This research was supported by the Basic Science Research Program (2016R1D1A1B03934418) and the Bio &#38; Medical Technology Development Program (2018M3A9H1023141) of the NRF, and funded by the Korean government, MSIT.

1. Centrifugal microfluidic-based spheroid (CMS) culture chip fabrication
<ol>
	<li>Make PC molds for the top and bottom layers of the CMS culture chip by CNC machining. Detailed dimensions of the chip are given in Figure 1.</li>
	<li>Mix PDMS base and PDMS curing agent at a ratio of 10:1 (w/w) for 5 min and place in a desiccator for 1 h to remove air bubbles.</li>
	<li>After pouring the PDMS mixture into the molds of the CMS culture chip, remove air bubbles for 1 h more and cure in a heat...

<ol>
	<li>Ravi, M., Paramesh, V., Kaviya, S. R., Anuradha, E., Paul Solomon, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+cell+culture+systems:+Advantages+and+applications.">3D cell culture systems: Advantages and applications.</a> Journal of Cellular Physiology. 230 (1), 16-26 (2015).</li><li>Tung, Y. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+three-dimensional+multicellular+spheroid+culture+for+biomedical+research.">Recent advances in three-dimensional multicellular spheroid culture for biomedical research.</a> Biotechnology Journal. 3 (9-10), 1172-1184 (2008).</li><li>Cesarz, Z., Tamama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+Culture+of+Mesenchymal+Stem+Cells.">Spheroid Culture of Mesenchymal Stem Cells.</a> Stem Cells International. 2016, (2016).</li><li>Li, Y., 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=Three-dimensional+spheroid+culture+of+human+umbilical+cord+mesenchymal+stem+cells+promotes+cell+yield+and+stemness+maintenance.">Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance.</a> Cell and Tissue Research. 360, 297-307 (2015).</li><li>Yamaguchi, Y., Ohno, J., Sato, A., Kido, H., Fukushima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+spheroids+exhibit+enhanced+in-vitro+and+in-vivo+osteoregenerative+potential.">Mesenchymal stem cell spheroids exhibit enhanced in-vitro and in-vivo osteoregenerative potential.</a> Bmc Biotechnology. 14 (1), 105 (2014).</li><li>Koh, C. 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(110), e1 (2016).</li><li>Lee, 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=All-in-one+centrifugal+microfluidic+device+for+size-selective+circulating+tumor+cell+isolation+with+high+purity.">All-in-one centrifugal microfluidic device for size-selective circulating tumor cell isolation with high purity.</a> Analytical Chemistry. 86 (22), 11349-11356 (2014).</li><li>Gorkin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugal+microfluidics+for+biomedical+applications.">Centrifugal microfluidics for biomedical applications.</a> Lab on a Chip. 10 (14), 1758-1773 (2010).</li><li>Park, J., Lee, G. H., Yull Park, J., Lee, J. C., Kim, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypergravity-induced+multicellular+spheroid+generation+with+different+morphological+patterns+precisely+controlled+on+a+centrifugal+microfluidic+platform.">Hypergravity-induced multicellular spheroid generation with different morphological patterns precisely controlled on a centrifugal microfluidic platform.</a> Biofabrication. 9 (4), (2017).</li><li>Rocca, 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=Barium+titanate+nanoparticles+and+hypergravity+stimulation+improve+differentiation+of+mesenchymal+stem+cells+into+osteoblasts.">Barium titanate nanoparticles and hypergravity stimulation improve differentiation of mesenchymal stem cells into osteoblasts.</a> International Journal of Nanomedicine. 10, 433-445 (2015).</li><li>Genchi, G. 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The CMS is a closed system in which all injected cells enter the microwell without waste, making it more efficient and economical than conventional microwell-based spheroid generation methods. In the CMS system, the media is replaced every 12&#8211;24 h through a suction hole designed to remove the media in the chip (Figure 3A). During the media suction process, barely any media escapes from inside the microwell due to the surface tension between the media and the wall of the microwell. A us...

It is easier to simulate biological in vivo microenvironments with three-dimensional (3D) spheroid cell culture than with two-dimensional (2D) cell culture (e.g., conventional Petri dish cell culture) to produce more physiologically realistic experimental results1. Currently available spheroid formation methods include the hanging drop technique2, liquid-overlay technique3, carboxymethyl cellulose technique4, magnetic force-based microfluidic technique5, and the use of bioreactors6. Although each method has its own benefits, further improvement in reproducibility, productivity, and generating coculture spheroids is necessary. For example, while the magnetic force-based microfluidic technique5 is relatively inexpensive, the effects of strong magnetic fields on living cells must be carefully considered. The benefits of spheroid culture, particularly in the study of mesenchymal stem cell differentiation and proliferation, have been reported in several studies7,8,9.
The centrifugal microfluidic system, also known as lab-on-a-CD (compact disc), is useful for easily controlling the fluid inside and exploiting the rotation of the substrate and has thus been utilized in biomedical applications such as immunoassays10, colorimetric assays for detecting biochemical markers11, nucleic acid amplification (PCR) assays, automated blood analysis systems12, and all-in-one centrifugal microfluidic devices13. The driving force controlling the fluid is the centripetal force created by rotation. Additionally, multiple functions of mixing, valving, and sample splitting can be done simply in this single CD platform. However, compared to the above-mentioned biochemical analysis methods, there have been fewer trials applying CD platforms to culture cells, especially spheroids14.
In this study, we show the performance of the centrifugal microfluidic-based spheroid (CMS) system by monoculture or coculture of human adipose-derived stem cells (hASC) and human lung fibroblasts (MRC-5). This paper describes in detail our group&#39;s research methodology15. Thus, the spheroid culture lab-on-a-CD platform can be easily reproduced. A CMS generating system comprising a CMS culture chip, a chip holder, a DC motor, a motor mount, and a rotating platform, is presented. The motor mount is 3D printed with acrylonitrile butadiene styrene (ABS). The chip holder and rotating platform are CNC (computer numerical control) machined with the PC (polycarbonate). The rotational speed of the motor is controlled from 200 to 4,500 rpm by encoding a PID (proportional-integral-derivative) algorithm based on pulse-width modulation. Its dimensions are 100 mm x 100 mm x 150 mm and it weighs 860 g, making it easy to handle. Using the CMS system, spheroids can be generated under various gravity conditions from 1 x g to 521 x g, so the study of cell differentiation promotion under high gravity can be extended from 2D cells16,17 to 3D spheroid. Coculture of various types of cells is also a key technology for effectively mimicking the in vivo environment18. The CMS system can easily generate monoculture spheroids, as well as coculture spheroids of various structure types (e.g., concentric, Janus, and sandwich). The CMS system can be utilized not only in simple spheroid studies but also in 3D organoid studies, to consider human organ structures.

<table border="0" cellpadding="0" cellspacing="0" style="width:1056px;" width="1056">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">3D printer</td>
			<td style="width:171px;">Cubicon</td>
			<td style="width:124px;">3DP-210F</td>
			<td style="width:416px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Adipose-derived mesenchymal stem cells (hASC)</td>
			<td style="width:171px;">ATCC</td>
			<td style="width:124px;">PCS-500-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Antibiotic-Antimycotic</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:124px;">15240-062</td>
			<td>Contained 1% of completed medium and buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">CellTracker Green CMFDA</td>
			<td style="width:171px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">C2925</td>
			<td>10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">CellTracker Red CMTPX</td>
			<td style="width:171px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">C34552</td>
			<td>10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Computer numerical control (CNC) rotary engraver</td>
			<td style="width:171px;">Roland DGA</td>
			<td style="width:124px;">EGX-350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">DC motor</td>
			<td style="width:171px;">Nurielectricity Inc.</td>
			<td style="width:124px;">MB-4385E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Dimethylsulfoxide (DMSO)</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:124px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Dulbecco&#39;s modified eaggle&#39;s medium (DMEM)</td>
			<td style="width:171px;">ATCC</td>
			<td style="width:124px;">30-2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Dulbecco&#39;s phosphate buffered saline (D-PBS)</td>
			<td style="width:171px;">ATCC</td>
			<td style="width:124px;">30-2200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Fetal bovine serum</td>
			<td style="width:171px;">ATCC</td>
			<td style="width:124px;">30-2020</td>
			<td>Contained 10% of completed medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">human lung fibroblasts (MRC-5)</td>
			<td style="width:171px;">ATCC</td>
			<td style="width:124px;">CCL-171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Inventor 2019</td>
			<td style="width:171px;">Autodesk</td>
			<td style="width:124px;"></td>
			<td>3D computer-aided design program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Petri dish &#934; 150 mm</td>
			<td style="width:171px;">JetBiofill</td>
			<td style="width:124px;">CAD010150</td>
			<td>Surface Treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Plasma cleaner</td>
			<td style="width:171px;">Harrick Plasma</td>
			<td style="width:124px;">PDC-32G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Pluronic F-127</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:124px;">11/6/9003</td>
			<td>Dilute with phosphate buffered saline to 4% (w/v) solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Polycarbonate (PC)</td>
			<td style="width:171px;">Acrylmall</td>
			<td style="width:124px;">AC15PC</td>
			<td>200 x 200 x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:171px;">Dowcorning</td>
			<td style="width:124px;">Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:345px;">Trypsin</td>
			<td style="width:171px;">Gibco</td>
			<td style="width:124px;">12604021</td>
			<td></td>
		</tr>
	</tbody>
</table>

lab-on-a-cd platform for generating multicellular three-dimensional spheroids

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 &#956;m microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

The 6 cm diameter CMS culture chip (Figure 2) was successfully made following the above protocol. First, the chip was made separately from a top layer and a bottom layer and then bonded together by plasma bonding. Resulting spheroids can be easily gathered by detaching the chip. The channel of the CMS culture chip comprises an inlet port and central, slide, and microwell regions (Figure 3). The cell, medium, and pluronic solutions are injected through an inlet h...

Watch this Scientific Journal Video about Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids at JoVE.com

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

This technique can be used to generate cell spheroids from individual and multiple cell types using a lab-on-a-CD platform, a pioneering system for the rapid generation of spheroids. The technique uses centrifugal force to deposit cells into designated microwells, allowing the sequential elation of multiple cell types for the production of diverse spheroids. For CMS culture chip fabrication, first make polycarbonate molds for the top and bottom layers of the culture chip by computer numerical control machining.Next mix PDMS base and PDMS curing agent at a 10 to one ratio for five minutes before placing the mixture in a desiccator for one hour to remove air bubbles. Pour the degassed PDMS mixture into the CMS culture chip molds, and place the molds into the desiccator for one more hour to remove any air bubbles. At the end of the second degassing, cure the mixture in a heat chamber at 80 degrees Celsius for two hours before placing the chips in a vacuumed plasma cleaner with the surfaces to be bonded facing up.Expose the culture chips to air-assisted plasma at a power of 18 watts for 30 seconds, and bond the two layers of the chip in the heat chamber at 80 degrees Celsius for 30 minutes to increase the adhesion strength. Then sterilize the CMS culture chip in an autoclave at 121 degrees Celsius for 15 minutes. To prepare the cells for spheroid formation, thaw a one-milliliter vial containing five to 10 times 10 to the five cells of interest in a 36.5 degrees Celsius water bath for two minutes before adding one milliliter of DMEM to the cells with gentle mixing.Next add the cells to a 150-millimeter diameter Petri dish containing 15 milliliters of 36.5-degree-Celsius medium, and place the dish in a cell culture incubator for 24 hours. The next day, replace the supernatant with 15 milliliters of fresh DMEM, and return the cells to the incubator. To detach the cells, create the culture with four milliliters of trypsin for four minutes at 36.5 degrees Celsius and 5%carbon dioxide, stopping the reaction with fresh medium when the cells have lifted from the bottom of the dish.To label the cells, warm an appropriate cell fluorescence dye to room temperature before adding 20 microliters of anhydrous dimethyl sulfoxide to the vial to obtain a one millimolar solution. Dilute the fluorescence to a final working concentration of one micromolar in DMEM, and add the dye to the cell suspension of interest with gentle mixing. Then place the cells in the cell culture incubator for 20 minutes.For monoculture spheroid formation, first add 2.5 milliliters of 4%Pluronic F-127 solution to the inlet hole of the CMS culture chip while rotating the chip at 500 to 1, 000 rotations per minute. When all of the solution has been added, rotate the chip at 4, 000 rotations per minute for three minutes using the CMS system. At the end of the rotation, incubate the culture chip overnight at 36.5 degrees Celsius at 5%carbon dioxide.The next morning, remove the Pluronic solution, and wash the culture chip with DMEM. After drying the chip for 12 hours on a clean bench, add 2.5 milliliters of the medium to the inlet port, and rotate the chip at 4, 000 rotations per minute for three minutes. At the end of the rotation, replace 100 microliters of the medium in the inlet port with 100 microliters of the prepared cell suspension while the chip rotates at 500 to 1, 000 rotations per minute.Then rotate the chip at 3, 000 rotations per minute for three minutes to trap the cells in each microwell by centrifugal force before placing the chip in the cell culture incubator for three days, refreshing the medium as demonstrated every day. For the generation of concentric spheroid coculture, add the first population of cells in 100 microliters of medium to the inlet port of the chip while the chip is rotating at 500 to 1, 000 rotations per minute followed by three minutes of rotation at 3, 000 rotations per minute. At the end of the rotation, add 100 microliters of the second cell population while the chip is rotating at 500 to 1, 000 rotations per minute, and rotate the chip at 3, 000 rotations per minute for another three minutes.When both volumes of cells have been injected, place the chip into the cell culture incubator at 1, 000 to 2, 000 rotations per minute for 24 hours. For the Janus spheroid formation, add 100 microliters of the first population of cells while the chip is rotating at 500 to 1, 000 rotations per minute followed by three minutes at 3, 000 rotations per minute. At the end of the rotation, place the chip in the cell culture incubator for rotation at 1, 000 to 2, 000 rotations per minute for three hours.At the end of the incubation, add 100 microliters of the second population of cells while the chip rotates at 500 to 1, 000 rotations per minute before rotating the chip at 3, 000 rotations per minute for three minutes. Then place the cells in the cell culture incubator at 1, 000 to 2, 000 rotations per minute for 24 hours. For sandwich spheroid formation, first add 100 microliters of the first cell population while the chip is rotating at 500 to 1, 000 rotations per minute followed by three minutes of rotation at 3, 000 rotations per minute.At the end of the rotation, place the chip on a rotator in the cell culture incubator for two hours at 1, 000 to 2, 000 rotations per minute before adding 100 microliters of the second population of cells while the chip is rotating at 500 to 1, 000 rotations per minute. Rotate the chip at 3, 000 RPM for three minutes, and place the chip back into the incubator for a three-hour incubation with rotation. Then add an additional 100 microliters of the first cell population while the chip rotates at 500 to 1, 000 rotations per minute, and rotate and culture the cells as just demonstrated for 12 hours.Following the protocol as demonstrated, a six-centimeter diameter CMS culture chip can be successfully fabricated. The channel of the CMS culture chip comprises an inlet port, and central, slide, and microwell regions. Time-lapse images of two types of cell culture taken at 2, 000 rotations per minute for the first 24 hours of culture within individual CMS chips, as demonstrated, show the successful formation of spheroids.Imaging of the microwells after three days of culture show that the spheroids demonstrate an excellent uniformity and sphericity. Cocultured spheroids with conentric, Janus, and sandwich structures can also be generated. In addition, cell cultures exposed to high gravity for seven days, followed by live/dead staining, demonstrate that most cells survive long-term culture within the chips.The top and bottom layers of the CMS culture chip should be carefully aligned when they are bonded, otherwise the number of cells in each microwell may not be equal. This study can lower the entry barrier for coculture spheroid research and can be widely used in coculture studies, for example, for screening new drugs of interest.

lab-on-cd-platform-for-generating-multicellular-three-dimensional

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JoVE Journal

Bioengineering

6.2K visualizações.  Chung-Ang University. Essa técnica pode ser usada para gerar esferoides celulares de tipos individuais e múltiplos de células usando uma plataforma lab-on-a-CD, um sistema pioneiro para a rápida geração de esferoides. A técnica utiliza força centrífuga para depositar células em microwells designadas, permitindo a euforia sequencial de vários tipos de células para a produção de diversos esferoides. Para a fabricação de chips de cultura CMS, primeiro faça moldes de policarbonato para as camadas supe...