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>

<ol>
	<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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+backscatter+microscopy+images+the+internal+structure+of+living+tumour+spheroids.">Ultrasound backscatter microscopy images the internal structure of living tumour spheroids.</a> Nature. 330 (6147), 493-495 (1987).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Hanging+Drop+Cell+Culture+Protocol+for+Generation+of+3D+Spheroids.">A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids.</a> J. Vis. Exp. (51), 2720 (2011).</li><li>Naber, H. P. 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 border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<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 <span id="docs-internal-guid-263ae0a0-aa3d-bddd-2a36-2ae1901f1604" 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>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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17.3K Views.  University of Calgary. 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.

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

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

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 ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was pretreated with NaOH and washed to a neutral pH. Cellulase was added to the pretreated bamboo powder to generate the hydrolysate, which contained 30 g/L glucose and 15 g/L xylose and was used as the carbon source to prepare a medium for chemical production. When cultured in microaerobic conditions, 12.7 g/L 2,3-butanediol was produced by wildtype K. pneumoniae. In aerobic conditions, 13.0 g/L R-acetoin was produced by the budC mutant of K. pneumoniae. A mixture of 25.5 g/L 2-ketogluconic acid and 13.6 g/L xylonic acid was produced by the budA mutant of K. pneumoniae in a two-stage, pH-controlled fermentation with high air supplementation. In the first stage of fermentation, the culture was maintained at a neutral pH; after cell growth, the fermentation proceeded to the second stage, during which the culture was allowed to become acidic.

Production of Chemicals by Klebsiella pneumoniae Using Bamboo Hydrolysate as Feedstock

Bamboo powder was pretreated with NaOH and enzymatically hydrolyzed. The hydrolysate of bamboo was used as the feedstock for 2,3-butanediol, R-acetoin, 2-ketogluconic acid, and xylonic acid production by Klebsiella pneumoniae.

Bamboo is an important biomass, and bamboo hydrolysate is used by Klebsiella pneumoniae as a feedstock for chemical production. Here, bamboo powder was ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...