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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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=Endothelial+cells+create+a+stem+cell+niche+in+glioblastoma+by+providing+NOTCH+ligands+that+nurture+self-renewal+of+cancer+stem-like+cells.">Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH ligands that nurture self-renewal of cancer stem-like cells.</a> Cancer Research. 71 (18), 6061-6072 (2011).</li><li>Calabrese, C., 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+perivascular+niche+for+brain+tumor+stem+cells.">A perivascular niche for brain tumor stem cells.</a> Cancer Cell. 11 (1), 69-82 (2007).</li><li>Charles, N., 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=Perivascular+nitric+oxide+activates+notch+signaling+and+promotes+stem-like+character+in+PDGF-induced+glioma+cells.">Perivascular nitric oxide activates notch signaling and promotes stem-like character in PDGF-induced glioma cells.</a> Cell Stem Cell. 6 (2), 141-152 (2010).</li><li>Seidel, 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+hypoxic+niche+regulates+glioblastoma+stem+cells+through+hypoxia+inducible+factor+2+alpha.">A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha.</a> Brain. 133, 983-995 (2010).</li><li>Yan, K., 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=Glioma+cancer+stem+cells+secrete+Gremlin1+to+promote+their+maintenance+within+the+tumor+hierarchy.">Glioma cancer stem cells secrete Gremlin1 to promote their maintenance within the tumor hierarchy.</a> Genes &amp; Development. 28 (10), 1085-1100 (2014).</li><li>Cheng, 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=Glioblastoma+stem+cells+generate+vascular+pericytes+to+support+vessel+function+and+tumor+growth.">Glioblastoma stem cells generate vascular pericytes to support vessel function and tumor growth.</a> Cell. 153 (1), 139-152 (2013).</li><li>Flavahan, W. 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. 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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.

<table><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 &amp;micro;m)</td><td>CellTreat Scientific</td><td>229483</td><td>Cell strainer (70 &amp;micro;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&amp;amp;D systems</td><td>236-EG-01M</td><td>Recombinant human EGF protein (250 &amp;micro;g/mL)</td></tr><tr><td>Recombinant human FGF basic</td><td>R&amp;amp;D systems</td><td>4144-TC-01</td><td>Recombinant human FGF basic (250 &amp;micro;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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3.2K Views.  Cleveland Clinic. 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.

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

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.

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

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.

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

Medicine

Derivation of 3D Glioma Organoids from Chopper-Processed Patient Tumors

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.

Author Spotlight: Enhanced Generation of Patient-Derived 3D Organoids for Glioblastoma and Glioma

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.

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

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

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.

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 ...

A Protocol for Explant Cultures of IDH1-mutant Diffuse Low-grade Gliomas

Diffuse gliomas, the most prevalent primary brain tumors, are categorized into low and high grades based on histopathological criteria and genetic mutations. Diffuse low-grade gliomas constitute a subset, predominantly afflicting young adults and predisposing to high-grade gliomas. There are two main types of diffuse low-grade gliomas: astrocytomas and oligodendrogliomas, each defined by distinct genetic and histological features. These tumors characteristically exhibit a mutated variant of the metabolic enzyme IDH1, disrupting cellular differentiation. They are heterogeneous, comprising both proliferative stem cells and more differentiated, quiescent glial cells. 
Patient-specific, in vitro tumor models hold significant potential for unraveling oncogenic mechanisms and tailoring drug selection for personalized therapy. However, the absence of reliable in vitro models specifically for IDH1-mutant low-grade gliomas has hindered fundamental and translational research. Here, we present a simple method for culturing IDH1-mutant tumors as explants in defined media without needing enzymatic dissociation. These explants can be sustained for extended periods, maintaining IDH1-mutant protein expression as well as markers for oligodendrocyte progenitor cells such as ASCL1 (MASH1), OLIG1, and OLIG2 and for astrocytes (GFAP, SOX9). They can also be established from frozen tumors in DMSO. These cultures can serve as a valuable platform for drug screening, videomicroscopy, and gene studies, thus facilitating advancements in understanding and treating these challenging tumors. They also offer the potential to derive IDH1-mutant cell lines.

IDH1-mutant diffuse low-grade gliomas are rare tumors that are difficult to culture. We introduce a simple, non-enzymatic explant method that preserves IDH1-mutant protein expression and sustains cultures for months. Importantly, this technique works with cryopreserved tumors, offering a valuable resource for glioma research.

Diffuse gliomas, the most prevalent primary brain tumors, are categorized into low and high grades based on histopathological criteria and genetic ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Bioengineering

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Generating and Maintaining Slice Cultures from Human Glioblastoma Tissue

Source: Parker, J. J., et al. <a href="https://app.jove.com/v/53557">A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs.</a> J. Vis. Exp. (2017).
The video demonstrates the generation and maintenance of organotypic slice cultures from glioblastoma tissue. The isolated tissue is embedded in an agarose matrix, and thin sections are obtained using a vibratome. These sections are then placed on permeable membrane inserts within a microplate and cultured in a slice culture maintenance media for long-term maintenance.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Pre-slicing Preparation 

	...

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Neuronal Culture Techniques

Advanced Neuronal Models

A Hyaluronic Acid-Based Hydrogel for 3-Dimensional Culture of Glioblastoma Cells

Source: Xiao, W.,&#160;et al. <a href="https://app.jove.com/v/58176">Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells.</a> J. Vis. Exp. (2018).
This video demonstrates how gliomaspheres, derived from brain tumors, are separated into single cells and encapsulated in a hyaluronic acid-based hydrogel. This process promotes cell-cell interactions, forming a structured, three-dimensional glioblastoma model.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Thiolation of Hyaluronic Acid
...

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

Summary

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Chapters in this video

A 3D Spheroid Model for Glioblastoma

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

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

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

A Protocol for Explant Cultures of IDH1-mutant Diffuse Low-grade Gliomas

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

Generating and Maintaining Slice Cultures from Human Glioblastoma Tissue

A Hyaluronic Acid-Based Hydrogel for 3-Dimensional Culture of Glioblastoma Cells