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.
Highlights
Liver cancer is the second most common cause of mortality from cancer worldwide and is estimated to have been responsible for nearly 781,000 deaths in 2018 [1]
Microwell size was incrementally adjusted by both changing the power and the location on the z-axis (Figure 2A)
The scanning electron microscopy (SEM) images of the top and side of the microwells, which had been fabricated to different depths and widths by varying the laser power and adjusting the focusing plane, showed a very smooth surface profile for all the parameters used (Figure 2B)
Summary
Liver cancer is the second most common cause of mortality from cancer worldwide and is estimated to have been responsible for nearly 781,000 deaths in 2018 [1]. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is associated with poor prognosis due to the lack of early diagnosis, and treatments are yet to be fully developed [2]. The pace of drug development for HCC has remained incredibly slow, underscoring the need for the development of new anticancer agents and therapeutic interventions [4]. The challenges of the lengthy cancer drug development process may be attributed, in part, to the lack of representative models [5]. Most drug screening programs have been routinely carried out in two-dimensional (2D) cell culture because it facilitates control of the working environment and ease of handling, rendering it the most common in vitro test platform in the fields of cancer biology and medicine. Multicellular tumor spheroids (MCTSs) or multicellular cancer aggregates (MCAs) have surfaced as one of the most commonly used models for recapitulating several crucial elements of the tumor microenvironment [7]
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