This work was funded by the University of Calgary, under a new investigator start-up grant to Dr. Ungrin.

NOTE: Microwell plates are available in different formats, depending on the desired results. Specifications as well as approximate guidelines for the minimum and maximum numbers of cells that should be used with each format are shown in Table 1. The smaller microwells taper to a sharp point, and thus there is no lower size limit, although variability between spheroids becomes more significant at smaller sizes. Routine production of spheroids from an average of as little as 20 cells each is straightforward8. The larger microwell size is not fully tapered, therefore attempts to form spheroids from small numbers of cells may result in multiple smaller spheroids in each microwell. Due to slight differences in surface properties, preparation with the surfactant solution (step 1.2) is not optional when using AggreWell 400Ex plates.
This protocol is based on the use of AggreWell 400 plates - for other formats consult Table 1. The number of cells to be loaded in each well is determined by multiplying the number of microwells it contains by the number of cells to be clustered for each spheroid. For example, to generate spheroids from 1000 cells apiece, each well must be loaded with (1,200 spheroids times 1,000 cells each = ) 1.2 x 106 cells. Cell density must be calculated given that the cells will be loaded in a volume of 0.4 ml. So for the example of spheroids formed from 1000 cells each, 1.2 x 106 cells in 0.4 ml translates to a density of 3 x 106 cells per ml.
1. Preparing Microwells to Receive Cells
<ol>
	<li>Microwell plates are sterile as provided, and should only be removed from their packaging in a sterile environment such as a laminar-flow biosafety cabinet. Sterility should be maintained throughout the protocol. Should sterility be compromised, the wells are resterilized using 70% ethanol in water using the following optional steps.
	<ol>
		<li>Add 0.5 ml of 70% ethanol to each unused well. Some microwells will trap air bubbles, although this can be reduced by adding liquid at one side, and allowing it to spread across the surface, rather than dropping it vertically onto the surface. Replace lid.</li>
		<li>While ethanol vapor should quickly permeate any bubbles, if there are large numbers it may be desirable to remove them. Centrifuge for 2 min at 2,000 x g. Note that it is important to ensure the rotor is properly balanced at this speed.</li>
		<li>Using a low-magnification inverted microscope, verify bubbles have been removed from the microwells.</li>
		<li>Incubate for 5 min with the lid on at room temperature.</li>
		<li>Aspirate ethanol. Plate can now be washed with sterile water or PBS for immediate use, or air-dried for storage.</li>
	</ol>
	</li>
	<li>In order to ensure that cells do not interact with the microwell surface, it may be prepared using a surfactant. In general it is advisable to perform this step initially, and subsequently compare spheroids generated with and without surfactant-coated microwells.
	<ol>
		<li>Add 0.5 ml of Rinsing Solution to each well. Some microwells will trap air bubbles, although this can be reduced by adding liquid at one side, and allowing it to spread across the surface, rather than dropping it vertically onto the surface. Replace lid.</li>
		<li>Centrifuge for 2 min at 2,000 x g to remove bubbles. Note that it is important to ensure the rotor is properly balanced at this speed.</li>
		<li>Using a low-magnification inverted microscope, verify bubbles have been removed from the microwells.</li>
		<li>Incubate for at least thirty minutes with the lid on at room temperature, or overnight at 4 &#176;C. Plates may be stored at four degrees with the surfactant solution in the wells until needed if properly sealed to prevent drying out.</li>
		<li>Wash plate with sterile water or PBS immediately prior to use. Do not allow plates to dry out after coating.</li>
	</ol>
	</li>
</ol>
2. Preparing Cells
NOTE: The details of cell preparation will vary somewhat with the specific cell type being studied. However we have observed these conditions to be effective with a broad range of cells, including the lines discussed here, as well as human and murine embryonic stem cells (ESC), human induced pluripotent stem cells (iPSC), fibroblasts, putative primitive endoderm, and stromal cells. Other groups have employed this system successfully for a wide range of applications including chondrogenesis from mesenchymal stem cells9, standardized generation of neural precursors from pluripotent stem cells10, toxicological analyses in hepatocytes11 and assessment of spinal cord regeneration in salamanders12. The specific protocol for working with HT29 cells is shown here.
<ol>
	<li>Start with a T75 flask of HT29 cells, cultured in DMEM + 10% FBS</li>
	<li>Aspirate growth medium from flask, and add 3 ml of trypsin.</li>
	<li>Incubate cells for 5 min at 37 &#176;C.</li>
	<li>Stop trypsin digestion by adding 3 ml of growth medium. Take an aliquot for cell counting and transfer the remainder into a 15 ml centrifuge tube.</li>
	<li>Centrifuge for 5 min at 200 x g.</li>
	<li>While the centrifugation is in progress, count cells.</li>
	<li>Aspirate trypsin/medium mixture and replace with fresh growth medium, volume determined by the required cell density as calculated above.</li>
	<li>(OPTIONAL) Pass the cell suspension through a strainer in order to remove any clumps. NOTE: Cell harvesting conditions will vary somewhat between lines. If a dissociation protocol for e.g. passaging the cells of interest is available, that should be used as a starting point. With sensitive cell lines use of trypsin may result in excessive cell death. In these cases we have observed good results using TrypLE as an alternative dissociation reagent8. If the cells become clumped during dissociation and lose viability, addition of DNAse to the enzyme solution may resolve this. Reducing dissociation times and employing a trituration step to break up cell clumps can also increase viability.</li>
</ol>
3. Spheroid Formation
NOTE: Spheroids may be generated in a variety of medium formulations, however initial trials should be carried out using the medium in which the cells were cultured, to distinguish consequences of transitioning to a 3-dimensional culture system from consequences of changing medium composition.
<ol>
	<li>Add 0.4 ml of growth medium to each well.</li>
	<li>Centrifuge for 2 min at 2,000 x g to remove bubbles. Note that it is important to ensure the rotor is properly balanced at this speed prior to centrifugation.</li>
	<li>Using a low-magnification inverted microscope, verify bubbles have been removed from the microwells.</li>
	<li>Add 0.4 ml cell suspension at the required density (calculated above). Gently mix by pipetting up and down, without introducing air bubbles into the microwells. It is important to ensure that the cells are evenly distributed throughout the well, in order to obtain consistent spheroids.</li>
	<li>Centrifuge for 5 min at 200 x g. If the plate does not self-level during centrifugation, convection currents may arise that result in uneven distribution of cells across the microwells. Therefore, the plate should be balanced internally as well as against the other plate, so that the weight is distributed evenly to both sides of the plate carrier.</li>
</ol>
NOTE: If evidence of uneven cell distribution is seen, it may be necessary to apply a small amount of lubrication to the pivot points on the plate carrier - consult with centrifuge manufacturer for instructions.
NOTE: Once the cells have been centrifuged into the microwells, they are reasonably resistant to washing out from minor motions of the plate. Abrupt movements that result in sloshing of the medium should be avoided, but transfer of the plate from centrifuge to microscope or incubator should not result in significant cell displacement.
<ol start="6">
	<li>Using an inverted microscope, verify that cells have clustered within the microwells, and that they are evenly distributed across the well.</li>
	<li>Incubate overnight at 37 &#176;C.</li>
	<li>Using an inverted microscope, observe the process of spheroid formation. Some cell lines will form compact spheroids within 24 hr, others may take longer. The progression from cluster to aggregate in HT29 cells is shown in Figure 2.</li>
	<li>Recover spheroids from the microwells and transfer to suspension culture by gently jetting them out with a pipette. Alternatively, maintain spheroids in situ with medium replacement8 - the duration depends on their initial size and growth rate, which define the time until they outgrow the microwells in which they were formed.
	<ol>
		<li>To replace medium without losing spheroids, aspirate medium at the edge of the well using a Pasteur pipette. Slowly bring the tip down until it begins to draw liquid from the meniscus, and follow the meniscus down as it drops. Then add fresh medium against the well wall in the same place. A few spheroids may be lost, however if medium is always aspirated and replaced in the same position, this number will remain small in comparison to the large numbers in the well.</li>
	</ol>
	</li>
</ol>

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	<li>Hirschhaeuser, F., Menne, H., Dittfeld, C., West, J., Mueller-Klieser, W., Kunz-Schughart, L. A. <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>Sherar, M. D., Noss, M. B., Foster, F. 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H., Wiercinska, E., Ten Dijke, P., Van Laar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+Assay+to+Measure+TGF-&#946;-induced+Invasion.">Spheroid Assay to Measure TGF-&#946;-induced Invasion.</a> J. Vis. Exp. (57), e3337 (2011).</li><li>Ungrin, M. D., Joshi, C., Nica, A., Bauwens, C., Reproducible Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ultra+high-throughput+formation+of+multicellular+organization+from+single+cell+suspension-derived+human+embryonic+stem+cell+aggregates.">ultra high-throughput formation of multicellular organization from single cell suspension-derived human embryonic stem cell aggregates.</a> PLoS ONE. 3 (2), e1565 (2008).</li><li>Bauwens, C. L., Song, 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=Geometric+control+of+cardiomyogenic+induction+in+human+pluripotent+stem+cells.">Geometric control of cardiomyogenic induction in human pluripotent stem cells.</a> Tissue Engineering. Part A. 17 (15-16), 1901-1909 (2011).</li><li>Bratt-Leal, A. M., Carpenedo, R. L., Ungrin, M. D., Zandstra, P. W., McDevitt, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+biomaterials+in+multicellular+aggregates+modulates+pluripotent+stem+cell+differentiation.">Incorporation of biomaterials in multicellular aggregates modulates pluripotent stem cell differentiation.</a> Biomaterials. 32 (1), 48-56 (2011).</li><li>Ungrin, M. D., Clarke, 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=Rational+bioprocess+design+for+human+pluripotent+stem+cell+expansion+and+endoderm+differentiation+based+on+cellular+dynamics.">Rational bioprocess design for human pluripotent stem cell expansion and endoderm differentiation based on cellular dynamics.</a> Biotechnology and Bioengineering. 109 (4), 853-866 (2012).</li><li>Markway, B. D., Tan, G. K., Brooke, G., Hudson, J. E., Cooper-White, J. J., Doran, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+chondrogenic+differentiation+of+human+bone+marrow-derived+mesenchymal+stem+cells+in+low+oxygen+environment+micropellet+cultures.">Enhanced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in low oxygen environment micropellet cultures.</a> Cell Transplantation. 19 (1), 29-42 (2010).</li><li>Kozhich, O., Hamilton, R., Mallon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+Generation+and+Differentiation+of+Neural+Precursor+Cells+from+Human+Pluripotent+Stem+Cells.">Standardized Generation and Differentiation of Neural Precursor Cells from Human Pluripotent Stem Cells.</a> Stem Cell Reviews and Reports. , 1-6 (2012).</li><li>Fey, S. J., Wrzesinski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Drug+Toxicity+Using+3D+Spheroids+Constructed+from+an+Immortal+Human+Hepatocyte+Cell+Line.">Determination of Drug Toxicity Using 3D Spheroids Constructed from an Immortal Human Hepatocyte Cell Line.</a> Toxicological Sciences. , (2012).</li><li>Mchedlishvili, L., Mazurov, V., 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=Reconstitution+of+the+central+and+peripheral+nervous+system+during+salamander+tail+regeneration.">Reconstitution of the central and peripheral nervous system during salamander tail regeneration.</a> Proceedings of the National Academy of Sciences. , (2012).</li><li>Butler, C. M., Foty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Aggregate+Cohesion+by+Tissue+Surface+Tensiometry.">Measurement of Aggregate Cohesion by Tissue Surface Tensiometry.</a> J. Vis. Exp. (50), e2739 (2011).</li><li>Xu, K., Buchsbaum, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mammary+Epithelial+Cells+from+Three-dimensional+Mixed-cell+Spheroid+Co-culture.">Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture.</a> J. Vis. Exp. (62), e3760 (2012).</li><li>Baraniak, P. R., Cooke, M. T., Saeed, R., Kinney, M. A., Fridley, K. M., McDevitt, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffening+of+human+mesenchymal+stem+cell+spheroid+microenvironments+induced+by+incorporation+of+gelatin+microparticles.">Stiffening of human mesenchymal stem cell spheroid microenvironments induced by incorporation of gelatin microparticles.</a> Journal of the Mechanical Behavior of Biomedical Materials. 11, 63-71 (2012).</li><li>Purpura, K. A., Bratt-Leal, A. M., Hammersmith, K. A., McDevitt, T. C., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+engineering+of+3D+pluripotent+stem+cell+niches+to+guide+blood+development.">Systematic engineering of 3D pluripotent stem cell niches to guide blood development.</a> Biomaterials. , (2012).</li></ol>

Dr. Ungrin has a financial interest in the AggreWell technology as an inventor.

We have established a system whereby large numbers of uniform spheroids may be generated from multiple cell lines from different sources. We have yet to encounter an adherent cell line that does not form spheroids under these conditions. We have previously observed cell loss in populations prone to anoikis5,8, however to date this issue has not arisen with tumor lines. The system is arbitrarily scalable with surface area, with behavior consistent across microwells in 24-well and 6-well format, as well as prototype bioreactors containing 50,000 microwells each presently under development.
Should spheroid asymmetry be a concern, the incubation time in step 4.8 may be increased to two or three days. Spheroids may also be incubated for a period after extraction from the microwells to increase symmetry, however in this case care must be taken to keep culture densities sufficiently low, as spheroids in contact with one another will often fuse into larger structures. For this reason, we have previously maintained cultures within the microwells in which the aggregates were formed, with the primary limitation being the size of the spheroid. This in turn is a function of both growth rate and initial size8, and if extended culture within the microwell plate is planned, this should be considered in advance. Options to prevent overgrowth, should it occur, include either starting with smaller spheroids, or employing the larger microwells of the AggreWell 800 plate.
Should spheroids fail to increase in coherence over time, one potential cause may be cell death. Particularly in larger aggregates of highly metabolically active cells, mass transport limitations on the delivery of oxygen and essential nutrients can result in a necrotic core2 - thus a non-cohesive spheroid may simply be a consequence of excessive cell death. Alternatively, measurements of the mechanical cohesion of spheroids have been used to assess intercellular binding forces, in relationship to the metastatic potential of a given cell line13. It would be interesting to investigate the relationship between spheroid shape and metastatic potential, perhaps using morphometric parameters such as roundness and perimeter to area ratio.
In addition to investigating the mechanical and morphometric properties of spheroids, assembly in the microwell system permits large-scale production of mixed-composition spheroids, consisting of combinations of multiple cell types and / or biomaterials6,7. The interactions of tumor cells with other cell types are important to more closely model the behavior of tumors in vivo14, thus it may be of interest to generate spheroids from tumor cells in combination with fibroblasts and endothelial cells, for example. Microparticles of various biomaterials may also be incorporated, and can affect spheroid properties both directly through their interactions with cells7,15, and also as reservoirs for the controlled release of growth factors and cytokines into the interior of the spheroid16.
If there is any concern about sterility, for example when working with an microwell plate in which some wells have previously been used, that may have spent some time in an incubator as part of a previous experiment, the wells may be resterilized with 70% ethanol in water.
Once spheroids have been formed, they may also be separated from residual unincorporated cells by passing the suspension over a cell strainer. Individual cells will pass through, while the spheroids will be retained. If the spheroid is suspected of shedding potentially metastatic cells over the course of culture, it may be desirable to perform this procedure multiple times - initially to remove unincorporated cells, and subsequently to isolate purified populations of the cells given off by the spheroids.

There is an increasing body of evidence that tumor cells behave differently in three dimensional cultures than they do when cultured on plastic, and that therapeutic agents identified on conventional tissue-culture platforms may lose efficacy when transitioned to a more physiologically-relevant system1. It is therefore desirable to study the behavior of cancer cells under these conditions, both to gain insights into their underlying biology, and also to increase the success rate of transitioning new therapeutic agents from the screening facility to the clinic. One useful model system with a long history employs three-dimensional clusters of cancer cells known as tumor spheroids1,2. Ideally, techniques for spheroid formation would permit production of large numbers of uniform spheroids whose size and composition are controlled by the experimenter. While hanging-drop and well-plate approaches3,4 are able to fulfill some of these requirements, throughput is generally limited, and generation of large numbers of spheroids becomes a labor-intensive task.
We have recently developed a system to resolve similar challenges in the regenerative medicine field5. Employing forced aggregation within a series of densely packed micron-scale wells (see Figure 1), this approach permits the generation of spheroids from arbitrary numbers of cells, including mixtures of multiple cell types6, as well as the incorporation of various biomaterials7. Spheroids are formed in large numbers - thousands to hundreds of thousands or more - and may be extracted immediately&#160;or maintained in the microwells in which they were formed with medium exchange for at least one8 to two (unpublished observation) weeks. This system is therefore well suited to the generation of large numbers of uniform and reproducible tumor spheroids for the assessment of the effectiveness of novel antitumor agents&#160;or basic biological investigations.

<table><tbody><tr><td>AggreWell 400 plate</td><td>StemCell Technologies Inc.</td><td>27845/27945</td><td></td></tr><tr><td>Rinsing Solution</td><td>StemCell Technologies Inc.</td><td>07010</td><td></td></tr><tr><td>Cell strainer (37 &lt;span id=&quot;docs-internal-guid-263ae0a0-aa3d-bddd-2a36-2ae1901f1604&quot; style=&quot;font-size:15px;font-family:Arial;color:#000000;background-color:#ffffff;font-weight:normal;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;&quot;&gt;&amp;micro;m&lt;/span&gt;)</td><td>StemCell Technologies Inc.</td><td>27215</td><td></td></tr><tr><td>PBS</td><td>VWR / LONZA</td><td>CA12001-676</td><td></td></tr><tr><td>Trypsin-Versene (EDTA)</td><td>VWR / LONZA</td><td>CA12001-660</td><td></td></tr><tr><td>DMEM</td><td>VWR / LONZA</td><td>CA12002-212</td><td></td></tr><tr><td>FBS</td><td>VWR / LONZA</td><td>CA-95042-112</td><td></td></tr><tr><td>TrypLE</td><td>Invitrogen / Life Technologies</td><td>12605010</td><td></td></tr><tr><td>Inverted microscope</td><td>VWR / Motic</td><td>CA19000-610</td><td></td></tr><tr><td>Allegra X15R centrifuge with carriers for standard well plates</td><td>VWR / Beckman</td><td>CABKA99465, CABK369704, CABK392806</td><td></td></tr><tr><td>Laminar flow biosafety cabinet</td><td>ESBE / Baker</td><td>BKR-SG603AHEUVSP</td><td></td></tr></tbody></table>

production of large numbers of size-controlled tumor spheroids using microwell plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

&#160;Spheroids from multiple tumor lines of differing anatomical origins may readily be generated and extracted into suspension culture. Figure 3 illustrates the consistent size control obtainable via this method, with highly uniform spheroid populations under each condition. Clear inter-line differences in behavior are also visible, with HT29 colon cancer cells and TE6 esophageal cancer cells forming densely packed spheroids with sharply defined boundaries, while LNCaP prostate cancer cells gave rise to less coherent spheroids with irregular boundaries (see Discussion for possible reasons). The stronger internal forces in the more coherent aggregates also resulted in collapse to a more symmetrical form even while still in the microwells in which they were formed, whereas the LNCaP aggregates, particularly at the larger size, visibly retain the square-pyramidal geometry of the microwells. The relationship between input cell numbers and the physical size of the resulting spheroid will depend on a number of variables. Theoretical volumes based on the product of the volume per cell and the number of cells employed may be reduced by cell loss, which in turn may include loss of cells during the single-cell suspension stage, as well as loss from excessively small aggregates due to insufficient numbers of neighbors, and from excessively large aggregates due to transport limitations and necrosis in the core. Size will also be influenced by any proliferation that may occur during the aggregation process. As these parameters will vary from cell line to cell line, if spheroids of specific physical dimensions are desired the correct number of cells to introduce must be determined experimentally. As a first approximation, spheroid diameter is expected to increase with the cube root of the number of cells incorporated - e.g. an 8-fold increase in number of cells should result in a doubling of spheroid diameter.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50665/50665fig1highres.jpg" src="/files/ftp_upload/50665/50665fig1.jpg" /> 
Figure 1. Schematic of spheroid production. The microwell system consists of an array of tightly packed square-pyramidal microwells (625 per cm2 for the 400 <span id="docs-internal-guid-263ae0a0-aa35-f9c7-69a1-8c5aa197db53" style="font-size:15px;font-family:Arial;color:#000000;background-color:#ffffff;font-weight:normal;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;">&#181;m size), into which cells may be centrifuged. The cells are brought together by the sloping sidewalls, and the size of the resulting spheroid is controlled by varying the density of the cell suspension employed.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50665/50665fig2highres.jpg" src="/files/ftp_upload/50665/50665fig2.jpg" /> 
Figure 2. Assembly of clustered cells into spheroids. Spheroids were formed from seven hundred HT29 cells each. Immediately after centrifugation (A), cells are loosely clustered in the bottom of each microwell. Note that the clusters are uniformly offset towards the lower right in this case. This is acceptable provided cluster sizes remain consistent across the array. If significant cluster size differences are seen from one side of the array to the other, see Note in step 3.5. After incubation for 24 hr (B), intercellular adhesive forces have caused the clusters to aggregate and form coherent spheroids, which may then be extracted (C).
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50665/50665fig3highres.jpg" src="/files/ftp_upload/50665/50665fig3.jpg" /> 
Figure 3. Spheroids generated in microwell plates. Spheroids were formed from seven hundred (A, C, E) or one thousand five hundred (B, D, F) HT29 colon cancer cells (A, B), LNCaP prostate cancer cells (C, D) or TE6 esophageal cancer cells (E, F). Note the consistency of spheroids within a preparation, and also the variation between cell lines - in particular the loose LNCaP aggregates as compared to the more densely packed HT29 and TE6 cells. Scale bar represents 200 &#956;m.
<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Microwell format</td>
		</tr>
		<tr>
			<td></td>
			<td>AggreWell 400</td>
			<td>AggreWell 40EX</td>
			<td>AggreWell 800</td>
		</tr>
		<tr>
			<td>Microwell width (<span id="docs-internal-guid-263ae0a0-aa38-43ed-6004-6ff57626f41a" style="font-size:15px;font-family:Arial;color:#000000;background-color:#ffffff;font-weight:normal;font-style:normal;font-variant:normal;text-decoration:none;vertical-align:baseline;">&#181;m)</td>
			<td>400</td>
			<td>400</td>
			<td>800</td>
		</tr>
		<tr>
			<td>Microwells per cm2</td>
			<td>625</td>
			<td>625</td>
			<td>156.25</td>
		</tr>
		<tr>
			<td>Microwells per well</td>
			<td>1,200</td>
			<td>4,700</td>
			<td>300</td>
		</tr>
		<tr>
			<td>Minimum cells per microwell*</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>2000</td>
		</tr>
		<tr>
			<td>Maximum cells per microwell*</td>
			<td>2,000</td>
			<td>2,000</td>
			<td>10,000</td>
		</tr>
		<tr>
			<td>Minimum cells per well*</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>6 x 105</td>
		</tr>
		<tr>
			<td>Maximum cells per well*</td>
			<td>2.4 x 106</td>
			<td>9.4 x 106</td>
			<td>3 x 106</td>
		</tr>
		<tr>
			<td>1.1.1 - 70% Ethanol volume (ml)</td>
			<td>0.5</td>
			<td>2.0</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>1.2.1 - Rinsing solution volume (ml)</td>
			<td>0.5</td>
			<td>2.0</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>3.2 - Medium preload volume (ml)</td>
			<td>0.4</td>
			<td>1.6</td>
			<td>0.4</td>
		</tr>
		<tr>
			<td>3.5 - Cell loading volume (ml)</td>
			<td>0.4</td>
			<td>1.6</td>
			<td>0.4</td>
		</tr>
		<tr>
			<td colspan="4">*Numbers of cells per microwell are only an approximation as this value will vary with the size of the specific cells employed, and should be determined empirically</td>
		</tr>
	</tbody>
</table>
Table 1. AggreWell options and protocol modifications.

Watch this Scientific Journal Video about Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates at JoVE.com

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

17.4K Views.  University of Calgary.  We introduceren een protocol voor het aanleggen van grote aantallen (duizenden tot honderdduizenden) van gelijke grootte-en-samenstelling gecontroleerde bolvormige tumoren, met behulp van in de handel verkrijgbare microputjesplaten. 

Video: Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Cancer Research

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

Biochemistry

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

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

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 ...

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

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