Generation and application of a new murine intestinal in vitro test system

Der Dünndarm ist an der Aufnahme von Nahrungsbestandteilen beteiligt und liefert den ersten Kontakt zu Pathogenen. Er stellt den größten Teil des Immunsystems dar und aus diesem Grund ist es besonders wichtig, dass die unterschiedlichen Zelltypen ihren verschiedenen spezialisierten Aufgaben nachkommen. Der Darm besteht aus Krypten, wobei die intestinalen Stammzellen in der Nische sitzen und in der Lage sind in alle Zelltypen des Dünndarms - Panethzellen, enteroendokrine Zellen, Gobletzellen und Enterozyten - zu differenzieren. In dieser Arbeit wurden ausgehend von murinen intestinalen Krypten, die in vitro als Organoidkulturen expandiert wurden, neuartige Zelllinien etabliert. Für diesen Prozess wurde zunächst eine Transduktionsstrategie für die Organoidkulturen optimiert und insgesamt 42 neue Zelllinien immortalisiert. Nachfolgend wurden die immortalisierten Zellen auf die Expression von verschiedenen Zelltyp-spezifischen Eigenschaften untersucht, um die vielversprechendsten Zelllinien zu identifizieren. Aus den initialen Charakterisierungen wurden ca. 10 Linien ausgewählt, die eingehender charakterisiert wurden. Dabei zeigte sich, dass die Zelllinien enge Zell-Zell-Kontakte aufwiesen, die durch Zonula Occludens-1 (ZO-1) und Ecadherin Expression sowie mittels einer Barrieremessung nachgewiesen wurden. Weiterhin konnte mithilfe der Elektronenmikroskopie gezeigt werden, dass die Zellen teilweise Mikrovilli ausbildeten. Außerdem zeichneten sich die Zelllinien durch eine für Gobletzellen-charakteristische Schleimproduktion aus, die sowohl auf RNA und Proteinebene als auch funktional nachgewiesen wurde. Zudem zeigten die Zelllinien Markerexpression von enteroendokrinen Zellen (Serotonin-Sekretion) und von Panethzellen (Defensin, Lysozym, CD24-Expression). Abschließend wurden die Zellen erneut in komplexe 3D-Umgebung überführt und bildeten Organoid-Strukturen aus. Die Daten deuten darauf hin, dass die neuartigen Zelllinien nicht terminal differenziert sind, sondern Vorläuferzellen. Um die generelle Anwendbarkeit dieser Zellen zu verbessern, wurde ein Medium neu entwickelt, welches die zellulären Eigenschaften erhält. Darüber hinaus konnte in einer ersten Studie mit einem enteropathogenen E. coli (EPEC) gezeigt werden, dass durch das TypIII-Sekretionssystem die zelluläre Barriere aufgelöst wird und der Erreger den Wirt infizieren kann.The small intestine is responsible for nutrient uptake und provides the first contact to invading pathogens. It represents the largest part of the immune system and that´s why it´s very important to have different cell types with specialized functions. The intestine consists of crypts which are localized at the bottom of the stem cell niche where they can differentiate in all other cell types – Paneth cells, enteroendocrine cells, Goblet cells and enterocytes. In this work new cell lines were established from murine intestinal crypts, which were expanded in vitro at the beginning. For this purpose the transduction strategy has to be optimized and adapted to the organoid culture. All in all 42 new cell lines were immortalized. Afterwards the immortalized cells were analyzed for their cell type specific functions and for the identification of the most promising cell lines. From the initial characterizations 10 cell lines were selected for the more detailed investigation. Some of the cells show strong cell-cell-contacts, verified by the expression of ZO-1 and Ecadherin as well as the measurement of the transepithelial electrical resistance (TEER). Moreover it was observed via electron microscopy that some cell lines show microvilli on top of their cell surfaces. Furthermore they show mucus production typical for goblet cells in their RNA as well as protein expression and in functional analysis. The immortalized cells express serotonin in a site-directed manner specific for enteroendokrine cells and also CD24, Defensin and Lysoyzym characteristic for Paneth cells. Finally the cells were seeded again in their complex 3D environment to check for stem cell capacity and it visible that some cells were able to form organoid structures. The data suggest that these novel cell lines are not terminal differentiated but precursor cells. To improve the general applicability a new defined medium was established by at the same time maintaining cellular properties. In addition an infection study with enteropathogenic Escherichia coli (EPEC) was performed to show the disruption of the epithelial barrier and infection of host cells because of the type III secretion system

Enhancing pre-clinical research with simplified intestinal cell line models

Two-dimensional culture remains widely employed to determine the bioavailability of orally delivered drugs. To gain more knowledge about drug uptake mechanisms and risk assessment for the patient after oral drug admission, intestinal in vitro models demonstrating a closer similarity to the in vivo situation are needed. In particular, Caco-2 cell-based Transwell® models show advantages as they are reproducible, cost-efficient, and standardized. However, cellular complexity is impaired and cell function is strongly modified as important transporters in the apical membrane are missing. To overcome these limitations, primary organoid-based human small intestinal tissue models were developed recently but the application of these cultures in pre-clinical research still represents an enormous challenge, as culture setup is complex as well as time- and cost-intensive. To overcome these hurdles, we demonstrate the establishment of primary organoid-derived intestinal cell lines by immortalization. Besides exhibiting cellular diversity of the organoid, these immortalized cell lines enable a standardized and more cost-efficient culture. Further, our cell line-based Transwell®-like models display an organ-specific epithelial barrier integrity, ultrastructural features and representative transport functions. Altogether, our novel model systems are cost-efficient with close similarity to the in vivo situation, therefore favoring their use in bioavailability studies in the context of pre-clinical screenings

The C0-C1f Region of Cardiac Myosin Binding Protein-C Induces Pro-Inflammatory Responses in Fibroblasts via TLR4 Signaling

Myocardial injury is associated with inflammation and fibrosis. Cardiac myosin-binding protein-C (cMyBP-C) is cleaved by µ-calpain upon myocardial injury, releasing C0-C1f, an N-terminal peptide of cMyBP-C. Previously, we reported that the presence of C0-C1f is pathogenic within cardiac tissue and is able to activate macrophages. Fibroblasts also play a crucial role in cardiac remodeling arising from ischemic events, as they contribute to both inflammation and scar formation. To understand whether C0-C1f directly modulates fibroblast phenotype, we analyzed the impact of C0-C1f on a human fibroblast cell line in vitro by performing mRNA microarray screening, immunofluorescence staining, and quantitative real-time PCR. The underlying signaling pathways were investigated by KEGG analysis and determined more precisely by targeted inhibition of the potential signaling cascades in vitro. C0-C1f induced pro-inflammatory responses that might delay TGFβ-mediated myofibroblast conversion. TGFβ also counteracted C0-C1f-mediated fibroblast activation. Inhibition of TLR4 or NFκB as well as the delivery of miR-146 significantly reduced C0-C1f-mediated effects. In conclusion, C0-C1f induces inflammatory responses in human fibroblasts that are mediated via TRL4 signaling, which is decreased in the presence of TGFβ. Specific targeting of TLR4 signaling could be an innovative strategy to modulate C0-C1f-mediated inflammation

sj-docx-1-tej-10.1177_20417314241228949 – Supplemental material for Enhancing pre-clinical research with simplified intestinal cell line models

Supplemental material, sj-docx-1-tej-10.1177_20417314241228949 for Enhancing pre-clinical research with simplified intestinal cell line models by Christina Fey, Theresa Truschel, Kristina Nehlsen, Spyridon Damigos, Julia Horstmann, Theresia Stradal, Tobias May, Marco Metzger and Daniela Zdzieblo in Journal of Tissue Engineering</p