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Microfluidic system for long-term spheroid culture and anticancer drug activity evaluation

Karina Ziółkowska, Radosław Kwapiszewski, Kamil Żukowski, Michał Chudy, Artur Dybko, Zbigniew Brzózka
Department of Microbioanalytics, Institute of Biotechnology, Faculty of Chemistry, Warsaw University of Technology

Current in vitro cellular models used in cancer research and preclinical trials (such as monolayer) do not correspond with the in vivo situation due to significant differences between in vitro and in vivo environments. Therefore novel cellular models suitable for drug screening are required. Coupling of a tumor spheroid model with the microfluidic chip technology is a prospective solution in this field.
In this work, we present a microfluidic array of microwells for long-term spheroid cultivation and anticancer drug activity evaluation. The three-dimensional system of 50µm deep and 1mm wide channels with arrays of 150µm deep microwells (diameters of 200µm) was fabricated using novel method of poly(dimethylsiloxane) (PDMS) double casting utilizing surface modification by thermal aging. PDMS was chosen due to its hydrophobicity promoting cell aggregation and gas permeability enabling culture oxygenation. The initial mold was fabricated by microdrilling in poly(methyl methacrylate). Then, a double casting in PDMS was performed, where a structure obtained in a first replica molding becomes a mold for a second replica molding step. Our group modified this technique to obtain high quality of replication with no chemical treatment of a PDMS master. We decided to apply thermal aging of a PDMS bulk to remove low molecular weight chains and minimize cross-liking between a mold and a cast.
The fabricated microsystem was used for three-dimensional culture of HT-29 human carcinoma cells. Tumor spheroids were cultured in the microsystem for four weeks and high viability was determined within this time. The spheroid growth rate obtained was significantly lower in the microsystem than in a non-adhesive Petri dish. After two weeks of the culture, growth slowdown was observed and high cell viability was kept. It is the evidence that microfluidic environment can mimicin vivo conditions, as a homeostasis-like state was achieved.
After 4 days of culture, a cytostatic drug (5-fluorouracil, 5-FU) was introduced into the microsystem with different frequencies (every-day or every-second-day) and different concentrations (2mM and 5mM). The geometry and construction of the microsystem enabled flushing away of unaggregated (including dead) cells while viable spheroids remained inside microwells. Therefore decreasing spheroid diameter could have been observed and measured as an indicator of decreasing cell viability. The results have shown differences in response of spheroids to different concentrations of 5-FU. It was also observed, that higher frequency of drug dosing resulted in more rapid spheroid diameter decrease. Observation of decreasing spheroid dimensions is a low-cost and easy-to-conduct mean of a quantitative determination of a three-dimensional cellular model response to an applied drug. It is suitable for long-term observation of spheroid response, in a contrary to other viability assays requiring termination of a culture. It also provides an unique possibility of evaluation of repeated doses of a drug.

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