MP40: A microfluidic chip for Multicellular Tumor Spheroid formation and analysis: a prospective system for anticancer drug screening

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Karina Ziolkowska, Radoslaw Kwapiszewski, Michał Chudy, Artur Dybko and Zbigniew Brzozka
Department of Microbioanalytics, Institute of Biotechnology, Warsaw University of Technology, POLAND

Nowadays drug development and pre-clinical tests are performed using animals and simple in vitro cellular models. Animal participation is questionable both for ethical reasons and differences between species occurrence. On the other hand, available in vitro models, based on monolayer cell culture, do not give answers for essential aspects concerning organism’s response to drugs. These limitations cause the long evaluation process of new drugs, which is expensive and may cost life of many patients suffering from cancer. Thus, there is a need for new, fast and reliable alternative drug testing methods.

The best cellular model for anticancer therapy testing developed so far is Multicellular Tumor Spheroid (MCTS). The MCTS presents morphology and physiology similar to tumor in vivo with the network of cell-cell interactions, three dimensional structure, ECM presence and nutrients, metabolites and oxygen gradients – factors inaccessible for monolayer cell culture. Number of methods of MCTS formation were described in literature and several found their final applications. However, most of them causes variation in size or is labor and energy consuming, which prevents automation and standardization of methods. First attempts of spheroid formation using microfluidic devices have been reported, but the solutions proposed are not fully suitable for MCTS cultivation and testing, e.g. rapid spheroid formation methods do not mimic original tumor genesis and microchambers of diameters of 70 200μm are too tight for MCTS, which diameter often exceeds 200μm.

In this work a 3D microfluidic system of microwells for MCTS cultivation is presented. The poly(dimethylsiloxane) was chosen for the fabrication, due to its hydrophobicity preventing cell adhesion. Separate layers were fabricated using low-cost soft lithography and replica molding method. An array of microwells (volumes of 0.2μL) was connected with the network of microchannels for medium supply. Alignment and plasma bonding of the three layers resulted in the 3D structure which enabled cultivation of MCTS of diameters up to 300μm with the medium perfusion around.

HT-29 cells’ suspension was introduced into the microsystem and after 16 hours of incubation cell aggregation was observed. Unaggregated cells were washed out from microchannels with the fresh medium flow. Within the next 24 hours loose cell aggregates remained in the microwells formed compact spheroids. In each experiment, 72 hour cultivation gave obtainment of uniform spheroids of required properties (diameter over 150μm, necrotic core presence). The growth rate of MCTS observed in the microsystem was lower than in the nonadhesive plates, which most likely corresponds with the tumor growth in vivo.

The presented MCTS microsystem can be an inexpensive and easy to handle alternative for current spheroids’ cultivation methods. The microfluidic array can be easily coupled with the concentration gradient generator forming an integrated system for anticancer drug screening.

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