Name: a paired bead and magnet array for molding microwells with variable concave geometries
Uploaded: 2018-01-28T13:00:00
Description: Watch this Scientific Journal Video about A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries at JoVE.com

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2014R1A1A2057527 and NRF-2016R1D1A1B03934418).

1. Preparation of through-hole array aluminum plate and magnet array
<ol>
	<li>Prepare two 50 mm x 50 mm (or larger) aluminum plates. The thickness of each plate was 300 &#181;m that is half of the bead diameter.</li>
	<li>Form a 30 x 30 through-hole array on one of the aluminum plates by using a CNC rotary engraver with a &#934;550-&#181;m micro drill bit with 30 mm/s of plunge rate and 8000 RPM of spindle speed. The distance between each hole (center to center) was 1 mm (Figure 1a and <s...

<ol>
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F., 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=Concave+microwell+based+size-controllable+hepatosphere+as+a+three-dimensional+liver+tissue+model.">Concave microwell based size-controllable hepatosphere as a three-dimensional liver tissue model.</a> Biomaterials. 32 (32), 8087-8096 (2011).</li><li>Yeon, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+concave+microwells+to+pancreatic+tumor+spheroids+enabling+anticancer+drug+evaluation+in+a+clinically+relevant+drug+resistance+model.">Application of concave microwells to pancreatic tumor spheroids enabling anticancer drug evaluation in a clinically relevant drug resistance model.</a> PloS one. 8 (9), (2013).</li><li>Park, J. Y., Hwang, C. M., Lee, S. 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The major challenge facing this fabrication method was the secure fixing of the beads in the through-hole array in the aluminum plate. To solve this challenge, magnetic force in the form of a 30 x 30 magnet array was used to fix the beads securely, as shown in Figures 6 and 7. The magnetic flux density of the magnet array, which has the opposite polarity, is strongest at the center of each magnet surface. Because the strength of the magnetic force depends on the flux density, the beads w...

Cells grown into a spheroid form are more similar to real tissue in the body than a two-dimensional planar culture1.&#160;Given this advantage, the use of spheroids has been adopted to improve the study of cell to cell interaction2,3, immune-activation4, drug screening5, and differentiation6. In addition, spheroids incorporating multiple cell types have recently been applied to organoids (near-physiological three-dimensional (3D) tissue), which are very useful for studying human development and disease7.&#160;Several methods have been used to produce spheroids. The simplest method involves the utilization of a non-adhesive surface, such that the cells aggregate with each other and form spheroids. A Petri dish can be treated with bovine serum albumin, pluronic F-127, or a hydrophobic polymer (e.g. poly 2-hydroxyethl methacrylate) to make its surface non-adhesive8,9. The spinner-flask method is another well-known means of producing large amounts of spheroids10,11. In this method, cells are held in suspension by stirring to prevent them from becoming attached to the substrate. Instead, the floating cells aggregate to form spheroids. Both the non-adhesive surface method and spinner flask method can produce large amounts of spheroids. However, they are subject to limitations including difficulties in controlling the spheroid size, as well as the tracking and monitoring of each spheroid. As a remedy for such problems, another spheroid production method, namely, the hanging drop method can be employed12. This involves depositing cell suspension drops onto the underside of the lid of a culture dish. These drops are usually 15 to 30 &#181;L in size and contain approximately 300 to 3000 cells13. When the lid is inverted, the drops are held in place by surface tension. The microgravity environment in each drop concentrates the cells, which then form single spheroids at the free liquid-air interface. The benefits of the hanging drop method are that it offers a well-controlled size distribution, while it is easy to trace and monitor each spheroid, relative to the non-adhesive surface and spinner flask methods. However, this method incurs one disadvantage in that the massive production of spheroids and the production process itself is excessively labor intensive.
A microwell array is a flat plate with many micro-size wells, each having a diameter ranging from 100 to 1000 &#181;m. The spheroid production principle when using microwells is similar to that of the non-adhesive surface method. Benefits include the fact that microwells provide spaces between the microwells for separating the cells or spheroids, such that it is easy to control the spheroid size, while also making it easy to monitor each single spheroid. With a large number of microwells, high-throughput spheroid production is also possible. Another advantage of microwells is the option to form wells of different shapes (hexahedral, cylindrical, trigonal prismatic) depending on the users&#39; unique experimental purposes. Generally, however, a three-dimensional (3D) concave (or hemispherical) shape is regarded as being the most suitable for producing uniform-sized single spheroids. Therefore, the usefulness of concave microwells has been reported for many cell biology studies such as those examining the cardiomyocyte differentiation of embryonic stem cells14, the insulin secretion of islet cell clusters15, the enzymatic activity of hepatocytes16, and the drug resistance of tumor spheroids17.
Unfortunately, the fabrication of microwells often requires specialized micropatterning facilities; conventional photolithography-based methods require exposure and developing facilities while reactive ion-etching-based methods need plasma and ion-beam equipment. Such equipment is costly which, together with the complicated fabrication process, presents a high barrier to entry for biologists who do not have access to microtechnology. To overcome these problems, other cost-effective methods such as ice lithography18 (using frozen water droplets) and the flexible membrane method14 (using a membrane, through-hole substrate, and a vacuum) have been suggested. However, these methods also incur serious drawbacks such as it being difficult to control the pattern sizes, the attainment of high aspect ratios, and the production of larger-area microwells.
To overcome the above issues, we are proposing a novel concave microwell fabrication method utilizing a through-hole substrate, steel beads, and a magnet array. Using this method, hundreds of concave spherical microwells can be fabricated by taking advantage of the mechanism of magnetic-force-assisted self-locking metallic beads (Figure 1). The fabrication process involves the use of very few expensive and complicated facilities and does not demand many advanced skills. As such, even unskilled persons can easily undertake this fabrication method. To demonstrate the proposed method, human-adipose-derived stem cells were cultured in the concave microwells to produce spheroids.

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CNC rotary engraver</td>
			<td style="width:111px;">Roland DGA</td>
			<td style="width:119px;">EGX-350</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro drill bit</td>
			<td style="width:111px;">HAM Pr&#228;zision</td>
			<td style="width:119px;">30-1301 TA</td>
			<td>&#934; 0.55 and 0.75 mm</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Sulfuric acid 98%</td>
			<td style="width:111px;">Daejung</td>
			<td style="width:119px;">7683-4100</td>
			<td style="width:167px;">For cleaning aluminum plate. 
			Dilute with distilled water with 15% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neodymium magnet</td>
			<td style="width:111px;">Supermagnete</td>
			<td>W-01-N</td>
			<td>1 x 1 x 1 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bearing ball</td>
			<td style="width:111px;">Agami Modeling</td>
			<td style="width:119px;">SUJ2</td>
			<td>&#934; 600 &#956;m steel bead</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:111px;">Dowcorning</td>
			<td style="width:119px;">Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pluronic F-127</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">p2443</td>
			<td>Dilute with phosphate buffered saline to 4% (w/v) solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s modified eaggle&#39;s medium (DMEM)</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">30-2002</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s phosphate buffered saline (D-PBS)</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">30-2200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal bovine serum</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">30-2020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adipose-derived mesenchymal stem cells</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">ATCC PCS-500-011</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A convex mold and microwell pattern were successfully fabricated by following steps 2.1 to 3.7. (Figure 4). The commercial steel beads were trapped in the 30 x 30 through-hole array. The beads were held tightly without any gaps between the beads and the corresponding through-holes (Figure 4a). The shape of fabricated concave microwell is concave hemispherical, with a diameter of 600 &#181;m, which is the same as that of the steel...

Watch this Scientific Journal Video about A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries at JoVE.com

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

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 ...

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