Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications.

Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell-cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers. V C 2013 AIP Publishing LLC. [http://dx

We use a microfluidic cell culture chip equipped with pneumatic microvalves to analyze the paracrine loop between lung cancer cells and fibroblasts. In order to assess the cellular responses in the paracrine loop, we measure the migration speeds of cancer cells and the aspect ratios of fibroblasts which reflect the phenotype of myofibroblasts. With well-controlled interaction sequences between these two types of cells, we verify that the cytokines from cancer cells effectively stimulate the fibroblasts into myofibroblasts. The cytokines from myofibroblasts, rather than fibroblasts, increase the migration speeds of cancer cells. We confirm that the transforming growth factor-β1 (TGF-β1) is involved in the interaction between cancer cells and fibroblasts, and we also interrupt this paracrine loop in the cell culture chip by inhibiting the TGF-β1 receptors on fibroblasts.

Physiological electric field (EF) plays a pivotal role in tissue development and regeneration. In vitro, cells under direct-current electric field (dcEF) stimulation may demonstrate directional migration (electrotaxis) and long axis reorientation (electro-alignment). Although the biophysical models and biochemical signaling pathways behind cell electrotaxis have been investigated in numerous normal cells and cancer cells, the molecular signaling mechanisms in CL1 lung adenocarcinoma cells have not been identified. Two subclones of CL1 cells, the low invasive CL1-0 cells and the highly invasive CL 1-5 cells, were investigated in the present study. CL1-0 cells are non-electrotactic while the CL 1-5 cells are anodally electrotactic and have high expression level of epidermal growth factor receptor (EGFR), in this study, we investigated the generally accepted hypothesis of receptor tyrosine kinase (RTK) activation in the two cell lines under dcEF stimulation. Erbitux, a therapeutic drug containing an anti-EGFR monoclonal antibody, cetuximab, was used to investigate the EGFR signaling in the electrotaxis of CL 1-5 cells. To investigate RTK phosphorylation and intracellular signaling in the CL1 cells, large amount of cellular proteins were collected in an airtight dcEF stimulation device, which has advantages of large culture area, uniform EF distribution, easy operation, easy cell collection, no contamination, and no medium evaporation. Commercial antibody arrays and Western blotting were used to study the phosphorylation profiles of major proteins in CL1 cells under dcEF stimulation. We found that electrotaxis of CL 1-5 cells is serum independent and EGFR independent. Moreover, the phosphorylation of Akt and S6 ribosomal protein (rpS6) in dcEF-stimulated CL1 cells are different from that in EGF-stimulated cells. This result suggests that CL1 cells’ response to dcEF stimulation is not through EGFR-triggered pathways. The new large-scale dcEF stimulation device developed in the present work will aid the sample preparation for protein-based experiments.

We combine a micro-fluidic electric-field cell-culture (MEC) chip with structured-illumination nano-profilometry (SINAP) to quantitatively study the variations of cancer cell filopodia under external direct-current electric field (dcEF) stimulations. Because the lateral resolution of SINAP is better than 150 nm in bright-field image modality, filopodia with diameters smaller than 200 nm can be observed clearly without fluorescent labeling. In the MEC chip, a homogeneous EF is generated inside the culture area that simulates the endogenous EF environment. With this MEC chip-SINAP system, we directly observe and quantify the biased growth of filopodia of lung cancer cells toward the cathode. The epidermal growth factor receptors around the cell edges are also redistributed to the cathodal side. These results suggest that cancer-cell filopodia respond to the changes in EFs in the microenvironment.

We measure the temporal evolution of three-dimensional membrane topography on living fibroblasts and characterize the propagation of membrane waves using a wide-field optical profiling technique. The measured membrane profiles are compared with the numerical results calculated by the active membrane model recently proposed by Shlomovitz and Gov. After the treatments of blebbistatin and latrunculin A separately, the membrane waves disappear and the membrane surfaces are flattened, verifying that the membrane waves are driven by the interactions between myosin II and actin polymerization.

Applying the principle of differential confocal microscopy to wide-field optically sectioning microscopy, we develop a noninterferometric optical profilometer without scanning mechanisms. Depth resolution of 2 nm is achieved with a power-regulated tungsten-halogen lamp as the light source and a 14-bit CCD camera as the detector. The effects of inhomogeneous surface reflectivity are removed from topographic measurements by arithmetic division. The whole system can be constructed on a single silicon chip for use as a miniaturized optical profiler.

We employ a microfluidic chip with three culture chambers to investigate the interactions among lung cancer cells, macrophages and myofibroblasts. By mixing the conditioned media of macrophages and myofibroblasts in this chip, we confirm that these two stromal cells have synergistic effects in accelerating the migration of cancer cells. However, as the myofibroblasts are pretreated with the conditioned medium of macrophages, the myofibroblasts' ability to enhance the migration of cancer cells is lowered. The tumour necrosis factor-α produced by macrophages reduces the expression of α-smooth muscle actin and the secretion of transforming growth factor-β1 in myofibroblasts. Once the tumour necrosis factor-α in the macrophage conditioned medium is neutralized, the macrophage medium-pretreated myofibroblasts can still accelerate the migration of cancer cells.