Name: evaporation-reducing culture condition increases the reproducibility of multicellular spheroid formation in microtiter plates
Uploaded: 2017-03-07T14:00:00
Description: Watch this Scientific Journal Video about Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates at JoVE.com

This work was supported by grants from the Czech Ministry of Education, Youth, and Sports (LO1304) and the Technological Agency of the Czech Republic (TE01020028). The authors would like to thank Dr. Lakshman Varanasi for taking the still images of environmental lids.

1. Preparation of Agarose-coated Plates
<ol>
	<li>Weigh 0.75 g of low-melting point agarose and add it to 100 mL of McCoy&#39;s 5A medium (with or without phenol red) without serum. Heat the solution in a microwave and swirl every 1-2 min to completely dissolve the agarose. Autoclave the solution to sterilize it.</li>
	<li>Cool the autoclaved agarose to about 70 &#176;C and filter it through a 500 mL, 0.22 &#181;m filter top by vacuum filtration in a laminar flow box. Aliquot the 0.75% filtered agarose solution (FAS) i...

<ol>
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M., Coutinho, P., Correia, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+liquid+overlay+technique+to+formulate+heterogenic+3D+co-cultures+models.">Optimization of liquid overlay technique to formulate heterogenic 3D co-cultures models.</a> Biotechnol. Bioeng. 111 (8), 1672-1685 (2014).</li><li>Walzl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+and+Cost+Efficient+Method+to+Avoid+Unequal+Evaporation+in+Cellular+Screening+Assays,+Which+Restores+Cellular+Metabolic+Activity.">A Simple and Cost Efficient Method to Avoid Unequal Evaporation in Cellular Screening Assays, Which Restores Cellular Metabolic Activity.</a> Int. J. Appl. Sci. Technol. 2 (6), 17-25 (2012).</li><li>Berthier, E., Warrick, J., Yu, H., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+evaporation+for+more+robust+microscale+assays.+Part+1.+Volume+loss+in+high+throughput+assays.">Managing evaporation for more robust microscale assays. Part 1. Volume loss in high throughput assays.</a> Lab Chip. 8 (6), 852-859 (2008).</li><li>Zanoni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tumor+spheroid+models+for+in+vitro+therapeutic+screening:+a+systematic+approach+to+enhance+the+biological+relevance+of+data+obtained.">3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained.</a> Sci. Rep. 6, 19103 (2016).</li><li>Zimmermann, H. F., John, G. T., Trauthwein, H., Dingerdissen, U., Huthmacher, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Evaluation+of+Oxygen+and+Water+Permeation+through+Microplate+Sealing+Tapes.">Rapid Evaluation of Oxygen and Water Permeation through Microplate Sealing Tapes.</a> Biotechnol. Prog. 19 (3), 1061-1063 (2003).</li><li>Sirenko, O., Mitlo, T., Hesley, J., Luke, S., Owens, W., Cromwell, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Content+Assays+for+Characterizing+the+Viability+and+Morphology+of+3D+Cancer+Spheroid+Cultures.">High-Content Assays for Characterizing the Viability and Morphology of 3D Cancer Spheroid Cultures.</a> Assay Drug Dev. Technol. 13 (7), 402-414 (2015).</li><li>Chen, W., Wong, C., Vosburgh, E., Levine, A. J., Foran, D. J., Xu, E. 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Coating 384-Well TC Plates with Filtered Agarose
The standard practice in LOT is to use a 1-1.5% low-melting point agarose to coat the plates, which requires the agarose and/or dispensing unit to be kept heated to prevent the gelling of the agarose6. The gelling of the agarose is of potential concern while preparing multiple plates using liquid dispensing cassettes with small tubing apertures ranging between 0.2 and 0.4 mm in diameter. To overcome the potential issue of cl...

Cancer cells in tumors are physiologically arranged in a complex, 3-dimensional (3D) structure surrounded by extracellular matrix and interacting cells. As nearly all cells in tissues reside in a 3D environment, the need for more physiologically relevant in vitro tumor models that mimic tumor traits has resulted in the development of several 3D culture techniques1,2,3. These models are now becoming fundamental research tools for studying the role of the tumor microenvironment on metastasis and cell response to therapeutics in 3D2. Moreover, compared to 2-dimensional (2D) cell cultures4, 3D models allow for a better understanding of tumor-stroma interactions, which affect cell signaling pathways.
Multicellular tumor spheroids (MCTSes) of cancer cell lines are frequently used in 3D cell culture models due to their relative closeness to in vivo tumors. Out of the several techniques in use, the liquid-overlay technique (LOT) of MCTS generation on agarose-coated plates has gained significant interest for lead optimization and target validation5,6,7,8,9,10,11. This is evident from the recent studies that were successfully able to run pilot screens of compound libraries in MCTS cultures using LOT6,7. However, well-to-well variability in MCTS morphology and growth due to the evaporation-induced uneven loss of medium are common hurdles that accompany the LOT using microtiter plates (MPs). Consequently, the formation of non-uniform MCTSes compromises the significance and relevance of data from pharmacological assays8,12,13. In addition to the reproducibility issues, another practical problem that affects LOT-based high-throughput assays is the coating of the MPs with agarose when using automatic liquid dispensing units. Although the dispensing unit can be kept heated to prevent the gelling of agarose, the clogging of the dispensing cassette and tubing is a potential concern for robotic systems6.
To overcome some of these challenges, we have recently devised a few modifications in the LOT for MCTS culture8. These modifications are mainly based on possible ways to prevent uneven medium loss from the MPs using instruments that are commonly found in high-throughput screening laboratories. A detailed procedure of the modified LOT for the generation of uniformly sized and reproducible MCTSes across 384-well plates (WPs) is presented here. The manuscript also presents a semi-automated routine for the evaluation of MCTS size, particularly in partially disintegrated, drug-treated MCTSes that do not have a clearly defined boundary for the measurement of cross-sectional area.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
			<td>Low-melting</td>
		</tr>
		<tr>
			<td>McCOY&#39;s 5A Medium</td>
			<td>Sigma-Aldrich</td>
			<td>M8403</td>
			<td></td>
		</tr>
		<tr>
			<td>&#8220;rapid&#8221; Filtermax filter</td>
			<td>TPP</td>
			<td>99505</td>
			<td>0.22 &#956;m, 500 mL</td>
		</tr>
		<tr>
			<td>Multidrop&#8482; Combi Reagent Dispenser&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Tube Dispensing cassette&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>24073295</td>
			<td>Metal tip&#160;</td>
		</tr>
		<tr>
			<td>384-well TC plate&#160;</td>
			<td>PerkinElmer</td>
			<td>6057308</td>
			<td>Plate type- CellCarrier</td>
		</tr>
		<tr>
			<td>Standard Tube Dispensing Cassette</td>
			<td>Thermo Fisher Scientific</td>
			<td>24072670</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroClime Environmental Lid</td>
			<td>Labcyte</td>
			<td>LLS-0310</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Incubator (SteriStore )</td>
			<td>HighRes Biosolutions</td>
			<td>23641</td>
			<td>Serial No.: D00384</td>
		</tr>
		<tr>
			<td>Microplate Washer Dispenser&#160;</td>
			<td>BioTek</td>
			<td>Unspecified</td>
			<td>Model: EL406&#160;</td>
		</tr>
		<tr>
			<td>High-Content Imaging System (CellVoyager )</td>
			<td>Yokogawa Electric Corporation</td>
			<td>Unspecified</td>
			<td>Model: CV7000</td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>New England Biolabs</td>
			<td>B7022S</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE&#8482; Express recombinant cell dissociation reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604021</td>
			<td>Phenol red free</td>
		</tr>
	</tbody>
</table>

evaporation-reducing culture condition increases the reproducibility of multicellular spheroid formation in microtiter plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium, particularly from peripheral wells, is a frequently encountered issue in MPs with small culture volumes. Substantially improved culture conditions, such as incubators with well-controlled temperature/humidification systems and evaporation-reducing MPs and plate lids, reduce the significant loss of medium across wells8. To measure the relative evaporation, equal volumes of Orange G (OG) were added to each well, and the change in OG absorba...

Watch this Scientific Journal Video about Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates at JoVE.com

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

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