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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

Cells grown into a spheroid form are more similar to real tissue in the body than a two-dimensional planar culture¹. Given this advantage, the use of spheroids has been adopted to improve the study of cell to cell interaction^2,3, 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. 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-adhesive^8,9. The spinner-flask method is another well-known means of producing large amounts of spheroids^10,11. 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 µL 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 µm. 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' 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.

Protokół

1. Preparation of through-hole array aluminum plate and magnet array

1. Prepare two 50 mm x 50 mm (or larger) aluminum plates. The thickness of each plate was 300 µm that is half of the bead diameter.
2. Form a 30 x 30 through-hole array on one of the aluminum plates by using a CNC rotary engraver with a Φ550-µm 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 Figure 2a, i).
3. Form a 30 x 30 array of Φ750-µm through-holes on the other aluminum plate, using the same procedure as that described in 1.2 (Figure 1a and Figure 2a, ii).
4. Attach the two plates each other by using a sticky tape and form Φ3 mm alignment holes at each of the four corners of both aluminum plates.
5. Soak the aluminum plates in 15% sulfuric acid for 12 h to clean their surfaces. Since the thin layer of aluminum oxide on the surface of the aluminum make it corrosion resistant, the hole diameter and thickness of the plate are not changed by this acid treatment.
6. Form a 30 × 30 array of 1 x 1 x 1 mm neodymium magnets (with a magnetic strength of 0.363 N). Ensure that each magnet is the opposite polarity to its neighbor. To prevent the breaking or scattering of the magnet array, attach a 30 x 30 mm aluminum plate to the bottom of the magnet array using double-sided tape (Figure 2a, iii and inset in Figure 2).

2. Bead trapping process

1. Align and stack the two aluminum plates (top plate: 750-µm-hole plate, bottom plate: 550-µm-hole plate) using the prepared alignment holes at the four corners of each plate (Figure 1b).
2. Lock the two plates together by inserting M3 bolts into the alignment holes, and then secure the bolts with nuts (Figure 1b).
3. Stack the aluminum plate assembly on the prepared magnet array (Figure 1b, 2b, and 2c). Align the array of magnets and the array of through holes in the aluminum plate during the stacking process. Then use a sticky tape to fix the position of the magnet array.
4. Place a sufficient number of Φ600 mm SUJ2 steel beads on the plate assembly and manipulate the beads using an acryl (or non-metallic) plate such that a bead becomes trapped in each hole (Figure 1c, 1d, and 1e) while simultaneously removing the excess beads which have not lodged in the holes.
5. Carefully remove the top plate to avoid unwanted scattering and dislocation of the trapped beads (Figure 1f).

3. Concave microwell fabrication

1. Move the concave microwell mold, produced in steps 2.1 to 2.5, above, to a Petri dish.
2. Mix polydimethylsiloxane (PDMS) monomer and curing agent according to the manufacturer's instructions¹⁹ with a PDMS monomer:curing agent ratio of 10:1.
3. De-gas the PDMS mixture by using a desiccator and vacuum pump to remove any bubbles trapped in the PDMS mixture.
4. Pour the PDMS mixture into the concave microwell mold and de-gas again using the same procedure as that described in 3.3 (Figure 1f).
5. Bake the PDMS mixture on a hotplate at 80 °C for 2 h to form a bead-embedded PDMS substrate (Figure 1g).
6. Remove the cured PDMS substrate from the mold (Figure 1g). In the removing process, spray methanol using washing bottle to detach PDMS substrate from the mold.
7. Using a Φ15 mm x 2 mm magnet, remove the trapped steel beads from the PDMS substrate (Figure 1h). For this process, any magnet that is sufficiently strong to extract the beads from the PDMS substrate can be used.

4. Spheroid culture

1. Cut the concave microwell-patterned PDMS substrate by using a Φ14 mm biopsy punch to be fitted in 24-well plate in this study.
2. Sterilize the resulting Φ14 mm PDMS substrate in an autoclave sterilizer at 121 °C and 15 psi.
3. Place the sterilized PDMS substrate into a 24 well plate.
4. Coat the entire PDMS substrate with 4% (w/v) pluronic F-127 solution overnight to prevent cell attachment to the microwell surface. During the coating process, remove any air bubbles entrapped in the concave microwells by pipetting or by using an ultrasonic cleaner.
5. Flush the F-127 solution three times by using phosphate-buffered saline (PBS).
6. Seed 1 mL of cell-medium (Dulbecco's Modified Eagle Medium) solution (which contains 2 x 10⁶ cells) on the PDMS substrate. Note that the seeding density can be changed according to the target spheroid size and/or target cell type. Here, adipose-derived stem cells (ASC) were used.
7. Aspirate the 1 mL of medium using a 1000 µL pipette to remove any excess cells that were not trapped in the microwells (Figure 3).
8. Incubate the cells at 36.5 °C, humidity of >95%, and 5% CO₂ condition. In the case of the ASCs used in our study, the cells aggregate to a spheroid in 48 h.

Wyniki

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 µm, which is the same as that of the steel...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
CNC rotary engraver	Roland DGA	EGX-350
Micro drill bit	HAM Präzision	30-1301 TA	Φ 0.55 and 0.75 mm
Sulfuric acid 98%	Daejung	7683-4100	For cleaning aluminum plate. Dilute with distilled water with 15% solution
Neodymium magnet	Supermagnete	W-01-N	1 x 1 x 1 mm
Bearing ball	Agami Modeling	SUJ2	Φ 600 μm steel bead
Polydimethylsiloxane (PDMS)	Dowcorning	Sylgard 184
Pluronic F-127	Sigma Aldrich	p2443	Dilute with phosphate buffered saline to 4% (w/v) solution
Dulbecco's modified eagle's medium (DMEM)	ATCC	30-2002
Dulbecco's phosphate buffered saline (D-PBS)	ATCC	30-2200
Fetal bovine serum	ATCC	30-2020
Adipose-derived mesenchymal stem cells	ATCC	ATCC PCS-500-011