1. Einrichtung Dreidimensionale Co-Kulturen <ol><li> Am Tag vor Gründung der Co-Kulturen, entfernen Sie das Matrigel aus dem Gefrierschrank und tauen auf Eis in einem 4 ° C Kühlschrank über Nacht. </li><li> Am Tag des Experiments, herzustellen Weg für die Co-Kultur, die mit dem Weg für den Epithelzellen zu verwenden (HMEC Medium in diesem Beispiel entspricht: DME/F12 Medium mit 10% bovinem Kälberserum, 5 mg / ml Insulin , 1 pg / ml Hydrocortison, und 1 ng / ml EGF). Halten Sie das Medium auf Eis mindestens 1 Stunde vor dem Mischen mit Matrigel. </li><li> Um das aufgetaute Matrigel verdünnen, bestimmen die Gesamtzahl der gewünschten Menge an Matrigel: mittel (300 ul pro Well für 24-Well-Platten) und fügen Sie die Hälfte dieses Volumens an eiskaltem HMEC Medium in ein steriles Röhrchen auf Eis. Dann fügen Sie das gleiche halbe Gesamtvolumen von aufgetaute Matrigel an das Rohr, um eine 1:1-Verdünnung zu erreichen. Halten Sie das Röhrchen auf Eis. </li><li> Platz 50 &amp;mgr; l Matrigel: Medium-Gemisch Mitte auch nur in dafür vorgesehenen Vertiefungen einer 24-Well-Platte undInkubation für 10 min in einem 37 ° C Inkubator mit befeuchteten Luft und 5% CO 2. </li><li> Fügen Sie eine weitere 450 ul Matrigel: Medium-Gemisch in die Vertiefungen pipettieren und für weitere 60 Minuten. </li><li> Während der Inkubation Herstellung einer 1:1-Suspension von Fibroblasten (hier h-TERT-immortalisierten Reduktion Mammary Fibroblasten, die GFP) und Epithelzellen (hier h-TERT-immortalisierten humanen Mamma-Epithelzellen exprimiert, RFP) in einer Konzentration von 0,5 x 10 5 Zellen jeweils in 0,5 ml Medium HMEC. </li><li> Nach der 60-minütigen Inkubation Schritt leicht fallen die Zellsuspension auf der Oberseite des erstarrten Gel, und zurück in den Inkubator Kultur. </li><li> Bitte nicht stören, die Kulturen für 2 Tage. Danach kann jedes Medium geändert werden 2-3 Tage mit sehr sanft Ansaugen Medium von der Seite des gut und leicht ersetzt mit frischem Medium HMEC. </li><li> Überwachen Sphäroid Bildung täglich unter dem Mikroskop. Kulturen sind in der Regel nach 10-14 Tagen reifen. Bild alsgegebenenfalls unter Verwendung von Licht-oder Fluoreszenz-Mikroskopie. Spheroid Größe und Auskraglänge kann unter dem Lichtmikroskop gemessen werden. </li></ol> 2. Isolierung von Zellen aus Spheroid Co-Kulturen <ol><li> Sehr sanft, pipettieren die Kultur auf und ab 10x mit einer sterilen 2-ml Glaspipette. Alternativ schneiden Sie die Spitze eines 1000 Mikroliter Kunststoff-Pipettenspitze mit einer Schere durch Autoklavieren oder Eintauchen in 70% Ethanol sterilisiert, um die Kultur pipettieren. Übertragen der Kultur mit entweder dem Glas der Pipette oder Pipettenspitze geschnittener Kunststoff in ein steriles 15 ml Zentrifugenröhrchen überführt und für 5 min bei 2000 rpm bei Raumtemperatur. Die zellulären Sphäroide wird Sediment am Boden. </li><li> Benutzen Sie einen sterilen Pasteurpipette Spitze zur schonenden Entfernung der gestörten Matrigel von der Spitze des zentrifugierten Röhrchen. </li><li> 1 ml Medium zu den HMEC pelletiert Sphäroide, und Abpipettieren 2-3 Mal mit einer sterilen 2 ml Glaspipette und übertragen der Kultur auf eine Mulde einer 24-WellGewebekulturplatte. </li><li> Lassen Sie die Sphäroide in die Schüssel für mindestens 48 Stunden vor Wert auf Wechsel des Mediums. Im Laufe der Zeit werden die Zellen verlassen die kugelige Strukturen und wachsen als Monolayer. </li><li> Passage der Zellkulturen, wenn die Zellen 50% Konfluenz erreichen. Erste Erweiterung der Kultur auf eine 35 mm-Schale (ca. 2-4 Tage), und dann zu einem 100 mm-Schale (ca. 3-5 Tage). Unmittelbar vor jedem Durchgang Schritt, verwenden Sie einen sterilen Pasteurpipette manuell so viele Rest-&quot;Ghost Sphäroiden&quot; wie möglich zu entfernen. </li><li> Analysieren Sie die Zellen mittels Durchflusszytometrie für Reinheit Bevölkerung mit geeigneten Zelloberflächenmarker und weiteren separaten unterschiedlichen Zellpopulationen durch Fluoreszenz-Zell-Sortierung, wenn nötig. Die isolierten Zellen sind nun für weitere Analysen bereit, wie gewünscht. </li></ol> 3. Repräsentative Ergebnisse Im dreidimensionalen Sphäroid Co-Kulturen, induzieren Fibroblasten mit technisch TIAM1 Silencing erhöht Invasion in mATrix durch Brustepithelzellen (Abbildung 1, vergleichen D, E, F bis A, B, C). Fibroblasten mit entwickelt Überexpression von TIAM1 induzieren verringerte Matrix Invasion durch die Brustkrebs-Zelllinie SUM159 (Abbildung 1, zu vergleichen, J, K, L bis G, H, I). Sobald co-kultivierten Sphäroide eingerichtet worden sind, können Zellpopulationen aus Sphäroiden durch erneutes Plattieren in einer zweidimensionalen Rahmen isoliert werden, wie in 2 gezeigt. Je nach den relativen Wachstumsraten der Zellen getestet, kann reine Populationen durch serielle Passage entstehen (wie in Abbildung 3 dargestellt) oder können sortiert mit geeigneten Zell-Marker werden. Nach der Isolierung von 3D Co-Kultur behalten epithelialen Zellen die Eigenschaften von Co-Kultur mit Fibroblasten über längere Zeit übertragenen, so dass reichlich Gelegenheit zur Analyse. In diesem Beispiel induziert Exposition gegenüber TIAM1-defizienten Fibroblasten erhöhte Migration in co-kultivierten HMECs, die für Wochen es weiterhin eineach isoliert von 3D-Co-Kultur (Abbildung 4). <img alt="1" src="/files/ftp_upload/3760/3760fig1.jpg" /> Abbildung 1. Imaging etablierter Sphäroid Co-Kulturen unter Licht-und Fluoreszenzmikroskopie. Spheroid Kulturen werden mit Brustepithelzellen und Fibroblasten-Zelllinien etabliert und man ließ sie in der Kultur für 10-14 Tage wachsen. AF: = HMEC-epithelialen Zellen mCherry. GL: Epithelzellen = SUM159 mCherry-Zellen. AC, GI: = Fibroblasten Kontrolle RMF-GFP-Zellen. DF: Fibroblasten = RMF-GFP mit TIAM1 Silencing. JL: Fibroblasten = RMF-GFP mit TIAM1 Überexpression. Die Pfeile zeigen Epithelzapfen Invasion in Matrix. <img alt="2" src="/files/ftp_upload/3760/3760fig2.jpg" /> Abbildung 2. Serial-Bildgebung der Kulturen unter Licht-und Fluoreszenzmikroskopie nach Isolierung aus Matrigel Kultur. Tag 0 darstellt Bildgebung unmittelbar nach der Isolierung, den Tagen 1, 2 und5 zeigen Tage nach Isolation. Die Korona der Zellen aus dem Sphäroid mit der Zeit zunimmt. Schließlich ein &quot;Geist&quot; Sphäroid mit sehr wenigen Zellen bleibt (Pfeil). Nur sehr wenige Sphäroiden bleiben nach Entfernung Geist und erste Durchgang. <img alt="Abbildung 3" src="/files/ftp_upload/3760/3760fig3.jpg" /> 3. Durchflusszytometrie-Analyse von Zellen nach der Isolierung aus Matrigel Co-Kultur. Nach Sphäroids Co-Kulturen gereift sind, werden die Zellen aus dem 3D-Co-Kultur mit Pipettieren und serielle Passage isoliert. Beispiel Aliquote mittels Durchflusszytometrie analysiert (in diesem Beispiel mit grüner Fluoreszenz und rote Fluoreszenz zu identifizieren GFP-exprimierende Fibroblasten und mCherry-exprimierenden Epithelzellen jeweils), um den relativen Häufigkeiten der Epithel und Fibroblasten-Zellen zu bestimmen. <img alt="Abbildung 4" src="/files/ftp_upload/3760/3760fig4.jpg" /> Abbildung 4. Transwell Migration von HMECs besteht, nachdem isolatiauf Matrigel von Co-Kultur. Sobald reine Populationen durch Kultur oder Fluoreszenz Zellsortierung gewonnen worden sein, können Zellen analysiert, wie gewünscht. Hier Migration von HMECs cokultiviert mit beiden Steuerkabel (C) oder TIAM1 schlecht (SHT) in Fibroblasten perforierten Transwells wurde nach 2, 4 und 8 Wochen nach der Isolierung aus Co-Kultur bewertet.

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Keine Interessenskonflikte erklärt.

Die hier vorgestellte Methode stellt einen einfachen Ansatz zur Analyse der Auswirkungen von stromalen Fibroblasten auf Epithelzellen, einschließlich Tumorzellen. Es ermöglicht die direkte Zell-Zell-Wechselwirkung in einem dreidimensionalen Rahmen durch extrazelluläre Basalmembran-Matrix unterstützt und gleichzeitig einfachem Gewinnung der Zellen aus der Matrix für die weitere Analyse. Dies ist auf die Untersuchung von Tumor-assoziiertem Stroma angewendet werden, wie Fibroblasten-Linien manipulieren kann, um die Si...

<table border="1"><tbody><tr><td> Name des Reagenz </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> BD Matrigel </td><td> BD Biosciences </td><td> 356237 </td><td> Phenolrot-frei </td></tr><tr><td> Hyclone klassischen flüssigen Medium: DMEM F12 </td><td> ThermoScientific </td><td> SH30023.01 </td><td></td></tr><tr><td> Hyclone Rinderkalbserum </td><td> ThermoScientific </td><td> SH3007303 </td><td></td></tr><tr><td> Insulin </td><td> EMD Chemicals </td><td> 407709 </td><td> Man löst in 0.005N HCl; 0,22 um Filter </td></tr><tr><td> Hydrocortison </td><td> Sigma-Aldrich </td><td> H4001 </td><td> Man löst in 50% EtOH; 0,22 um Filter </td></tr><tr><td> EGF </td><td> Sigma-Aldrich </td><td> E9644 </td><td> Man löst in 10 mM Essigsäure, 0,22 um Filter </td></tr></tbody></table>

isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture

Während die enormen Anstrengungen haben sich identifizieren Signalwege und Moleküle in normalen und malignen Zelle Verhaltensweisen 1-2, einen Großteil dieser Arbeit geleistet worden ist mit klassischen zweidimensionalen Zellkultur-Modellen, die für eine einfache Handhabung ermöglichen Zelle beteiligt sind verschwunden. Es ist deutlich geworden, dass intrazelluläre Signalwege durch extrazelluläre Kräfte, einschließlich der Dimensionalität und Zelle Oberflächenspannung 4.3 sind betroffen. Mehrere Ansätze wurden ergriffen, um dreidimensionale Modelle zu entwickeln, dass mehr genau darstellen biologische Gewebe-Architektur 3. Während diese Modelle verfügen über Multi-Dimensionalität und architektonischen betont, ist der Studie die resultierenden Auswirkungen auf Zellen weniger leicht als in zweidimensionalen Zellkultur aufgrund der Einschränkungen der Modelle und der Schwierigkeiten bei der Gewinnung von Zellen für die anschließende Analyse. Die wichtige Rolle der Mikroumgebung um Tumoren in der Tumorgenese und Tumor-Verhalten is zunehmend erkannt 4. Tumor-Stroma besteht aus mehreren Zelltypen und extrazellulären Molekülen zusammengesetzt. Während der Tumor-Entwicklung gibt es bidirektionale Signale zwischen Tumorzellen und Stromazellen 5. Obwohl einige Faktoren, die an Tumor-Stroma-Co-Evolution identifiziert wurden, gibt es immer noch ein Bedarf an einfachen Techniken systematisch zu identifizieren und untersuchen die volle Palette von diesen Signalen 6 zu entwickeln. Fibroblasten sind die am häufigsten vorkommende Zelltyp im normalen oder Tumor-assoziierten Stroma-Gewebe, und tragen zur Abscheidung und Wartung der Basalmembran und parakrine Wachstumsfaktoren 7. Viele Gruppen haben dreidimensionale Kultur-Systemen verwendet werden, um die Rolle der Fibroblasten auf verschiedenen zellulären Funktionen, einschließlich Ansprechen des Tumors auf Therapien, Rekrutierung von Immunzellen, Signalmolekülen, Proliferation, Apoptose, Angiogenese und Invasion 8-15 studieren. Wir haben eine einfache Methode zum Arsch optimiertEsser die Auswirkungen von Mamma-Fibroblasten auf Milchdrüsenepithelzellen Verwendung eines kommerziell erhältlichen extrazellulären Matrix Modell dreidimensionalen Kulturen von gemischten Zellpopulationen (Co-Kulturen) 16-22 erstellen. Mit weiterem Co-Kultur wurden die Zellen bilden Sphäroide mit dem Fibroblasten-Clustering im Inneren und die Epithelzellen überwiegend auf der Außenseite der Sphäroide und Bilden mehrzelligen Vorsprünge in die Matrix. Die Manipulation der Fibroblasten, die zu veränderten Epithelzellen führt Invasivität kann leicht durch Änderungen in Zahlen und Länge der epithelialen Vorsprünge 23 quantifiziert werden. Darüber hinaus haben wir ein Verfahren zur Isolierung von Epithelzellen aus der dreidimensionalen Co-Kultur, die Analyse der Auswirkungen von Fibroblasten-Exposition auf epithelialen Verhalten ermöglicht entwickelt. Wir haben gefunden, dass die Wirkungen der Co-Kultur für Wochen noch nach Epithelzelle Isolation ermöglicht ausreichend Zeit, um mehrere Tests durchzuführen. Diese Verfahren ist an Zellenunterschiedlichen malignen Potential und benötigt keine spezielle Ausrüstung. Diese Technik ermöglicht eine schnelle Auswertung der In-vitro-Zell-Modelle unter mehreren Bedingungen, und die entsprechenden Ergebnisse können in in vivo Modellen tierischem Gewebe sowie menschlichen Gewebeproben verglichen werden.

Watch this Scientific Journal Video about Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture at JoVE.com

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

Eine einfache Methode ist für die Analyse von Auswirkungen von Fibroblasten aus dem Bindegewebe auf verbundene Epithelzellen beschrieben. Die Kombination dieser Verfahren und dreidimensionalen Gewebekultur Analyse von Zellen nach der Isolierung aus 3D zu erleichtern. Die Technik ist für Zellen unterschiedlicher maligne Potential, so dass systematische Untersuchung der Wirkungen von Tumor-assoziiertem Stroma auf Tumorzellen.

Isolierung von Brustepithelzellen aus dreidimensionalen Mixed-Zelle Spheroid Co-Kultur

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

isolation-mammary-epithelial-cells-from-three-dimensional-mixed-cell

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Biology

16.0K Aufrufe.  Tufts Medical Center. Eine einfache Methode ist für die Analyse von Auswirkungen von Fibroblasten aus dem Bindegewebe auf verbundene Epithelzellen beschrieben. Die Kombination dieser Verfahren und dreidimensionalen Gewebekultur Analyse von Zellen nach der Isolierung aus 3D zu erleichtern. Die Technik ist für Zellen unterschiedlicher maligne Potential, so dass systematische Untersuchung der Wirkungen von Tumor-assoziiertem Stroma auf Tumorzellen.

Serie: Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

Maus Brustepithelzellen Form Mammospheres Während lactogenic Differenzierung

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Forschung

Biologie

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Mikrofluidik-Co-Kultur von Epithelzellen und Bakterien für die Untersuchung Löslich Signal-vermittelte Wechselwirkungen

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

Brustepithelzellen Transplantation

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolierung und Kultivierung von Adult epithelialen Stammzellen aus menschlicher Haut

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Chromatin Isolierung von RNA-Reinigung (Chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induktion und Analyse von epithelialen zu mesenchymalen Transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolation und Kultur der Dental epithelialen Stammzellen aus dem erwachsenen Maus Schneidezahn

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Bewertung der angiogenetischen Eigenschaften von Stammzellen von Eierstockkrebs mit dem dreidimensionalen Kokultursystem NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

Färbung und hochauflösende Bildgebung von dreidimensionalen Organoid- und Sphäroidmodellen

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

Isolierung von Epithelzellen aus dem menschlichen Zahnfollikel

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...