayın. </li><li> % 70 Etanol Yıkama: RT, 15 dakika. Bir kez tekrarlayın. </li><li> RT, her biri 30 dakika% 85 Etanol ve% 95 Etanol yıkayın. </li><li> % 100 Etanol Yıkama: RT, 30 dakika. Bir kez tekrarlayın. </li><li> Mağaza, 4 ° C veya devam. </li><li> % 100 ksilen Yıkama: RT, 30 dakika. Bir kez tekrarlayın. </li><li> 60 ° C, 45 dakika ksilen / parafin (1:1) ile tedavi. </li><li> 60 ° C&#39;de 20 dakika:% 100 Parafin ile şımartın. Iki kez daha tekrarlayın. </li><li> Kalıpları gömme gömün. </li></ol> 5. Kesit <ol><li> Gömülü göz 15 mikron bölümleri mikrotom kullanarak hazırlayın. </li><li> Jelatin kaplı bir slayt göz bölümleri yerleştirin ve sıcak bir slayt bırakın. </li></ol> 6. Boyanma <ol><li> Place 10 dakika için ksilen slaytlar. Bir kez daha tekrarlayın. </li><li> Hidrat:% 100,% 95,% 80,% 70,% 30 etanol her biri için 1 dakika </li><li> Bir dakika için DI suyla durulayın. </li><li> PBS içinde 15 dakika boyunca yıkayın. </li><li> PBST (% 0.2 Triton-X 100 PBS içinde) 15 dakika boyunca yıkayın. </li><li> PBS içinde 15 dakika boyunca yıkayın. </li><li> Solüsyonu (% 10 keçisi sekonder antikoru) bir saat boyunca bloke yerleştirin. </li><li> Gece için primer antikor yerleştirin. </li><li> Primer antikor PBS içinde 15 dakika boyunca yıkayın. </li><li> PBST 15 dakika boyunca yıkayın. </li><li> PBS içinde 15 dakika boyunca yıkayın. </li><li> Yeri karanlıkta 2 saat için sekonder antikoru (1:100 seyreltme PBS). </li><li> Her biri ve daha sonra montaj için 15 dakika PBS, PBST ve PBS içinde yıkayın. </li><li> Floresan mikroskop altında slaytlar uyun. </li></ol> 7. Temsilcisi Sonuçlar: Kültür newt ir Bu prosedürolan hücreler dorsal ve ventral IPE hücrelerin yenilenme potansiyelini incelemek için kullanılmıştır. Ayrıca, newt gözünde lens yenilenme mekanizması katkıda belirli genlerin çalışma da mümkündür. Hücreleri, 2 hafta boyunca kültüre edilmiştir (Şekil 1) objektif yenilenme fonksiyonunu incelemek için genler tarafından transfekte olabilir . Özellikle ilgi çekici olan ventral iris neden olabilir genler. Ventral iris hücrelerinin lens transdifferentiate olamayacağına göre (Şekil 2) bir aday gen endüktif işlevi incelenebilir. Geçmişte, bu tekniği kullanarak altı-3 üzerinden retinoik asit lens indüksiyon huzurunda ifade edildi ventral iris 6 gözlenen olduğunu göstermiştir. Şekil 3&#39;te ventral dorsal iris (ok) ev sahibi lens farklı değil, tam olarak gelişmiş bir ve farklılaşmış bir lens (ok ucu), iris agrega sebebiyet verdiğini görebilirsiniz. <img src="/files/ftp_upload/2713/2713fig1.jpg" alt="Şekil 1" /> Şekil 1. a) Dorsal iris pigmente epitel hücreleri 2 haftalık bir süre için in vitro kültür. b) Ventral iris pigmente epitel hücreleri, 2 haftalık bir süre için in vitro kültüre . Pigmentasyon hem dorsal ve ventral iris hücreleri bu aşamada devam edin. <img src="/files/ftp_upload/2713/2713fig2.jpg" alt="Şekil 2" /> Şekil 2. Kültürlü IPE hücrelerin yenilenmesi yeteneği. a) Objektif dorsal IPE hücre agrega (ok ucu) indüksiyon. Objektif indüksiyon dorsal iris (ok), di Host: dorsal iris, vi: ventral iris le: lens epitel, lf: lens lifleri. b) objektif indüksiyon ventral IPE hücre agrega (ok ucu) olmaması. Dorsal iris (ok) lens indüksiyon ev sahipliği yapmaktadır. <img src="/files/ftp_upload/2713/2713fig3.jpg" alt="Şekil 3" /> Şekil 3. Lens indüksiyon ventral toplam altı-3 ile transfekte ve retinoik asit ile tedavi. Dorsal iris (ok) lens indüksiyon ev sahipliği yapmaktadır. Ventral agrega (ok ucu) Lens indüksiyon.

<ol>
	<li>Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+in+vertebrates.">Regeneration in vertebrates.</a> Dev Biol. 221, 273-284 (2000).</li><li>Tsonis, P. A., Madhavan, M., Tancous, E. E., Del Rio-Tsonis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+newt's+eye+view+of+lens+regeneration.">A newt's eye view of lens regeneration.</a> Int. J. Dev. Biol. 48, 975-980 (2004).</li><li>Del Rio-Tsonis, K., Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+regeneration+at+the+molecular+age.">Eye regeneration at the molecular age.</a> Dev. Dyn. , 226-2211 (2003).</li><li>Okamoto, M., Ito, M., Owaribe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difference+between+dorsal+and+ventral+iris+in+lens+producing+potency+in+normal+lens+regeneration+is+maintained+after+dissociation+and+reaggregation+of+cells+from+the+adult+newt,+Cynops+pyrrhogaster.">Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells from the adult newt, Cynops pyrrhogaster.</a> Develop Growth Differ. 40, 11-18 (1998).</li><li>Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+transfection+system+for+functional+gene+analysis+in+adult+amphibian+lens+regeneration.">Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration.</a> Develop Growth Differ. 43, 361-370 (2001).</li><li>Grogg, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+inhibition-driven+regulation+of+six-3+underlies+induction+of+newt+lens+regeneration.">BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration.</a> Nature. 438, 858-862 (2005).</li><li>Nakamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+in+Newt+Lens+Regeneration:+Specific+Control+of+Proliferation+and+Evidence+for+miRNA+Networking.">miRNAs in Newt Lens Regeneration: Specific Control of Proliferation and Evidence for miRNA Networking.</a> PLoS One. 5, e12058-e12058 (2010).</li><li>Madhavan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Pax-6+in+lens+regeneration.">The role of Pax-6 in lens regeneration.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 14848-14853 (2006).</li><li>Maki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-type+linker+histone+B4+is+required+for+transdifferentiation+of+somatic+cells+in+vivo.">Oocyte-type linker histone B4 is required for transdifferentiation of somatic cells in vivo.</a> FASEB J. 24, 3462-3467 (2010).</li></ol>

Bu protokol SBD lens yenilenme mekanizmasını çalışma in vitro sistemi kurmuştur. Agrega bu yana (ya dorsal veya ventral sadakatle rejenerasyon bu tekniği sırasında in vivo davranış SBD yılında transgenesis için gerekli ve fonksiyon kazanç yanı sıra, fonksiyon deneyler 7,8,9 kaybı için kullanılabilir büyük çaba hafifletmek izleyin . , bir bütün olarak agrega veya süsen, büyüme faktörleri ile kolayca tedavi edilen ve bu protokol açıklandığı gibi bunların etkilerini incelemek. Örneğin BMP yolun rol, bu tekniği 6 kullanılarak incelenmiştir olabilir.

<table><tbody><tr><td>Lugol&amp;rsquo;s solution</td><td>Sigma-Aldrich</td><td>L-6146</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H-4034</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonic acid</td><td>Sigma-Aldrich</td><td>E-10521</td><td></td></tr><tr><td>Dispase</td><td>GIBCO, by Life Technologies</td><td>17105-041</td><td></td></tr><tr><td>Trypsin 1:250</td><td>GIBCO, by Life Technologies</td><td>27250018</td><td></td></tr><tr><td>L-15 ( Leibovitz)</td><td>Sigma-Aldrich</td><td>L-4386</td><td></td></tr><tr><td>DNase 1</td><td>Sigma-Aldrich</td><td>D5025-150KU</td><td></td></tr><tr><td>Kanamycin Sulfate</td><td>GIBCO, by Life Technologies</td><td>100x, 15160</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F4135</td><td></td></tr><tr><td>Fungizone (amphotericin B solution)</td><td>Sigma-Aldrich</td><td>A2942</td><td></td></tr><tr><td>24 well collagen coated plate</td><td>BD Biosciences</td><td>354408</td><td></td></tr></tbody></table>

a system for culturing iris pigment epithelial cells to study lens regeneration in newt

Newt ve axolotl gibi Semenderler kayıp vücut parçaları gibi uzuvların, omurilik, göz, beyin, kalp, çene 1 ile kuyruk gibi pek çok yeniden yeteneğine sahip. Özellikle, SBD objektifi rejenerasyon yeteneği için benzersiz. Objektif kaldırılması üzerine, dorsal iris IPE hücrelerinin lens hücrelerinin transdifferentiate ve sonunda, yaklaşık bir ay 2,3 yeni bir mercek formu . Bu özellik, rejenerasyon ventral iris hücreleri tarafından sergilenen asla. Iris hücrelerinin yenilenme potansiyeli, in vitro kültür IPE hücrelerinin nakli yaparak incelenebilir. Kültür, dorsal ve ventral iris hücreleri ilk göz izole edilir ve 2 hafta (Şekil 1) bir süre için ayrı ayrı kültür. Bu kültür hücreleri reaggregated ve geri newt göz implante. Geçmişte yapılan çalışmalar, ventral toplam 4,5 (Şekil 2), in vivo sürecinde böylece yansıtan bir mercek formu gelmez ise dorsal reaggregate objektif oluşturan kapasitesi koruduğunu göstermiştir. Dorsal ve ventral iris hücrelerinin yenilenme potansiyeli belirleme sistemi objektif rejenerasyon ilgili genler ve proteinlerin rolü eğitim çok yararlı olur.

Watch this Scientific Journal Video about A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt at JoVE.com

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Newt, lens, iris pigmente epitel hücreleri (IPEs) transdiferansiye dorsal iris her zaman yeniler. Burada newt göz kültür dorsal ve ventral newt IPE hücreleri ve implantasyon için bir prosedür açıklanmaktadır. Implante edilen hücreler daha sonra doku kesit ve immünohistokimya tarafından incelenir.

Newt Lens Rejenerasyon Eğitim Kültürleme İris Pigment Epitel Hücreler için Sistem

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

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Research

JoVE Journal

Biology

11.7K Görüntüleme.  University of Dayton. Newt, lens, iris pigmente epitel hücreleri (IPEs) transdiferansiye dorsal iris her zaman yeniler. Burada newt göz kültür dorsal ve ventral newt IPE hücreleri ve implantasyon için bir prosedür açıklanmaktadır. Implante edilen hücreler daha sonra doku kesit ve immünohistokimya tarafından incelenir.

Video: A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

Retina Pigment Epitel Fizyoloji ve Patofizyoloji Çalışma Deneysel Modeller

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Nörobilim

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Yetişkin Tavşan Retina Organotipik Kültürü

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Biyoloji

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Bir Ex Vivo modeli yaşlı insan Bruch'ın membran bir retina Pigment epitel hücrelerinin kültürü

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

Gelişim Biyolojisi

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

Terminal Farklılaşma incelenmesi için hazırlanması ve Rat Lens Epitel eksplantlar Kültür

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

Primary Cell Cultures to Study the Regeneration Potential of Murine M&#252;ller Glia after MicroRNA Treatment

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates such as fish, MG-driven regeneration occurs naturally; in mammals, however, stimulation with certain factors or genetic/epigenetic manipulation is required. Since MG comprise only 5% of the retinal cell population, there is a need for model systems that allow the study of this cell population exclusively. One of these model systems is primary MG cultures that are reproducible and can be used for a variety of applications, including molecule/factor screening and identification, testing of compounds or factors, cell monitoring, and/or functional tests. This model is used to study the potential of murine MG to convert into retinal neurons after supplementation or inhibition of microRNAs (miRNAs) via transfection of artificial miRNAs or their inhibitors. The use of MG-specific reporter mice in combination with immunofluorescent labeling and single-cell RNA sequencing (scRNA-seq) confirmed that 80%-90% of the cells found in these cultures are MG. Using this model, it was discovered that miRNAs can reprogram MG into retinal progenitor cells (RPCs), which subsequently differentiate into neuronal-like cells. The advantages of this technique are that miRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications.

M&#252;ller glia primary cultures obtained from mouse retinas represent a very robust and reliable tool to study the glial conversion into retinal progenitor cells after microRNA treatment. Single molecules or combinations can be tested before their subsequent application of in vivo approaches.

MikroRNA Tedavisi Sonrası Murine Müller Glia'nın Rejenerasyon Potansiyelini İncelemek için Birincil Hücre Kültürleri

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Beyazlatılmış Sıçan Retinal Ganglion Nöronlarının Koku Alma Ensheathing Glia ile Birlikte, Yetişkin Aksonal Rejenerasyonun In Vitro Modeli Olarak

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

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 ...

Lens Epitel Hücrelerinin Yetiştirilmesi ve Karakterizasyonu

Optimizing Mouse Primary Lens Epithelial Cell Culture: A Comprehensive Guide to Trypsinization

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, development, size, and transparency. Conversely, dysfunctional LECs can lead to cataract formation and posterior capsule opacification (PCO). Consequently, establishing a robust primary LEC culture system is important to researchers engaged in lens development, biochemistry, cataract therapeutics, and PCO prevention. However, cultivating primary LECs has long presented challenges due to their limited availability, slow proliferation rate, and delicate nature. 
This study addresses these hurdles by presenting a comprehensive protocol for primary LEC culture. The protocol encompasses essential steps such as the formulation of an optimized culture medium, precise isolation of lens capsules, trypsinization techniques, subculture procedures, harvest protocols, and guidelines for storage and shipment. Throughout the culture process, cell morphology was monitored using phase-contrast microscopy. 
To confirm the authenticity of the cultured LECs, immunofluorescence assays were conducted to detect the presence and subcellular distribution of critical lens proteins, namely &#945;A- and &#947;-crystallins. This detailed protocol equips researchers with a valuable resource for cultivating and characterizing primary LECs, enabling advancements in our comprehension of lens biology and the development of therapeutic strategies for lens-related disorders.

Yazar Spotlight: Kataraktın Sırlarını Çözmek – Lens Epitel Hücrelerinde Redoks Onarım Enzimlerinin Araştırılması

This manuscript outlines a detailed video protocol for culturing primary lens epithelial cells (LECs), aiming to improve reproducibility and aid research in cataracts and posterior capsule opacification (PCO). It offers step-by-step instructions on lens dissection, LECs isolation, and validation, serving as a valuable guide, especially for newcomers in the field.

Fare Primer Lens Epitel Hücre Kültürünü Optimize Etme: Tripsinizasyon için Kapsamlı Bir Kılavuz

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, ...

Newt Lens Rejenerasyon Eğitim Kültürleme İris Pigment Epitel Hücreler için Sistem

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Fare retina Pigment epiteli birincil hücre kültürleri

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

Fare Primer Lens Epitel Hücre Kültürünü Optimize Etme: Tripsinizasyon için Kapsamlı Bir Kılavuz

Fare Primer Lens Epitel Hücre Kültürünü Optimize Etme: Tripsinizasyon için Kapsamlı Bir Kılavuz