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>
	<li>Stupp, R., 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=Radiotherapy+plus+concomitant+and+adjuvant+temozolomide+for+glioblastoma.">Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma.</a> New England Journal of Medicine. 352 (10), 987-996 (2005).</li><li>Heffernan, J. M., Sirianni, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+microenvironmental+regulation+of+glioblastoma+stem+cells:+a+biomaterials+perspective.">Modeling microenvironmental regulation of glioblastoma stem cells: a biomaterials perspective.</a> Frontiers in Materials. 5, 7 (2018).</li><li>Zanders, E. D., Svensson, F., Bailey, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapy+for+glioblastoma:+is+it+working.">Therapy for glioblastoma: is it working.</a> Drug Discovery Today. 24 (5), 1193-1201 (2019).</li><li>Hubert, C. G., 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+Three-dimensional+organoid+culture+system+derived+from+human+glioblastomas+recapitulates+the+hypoxic+gradients+and+cancer+stem+cell+heterogeneity+of+tumors+found+in+vivo.">A Three-dimensional organoid culture system derived from human glioblastomas recapitulates the hypoxic gradients and cancer stem cell heterogeneity of tumors found in vivo.</a> Cancer Research. 76 (8), 2465-2477 (2016).</li><li>Lathia, J. D., Heddleston, J. M., Venere, M., Rich, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deadly+teamwork:+neural+cancer+stem+cells+and+the+tumor+microenvironment.">Deadly teamwork: neural cancer stem cells and the tumor microenvironment.</a> Cell Stem Cell. 8 (5), 482-485 (2011).</li><li>Meyer, 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=Single+cell-derived+clonal+analysis+of+human+glioblastoma+links+functional+and+genomic+heterogeneity.">Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (3), 851-856 (2015).</li><li>Bao, 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=Stem+cell-like+glioma+cells+promote+tumor+angiogenesis+through+vascular+endothelial+growth+factor.">Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor.</a> Cancer Research. 66 (16), 7843-7848 (2006).</li><li>Yu, S. C., Bian, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+cancer+stem+cells+based+on+heterogeneity+of+invasiveness.">Enrichment of cancer stem cells based on heterogeneity of invasiveness.</a> Stem Cell Reviews and Reports. 5 (1), 66-71 (2009).</li><li>Bao, 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=Glioma+stem+cells+promote+radioresistance+by+preferential+activation+of+the+DNA+damage+response.">Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.</a> Nature. 444 (7120), 756-760 (2006).</li><li>Vescovi, A. L., Galli, R., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumour+stem+cells.">Brain tumour stem cells.</a> Nature Reviews. Cancer. 6 (6), 425-436 (2006).</li><li>Zhu, T. 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. 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. A., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cerebral+organoids+from+human+pluripotent+stem+cells.">Generation of cerebral organoids from human pluripotent stem cells.</a> Nature Protocols. 9 (10), 2329-2340 (2014).</li><li>Sundar, S. 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=Three-dimensional+organoid+culture+unveils+resistance+to+clinical+therapies+in+adult+and+pediatric+glioblastoma.">Three-dimensional organoid culture unveils resistance to clinical therapies in adult and pediatric glioblastoma.</a> Translational Oncology. 15 (1), 101251 (2021).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ...

maintaining-human-glioblastoma-cellular-diversity-ex-vivo-using-three

Research

JoVE Journal

Cancer Research

3.1K 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

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

We describe a protocol for the production, culture, and visualization of human cancers, which have metastasized to the peritoneal surfaces. Resected tumor specimens are cut using a vibratome and cultured on permeable inserts for increased oxygenation and viability, followed by imaging and downstream analyses using confocal microscopy and flow cytometry.

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

Establishing a 3D Ex Vivo Model for Ovarian Cancer-Omentum Interaction

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

Author Spotlight: Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...