Name: method of studying palatal fusion using static organ culture
Uploaded: 2015-09-19T14:00:00.000+00:00
Duration: 4 min 58 s
Description: Watch this Scientific Journal Video about Method of Studying Palatal Fusion using Static Organ Culture at JoVE.com

The authors do not have any acknowledgements.

Açıklanan tüm prosedürler yerel Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından onayı dahil olmak üzere, omurgalı hayvanlar, kullanımına yönelik kurallar ve yönetmeliklere uygun olarak yapılmalıdır. Kesme Aletleri ve Kültür Medya 1. Hazırlık <ol><li> Ön-yıkama, bütün araçların% 3 hidrojen peroksit ve 20 dakika boyunca 121 ° C&#39;de daha sonra otoklav ile diseksiyon kullanılmak üzere. Bir kaputu vakum emme ile 0.22 mikron gözenek boyutlu filtre kullanılarak antibiyotik ve antimikotik çözüm ile edilebilen BGJb ortamı Filtre. </li></ol> Kültür Ekipman 2. Hazırlık <ol><li> % 70 alkol ile sprey, küçük üçgenler halinde damak organları destekleyecek polikarbonat membran filtreler kesin ve kurumaya bırakın. Organ kültürü plakası üzerinde temiz ve steril eşkenar üçgen şekilli tel ızgara (20 mm) koyarak Merkez-Well Organ Kültür Dish (60x15 mm) hazırlayın. Sonra B 1 ml kuyu doldurunGJB medya kültür ortamı. NOT: Ortam damak batış olmadan ızgara seviyesine ulaşmak için yeterli olmalıdır. Deneye .Depending ızgara üstündeki zar filtreler, parlak aşağı tarafı, (grid başına 3-4 membranlar) koyun, tedavi (proteinler, antikorlar veya inhibitörleri) ortama eklenebilir </li></ol> 3. Fare Embriyo Toplama ve Kültür Hazırlık <ol><li> Vahşi tip fare embriyoları elde etmek için, kullanım 13.5 gebe CD-1 fare genetik arka plan gerginlik zaman aşımına. % 70 etanol ile diseksiyon alanı temizleyin. Not: Farelerin diğer suşları kullanılabilir. Nedeniyle büyük çöp sayısının CD-1 fareleri tercih ederim. </li><li> Onaylı protokolleri kullanarak ölüm sigorta servikal dislokasyon tarafından takip% 5 İsofluranın ile E13.5 DPC (gün çiftleşme sonrası) hamile fareler Euthanize. </li><li> Araçlara karın saç sopa önlemek için ventral karın yüzeyi üzerinde% 70 etanol püskürtün. Sonra ventral karın boşluğuna açılırkeskin künt bir işletim makas ve mikro-diseksiyon forseps bir kullanarak rahim bulun. Forseps kullanarak, tüm rahim kaldırın ve rahim vücutta kesilerek ve hafif bir işletim makas ile rahim boynuzları ucunda gövdesinden ayırın. </li><li> (Bakım ve laboratuar hayvanlarının kullanımı için protokol kılavuzuna göre) buz üzerinde temiz bir kağıt havlu üzerine rahim yerleştirin. Kontaminasyonu önlemek için başka bir temiz kağıt havluyla örtün rahim. Önce daha fazla kullanmak% 70 etanol sprey kullanarak aletleri sterilize edin. Uterus ve embriyolar, buz üzerinde laboratuara nakledilebilir. </li><li> Açık laminer akış kaputu, dikkatlice keskin künt bir işletim makas ve mikro diseksiyon forseps kullanarak yumurta kesesinde küçük bir açıklık yapmak. Açılış geniş yapın ve ardından yavaşça yolk kesesi embriyo exteriorize itin. </li><li> Soğutulmuş fosfat tamponlu salin 1X (PBS), pH 7.4 içeren bir Petri kabı hemen embriyolar aktarın. Doğru sahne, ex sağlamak içinvücut, kafa büyüklüğü ve uzuv morfolojisi için bir mikroskop altında embriyo amin. Not: Doğru gelişim aşamasında olmayan embriyolar deney için kullanılmamaktadır. </li></ol><img alt="Şekil 2," src="/files/ftp_upload/53063/53063fig2.jpg" /> Şekil 2. Ekstremite Bud Morfoloji. Ön ve arka bacaklarda basamak arasında dokuma girinti derecesi için kontrol edilir. E13.5, tüm haneli kondensasyonları rakamlar arasında dokuma ve distal çentikler çok farklı görünen ile belirgindir. Arka basamaklı yarım gün arkasında 12 Üst sıra:. Ön ayakları, alt sıra: arka bacak. 4. damak Diseksiyon <ol><li> Mikroskop altında PBS ile bir petri çanağı bir seferde bir embriyo yerleştirin. Kullanımı küçük kesme makası ve cımbız embriyo gövdesinden başını ayırın. </li><li> Dikkatle forseps ile göz seviyesinden fare kafatası tutarak (kaçınarakmaksiller işlemi) dudak çizgisinin her iki tarafında iki kesiler yapmak. Ardından mandibula ve dilini çıkarın. Göz hizasında bir kesim yaparak maksiller süreç bölgeyi izole edin ve beyin, gözler, burun septum ve bitişiğindeki dokuyu. </li></ol><img alt="Şekil 3," src="/files/ftp_upload/53063/53063fig3.jpg" /> Şekil 3. Damak doku damak raflar yüksek olan 13.5 embriyolar, toplanan ancak temas sergiledi. Damak raflar statik modeli 72 st için hava ortam maddesi ara kısmında bulunan bir filtrede bir organ kültür içine yerleştirildi. (A) Düzey E13.5 embriyo üzerinde diseksiyon. (B) Mavi yıldız Diseksiyon sırasında forseps ile doku tutmak için sınır alanları. Bir yamuk filtre zarı üzerine yerleştirilir disseke damak raflar (C) Görünüm. kültür çanak (D) Side view yerleştirildikten sonra Diseke damak ve kültür ortamı eklenmesi. <ol start="3"><li> Posterior anterior gelen yönlendirmek ona yardım bağlı primer damak tutarak, damak ağız tarafı yukarı yerleştirin ve burun septum ve çevresindeki doku kaldırmak. Bir kaşık spatula kullanın ve çok dikkatli bir şekilde kültür yemekleri ızgaraları Hazırlanan filtreler (burun tarafı aşağı) damak raflar aktarın. NOT: Doğru hava medya arayüzü sağlamak için medyanın sesini ayarlayın. </li><li> Stereo mikroskop altında birincil damak incelemek ve birbirine yakın damak raflara yerleştirmek için cımbız kullanın. Onlar temas edecek şekilde yerleştirilir değilse damak raflar kaynaştırmak olmaz. Damak ön kısmı bulunan üçgen membran filtre kesin. Yamuk şekli damak ön kısmını lokalize yardımcı olacaktır. </li><li> Kültür damak raflar tek tek veya iki veya edilebilen BGJb kültür ortamı kullanarak aynı organ kültür çanak üç ile. </li></ol> 5. Kültürleme Fare damak Raflar <ol><li> Tissu inkübe72 saat boyunca nemlendirilmiş bir gaz karışımı (% 5 CO2 ve% 95 hava), 37 ° C &#39;de es ve ortam her 24 saatte değiştirin. </li></ol> Histolojik Analizi 6. Damak İşleme <ol><li> / N 4 ° &#39;de% 4 formaldehit / fosfat tamponlu tuz O ile kültür içinde 72 saat sonra damak saptamak &#160; Daha sonra, yumuşak bir transfer pipeti ile PBS ile kültürü çanağı içinde doku yıkayın. Kasetleri gömme laboratuvar mendil ve yerde bir parça damakları sarın. NOT: Laboratuvar mendil içinde damak Ambalaj dehidrasyon sürecinde kendi kaybını önlemek. </li><li> 70,% 80,% takip eden ve düzenli parafine gömme prosedürü takiben 1 saat boyunca% 100 etanol yıkamadan kadar doku kurutmak. Gömmek için, tepsiyi bakan damak ön kısmını yerleştirin. </li><li> Posterior anterior koronal yönde Seri 6 mikron bölümleri kesin. Tam bir bölüm, damak 350-450 SECTI verecektirons. Hematoksilen ve eozin (H &amp; E). 10 Skor daha önce tarif Mean Fusion skoru kullanılarak her 20 inci bölümü (MFS) Ölçek (Tablo 1) bölümleri Leke. 8 </li></ol>

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The authors do not have any competing financial interests.

Bu makalede protokolü embriyonik gün 13,5 olarak embriyolardan damak raflar kesme için bir yöntem sağlar. Fare embriyosu Palatal raflar 37 ° C &#39;de 95% O 2,% 5 CO2 atmosferi içinde serumsuz kültür ortamı içinde kültürlenmiştir. Başarılı damak diseksiyonu kültürünün tamamlanmasına ötenazi farelerin embriyonik zaman işlem sırasında her adımda birçok faktöre kritik bağlıdır. Damak füzyon etkileyen en önemli faktörlerden biri, organ kültürü başlatmak için harcanan zaman ...

Orofasial yarıklar en baskın kraniofasyal doğum kusurları vardır. Ayrıca, göz olası tüm kraniofasial kusurları alarak, bu yenidoğanların 2 en sık görülen ikinci doğum anomalisi bulunmaktadır. Yarık damak, yaklaşık 1 Amerika Birleşik Devletleri&#39;nde (ABD) her yıl her 700 doğumda meydana, yarık damak insidansı yarık ayda damak veya gün başına 3 yarıkların 15 çocuklu doğan 475 çocuk eşittir. Çocuklar yaklaşık% 1, her yıl sergilediğini dünyada kraniyofasiyal dismorfoloji çeşit doğdu. Damak ve dudak yarıkları, bu anomali olan hastalarda yaşam boyu etkileri olan bir çok pahalı ve karmaşık prosedür gerekiyor. Oral yarıklı her hasta için tahmini maliyeti yaklaşık 100.000 $ 4&#39;tür. Dudak ve damak bir hastanın tedavisi, kraniofasial cerrahlar, kulak burun boğaz, genetikçiler, anestezist de dahil olmak üzere doktorların bir ekip gerektirirkonuşma-dil patologlar, beslenme, ortodontist, prostodontist, psikologlar, beyin cerrahları ve göz doktoru. Palatogenesis, ikincil damak başlangıçta dikey büyümeye ve dil sırtında üzerinde damak raf yüksekliği geçmesi eşleştirilmiş çıkıntılar olarak ortaya çıkar. Yükseklik ardından, eşleştirilmiş damak raflar (insanlarda, farelerde E14.5 -E15 ve haftanın 9) orta hat doğru büyür. Raf ucu kapakları medial kenar epiteli (MEE) orta hat epitel dikişi oluşturan yapışır. Bu mezenkimal izdiham izin vermek için mezenkimal geçiş ve / veya apoptoza epitelyal izlemektedir. Karşıt MEE yapışması olan değişiklik yarık damak neden temel bir olaydır. Ancak, sadece birkaç çalışma damak raflar 5 başlangıç ​​yapışma araştırdık. Osteoblast içine mezenkimal hücrelerin farklılaşması ile sert damak formları. Clef üretebilir damak anormal gelişimt veya dudak katılımı olmadan damakları. Damak organ kültürü teknikleri, son 30 yılda 6,7 boyunca birçok laboratuvarlar için yaygın olarak kullanılır olmuştu. Bu protokolde ayrıntıları damak diseksiyon ve statik organ kültürü bir yöntem açıklanmaktadır. Hareketsiz bir organ kültürü avantajı damak raflar kaynaştırmak olanak sağlamasıdır. Bu teknik pek çok füzyon ve sinyalizasyon deneyler 8,9 bizim laboratuvarda başarıyla kullanılmıştır. Tekniğin kapsamı geniş ve statik organ kültürü sistemi eksojen kimyasal maddelere yanıtların değerlendirilmesi de dahil olmak üzere, gerekli olduğunda kullanılabilir Ancak, farklı yollar ve spesifik proteinlerin düzenleyici büyüme faktörlerinin etkileri. <img alt="figür 1" src="/files/ftp_upload/53063/53063fig1.jpg" /> Şekil 1. Fare Palatogenesis. Fareler damağın Gelişim aşamaları. (BF) Tarama eletemsili gelişim zamanlarda ikincil damak cTRON mikrograflan (SEM). Kırmızı oklar: damak yapışma ve füzyon ilk bölümünü gösterir. Sarı oklar: füzyon sonra kaybolur birincil ve ikincil damak arasındaki boşluğa gelin (PLoS biri izni ile Kaufman 11 yeniden basıldı).

<table><tbody><tr><td>BGJ&lt;sub&gt;b&lt;/sub&gt;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Lonza Inc.</td><td>17-602E</td><td>100X</td></tr><tr><td>Petri dishes</td><td>VWR</td><td>25384-088</td><td>100 x15 mm</td></tr><tr><td>Center-well organ culture dish</td><td>Falcon&amp;nbsp;</td><td>#353037</td><td>60 x15 mm</td></tr><tr><td>Wire triangular grid</td><td>Custom made with stainless steel wire mesh to fit the organ culture dish well.&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Polycarbonate filter</td><td>GE&amp;amp; Water and Process Technologies</td><td>K04BP04700</td><td>Black 0.4 micron, 47 mm</td></tr><tr><td>Stereo microscope</td><td>ZEISS&amp;nbsp; Stemi SR</td><td></td><td></td></tr><tr><td>Culture hood</td><td>NUAIRE Laminar Flow</td><td></td><td></td></tr><tr><td>Microdissecting forceps</td><td>Dumont Medical&amp;nbsp;</td><td></td><td>#5</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS14003-G</td><td>Vannas Scissors, straight, 8 cm long, 0.1mm tips, 5 mm blades, German made</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS500086</td><td>Vannas Scissors, straight, 8.5 cm long, 0.025 mm x 0.015 mm tips, 7 mm blades</td></tr><tr><td>Microdissecting scissors</td><td>Kent Scientific Corporation</td><td>INS14127-G</td><td>Spring scissors curved curved, 10.5 cm long, 8 mm blades, German made</td></tr><tr><td>Surgical currete (spoon spatula)</td><td>Hu-friedy</td><td></td><td>CM 2/4</td></tr><tr><td>Protector laboratory hood</td><td>Labconco</td><td></td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td></td><td>Farma</td></tr><tr><td>Series11 water Jacket</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>GIBCO</td><td>10010-023&amp;nbsp;</td><td>1X</td></tr><tr><td>Fiber optic Light source</td><td>Fiber-light&amp;nbsp; Dolan-Jenner</td><td>PL-750</td><td></td></tr><tr><td>Embedding cassette&amp;nbsp;</td><td>Statlab</td><td>EC301</td><td></td></tr><tr><td>Kim Wipes</td><td>VWR</td><td>470173-504</td><td></td></tr></tbody></table>

method of studying palatal fusion using static organ culture

Yarık dudak ve damak, tüm doğum kusurları en yaygın arasındadır. Orta hat epitel dikiş oluşturmak yapışır epitel ile örtülü mezenkimal raflardan ikincil damak formları (MES). Teoriler MES hücreler kaynaşmış damak 1 yaparak, mezenkimal geçiş (EMT), apoptoz ve göç bir epitel izleyin öneririz. MES tam parçalanma mezenkimal hücreler çevredeki damak izdiham son temel aşamasıdır. Bu damak organ kültürü için bir yöntem sağlamaktır. In vitro protokol geliştirilen füzyon sırasında biyolojik ve moleküler proseslerin çalışma sağlar. Bu tekniğin uygulamalar harici kimyasal ajanlara değerlendirilmesi tepkiler, düzenleyici ve büyüme faktörleri ve spesifik proteinlerin etkileri de dahil olmak üzere, çok sayıda bulunmaktadır. Palatal organ kültür in vivo çalışmalar ile mümkün değildir farklı gelişim safhalarında manipülasyon da dahil olmak üzere bir dizi avantaj vardır.

Tekniği experiments.The Aşağıdaki çalışma farklı uygulanabilir damak füzyon üzerinde in vitro nikotin teratojenik etkisini test etmek için tasarlanmıştır. Farelerin iki suş Bu deney için kullanılmıştır, CD1 ve C57.Palatal dokusu nikotin hemisülfatın 0.06 6 mM konsantrasyonları ile muamele edilmiştir. Bu damak füzyon desen ve zamanlama iki farklı suşları farklı olduğu görülmüştür. CD1 fareleri, kontrol grubundan damak raflar zamana bağlı bir şekilde füzyon devam et...

Watch this Scientific Journal Video about Method of Studying Palatal Fusion using Static Organ Culture at JoVE.com

Method of Studying Palatal Fusion using Static Organ Culture

Damak gelişimi çalışmalar yarık damak, muazzam bir sağlık yük getirmektedir ve şekil bozukluğuna kalıcı bırakabilirsiniz doğum defekti insidansının motive etmektedir. Biz burada damak gelişimi ve füzyon içinde yer alan farklı sinyal yollarını incelemek için kullanılabilecek kültür damak raflarına bir tekniği göstermek.

Statik Organ Kültürü kullanarak Palatal Fusion incelenmesi yöntemi

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

method-of-studying-palatal-fusion-using-static-organ-culture

Research

JoVE Journal

Developmental Biology

8.7K Görüntüleme.  Texas A&M University Baylor College of Dentistry.  Damak gelişimi çalışmalar yarık damak, muazzam bir sağlık yük getirmektedir ve şekil bozukluğuna kalıcı bırakabilirsiniz doğum defekti insidansının motive etmektedir. Biz burada damak gelişimi ve füzyon içinde yer alan farklı sinyal yollarını incelemek için kullanılabilecek kültür damak raflarına bir tekniği göstermek. 

Video: Method of Studying Palatal Fusion using Static Organ Culture

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

Faringeal Arches Ex Vivo Kültür Kalp ve kas atalarıdır ve Bunların Niche Eğitim için

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Gelişim Biyolojisi

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

Floresan In-Situ Hibridizasyon ve Konfokal Lazer Tarama Mikroskopi Palatal Genişleticilerden Oral Biyofilm Analizi

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

Tıp

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

İnsan Mantar şeklinde Lezzet papilla hücrelerinin izolasyonu ve Kültürü

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Nörobilim

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 ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

İlköğretim Kültür ve Corti murin Organ Plazmid Elektroporasyon.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Biyoloji

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Kolektif Hareket Niteliklerini Analiz Etmek için Birincil Fare Embriyonik Palatal Mezenkim Hücrelerinin İzolasyonu ve Zaman Atlamalı Görüntülenmesi

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Trowell Tipi Organ Kültürünün Diş Kök Hücrelerinin Düzenlenmesini Incelemek İçin Kullanılması

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Mantar Formu, Sirkülasyon ve Damak Tat Tomurcuklarının Bütün Montajlı Boyama, Görselleştirme ve Analizi

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

İnsan Nazal Epitel Organoidlerinin Kültürü ve Görüntülenmesi

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Yetişkin Fare Damak Tadından Birincil Oral Keratinositlerin İzolasyonu ve Kültürü

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...