Shell engineering in soft alginate‐based capsules for culturing liver spheroids

Abstract

Functional interaction between cancer cells and the surrounding microenvironment is still not sufficiently understood, which motivates the tremendous interest for the development of numerous in vitro tumor models. Diverse parameters, for example, transport of nutrients and metabolites, availability of space in the confinement, etc. make an impact on the size, shape, and metabolism of the tumoroids. We demonstrate the fluidics-based low-cost methodology to reproducibly generate the alginate and alginate-chitosan microcapsules and apply it to grow human hepatoma (HepG2) spheroids of different dimensions and geometries. Focusing specifically on the composition and thickness of the hydrogel shell, permeability of the microcapsules was selectively tuned. The diffusion of the selected benchmark molecules through the shell has been systematically investigated using both, experiments and simulations, which is essential to ensure efficient mass transfer and/or filtering of the biochemical species. Metabolic activity of spheroids in microcapsules was confirmed by tracking the turnover of testosterone to androstenedione with chromatography studies in a metabolic assay. Depending on available space, phenotypically different 3D cell assemblies have been observed inside the capsules, varying in the tightness of cell aggregations and their shapes. Conclusively, we believe that our system with the facile tuning of the shell thickness and permeability, represents a promising platform for studying the formation of cancer spheroids and their functional interaction with the surrounding microenvironment.

Impact of Viscosity on Human Hepatoma Spheroids in Soft Core–Shell Microcapsules

“…Increased viscosity of liquid MC core may affect the character of collective cell behavior, which is described in Figures 4 and Figure 5. In accordance with our previous results, [29] in this study we also observed large-scale cell spheroids within MCs with 0% CMC in the core and highly dispersed small cell clusters within GBs (Figure 4A). Optical and fluorescent images recorded during the first 9 d of culturing (Figure 4A; Figure S3, Supporting Information) reveal that cells aggregate faster in pristine AL MCs than in AL-CMC MCs with more viscous core or AL GBs.…”

“…Before the culturing of cells in MCs, we confirmed that the capsule structure allows for efficient mass transfer of nutrients and metabolites through the shell. [29] Higher viscosity of the fluid is expected to reduce the diffusion rate of the molecules. To assess the impact of increased viscosity on the rate of nutrient exchange (e.g., glucose, molecular mass (M r ) of 180 Da and amino acids, M r = 80-210 Da) within the MCs during a specific period, we conducted a diffusion experiment using similarly sized model molecule -sodium fluorescein (FSC, M r = 376.27 Da, 5 × 10 −3 m solution), and visualized FSC permeation using a confocal microscope.…”

“…This technology provides large-scale quantitative 3D extracellular environments for high-throughput analysis, including liquid droplets, [25,26] gel beads, [27,28] and capsules. [29,30] In addition, the droplet-based microfluidic approach enables fabrication suitable for engineering cell spheroids and organoids. [30][31][32][33] In our previous study, [29] we utilized this approach to optimize microcapsule generation and investigate the differences in proliferation and aggregation behaviors of HepG2 cells caused by the changes in capsule shell properties (thickness, chemical composition, and gelation time) which affect shell permeability.…”

“…[29,30] In addition, the droplet-based microfluidic approach enables fabrication suitable for engineering cell spheroids and organoids. [30][31][32][33] In our previous study, [29] we utilized this approach to optimize microcapsule generation and investigate the differences in proliferation and aggregation behaviors of HepG2 cells caused by the changes in capsule shell properties (thickness, chemical composition, and gelation time) which affect shell permeability. Furthermore, a method utilizing cancer spheroids has been employed to study the immunological behavior of antigen-specific T cells in vitro.…”

“…To perform the investigation, we generated core-shell microcapsules (MCs) with diameters of ≈500 μm as bioreactors for cell culturing using the microfluidic platform that we designed previously. [29] The shell of the MCs is composed of a soft alginate (AL), while the core of the MCs contains a cell culture medium with adjusted viscosity. The viscosity of the core liquid is modulated by incorporating a predetermined amount of carboxymethylcellulose (CMC).…”