Bu çalışmada kısmen (DS için) BrightFocus Vakfı tarafından finanse edildi.

Hayvan konular içeren yordamlar kurumsal hayvan bakım ve kullanım Komitesi (IACUC), Pittsburgh Üniversitesi tarafından onaylanmıştır
1. çözümleri hazırlayın
<ol><li>Büyüme orta tarafından getirilerini Dulbecco modifiye kartal orta (DMEM), yüksek glikoz % 10 fetal sığır serum (FBS), % 1 penisilin/streptomisin, 2.5 mM L-glutamin ve 1 x MEM önemli olmayan amino asitler ile hazır olun. 37 ° C kuluçka kullanmadan önce medya önceden ısıtmak.</li>
	<li>%2 (wt/vol) dispase II çalışma çözüm hazırlayın:<ol>
<li>Dispase II 300 µL HEPES arabelleğe alınmış serum (50 mM HEPES/KOH pH 7.4, 150 mM NaCl) içinde 30 mg çözülerek 100 mg/mL dispase II stok çözeltisi hazırlamak. Bu birim için 3-4 fareler, ölçekli veya aşağı fareler için deneme sayısını temel alan). Bu hisse senedi en az 1 hafta 4 ° C'de depolanan</li>
		<li>% 2 dispase çalışma çözüm olarak 100 mg/mL 300 µL ekleyerek dispase II stok 1.2 mL steril DMEM, yüksek glikoz ve çözüm 0,22 µm filtre filtreleme için hazırlayın. Bu adım laminar akışı steril koşullarda kabine yapılmalıdır. 37 ° C kuluçka kullanmadan önce hazırlanan dispase II önceden ısıtmak.</li>
	</ol>
</li>
</ol>2. fare gözler elde etme
<ol><li>Onaylanmış herhangi bir yöntem kullanarak fare ötenazi ve emici bir yastık yerleştirin.</li>
	<li>Eldivenler, başparmak ve işaret parmağı gözün çevresinde koyun ve cilt proptose gözü yavaşça itin.</li>
	<li>Cilt ve göz küresi arasında açılı makas ucunu takın ve dikkatle göz kesin. Kirlenmesini önlemek için kısaca %70 etanol gözüne batır.</li>
	<li>Hemen göz içinde fosfat tamponlu tuz (PBS) içinde Petri kabına yerleştirin.</li>
	<li>İkinci da gözü çıkarmak için 2.2-2.4 adımları yineleyin.</li>
</ol>3. göz diseksiyon
<ol><li>Dikkatli bir şekilde bağ dokusu diseksiyon mikroskop altında Eye'dan çıkarın. Bu PBS veya kuru Petri kabına üzerinde yapılabilir.<ol>
<li>Dikiş forseps bağ dokusu göz küresi üzerinden Kaldır ve kenasen makas kullanılarak kesin için kullanın. Sklera kesersen sağlam posterior eyecups elde etmek pratik olarak mümkün değildir çünkü sklera kesme.</li>
		<li>Bağ dokusu temizlendikten sonra elde edilen göz yıkama adım için PBS içinde tutun. Not: Aşağıdaki adımların tümünü laminar akış steril koşullarda kabine içinde gerçekleştirilmelidir.</li>
	</ol>
</li>
	<li>Gözleri iki kez Önceden ısıtılmış 37 ° C'de DMEM (yüksek glikoz) yıkayın. Dikkatle forseps kullanarak gözbebekleri Önceden ısıtılmış 37 ° C DMEM (yüksek glikoz) içine aktarın. Yeni bir tabak taze orta orta ve transfer gözbebekleri Aspire edin.</li>
	<li>DMEM (yüksek glikoz) orta Aspire edin ve Önceden ısıtılmış %2 (wt/vol) dispase II çalışma çözüm için 45 dk 37 ° C kuluçka gözünde kuluçkaya. Not: Aşağıdaki adımlarda, yerleştirin ve gözleri veya dokuyu korumak ve iyi bir aracı diseksiyon için sağlar eyecups büyüme orta işlemek.</li>
	<li>Dispase II çözüm kaldırın ve eski orta aspirating ve yeni Önceden ısıtılmış 37 ° C büyüme orta (Şekil 1a) ekleyerek iki kez büyüme aracı olarak gözleri yıkayın. Not: dispase II tarafından ayrılma sonra gözbebekleri yumuşak olmak.</li>
	<li>Forseps ile göz tutun ve ora serrata çevresinde kesik laminar akış kabine yerleştirilmiş diseksiyon mikroskop altında kenasen makas kullanarak her göz olun.<ol>
<li>Göz küresi çözüm içinde tutmak, dikkatlice ön kornea ora serrata çevresi kesme uzanan ve kesme tam edildikten sonra ön kornea uzak çekerek çıkarın.</li>
		<li>Lens kapsülü ve ilişkili Iris Pigmentli epitel yavaşça onları dışarı kadeh teethed forseps (Şekil 1b, 1 c) ile çekerek çıkarın.</li>
	</ol>
</li>
	<li>Elde edilen posterior eyecups 20 dk 37 ° C'de sinirsel retina ayrılması RPE kolaylaştırmak için büyüme orta kuluçkaya.</li>
	<li>Laminar akış kabine yerleştirilmiş diseksiyon mikroskop altında 4 yaprakları posterior eyecups kesilmiş. Kesikler bağlı yaprakları (şekil 1 d) tutmak için kısa ama kadeh düzleştirmek yeterince uzun olmalıdır.<ol>
<li>Kadeh dümdüz ve sinirsel retina çok yavaşça o Forseps ile kalan tutarken çekerek çıkarın RPE-koroid-sklera kompleksi ile başka bir forseps (1e rakam). Kenarları yerine merkezi çekerek başlayın.</li>
	</ol>
</li>
	<li>Önceden ısıtılmış 37 ° C büyüme orta ile dolu yeni bir steril kültür çanak içine katlanmış mendil aktarın.</li>
</ol>4. yalıtım birincil RPE hücrelerinin
<ol><li>Tutan bir yaprağı ile katlanmış RPE-koroid-sklera kompleks forseps süper güzel, yavaşça RPE sağlam yaprak alttaki Bodrum zar (Bruch'ın membran) diseksiyon mikroskop altında mikrocerrahi Hilal bıçak kullanarak akasındaki laminar akış içinde kabine (ekil 1f) yerleştirilir.<ol>
<li>RPE sayfaları içeren büyüme orta P200 micropipette kullanarak yeni steril kültür çanak içine aktarın. RPE sayfaları aktarırken, pipet ipuçları büyüme orta ile birkaç kez pipetting tarafından ıslak doku içinde belgili tanımlık uç yapışmasını önlemek için. RPE-ecek var olmak--dan koroid açıkça ayırt: koroid daha koyu ve yapışkan ve kabarık görünüyor iken RPE daha kahverengi görünüyor.</li>
		<li>Alternatif olarak, Enzimatik sindirim ve forseps olmadan nazik ayrılma choroid14RPE ayırmak için kullanın.</li>
	</ol>
</li>
	<li>2 - 3 kez Önceden ısıtılmış 37 ° C kullanarak RPE çarşafları büyüme orta steril kültür tabağına. Her yıkama adımdan sonra dikkatle retina enkaz veya koroid gibi diğer dokulara kaçınırken RPE sayfaları toplamak. Son yıkama sonunda P200 micropipette RPE sayfalarıyla toplamak ve yeni bir 1.5 mL microcentrifuge tüpü koyun. Not: RPE sayfaları santrifüj kapasitesi için gerek yoktur: 1 dk içinde yerçekimi tarafından tortu olacaktır.</li>
	<li>P200 micropipette kullanarak ilave büyüme orta kaldırın. Taze Önceden ısıtılmış büyüme orta hücrelerde resuspend ve yavaşça P200 micropipette kullanarak triturate. Kabarcıklar oluşumunu önlemek. Bir tek hücre süspansiyon idealdir ama aynı zamanda çok fazla toz önlemek, aksi takdirde RPE hücrelerinin hayatta mümkün değildir. Çok yavaşça toz için 40 - 50 kez öneririz.</li>
</ol>5. Kültür RPE hücrelerinin
<ol><li>Hücre kültür plaka üzerinde plaka.<ol>
<li>RPE hücrelerinin aşağı akım uygulamaları onların polarite istinat gerektiriyorsa, 2 adet 12 mm polyester membran insert 12-şey kültür levha önceden yüklenmiş farelerde dan plaka RPE hücreleri. Bu durumda RPE hücrelerinin altıgen şekil ve pigmentasyon gibi orijinal özelliklerini devam edebiliyoruz çünkü hücreleri yüksek yoğunluklu, kaplama isteniyorsa.</li>
	</ol>
</li>
	<li>Plaka taşımayın veya büyüme orta kültür ilk 72 h için değiştirin. 3 gün sonra eski orta ile taze Önceden ısıtılmış büyüme orta yerine. Bundan sonra her gün kültür orta değiştirin. RPE hücrelerinin confluency çıkınca, FBS büyüme orta % 2'ye azaltmak. Not: % 0.25 tripsin kullanarak hücreleri bölebilirsiniz. Ancak, bölme sonra hücreleri kendi altıgen şekil ve pigmentasyon sonraki pasajlar kaybedebilirsiniz. Biz hücreleri bölme tavsiye etmiyorum, ama kültür, aşağı akım uygulamaları tarafından gerekirse Primer hücre süresiz olarak passaged olamaz unutmayın: de-5-7 pasajlar sonra ayırt etmek için bilinir.</li>
</ol>

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DS Araştırma fon Bayer HealthCare, Almanya ve F. Hoffmann La Roche Merkezi, İsviçre aldı. Kalan yazarlar hiçbir rakip mali çıkarlarının bildirin. Bu eser araştırma önlemek körlük (sınırsız hibe Wilmer göz Enstitüsü ve Pittsburgh Üniversitesi) tarafından desteklenmektedir.  Bu eser DS için start-up fonlarından Oftalmoloji, Pittsburgh Üniversitesi tarafından da desteklenir.

Kültürlerin confluency 1 hafta sonra ulaşmak ve altıgen şekil ve pigmentasyon 2 hafta sonra ana RPE niteliklerinden fare birincil RPE güvenilir kuruluş sunulan ayrıntılı iletişim kuralı sağlar. Elde edilen RPE hücrelerinin aşağı akım kullanma öyle aynı derecede A vitamini metabolizma9, photoreceptor dış segmentleri10 fagositoz ve iyon taşıma11, epitel hücre polarite2, lizozomal bir dizi için kullanıl...

Retina pigment epiteli (RPE) sinir retina ve koroid göz arasında yer alan tek bir katman polarize epitel hücresi var. RPE hücrelerinin işlevsellik ve RPE monolayer bütünlüğünü vizyon için kritik çünkü RPE dış kan-retina bariyeri, taşıma su ve retina arasında iyonları bakımı gibi birden çok işlem önemli bir rol oynar ve Choroid, ışık emilimi, oksidatif stres, retinoid metabolizma kontrolünü ve fagositoz photoreceptors1,2dış segmentleri koruma. RPE göz gibi sistemik kan üzerinden geçişine Vitre mizah yönetilmektedir uyuşturucu önleme bariyer fonksiyonu arkasında konumunu RPE işlev içinde vivokarmaşıklığı çalışmaya zor yapmak. Böylece, esnek, kontrollü bir ortamda3,4RPE hücrelerinde çalışmaya RPE hücre kültürleri kurulması için büyük bir ihtiyaç vardır.
Kolay ve rahat bir şekilde elde etmek ve hücre depolama sağlayan birkaç kurulan RPE hücre satır yok; Ancak, pasajlı hücreleri Primer hücre2,3,4' e göre bazı dezavantajları var. İlk olarak, onlar kez hücre morfolojisi değişimler ile karakterizedir. Örneğin, varolan hücre hatlarda RPE bariyer özellikleri hücre polarite fenotip kaybı ve sıkı kavşak4kısmi kaybolması nedeniyle, güvenilir bir çalışma için uygun bulunmuştur. Polarite ve uygun hücre hücre bağlantıları kaybı ek olarak, RPE hücre satırlarını hızlı bir şekilde olmaması nedeniyle kendi pigmentasyon yetişkin RPE5anahtar melanogenesis enzimlerin kaybetmek. Pigmentasyon geri yüklenebilir, ancak iletim elektron mikroskobu bir arada içeren yeniden pigmentasyon mekanizmasının kapsamlı analiz, melanin varlığını doğrulamak için gen ifade analizi ve kimyasal deneyleri vardır asla gerçekleştirilen6oldu. Bir daha fazla sınırlama RPE hücre hatları genişletilmiş hücre yaşam (bazen - ölümsüzlük) ve belirli koşullar altında substrat ve koloniler7, yüzen formu ayırmak kendini yenileyerek multipotent kök hücre içine dönüştürebilirsiniz olması 8. bu sınırlama nakli3deneyler için hücre hatları kullanmak imkansız hale getiriyor.
Kurulan RPE hücre hatları dezavantajları göz önüne alındığında, taze dokulardan alınan birincil RPE hücre kültürleri RPE çalışmaya daha biyolojik olarak uygun bir model olarak hizmet verebilir. A vitamini metabolizma9, photoreceptor dış segmentleri10 ve iyon fagositoz11taşıma gibi sadece RPE özgü işlevleri çalışmaya, aynı zamanda temel hücre biyolojisi gibi çalışmaya birincil RPE hücrelerinin kullanıldığını epitel hücre polarite2 , lizozomal homeostazı ve autophagy12,13.
Birincil RPE kültürler oluşturma konusunda yayınlar bir dizi olmuştur yıl son çift içinde bu alanda araştırma3,14,15büyüyen bir ilgi gösteren. Yayınlanan16,insan RPE hücrelerinin ve insan olmayan RPE hücrelerinin sığır ve domuz RPE hücrelerinin gibi çok sayıda protokollerde idi17,18,19. Ancak, fare RPE hücrelerinin çok daha küçük boyutlarından dolayı işlemek daha zordur. Tamamen a sayı yayınların RPE hücrelerinin fare14,20,21yalıtmak için protokolleri tarif var olsa bile, orada hala pek çok araştırmacı RPE hücrelerinin olmadan yalıtmak için mücadele kirlenme choroid hücre veya hücreler nöral retina enkaz üzerinden. Burada birincil fare RPE hücre kültürü, gözleri elde etme fare, gözleri diseksiyon ve yalıtım hücre kültürü çalışmalarının için vermeye RPE sayfaların dahil kurmak için iletişim kuralı mevcut. Bu video protokol fare birincil RPE kültürleri ile çalışmak ve diseksiyon teknikleri konusunda yardıma gereksinim başlayan araştırmacılar için özellikle yararlı olacaktır.

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	</tbody>
</table>

primary cell cultures from the mouse retinal pigment epithelium

Retina pigment epiteli (RPE) sinir retina ve koroid göz arasında yer alan bir çok polarize çok fonksiyonlu epitel var. Hexagonally Paketli ve sıkı kavşaklar tarafından bağlı Pigmentli hücreleri tek bir sayfadır. RPE ana işlevleri ışık, fagositoz döken photoreceptor dış segmentleri, kayma iyonları tampon, besin, iyonlar ve su yanı sıra aktif katılımı taşımacılığının emilimini görsel döngüsünde içerir. Böyle önemli ve çeşitli fonksiyonları ile biyoloji RPE hücrelerinin çalışma kritik önemlidir. RPE hücre satır sayısını kurduk; Ancak, bazı morfolojik ve fizyolojik özellikleri doğal RPE hücrelerinin hızla kaybetmeye pasajlı ve ölümsüzleştirdi hücreler denir. Böylece, Primer hücre RPE hücre biyolojisi ve işlev farklı yönlerini eğitim için daha uygundur. Fare modelleri biyolojik çalışmalarda yaygın olarak kullanıldığından fare birincil RPE hücre kültürü araştırmacılar için çok yararlı, ancak RPE toplama fare hücreleri de kendi küçük boyutu nedeniyle çok zor. Burada, enükleasyon ve gözleri diseksiyon ve yalıtım RPE yaprak hücre kültürü çalışmalarının için verim içeren birincil fare RPE hücre kültürleri kurmak için bir protokol mevcut. Bu yöntem etkili hücre kurtarma sağlar. İki fare elde RPE hücrelerinin kültür tabağına sonra kültür ve bazı iyi niyetli RPE orijinal özelliklerini hücreleri altıgen şekil ve pigmentasyon iki sonra gibi görünen bir hafta önceden yüklenmiş bir 12 mm polyester membran Ekle confluency ulaşabilirsiniz Kültür Haftası.

2 fareler kültür 1 hafta sonra 12 mm polyester membran Ekle ulaştığı % 90-95 confluency kurulan fare birincil RPE kültür. Kültür, % 100 confluency vardım ve bir mozaik altıgen, Pigmentli kurmaya başladım hücreleri ve hücre BI parçalanmayabilir 2 hafta sonra. Kültür, şekil ve pigmentasyon, kurmaya devam etti hücreleri tarafından 3 hafta ancak, 4 hafta sonra hücrelerin bir kısmı hiperpigmente (Şekil 2) var.
<p class="jove_content" ...

Watch this Scientific Journal Video about Primary Cell Cultures from the Mouse Retinal Pigment Epithelium at JoVE.com

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Retina pigment epiteli (RPE) çok fonksiyonlu bir epitel göz olduğunu. Burada bir iletişim kuralı fare RPE türetilmiş birincil hücre kültürleri kurmak için mevcut.

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 ...

primary-cell-cultures-from-the-mouse-retinal-pigment-epithelium

Research

JoVE Journal

Biology

20.9K Görüntüleme.  University of Pittsburgh School of Medicine. Retina pigment epiteli (RPE) çok fonksiyonlu bir epitel göz olduğunu. Burada bir iletişim kuralı fare RPE türetilmiş birincil hücre kültürleri kurmak için mevcut.

Video: Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Geç Stage Drosophila pupa Brain İlköğretim Nöronal Kültürler

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Biyoloji

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Kemirgen Yenidoğanlar İlköğretim grubuyla orta beyin dopamin Hücre Kültürleri

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Birincil Hücre Kültürleri Drosophila Gastrula Embriyolar

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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, ...

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 ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

Zebra Balığı Retinal Pigment Epitelinin Rejenerasyonunu İncelemek için Nitroredüktaz / Metronidazol Aracılı Ablasyon ve Bir MATLAB Platformu (RpEGEN)

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

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. ...

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 ...