Development and refinement of an aqueous two-phase system three-dimensional cell culture method for MDA-MB-231 cancer cell spheroid analysis

Abstract

Aqueous two-phase systems (ATPS) are generated by mixing immiscible solutions of polymer-polymer combinations above certain concentrations forming two phases, allowing selective partition of biological particles, such as cells. Leveraging this, novel biomedical applications have emerged using ATPS for three-dimensional (3D) cell culture due to their ability to recapitulate the tumor in vivo conditions. Current approaches require sophisticated equipment and highly trained personnel for their production. To address this, a 3D cell culture method using ATPS and basic laboratory equipment for culturing cancer cell spheroids (CCS) has been designed. Nevertheless, to produce CCS of well-defined baseline characteristics with this approach, refinement of culture conditions is necessary for accurate drug-response evaluations. CCS configuration is achieved through the confinement of cells within droplets of 6.4% w/w dextran (DEX) 500,000 g/mol that are subsequently submerged in 5% w/w polyethylene glycol (PEG) 35,000 g/mol. Confining cells in DEX at different cell densities (5.0x103 -1.6x104 cells) produced CCS of diameters ranging from 419.51±64.16 μm to 818.11±83.27 μm after 48 h of culture, indicating that CCS of increasing size are achieved. Furthermore, 8-day examination of CCS viability showed reduced cell metabolic activity compared to their 2D culture (p-value <0.05) and consistent viability onwards day 4 of culture (p-value >0.05), reflecting diminished diffusion of nutrients and enabling assessing long-term biological processes. Finally, CCS showed a deviation from a sigmoidal response to paclitaxel (PTX), displaying an increase of 31% in maximum cell viability inhibition (Emax) in contrast with their 2D counterpart, signifying drug resistance. In conjunction, these results indicate that the ATPS-3D approach can produce in vitro models of consistent, predictable signals for end-point assays and biologically complex responses to drugs.