Dr. Ben Fowler sayesinde bizim samimi-TEM deneyleri ile onun teknik yardım için genişletmek istiyorum.

Bu iletişim kuralı Üniversitesi Oklahoma Sağlık Bilimleri Merkezi/Kurumsal inceleme Kurulu (IRB #4509) kuralları izler. İletişim kuralının tüm parçaları Helsinki Bildirgesi ilkelerinin bir araya geldi. Kornea örnekleri ulusal kalkınma ve Araştırma Enstitüsü (NDRI) ve Oklahoma Lions göz Bankası elde edilmiştir.
1. Primer hücre izolasyon
<ol><li>İnsan kornea doku örneği alınması üzerine, dokularda bir 21,5 cm2 Petri dish 2 mL steril Dulbecco'nın fosfat tamponlu tuz (DPBS) içeren naklet (1 x).</li>
	<li>Scrape ve kornea epitel kornea kubbe yukarı doğru konumlandırma tarafından kaldırın. Tümüyle kaldırılmasını sağlamak için iyice bir 45 ° açıyla bir jilet kullanarak epitel tabaka kapalı kazımak. Ardından, kornea kubbe aşağı doğru çevirmek ve iyice endotel stroma tümüyle kaldırılmasını sağlamak için bir 45 ° açıyla bir tıraş bıçağı ile gelen kapalı kazımak. Stromal doku DPBS ile yıkayın.</li>
	<li>Stromal dokuları at DPBS kullanarak sürekli ıslak stromal parçaları tutmak bir 10 numara cerrahi bıçak kullanarak küçük parçalar halinde kesin. Stromal adet steril forseps ve yer ile bir T25 kültür şişesi ezelî kitle iletişim araçları (4 veya 5 adet şişe başına 2 x 2 mm dokusunun) içine al.</li>
	<li>Explants şişesi yaklaşık 30-40 dk 37 ° C'de altına bağlı kalmak izin verir. Bir kez yapıştırılır, yavaş yavaş % 10 fetal sığır serum (FBS) ve % 1'i içeren kartal'ın en az gerekli orta (EMEM) eklemek antibiyotik/Antimycotic explants bozmadan şişesi için.</li>
	<li>Yavaşça kuluçka 37 ° c, % 5 CO2şişeye koyun. Hücreleri izole başlayana explants rahatsız bekletin ve şişeye geçiş yapma. Yaklaşık 2-4 hafta sonra hücrelerin % 100 izdiham ulaştırılması gerekmektedir. % 100 izdiham, üzerine hücreleri doku kültürü medya kaldırarak geçiş ve DPBS hücrelerle yıkayın.</li>
	<li>Tripsin hücrelere ekleme ve sonra onları 37 ° C'de kuluçkaya Yaklaşık 5 dk sonra onlar ayırdıktan onaylamak için ışık mikroskobu altında hücreler görüntüleyin ve sonra taze medya ekleyerek proteolizis durdurmak.</li>
	<li>Santrifüjü 1000 x g. adlı için çıkarmak süpernatant dikkatle Pelet bozmadan hücre süspansiyon 15 mL konik tüp içine aktarmak ve taze EMEM hücrelerde % 10 askıya FBS % 1 antibiyotik. Hücreleri saymak ve T75 kültür şişesi için daha fazla genişleme ile taze EMEM hücrelerde yaklaşık 1 x 106 %1 tohum FBS % 1 antibiyotik.</li>
</ol>2. birincil HCFs kültür ve 3D birleştirme oluşturur
<ol><li>3D bir araya gelen yapıları explants daha önce yetişen EMEM % 10 FBS %1 antibiyotik/Antimycotic, geçit ve sayısı HCFs.</li>
	<li>Yaklaşık 1 x 106 hücreler/kuyu polikarbonat membran ekler ile birlikte 1.5 mL taze EMEM 3D yapılar üzerinden 0,4-µm gözenekleri üzerinde birincil HCFs % 10 tohum FBS % 1 antibiyotik/Antimycotic yapı dön. Aynı medya başka bir 1,5 mL her yapı altýna ekleyin.</li>
	<li>Hücre tohum 24 saat sonra % 10 EMEM hücrelerle kültür FBS % 1 antibiyotik/Antimycotic, ekleyerek 1,5 mL üstünde belgili tanımlık tepe ve her yapı altındaki 1,5 mL 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic asit (C vitamini) ile uyarılmış.</li>
	<li>37 ° c, % 5 CO2 3 hafta için bir kuluçka kültürlerde korumak ve taze medya tüm çalışma süresi boyunca her gün verin. Daha fazla geçiş yok gereklidir.</li>
</ol>3. hücre farklılaşma ve ortak kültürler
<ol><li>3 hafta sonra 8 x 103 hücreleri/iyi SH-SY5Y insan Nöroblastom hücre önceden birleştirilmiş 3D üst yapıları içeren 1,5 mL % 10 içeren EMEM eklemek FBS, % 1 antibiyotik/Antimycotic ve 0,5 mM 2-O-α-D-glucopyranosyl-L-ascorbic asit (C vitamini).<ol>
<li>SH-SY5Y Nöroblastom hücreleri hücre farklılaşması işlemini başlatmadan önce en az 24 saat için kornea stromal hücreler üzerine büyümeye izin.</li>
	</ol>
</li>
	<li>SH-SY5Y hücre farklılaşması ile EMEM %1 FBS ve 10 µM 1,5 mL içeren hücreleri uyararak başlatmak retinoik asit üzerine inşa. EMEM % 10 FBS % 1 antibiyotik/Antimycotic 0,5 mM 2-O-α-D-glucopyranosyl-L-ascorbic asit (C vitamini) 1,5 mL yapı altýna ekleyin. Retinoik asit adım karanlıkta içermesi gerektiğini unutmayın.<ol>
<li>Bu farklılaşma medya hücrelerde 5 gün boyunca kültür devam ediyor.</li>
	</ol>
</li>
	<li>Hücrelere farklılaşma aşama ulaşmak, hücreleri için 1,5 mL serum-Ücretsiz medya içeren 2 geçiş beyin kaynaklı Nörotrofik faktör (BDNF) ve % 1 nM antibiyotik/Antimycotic eklendi EMEM için inşa en üstüne ekleyerek. EMEM % 10 FBS % 1 antibiyotik/Antimycotic 0,5 mM 2-O-α-D-glucopyranosyl-L-ascorbic asit (C vitamini) 1,5 mL yapı altýna ekleyin. BDNF adım karanlıkta içermesi gerektiğini unutmayın.</li>
	<li>Tablo 1' de gösterildiği gibi başka herhangi bir analiz için 2 ek gün önce işleme için hücreleri kültür.</li>
</ol>

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Yazarlar hiçbir rakip mali çıkarlarının bildirin.

Çeşitli çalışmalarda de kornea hastalıkları daha iyi bir anlayış geliştirmelerine yardımcı olarak tedavileri keşfetmek çeşitli hayvan modelleri geliştirilmesi üzerinde duruldu. Ancak, bu çalışmalar insanlar için önemli bir değer doğrulanmadı. Bugüne kadar çeşitli vitro modelleri geliştirilmiş ve onların olağanüstü klinik önemi nedeniyle yaygın olarak araştırıldı. Bizim daha önce kurulan 3D vitro modeli önemli ölçüde vizyon araştırma gelişmeler tıp bilimin...

İnsan vücudunda kornea en yoğun innervated dokudur. Sinirleri dokunma, ağrı, sıcaklık gibi çeşitli duyumlar sorumludur ve iyileşmesi, refleksleri yanıp, gözyaşı üretim ve salgı5,6,7önemli bir rol olarak da mevcuttur. Kornea, stromal sinir mayo limbal pleksus ortaya ve periferik Korneal stroma Radyal girin. Stromal sinir organizasyonu için kollajen lamellae paralel ve daha fazla içine doğru yüzeysel stroma5,8devam ederken daha küçük fascicles şube. Sinir lifleri daha da epitel tabakası nüfuz ve böylece, innervasyon yaygın olarak kornea epitel ve stroma üzerinden dışarı yayılır. Bu nedenle, innervasyon kornea sağlıklı ve hastalıklı durumda önemli bir rol vardır. Bu protokol için in vivo sinir stromal etkileşimleri taklit etmek için kendi türünde ilk roman 3D vitro manken ilerlemesi ortaya koyuyor. Nöronal büyüme çalışırdım en köklü, iyi karakterize satırlardan birini olduğu gibi SH-SY5Y hücre kültürünü bu çalışma için kullanıldı. SH-SY5Y hücre kültürünü her iki substrat yapışık (S-type) üretmek için tanımlanmıştır ve neuroblastic (N-tipi) hücreleri transdifferentiation9tabi olabilir. Sonuç olarak, bu hücre kültürünü N-tipi hücre Çift Kişilik birbirini izleyen subclone seçimden türetilmiştir olsa bile, ayrıca S-type hücrelerin nöronlar aracılığıyla retinoik asit ve beyin kaynaklı içine farklılaşma geçiren küçük bir dizi içerir Nörotrofik faktör9. Bu kornea komplikasyon diabetik retinopati (DM) ve diğer göz hastalıkları ile ilgili daha iyi bir anlayış neden olabilir bir araç sağlar. Alma ve oküler hastalığı olan hastalarda neurons kültür ile ilgili nedenlerden dolayı nöronal etkileşimleri eğitim ve Korneal stroma ile sinyal önemli etkileri bu 3D vitro modeli sağlar.
Hastalıklı koşulları kez güvenliği aşılan bir yaşam kalitesi için önde gelen bir çok büyük ölçekli, vücudun çeşitli dokuları etkiler. Oküler dystrophies ortak komplikasyonlar genellikle sistemik hastalıklar ve kurşun görme keskinliği kaybı veya hatta kalıcı görme kaybı ile ilişkili vardır. Kapsamlı çalışmalar çoğu kez daha iyi hastalık durumu gibi Bazal hücresel düzeyde etkilerini anlamak için gerekli. Gibi hastalıklar etkileri çalışmaya, doku mühendisliği uygulamaları yardımıyla çeşitli in vivo ve in vitro modelleri geliştirilmiştir. Kornea doku mühendisliği uygulamaları bilim10,11,12,13,14çeşitli alanlarda büyük ilgi topladı var ama sırasında hala önemli sınırlamaları vardır fiili uygulama, kornea dahil greft reddi, enfeksiyonlar ve skarlasma10,11,12,13,14. Başarılı bir şekilde geliştirilen ve çeşitli vitro modelleri3,15,16,17,18, kurulan birçok çalışma vardır 19,20,21,22,23,24,25,26. 3D vitro en umut verici ve büyük bilimsel ilgi modeller. 3D modelleri daha iyi fibrozis sırasında kritik olan ve şifa15,27,28,29yara vivo içinde hücresel ve fizyolojik olaylar ayna olduğu bilinmektedir. Bu vitro modelleri kornea komplikasyonlar da dahil olmak üzere farklı hastalık tedavi için yeni tedavi yaklaşımları bulma konusunda önemli bir rol oynamak. İnnervasyon kornea işlevlerinde önemli rol rağmen küçük bir çaba periferik sinirleri yayılması kornea dokusu mühendislik yapıları2,3içinde tanıtmak için yapılmıştır. Ancak, önerilen 3D vitro hücre yapıları istenen doku işlevselliği elde etmek için hedef doku taklit.
Diyabetik keratopathy nöronal kusurları nedeniyle burada açıklanan model için bariz bir uygulama olmakla birlikte, Keratokonus ve Fuchs dystrophies de dahil olmak üzere bir insan vitro modelinden yararlanabilir birçok kornea hastalıkları vardır. Bizim 3D modeli bu umudu ortaya çıkar ve vitro gösterilişinin ilaç dağıtım değerlendirmek için kornea dokusunun ve Emanet yeni oküler ilaçların geliştirilmesi öneriyor.

<table>
	<tbody>
		<tr>
			<td>Healthy corneal tissue</td>
			<td>NDRI</td>
			<td></td>
			<td>Samples from donors with no ocular trauma or systemic disease</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Solution (1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile forceps</td>
			<td>Fischer Scientific</td>
			<td>13-812-42</td>
			<td>Fisherbrand Dissecting Extra-Fine-Pointed Splinter Forceps</td>
		</tr>
		<tr>
			<td>Single edge razor blades</td>
			<td>Personna</td>
			<td>270100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical scalpel blades No.10</td>
			<td>Feather Surgical Blade</td>
			<td>2976#10</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#8217;s Minimum Essential Medium</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td>10% FBS is required for media preparation</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco by Life Technologies</td>
			<td>15240-062</td>
			<td>1% Antibiotic-Antimycotic is required for media preparation</td>
		</tr>
		<tr>
			<td>0.05% Trypsin EDTA(1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane inserts with 0.4-&#956;m pores</td>
			<td>Corning Costar</td>
			<td>3412</td>
			<td></td>
		</tr>
		<tr>
			<td>2-O-&#945;-Dglucopyranosyl-L-ascorbic acid (Vitamin C)</td>
			<td>Sigma-Aldrich</td>
			<td>SMB00390-14</td>
			<td>A concentration of 0.5 mM should be used for the study</td>
		</tr>
		<tr>
			<td>Wax block</td>
			<td>VWR</td>
			<td>50-949-027</td>
			<td></td>
		</tr>
		<tr>
			<td>SH-SY5Y Neuroblastoma cells</td>
			<td>ATCC</td>
			<td>SHSY5YATCC CRL-2266</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>SRP3014-10UG</td>
			<td>Final concentration of 10uM needs to be used</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-100MG</td>
			<td>Final concentration of 2nM needs to be used</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide(DMSO)</td>
			<td>VWR-Alfa Aesar</td>
			<td>67-68-5</td>
			<td>Ultra Pure Grade-Sterile DMSO to be used</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T25)</td>
			<td>Fisher Scientific</td>
			<td>12-565-351</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T75)</td>
			<td>Fisher Scientific</td>
			<td>12-565-349</td>
			<td></td>
		</tr>
	</tbody>
</table>

corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea

Doku Mühendisliği ile yaklaşık 10 milyon kişi kornea vizyon kaybı1ile acı dünya çapında insan kornea replasmanları için yüksek talep nedeniyle önemli tanıma kazanmıştır. Uygun insan kornealar, üç boyutlu (3D) doku içinde önemli bir ilerleme için talep adrese Mühendisliği2,3,4yapılmıştır. Bu kornea modelleri arasında değişir basit monolayer sistemleri çok katmanlı modelleri, 3D tam-kalınlık kornea eşdeğerleri2' ye lider.
Ancak, çok katmanlı 3D kornea dokusu yapı, fonksiyon ve farklı hücre türleri (örneğin, sinir, ağ için tarihi yoksun benzerlik için bir 3D doku Mühendisliği kornea vitro hastalık modelleri bağlamında kullanımı eğitimi epitel, stroma ve endotel)2,3. Buna ek olarak, talep vitro kornea dokusu modelleri için bir girişim için eczacılık ürünleri hayvan test azaltmak için artmıştır. Böylece, daha gelişmiş modeller daha iyi sistemleri insan fizyolojik gereksinimleri için maç için gerekli olan ve daha çok hasta popülasyona ilgili bir model geliştirilmesi kesinlikle gereklidir. Birden fazla hücre türü kornea hastalıkları ve dystrophies, Keratokonus, diyabetik Keratopathy ve Fuchs, gibi etkilenir verilen bu birincil insan kornea fibroblastlar (HCFs) sağlıklı bağış ve neurons 3D ortak kültür modeli bu modeli içerir SH-SY5Y hücre satırı. Bu bizim için ilk kez insan kornea dokusu içinde iki hücre tipleri arasındaki etkileşimler araştırmak için izin verir. Biz bu model potansiyel sinir hasar oluşturan kornea hastalıkları sinir stromal etkileşimleri ile ilgili temel mekanizmaları incelemek inanıyoruz. Bu 3D model kornea dokusu içinde vivo temel anatomik ve fizyolojik doğasını yansıtır ve gelecekte kornea kusurları araştıran yanı sıra çeşitli ajanların etkinliği hayvan test etmeden önce eleme için bir araç olarak kullanılabilir.

Şekil 1 çalışma 3D vitro modeli hakkında adım adım bir temsilcisi görüntüdür. İlk adımda, insan kornealar izole hücrelerdir. O zaman, onlar bir polikarbonat membran üzerinde yetiştirilen ve kendini salgılanan 3D matris montajı için C vitamini ile uyarılmış. Bu 3D yapı sistemi çok katmanlı bir hücresel vivo içindesentezi indükler-stromal matris gibi. Bundan sonra Nöroblastom hücreleri nöronal hücre farklılaşm...

Watch this Scientific Journal Video about Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea at JoVE.com

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Bu iletişim kuralı muayene ve etkileşimleri iki hücre tiplerinin anlayış yardımcı olmak için nerede kornea stromal hücreler ve farklılaştırılmış nöronal hücre birlikte kültürlü bir roman üç boyutlu vitro modeli, açıklar.

Kornea doku Mühendisliği: İnsan kornea sinir Stromal etkileşimlerin bir Vitro modeli

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

corneal-tissue-engineering-an-vitro-model-stromal-nerve-interactions

Research

JoVE Journal

Developmental Biology

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

8.7K Görüntüleme.  University of Oklahoma Health Sciences Center. Bu iletişim kuralı muayene ve etkileşimleri iki hücre tiplerinin anlayış yardımcı olmak için nerede kornea stromal hücreler ve farklılaştırılmış nöronal hücre birlikte kültürlü bir roman üç boyutlu vitro modeli, açıklar.

Video: Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Evans Blue Dye ile Zebra balığı Larva İskelet Kası Bütünlüğü Analizi

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Gelişim Biyolojisi

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

SH-SY5Y İnsan Nöroblastom Hücre Hattı Farklılaşma

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Çok renkli Floresan Reporter Fare kullanma Kornea Hücre Lineage İzleme Bir Yöntem

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Bir Gelişimi<em&gt; İn Vitro</em&gt; Deney Mezenkimal Hücreleri Uyguladığımız Epitel-Mezenkimal Geçiş kasılma Fonksiyonu değerlendirin

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

Doku Mühendisliği ve Hastalık Soruşturma Kullanılmak Üzere Pediatrik İnsan Özofagus Epitel Hücreleri koşullu yeniden programlanması

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

5-mC Nokta-Blot Testi Kondrosit Farklılaşmasımın DNA Metilasyon Seviyesinin Nicelendirilmesi<em&gt; In vitro</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

Vitro Simüle mikrogravite altında fare Preantral follikül gelişimi

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Fare İkincil Palin Füzyonunun Canlı Görüntüsü

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Diseksiyon ve mantar vücut immünfloresan boyama ve yetişkin Drosophila melanogaster beynindeki nöronlar Photoreceptor

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Hücre toplama Trans Drosophila çentikle bağlama değerlendirmek için deneyleri-ligandlar ve onun inhibisyon CIStarafından-ligandlar

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...