<meta charset="utf8"></head><body>LEGO: Lipid Düzensizliği ve İlaç Hedeflerini Ortaya Çıkaran Bir İnsan GBM Organoid Modeli

<ol>
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<table><tbody><tr><td>Accutase</td><td>A6964</td><td>Sigma-Aldrich&amp;nbsp;</td><td>Passaging reagent B</td></tr><tr><td>Antibiotic-Antimycotic</td><td>15240096</td><td>Life-Technologies</td><td></td></tr><tr><td>B27&amp;nbsp;</td><td>17504044</td><td>Life Technologies&amp;nbsp;</td><td></td></tr><tr><td>B27 without vitamin A&amp;nbsp;</td><td>12587010</td><td>Life Technologies&amp;nbsp;</td><td></td></tr><tr><td>Bbs1-HF</td><td>R3539S</td><td>New England Biolabs</td><td></td></tr><tr><td>Bsa1-HF</td><td>R3535S</td><td>New England Biolabs</td><td></td></tr><tr><td>CHIR99021</td><td>4423</td><td>Tocris Bioscience</td><td></td></tr><tr><td>CloneR</td><td>#05889</td><td>STEMCELL Technologies&amp;nbsp;</td><td>Reagent to&amp;nbsp;facilitate single-cell survival</td></tr><tr><td>D-Luciferin</td><td>L2916</td><td>Invitrogen</td><td>Luciferase Substrate</td></tr><tr><td>DMEM/F-12</td><td>11330032</td><td>Life Technologies&amp;nbsp;</td><td></td></tr><tr><td>EcoRl-HF</td><td>R3101T</td><td>New England Biolabs</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>30-2020</td><td>ATCC&amp;nbsp;</td><td></td></tr><tr><td>FGF-2</td><td>100-18B</td><td>PeproTech&amp;nbsp;</td><td></td></tr><tr><td>GlutaMAX</td><td>35050038</td><td>Life Technologies&amp;nbsp;</td><td></td></tr><tr><td>Heparin</td><td>H3149</td><td>Sigma-Aldrich&amp;nbsp;</td><td></td></tr><tr><td>hES-quality Fetal Bovine Serum</td><td>10270106</td><td>Life Technologies&amp;nbsp;</td><td></td></tr><tr><td>Insulin Solution</td><td>I9278</td><td>Sigma-Aldrich&amp;nbsp;</td><td></td></tr><tr><td>IVIS Lumina II</td><td>NA</td><td>PerkinElmer</td><td></td></tr><tr><td>Knockout serum replacement</td><td>10828-028</td><td>Life Technologies&amp;nbsp;</td><td></td></tr><tr><td>L-Ascorbic Acid</td><td>A4544-25G</td><td>Sigma-Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Matrigel&amp;reg; hES qualified</td><td>354277</td><td>Corning</td><td>Basement matrix membrane</td></tr><tr><td>MEM-NEAA</td><td>11140050</td><td>Life Technologies&amp;nbsp;</td><td></td></tr><tr><td>mTeSR Plus</td><td>100-0276</td><td>STEMCELL Technologies&amp;nbsp;</td><td>Human embryonic cell qualified culture medium</td></tr><tr><td>N2&amp;nbsp;</td><td>17502048</td><td>Life Technologies&amp;nbsp;</td><td></td></tr><tr><td>Neon Electroporation System</td><td>NA</td><td>Thermo Fisher Scientific</td><td>Electroporation system</td></tr><tr><td>Neon Transfection System 100 &amp;micro;L Kit</td><td>MPK10096</td><td>Invitrogen</td><td>Electroporation kit</td></tr><tr><td>Neurobasal medium</td><td>21103049</td><td>Life Technologies&amp;nbsp;</td><td></td></tr><tr><td>Online knockout efficiency analysis</td><td>NA</td><td></td><td>http://shinyapps.datacurators.nl/tide/</td></tr><tr><td>Organoid Embedding Sheet&amp;nbsp;</td><td># 08579</td><td>STEMCELL Technologies&amp;nbsp;</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>15140122</td><td>Life-Technologies</td><td></td></tr><tr><td>Polybrene (10mg/mL)</td><td>TR-1003-50UL</td><td>Merck Millipore</td><td></td></tr><tr><td>Puromycin</td><td>P8833</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>px330-A-1x2</td><td>https://www.addgene.org/58766/</td><td>Addgene&amp;nbsp;</td><td></td></tr><tr><td>px330-S-2&amp;nbsp;</td><td>https://www.addgene.org/58778/</td><td>Addgene&amp;nbsp;</td><td></td></tr><tr><td>px459</td><td>https://www.addgene.org/48139/</td><td>Addgene&amp;nbsp;</td><td></td></tr><tr><td>ReleSR</td><td>5872</td><td>STEMCELL Technologies&amp;nbsp;</td><td>Passaging reagent A</td></tr><tr><td>Rock Inhibitor</td><td>72304</td><td>STEMCELL Technologies&amp;nbsp;</td><td></td></tr><tr><td>sgRNA design</td><td>NA</td><td></td><td>https://zlab.bio/guide-design-resources</td></tr><tr><td>sgRNA design</td><td>NA</td><td>NA</td><td>www.benchling.com</td></tr><tr><td>T4 DNA ligase</td><td>M0202S</td><td>New England Biolabs</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol&amp;nbsp;</td><td>M3148</td><td>Sigma-Aldrich&amp;nbsp;</td><td></td></tr></tbody></table>

laboratory-engineered glioblastoma organoid culture and drug screening

<meta charset="utf8"></head><body>Laboratuvar Mühendisliği: Glioblastoma Organoid Kültürü ve İlaç Taraması

Laboratuvar Mühendisliği: Glioblastoma Organoid Kültürü ve İlaç Taraması

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and ...

laboratory-engineered-glioblastoma-organoid-culture-and-drug-screening

Research

JoVE Journal

Cancer Research

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

503 Görüntüleme.  German Cancer Research Center (DKFZ). Burada, genotip-fenotip bağımlılıklarını araştırmak ve glioblastoma tedavisi için potansiyel ilaçları taramak için laboratuvar mühendisliği glioblastoma organoidleri (LEGO) üretmek ve kullanmak için kapsamlı bir protokolün ana hatlarını çiziyoruz.

Video: Laboratuvar Mühendisliği: Glioblastoma Organoid Kültürü ve İlaç Taraması

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Glioblastoma için 3D Spheroid Model

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Kanser Araştırmaları

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

<meta charset="utf8"></head><body>Hasta Kaynaklı Organoidler Kullanılarak Orta Verimli İlaç ve Radyoterapi Tarama Testi

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

Hasta Kaynaklı Organoidler Kullanılarak Orta Verimli İlaç ve Radyoterapi Tarama Testi

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Klinik Olarak İlgili Hayvan Modellerinde ve 3D İmmün entrikalarda Kullanılmak Üzere Cerrahi Örneklerden Yüksek Dereceli Glioma Hücre Kültürünün Optimizasyonu

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Tıp

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular diversity, as well as complex interactions between tumor microenvironments, can make finding effective treatments a challenge. Traditional adherent or sphere culture methods can mask such complexities, whereas three-dimensional organoid culture can recapitulate regional microenvironmental gradients. Organoids are a method of three-dimensional GBM culture that better mimics patient tumor architecture, contains phenotypically diverse cell populations, and can be used for medium-throughput experiments. Although three-dimensional organoid culture is more laborious and time-consuming compared to traditional culture, it offers unique benefits and can serve to bridge the gap between current in vitro and in vivo systems. Organoids have established themselves as invaluable tools in the arsenal of cancer biologists to better understand tumor behavior and mechanisms of resistance, and their applications only continue to grow. Here, details are provided about methods for generating and maintaining GBM organoids. Instructions of how to perform organoid sample embedding and sectioning using both frozen and paraffin-embedding techniques, as well as recommendations for immunohistochemistry and immunofluorescence protocols on organoid sections, and measurement of total organoid cell viability, are all also described.

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Here, we describe a method of generating glioblastoma (GBM) organoids from primary patient specimens or patient-derived cell cultures and maintaining them to maturity. These GBM organoids contain phenotypically diverse cell populations and recreate tumor microenvironments ex vivo.

Üç Boyutlu Organoid Kültür Kullanarak İnsan Glioblastoma Hücresel Çeşitliliğinin Ex vivo Korunması

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Toplu ve Tek-Organoid Analizi için Geniş Alan Canlı Hücre Görüntüleme Kullanılarak Multiparametrik Tümör Organoid İlaç Taraması

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Doğrayıcıyla İşlenmiş Hasta Tümörlerinden 3D Glioma Organoidlerinin Türetilmesi

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing realistic ex vivo models remain challenging. Patient-derived 3-dimensional organoid (PDO) models offer innovative platforms that capture the phenotypic and molecular heterogeneity of GBM, while preserving key characteristics of the original tumors. However, manual dissection for PDO generation is time-consuming, expensive and can result in a number of irregular and unevenly sized PDOs. This study presents an innovative method for PDO production using an automated tissue chopper. Tumor samples from four GBM and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients were processed manually as well as using the tissue chopper. In the manual approach, the tumor material was dissected using scalpels under microscopic control, while the tissue chopper was employed at three different angles. Following culture on an orbital shaker at 37 &#176;C, morphological changes were evaluated using bright field microscopy, while proliferation (Ki67) and apoptosis (CC3) were assessed by immunofluorescence after 6 weeks. The tissue chopper method reduced almost 70% of the manufacturing time and resulted in a significantly higher PDOs mean count compared to the manually processed tissue from the second week onwards (week 2: 801 vs. 601, P = 0.018; week 3: 1105 vs. 771, P = 0.032; and week 4:1195 vs. 784, P &lt; 0.01). Quality assessment revealed similar rates of tumor-cell apoptosis and proliferation for both manufacturing methods. Therefore, the automated tissue chopper method offers a more efficient approach in terms of time and PDO yield. This method holds promise for drug- or immunotherapy-screening of GBM patients.

Yazar Spotlight: Glioblastoma ve Glioma için Hastadan Elde Edilen 3D Organoidlerin Gelişmiş Üretimi

This study introduces an automated method to generate patient-derived glioblastoma 3-dimensional organoids utilizing a tissue chopper. The method provides a suitable and effective approach to obtain such organoids for therapeutical testing.

Ex Vivo Hastadan Elde Edilen Glioma Organoidlerinin Bir Doku Kıyıcı Kullanılarak Yetiştirilmesi

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing ...

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

<meta charset="utf8"></head><body>Glioma Kök Hücre Hatlarında Sferoid İlaç Duyarlılık Taraması

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

Glioma Kök Hücre Hatlarında Sferoid İlaç Duyarlılık Taraması

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

Laboratuvar Mühendisliği: Glioblastoma Organoid Kültürü ve İlaç Taraması

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