Name: maintaining human glioblastoma cellular diversity ex vivo using three-dimensional organoid culture
Uploaded: 2022-08-25T13:10:00
Duration: 7 min 11 s
Description: Watch this Scientific Journal Video about Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture at JoVE.com

We would like to thank Dr. Justin Lathia for his&#160;invaluable advice and ongoing support. We also thank Katrina Fife, Lisa Wallace, and Maya Camhi for their excellent technical support.

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumor+initiating+cells+adapt+to+restricted+nutrition+through+preferential+glucose+uptake.">Brain tumor initiating cells adapt to restricted nutrition through preferential glucose uptake.</a> Nature Neuroscience. 16 (10), 1373-1382 (2013).</li><li>Hjelmeland, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidic+stress+promotes+a+glioma+stem+cell+phenotype.">Acidic stress promotes a glioma stem cell phenotype.</a> Cell Death and Differentiation. 18 (5), 829-840 (2011).</li><li>Bar, E. E., Lin, A., Mahairaki, V., Matsui, W., Eberhart, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+increases+the+expression+of+stem-cell+markers+and+promotes+clonogenicity+in+glioblastoma+neurospheres.">Hypoxia increases the expression of stem-cell markers and promotes clonogenicity in glioblastoma neurospheres.</a> The American Journal of Pathology. 177 (3), 1491-1502 (2010).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factors+regulate+tumorigenic+capacity+of+glioma+stem+cells.">Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells.</a> Cancer Cell. 15 (6), 501-513 (2009).</li><li>Azzarelli, R., Ori, M., Philpott, A., Simons, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+model+of+glioblastoma+by+co-culturing+tumor+stem+cells+with+human+brain+organoids.">Three-dimensional model of glioblastoma by co-culturing tumor stem cells with human brain organoids.</a> Biology Open. 10 (2), 056416 (2021).</li><li>Linkous, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+patient-derived+glioblastoma+with+cerebral+organoids.">Modeling patient-derived glioblastoma with cerebral organoids.</a> Cell Reports. 26 (12), 3203-3211 (2019).</li><li>da Silva, B., Mathew, R. K., Polson, E. S., Williams, J., Wurdak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+glioblastoma+spheroid+infiltration+of+early-stage+cerebral+organoids+models+brain+tumor+invasion.">Spontaneous glioblastoma spheroid infiltration of early-stage cerebral organoids models brain tumor invasion.</a> SLAS Discovery: Advancing Life Sciences R &amp; D. 23 (8), 862-868 (2018).</li><li>Jacob, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Patient-derived+glioblastoma+organoid+model+and+biobank+recapitulates+inter-+and+intra-tumoral+heterogeneity.">A Patient-derived glioblastoma organoid model and biobank recapitulates inter- and intra-tumoral heterogeneity.</a> Cell. 180 (1), 188-204 (2020).</li><li>Boretto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+organoids+from+endometrial+disease+capture+clinical+heterogeneity+and+are+amenable+to+drug+screening.">Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening.</a> Nature Cell Biology. 21 (8), 1041-1051 (2019).</li><li>Huang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductal+pancreatic+cancer+modeling+and+drug+screening+using+human+pluripotent+stem+cell-and+patient-derived+tumor+organoids.">Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell-and patient-derived tumor organoids.</a> Nature Medicine. 21 (11), 1364-1371 (2015).</li><li>Ledur, P. F., Onzi, G. R., Zong, H., Lenz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+conditions+defining+glioblastoma+cells+behavior:+what+is+the+impact+for+novel+discoveries.">Culture conditions defining glioblastoma cells behavior: what is the impact for novel discoveries.</a> Oncotarget. 8 (40), 69185-69197 (2017).</li><li>Lenin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drug+screening+pipeline+using+2D+and+3D+patient-derived+in+vitro+models+for+preclinical+analysis+of+therapy+response+in+glioblastoma.">A drug screening pipeline using 2D and 3D patient-derived in vitro models for preclinical analysis of therapy response in glioblastoma.</a> International Journal of Molecular Sciences. 22 (9), 4322 (2021).</li><li>Rybin, M. J., Ivan, M. E., Ayad, N. G., Zeier, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+models+of+glioblastoma+and+their+role+in+drug+discovery.">Organoid models of glioblastoma and their role in drug discovery.</a> Frontiers in Cellular Neuroscience. 15 (4), 605255 (2021).</li><li>Shakya, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+lipid+metabolism+marks+glioblastoma+stem+and+non-stem+cells+in+separate+tumor+niches.">Altered lipid metabolism marks glioblastoma stem and non-stem cells in separate tumor niches.</a> Acta Neuropathologica Communications. 9 (1), 101 (2021).</li><li>Lee, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+stem+cells+derived+from+glioblastomas+cultured+in+bFGF+and+EGF+more+closely+mirror+the+phenotype+and+genotype+of+primary+tumors+than+do+serum-cultured+cell+lines.">Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines.</a> Cancer Cell. 9 (5), 391-403 (2006).</li><li>Bian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+cerebral+organoids+model+brain+tumor+formation.">Genetically engineered cerebral organoids model brain tumor formation.</a> Nature Methods. 15 (8), 631-639 (2018).</li><li>Lancaster, M. 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The authors have no conflicts to disclose.

GBM organoids are a complementary culture method to traditional spheres that include greater cellular and microenvironmental heterogeneity4,22,30. Although more time and resource-intensive, organoid culture can offer valuable insight into intra-tumoral behavior and mechanisms of drug resistance. 
GBM is driven by a population of CSCs5,31, and these methods were developed to allow continued growth and self-renewal of this CSC population. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) are known to enhance stem cell maintenance and growth and provide active receptor tyrosine kinase (RTK) signaling. The formation of heterogeneous cellular populations and distinct tumor microenvironments within GBM tumors relies on supporting CSC behaviors. The selection of an lrECM mimics the laminin-rich brain environment and supports the cells in organoid culture to self-organize and migrate by invasion. Although some groups have established organoid culture without the use of an lrECM or EGF/FGF enriched media24,28, which may offer a more time-efficient manner of this culture method and stronger selection of oncogenic signaling to drive growth, these methods were chosen to optimize the pro-stem cell environment to best establish the cellular heterogeneity of organoids. Both cerebral organoids and GBM organoids have been made with lrECM in the literature previously21,32,33. Although we have established data regarding the tumor populations found within organoids and the spatial variation, less is known about non-tumor populations within the organoids and how long they survive from the original patient specimens. Certain IHC stains (such as CD45) may provide this data, and could be an interesting point of research in the future with organoids. 
Knowing the intended use for organoid culture is important for selecting appropriate methods. Establishing organoids from primary specimens versus growing uniform organoids for specific experiments have slightly different procedures. Having an appreciation for how organoids mature and visually fill in the lrECM scaffold is important for being able to allocate proper time and resources towards organoid culture. Sparse regions of cells in organoids will slowly expand and grow to fill the lrECM, which can take anywhere from 2-8 weeks, depending on the specimen's behavior. This rate of growth is somewhat intrinsic to each specimen; it is preserved across different batches of organoids and fairly consistent with the relative rate of sphere growth. Organoids can be maintained for over 1 year and retain tumor formation capabilities on xenograft into mice; however, it is recommended to grow them with a distinct purpose as to not waste lab resources (both materials and time)4. Organoid growth has been tested in multiple well sizes and formats, and shows that a 10 cm plate is the ideal setting for maintaining optimal cell viability, followed by a 6-well plate with three organoids per well34. Organoids consume more media compared to their two-dimensional culture counterparts, and using a smaller well format does not lead to proper maintenance. For example, one well of a 96-well format plate does not have enough space or media volume relative to the size of an organoid to sustain organoid growth.
As organoids establish, being observant is important for increasing success. Initially, organoids will consume media slowly, but as they become denser and more mature, they will consume media more quickly. Adding phenol red to the media can help serve as an indicator of media consumption. When the media is more yellow, it may prompt us to adjust the feeding pattern, whether it is to exchange a higher volume of media, increase the total media amount in the plate, divide organoids amongst multiple cell culture plates to keep up with their growth, or even adjust the timeline for experiments. 
In many ways, organoids are an inefficient way of conducting cancer research. They involve long time scales, and are expensive and resource-heavy compared to GBM sphere culture. However, compared to patient-derived xenografts, an alternative method for recreating cellular and microenvironmental diversity, they are more straightforward, less expensive, and controllable. Selection of when to best use organoids is important for cancer researchers. They are not intended to replace traditional sphere or adherent culture and not to replace xenograft models. Organoids can, when applied to the right scientific question, combine the benefits of both these systems and may allow us to observe tumor cell biology that would otherwise remain hidden. The scientific community is only beginning to understand what learning opportunities organoids offer, but it is clear they will be an invaluable tool in the future for understanding the complex biology of GBM.

Glioblastoma (GBM) is the most commonly occurring primary brain tumor with a grim prognosis of approximately 15 months from diagnosis1. Treatments that are effective in preclinical studies can often be poorly effective in patients2,3. Poor clinical response is attributed to many factors, including the microenvironmental heterogeneity of GBM and complex intra-tumoral interactions. These can be difficult to recreate in the laboratory setting with traditional adherent or sphere culture methods4. The presence of a subset of self-renewing cancer stem cells (CSCs) within GBM may also contribute to this complexity5,6. CSCs are crucial for tumor propagation and maintain tumor growth by promoting active angiogenesis, cancer invasion, and resistance to therapies including radiation7,8,9. CSCs are not uniformly distributed throughout tumors but rather are enriched within specific microenvironments, including the perivascular niche and perinecrotic regions, which each provide distinct molecular regulation of their cellular states10,11,12,13,14. CSCs are not passive recipients of microenvironmental cues but instead possess the ability to remodel their own microenvironments7,15,16. The microenvironment of a CSC can promote maintenance of stem cell state in response to pressures such as nutrient scarcity, pH, and hypoxia17,18,19,20, suggesting the importance of these conditions in a model system. Recapitulation of the diverse cellular microenvironment within tumors is therefore critical to understanding therapeutic resistance and identifying novel therapies.
Three-dimensional culture has increased in popularity in recent years21,22. Organoids have been used in other types of cancer, and the primary goal of maintaining cells as organoids is to allow for growth of heterogeneous cell populations (many of which may normally be outcompeted in more homogeneous sphere culture) and spatial diversity, seen as regional tumor microenvironments with genetic specificity4,23,24,25,26. There are many methods for three-dimensional culture of cancer cells, which each have advantages and disadvantages27,28,29. Organoid culture is not intended to be a replacement for traditional adherent or sphere culture. It is best used as a complementary technique to two-dimensional methods when there are specific questions where the interaction between cell microenvironment and tumor cell responses is critical.
This article describes reliable and repeatable methods to generate GBM organoids from either primary patient samples or patient-derived cultures. We address&#160;two different objectives for three-dimensional organoid culture: (1) establishing organoids from primary patient tissue, with maximal engraftment potential regardless of uniformity, or (2) growing uniform organoids for more quantitative experimental use. When establishing a primary specimen as organoids, it is not necessary to filter for single cells or count cells, because keeping maximum cell numbers and types to establish the initial culture is a priority. When growing organoids for comparative experiments, however, single-cell filtration and cell counting are needed to ensure replicate organoids are comparable for experimental consistency. This protocol details how to establish organoid cultures and create uniform organoids, as well as refined methods for embedding and preserving organoids and standard cell culture experiments, including immunohistochemistry, immunofluorescence, and assessment of total cell viability in GBM organoids.

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	<tbody>
		<tr>
			<td>3,3-Diaminobenzidine (DAB) tablets</td>
			<td>MP Biomedicals</td>
			<td>08980681</td>
			<td>3,3-Diaminobenzidine (DAB) tablets</td>
		</tr>
		<tr>
			<td>96-well PCR plates</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>96-well PCR plates</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>ThermoFisher Scientific</td>
			<td>SCR005</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic</td>
			<td>ThermoFisher Scientific</td>
			<td>15240062</td>
			<td>Antibiotic-antimycotic</td>
		</tr>
		<tr>
			<td>B-27 supplement minus vitamin A</td>
			<td>ThermoFisher Scientific</td>
			<td>12587001</td>
			<td>B-27 supplement minus vitamin A (50x)</td>
		</tr>
		<tr>
			<td>GelCode Blue Stain Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>24590</td>
			<td>Bluing reagent</td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#181;m)</td>
			<td>CellTreat Scientific</td>
			<td>229483</td>
			<td>Cell strainer (70 &#181;m)</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D</td>
			<td>Promega</td>
			<td>G9681</td>
			<td>Luminescent cell viability assay</td>
		</tr>
		<tr>
			<td>Clarifier 2</td>
			<td>ThermoFisher Scientific</td>
			<td>7301</td>
			<td>Nuclear hematoxylin clarifying reagent</td>
		</tr>
		<tr>
			<td>Clear-Rite 3</td>
			<td>ThermoFisher Scientific</td>
			<td>6901</td>
			<td>xylene substitute</td>
		</tr>
		<tr>
			<td>Epredia Gill 2 Hematoxylin</td>
			<td>ThermoFisher Scientific</td>
			<td>72504</td>
			<td>Hematoxylin</td>
		</tr>
		<tr>
			<td>Glutamine in 0.85% NaCl</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td>Glutamine in 0.85% NaCl (200 mM)</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>ThermoFisher Scientific</td>
			<td>354234</td>
			<td>Laminin-enriched extracellular matrix</td>
		</tr>
		<tr>
			<td>Mini PAP pen</td>
			<td>ThermoFisher Scientific</td>
			<td>008877</td>
			<td>Hydrophobic barrier pen</td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>ThermoFisher Scientific</td>
			<td>22-050-102</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>Neurobasal media minus phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>12349015</td>
			<td>Neurobasal media minus phenol red (500 mL)</td>
		</tr>
		<tr>
			<td>Normal donkey serum (NDS)</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td>Normal donkey serum (NDS)</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 4% in PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>AAJ19943K2</td>
			<td>Paraformaldehyde 4% in PBS</td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Sigma</td>
			<td>P0290</td>
			<td>Phenol red (0.5%)</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>P10144</td>
			<td>Liquid curing mountant</td>
		</tr>
		<tr>
			<td>Recombinant human EGF protein</td>
			<td>R&#38;D systems</td>
			<td>236-EG-01M</td>
			<td>Recombinant human EGF protein (250 &#181;g/mL)</td>
		</tr>
		<tr>
			<td>Recombinant human FGF basic</td>
			<td>R&#38;D systems</td>
			<td>4144-TC-01</td>
			<td>Recombinant human FGF basic (250 &#181;g/mL)</td>
		</tr>
		<tr>
			<td>SignalStain Antibody Diluent</td>
			<td>Cell Signaling</td>
			<td>8112</td>
			<td>Antibody diluent</td>
		</tr>
		<tr>
			<td>SignalStain Boost IHC Detection Reagent</td>
			<td>Cell Signaling</td>
			<td>8114</td>
			<td>Immunohistochemistry detection reagent</td>
		</tr>
		<tr>
			<td>SignalStain Citrate Unmasking Solution</td>
			<td>Cell Signaling</td>
			<td>14746</td>
			<td>Citrate unmasking solution</td>
		</tr>
		<tr>
			<td>Single edge razor blade</td>
			<td>Uline</td>
			<td>H-595B</td>
			<td>Single edge razor blade</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>11360070</td>
			<td>Sodium pyruvate (100 mM)</td>
		</tr>
		<tr>
			<td>Tissue-Tek Cryomolds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Disposable cryomolds</td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Optimal cutting temperature compound</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>T10282</td>
			<td>Cell impermeant stain (0.4%)</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>ThermoFisher Scientific</td>
			<td>X3P-1GAL</td>
			<td>Xylene</td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows early organoid growth seen via light microscopy at 10x magnification. Figure 1A shows migration and invasion of single cells through lrECM in the center view. The cells will continue to expand and &#39;colonize&#39; the lrECM, and they will appear more dense and eventually opaque by visual inspection. Figure 1B shows several mature organoids (at 7 weeks) without magnification, relative to the size of a dime.
Figure 2 demonstrates immunohistochemical staining of GBM organoids for phospho-histone H3, a marker of active proliferation. Most highly proliferative cells are seen in the organoid perimeter compared to the organoid core. Positive staining will have a brown/copper appearance.
Figure 3 describes the process to homogenize and measure total cell number in GBM organoids using a 3D-specific luminescent cell viability assay. Due to the high number of cells present in GBM organoids, the larger organoid structure is initially homogenized by triturating in luminescent assay reagent. Then fractions of the total organoid lysate are loaded into individual wells and diluted with additional luminescent assay reagent prior to incubation and reading on an appropriate multi-well plate reader.
Figure 4 represents DMSO (common vehicle) control data for organoids. Plotted data demonstrate intra-organoid and inter-organoid consistency. Luminescent viability data will typically be normalized to controls for each specimen when generating experimental data.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63745/63745fig01.jpg" /> 
Figure 1: View through a light microscope (10x). (A) Early organoid growth demonstrating migration/invasion of GBM cells throughout the laminin-rich extracellular matrix. (B) Mature organoids relative to the size of a dime. <a href="https://www.jove.com/files/ftp_upload/63745/63745fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63745/63745fig02.jpg" /> 
Figure 2: Immunohistochemistry of GBM organoids. (A, B) GBM organoids showing phospho-histone H3 stained cells for active proliferation. Scalebars are 600 &#181;m and 300 &#181;m for A and B, respectively. <a href="https://www.jove.com/files/ftp_upload/63745/63745fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63745/63745fig03.jpg" /> 
Figure 3: Luminescent cell viability protocol for organoids. (A) Move individual organoids to small centrifuge tubes and remove excess media. (B) Add 500 &#181;L of 1:1 PBS and luminescent cell viability assay mixture to each tube and pipette aggressively to break down the organoid. (C) Add 25 &#181;L of this organoid mixture and 75 &#181;L of the same 1:1 PBS and luminescent cell viability assay mixture to each well of a 96-well plate. (D) Place on a shaker for 2 min, followed by incubation for 20 min at RT, and then read luminescence on a plate reader. Figure made using BioRender.com. <a href="https://www.jove.com/files/ftp_upload/63745/63745fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63745/63745fig04.jpg" /> 
Figure 4: Cell viability. Cell viability data for organoids in 0.1% dimethyl sulfoxide (DMSO) for six technical replicates of four organoids for four patient specimens. <a href="https://www.jove.com/files/ftp_upload/63745/63745fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td>Neurobasal medium minus phenol red</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>B-27 supplement minus vitamin A (50x)</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic (100x)</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM)</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Glutamine in 0.85% NaCl (200 mM)</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Recombinant human FGF basic (250 &#181;g/mL)</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>Recombinant human EFG protein (250 &#181;g/mL)</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>500 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Neurobasal medium complete (NBMc) formulation
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Time</td>
		</tr>
		<tr>
			<td>50% Ethanol</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>75% Ethanol</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>4 min</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>4 min</td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>4 min</td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>15 min</td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>15 min</td>
		</tr>
	</tbody>
</table>
Table 2: Processing schedule for small organoids (below 3 mm in diameter)
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Time</td>
		</tr>
		<tr>
			<td>50% Ethanol</td>
			<td>6 min</td>
		</tr>
		<tr>
			<td>75% Ethanol</td>
			<td>6 min</td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>8 min</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>8 min</td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>5 min</td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>8 min</td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>30 min</td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>30 min</td>
		</tr>
	</tbody>
</table>
Table 3: Processing schedule for large organoids (above 3 mm in diameter)
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Time</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>Running diH2O</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>Nuclear hematoxylin clarifying reagent</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>Running diH2O</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>Bluing Reagent</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>Running diH2O</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>Xylene substitute</td>
			<td>2 min</td>
		</tr>
	</tbody>
</table>
Table 4: Hematoxylin counterstain

Watch this Scientific Journal Video about Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture at JoVE.com

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.

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 ...

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