Reproducible Construction of Surface Tension-Mediated Honeycomb Concave Microwell Arrays for Engineering of 3D Microtissues with Minimal Cell Loss

Abstract

The creation of engineered 3D microtissues has attracted prodigious interest because of the fact that this microtissue structure is able to mimic in vivo environments. Such microtissues can be applied extensively in the fields of regenerative medicine and tissue engineering, as well as in drug and toxicity screening. Here, we develop a novel method of fabricating a large number of dense honeycomb concave microwells via surface tension-mediated self-construction. More specifically, in order to control the curvature and shape of the concavity in a precise and reproducible manner, a custom-made jig system was designed and fabricated. By applying a pre-set force using the jig system, the shape of the honeycomb concave well was precisely and uniformly controlled, despite the fact that wells were densely packed. The thin wall between the honeycomb wells enables the minimization of cell loss during the cell-seeding process. To evaluate the performance of the honeycomb microwell array, rat hepatocytes were seeded, and spheroids were successfully formed with uniform shape and size. Liver-specific functions such as albumin secretion and cytochrome P450 were subsequently analyzed. The proposed method of fabricating honeycomb concave wells is cost-effective, simple, and reproducible. The honeycomb well array can produce multiple spheroids with minimal cell loss, and can lead to significant contributions in tissue engineering and organ regeneration.

Fig 1. Fabrication of a honeycomb concave microwell chip.

(a) Poly(dimethylsiloxane) (PDMS) honeycomb structures are fabricated using standard lithography. (b) A PDMS prepolymer is poured onto a PDMS base chip to fill the microwell, (c) and is then raked out using a flat plate with trimmed edges. (d, e) Surface tension-mediated honeycomb concave microwells are thermally cured on a hot plate. (f) A custom-designed jig system is used to fabricate honeycomb concave microwells. (g) Constant force is applied to the chip on the linear stage. (h) The PDMS prepolymer is raked out from the chip as the motorized linear stage moves in one direction.

Fig 2. Honeycomb concave microwell and custom-made jig system.

(a) Schematic diagram of a honeycomb concave microwell array. W: width; D: diameter; T: wall thickness. (b) A picture of various sizes of honeycomb concave microwell chambers. (c) Pictures of the jig system pressing a chip. Scanning electron microscope (SEM) images of wells formed at a force of (d) 3 N and (e) 7 N showing top views and cross-sectional side views of microwells. Scale bars: 500 μm.

Fig 3. Cross-sectional scanning electron microscopy (SEM) images of honeycomb concave microwell.

Curvatures of honeycomb concave microwells formed by applying various forces to the PDMS prepolymer. The black dotted lines and black triangles show the original microwell before pouring the PDMS prepolymer. The blue-dotted circle depicts the radius of the honeycomb concave microwell. Scale bars: 200 μm.

Fig 4. Depth and curvature of honeycomb concave microwells formed by applying various forces to the PDMS prepolymer.

(a) Schematic depiction of side and top views of honeycomb concave microwells. (b) Relationship between well depth and radius of curvature of honeycomb concave microwells.

Fig 5. Hepatocytes cultured in (1) circular concave microwells and (2) honeycomb concave microwells.

(a, e) Scanning electron microscopy (SEM) images showing the smooth bottom profile of concave microwells 1 and 2. (b, f) Images of hepatocyte spheroids formed in concave microwells 1 and 2. (c, g) Merged images of Calcein AM- and Ethidium homodimer-1-stained spheroids. Calcein AM-stained hepatocyte spheroids (green) cultured for 3 days showing cell viability in concave microwells 1 and 2. Ethidium homodimer-1 fluorescence images (red), which indicate dead cells, are weakly appeared on the images. Scale bars: 500 μm. (d, h) SEM images of spheroids showing formation of tight cell-to-cell junctions in spheroids (black arrows) formed in concave microwells 1 and 2. Confocal microscopic images of cell viability in circular concave microwells (i) and honeycomb concave microwells (j). Scale bars: 250 μm.

Fig 6. Evaluation of hepatocyte spheroids.

(a) Spheroid formation in two types of concave microwells. Scale bars: 500 μm. (b) Size distribution of hepatocyte spheroids in circular and honeycomb concave microwells.

Fig 7. Hepatocyte spheroids in honeycomb concave microwells exhibit greater liver-specific function than those in circular concave microwells.

(a) Analysis of metabolic functions of spheroids in the two types of microwells, as measured by the secretion of albumin. (b) Immunostaining of hepatocyte spheroids for albumin (primary hepatocytes; green in first row), DAPI (nuclei; blue), cytochrome P450 activity (CYP450; green in second row) and microfilaments (F-actin; red). (c) Quantified analysis of hepatocytes function test. Scale bars: 300 μm.