eoe sub articles

EoE Sub Articles

1. Generation of uniform size tumor spheroidsNOTE: Stem-like cells are cultured in neurobasal medium complemented with B27 supplement, heparin, Fibroblast growth factor&#160;2&#160;(FGF-2), penicillin, and streptomycin. These cells spontaneously form spheroids in culture.<ol><li>Wash the tumor cells with 5 mL phosphate-buffered saline (PBS) and incubate the cells with 0.5-1 mL dissociation enzyme for 5 minutes at 37 &#176;C.</li><li>Wash with 4-4.5 mL PBS and add 10 mL of the complete growth medium (complete neurobasal medium, cNBM).</li><li>Count the cells using an automatic counting technique with trypan blue and a cell counting chamber slide.</li><li>To generate 100 spheroids with 104&#160;cells per spheroid (according to preferred size), mix 106&#160;cells in 8 mL of NBM with 2 mL of 2% methylcellulose.</li><li>Transfer the suspension to a sterile system container and dispense 100 &#181;L/well with a multichannel pipette into a 96 well round bottom plate.</li><li>Incubate the plate at 37 &#176;C, 5% CO2&#160;and 95% humidity. Equal sized spheroids will form and can be used after 3-4 days.</li></ol>2. Three-dimensional experiments<ol><li>Invasion<ol style='list-style-type:decimal;'><li>Preparation<ol style='list-style-type:decimal;'><li>Prepare the collagen matrix in a tube on ice with type I collagen at 1 mg/mL final concentration, 1x PBS, 0.023xVcollagen, 1 M sodium hydroxide, and sterile H2O. Incubate the solution on ice for 30 min.</li><li>Collect spheroids from the round bottom well plate in 500 &#181;L tubes and wash 2x with 200 &#181;L 1x PBS.</li><li>Pipette the spheroids carefully into 100 &#181;L of the collagen matrix and insert in the center of a well in normal 96 well plates.</li><li>Incubate the collagen gel for 30 min at 37 &#176;C and then add cNBM on top of the gel. Inhibitors or activators (e.g., hydrogen chloride as shown in&#160;Figure 1B, 1C) can be added to the medium at this step.</li></ol></li><li>Image acquisition and analysis<ol style='list-style-type:decimal;'><li>Take pictures sequentially with a video microscope in brightfield mode 24 h after collagen inclusion.</li><li>Use Fiji to analyze pictures either manually or in a semiautomated manner. To do so manually, draw around the core and the total area of the spheroid with the freehand selection tool and measure the invasive area of each spheroid by subtract total area with core area. To analyze the images in a semiautomated manner, use a macro to determine the invasive area.</li></ol></li></ol></li></ol>

<figure class='table' style='width:727pt;'><table class='ck-table-resized'><colgroup><col style='width:23.66%;'><col style='width:12.52%;'><col style='width:12.93%;'><col style='width:50.89%;'></colgroup><tbody><tr><td style='height:14.5pt;'>96 well round-bottom plate</td><td>Falcon</td><td>08-772-212</td><td>&#160;</td></tr><tr><td style='height:14.5pt;'>Accutase</td><td>Gibco</td><td>A11105-01</td><td>Stored at 4 &#176;C, sphere dissociation enzyme</td></tr><tr><td style='height:14.5pt;'>B27</td><td>Gibco</td><td>12587</td><td>Stored at -20 &#176;C, defrost before use</td></tr><tr><td style='height:14.5pt;'>Basic Fibroblast Growth Factor</td><td>Peprotech</td><td>100-18B</td><td>Stored at -20 &#176;C, defrost before use</td></tr><tr><td style='height:14.5pt;'>DPBS 10X</td><td>Pan Biotech</td><td>P04-53-500</td><td>Stored at 4 &#176;C</td></tr><tr><td style='height:14.5pt;'>Methylcellulose</td><td>Sigma</td><td>M0512</td><td>Diluted in NBM for a 2% final concentration</td></tr><tr><td style='height:14.5pt;'>NBM</td><td>Gibco</td><td>21103-049</td><td>Stored at 4 &#176;C</td></tr><tr><td style='height:14.5pt;'>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td>Stored at 4 &#176;C</td></tr><tr><td style='height:14.5pt;'>Penicillin - Streptomycin</td><td>Gibco</td><td>15140-122</td><td>Stored at 4 &#176;C</td></tr><tr><td style='height:14.5pt;'>Type I Collagen</td><td>Corning</td><td>354236</td><td>Stored at 4 &#176;C</td></tr></tbody></table></figure>

generation of a three-dimensional spheroid model of tumor cell invasion

<a href='https://app.jove.com/v/60998/a-3d-spheroid-model-for-glioblastoma'>Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma.&#160;J. Vis. Exp.&#160;(2020)</a>The video demonstrates the generation of a three-dimensional spheroid model of tumor cell invasion. Spheroids are formed from human brain tumor cells and embedded in a collagen matrix. The tumor cells at the spheroid periphery adopt an invasive phenotype, secreting proteases to degrade the collagen and advancing to invade the surrounding matrix.

<img src='https://cloudfront.jove.com/files/ftp_upload/23265/23265_Figure_1_editor_23265.jpg' alt='23265_Figure_1.jpg' />Figure 1: Glioblastoma P3 spheroid in proliferation, invasion, or migration assays.&#160;(A) Proliferation assay. Left panel: Representative pictures with DMSO as control or with 20 &#181;M of rotenone (respiratory chain complex I inhibitor) at time 0 or 72 h. Right panel: Spheroid area quantification represented as dashed lines in the images. Scale = 250 &#181;m. (B) Invasion assay in collagen matrix. Left panel: Representative pictures with or without 20 mM hydrogen chloride, at time 0 or 24 h. Right panel: Quantification of invasive areas. Scale = 100 &#181;m. (C) Migration assay on Matrigel coating. Left panel: Representative images in brightfield mode at time 0 or 24 h. Magnified areas are represented in the bottom panels. Right panel: Quantification of migratory areas. Scale = 250 &#181;m. (D) Z-stack representation of the spheroid (40 &#181;m step) in (a) spheroid dynamic tracked over 18 h (image with 3 h interval) in (b). Cells stably expressed nuclear mCherry (orange) and cytoplasmic GFP (blue). Scale = 100 &#181;m.

Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion

Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma.&#160;J. Vis. Exp.&#160;(2020)The video demonstrates the generation of a three-dimensional ...

Take a culture of human brain tumor cells.Treat with a dissociation enzyme to disrupt cell junctions, separating aggregated cells.Wash with a buffer to stop enzyme activity, then add a culture medium to maintain cell viability.Introduce methylcellulose, a suspending agent.Transfer the suspension into a round-bottom microplate and incubate.The viscous methylcellulose keeps the cells suspended, promoting cell-cell interactions for aggregation into spheroids.Mix the spheroids with a collagen matrix that mimics the physiological extracellular environment.Transfer the suspension into a microplate and incubate, allowing the matrix to form a gel that entraps the spheroids.Add the growth medium on top of the gel.At the spheroid periphery, tumor cells lose cell-cell adhesion and adopt an invasive phenotype.These cells secrete proteases to degrade the matrix, extend protrusions to anchor themselves, and move forward, invading the matrix.The spheroid model exhibiting tumor invasion is ready for analysis.

generation-three-dimensional-spheroid-model-tumor-cell

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Tumors

21 vistas. Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma. J. Vis. Exp. (2020) The video demonstrates the generation of a three-dimensional spheroid model of tumor cell invasion. Spheroids are formed from human brain tumor cells and embedded in a collagen matrix. The tumor cells at the spheroid periphery adopt an invasive phenotype, secreting proteases to degrade the collagen and advancing to invade the surrounding matrix. 

Video: Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion - Concepto

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Un modelo de piel Organotypic Melanoma 3D esferoide

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Investigación

JoVE Journal

Investigación sobre el cáncer

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Evaluación de la viabilidad celular y la muerte en cultivos esferoides 3D de células cancerosas

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Un novedoso modelo de esferoides de tumor 3D modulado por fibroblastos para el estudio de la interacción tumor-Stroma y el descubrimiento de fármacos

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion

Transcripción

Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion

Un modelo de piel Organotypic Melanoma 3D esferoide

Evaluación de la viabilidad celular y la muerte en cultivos esferoides 3D de células cancerosas

Un novedoso modelo de esferoides de tumor 3D modulado por fibroblastos para el estudio de la interacción tumor-Stroma y el descubrimiento de fármacos