Simple In-House Fabrication of Microwells for Generating Uniform Hepatic Multicellular Cancer Aggregates and Discovering Novel Therapeutics

Abstract

Three-dimensional (3D) cell culture models have become powerful tools because they better simulate the in vivo pathophysiological microenvironment than traditional two-dimensional (2D) monolayer cultures. Tumor cells cultured in a 3D system as multicellular cancer aggregates (MCAs) recapitulate several critical in vivo characteristics that enable the study of biological functions and drug discovery. The microwell, in particular, has emerged as a revolutionary technology in the generation of MCAs as it provides geometrically defined microstructures for culturing size-controlled MCAs amenable for various downstream functional assays. This paper presents a simple and economical microwell fabrication methodology that can be conveniently incorporated into a conventional laboratory setting and used for the discovery of therapeutic interventions for liver cancer. The microwells were 400–700 µm in diameter, and hepatic MCAs (Huh-7 cells) were cultured in them for up to 5 days, over which time they grew to 250–520 µm with good viability and shape. The integrability of the microwell fabrication with a high-throughput workflow was demonstrated using a standard 96-well plate for proof-of-concept drug screening. The IC50 of doxorubicin was determined to be 9.3 µM under 2D conditions and 42.8 µM under 3D conditions. The application of photothermal treatment was demonstrated by optimizing concanavalin A-FITC conjugated silica-carbon hollow spheres (SCHSs) at a concentration of 500:200 µg/mL after a 2 h incubation to best bind with MCAs. Based on this concentration, which was appropriate for further photothermal treatment, the relative cell viability was assessed through exposure to a 3 W/cm2 near-infrared laser for 20 min. The relative fluorescence intensity showed an eight-fold reduction in cell viability, confirming the feasibility of using photothermal treatment as a potential therapeutic intervention. The proposed microwell integration is envisioned to serve as a simple in-house technique for the generation of MCAs useful for discovering therapeutic modalities for liver cancer treatment.

Development of an In Vitro 3D Model for Investigating Ligamentum Flavum Hypertrophy

“…Compared with the aforementioned traditional 2D culture systems, in vitro three-dimensional (3D) culture systems [16,17] may better recapitulate the cell-cell and cell-matrix interactions occurring in LF tissue [18]. However, although 3D culture systems have been widely applied in many disease models, such as cancer [19][20][21] cardiovascular disease [22] and wound healing [23], limited information regarding in vitro 3D LF models is available. Given that clinical observations of LFH tissue have revealed that elastic fibers decrease and degenerate while collagen increases in hypertrophic tissue [24] developing a pure cell model without adding the extracellular matrix (ECM) could help us understand the changes in the ECM composition over time.…”

A Facile Approach for Rapid Prototyping of Microneedle Molds, Microwells and Micro-Through-Holes in Various Substrate Materials Using CO2 Laser Drilling

“…Rapid CO 2 laser prototyping has been previously demonstrated as an effective alternative for the fabrication of microwells for the formation of size-controlled 3D spheroids [ 3 , 11 , 13 , 16 ]. However, one major obstacle that has remained non-demonstrated in PS microwells is the inevitable loss of cells after cell seeding because of the required washing step to ensure that cells are not lodged into the microwells and can be efficiently removed without perturbing the size-uniformity of the MCTSs [ 27 ].…”

“…Additionally, choosing PS as the microwell substrate is favorable from a biologist’s perspective because PS is the most commonly utilized plastic for in vitro cell culture research. Compared to PDMS [ 28 ] or polyester [ 13 ] microwells, PS is hassle-free for the researchers in terms of the material accessibility, and more amenable in terms of compatibility with different forms of conventional culture plasticwares, such as Petri dishes [ 3 ] or microtiter plates [ 16 ]. In addition, the PS microwell displayed a gentle concave bottom, and curved microwell structures have been reported to promote the aggregation of spheroids [ 29 ].…”

“…However, a major barrier to fabricating microstructure arrays using CO 2 laser drilling is that different studies have only been reported sporadically, and these studies have involved different systems, substrate materials, and applications [ 3 , 8 , 9 , 16 , 17 ]. On the one hand, these outcomes have prohibited this technique from reaching the greater scientific community because of the concern that specific and unique protocols as well as meticulous pre/postmaterial processes are required for different applications.…”

“…On the one hand, these outcomes have prohibited this technique from reaching the greater scientific community because of the concern that specific and unique protocols as well as meticulous pre/postmaterial processes are required for different applications. On the other hand, varying microstructure geometries and qualities from different studies [ 13 , 14 , 16 ] suggest ample room for further improvement based on a comprehensive and systematic understanding of the choice of both the materials and the laser system. With these notions, we hope to generate a holistic viewpoint of the simplicity and consistency of the method as well as of the diverse microstructures and applications that can be achieved through the integration of various common laboratory-accessible consumables.…”