Name: three-dimensional inflammatory human tissue equivalents of gingiva
Uploaded: 2018-04-03T14:00:00.000+00:00
Duration: 8 min 43 s
Description: Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

Diese Arbeit wurde zum Teil von der Japan Society für die Promotion of Science (JSPS) Beihilfe für wissenschaftliche Forschung (26861689 und 17 K 11813) unterstützt. Die Autoren möchte Herr Nathaniel Green für das Korrekturlesen zu danken.

Dieses Protokoll soll menschliche Gingiva-Äquivalente, gingival Wunde Modelle und Gingivitis-Modelle zu erstellen. Menschliche Haut epidermalen Keratinozyten (HaCaT) wurden freundlicherweise zur Verfügung gestellt von Professor Norbert E. Fusenig Deutsches Krebsforschungszentrum (Heidelberg, Deutschland)7. Menschliche gingivale Fibroblasten (HGFs) waren von gingival Gewebe gemäß den zuvor veröffentlichten Protokolle8isoliert. Einwilligung war vorher, und die Studie wurde nach den Richtlinien der Ausschuss Ethik, die Nippon Dental University School of Life Dentistry in Tokio zugelassen (Zulassungsnummer: NDU-T2012-35). Protokoll-Schritte 1 – 3 sollte in einer Zelle Kultur Haube durchgeführt werden.
1. Vorbereitung des 3D menschliches Gewebe Äquivalente der Gingiva (GTE) (Abbildung 1A)
<ol><li>MEM-Alpha und 10 % Wiederaufbau Puffer Mix Kollagen Typ-A-Gel mit 10 % konzentriert (2,2 g Nahco33 und 4,47 g HEPES in 100 mL 0,05 N NaOH) in einem 15 mL steriles Röhrchen durch pipettieren. Halten Sie die gemischten Kollagen-Lösung auf Eis bis verwendet.</li>
	<li>24-Well-Kultur-Einsätze (Pore Größe 3,0 µm) in einer 24-Well-Platte zu platzieren.</li>
	<li>
HGFs von der Kultur-Oberfläche mit 0,25 % Trypsin-EDTA-Lösung entfernen. Aussetzen der Zellen in 10 mL Kulturmedium A (MEM-Alpha + 20 % FBS + 1 % GlutaMAX). Zählen der Zellen durch einen Bürker-Türk-Zelle-Zähler.
	<ol>
<li>Extrahieren Sie die gewünschte Menge an Zellen (1-5 X105 Zellen/mL), dann 4 Minuten bei 190 X g zentrifugieren.</li>
		<li>Unterbrechen Sie die Zellen in der gemischten Kollagen-Lösung. Halten Sie die Mischung auf dem Eis erst im nächsten Schritt.</li>
	</ol>
</li>
	<li>Fügen Sie 0,5 mL der Zelle-Kollagen-Mischung (0,5 – 2,5 x 105 Zellen/gut) in der Kultur einfügen ohne Bläschen zu produzieren. Inkubieren Sie die Mischung für 10-30 min in eine feuchte Atmosphäre mit 5 % CO2 bei 37 ° C zu gerinnen die Mischung in ein Gel. Hinweis: Fügen Sie die Kollagen-Mischung in die Mitte der Kultur einfügen ungleiche Bildung von Kollagen Gel zu vermeiden.</li>
	<li>Fügen Sie 1 mL Kulturmedium in den Brunnen und 0,5 mL in den Einsatz. Inkubation der Kultur-Platte für 24 h oder über Nacht in eine feuchte Atmosphäre mit 5 % CO2 bei 37 ° C.</li>
	<li>Entfernen Sie HaCaT-Zellen von der Kultur-Oberfläche mit 0,25 % Trypsin-EDTA-Lösung. Die Zellen in 10 mL Medium B auszusetzen (DMEM + 10 % FBS + 1 % GlutaMAX) und die Zellen zu zählen. Extrakt die benötigte Menge von Zellen (1 – 5 x 105 Zellen/mL) mit einer Pipette, Zentrifuge für 4 min bei 190 X g. auszusetzen, die Zellen in der mittleren B.</li>
	<li>Entfernen Sie das Medium aus der Kultur-Einsätzen, die durch Absaugen die HGF-Kollagen-Mischung enthalten. 0,5 mL HaCaT Zellsuspension (0,5 – 2,5 x 105 Zellen/Brunnen) auf der Oberseite der HGF-Kollagen-Mischung zugeben. Inkubation der Kulturen für 24 Stunden oder über Nacht. Hinweis: Zu diesem Zeitpunkt sollte das Medium in der Kultur gut mittlere A, werden während des Mediums in der Kultur einfügen sollte mittlere B.</li>
	<li>Ersetzen Sie das Kulturmedium mit KSR Medium (Co Kulturmedium) (MEM-Alpha + 15 % Ko-Serum Ersatz + 1 % GlutaMAX) + 5 % FBS. Fügen Sie 1 mL Kulturmedium in die Kultur gut und 0,5 mL in der Kultur einfügen. Inkubation der Kulturen für 24 – 48 h.</li>
	<li>Ersetzen Sie das Kulturmedium mit KSR Medium + 1 % FBS. Fügen Sie 1 mL in die Kultur gut und 0,5 mL in der Kultur einfügen. Inkubation der Kulturen für 24 Stunden oder über Nacht.</li>
	<li>Ersetzen Sie das Kulturmedium in der Kultur gut mit 0,7 – 1 mL KSR Medium. Entfernen Sie das Medium vollständig aus dem Kultur einfügen (Kultur-Oberfläche sollte Luft ausgesetzt werden). 1 – 2 Wochen inkubieren Sie der Kulturen. Ändern Sie das Medium in der Kultur auch zwei Mal pro Woche. Hinweis: Lassen Sie nicht die mittlere Ebene steigen über der Kultur-Oberfläche (die oberste Schicht aus Keratinozyten). Immer halten Sie die Kultur Oberfläche trocken.</li>
</ol>2. Vorbereitung des Verwundeten GTE (Abbildung 1 b)
<ol><li>Bereiten Sie eine 1 – 3 mm Durchmesser Gewebe Puncher. Mit einem Autoklaven bei 121 ° C für 20 min zu sterilisieren.</li>
	<li>Der Gewebe-Puncher senkrecht in GTE über 300 µm Tiefe schieben, und ein Loch in das Gewebe um eine Wunde zu simulieren.</li>
	<li>Verwenden Sie in diesem Schritt die Verwundeten GTE für die Untersuchung von Wunde heilen und Geweberegeneration. Alternativ zu beheben GTE mit 10 % Formalin neutraler Pufferlösung histologische Analysen durchzuführen.</li>
</ol>3. Vorbereitung des entzündlichen GTE (iGTE) (Abbildung 1 b)
<ol><li>THP-1-Zellen nach dem vorherigen Bericht9zu kultivieren.</li>
	<li>Mix Kollagen Typ-A-Gel mit 10 % konzentriert RPMI 1640, 10 % Wiederaufbau Puffer (2,2 g Nahco33 und 4,47 g HEPES in 100 mL 0,05 N NaOH) und 10 ng/mL PMA. Halten Sie die gemischten Kollagen-Lösung auf dem Eis bis zur Verwendung.</li>
	<li>Anzahl 1 – 2 x 106 THP-1 Zellen und Zentrifuge für 4 min bei 190 X g. aussetzen der Zellen in 1 mL der Lösung gemischt Kollagen. Halten Sie die Mischung auf dem Eis erst im nächsten Schritt.</li>
	<li>Fügen Sie 10 – 30 µL THP-1-Kollagen Mischung in den verletzten Bereich der GTE. Ersetzen Sie das Kulturmedium 0,7 – 1 mL KSR 10 ng/mL von PMA-haltigem Medium.</li>
	<li>Inkubieren Sie die Mischung in eine feuchte Atmosphäre mit 5 % CO2 bei 37 ° C für 72 h.</li>
	<li>Verwenden Sie das Modell für die Untersuchung von Gingivitis oder Parodontitis (z. B. hinzufügen potenzielle entzündungshemmendes Medikament iGTE zu sehen, seine Auswirkungen auf die Cytokine Release10). Alternativ lassen Sie 72 h PMA-Behandlung Weg, fügen Sie 2 µg/mL LPs in die Kultur hinzu und inkubieren Sie das Gewebe in eine feuchte Atmosphäre mit 5 % CO2 bei 37 ° C für 48 h11,12.</li>
</ol>4. Fixierung und ganze montieren Immunostaining GTE und iGTE Kulturen
<ol><li>
Fixierung der Kulturen.
	<ol>
<li>Entfernen Sie das Kulturmedium aus 24-Well-Platte. Waschen Sie das Gewebe 2 Mal mit Phosphat gepufferte Kochsalzlösung (PBS).</li>
		<li>Die Kultur gut 1 mL 10 % Formalin neutraler Pufferlösung, die Einlage und 1 mL hinzufügen. Lassen Sie die 24-Well-Platte über Nacht im Kühlschrank bei 4 ° C.</li>
		<li>Entfernen Sie die 10 % Formalin neutraler Pufferlösung am folgenden Tag.</li>
		<li>Waschen Sie für ganze Berg Immunostaining Gewebe 3-4 Mal mit PBS nach der Fixierung.</li>
		<li>Gehen Sie für Hämatoxylin und Eosin (H & E) Färbung und regelmäßige Immunostaining mit Austrocknung, Paraffin einbetten und schneiden. Hinweis: Für ganze Berg Immunostaining kann dieser Schritt entfallen.</li>
	</ol>
</li>
	<li>
Ganzen Berg immunostaining Hinweis: Dieses Protokoll wurde von der Referenz 13 geändert.<ol>
<li>Waschen Sie das Gewebe 3 X in PBS 0,5 – 1 % Triton, 10-30 min jedes Mal.</li>
		<li>Inkubieren Sie das Gewebe zweimal für 30 min in blocking-Puffer (PBS 1 % Triton + 10 % BSA), bei Raumtemperatur.</li>
		<li>Waschen Sie das Gewebe 2 × in blockierende Puffer, 5 – 10 Minuten jedes Mal.</li>
		<li>Fügen Sie 50 µL Primärantikörper Lösung am erforderlichen Verdünnung/Konzentration auf die Einsätze. Verwenden Sie eine dünne Kunststoff Frischhaltefolie wickeln die Einsätze um Austrocknung des Gewebes zu vermeiden.</li>
		<li>Inkubieren Sie das Gewebe für 1 bis 2 Tage bei 4 ° c</li>
		<li>Waschen Sie das Gewebe 3 Mal für 30 min in PBS 1 % Triton + 10 % BSA. Waschen wieder 3 Mal für 5 min in PBS 1 % Triton.</li>
		<li>Fügen Sie 50 µL Sekundärantikörper in blocking-Puffer (PBS 1 % Triton + 10 % BSA) und 5 µL Hoechst 33342 Lösung (NucBlue Live Cell Fleck) für jeden Einsatz. Verwenden Sie eine dünne Kunststoff Frischhaltefolie wickeln die Einsätze um Austrocknung des Gewebes zu vermeiden.</li>
		<li>1 bis 2 Tage bei 4 ° c inkubieren Wickeln Sie die 24-Well-Platte in einem feuchten Papiertuch, und steckte sie dann in ein Kunststoff-Pack im gesamten Inkubation verwendet werden.</li>
		<li>Waschen Sie das Gewebe 3 Mal für 10 min in PBS 1 % triton</li>
		<li>Verwenden einer spitzen Nadel, schneiden Sie die Membran von der Kultur einfügen, um das Gewebe zu erhalten. Platzieren Sie die Membran auf einer Folie. Hinweis: Das Gewebe sollte auf der Membran.</li>
		<li>Fügen Sie 2 – 3 Tropfen Fluoreszenz Mount Medium. Decken Sie das Gewebe mit einem Deckglas und Speicher bei 4 ° C im Dunkeln bis zur Analyse.</li>
		<li>Zeigen Sie Gewebe mit einem konfokalen Laser-scanning-Mikroskopie (LSM an).</li>
	</ol>
</li>
</ol>

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Die Autoren erklären keinen Interessenskonflikt.

Dieses Protokoll basiert auf Methoden der Schaffung von Gingiva-äquivalente und Subkutane Fettgewebe Äquivalente von vorherigen Berichte8,21,22beschrieben. Obwohl dies eine einfache und einfache Methode, erfordern einige Schritte besondere Aufmerksamkeit. Beispielsweise sollte die Kollagen-Mischung auf dem Eis bis zur Verwendung, Gelbildung in der Lösung zu vermeiden gehalten werden. Wenn die Kollagen-Mischung in das Kultur e...

Parodontale Erkrankungen sind die häufigste Ursache für Zahnverlust bei Erwachsenen. Gingivitis und Parodontitis sind die häufigsten parodontalen Erkrankungen. Die beiden präsentieren Biofilm-vermittelten akute oder chronische entzündliche Veränderungen im Zahnfleisch. Gingivitis ist geprägt von akuten Entzündungen, während Parodontitis in der Regel als chronische Entzündung präsentiert. Bei der histologischen Ebene ausgelöst bakterielle Komponenten die Aktivierung von Immunzellen, wie Makrophagen, Lymphozyten, Plasmazellen und Mastzellen1,2. Diese Immunzellen, vor allem Makrophagen, interagieren mit lokalen Zellen (einschließlich Zahnfleisch Epithelzellen, Fibroblasten, Endothelzellen und Osteoblasten) wodurch entzündliche Läsionen im parodontalen Gewebe3,4. Experimentelle Modelle der Parodontalerkrankungen wurden in verschiedene Arten von Tieren, wie Ratten, Hamster, Kaninchen, Frettchen, Eckzähne und Primaten. Die Physiopathologie von Tiermodellen ist jedoch anders als die des Menschen, macht es schwierig, zelluläre und molekulare Mechanismen zu analysieren und bewerten neue Medikamente von Parodontalerkrankungen5. Co-Kultur von parodontale Bakterien und menschlichen mündliche Epithelzellen Monolage wurde verwendet, zu untersuchen, den Mechanismus der parodontalen Infektionen6. Dennoch fehlt Monolayer-Kulturen der mündlichen Zellen die dreidimensionale (3D) zelluläre Architektur von intaktem Gewebe; Daher können nicht sie die in-vitro- Situation imitieren.
Hier sind 3D entzündliche menschliches Gewebe Äquivalente der Gingiva (iGTE) gegründet, um parodontale Erkrankungen in Vitrodarstellen. Das 3D-Modell der Parodontalerkrankungen nimmt eine Zwischenstellung zwischen Monolage Zellkulturen und Tiermodellen. Drei Arten von menschlichen Zellen, einschließlich HaCaT Keratinozyten, gingival Fibroblasten und Makrophagen THP-1, sind Co auf Kollagen Gel angebaut und durch entzündliche Initiatoren iGTE bauen stimuliert. IGTE simuliert eng die in-Vivo -Bedingungen der Zelldifferenzierung, Zell-Zell-Interaktion und Makrophagen Aktivierung im Zahnfleisch. Dieses Modell hat viele Einsatzmöglichkeiten für Drug screening und testen neue pharmakologische Ansätze bei parodontalen Erkrankungen sowie zur Analyse von zellulärer und molekularer Mechanismen in der Wundheilung, Entzündung und Regeneration des Gewebes.

<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

Parodontale Erkrankungen (z.B. Gingivitis, Parodontitis) sind die führenden Ursachen für Zahnverlust bei Erwachsenen. Entzündung im Zahnfleisch ist die grundlegende physiopathologie der Parodontalerkrankungen. Aktuelle experimentelle Modelle der Parodontalerkrankungen entstanden in verschiedenen Arten von Tieren. Die Physiopathologie von Tiermodellen ist jedoch anders als die des Menschen, macht es schwierig, zelluläre und molekulare Mechanismen zu analysieren und bewerten neue Medikamente für parodontale Erkrankungen. Hier präsentieren wir Ihnen ein detailliertes Protokoll zur Rekonstruktion von entzündlichen Gewebe Entsprechungen des Zahnfleisches (iGTE) in Vitro. Wir erstellen zunächst menschliches Gewebe Äquivalente der Gingiva (GTE) durch die Verwendung von zwei Arten von menschlichen Zellen, einschließlich menschliche gingival Fibroblasten (HGF) und menschliche Haut epidermalen Keratinozyten (HaCaT), dreidimensionale Bedingungen. Wir erstellen eine Wunde Modell mithilfe eine Gewebe Puncher, ein Loch in der GTE Punsch. Nächste, menschliche THP-1 Monozyten mit Kollagen Gel gemischt werden in das Loch in der GTE injiziert. Durch Adimistration von 10 ng/mL Phorbol Myristate-12 13-Acetat (PMA) für 72 h, THP-1-Zellen unterschieden in Makrophagen in Form entzündlicher Herde in GTE (iGTE) (IGTE auch kann sein Stumilated mit 2 µg/mL von Lipopolysacchariden (LPS) für 48 h, Entzündung zu initiieren ). IGTE ist das erste in-vitro- Modell der entzündlichen Gingiva mit menschlichen Zellen mit einer dreidimensionalen Architektur. IGTE spiegelt die wichtigsten pathologische Veränderungen (Immunzellen Activition, intrazellulären Interaktionen zwischen Fibryoblasts, Epithelzellen, Monozyten und Makrophagen) in parodontale Erkrankungen. GTE, Verwundeten GTE und iGTE dient als vielseitige Werkzeuge, um die Wundheilung zu studieren, Geweberegeneration, Entzündungen, Zell-Zell-Interaktion und Bildschirm mögliche Medikamente für parodontale Erkrankungen.

HaCaT-Zellen angezeigt typische Keratinozyten Morphologie unter Phasenkontrast-mikroskopische Beobachtung (Abbildung 2A). Scanner Elektron mikroskopische (SEM) Bilder zeigten, dass viele Mikrovilli HaCaT Zelloberflächen abgedeckt wurden. Interzelluläre Verbindungen zwischen HaCaT-Zellen wurden durch Membranverfahren (Abb. 2 b) vermittelt. HaCaT-Zellen ausgedrückt gingivalen Epithel Marker K8/1814, darau...

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

Das Ziel des Protokolls ist eine entzündliche menschlichen Gingiva Modell in Vitrozu bauen. Dieses Gewebe Modell pflegt Co drei Arten von menschlichen Zellen, HaCaT Keratinozyten, gingival Fibroblasten und THP-1 Makrophagen, dreidimensionalen Bedingungen. Dieses Modell kann angewendet werden, für die Untersuchung von parodontaler Erkrankungen wie Gingivitis und Parodontitis.

Dreidimensionale entzündliche menschliches Gewebe Äquivalente der Gingiva

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 Aufrufe.  The Nippon Dental University. Das Ziel des Protokolls ist eine entzündliche menschlichen Gingiva Modell in Vitrozu bauen. Dieses Gewebe Modell pflegt Co drei Arten von menschlichen Zellen, HaCaT Keratinozyten, gingival Fibroblasten und THP-1 Makrophagen, dreidimensionalen Bedingungen. Dieses Modell kann angewendet werden, für die Untersuchung von parodontaler Erkrankungen wie Gingivitis und Parodontitis.

Serie: 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.

Tissue Engineering eines Menschen 3D<em&gt; In-vitro-</em&gt; Tumor-Testsystem

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

Forschung

Biotechnik

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.

Isolation, Verarbeitung und Analyse von Murine Gingivazellen

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

Immunologie und Infektion

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.

Erzeugung eines dreidimensionalen Vollhaut Equivalent und automatisierte Verwundung

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.

Herstellung, Charakterisierung und Einsatzmöglichkeiten von 3D-Tissue-Engineering Menschen Ösophagus-Schleimhaut-Modell

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.

Technik Dreidimensionale Epithelgewebe Eingebettet in extrazellulären Matrix

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.

Isolierung, Charakterisierung und funktionelle Untersuchung des Zahnfleisch 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 ...

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.

Fibroblasten-abgeleitetes Human Engineered Connective Tissue für Screening-Anwendungen

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.

Ein neuer Ansatz zur Überwachung Graft Neovaskularisation in der menschlichen 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 ...

Medizin

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.

Organotypische Gewebemodellsysteme zur Untersuchung von Wirt-Erreger-Interaktionen in vitro

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

Erstellung eines Mausmodells für eine Pulpaentzündung unter Verwendung eines neuartigen Mundknebels 

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.

Autor im Rampenlicht: Verbesserung der Zahnpulpaforschung mit verbesserten Mausmodellen

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

Dreidimensionale entzündliche menschliches Gewebe Äquivalente der Gingiva

Zusammenfassung

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Tissue Engineering eines Menschen 3D