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

Summary

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.

Abstract

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.

Introduction

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 disease

Protocol

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 pl....

Results

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 bead.......

Discussion

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.......

Materials

List of materials used in this article

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