Name: Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening (Video) | JoVE Journal
Uploaded: 2025-01-10T14:50:00.000+00:00
Duration: 10 min 49 s
Description: Learn laboratory-engineered glioblastoma organoid culture for drug screening and genotype-phenotype analysis. This method enables personalized treatment strategies through accurate tumor modeling.

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This work is supported by Deutsche Forschungsgemeinschaft grant SFB1389 (H-K.L.), Deutsche Krebshilfe grant 110227 (H-K.L.), European Research Council (ERC) grant 647055 (H-K.L.), Deutschen Konsortium f&#252;r Translationale Krebsforschung (DKTK) grant AIM2GO (H-K.L.).

1. Luciferase labeling of the iPSCs
NOTE: For detailed instructions on culturing iPSCs, refer to the protocol provided by the institutions or the companies from which the cells were obtained. In all experiments described in this protocol, the iPSCs at passages 3-10 post-recovery were used, and any cells showing signs of differentiation were discarded. For the virus production, plasmids containing an EF1&#945; promoter driving Luc2, along with a puromycin-resistant cassette, were used.
<ol>
	<li>When the cells reach ~70% confluency, digest iPSCs with passaging reagents A and B (see Table of Materials), respectively, for 4 min at 37 &#176;C, centrifuge at 120 x g for 5 min, and resuspend into a single cell suspension by gentle pipetting.</li>
	<li>Count the cells and seed 1 x 104 single cells of iPSC in each well of a basement matrix membrane (BMM)-coated 48 well plate, culture for 24 h with human embryonic cell qualified culture medium containing 10 &#181;M rho kinase (ROCK) inhibitor (see Table of Materials).</li>
	<li>Infect the cells with lentivirus-containing luciferase-expressing plasmids (MOI around 10) and 8 &#181;g/mL Polybrene (see Table of Materials). Replace the medium with a fresh human embryonic cell-qualified culture medium 6 h later.</li>
	<li>After 24 h, add 150 &#181;g/mL Luciferase Substrate (see Table of Materials) to the cell culture medium and check the bioluminescence (BLI) signal with bioluminescence imaging equipment (see Table of Materials) with a 5 s exposure.</li>
	<li>Add 2 &#181;g/mL puromycin to the culture medium for 2 days, then change the medium every other day for 1 week to expand the cells.</li>
	<li>When the cells reach ~70% confluency, digest iPSCs with passaging reagents A and B (see Table of Materials), respectively, for 4 min at 37 &#176;C, centrifuge at 120 x g for 5 min, and resuspend into a single cell suspension by gentle pipetting.</li>
	<li>Count the cells and seed the single cells at a concentration of 50 cells per 10 cm dish for 2 dishes. Change the medium every other day until the clones reach 3-5 mm in diameter (Figure 1B).</li>
	<li>Check the BLI signal as described above and pick the clones with strong BLI signals under the microscope with sterilized 10 &#181;L pipette tips.</li>
	<li>Expand the cells for further usage.</li>
</ol>
2. gRNA design and plasmid cloning
NOTE: These steps can be carried out simultaneously with Section 1. CRISPR/Cas9 plasmids with a proper selection system should work. These specific plasmids were used because they enable simultaneous knockout of several target genes. This section demonstrates how to clone a plasmid with two gRNA scaffolds; more gRNA scaffolds can be cloned in one plasmid following the same principle. The original protocol was previously published13.
<ol>
	<li>Design gRNAs targeting the respective genes of interest with online web tools (see Table of Materials). Select the gRNAs targeting the exons presented in all splicing variants and locate the closest to the transcription starting site.</li>
	<li>Anneal gRNA oligos (95 &#176;C for 5 min, cool down to 25 &#176;C at a speed of 1 &#176;C/s) and linearize pX330A-1x2 (see Table of Materials) and pX330S-2 (see Table of Materials) plasmids with Bbs1 enzyme.</li>
	<li>Ligate one of the gRNAs with pX330A-1x2 or pX330S-2, respectively, with T4 ligase at room temperature (RT) for 4 h.</li>
	<li>Digest the two resulting plasmids with BsaI enzyme (37 &#176;C for 1 h) and purify the products by gel extraction.</li>
	<li>Ligate the gRNA scaffolds from the pX330S-2-gRNA plasmid to the linearized pX330A-1x2-gRNA plasmid with T4 ligase at RT for 4 h.</li>
	<li>Clone puromycin resistance gene (PuroR) from the pX459 (see Table of Materials) plasmid to the resulting plasmids by EcoRI digestion (37 &#176;C for 1 h) and subsequential T4 ligation at RT for 4 h.</li>
	<li>Transform the plasmids in DH5alpha-competent cells and extract the plasmids after expanding.</li>
</ol>
3. Knockout generation and validation on iPSCs
NOTE: Follow the standard CRISPR/Cas9 knockout protocols published before13,14. This section describes the method used to perform the knockouts via electroporation.
<ol>
	<li>Digest the iPSCs with passaging reagents A and B (see Table of Materials) for 4 min at 37 &#176;C each, and spin down the cells for 5 min at 120 x g and resuspend in human embryonic cell qualified culture medium (see Table of Materials) to generate single-cell suspensions.</li>
	<li>Count the cells and divide them into several tubes to achieve 1.2 x 106 cells per sample and spin down the cells at 120 x g for 5 min.</li>
	<li>Meanwhile, mix 15 &#181;g of the plasmids in 135 &#181;L of resuspension buffer from the transfection kit (see Table of Materials).</li>
	<li>Use 120 &#181;L of the mixture to resuspend the cell pellet.</li>
	<li>Electroporate the cells for two pulses at 1200 V for 20 ms with an electroporation system (see Table of Materials) and plate the resulting cells in one 6 well plate well with a human embryonic cell qualified culture medium containing reagent to facilitate single-cell survival (see Table of Materials).</li>
	<li>Add 2 &#181;g/mL puromycin to the culture medium every 12 h for 2 days. Then, expand the cells for 1 week before harvest.</li>
	<li>Check the efficiency of the knockout by western blotting or webtool analysis (see Table of Materials) and select the knockout population with the highest efficiency.</li>
	<li>Digest the iPSCs with the highest knockout efficiency with passaging reagents A and B for 4 min at 37 &#176;C each, and spin down the cells for 5 min at 120 x g. Resuspend in human embryonic cell qualified culture medium to generate single-cell suspensions.</li>
	<li>Count the cells and seed approximately 100 cells in two 10 cm dishes (50 cells in each dish) containing pre-warmed human embryonic cell qualified culture medium supplied with a reagent to facilitate single-cell survival.</li>
	<li>Change the medium every other day and culture the cells for 2 weeks.</li>
	<li>When the single-cell colonies are 3-5 mm in diameter, pick the colonies with 10 &#181;L pipette tips under the microscope and expand the cells in two wells of 48 well plates.</li>
	<li>When the cells reach 30%-50% confluency, extract the DNA from one well of the 48 well plate and perform PCR.</li>
	<li>Sequence the PCR products and analyze the results with the web tool (see Table of Materials).</li>
	<li>Select 5 positive clones to expand and further validate the knockout results with TA cloning and western blots.</li>
</ol>
4. Organoid Culture
NOTE: The organoids were generated following previously published protocols with minor adaptations15,16,17. The optimal age of the organoids (the culture duration) should be determined based on the specific purpose of the study. For instance, when investigating genotype-to-phenotype associations or observing organoid growth patterns, analyzing multiple time points ranging from 1-4 months or longer is recommended. Organoids aged 2-2.5 months were used for drug screening, as they tended to develop a necrotic core in the center due to nutrient limitations at larger sizes, which could impact the accuracy of drug screening results.
<ol>
	<li>On day 0, digest iPSCs with passaging reagents A and B (see Table of Materials) respectively for 4 min at 37 &#176;C, centrifuge at 120 x g for 5 min, and resuspend into a single cell suspension using low basic fibroblast growth factor (bFGF) hES media (Table 1).</li>
	<li>Count the cells and add an appropriate number of cells to reach a concentration of 6 &#215; 104 cells/mL.</li>
	<li>Add 50 &#181;M ROCK inhibitor and 6 ng/mL bFGF to the low bFGF hES medium-containing cells.</li>
	<li>Seed 150 &#181;L of cell suspension (9000 cells) into each well of the 96-well ultra-low attachment plate for both control (WT organoids) and experimental (knockout organoids) groups.</li>
	<li>On day 3, carefully remove 80 &#181;L of old culture medium from each well and add 150 &#181;L fresh low bFGF hES medium.</li>
	<li>On day 5, when embryonic bodies (EBs) begin to brighten and have smooth edges, remove as much old culture medium as possible from each well and add 150 &#181;L to 200 &#181;L of fresh neural induction medium (Table 1).</li>
	<li>On day 7, remove as much old culture medium as possible from each well and add 150-200 &#181;L of neural induction medium.</li>
	<li>On day 9, observe the EBs under the tissue culture microscope and select the EBs that are brighter around the outside.</li>
	<li>Thaw BMM at 4 &#176;C or on ice for 30 min before starting the following steps.</li>
	<li>Use a cut 200 &#181;L wide pipette tip to transfer the EBs to the organoid embedding sheet (see Table of Materials), remove the excessive medium, drop 30 &#181;L of BMM on the organoids, position the EBs in the middle of the droplet with a 10 &#181;L pipette tip, and let it solidify in the 37 &#176;C incubator for 20 min.</li>
	<li>Wash off the BMM droplets to a 6-well plate containing 3 mL of NeuroDMEM - A medium (Table 1) and 3 &#181;M CHIR99021 (see Table of Materials).</li>
	<li>On day 11, remove as much of the old culture medium from each well and add 3 mL of fresh NeuroDMEM -A medium containing 3 &#181;M CHIR99021.</li>
	<li>On day 13, remove as much of the old culture medium from each well as possible and add 3 mL of NeuroDMEM + A culture medium (Table 1). Place the 6-well plate on a shaker rotating at 75 rpm in the incubator.</li>
	<li>Refresh NeuroDMEM + A medium every 3 days until organoids are used.</li>
	<li>Use the organoids for multi-omic analysis following the standard protocols or drug screen as performed in the previous publication12.</li>
</ol>
5. Drug screen with LEGOs
NOTE: The exposure time for iPSCs or organoids BLI should be evaluated using various equipment or laboratory environments.
<ol>
	<li>Transfer organoids at the desired age to a 24-well plate containing 1 mL of NeuroDMEM + A medium, one organoid per well, 2 days before the experiment.</li>
	<li>Add 150 &#181;g/mL D-luciferin and incubate on the shaker for 15-30 min.</li>
	<li>Perform BLI as described before with an exposure time of 1 s-5 s.</li>
	<li>Select the organoids with similar signal strengths for subsequent experiments (at least 3 organoids for each group).</li>
	<li>On day 0, repeat steps 5.2 and 5.3. Then apply dimethyl sulfoxide (DMSO) or interested drugs to the culture medium of the organoids. Start with an initial drug concentration of 10 &#181;M, then perform an IC50 assay or adjust the concentration further according to different purposes.</li>
	<li>Repeat step 5.5 every 2 days. Treat the organoids for 6-10 days. 
	NOTE: The duration can be changed according to different purposes or the different nature of the drugs.</li>
	<li>Add 100 &#181;M BrdU to the medium and incubate it in a CO2 incubator for 2 h before harvesting the organoids, if needed, for subsequent proliferation analysis.</li>
	<li>Normalize the BLI signal to the DMSO control measured on the same day and then compare it with before drug treatment to evaluate the drug treatment effect. Consider drugs with a P value less than 0.05 and a signal decrease of more than 50% effective.</li>
</ol>

LEGO: A Human GBM Organoid Model Revealing Lipid Dysregulation and Drug Targets

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</ol>

The authors have no conflicts of interest to disclose

The lack of personalized treatment in human GBM could largely be attributed to the fact that many GBM models, such as human cell lines or mouse models, cannot faithfully recapitulate the human GBM. Consequently, the treatment strategies selected based on these model systems cannot be transferred into clinical applications. Instead, organoids can tackle these translational problems with the presence of human physiological conditions. To this end, we have generated LEGO and shown that LEGOS can faithfully recapitulate many...

Glioblastoma (GBM) accounts for more than 60% of all diagnosed primary brain tumors in adults, with a median survival of less than two years1,2,3. Despite considerable efforts in deciphering the underlying complexity of this disease, continuous attempts with targeted therapies or immunotherapies, and screening for potential anti-cancer drugs, the treatment strategy for newly diagnosed GBM patients remains to be the maximal safe surgical resection followed by radiotherapy (RT) combined with temozolomide (TMZ) and then adjuvant TMZ4.
The highly heterogeneous mutational nature of glioblastoma within and among tumors makes it extremely difficult to dissect the molecular properties of this tumor, which eventually leads to treatment failure. Thus, it is essential to decomplexify the disease by reducing the variety of genetic mutations to the modules of mutations that occur most often in GBM patients5,6,7,8 and to investigate the molecular consequences of each mutational subclone individually.
Recently, organoids have emerged as a promising model for cancer research. Organoids surpass the canonical cancer models since they exhibit a human microenvironment and complex cellular components while being easy to generate and expand with a relatively low cost9. There were also pioneers of genetically engineered organoids with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to study glioma10,11. Following this direction, we generated a set of induced pluripotent stem cell (iPSC)-based human GBM organoid models (LEGO: Laboratory Engineered Glioblastoma Organoid) based on CRISPR/Cas9 genetic engineering of the frequent mutations identified in GBM patients. With detailed characterization of the single-cell transcriptome, DNA methylome, metabolome, lipidome, proteome, and phospho-proteome of LEGOs, we have demonstrated the high resemblance of LEGOs with human GBM and discovered major milestones during the progression of GBM, and with luciferase-based drug screen, we have identified several potential drugs for GBM treatment, the details of such results have been published previously12. This article describes the detailed protocol for the generation and drug screening of LEGOs (Figure 1A).

<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

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

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 cellular characteristics of GBM have been a focal point of ongoing research, revealing that it is a group of heterogeneous diseases with variations in RNA expression, DNA methylation, or cellular composition. Despite the wealth of molecular data available, the lack of transferable pre-clinic models has limited the application of this information to disease classification rather than treatment stratification. Transferring the patients' genetic information into clinical benefits and bridging the gap between detailed descriptions of GBM, genotype-phenotype associations, and treatment advancements remain significant challenges. In this context, we present an advanced human GBM organoid model, the Laboratory Engineered Glioblastoma Organoid (LEGO), and illustrate its use in studying the genotype-phenotype dependencies and screening potential drugs for GBM. Utilizing this model, we have identified lipid metabolism dysregulation as a critical milestone in GBM progression and discovered that the microsomal triglyceride transfer protein inhibitor Lomitapide shows promise as a potential treatment for GBM.

In our hands, LEGOs derived from mutant iPSCs displayed increased expansion compared to the WT isogenic control (Figure 1C) and showed atypical nuclear after 4 weeks of culture (Figure 1D), which supports the potential malignant transformation of the cells in mutant organoids18. The tumorigenic potential of the organoids can be further verified with xenografted experiments if required, as we have shown previously12...

Here, we outline a comprehensive protocol for generating and utilizing laboratory-engineered glioblastoma organoids (LEGO) to investigate genotype-phenotype dependencies and screen potential drugs for glioblastoma treatment.

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

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

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Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

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Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

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.

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

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.

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

We present a protocol that can be used to conduct therapeutic drug testing with patient-derived ovarian cancer organoids.

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

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.

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

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Learn laboratory-engineered glioblastoma organoid culture for drug screening and genotype-phenotype analysis. This method enables personalized treatment strategies through accurate tumor modeling.

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

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