A novel approach to producing uniform 3-D tumor spheroid constructs using ultrasound treatment

Abstract

Producing three-dimensional (3-D) multicellular tumor spheroids (TSs) is valuable for characterizing anticancer drugs since they provide a more representative model of the 3-D in vivo tumor than conventional two-dimensional (2-D) monolayer culture. The interaction of tumor cells with the extracellular matrix (ECM) in a 3-D culture environment is more similar to a tumor in vivo than in a 2-D environment; cell-cell and cell-ECM interaction can influence cell behaviour, such as in response to drug treatment. In vitro tumor spheroid models have been developed using microfluidic systems to generate 3-D hydrogel beads containing components of alginate and ECM protein, such as collagen, with high uniformity and throughput. Cell-laden hydrogel droplets are formed using a flow focusing process wherein the hydrogel precursors should be a homogeneous mixture. During gelation of the droplets into beads, the alginate acts as a fast gelling component helping to maintain the spherical shape of beads and preventing coalescence as the temperature-sensitive collagen I component gels more slowly. To produce uniform hydrogel droplets using the microfluidic flow focusing system, the mixtures must be homogeneous. However, collagen’s sensitivity to temperature can lead to formation of chunks of collagen gel inside of the mixture, causing the mixture to become non-uniform and risking chip clogging. In order to overcome this limitation, previous approaches have used a cooling system during bead encapsulation while tumor cells were also present in the mixture, but this procedure can contribute to a delay in cell proliferation. Here a novel yet simple method is developed to prepare homogeneous pre-bead-encapsulation-mixtures containing collagen type I through ultrasonication. This method allows the cultivation of homogenous TS cultures with high uniformity and compact structure, and not only maintains cell viability but also the proliferation of cells in alginate/collagen hydrogel bead cultures. Depending on the sonication parameters, time and temperature, collagen can form small sized fibrils to thick fibers. Here, the mixtures containing collagen are assessed for morphology of collagen fibers/fibrils, cell viability, and proliferation. Human source Michigan Cancer Foundation-7 (MCF-7) breast cancer cells are successfully incorporated into alginate/collagen mixtures, followed by sonication, and then bead production. After bead gelation, the encapsulated MCF-7 cells remained viable and proliferated to form uniform TSs when the beads contained alginate and collagen. Results indicate that ultrasound treatment (UST) provides a powerful technique to change the structure of collagen from fiber to fibril, and to disperse collagen fibers in the mixture homogeneously for an application to generate uniform hydrogel beads and spheroids while not inhibiting cell proliferation.