This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) (26461185 and 25460137).

HINWEIS: Diese Studie wurde von den zuständigen Ethikkommissionen genehmigt. 1. Primär Kultur der menschliche Lungen-Fibroblasten <ol><li> Sammlung von Lungengewebe: <ol><li> Erhalten menschlichen Lungengewebeproben aus dem Operationsraum unmittelbar. Sammeln Sie ca. 1 cm 3 Blocks von krebsartigen und nicht krebsartigen Lungengewebe, die nicht krebsartigen Probe so weit weg von den Tumor wie möglich gesammelt. </li><li> Aussetzung der Proben in Dulbeccos modifiziertem Eagle-Medium (DMEM) mit 100 Einheiten / ml Penicillin, 100 ug / ml Streptomycin und 0,25 ug / ml Amphotericin B (serumfreies Medium) ergänzt. Pflegen Sie die suspendierten Proben in sterilen 50-ml-Röhrchen und übertragen an das Labor. HINWEIS: Die Fibroblasten sind hoch Migration und Proliferation im Vergleich zu anderen Zelltypen, wie Epithelzellen, Endothelzellen und glatten Muskelzellen. Der Auswuchs Verfahren nutzt diese Features von Fibroblasten und outgroFlügelzellen aus Gewebeschnitten sind reichlich in Fibroblasten. Nach mehreren Zellpassagen anderen Zelltypen nicht zu überleben und vermehren sich in DMEM mit 10% fötalem Rinderserum (FBS), wodurch gereinigte Fibroblasten-Zellkulturen. Die Zellen könnten 3-7 Passagen folgenden Primärkultur verwendet werden. </li></ol></li><li> Explantatkultur und Anlage für Zell Auswuchs: <ol><li> Führen Primärkultur von Fibroblasten mit einem zuvor berichtet Protokoll mit Modifikationen 5. </li><li> Im Labor, legen Sie die Gewebeprobe auf einer 10 cm Gewebekulturschale ohne Kulturmedium und in kleine Stücke geschnitten ~ 2-3 mm Größe mit einer sterilen Pinzette und Skalpell. Dabei verhindern, dass die trockenen Abschnitt, da sonst die Wirksamkeit der Zellauswuchs beeinträchtigt wird. </li></ol></li><li> Trennung der epithelialen Zellschicht und Bindegewebe: <ol><li> Tränken der geschnittenen Gewebeabschnitte in Kulturmedium, das 2000 PU / ml Dispase I und die Kultur für 16 Stunden bei 4 ° C. &lt;/ Li&gt; <li> Übertragen Sie die Gewebeschnitte in eine Schale, und trennen Sie die Epithelzellschicht vom benachbarten Bindegewebe. Verarbeiten Sie die Abschnitte in einer Sterilbank, um eine Kontamination von Mikroorganismen zu vermeiden. HINWEIS: Wenn der Primärkultur von Epithelzellen ist erforderlich, führen Sie Schritt 1.3. Wenn nur Fibroblasten isoliert werden sollen, lassen Sie Schritt 1.3, da die nachfolgenden Explantatkultur und Zellpassagen ungünstig für Epithelzellen, die gereinigte Fibroblasten-Populationen zu erhalten sind. </li></ol></li><li> Befestigung von Gewebeschnitten: <ol><li> Hackfleisch das Gewebe in 1 mm Stücke mit zwei Skalpelle. Wenn die Stücke nicht klein genug sind, leicht löst sie aus der Schale, und die Zellen werden nicht auf der Geschirroberfläche hinauswachsen. </li><li> Legen Sie kleine Lungengewebe Abschnitte voneinander auf einen leeren Bereich jedes Stück Umgebung aufrecht zu erhalten. Make Kratzer auf der Oberfläche der Gewebekulturschale mit einem Skalpell, um die Befestigung von Gewebeschnitten zu erleichtern. HINWEIS: Scratchingscheint auch Zellauswuchs erleichtern, wodurch Spuren entlang dem Zellen migrieren können. <ol><li> Alternativ stellen Hackfleisch Gewebestücke einzeln in jede Vertiefung einer Platte mit 6 Vertiefungen, und sie mit einem Deckglas. Befestigen Sie das Deckglas auf die Oberfläche der Platte mit Silikonfett. </li><li> Zeigen Silikonfett an zwei Punkten 1,5 cm Abstand in jedem Well mit einem sterilen Stift. Coverage der Gewebestücke erhöht die Wirksamkeit der Zell Auswuchs und Zellvermehrung nach mehreren Durchgängen. </li></ol></li></ol></li><li> Nachfolgende Explantatkultur: <ol><li> Kulturmedium hinzufügen, vorsichtig in die Schale, bevor die Gewebeschnitte austrocknen, so effizient Zellauswuchs wird zum größten Teil verloren, wenn die Proben trocknen. Stellen Sie das Medium strömen nicht zu schnell, wie die Gewebeschnitte würde abnehmen. </li><li> Kulturzellen in DMEM mit 10% FBS. Verwenden Sie ausreichend Medium nur das Gewebe zu bedecken, aber nicht zu viel, dass es ermöglicht, schwebend, als zusätzliche Auftriebs fördert Loslösung von derGericht. </li></ol></li><li> Zellpassage: <ol><li> Gehen Sie mit dem Kulturmedium mit Sorgfalt zu allen Zeiten als wiederholte Bewegungen der Kulturschale kann zur Ablösung der Gewebeschnitte führen. </li><li> Legen Sie die Kulturschalen in einem Brutschrank bei 37 ° C für 5-7 Tage. Aktualisieren Sie das Medium jeden zweiten Tag, und übernehmen die Kulturschalen minimal während des Mediums ändert. Fibroblasten entwachsen von der Kante der Gewebeschnitte in den nächsten Wochen. HINWEIS: Nach ~ 2-3 Wochen entwachsen Fibroblasten fast bis Konfluenz in Abhängigkeit von der Höhe der Oberfläche. </li><li> Waschen Sie die Zellen mit 1 × PBS und 1 ml Trypsin ich auf den Teller. </li><li> Nach Trypsinierung lösen sich die Zellen unter Verwendung von 9 ml DMEM, ergänzt mit 10% FBS. Sammeln Zellen in dem Medium in einer 10-ml-Röhrchen suspendiert, zusammen mit den Gewebeschnitten. Nach der Zentrifugation, um Zellpellets zu bilden, Resuspendieren der resultierenden Pellets in dem neuen Medium. </li><li> Seed die Zellen und Gewebeschnitte auf eine neue cultur Schüssel, an welchem ​​Punkt das Gewebe nicht an den Teller zu befestigen, während die Zellen anhaften und wachsen. Am folgenden Tag, nehmen Sie die schwimmenden Gewebestücke durch Aspiration, und ändern Sie das Medium. Angehängte Fibroblasten breiten sich mit nachfolgenden Kulturen. </li></ol></li></ol> 2. Dreidimensionale Co-Kultur der menschliche Lungen-Fibroblasten und Krebszellen <ol><li> Herstellung von Zellen und Reagenzien: <ol><li> Etwa verwenden 2 × 10 5 Epithelzellen und 2,5 x 10 5 Fibroblasten pro Vertiefung. Bereiten Kollagen Typ IA (3 mg / ml, pH 3), FBS (100%), Rekonstitutionspuffer (50 mM NaOH, 260 mM NaHCO 3, 200 mM HEPES), 5x DMEM, DMEM mit 10% FBS für Fibroblastenkultur ergänzt, und ein 3D-Co-Kulturmedium (eine 1: 1-Mischung von Fibroblastenkulturmedium und das Kulturmedium für die Krebszellen). </li><li> Kultur A549 Lungenadenokarzinomzellen, die Fibroblasten und die A549 3D Kokultur in DMEM, ergänzt mit 10% FBS. </li></ol></li><li> Kollagen-Gel formationen: <ol><li> Fibroblasten auf 10 cm Schüssel mit 1x PBS kultiviert waschen und mit 1 ml Trypsin ich auf den Teller. Nach Trypsinisierung für ~ 5 min bei 37 ° C lösen sich die Zellen unter Verwendung von 9 ml DMEM mit 10% FBS ergänzt. Sammeln Zellen in dem Medium in einer 10-ml-Röhrchen suspendiert und zentrifugieren bei 200-300 xg Zellpellets zu bilden. </li><li> Resuspendieren der resultierenden Pellets in 100% FBS bei einer Dichte von 5 × 10 5 / ml. </li><li> Bereiten Sie die Kollagen-Gel auf Eis. Kühlung der Reagenzien, Pipetten und Röhren in einem Kühlschrank vor dem folgenden Verfahren. Auch auf Eis erstarrt die Mischung zu einem gewissen Grad, und das Gesamtvolumen weniger als die Summe aller Bestandteile ist. </li><li> In jede Vertiefung, bereiten Sie die Kollagen-Gel durch Mischen von 0,5 ml Fibroblastensuspension (2,5 x 10 5 Zellen) in FBS, 2,3 ml Typ IA Kollagen, 670 ul 5x DMEM, und 330 &amp; mgr; Rekonstitutionspuffer. Lassen Sie die Gele leicht bei Raumtemperatur eingestellt. <ol><li> Kräftig pipettieren, um eine homogene mi gewährleistenBefestigung. Vermeiden Sie Luftblasen beim Pipettieren. </li></ol></li><li> Hinzufügen der Mischung (3 ml) zu jedem Well einer 6-Well-Platte und zu ermöglichen, in den Inkubator bei 37 ° C für 30-60 min gelatinieren ohne Störung. </li></ol></li><li> Krebszellkultur auf das Kollagengel: <ol><li> Führen dreidimensionalen Co-Kultur mit einem zuvor berichtet Protokoll mit Modifikationen 6. </li><li> Resuspendieren Krebszellen in der 3D-Co-Kulturmedium (oder in DMEM mit 10% FBS bei der A549-Zellen) in einer Dichte von 1 x 10 5 / ml. </li><li> Pour 2 ml des resuspendierten Zelllösung (2 x 10 5 Zellen) auf die Oberfläche eines jeden Gels. Ändern der Zellzahl der Krebszellen oder Fibroblasten in Kokultur je nach Versuchsbedingungen. </li></ol></li><li> Gelkontraktion: <ol><li> Gele über Nacht bei 37 ° C, so dass die Krebszellen zu dem Kollagengel haften. Trennen Sie jedes Gel, und erzeugen einen &quot;schwebenden Kultur&quot; in jede Vertiefung of 6-Well-Platte. HINWEIS: Durch die Verwendung eines schwimmenden Kultur, sind verschiedene Änderungen in Gelgröße durch mechanische Spannung und Kollagenabbau, die von zellulären Wechselwirkungen zwischen co-kultivierten Krebszellen und Fibroblasten vermittelt werden induziert. </li><li> Verwenden eine abgewinkelte 21 G Nadel oder Spachtel, jedes Gel, das aus dem Rand des Bohrlochs abzutrennen. Alle 2-3 Tage, aktualisieren Sie die Vertiefungen mit 2 ml 3D-Co-Kulturmedium. Messen der Größe jedes Gel täglich für 5 Tage. </li></ol></li><li> Analyse Kollagengelen: <ol><li> Messung der Kollagengehalt von jeweils unter Verwendung von Kollagen-Quantifizierungsverfahren, wie dem Sircol Assay. Führen Transkriptom-Analyse oder quantitativer RT-PCR nach dem Sammeln von RNA aus den Gelen 5. </li></ol></li></ol> 3. Luft-Flüssigkeit-Grenzfläche, Kultur und Invasion Assay <ol><li> Nach Kultivieren der Kollagengele bei 37 ° C für 5 Tage, setzen die kontrahierten Gele Luft, indem sie auf ein Sieb (70 &amp; mgr; m Porengrße) in neue 6-Well-Platten. Stellen Sie das Netz aus kommerziell erhältlichen Zellsiebe für die Durchflusszytometrie verwendet. </li><li> Vorsichtig füllen jede Vertiefung mit 11 ml 3D Co-Kulturmedium. </li><li> Positionieren Sie die Gele auf das Netz mit einem sterilen Löffel, und entfernen Sie alle verbleibenden Medium aus den Gelen. Unter dieser Bedingung, tauchen Gele in dem Medium während setzen die obere Oberfläche des Gels in die Luft. Definieren Sie diesen Kulturbedingungen wie Luft-Flüssigkeit-Grenzfläche. HINWEIS: Nach 5-7 Tagen der Beibehaltung des Luft-Flüssigkeit-Grenzfläche bei 37 ° C im Brutschrank migrieren invasive Krebszellen in das Gel. Je nach Zelltyp und als Ergebnis zellulärer Wechselwirkungen sind variable Grad der Zell morphologische Veränderung in den Krebszellen durch die Luft-Flüssigkeits-Schnittstelle Kultur induziert. </li><li> Für die histologische Beurteilung, beheben Sie das Gel in Formalin-Lösung über Nacht bei Raumtemperatur und in Paraffin eingebettet werden. Fleck Vertikalschnitte (4 &amp; mgr; m) mit Hämatoxylin und Eosin (H &amp; E). Führen immunhistochemische Analyse on die Abschnitte 4. </li></ol>

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The authors declare that they have no competing financial interests.

CAF bilden eine wichtige Komponente der ECM Krebszellen umgibt, und nicht nur ein Gerüst für den Tumor, sondern auch aktiv in Tumorentwicklung 7 beteiligen. Immer mehr deutet entwirrt die Auswirkungen der CAF oder die entsprechenden Moleküle auf der eventuelle Prognose, Hervorhebung der kritischen Rolle der CAF-vermittelte Tumorprogression 8. In der vorangegangenen Studie beschäftigten wir Auswuchs Methode zur Lungen CAF 4 zu isolieren. In diesem Experime...

Cancer tissue can be perceived as a type of organ, which evolves through close interactions between the cancer and the tumor stromal microenvironment, composed of cancer-associated fibroblasts (CAFs), immune cells, tumor vessels and the extracellular matrix (ECM). CAFs are the major source of soluble factors (cytokines, growth factors and chemokines) that exert mitogenic, pro-migratory and pro-invasive effects on cancer cells. They also stimulate tumor vessel formation and recruit precursor cells, such as bone marrow-derived cells (BMDC). Activated CAFs are involved in the production and remodeling of the ECM, thereby promoting the growth and spread of cancer cells1. CAFs also provide a niche that facilitates tumor cell colonization and metastasis and are capable of conferring stem cell phenotypes onto neighboring cancer cells. Pathological observations suggest that stromal reactions or fibrotic changes in cancer tissues are indicative of a poor prognosis. Recent studies have also demonstrated that tumor stromal features, such as the gene signature, can predict patient prognosis. Furthermore, CAF-derived factors can modulate sensitivity to chemotherapy, highlighting the role of CAFs in determining drug sensitivity and resistance2.
As CAFs play a multifaceted role in the promotion of tumor progression through signaling pathways that mediate interactions between CAFs and different cell types within the tumor microenvironment, they have attracted increasing attention as novel targets for cancer therapies. The heterogeneity of the cell populations within the cancer microenvironment presents an obstacle for targeting CAFs. Several markers for CAFs have been proposed, such as &#945;-smooth muscle actin (&#945;-SMA), fibroblast activation protein (FAP), and fibroblast specific proten-1 (FSP-1: also called S100A4); however, these molecular markers are not specific for distinguishing CAFs from other cells present in non-cancerous tissues3. Therefore, further studies are needed to obtain more knowledge about the specific properties of CAFs. To this end, it is informative to characterize primary cultured CAFs compared with patient-matched normal fibroblasts.
Recently, analyses on patient-derived CAFs have been reported in several cancer types, revealing unique gene expression patterns and cell behaviors compared with fibroblasts derived from non-cancerous tissues. Using isolated CAFs from human lung cancer tissues, we developed a three-dimensional co-culture method, enabling us to evaluate the properties of lung CAFs. In this model, we investigated the effects of the CAFs on lung cancer cell invasion, proliferation and collagen gel contraction, which experimentally recapitulated the tumor-promoting roles of lung CAFs4.

<table border="0" cellpadding="0" cellspacing="0" width="1035">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:267px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:471px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">DMEM</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td style="width:471px;">D5796</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FBS</td>
			<td style="width:297px;">GIBCO</td>
			<td style="width:471px;">10437</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Collagen type IA</td>
			<td style="width:297px;">Nitta gelatin Inc.</td>
			<td style="width:471px;">CELL-1A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Reconstitution buffer</td>
			<td style="width:297px;">Nitta gelatin Inc.</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cover slip</td>
			<td style="width:297px;">NUNC</td>
			<td style="width:471px;">174934</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Silicone grease</td>
			<td style="width:297px;">Dow Corning Toray</td>
			<td style="width:471px;">High vacuum grease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dispase I</td>
			<td style="width:297px;">WAKO</td>
			<td style="width:471px;">386-02271</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">6-well plate</td>
			<td style="width:297px;">BD Falcon</td>
			<td style="width:471px;">353046</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cell strainer (70 &#956;m)</td>
			<td style="width:297px;">BD Falcon</td>
			<td style="width:471px;">352350</td>
		</tr>
	</tbody>
</table>

three-dimensional co-culture model for tumor-stromal interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Diese Co-Kulturverfahren, imitiert der Tumor-Mikroumgebung ist ein nützliches Werkzeug, um die Wechselwirkungen zwischen den Krebszellen und Fibroblasten in Kollagengele eingebettet untersuchen. Kollagengel Kontraktion, Invasion von Krebszellen und morphologische Veränderungen: In der früheren Studie wurden drei Parameter in diesem experimentellen Modell bewertet. Krebszellproliferation wurde ebenfalls mit Ki67 Immunfärbung 4 geschätzt. Lungengewebeproben wurden von krebsartigen und nicht-krebsartigen Ab...

Watch this Scientific Journal Video about Three-dimensional Co-culture Model for Tumor-stromal Interaction at JoVE.com

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Dreidimensionale Co-Kultur-Modell für Tumor-Stroma-Interaktion

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

three-dimensional-co-culture-model-for-tumor-stromal-interaction

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17.2K Aufrufe.  The University of Tokyo. Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Serie: Three-dimensional Co-culture Model for Tumor-stromal Interaction

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Experimentelle Erzeugung von Karzinom-assoziierte Fibroblasten (CAF) von menschlichen Brustkrebszellen Fibroblasten

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Forschung

Medizin

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Menschliche neuroendokrinen Tumor-Zelllinien als dreidimensionales Modell für das Studium der menschlichen neuroendokrinen Tumor-Therapie

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Eine dreidimensionale Zellkulturmodell zu primären humanen Knochenmark und seine Malignome Studieren

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantifizierung der Brustkrebs-Zelle Invasivität Mit einem Drei-dimensionale (3D) Modell

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Dreidimensionale (3D) Tumor Spheroid Invasionsassay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Dreidimensionale Alginat-Perle Kultur der Menschen Hypophysenadenom Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Messung der isometrischen Kontraktilität der Maus mesenterialen Arterie mit Draht Myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

Drei-dimensionale Druck einer komplexen Aorten Anomalie

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The accuracy of PVP mainly depends on the surgeons&#39; experience and multiple fluoroscopes during a traditional procedure. Puncture related complications were reported all over the world. To make the surgical procedure more precise and decrease the rate of puncture-related complications, our team applied a three-dimensional printing guide template to PVP to modify the traditional procedure. This protocol introduces how to model target vertebrae DICOM imaging data into three-dimensions in the software, how to simulate operation in this 3-D model, and how to use all of the surgical data to reconstruct a patient specific template for application. Using this template, surgeons can identify suitable puncture points accurately to improve the accuracy of the operation. The whole protocol includes: 1) diagnosis of the osteoporotic vertebral compression fracture; 2) acquisition of CT imaging of the target vertebra; 3) simulation of the operation in the software; 4) design and fabrication of the 3-D printing guide template; and 5) application of the template into an operation procedure.

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty PVP

Herein, we present a three-dimensional printing guide template for percutaneous vertebraplasty. A patient with a T11 vertebral compression fracture was selected as a case study.

Dreidimensionale Druckanleitung Vorlage Assistierte Perkutane Vertebropplastik (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Hochauflösende dreidimensionale Abbildung der Fußpolstervaskulatur in einem murinen Hinterbein-Gangränmodell

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...