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...

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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 ...

The overall goal of this method is to fabricate concave spherical micro wells by utilizing magnetic force assisted, self locking metallic beads. Hi, my name is Gi Hun Lee, and I'm a teaching assistant at Chung-Ang University. What we are interested in our lab is fabricating various micro scale devices and platforms to enhance our studies regarding the development of a bio instrument, their culture and novel micro structures.One of our research interest is developing novel micro devices and methodologies to contract this in a three dimensional environment. We call this culture. Culture is very useful for understand three dimensional response, because it is more similar to the actual tissue, than the conventional two dimensional planar culture.Several methods for spread production have been attempted, such as in a flask or tubes, in a drum net and microwave. Among these methods, the concave micro method is very powerful tool to produce three D produce. Any pitch of the micro ware are each monitoring and the ability to control spherical sizes and distribution.Here we propose our method for fabricating concave micro wares with a bead and an array of small magnets. Using this technique, 100 of concave micro wares can be fabricated by mechanism of magnetic force assisting still beads. The augmentations are in disparate of process in each concave and no recurring proposition scales.To start off, 230 by 30 through hole array aluminum plates were made by using a CNC rotary engraver. The place provide a guide roll for the beads, as will be shown in the following sections. The placer drilled with different diameter drill bits and the plate with the smaller hole was placed at the bottom.The outer corners are also drilled in order to align the plates. The two plates are then aligned, stacked and locked together by inserting N three bolts into the limen holes and are fastened with nuts. As mentioned before, it is important for the manufacturer to place the plate with a smaller hole on the bottom, so that the beads may be fixed inside the guide wells.The next step is preparing a 30 by 30 array of neodymium magnets. We begin by manually isolating the magnets and arrange them into a two by two array. Then we align them into a larger array using tweezers.In order to make a proper magnet array, each magnet must be of the opposite polarity to its neighbor. Furthermore, to prevent breaking or scattering of the array, an aluminum plate is attached to the bottom of the magnet array using double sided tape. Now, we are ready to assemble each piece we made into micro well mold.Magnet array is carefully attached to the bottom plate using ordinary tape. It is important, that the magnet array is attached with the magnets facing downwards. After the aluminum plate and the magnet array are properly ordered, steel beads are placed on the plate assembly.the beads used in our paper are SUJ two steel beads with 600 micron diameters. Our alignment process is very simple. We pour a sufficient number of beads on the assembly and scrape them off using an acrylic plate.The magnet at the bottom traps the beads, that get into the holes of the aluminum plate and the excessive beads, which have not lodged in the holes are removed during the scraping process. The excessive beads are removed using another magnet. When the beads are properly lodged inside the guide wells, we proceed to removing the top plate.During this process, there are some cases, where the beads fall out and become displaced. In this case, manually insert the bead back into the hole using tweezers. Now the concave micro well mold is ready.Move the platform to another petri dish and prepare for the next step. We prepare PDMS by following the manufacturer's instructions by mixing PDMS monomer and the curing agent with the ration of 10 to one. To remove air bubbles trapped in the PDMS mixture, we degas the mixture by placing it in a desiccator for approximately one hour.When this is done, we take the degassed PDMS mixture and pour it onto the concave micro well mold and degas it again. Then we bake the PDMS mixture on a hot plate at 80 degrees Celsius for two hours to form imbued embedded PDMS sub tray. Next process must be carried out with great care.In order to ease the removal process, methanol is poured on the sub tray and the peripheral PDMS sub tray is removed in sequence. It should be noted, that the manufacturer should make sure, that the methanol is well permeated into the gaps to ensure a clean removal. The PDMS sub tray is then detached from the aluminum plate, it is easier to cut the edges before peeling the sub tray off.Finally, we remove the beads from the PDMS sub tray. Before removal, we stretch and flew the sub tray for some time to loosen the bonds between the beads and the PDMS sub tray. The actual removal of the beads is very easy and can be done by using a large magnet.The fabrication of concave micro wells is finished and the micro wells are ready for cell culture. To test whether our PDMS platform is suitable for culturing cells, we conducted the following procedures. First, we used a 14 millimeter diameter biopsy punch to cut the concave micro well pattern PDMS sub tray.After sterilizing the PDMS sub tray, we place it into a 24 well plate. We then coat the entire PDMS sub tray with a four percent pluronic F-127 solution overnight to prevent cells from adhering to the micro well surface. During the coating process, any air bubbles entrapped in the micro wells should be removed, either by pipetting or by using an ultra sonic cleaner.After making sure, that there are no air bubbles remaining, we seed one milliliter of cell medium solution, which will contain an approximate of two million cells onto the PDMS sub tray. Next, aspirate the medium using a 1000 microliter pipet to remove any excess cells, that were not trapped in the micro wells and everything is set for the cells to go into the incubator. A convex mold in micro well pattern was successfully fabricated, commercial steel beads were trapped in the 30 by 30 through hole array.The beads were held tightly without any gas between the beads and the corresponding through holes. The shape of the fabricated concave micro well is concave hemispherical with a diameter of 600 microns, which is the same of that of the steel bead. A cross section of a concave micro well shows, that the distance from the neighboring micro well was one millimeter, which was the same as that of the through holes.Adipose derived stem cells were cultured in the concave micro wells. We seeded two million cells on the 14 millimeter diameter concave micro well array. After 24 hours, the cells had aggregated into spheroids.The average diameter of the spheroids formed in a micro well array was 185.68 microns. At day three, the cells had become more aggregated, but the average diameter of the spheroids falling to 147 microns. Following this procedure, 100 of concave spherical micro wells can be fabricated in our cost efficient and relatively simple method.

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Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

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Conducting Multiple Imaging Modes with One Fluorescence Microscope

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