Design of microfluidic chip for tumor spheroid-fibroblast co-culture.

<p>Structure and organization of a microfluidic chip used for 3D co-culture of human colorectal cancer cells (HT-29) and normal colorectal fibroblasts (CCD-18Co). One chip contained 4 units and one unit consisted of 7 channels for either cell loading or media fill. Channel designation for co-culture: cancer cells and fibroblast cells were loaded in channel 4 and 2, respectively, and other channels (1 and 3) were used for media fill. A cell loading channel is shown with detailed structure and dimension (left-bottom).</p

Effect of co-culture on growth and size distribution of spheroids.

<p>Cells were grown in collagen-supported microfluidic channels with or without fibroblast co-cultures and number and size distribution was determined at day 5. (A) Growth of HT-29 spheroids in size and number (scale bar = 200 μm). (B) Comparison of mean diameter of HT-29 spheroids over 5 days. (C) Comparison of size distribution of tumor spheroids on day 5. Diameter of spheroids was calculated using bright field images and Image J program. Cell aggregates of diameter larger than 50 μm were considered as spheroids. Student t-test as well as χ² test were used for the statistical significance. Data are expressed as the mean ± SE of 3 replicates. * p<0.05, **p<0.01.</p

Activation of fibroblasts under co-culture with 3D tumor spheroids.

<p>(A) Fluorescence images of fibroblasts stained for F-actin and α-SMA showing differential expression levels under mono- and co-culture conditions. (B) Increased migration ability of fibroblasts towards 3D tumor compartment. A picture for a representative regions showing fibroblast migrated out of the designated channel and comparison of migration distance. The migration distance of fibroblasts was measured between the nucleus of fibroblast in medium channel and end of fibroblast culture channel. Cells were grown for 6 days before all measurements. Optical sections were acquired at 3 μm intervals and stacked into a z-projection from which fluorescence intensity was calculated. Student t-test was used to test the statistical significance. Data are expressed as the mean ± SE of 3 replicates. (Scale bars 200 μm, 100 μm). * p<0.05.</p

General schematic of the device.

<p>Microfluidic system consisting of three independently addressable media channels, separated by chambers into which an ECM-mimicking gel can be injected (a). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056910#pone-0056910-g001" target="_blank">Figure 1b</a> shows the inside view of the device with endothelial monolayer (blue) covering the center channel. This channel acts as cell channel where both endothelial cells and cancer cells are introduced to form monolayer and transmigrate respectively (b). The green region indicates the 3D space filled with collagen gel and the pink regions indicate the channel filled with medium. Cancer cells which adhere to endothelial monolayer can extravasate into the collagen gel region as shown in (c).</p

Differential protein expressions in HT-29 tumor spheroids with or without fibroblast co-cultures.

<p>When co-cultured with fibroblasts, TS showed up-regulation of 7 angiogenesis-related proteins with 1.5-fold or greater changes (A) and down-regulation of 5 apoptosis-related proteins with greater than 30% changes (B). HT-29 TSs were grown for 6 days with or without fibroblasts in microfluidic channels and harvested for analysis using Proteome Profiler™ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159013#sec002" target="_blank">Materials and Methods</a> for details).</p

Optimization of tumor cell seeding density.

<p>The tumor cell seeding density was optimized to have only a limited number of tumor cells in ROI while maintaining as many experimental ROIs as possible that contain at least one tumor cell so tumor cell events can be observed. Histograms of number of total tumor cells present in each ROI (250 µm×250 µm×120 µm) show different trends in distribution of tumor cells for three different tumor seeding densities: 20,000 cells/ml, 50,000 cells/ml, and 200,000 cells/ml (a). The average value and the histogram can be used for choosing the optimal tumor seeding condition (b). Seeding density of 50,000 cells/ml was chosen as a compromise between mimicking the low number of tumor cells of the <i>in vivo</i> of extravasation condition and increasing the chance to have at least one tumor cell to analyze in any given ROI. The statistical significance was tested with one way ANOVA (p<0.05).</p

Confirmation of endothelial monolayer integrity.

<p>The integrity of the endothelial monolayer was confirmed by both fluorescence imaging of the dextran distribution and confocal microscopy of fixed and labeled cells. An intact endothelial monolayer gives rise to an abrupt intensity drop between the channel and the gel region once the fluorescently-labeled dextran is introduced. Three hours after dextran injection, a sharp drop in fluorescence intensity is seen across the endothelial layer demonstrating its function as a barrier to macromolecules (a). Fluorescence intensity is quantified using Matlab (b). The dashed arrow in (a) the location and direction for the quantification.The intensity value drops to 15% of is peak value due to the barrier effect. The endothelial monolayer is located near the 400 µm point on the plot (shown with dashed line). Samples fixed on the third day after cell seeding and stained for VE-cadherin and nuclei (DAPI-blue) exhibit well-defined junctions with no apparent gaps in the confluent monolayer (c). The confocal image shows the front view of the microfluidic device.</p

Simple and Highly Sensitive Molecular Diagnosis of Zika Virus by Lateral Flow Assays

We have developed a simple, user-friendly, and highly sensitive Zika virus (ZIKV) detection method by incorporating optimized reverse transcription loop-mediated isothermal amplification (RT-LAMP) and a lateral flow assay (LFA). The optimized RT-LAMP reaction was carried out using <i>Bst</i> 3.0 polymerase, which has robust and fast isothermal amplification performance even in the presence of high concentrations of inhibitors; this permitted the amplification of ZIKV RNA in pure water and human whole blood. In addition, the strong reverse transcription activity of <i>Bst</i> 3.0 polymerase enabled specific ZIKV RNA amplification without extra addition of reverse transcriptase. The RT-LAMP condition was optimized by adjusting the Mg²⁺ and dNTP mix concentration to extirpate nontarget amplification, which is caused by nonspecific primer dimers amplification. After 30 min of RT-LAMP reaction, the resultant amplicons were simply and rapidly analyzed by the LFA test in less than 5 min. The optimized RT-LAMP combined with the LFA allowed specific ZIKV RNA detection down to the single copy level within 35 min

Depiction of hepatic bile duct and CCA.

<p>(A) Upon infection of the common bile duct, <i>C. sinensis</i> produces ESPs, which stimulate CCA. (B) CCA and ESPs stimuli can be simulated using a microfluidic platform, culturing HuCCT1 cells (in the bile duct channel) on a COL1 hydrogel incorporated in the ECM channel. The cultured HuCCT1 cells form aggregates on COL1 in the bile duct channel and invade into the COL1 in response to ESPs stimuli.</p

Effect of ESPs on MMP mRNA and protein expression.

<p>HuCCT1 cells were treated with 800 ng/ml ESPs or an equivalent volume of PBS, harvested after 24 hours, and analyzed for the expression of MMP1, −2, −9 and −13 mRNA and protein. (A) Expression of MMP1, −2, −9, and −13 mRNA normalized to 18 S rRNA was assessed by qRT-PCR. *<i>P</i><0.05, ** p<0.01. Significance was analyzed by Student's <i>t</i>-test. Error bars, ± SEM. (B) Representative immunoblots of MMP1, −2, −9, and −13. The intensity of individual protein bands was determined by scanning densitometry and normalized to that of GAPDH. Data in graphs are expressed as a percentage of control values (in densitometric units).</p