Bu eser kısmen bilim promosyon (JSP'ler) Grant-in-Aid için bilimsel araştırmalar (26861689 ve 17 K 11813) Japonya Derneği tarafından desteklenmiştir. Yazarlar Bay Nathaniel Green proofreading için teşekkür etmek istiyorum.

Bu iletişim kuralı insan dişeti dokusu karşılıkları, diş eti yara modelleri ve gingivitis modelleri oluşturmak için tasarlanmıştır. İnsan derisi epidermal keratinositler (HaCaT) tür Deutsches Krebsforschungszentrum (Heidelberg, Almanya)7Profesör Norbert E. Fusenig temin edilmiştir. İnsan dişeti fibroblastlar (HGFs) daha önce yayımlanan protokolleri8göre dişeti dokuları yalıtılmış. Aydınlatılmış onam önceden elde ve çalışma Etik Komitesi, Nippon diş Üniversitesi School of hayat diş hekimliği Tokyo tarafından belirlenen esaslarına göre kabul edildi (yetki numarası: NDU-T2012-35). Protokol 1-3 adımları bir hücre kültür mahallede yapılmalıdır.
1. hazırlanması jinjiva (GTE) (şekil 1A) 3D insan dokusu karşılıkları
<ol><li>Mix kollajen tipi ı-A jel % 10 ile konsantre MEM-Alfa ve % 10 imar arabellek (2,2 g NaHCO3 ve 100 ml 0,05 4,47 g HEPES N NaOH) pipetting tarafından bir 15 mL steril tüp. Karışık kollajen çözüm kullanana kadar buz üzerinde tutun.</li>
	<li>24-şey kültür ekler (gözenek boyutu 3.0 µm) 24-şey plaka yerleştirin.</li>
	<li>
HGFs % 0.25 tripsin-EDTA çözüm kullanarak kültür yüzeyden kaldırın. Kültür orta A 10 mL hücrelerde askıya alma (MEM-Alfa + %20 FBS + %1 GlutaMAX). Hücreleri tarafından Bürker-Türk hücre sayaç sayar.
	<ol>
<li>Hücre (1-5 x105 hücre/mL) istenen miktar ayıklamak sonra 190 x g de 4 dk santrifüj kapasitesi.</li>
		<li>Karışık kollajen çözüm hücrelerde askıya alma. Buz üzerinde karışımı sonraki adım kadar tutun.</li>
	</ol>
</li>
	<li>Hücre-kollajen karışımı (0.5-2.5 x 105 hücreleri/de) 0.5 mL herhangi bir kabarcıklar üretmeden kültür sokmak içine ekleyin. 10 – 30 dk % 5 CO2 37 ° C'de karışımı bir jel koagüle oksijen bir ortamda karışımı kuluçkaya. Not: eşit olmayan kollajen jel oluşumunu önlemek için kültür ekleme merkezi haline kollajen karışımı ekleyin.</li>
	<li>1 mL kültür ortamının iyi ve 0.5 mL eklemek içine içine ekleyin. Kültür plaka 24 h için kuluçkaya veya bir oksijen bir atmosferde 37 ° C'de % 5 CO2 gece</li>
	<li>HaCaT hücre kültür yüzeyden %0.25 tripsin-EDTA çözeltisi kullanarak kaldırma. 10 mL orta B hücreleri askıya alma (DMEM + %10 FBS + %1 GlutaMAX) ve hücreleri saymak. Özü hücreleri (105 hücre/mL x 1 – 5) bir pipet, 190 x g., 4 dk santrifüj ile gerekli miktar askıya orta b hücreleri</li>
	<li>Orta aspirasyon tarafından HGF-kollajen karışımı içeren kültür ekler kaldırın. 0.5 mL HaCaT hücre süspansiyon (0.5-2.5 x 105 hücreleri/de) üst kısmında HGF-kollajen karışımı ekleyin. 24 saat veya gece boyunca kültürler kuluçkaya. Not: Kültür eklemek içinde orta orta B. gerekirken bu zaman noktada orta kültür de orta A, olmalıdır</li>
	<li>KSR orta (ortak kültür orta) ile Kültür orta yerine (MEM-Alfa + %15 nakavt Serum değiştirme + %1 GlutaMAX) + % 5 FBS. Kültür ortamının 1 mL de ve kültür Ekle içine 0.5 mL kültürün içine ekleyin. 24-48 h için kültürler kuluçkaya.</li>
	<li>Kültür orta yerine KSR orta + % 1 ile FBS. İyi kültür içine 1 mL ve kültür Ekle içine 0.5 mL ekleyin. 24 saat veya gece boyunca kültürler kuluçkaya.</li>
	<li>Kültür Kültür orta de 0.7-1 ile yerine mL KSR orta. Orta tamamen (kültür yüzey havaya maruz) kültür Ekle Kaldır. Kültürler için 1 – 2 hafta kuluçkaya. Haftada iki kez de kültür orta değiştirin. Not: Orta seviye Yükselişi (üst katman keratinositler oluşan) kültür yüzey üzerinde izin vermeyin. Her zaman kültür yüzey kuru tutun.</li>
</ol>2. yaralı GTE (şekil 1B) hazırlanması
<ol><li>1-3 mm çap doku zımba hazırlayın. 20 dk için 121 ° C'de bir otoklav ile sterilize.</li>
	<li>Doku zımba dik GTE 300 µm hakkında derin bas. ve doku bir yara benzetimini yapmak için bir delik yumruk.</li>
	<li>Bu adımda, yaralı GTE yara iyileşmesi ve doku rejenerasyonu soruşturma için kullanın. Alternatif olarak, histolojik Çözümlemeyi gerçekleştirmek için % 10 formalin tarafsız tampon çözüm kullanarak GTE düzeltmek.</li>
</ol>3. inflamatuar GTE (iGTE) (şekil 1B) hazırlanması
<ol><li>THP-1 hücreleri önceki rapor9göre yetiştirmek.</li>
	<li>Mix kollajen tipi ı-A jel % 10 ile konsantre RPMI 1640, % 10 imar arabellek (2,2 g NaHCO3 ve 100 ml 0,05 4,47 g HEPES N NaOH) ve 10 ng/mL PMA. Karışık kollajen çözüm buz üzerinde kullanımda kadar devam.</li>
	<li>Sayısı 1-2 x 106 THP-1 hücreleri ve 190 x g., 4 dk santrifüj 1 mL karışık kollajen çözeltisi hücrelerde askıya alma. Buz üzerinde karışımı sonraki adım kadar tutun.</li>
	<li>10-30 µL THP-1-kollajen karışımı GTE yaralı bölgeye ekleyin. 0.7-1 Kültür ortamına yerine mL KSR orta PMA 10 ng/mL içeren.</li>
	<li>72 h 37 ° C'de % 5 CO2 ile oksijen bir ortamda karışımı kuluçkaya.</li>
	<li>Dişeti iltihabı veya periodontal hastalık araştırma modeli kullanın (örneğin, potansiyel anti-inflamatuar ilaç iGTE sitokin yayın10üzerindeki etkilerini görmek için ekleyin). Alternatif olarak, 72 h PMA-tedavi atlarsanız, 2 µg/mL LPS kültür içine ekleyin ve doku oksijen bir atmosferde % 5 CO2 48 h11,1237 ° C'de kuluçkaya.</li>
</ol>4. fiksasyon ve GTE ve iGTE bütün Mount Immunostaining kültürler
<ol><li>
Fiksasyon kültürlerin.
	<ol>
<li>Kültür orta 24-şey plaka kaldırın. Doku 2 kez fosfat tamponlu tuz (PBS) ile yıkayın.</li>
		<li>1 mL % 10 formalin tarafsız tampon çözeltisi için INSERT ve 1 mL de kültür için ekleyin. 24-şey plaka gece 4 ° C'de bir buzdolabında bırakın.</li>
		<li>% 10 formalin tarafsız tampon çözüm ertesi gün kaldırın.</li>
		<li>Bütün Dağı immunostaining için 3 - 4 kez PBS ile doku fiksasyon sonra yıkayın.</li>
		<li>Hematoksilen Eozin (H & E) Boyama ve düzenli immunostaining için dehidratasyon, parafin gömme ve kesit ile devam edin. Not: Bütün Dağı immunostaining için bu adımı ihmal edilebilir.</li>
	</ol>
</li>
	<li>
Bütün Dağı immunostaining Not: Bu protokol başvurusu 13 bir değişiklik yapıldı.<ol>
<li>Doku 3 yıkama PBS 0.5-1 x % Triton, 10-30 dakika her zaman.</li>
		<li>Belgili tanımlık doku 30 dk içinde engelleme arabellek için iki kez kuluçkaya (PBS %1 Triton + % 10 BSA), oda sıcaklığında.</li>
		<li>Doku 2 × engelleme arabellekte, 5-10 dk her zaman yıkama.</li>
		<li>Birincil antikor çözüm 50 µL gerekli seyreltme/toplama Ekle için ekler. Bir ince plastik sarılmak film doku kurutma önlemek için ekler sarmak için kullanın.</li>
		<li>1-2 gün 4 ° C'de doku kuluçkaya</li>
		<li>Dokular yıkama 3 kez, 30 dk içinde PBS için % 1 Triton + % 10 BSA. Tekrar 3 kez, 5 dakika içinde PBS için % 1 yıkama Triton.</li>
		<li>İkincil antikor 50 µL engelleme arabellekte ekleyin (PBS %1 Triton + % 10 BSA) ve 5 µL Höchst 33342 çözüm (NucBlue canlı hücre leke) her ekleme. Bir ince plastik sarılmak film doku kurutma önlemek için ekler sarmak için kullanın.</li>
		<li>1-2 gün 4 ° C'de kuluçkaya 24-şey plaka bir ıslak kağıt havlu sarın ve daha sonra kuluçka kullanılacak plastik bir paket içine koydu.</li>
		<li>Dokular için PBS % 1 triton 10 min 3 kez, yıkama</li>
		<li>Keskin iğne doku almak için kültür Ekle membran kesmek için kullanın. Membran bir slayt üzerine yerleştirin. Not: Doku membran üzerinde olmalıdır.</li>
		<li>Floresans Dağı orta 2-3 damla ekleyin. Bir cam ve karanlıkta 4 ° C'de mağaza ile doku analizi kadar kapsar.</li>
		<li>Doku mikroskobu (LSM) tarama confocal lazer ile görüntüleyin.</li>
	</ol>
</li>
</ol>

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Yazarlar hiçbir çıkar çatışması beyan ederim.

Bu iletişim kuralı dişeti doku eşdeğerleri ve deri altı yağ dokusu eşdeğerleri önceki raporlar8,21,22tarafından açıklanan oluşturma yöntemleri temel alır. Bu basit ve kolay bir yöntem olmasına rağmen bazı adımlar özel dikkat gerektirir. Örneğin, kollajen karışımı buz üstünde jel oluşumu çözüm önlemek için kullanım kadar tutulmalıdır. Kollajen karışımı içine kültür ekleme eklerken, ç...

Yetişkin diş kaybına önde gelen nedenidir periodontal hastalıklardır. Dişeti iltihabı ve periodontitis en yaygın periodontal hastalıklardır. Her iki mevcut biyofilm aracılı akut veya kronik inflamatuar değişiklikler jinjiva. Periodontitis genellikle kronik inflamasyon sunar, ancak dişeti iltihabı akut iltihabı ile karakterizedir. Histolojik düzeyde, örneğin makrofajlar, lenfositler, plazma hücreleri ve mast hücreleri1,2bağışıklık hücrelerinin aktivasyonu bakteriyel bileşenleri tetikler. Bu bağışıklık hücreleri, özellikle makrofajlar, (dişeti epitel hücreleri, fibroblastlar, endotel hücreleri ve dokusunu da dahil olmak üzere) yerel hücreleri ile etkileşim periodontal doku3,4inflamatuar lezyonlar sonuçlanan. Periodontal hastalıkların deneysel modeller, fare gibi hayvanlar çeşitli kurulmuştur hamster, tavşan, yaban gelinciği, köpek dişleri ve primatlar. Ancak, hayvan modelleri Fizyopatoloji insanların, hücresel ve moleküler mekanizmaları analiz etmek ve periodontal hastalıklar5yeni ilaçların değerlendirmek üzere farklıdır. Periodontal bakteri ve monolayer insan sözlü epitel hücreleri co ekimi periodontal hastalık6mekanizmasının araştırmak için kullanılmıştır. Yine de, sözlü hücre kültürleri monolayer üç boyutlu (3D) hücresel mimarisi sağlam doku eksikliği; Bu nedenle, vitro durumu taklit edemez.
Burada, jinjiva (iGTE) 3D inflamatuar insan dokusu karşılıkları periodontal hastalıklar vitrotemsil etmek üzere oluşturulur. Bu 3D model periodontal hastalıkların monolayer hücre kültürleri ve hayvan modelleri arasında orta bir konuma sahiptir. İnsan hücreleri HaCaT keratinositler, dişeti fibroblastlar ve THP-1 makrofajlar, dahil olmak üzere, üç tür ortak kollajen jel üzerinde ekili ve iGTE oluşturmak için inflamatuar başlatıcıları tarafından uyarılmış. IGTE yakından hücre farklılaşması, hücre-hücre etkileşim ve makrofaj etkinleştirme jinjiva ' vivo şartları simüle. Bu model birçok olası uygulama tarama ve yeni farmakolojik yaklaşımlar periodontal hastalıklarda test ilaç için hem de hücresel ve moleküler mekanizmaları yara iyileşmesi, inflamasyon ve doku rejenerasyonu analiz etmek için vardır.

<table>
	<tbody>
		<tr>
			<td>Collagen type I-A</td>
			<td>Nitta Gelatin Inc</td>
			<td></td>
			<td>For making dermis of GTE</td>
		</tr>
		<tr>
			<td>MEM-alpha</td>
			<td>Thermo Fisher Scientific</td>
			<td>11900073</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Cell Culture Insert (for 24-well plate), pore size 3.0 &#956;m</td>
			<td>Corning, Inc.</td>
			<td>353096</td>
			<td>For tissue culture</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31600034</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>Tissue puncher</td>
			<td>Shibata system service co., LTD</td>
			<td>SP-703</td>
			<td>For punching holes in GTE</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>31800022</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A3294</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Hoechst 33342 (NucBlue Live Cell stain)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37605</td>
			<td>For labeling nuclei</td>
		</tr>
		<tr>
			<td>Fluorescence mount medium</td>
			<td>Dako</td>
			<td></td>
			<td>For mounting samples after immunostaining</td>
		</tr>
		<tr>
			<td>Anti-Cytokeratin 8+18 antibody [5D3]</td>
			<td>abcam</td>
			<td>ab17139</td>
			<td>For identifying epithelium</td>
		</tr>
		<tr>
			<td>Scaning electron microscope</td>
			<td>Hitachi, Ltd.</td>
			<td>HITACHI S-4000</td>
			<td>For observing samples&#39; surface topography and composition</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscopy</td>
			<td>LSM 700; Carl Zeiss Microscopy Co., Ltd.</td>
			<td>LSM 700</td>
			<td>For observing samples&#39; immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Anti-Cytokeratin 19 antibody</td>
			<td>abcam</td>
			<td>ab52625</td>
			<td>For identifying epithelium</td>
		</tr>
		<tr>
			<td>Anti-vimentin antibody</td>
			<td>abcam</td>
			<td>ab92547</td>
			<td>For identifying fibroblasts and activated macrophages</td>
		</tr>
		<tr>
			<td>Anti-TE-7 antibody</td>
			<td>Millipore</td>
			<td>CBL271</td>
			<td>For identifying fibroblasts in the dermis</td>
		</tr>
		<tr>
			<td>Anti-CD68 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>SAB2700244</td>
			<td>For identifying macrophages</td>
		</tr>
		<tr>
			<td>Human CD14 Antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB3832-SP</td>
			<td>For identifying macrophages</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594-conjugated secondary goat anti-rabbit antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11012</td>
			<td>For immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated secondary goat anti-mouse antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11001</td>
			<td>For immunofluorescence staining</td>
		</tr>
		<tr>
			<td>EVOS FL Cell Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>For observing the sample&#39;s immunofluorescence staining</td>
		</tr>
		<tr>
			<td>THP-1 cells</td>
			<td>Riken BRC cell bank</td>
			<td>RCB1189</td>
			<td>For making iGTE</td>
		</tr>
		<tr>
			<td>PMA(Phorbol 12-myristate 13-acetate)</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td>For differentiatting THP-1 cells</td>
		</tr>
	</tbody>
</table>

three-dimensional inflammatory human tissue equivalents of gingiva

Periodontal (dişeti iltihabı ve periodontitis gibi) Yetişkin diş kaybına önde gelen nedenleri hastalıklardır. Periodontal hastalıkların temel Fizyopatoloji jinjiva içinde iltihabıdır. Geçerli modeller periodontal hastalıkların hayvan türleri içinde kurulmuştur. Ancak, hayvan modelleri Fizyopatoloji insanların, hücresel ve moleküler mekanizmaları analiz etmek ve periodontal hastalıklar için yeni ilaçların değerlendirmek üzere farklıdır. Burada, jinjiva (iGTE) vitroinsan inflamatuar doku karşılıkları yeniden inşa için detaylı bir iletişim kuralı mevcut. İlk insan dokusu karşılıkları jinjiva (GTE) insan hücreleri, insan dişeti fibroblastlar (HGF) ve insan derisi epidermal keratinositler (HaCaT), üç boyutlu koşullar altında da dahil olmak üzere iki tür kullanarak oluşturmak. Biz bir yara modeli GTE içinde bir delik açacak bir doku zımba kullanarak oluşturun. Kollajen jel ile karışık sonraki, insan THP-1 monosit GTE delik içine enjekte edilir. Tarafından adimistration 10 ng/mL phorbol 12-myristate 13-asetat (PMA) için 72 h THP-1 hücreleri ayrıştırılan GTE (iGTE) formu inflamatuar foci için makrofajlar içine (IGTE da-ebilmek var olmak stumilated ile 2 µg/mL lipopolisakkaritler (LPS) inflamasyonu başlatmak 48 saat ). IGTE inflamatuar jinjiva insan hücreleri ile üç boyutlu bir mimarisi kullanarak ilk vitro modeldir. IGTE periodontal hastalıklarda önemli patolojik değişiklikler (immunocytes hareket, fibryoblasts, epitel hücreleri, monosit ve makrofajlar arasında hücre içi etkileşimleri) yansıtır. GTE, yaralı GTE ve iGTE yara iyileşmesi çalışmaya çok yönlü araçlar, doku yenilenmesi, iltihap, hücre-hücre etkileşim ve ekran potansiyel ilaç periodontal hastalıklar için kullanılabilir.

HaCaT hücreler tipik keratinosit Morfoloji faz kontrast mikroskobik gözlem (şekil 2A) altında görüntülenir. Tarayıcı elektron mikroskobik (SEM) görüntüleri HaCaT hücre yüzeyler tarafından birçok microvilli kaplıydı gösterdi. Hücreler arası bağlantıları HaCaT hücreleri arasındaki membran işlemler (şekil 2B) tarafından aracılık. HaCaT hücreleri HaCaT hücreleri jinjiva imar (şek...

Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Protokol amacı bir enflamatuar insan jinjiva modeli içinde vitrooluşturmaktır. Bu doku model insan hücreleri, HaCaT keratinositler, dişeti fibroblastlar ve THP-1 makrofajlar, üç boyutlu koşullar altında üç tür ortak yetiştirerek. Dişeti iltihabı ve periodontitis gibi periodontal hastalıkların araştırılması için bu modeli uygulayabilirsiniz.

Üç boyutlu inflamatuar insan dokusu jinjiva karşılıkları

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

three-dimensional-inflammatory-human-tissue-equivalents-of-gingiva

Research

JoVE Journal

Developmental Biology

9.7K Görüntüleme.  The Nippon Dental University. Protokol amacı bir enflamatuar insan jinjiva modeli içinde vitrooluşturmaktır. Bu doku model insan hücreleri, HaCaT keratinositler, dişeti fibroblastlar ve THP-1 makrofajlar, üç boyutlu koşullar altında üç tür ortak yetiştirerek. Dişeti iltihabı ve periodontitis gibi periodontal hastalıkların araştırılması için bu modeli uygulayabilirsiniz.

Video: Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Üç Boyutlu Kültürleri, mıkroRNA Analizi ve Putatif Hedef Genler üzerinde bir İnsan Sinir Kök Hücre Hattı Farklılaşma

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Gelişim Biyolojisi

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

İnsan uyarılmış pluripotent kök hücrelerin türetilmiş Piramidal Nöronlar gelen Dendritik Spine Üç boyutlu Niceleme

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

İnsan Kardiyak Doku perivasküler Multipotent Öncü hücre popülasyonlarının izolasyonu

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

İki ve Üç boyutlu Embriyoid Oluşumu Fare Embriyonik Kök Hücreler Retinoik Asit kaynaklı sinir Farklılaşma analizi

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

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 ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

Yatay Dağ Dağı: Derinin kalın, üç boyutlu doku kesitleri için yeni bir İşleme ve Görüntüleme Protokolü

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

Üç boyutlu yüksek frekanslı Ultrasonografi romanı kullanımı için fare erken gebelik karakterizasyonu

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Üç boyutlu görüntü oluşturma ve çözümleme-in Immunolabeled, açıklık insan plasental Villous damar ağları

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

Vestibüler ve işitme duyu organları kültürleri üç boyutlu Organotypic

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

İskele-free, üç boyutlu insülin ifade Pancreatoids fare pankreas ataları Vitro gelen nesil

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...