This work was funded by start-up funds provided to Dr. Silviya P Zustiak by Saint Louis University as well as by a seed grant from the Henry and Amelia Nasrallah Center for Neuroscience at Saint Louis University awarded to Dr. Silviya P Zustiak.

1. Solutions preparation
<ol>
	<li>Preparation of polydimethylsiloxane (PDMS) precursor solution
	<ol>
		<li>Prepare the negative PDMS precursor solution (also used for the glue precursor solution). Scoop the elastomer into a weigh boat using a spatula and weigh it. Add the curing agent to the elastomer base at a 1:10 ratio. Mix the PDMS and curing agent gently and thoroughly using the spatula in the plastic weigh boat. 
		NOTE: This PDMS precursor solution is poured into the 6-well square pyramid...

Fabrication Followed by 3D Encapsulation of Tumor Spheroids in PEG Hydrogels

<ol>
	<li>Hirschhaeuser, 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=Multicellular+tumor+spheroids:+an+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: an underestimated tool is catching up again.</a> Journal of Biotechnology. 148 (1), 3-15 (2010).</li><li>Costa, E. C., de Melo-Diogo, D., Moreira, A. F., Carvalho, M. P., Correia, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroids+formation+on+non-adhesive+surfaces+by+liquid+overlay+technique:+Considerations+and+practical+approaches.">Spheroids formation on non-adhesive surfaces by liquid overlay technique: Considerations and practical approaches.</a> Biotechnology Journal. 13 (1), 1700417 (2018).</li><li>Li, Y., Kumacheva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+microenvironments+for+cancer+spheroid+growth+and+drug+screening.">Hydrogel microenvironments for cancer spheroid growth and drug screening.</a> Science Advances. 4 (4), eaas8998 (2018).</li><li>Kamatar, A., Gunay, G., Acar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+and+synthetic+biomaterials+for+engineering+multicellular+tumor+spheroids.">Natural and synthetic biomaterials for engineering multicellular tumor spheroids.</a> Polymers. 12 (11), 2506 (2020).</li><li>Wang, C., Tong, X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+3D+brain+tumor+model+to+elucidate+the+effects+of+matrix+stiffness+on+glioblastoma+cell+behavior+using+PEG-based+hydrogels.">Bioengineered 3D brain tumor model to elucidate the effects of matrix stiffness on glioblastoma cell behavior using PEG-based hydrogels.</a> Molecular Pharmaceutics. 11 (7), 2115-2125 (2014).</li><li>Nakod, P. S., Kim, Y., Rao, S. S. <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+biomimetic+hyaluronic+acid+hydrogels+to+investigate+glioblastoma+stem+cell+behaviors.">Three-dimensional biomimetic hyaluronic acid hydrogels to investigate glioblastoma stem cell behaviors.</a> Biotechnology and Bioengineering. 117 (2), 511-522 (2020).</li><li>Xiao, W., 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=Bioengineered+scaffolds+for+3D+culture+demonstrate+extracellular+matrix-mediated+mechanisms+of+chemotherapy+resistance+in+glioblastoma.">Bioengineered scaffolds for 3D culture demonstrate extracellular matrix-mediated mechanisms of chemotherapy resistance in glioblastoma.</a> Matrix Biology. 85, 128-146 (2020).</li><li>Pedron, 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=Hyaluronic+acid-functionalized+gelatin+hydrogels+reveal+extracellular+matrix+signals+temper+the+efficacy+of+erlotinib+against+patient-derived+glioblastoma+specimens.">Hyaluronic acid-functionalized gelatin hydrogels reveal extracellular matrix signals temper the efficacy of erlotinib against patient-derived glioblastoma specimens.</a> Biomaterials. 219, 119371 (2019).</li><li>Hill, L., Bruns, J., Zustiak, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+matrix+presence+and+composition+influence+drug+responses+of+encapsulated+glioblastoma+spheroids.">Hydrogel matrix presence and composition influence drug responses of encapsulated glioblastoma spheroids.</a> Acta Biomaterialia. 132, 437-447 (2021).</li><li>Chen, J. -. W. E., 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=Crosstalk+between+microglia+and+patient-derived+glioblastoma+cells+inhibit+invasion+in+a+three-dimensional+gelatin+hydrogel+model.">Crosstalk between microglia and patient-derived glioblastoma cells inhibit invasion in a three-dimensional gelatin hydrogel model.</a> Journal of Neuroinflammation. 17 (1), 346 (2020).</li><li>Thakuri, P. S., Liu, C., Luker, G. D., Tavana, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials-based+approaches+to+tumor+spheroid+and+organoid+modeling.">Biomaterials-based approaches to tumor spheroid and organoid modeling.</a> Advanced Healthcare Materials. 7 (6), 1700980 (2018).</li><li>Shin, 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=Alginate-marine+collagen-agarose+composite+hydrogels+as+matrices+for+biomimetic+3D+cell+spheroid+formation.">Alginate-marine collagen-agarose composite hydrogels as matrices for biomimetic 3D cell spheroid formation.</a> RSC Advances. 6 (52), 46952-46965 (2016).</li><li>Pradhan, S., Clary, J. M., Seliktar, D., Lipke, E. A. <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+spheroidal+cancer+model+based+on+PEG-fibrinogen+hydrogel+microspheres.">A three-dimensional spheroidal cancer model based on PEG-fibrinogen hydrogel microspheres.</a> Biomaterials. 115, 141-154 (2017).</li><li>Imaninezhad, M., Hill, L., Kolar, G., Vogt, K., Zustiak, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Templated+macroporous+polyethylene+glycol+hydrogels+for+spheroid+and+aggregate+cell+culture.">Templated macroporous polyethylene glycol hydrogels for spheroid and aggregate cell culture.</a> Bioconjugate Chemistry. 30 (1), 34-46 (2018).</li><li>Mirab, F., Kang, Y. J., Majd, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+size-controlled+glioma+spheroids+using+agarose+hydrogel+microwells.">Preparation and characterization of size-controlled glioma spheroids using agarose hydrogel microwells.</a> PLoS One. 14 (1), e0211078 (2019).</li><li>Razian, G., Yu, Y., Ungrin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+large+numbers+of+size-controlled+tumor+spheroids+using+microwell+plates.">Production of large numbers of size-controlled tumor spheroids using microwell plates.</a> Journal of Visualized Experiments: JoVE. 81, e50665 (2013).</li><li>Timmins, N. E., Nielsen, L. K., Hauser, H., Fussenegger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Generation of Multicellular Tumor Spheroids by the Hanging-Drop Method. 140, (2007).</li><li>Zhao, 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=A+3D+printed+hanging+drop+dripper+for+tumor+spheroids+analysis+without+recovery.">A 3D printed hanging drop dripper for tumor spheroids analysis without recovery.</a> Scientific Reports. 9 (1), 19717 (2019).</li><li>Amaral, R. L., Miranda, M., Marcato, P. D., Swiech, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+3D+bladder+tumor+spheroids+obtained+by+forced+floating+and+hanging+drop+methods+for+drug+screening.">Comparative analysis of 3D bladder tumor spheroids obtained by forced floating and hanging drop methods for drug screening.</a> Frontiers in Physiology. 8, 605 (2017).</li><li>Gencoglu, M. 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=Comparative+study+of+multicellular+tumor+spheroid+formation+methods+and+implications+for+drug+screening.">Comparative study of multicellular tumor spheroid formation methods and implications for drug screening.</a> ACS Biomaterials Science &amp; Engineering. 4 (2), 410-420 (2018).</li><li>Zhang, C., Liu, C., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+brain+tissue+based+on+microstructure.">Mechanical properties of brain tissue based on microstructure.</a> Journal of the Mechanical Behavior of Biomedical Materials. 126, 104924 (2021).</li><li>Alifieris, C., Trafalis, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+multiforme:+Pathogenesis+and+treatment.">Glioblastoma multiforme: Pathogenesis and treatment.</a> Pharmacology &amp; Therapeutics. 152, 63-82 (2015).</li><li>Zustiak, S. P., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrolytically+degradable+poly+(ethylene+glycol)+hydrogel+scaffolds+with+tunable+degradation+and+mechanical+properties.">Hydrolytically degradable poly (ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties.</a> Biomacromolecules. 11 (5), 1348-1357 (2010).</li><li>Zustiak, S. P., Wei, Y., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-hydrogel+interactions+in+tissue+engineering:+Mechanisms+and+applications.">Protein-hydrogel interactions in tissue engineering: Mechanisms and applications.</a> Tissue Engineering Part B: Reviews. 19 (2), 160-171 (2013).</li><li>Kroger, S. 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=Design+of+hydrolytically+degradable+polyethylene+glycol+crosslinkers+for+facile+control+of+hydrogel+degradation.">Design of hydrolytically degradable polyethylene glycol crosslinkers for facile control of hydrogel degradation.</a> Macromolecular Bioscience. 20 (10), 2000085 (2020).</li><li>Raeber, G., Lutolf, M., Hubbell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecularly+engineered+PEG+hydrogels:+a+novel+model+system+for+proteolytically+mediated+cell+migration.">Molecularly engineered PEG hydrogels: a novel model system for proteolytically mediated cell migration.</a> Biophysical Journal. 89 (2), 1374-1388 (2005).</li><li>Bruns, J., Egan, T., Mercier, P., Zustiak, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+spheroid+growth+and+chemotherapeutic+responses+in+single+and+dual-stiffness+hydrogels.">Glioblastoma spheroid growth and chemotherapeutic responses in single and dual-stiffness hydrogels.</a> Acta Biomaterialia. 163, 400-414 (2023).</li><li>Kuwajima, T., 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=ClearT:+a+detergent-and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue.">ClearT: a detergent-and solvent-free clearing method for neuronal and non-neuronal tissue.</a> Development. 140 (6), 1364-1368 (2013).</li><li>Holt, S. E., Ward, E. S., Ober, R. J., Alge, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shooting+for+the+moon:+using+tissue-mimetic+hydrogels+to+gain+new+insight+on+cancer+biology+and+screen+therapeutics.">Shooting for the moon: using tissue-mimetic hydrogels to gain new insight on cancer biology and screen therapeutics.</a> MRS Communications. 7 (3), 427-441 (2017).</li><li>Jain, E., Scott, K. M., Zustiak, S. P., Sell, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+polyethylene+glycol-based+hydrogel+microspheres+through+electrospraying.">Fabrication of polyethylene glycol-based hydrogel microspheres through electrospraying.</a> Macromolecular Materials and Engineering. 300 (8), 823-835 (2015).</li><li>Raghavan, 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=Comparative+analysis+of+tumor+spheroid+generation+techniques+for+differential+in+vitro+drug+toxicity.">Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity.</a> Oncotarget. 7 (13), 16948 (2016).</li><li>Lee, K. -. H., Kim, T. -. 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Hydrogel-based multicellular tumor spheroid models are increasingly being developed to advance cancer therapeutic discoveries11,13,29. They are beneficial because they emulate key parameters of the tumor microenvironment in a controlled manner and, despite their complexity, are simpler and cheaper to use than in vivo models, and many are compatible with high-throughput screening technologies. The hydrogel biomaterials can be tun...

Tumor spheroids have emerged as useful in vitro tools in studying cancer etiology, pathology, and drug responsiveness1. Traditionally, spheroids have been cultured in conditions such as low adhesion plates or bioreactors, where cell-cell adhesion is favored over cell-surface adhesion2. However, it is now recognized that to recapitulate the tumor microenvironment more faithfully, in vitro spheroid models should capture both cell-cell and cell-matrix interactions. This has prompted multiple groups to design scaffolds, such as hydrogels, where spheroids can be encapsulated3,4. Such hydrogel-based spheroid models enable the elucidation of cell-cell and cell-matrix interactions on various cell behaviors, such as viability, proliferation, stemness, or therapy responsiveness3.
Here, we describe a protocol for the encapsulation of glioblastoma spheroids in polyethylene glycol (PEG) hydrogels. There are multiple literature reports of glioblastoma cell spheroid encapsulation in hydrogels. For example, spheroids were formed by encapsulating U87 cells in PEG hydrogels decorated with an RGDS adhesive ligand and crosslinked with an enzymatically cleavable peptide to determine the effect of hydrogel stiffness on cell behavior5. U87 cells have also been formed in other PEG-based or hyaluronic acid-based hydrogels to expand the cancer stem cell population6 or to explore matrix-mediated mechanisms of chemotherapy resistance7,8,9. Glioblastoma spheroids have also been encapsulated in gelatin hydrogels to study the crosstalk between microglia and cancer cells and its effect on cell invasion10. Overall, such studies have demonstrated the utility of hydrogel-based in vitro models in understanding glioblastoma pathology and devising treatments.
Further, there are different methods for tumor spheroid fabrication and hydrogel encapsulation11. For example, dispersed cells could be seeded in hydrogels and allowed to form spheroids over time5,12. One drawback of such a method is the polydispersity of the formed spheroids, which could lead to differential cell responses. To produce uniform spheroids, cells could be encapsulated in microgels and cultured for extended periods until they invade and remodel the gel13, or cells could be deposited in templated gels with spherical &#39;holes&#39; and allowed to aggregate14. The drawback of these methods is their relative complexity, the need for a droplet generator or other means to form microgels or the &#39;holes&#39; in the gel, and the time it takes for spheroids to grow and mature. Alternatively, spheroids could be pre-formed in microwells9,15,16 or in hanging-drop plates17,18 and then encapsulated in a hydrogel, similar to the technique described here. These methods are simpler and can be done in a higher throughput fashion. Interestingly, it has been shown that the method of spheroid formation can affect spheroid cell behaviors, such as gene expression, cell proliferation, or drug responsiveness19,20.
Here, we focus on glioblastoma since it is a solid tumor whose native environment is the soft, nanoporous brain matrix21, which can be mimicked by a soft, nanoporous hydrogel. Glioblastoma is also the deadliest brain cancer for which there is no available cure22. However, the protocol described here can be used for the encapsulation of spheroids representing any solid tumor. We chose to use PEG hydrogels that are formed through a Michael-type addition reaction23. PEG is a synthetic, non-degradable, and biocompatible hydrogel that is inert and serves as scaffolding and physical cell support but does not support cell attachment23. Cell adhesiveness can be added separately via tethering of whole proteins or adhesive ligands24, and degradability can be added via chemical modifications of the PEG polymer chain or hydrolytically or enzymatically degradable crosslinkers25,26. This allows for biochemical properties to be tuned independently of mechanical or physical hydrogel properties, which could be advantageous in studying cell-matrix interactions. The Michael-type gelation chemistry is selective and happens at physiological conditions; hence, it allows for spheroid encapsulation by simply mixing the spheroids with the hydrogel precursor solution.
Overall, the methodology presented here has several notable characteristics. First, fabricating tumor spheroids in a multiwell assembly is efficient, quick, and the cost of the required materials is low. Second, the spheroids are produced in large batches in a variety of sizes with low polydispersity. Finally, only commercially available materials are required. The utility of the methodology is illustrated by exploring the effect of substrate properties on spheroid cell viability, circularity, and cell stemness.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>70% Ethanol</td>
			<td>Fisher Scientific&#160;</td>
			<td>LC22210-4</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conicals</td>
			<td>FALCON</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Plate Ultra Low Attachment plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-602</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri Dish</td>
			<td>Amazon</td>
			<td>706011</td>
			<td></td>
		</tr>
		<tr>
			<td>4-arm poly(ethylene glycol)-acrylate (4-arm PEG-Ac; 10 kDa)</td>
			<td>Laysan Bio</td>
			<td>ACRL-PEG-ACRL-10K-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conicals</td>
			<td>Fisher Scinetific</td>
			<td>3181345107</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well AggreWell 400&#160;</td>
			<td>StemCell Technologies, Vancouver, Canada</td>
			<td>34421</td>
			<td>Square pyramidal microwells&#160;</td>
		</tr>
		<tr>
			<td>anti-adherence rinsing solution</td>
			<td>StemCell Technologies, Vancouver, Canada</td>
			<td>Cat #: 07010</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspartic Acid-Arginine-Cysteine-Glycine-Valine-Proline-Methionine-Serine-Methionine-Arginine-Glycine-Cysteine-Arginine- Aspartic Acid (DRCG-VPMSMR-GCRD) peptide</td>
			<td>Genic Bio, Shanghai, China</td>
			<td>n/a</td>
			<td>Custom synthesis</td>
		</tr>
		<tr>
			<td>Chemical Fume Hood</td>
			<td>KEWAUNEE</td>
			<td>99151</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel Basement Membrane Matrix, LDEV Free&#160;</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole, dihydrochloride)</td>
			<td>Thermo Scientific</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr>
			<td>Detergent - Triton-X</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td>Nonionic surfactant</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pipettes (1 mL, 2 mL, 5 mL, 10 mL, 25 mL, 50 mL)</td>
			<td>Fisher Scinetific</td>
			<td>1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH30073-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 37% Solution</td>
			<td>Sigma Aldrich</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Plates</td>
			<td>Slumpys</td>
			<td>GBS4100SFSL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Transfer Pipettes</td>
			<td>Fisher Scinetific</td>
			<td>5 3/4&#34;: 1367820A, 9&#34;:136786B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine-Arginine-Cysteine-Aspartic Acid-Arginine-Glycine-Aspartic Acid-Serine (GRCD-RGDS) peptide</td>
			<td>Genic Bio, Shanghai, China</td>
			<td>n/a</td>
			<td>Custom synthesis</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Bright-Line</td>
			<td>383684</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic solution - Repel Silane&#160;</td>
			<td>GE Healthcare Bio-Sciences</td>
			<td>17-1332-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>NUAIRE</td>
			<td>NU-8500</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope (Axiovert 25)</td>
			<td>Zeiss</td>
			<td>663526</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen DiOC16(3) (3,3&#39;-Dihexadecyloxacarbocyanine Perchlorate)</td>
			<td>Fisher Scientific&#160;</td>
			<td>D1125</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Confocal SP8</td>
			<td>Leica Microsystems Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light and Flourescent Microscope (Axiovert 200M)</td>
			<td>Zeiss</td>
			<td>3820005619</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>2 mL: 02681258</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Software</td>
			<td>Zeiss</td>
			<td>AxioVision Rel. 4.8.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin Alexa Fluor 594&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-23927</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>PARAFILM&#160;</td>
			<td>PM992</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1x), pH 7.4</td>
			<td>HyClone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>MP Biomedicals</td>
			<td>1670046</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Aid</td>
			<td>Drummond Scientific Co.</td>
			<td>P-76864</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (1&#8211;200 &#181;L, 101&#8211;1000 &#181;L)</td>
			<td>Fisher Scinetific</td>
			<td>2707509</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Standard Disposable Transfer Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-9D</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Weigh Boats (100 mL)</td>
			<td>Amazon&#160;</td>
			<td>mdo-azoc-1030</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(ethylene glycol)-dithiol (PEG-diSH; 3.4 kDa)</td>
			<td>Laysan Bio</td>
			<td>SH-PEG-SH-3400-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]</td>
			<td>Elsworth Adhesives</td>
			<td>3097358-1004</td>
			<td>Polydimethylsiloxane</td>
		</tr>
		<tr>
			<td>Powder Free Examination Gloves</td>
			<td>Quest</td>
			<td>92897</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide, 1 mg/mL aqueous soln.&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>AAJ66584AB</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium (1x)</td>
			<td>HyClone</td>
			<td>SH30027-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone spacers - Silicone sheet, 0.5 mm thick/13 cm x 18 cm</td>
			<td>Grace Bio-Labs</td>
			<td>JTR-S-0.5</td>
			<td></td>
		</tr>
		<tr>
			<td>SOX2 Alexa Fluor 488&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-365823</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>NUAIRE</td>
			<td>NU-425-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine, &#8805;99.0% (GC)&#160;</td>
			<td>Sigma Aldrich</td>
			<td>90279</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1x)&#160;</td>
			<td>Sigma Aldrich</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>U-87 MG human glioblastoma cells</td>
			<td>American Type Culture Collection&#160;</td>
			<td>HTB-14</td>
			<td></td>
		</tr>
	</tbody>
</table>

tumor spheroid fabrication and encapsulation in polyethylene glycol hydrogels for studying spheroid-matrix interactions

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Author Spotlight: In Vitro Hydrogel Model for Glioblastoma Microenvironment Study

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Our lab focuses on designing in vitro hydrogel models for glioblastoma steroid encapsulation to mimic the tumor extracellular microenvironment. The effective substrate, mechanical and biochemical properties on steroid cell viability, circularity, stemness, and spreading are investigated with such models. Current experimental challenges in tumor module development revolve around a desire for complexity to mimic all aspects of the tumor microenvironment for physiological relevance, and also a desire for simplicity to make the model accessible, robust, low cost, and easy to use, and compatible with multiplex screening approaches.We present a protocol that enables fast, robust, and low-cost fabrication of tumor steroids and encapsulation in hydrogels made to mimic the tumor microenvironment. The model does not require any specialized equipment. It will be particularly useful for exploring steroid matrix interactions in building in vitro tissue physiology or pathology models.Understanding the interactions between glioblastoma cancer cells and their microenvironment can encourage development of new therapies to better treat this cancer. Furthermore, development of an in vitro model for glioblastoma cancer can enhance development of this model for other cancer diseases.

Spheroid-based drug screening platforms to study chemotherapeutic effects are increasingly sought after due to the emphasis on modulating the tumor microenvironment upon spheroid encapsulation in biomaterials replicating native tissue. Here we developed a method for multicellular tumor spheroid preparation and subsequent encapsulation and imaging in a 3D hydrogel. The spheroids are prepared in microwell molds (Figure 3A,B), which result in spheroids with spherical shapes and...

Watch this Scientific Journal Video about Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions at JoVE.com

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth.  Here, we ...

tumor-spheroid-fabrication-encapsulation-polyethylene-glycol