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.
