Multi-cellular tumor spheroids ͑MCTSs͒ have been established as a 3D physiologically relevant tumor model for drug testing in cancer research. However, it is difficult to control the MCTS testing parameters and the entire process is timeconsuming and expensive. To overcome these limitations, we developed a simple microfluidic system using polydimethylsiloxane ͑PDMS͒ microbubbles to culture tumor spheroids under physiological flow. The flow characteristics such as streamline directions, shear stress profile, and velocity profile inside the microfluidic system were first examined computationally using a COMSOL simulation. Colo205 tumor spheroids were created by a modified hanging drop method and maintained inside PDMS microbubble cavities in perfusion culture. Cell viability inside the microbubbles was examined by live cell staining and confocal imaging. E-selectin mediated cell sorting of Colo205 and MDA-MB-231 cell lines on functionalized microbubble and PDMS surfaces was achieved. Finally, to validate this microfluidic system for drug screening purposes, the toxicity of the anti-cancer drug, doxorubicin, on Colo205 cells in spheroids was tested and compared to cells in 2D culture. Colo205 spheroids cultured in flow showed a threefold increase in resistance to doxorubicin compared to Colo205 monolayer cells cultured under static conditions, consistent with the resistance observed previously in other MCTS models. The advantages presented by our microfluidic system, such as the ability to control the size uniformity of the spheroids and to perform real-time imaging on cells in the growth platform, show potential for high throughput drug screening development.

The hematogenous metastatic cascade is mediated by the interaction of cancer cells and the endothelial cell lining of blood vessels. In this work, we examine the colon cancer cell line COLO 205, which grows simultaneously in both adherent and suspended states in culture and can serve as a good model for studying tumor heterogeneity. The two subpopulations of cells have different molecular characteristics despite being from the same parent cell line. We found that the ratio of adherent to suspended cells in culture is maintained at 7:3 (equilibrium ratio). The ratio was maintained even when we separate the two populations and culture them separately. After 8 h in culture the equilibrium was achieved only from either adherent or suspended population. The adherent cells were found to express less E-selectin binding glycans and demonstrated significantly weaker interaction with E-selectin under flow than the suspended cells. Manipulation of the epithelial–mesenchymal transition (EMT) markers β-catenin and E-cadherin expression, either by siRNA knockdown of β-catenin or incubation with E-cadherin antibody-coated microbeads, shifted the ratio of adherent to suspended cells to 9:1. Interestingly, human plasma supplemented media shifted the ratio of adherent to suspended cells in the opposite direction to 1:9, favoring the suspended state. The dynamic COLO 205 population switch presents unique differential phenotypes of their subpopulations and could serve as a good model for studying cell heterogeneity and the EMT process in vitro.

Here we show that dielectrophoretic (DEP) liquid actuation can be used to dispense arrays of nanoliter-sized droplets loaded with biomolecules. Size-based enrichment of these biomolecules occurs rapidly and simultaneously with the droplet dispensing. The physical mechanism responsible for the effect is the positive DEP force directed toward the electrodes that is imposed by the non-uniform electric field during the very rapid DEP actuated flow before droplet formation. Experiments conducted with a suspension of lambda DNA (molecular weight: 31.5 x 10(3) kDa) and lectin protein (120 kDa) containing identical molar concentration shows separation of DNA and protein within the nanolitre sized droplets formed along the electrode. The density ratio of protein to DNA varies smoothly from 1 : 1 in the parent droplet to approximately 3 : 1, favoring the smaller sized protein in the daughter droplet dispensed furthest from the parent droplet, approximately 2.4 mm from the parent droplet. Experiments conducted with binary protein solutions containing identical molar concentrations of bovine serum albumin (66 kDa) and fibrinogen (340 kDa) reveal that enrichment is enhanced as the length of the electrodes is increased. The density ratio of BSA to fibrinogen varied from 1 : 1 in the parent droplet to approximately 1.97 : 1 at the last (tenth) droplet, located approximately 4.2 mm from the parent droplet. The entire process, consisting of droplet dispensing and particle separation, occurs in less than one second.

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