Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Abstract

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

Engineered Breast Cancer Cell Spheroids Reproduce Biologic Properties of Solid Tumors

“…Each well from a column of a 384 well plate (source plate) was filled with ≈20 μL of the aqueous DEX phase containing cells. A liquid handling robot (Bravo SRT, Agilent) was used to mix the content of the wells of the source plate and then aspirate 0.3 μL from each well . Then the solution was dispensed as a single drop into each well of one column of the PEG phase‐containing destination plate.…”

Micropocket hydrogel devices for all-in-one formation, assembly, and analysis of aggregate-based tissues

“…This effect was enhanced by the addition of HGF, resulting in larger invasion areas ( figure 6(b)). These images confirm that aggregates are embedded within a matrix, and remain viable and [11], and aqueous two-phase systems (ATPS) are immiscible liquids that can be used to confined cells in small spaces, to encourage them to aggregate [19,20]. (b) Representative images of similarly-sized MDA-MB-231, MCF-7, and T-47D human breast cancer cell lines formed into aggregates using each of the three techniques (scale bar=200 μm).…”

“…We sought to benchmark our technology against two other technological approaches to forming aggregates in terms of applicability to various cell types. We compared our system with hanging drop cultures, which prevent cells from attaching to any other surface, and is hence similar in mechanism of action to pipette-able hanging drop plates [63] and non-adhesive microwells [21,36]; and to an aqueous two-phase bioprinting strategy [19,20], in which cells are physically confined close to each other, and can be considered similar in mechanism to microcavity-based strategies [27] ( figure 5(a)). Both strategies have proven advantages for spheroid culture, most notably the ability to individually stimulate each spheroid individually, as they are separated by a physical barrier.…”

Review on biofabrication and applications of heterogeneous tumor models

“…The results showed higher drug resistance in 3D cospheres compared with monospheres. Ham, Atefi, Fyffe, and Tavana () established a robotic heterogeneous tumor spheroid fabrication system and used it for drug testing. Increased proliferation rate and drug resistance to mitomycin, doxorubicin hydrochloride, and 5‐azacytidine of tumor cells were found in Wang et al's () study, where MCTSs were constructed in the fibrous scaffold.…”