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 &amp;mu;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&#039; 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&#039; 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&amp;amp;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&#039;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

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

İnsan 3D Doku Mühendisliği<em&gt; In vitro</em&gt; Tümör Test Sistemi

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Biyomühendislik

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

Murin Dişeti Hücre İzolasyonu, İşleme ve Analiz

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

İmmünoloji ve Enfeksiyon

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Üç-boyutlu Tam Kalınlığı Deri Üretimi Eşdeğer ve Otomatik Yaralama

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

3D Doku mühendisliği İnsan Özofagus Mukozal Modeli Üretimi, Karakterizasyonu ve Potansiyel Kullanımları

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Ekstraselüler Matrix içinde Gömülü Mühendislik Üç boyutlu Epitel Dokular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

İzolasyon, Karakterizasyonu ve Dişeti Bağışıklık Hücre Ağı Fonksiyonel Sınav

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblast Türetilmiş İnsan Tarafından Tasarlanmış Bağ Dokusu Tarama Uygulamaları için

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

İnsan jinjiva greft neovaskülarizasyon izleme için yeni bir yaklaşım

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

Tıp

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

Konak-Patojen Etkileşimlerinin İn Vitro Araştırılması için Organotipik Doku Model Sistemleri

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...

Yeni Bir Ağız Tıkaç Kullanarak Pulpa İltihabı Fare Modeli Oluşturma 

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

Yazar Spotlight: Geliştirilmiş Fare Modelleri ile Dental Pulpa Araştırmalarını Geliştirme

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

Üç boyutlu inflamatuar insan dokusu jinjiva karşılıkları

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İnsan 3D Doku Mühendisliği