The authors acknowledge the support of an infrastructure research grant from Science Foundation Ireland (SFI) (16/RI/3745) to JCS. Work in the UCD Cell Screening Laboratory is supported by the UCD College of Science. ASC is funded by an Irish Research Council (IRC) Government of Ireland Postgraduate Scholarship (GOIPG/2019/68). The authors also thank all members of the lab for their input and helpful discussions. The artwork in Figure 1 was generated in BioRender.

1. Cell culture
<ol>
	<li>Prepare media
	<ol>
		<li>Prepare specific cell culture media depending on the type of cell line. Ensure that all media for cell maintenance contains 10% Foetal Bovine Serum (FBS). 
		NOTE: Different cell lines use different media. HT-29 colon carcinoma cells (ATCC HTB-38) are grown in McCoys 5A + 10% FBS. HepG2 hepatocellular carcinoma cells (ATCC HB-8065) are grown in Minimum Essential Medium + 1% L-glutamine + 10% FBS. H358 bronchoalveolar carcinoma cells (ATCC CRL-580...

<ol>
	<li>Jensen, C., Teng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+time+to+start+transitioning+from+2D+to+3D+cell+culture.">Is it time to start transitioning from 2D to 3D cell culture.</a> Frontiers in Molecular Biosciences. 7, 33 (2020).</li><li>Langhans, 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=Three-dimensional+in+vitro+cell+culture+models+in+drug+discovery+and+drug+repositioning.">Three-dimensional in vitro cell culture models in drug discovery and drug repositioning.</a> Frontiers in Pharmacology. 9, 6 (2018).</li><li>Edmondson, R., Broglie, J. J., Adcock, A. F., Yang, L. <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+cell+culture+systems+and+their+applications+in+drug+discovery+and+cell-based+biosensors.">Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors.</a> Assay and Drug Development Technologies. 12 (4), 207-218 (2014).</li><li>Lv, D., Hu, Z., Lu, L., Lu, H., Xu, X. <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+cell+culture:+A+powerful+tool+in+tumor+research+and+drug+discovery.">Three-dimensional cell culture: A powerful tool in tumor research and drug discovery.</a> Oncology Letters. 14 (6), 6999-7010 (2017).</li><li>Zhang, X., Jiang, T., Chen, D., Wang, Q., Zhang, L. W. <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+liver+models:+state+of+the+art+and+their+application+for+hepatotoxicity+evaluation.">Three-dimensional liver models: state of the art and their application for hepatotoxicity evaluation.</a> Critical Reviews in Toxicology. 50 (4), 279-309 (2020).</li><li>Dutta, D., Heo, I., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease+modeling+in+stem+cell-derived+3D+organoid+systems.">Disease modeling in stem cell-derived 3D organoid systems.</a> Trends in Molecular Medicine. 23 (5), 393-410 (2017).</li><li>Nunes, A. S., Barros, A. S., Costa, E. C., Moreira, A. F., 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=3D+tumor+spheroids+as+in+vitro+models+to+mimic+in+vivo+human+solid+tumors+resistance+to+therapeutic+drugs.">3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs.</a> Biotechnology and Bioengineering. 116 (1), 206-226 (2019).</li><li>Kleinman, H. K., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrigel:+Basement+membrane+matrix+with+biological+activity.">Matrigel: Basement membrane matrix with biological activity.</a> Seminars in Cancer Biology. 15, 378-386 (2005).</li><li>Caliari, S. R., Burdick, 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=A+practical+guide+to+hydrogels+for+cell+culture.">A practical guide to hydrogels for cell culture.</a> Nature Methods. 13 (5), 405-414 (2016).</li><li>Foglietta, F., Canaparo, R., Muccioli, G., Terreno, E., Serpe, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+aspects+and+pharmacological+applications+of+three-dimensional+cancer+cell+cultures+and+organoids.">Methodological aspects and pharmacological applications of three-dimensional cancer cell cultures and organoids.</a> Life Sciences. 254, 117784 (2020).</li><li>Bardsley, K., Deegan, A. J., El Haj, A., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+state-of-the-art+3D+tissue+models+and+their+compatibility+with+live-cell+imaging.">Current state-of-the-art 3D tissue models and their compatibility with live-cell imaging.</a> Advances in Experimental Medicine and Biology. 1035, 3-18 (2017).</li><li>Darrigues, 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=Tracking+gold+nanorods'+interaction+with+large+3D+pancreatic-stromal+tumor+spheroids+by+multimodal+imaging:+Fluorescence,+photoacoustic,+and+photothermal+microscopies.">Tracking gold nanorods' interaction with large 3D pancreatic-stromal tumor spheroids by multimodal imaging: Fluorescence, photoacoustic, and photothermal microscopies.</a> Scientific Reports. 10 (1), 3362 (2020).</li><li>Boutros, M., Heigwer, F., Laufer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopy-based+high-content+screening.">Microscopy-based high-content screening.</a> Cell. 163 (6), 1314-1325 (2015).</li><li>Mysior, M. M., Simpson, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell3:+A+new+vision+for+study+of+the+endomembrane+system+in+mammalian+cells.">Cell3: A new vision for study of the endomembrane system in mammalian cells.</a> Bioscience Reports. , (2021).</li><li>N&#252;rnberg, 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=Routine+optical+clearing+of+3D-cell+cultures:+Simplicity+forward.">Routine optical clearing of 3D-cell cultures: Simplicity forward.</a> Frontiers in Molecular Biosciences. 7 (20), (2020).</li><li>Cutrona, M. B., Simpson, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+High-throughput+automated+confocal+microscopy+platform+for+quantitative+phenotyping+of+nanoparticle+uptake+and+transport+in+spheroids.">A High-throughput automated confocal microscopy platform for quantitative phenotyping of nanoparticle uptake and transport in spheroids.</a> Small. 15 (37), 1902033 (2019).</li><li>Kelly, S., Byrne, M. H., Quinn, S. J., Simpson, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+nanoparticle-induced+toxicity+readouts+with+single+cell+resolution+in+HepG2+multicellular+tumour+spheroids.">Multiparametric nanoparticle-induced toxicity readouts with single cell resolution in HepG2 multicellular tumour spheroids.</a> Nanoscale. 13 (41), 17615-17628 (2021).</li><li>Sirenko, O., Mitlo, T., Hesley, J., Luke, S., Owens, W., Cromwell, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-content+assays+for+characterizing+the+viability+and+morphology+of+3D+cancer+spheroid+cultures.">High-content assays for characterizing the viability and morphology of 3D cancer spheroid cultures.</a> Assay and Drug Development Technologies. 13 (7), 402-414 (2015).</li><li>Redondo-Castro, E., Cunningham, C. J., Miller, J., Cain, S. A., Allan, S. M., Pinteaux, E. <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+human+mesenchymal+stem+cell+3D+spheroids+using+low-binding+plates.">Generation of human mesenchymal stem cell 3D spheroids using low-binding plates.</a> Bio-protocol. 8 (16), (2018).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <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+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nature Methods. 4 (4), 359-365 (2007).</li><li>Eismann, 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=Automated+3D+light-sheet+screening+with+high+spatiotemporal+resolution+reveals+mitotic+phenotypes.">Automated 3D light-sheet screening with high spatiotemporal resolution reveals mitotic phenotypes.</a> Journal of Cell Science. 133 (11), 245043 (2020).</li><li>Alsehli, H., 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=An+integrated+pipeline+for+high-throughput+screening+and+profiling+of+spheroids+using+simple+live+image+analysis+of+frame+to+frame+variations.">An integrated pipeline for high-throughput screening and profiling of spheroids using simple live image analysis of frame to frame variations.</a> Methods. 190, 33-43 (2021).</li><li>Stirling, D. 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=CellProfiler+4:+improvements+in+speed,+utility+and+usability.">CellProfiler 4: improvements in speed, utility and usability.</a> BMC Bioinformatics. 22 (1), 1-11 (2021).</li><li>Renner, H., 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+fully+automated+high-throughput+workflow+for+3D-based+chemical+screening+in+human+midbrain+organoids.">A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids.</a> eLife. 9, 52904 (2020).</li><li>Powley, I. 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=Patient-derived+explants+(PDEs)+as+a+powerful+preclinical+platform+for+anti-cancer+drug+and+biomarker+discovery.">Patient-derived explants (PDEs) as a powerful preclinical platform for anti-cancer drug and biomarker discovery.</a> British Journal of Cancer. 122 (6), 735-744 (2020).</li><li>Collins, A., Miles, G. J., Wood, J., MacFarlane, M., Pritchard, C., Moss, E. <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+explants,+xenografts+and+organoids:+3-dimensional+patient-relevant+preclinical+models+in+endometrial+cancer.">Patient-derived explants, xenografts and organoids: 3-dimensional patient-relevant preclinical models in endometrial cancer.</a> Gynecologic Oncology. 156 (1), 251-259 (2020).</li><li>Miles, G. 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The approach described here details a platform for generating several hundred spheroids per well in a manner that is suitable for HCS and HCA. In comparison to other popular methods, such as the use of flat-bottomed and round-bottomed ULA plates, which allow the formation of only one spheroid per well18,19, this method provides the opportunity for high-resolution information to be extracted from large numbers of spheroids in a screening format. Notably, this meth...

Traditionally, cell-based assays have been performed in monolayers growing on a solid substrate, which effectively can be considered as a two-dimensional (2D) environment. However, it is becoming increasingly recognized that 2D cell culture models lack physiological relevance in some contexts and cannot replicate many of the complex interactions that occur between cells1. Three-dimensional (3D) cell culture methods are rapidly becoming popular among researchers, and 3D cell models show high potential to better mimic the physiological conditions encountered by cells in the tissue environment2. There are several different types of 3D cell assemblies that have been employed, but the two most common types are spheroids and organoids. Spheroids can be grown from many different cell lines, and they can adopt various shapes and sizes depending on the cell type used and their method of assembly3. Moreover, spheroids can also be referred to as multicellular tumor spheroids (MCTS) when they are grown from cancer cell lines, and these models have found particular use for preclinical in vitro drug delivery and toxicity studies4,5. Organoids, on the other hand, aim to better mimic the tissues and organs in our body and can adopt more complex morphological arrangements. The production of organoids involves the use of adult stem cells or pluripotent stem cells, which can be reprogrammed into the appropriate cells to resemble the tissue or organ of interest. They are primarily used to investigate the development of organs and to model diseases and host-pathogen interactions6.
There is a range of different methods used to generate 3D cell assemblies. Scaffold-based methods provide a substrate or support to which cells can either attach or grow within. These scaffolds can have various shapes and can be made from a variety of different materials. The most common are extracellular matrix (ECM) components and hydrogels, and they are designed to resemble the natural extracellular environment of cells and thereby facilitate physiological interactions4,7. ECM basement material has been extracted from Engelbreth-Holm-Swarm mouse sarcoma tumor and shown to contain a rich mixture of ECM components, including laminin, type IV collagen, and perlecan8. However, despite its advantageous composition, there are two main challenges with its use, namely its batch-to-batch variability and that it has two different aggregate states below and above 10 &#176;C8,9. In contrast, hydrogels have the advantage of being flexible with respect to their components and rigidity, and they can be customized to suit the specific 3D cell assembly desired7,10. Scaffold-based methods are essential for organoid growth but are also widely used for spheroids. Scaffold-free methods, which work by preventing cells from attaching to the surface they are growing on, are usually only compatible with spheroid assembly. Examples include ultra-low attachment (ULA) plates, with either a flat bottom or U-bottom, which allow aggregation of the cells into spheroids, or the use of continuous agitation of the cells in spinner/rotation flasks10.
The use of 3D cell assemblies to study a wide variety of biological events is rapidly gaining popularity; however, it is essential that the method chosen for their culture is appropriate and compatible with the plans for their downstream analysis. For example, the use of ULA plates generates spheroids of high consistency; however, this method is restricted to the production of a single spheroid per well, thereby limiting throughput. Particular consideration is needed when fluorescence imaging of the 3D structure is planned. The substrate or plate on which the assembly is grown needs to be optically compatible, and care must be taken to minimize the effects of light scattering caused by any scaffolds that may have been used11. This particular problem becomes more acute as the numerical aperture of the microscope objective lenses increases.
Arguably one of the major reasons for selecting to work with a 3D cell model is to extract volumetric imaging data about not only the entire assembly but also the individual cells within it. MCTS models, in particular, are beginning to prove very powerful for deepening our understanding of how therapeutics transit from the outside to central cells (as they would need to in a tumor)12, and so gaining knowledge from individual cells at different layers is essential. The imaging technology that extracts quantitative information from individual cells is termed high-content analysis (HCA) and is a powerful approach in the context of screening13. To date, HCA has almost exclusively been applied to monolayer cultures, but there is an increasing realization that this approach has the power to be applied to 3D cultures enabling a wide range of cellular functions and processes to be studied14. It would have the clear advantage that large numbers of 3D assemblies could be analyzed, potentially providing cell-level data from across each structure. However, challenges associated with imaging of potentially thick cell assemblies, as well as the large data sets generated, need to be overcome.
In this article, a robust scaffold-based method for the large-scale production of MCTS in a 96-well format is presented. The method facilitates the production of several hundred 3D cell assemblies in each well. Examples are shown for three different cell types, representing solid tumor models of the liver, lung, and colon. The spheroids that form can be of a variety of sizes, and so HCA is used to select structures of a particular size and/or morphology. This feature provides the additional advantage that any phenotypes observed can be compared across spheroids of different sizes, but all treated in the same way in the same well. This approach is compatible with high-resolution imaging, importantly providing both cell-level and subcellular level quantitative data from the same cellular assemblies. This method of spheroid production has the additional advantage over methods that generate a single spheroid per well, that the large numbers of spheroids produced in each well potentially provide sufficient biomass for other downstream analyses, such as transcriptome and proteome profiling.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x), phenol red</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma Aldrich</td>
			<td>A6003</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>10050070</td>
			<td></td>
		</tr>
		<tr>
			<td>CellCarrier-96 Ultra Microplates, tissue culture treated, black, 96-well with lid</td>
			<td>Perkin Elmer</td>
			<td>6055302</td>
			<td>These plates have been renamed as Phenoplates</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum (FBS), qualified, EU approved, South America origin, heat inactivated</td>
			<td>Gibco</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP381-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution, 200 mM</td>
			<td>Gibco</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>14533</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher Scientific</td>
			<td>10647032</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix, Phenol Red-free, LDEV-free, 10 mL</td>
			<td>Corning</td>
			<td>356237</td>
			<td>This Matrigel formulation can be also found with the same catalogue number at BD Biosciences</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced Matrigel</td>
			<td>BD Biosciences</td>
			<td>356231</td>
			<td>This Matrigel formulation can be also found with the same catalogue number at Corning</td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5A medium</td>
			<td>Gibco</td>
			<td>26600023</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5A medium with L glutamine and sodium bicarbonate, without phenol red</td>
			<td>Hyclone</td>
			<td>10358633</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM)</td>
			<td>Gibco</td>
			<td>21090022</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM), without glutamine, without phenol red</td>
			<td>Gibco</td>
			<td>51200046</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-LAMP1 antibody (concentrate)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>H4A3-a</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Hirschmann</td>
			<td>8100203</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunclon tissue culture dish with lid, polystyrene, 92 mm x 17 mm</td>
			<td>ThermoFisher Scientific</td>
			<td>150350</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera Phenix HCS System and Harmony HCA software</td>
			<td>Perkin Elmer</td>
			<td>HCSHH14000000</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) tablets</td>
			<td>Sigma Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>Sigma Aldrich</td>
			<td>P5927</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>61870010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, without glutamine, without phenol red</td>
			<td>Gibco</td>
			<td>11835063</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup sterile vacuum filter units</td>
			<td>Millipore</td>
			<td>SCGVU05RE</td>
			<td></td>
		</tr>
	</tbody>
</table>

a robust method for the large-scale production of spheroids for high-content screening and analysis applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

In this protocol, a robust method to produce 3D cell culture assemblies in the form of spheroids, using different cell types to represent various tumor tissues, is detailed. This method allows for the generation of hundreds of spheroids per well, which enables cell-based assays to be performed in a high-content manner (Figure 1). This approach has previously been used to study nanoparticle uptake in HT-29 spheroids16 and nanoparticle-induced toxicity in HepG2 spheroid...

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A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale ...

This protocol provides a method to produce several hundred multicellular tumorous spheroids in each well of a multi well plate suitable for automated microscopy. Spheroid produced can be categorized into different size classes, and subcellular information can be extracted from every cell. The technique produces sufficient serums that can be used in direct screening and RNA interference studies.Dilute ECM basement material in cold phenol red serum free medium using pre chilled tips kept at minus 20 degrees Celsius. I bet 15 microliters of the ECM basement material and medium solution into a 96 well imaging plate. Centrifuge the plate for 20 minutes at 91 times G at four degrees Celsius.Incubate the plate for no longer than 30 minutes at 37 degrees Celsius. Add trips and EDTA to the cells. Incubate the cells at 37 degrees Celsius at the end of incubation, we suspend the cells in a complete medium containing 10%FBS.Pipette 10 microliters of the cell suspension, into a hemocytometer counting chamber. Count the number of cells in four quadrants of the chamber. Centra views the cells at 135 times G for four minutes at room temperature.One this cells are pelleted, aspirate the supernatant and resuspend the cells in a phenol red free complete medium to obtain a concentration of 10 to the six cells per milliliter. Dilute the cells in a phenol red free complete medium. See the cells dropwise in a circular motion in a total volume of 35 microliters per well.Incubate the cells for one hour at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide. At 1.2 microliters of ECM basement material to 23.8 microliters of phenol red free complete medium per well, resulting in a final volume of 60 microliters per well. Incubate the cells at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide.The following day, replace the medium with 100 microliters of fresh phenol red free complete medium. Insert the 96 well plate containing spheroids, into a confocal high content screening microscope. Select the appropriate lasers to excite the floors used.Acquire the channels sequentially to avoid crosstalk, image the spheroids using appropriate objectives. To analyze the morphology of spheroids, segment the spheroids based on the fluorescently conjugated FLoid and staining. Segment the nuclei based on the hex three, three, three, four, two staining.Segment the cytoplasm of each cell by the residual hex three, three, four, two, stain that is present in the cell cytoplasm. Calculate different morphological properties of the spheroids including volume, surface area, sphericity and the number of nuclei per his spheroids. Ross has images further to remove spheroids smaller than 75 cubic micrometers and a larger than 900, 000 cubic micrometers.To analyze a single cell within spheroids, segment the spheroids based on the fluorescently conjugated spheroid staining. Segment the nuclei based on the hex three, three, three, four, two staining. Segment the cytoplasm of each cell by the residual hex three, three, three, four, two stain that is present in the cell cytoplasm.Segment the lysosomes based on the secondary antibody staining. Calculate different morphological properties of the cells within each spheroid, including the number of lysosomes per cell, cell volume and cell surface area. The figure shows well overviews of three different types of spheroids, including images at different confocal planes.A volumetric view of the entire spheroids is also shown. The figure shows the pH segmentation process, panel A shows the entire spheroids in a volumetric view. Panel B displays the set segmentation of the cytoplasm, the nuclei and the lysosomes within all cells of a single spheroids.The cytoplasm and nuclei are segmented based on the hex three, three, three, four, two staining. And lysosomes are segmented based on the anti lab one antibody staining. Panels C shows the same segmented as panel B, but in a three plain view.Various morphological measurements at the spheroids level can be made. These measurements include calculation of the number of nuclei per spheroids, as well as these Felicity, volume, and surface area of the pH. The typical volume of each cell in the three spheroid types.As well as the number of lysosomes per cell are shown in this figure. Optimizing the initial cell seating density at the start of SP generation is a critical step. The figure shows that adding too many cells still allows spheroids formation.However, this can result in the fusion if spheroids. Spheroids produce in this manner can be subjected to transcriptomic and proteomic analysis. This technique is used to quantitatively analyze the uptake of drug delivery such nano particles and assess their toxicity to individual cells in the spheroids.

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3.0K Views.  University College Dublin. This protocol provides a method to produce several hundred multicellular tumorous spheroids in each well of a multi well plate suitable for automated microscopy. Spheroid produced can be categorized into different size classes, and subcellular information can be extracted from every cell. The technique produces sufficient serums that can be used in direct screening and RNA interference studies.Dilute ECM basement material in cold phenol red serum free medium using pre chilled tips kept...