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When breast cancer patients start to exhibit resistance to hormonal therapy or chemotherapy, the mTOR inhibitor everolimus can be considered as an alternative therapeutic agent. Everolimus can deregulate the PI3K/AKT/mTOR pathway and affect a range of cellular functions. In some patients, the agent does not exhibit the desired efficacy and, even worse, not without the associated side effects. This study assessed the use of immunofluorescence (IF) as a modality to fill this unmet need of predicting the efficacy of everolimus prior to administration. Cell viability and MTT assays based on IF intensities of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 on breast cancer cells (Hs578T, MCF7, BT474, MDA-MB-231) and patient-derived cell culture from metastatic sites (ABC-82T and ABC-16TX1) were interrogated. Results show that independent pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 IF expressions can classify data into different groups: everolimus sensitive and resistant. The combined IF baseline intensity of these proteins is predictive of the efficacy of everolimus, and their intensities change dynamically when cells are resistant to everolimus. Furthermore, mTOR resistance is not only consequence of the AKT/mTOR pathway but also through the LKB1 or MAPK/ERK pathway. The LKB1 and pho-GSK3β may also be potential predictive markers for everolimus.

Therapeutic drug synergism intervened in cancer treatments has been demonstrated to be more effective than using a single effector. However, it remains inherently challenging, with a limited cell count from tumor samples, to achieve potent personalized drug cocktails. To address the issue above, we herein present a nanodroplet cell processing platform. The platform incorporates an automatic nanodroplet dispenser with cell array ParaStamp chips, which were fabricated by a new wax stamping approach derived from laser direct writing. Such approach enables not only the on-demand de-wetting with hydrophobic wax films on substrates but also the mask-less fabrication of non-planar microstructures (i.e. no photolithography process). The ParaStamp chip was pre-occupied with anti-cancer drugs and their associate mixtures, enabling for the spatially addressable screening of optimal drug combinations simultaneously. Each droplet with a critical volume of 200 nl containing with 100 cells was utilized. Results revealed that the optimal combination reduces approximate 28-folds of conducted doses compared with single drugs. Tumor inhibition with the optimally selected drug combination was further confirmed by using PC-3 tumor-bearing mouse models. Together, the nanodroplet cell processing platform could therefore offer new opportunities to power the personalized cancer medicine at early-stage drug screening and discovery.

When breast cancer patients start to exhibit resistance to hormonal therapy or chemotherapy, the mTOR inhibitor everolimus can be considered as an alternative therapeutic agent. Everolimus can deregulate the PI3K/AKT/mTOR pathway and affect a range of cellular functions. In some patients, the agent does not exhibit the desired efficacy and, even worse, not without the associated side effects. This study assessed the use of immunofluorescence (IF) as a modality to fill this unmet need of predicting the efficacy of everolimus prior to administration. Cell viability and MTT assays based on IF intensities of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 on breast cancer cells (Hs578T, MCF7, BT474, MDA-MB-231) and patient-derived cell culture from metastatic sites (ABC-82T and ABC-16TX1) were interrogated. Results show that independent pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 IF expressions can classify data into different groups: everolimus sensitive and resistant. The combined IF baseline intensity of these proteins is predictive of the efficacy of everolimus, and their intensities change dynamically when cells are resistant to everolimus. Furthermore, mTOR resistance is not only consequence of the AKT/mTOR pathway but also through the LKB1 or MAPK/ERK pathway. The LKB1 and pho-GSK3β may also be potential predictive markers for everolimus.

With respect to male fertility, sperm play a crucial role in the whole process of fertilization. Many technologies for manipulating sperm have been developed over the past few decades and these have focused on counting and sorting sperm. Microfludic technologies were introduced into this field at the beginning of the 21 st century. This paper reviews the history of microfluidic technologies and their application to forensic medicine and sperm counting and sorting. An attempt is made to establish an automatic platform for in vitro fertilization using microfluidic technologies.

BACKGROUND Circulating endothelial cells (CECs) in the blood are rare but have been shown to be associated with various diseases. With the ratio of CECs to peripheral blood mononuclear cells (PBMCs) less than 1 part per thousand, their separation from PBMCs and detection are challenging. We present a means of detecting CECs from PBMCs via an economical microfluidic disk with a model cell system [human umbilical vein endothelial cells (HUVECs) in PBMCs], along with demonstration of its efficacy clinically. METHODS To enrich these rare cells, we used immunomagnetic beads and a tailor-made magnet on the disk. CEC-simulating HUVECs, as target cells, were stained with primary anti–CD146-phycoerythrin antibody and bound with secondary antibody on antiphycoerythrin magnetic beads. PBMCs served as nontarget cells and were labeled with anti–CD45-FITC antibody. RESULTS When hundreds of HUVECs were mixed in 106 PBMCs, 95% of spiked HUVECs were detected. This yield also held for 60 HUVEC in CONCLUSIONS The count of CECs is a suitable marker for symptoms of systemic lupus erythematosus. The microfluidic disk system should be a viable platform for detection of CECs.

Microenvironment mimicking biological situation is a vital issue in tissue regeneration. With much progress being made, one of the major challenges remains to develop a convenient method to fabricate the scaffold microenvironment suitable for cell attachment and proliferation. This article demonstrates the efficacy of microcontact printed laminin, an extracellular matrix protein, on three different oxygen plasma treatment substrates-tissue culture polystyrene, poly(methyl methacrylate) films, and chitosan films-for alignment and growth of the Schwann cells in in vitro culturing. Replica molding of polydimethylsiloxane elastomeric stamps, fabricated from patterned SU-8 structure on silicon master, was used to print laminin on the three substrates. Pattern and growth of Schwann cells for low (10(3) cells/cm(2)) and increased cell density (2 x 10(4) cells/cm(2)) on the varied substrates with and without microcontact printed laminin were characterized. Results of in vitro cell culture of Schwann cells showed a high degree of cell orientation on the laminin-micropatterned substrates for both cell densities. However, different cell seeding densities will strongly impact the morphology and orientation of Schwann cells. Microcontact printing proves to be a convenient means to pattern cell-recognition molecules on scaffold for cell-guilded growth in tissue regeneration.

Drug-based treatments are the most widely used interventions for cancer management. Personalized drug response profiling remains inherently challenging with low cell count harvested from tumour sample. We present a 96well-formatted microfluidic plate with built-in micro-gap that preserves up to 99.2% of cells during multiple assay/wash operation and only 9,000 cells needed for a single reagent test (i.e. 1,000 cells per test spot x 3 selected concentration x triplication), enabling drug screening and compatibility with conventional automated workstations. Results with MCF7 and MDA-MB-231 cell lines showed that no statistical significance was found in dose-response between the device and conventional 96-well plate control. Primary tumour samples from breast cancer patients tested in the device also showed good IC50 prediction. With drug screening of primary cancer cells must consider a wide range of scenarios, e.g. suspended/attached cell types and rare/abundant cell availability, the device enables high throughput screening even for suspended cells with low cell count since the signature microfluidic cell-trapping feature ensures cell preservation in a multiple solution exchange protocol.

Microfabricated devices for cell lysis have demonstrated many advantages over conventional approaches. Among various design of microdevices that employ electroporation for cytolysis, most utilize Ag/AgCl wires or 2D planar electrodes. Although, simple in fabrication the electric field generated by 2D electrodes decays exponentially, resulting in rather non-uniform forcing on the cell membrane. This paper investigates the effect of electric field generated by 3D cylindrical electrodes to perform cell lysis via electroporation in a microfluidic platform, and compared with that by 2D design. Computational results of the electric field for both 2D and 3D electrode geometries showed that the 3D configuration demonstrated a significantly higher effective volume ratio-volume which electric field is sufficient for cell lysis to that of net throughflow volume. Hence, the efficacy of performing cell lysis is substantially greater for cells passing through 3D than 2D electrodes. Experimentally, simultaneous multi-pores were observed on leukocytes lysed with 3D electrodes, which is indicative of enhanced uniformity of the electric field generated by 3D design. Additionally, a single row of 3D electrode demonstrated a substantially higher lysing percentage (30%) than that of 2D (8%) under that same flow condition. This work should aid in the design of electrodes in performing cell lysis via electroporation.

This paper addresses the vibratory mechanics associated with frequency discrimination of basilar membrane within the cochlear of the inner ear. Periodic excitation is provided to the oval window, which results in generation of waves within the fluid-filled cochlear traveling towards the apex. These waves interact with the compliant basilar membrane structure causing its vibratory motion. Solution procedure of the fluid/structure model consists of a two-step process. First, a finite element calculation (ANSYS) solves for the membrane vibration with an initial harmonic pressure distribution. Second, a control volume analysis links the resultant vibratory motion with the fluid pressure acting on the basilar membrane, thus a pressure feedback loop is accomplished. Results show that dominant factors affecting vibratory characteristics of the basilar membrane are its structural geometry and attenuation of pressure wave as it travels away from the oval window. Calculations clearly capture the designed function of the basilar membrane, principally its frequency discrimination behavior.

Circulating tumor cells (CTC) are believed to be the culprit of metastasis and studies have shown that their enumeration has prognostic value in wide range of solid tumors. Although much progress has been made in CTC technology, their rarity in blood and their inherent heterogeneity still provide much challenge towards maturity of the technology. This paper presents semi-automated enrichment of CTC via density-based approach in a novel microfluidic disk, followed by multi-step on-disk immunofluorescence staining (pan-CK, EpCAM, Hoechst and CD45), fluorescence microscopy for image capture, and software image analysis. To characterize the performance of the system, we spiked 1 to 300 cells (mean 105, median 125) from six cell lines (DLD-1, Huh-7, MCF7, PC3, MDA-MB-231 and PC-9) from five cancers into whole blood from healthy donors. The recovery rate of the system is 87.4%±3.7% (R2=0.958) regardless of EpCAM expression levels of the cell lines. To interrogate the limitation of the technology, this data set included ultra-low cell counts spiked of 1 to 13 cells (mean 5.7, median 5) which might be indicative of CTC from metastatic breast cancer (MBC) patients. The recovery rate for this low cell count is 90%±10%. Notably, one single cell was spiked into healthy whole blood, processed via the technology, and one target cell was detected. This test was repeated in triplicate with all three tests recovered one cell each. Furthermore, the microfluidic disk technology enables operation over a range of blood volume (from 2 to 7.5ml) with no statistically significant difference in recovery rate. Recovered MCF-7 cells spiked in blood were subsequently cultured for 6 days and showed good viability with cell proliferation. We tested the technology in MBC patients along with CA15-3 and CT imaging. A total 34 of blood samples were collected from 21 patients over the course of their systemic treatment. Results showed CTC were detected in 28 samples (82%). The target CTC detected ranges from 0 to 138 (mean 16, median 4 CTCs per 7.5mL). 15 out of 34 samples (44%) had CTC number ≥ 5/7.5mL). In one patient with triple negative diagnosis, CTC count, CA15-3 and CT imaging were monitored during the course of chemotherapy. The CTC count remained zero at the end of the first and second treatment course, elevated to 24 CTCs at the end of the third course, and continued its elevation to 31 by the end of the fourth treatment course. During this entire treatment course, CA15-3 did not vary significantly. However, CT image confirmed the metastasized liver tumor grew from 6.15cm at the beginning of the second chemo course to 8.09cm at the beginning of the fourth course. Taken together, our label-free CTC enrichment technology has high analytical sensitivity (87%) over a wide range of cancers, able to handle a flexible blood volume (2mL to 7.5mL), and amenable to detect a high percentage (82%) of CTC in MBC patients. Citation Format: Yu-Jen Chang, Chen-Lin Chen, Wei-Fan Hsu, Meng-Ze Li, Wei-Yuan Ma, Ken-Chao Chen, Guan-Syuan Huang, Wai-Sang Wong, Jhan-Yu Syu, Thomas, Yo-Yan Huang, Ching-Hung Lin, Andrew M. Wo, Chiun-Sheng Huang. Label-free enrichment and detection of circulating tumor cells in metastatic breast cancer patients show 82% of cohorts have detectable targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 798. doi:10.1158/1538-7445.AM2017-798

Three-dimensional (3D) tissue culture platforms that are capable of mimicking in vivo microenvironments to replicate physiological conditions are vital tools in a wide range of cellular and clinical studies. Here, learning from the nature of cilia in lungs - clearing mucus and pathogens from the airway - we develop a 3D culture approach via flexible and kinetic copolymer-based chains (nano-cilia) for diminishing cell-to-substrate adhesion. Multicellular spheroids or colonies were tested for 3-7 days in a microenvironment consisting of generated cells with properties of putative cancer stem cells (CSCs). The dynamic and reversible regulation of epithelial-mesenchymal transition (EMT) was examined in spheroids passaged and cultured in copolymer-coated dishes. The expression of CSC markers, including CD44, CD133, and ABCG2, and hypoxia signature, HIF-1α, was significantly upregulated compared to that without the nano-cilia. In addition, these spheroids exhibited chemotherapeutic resistance in vitro and acquired enhanced metastatic propensity, as verified from microfluidic chemotaxis assay designed to replicate in vivo-like metastasis. The biomimetic nano-cilia approach and microfluidic device may offer new opportunities to establish a rapid and cost-effective platform for the study of anti-cancer therapeutics and CSCs.

BACKGROUND Semen analysis is essential for evaluating male infertility. Besides sperm concentration, other properties, such as motility and morphology, are critical indicators in assessing sperm quality. Nevertheless, rapid and complete assessment of these measures still presents considerable difficulty and involves a range of complex issues. Here we present a microfluidic device capable of quantifying a range of properties of human sperm via the resistive pulse technique (RPT). METHODS An aperture, designed as a long channel, was used to allow the quantification of various properties as sperm swam through. RESULTS The time trace of the voltage drop across the aperture during sperm passage contained a wealth of information: the sperm volume was presented by the amplitude of the induced pulse, the swim velocity was evaluated via the duration, and the beat frequency was calculated from the voltage undulation superposed on the pulse signal. The RPT measurement of swim velocity and beat frequency showed a correlation with the same observation in a microscope (R2 = 0.94 and 0.70, respectively). CONCLUSIONS The proposed proof of principle enables substantial quantification of the motion-dependent properties of sperm. Because this approach requires only a current/voltage source and data analysis, it is economically advantageous compared with optical methods for characterizing sperm motion. Furthermore, this approach may be used to characterize sperm morphology.

This paper reports a hydrodynamic single-cell trapping platform for cellular assays. This approach utilizes an oscillatory microplate (100μm × 100μm) via Lorentz force to generate microvortices for cell trapping. The microdevice can trap a single cell in suspended state, which should aid analysis of a single cell. Results obtained with this platform show a single Jurkat cell can be trapped and labeled on CD45 antibodies under real-time monitoring.

We present a dielectrophoresis (DEP)-based cellular microarray chip for cell-based anticancer drug screening in perfusion microenvironments. Human breast cancer cells, MCF7, were seeded into the chip and patterned via DEP forces onto the planar interdigitated ring electrode (PIRE) arrays. Roughly, only one third of the cell amount was required for the chip compared to that for a 96-well plate control. Drug concentrations (cisplatin or docetaxel) were stably generated by functional integration of a concentration gradient generator (CGG) and an anti-crosstalk valve (ACV) to treat cells for 24 hours. Cell viability was quantified using a dual staining method. Results of cell patterning show substantial uniformity of patterned cells (92 ± 5 cells per PIRE). Furthermore, after 24 hour drug perfusion, no statistical significance in dose-responses between the chip and the 96-well plate controls was found. The IC(50) value from the chip also concurred with the values from the literature. Moreover, the perfusion culture exhibited reproducibility of drug responses of cells on different PIREs in the same chamber. The chip would enable applications where cells are of limited supply, and supplement microfluidic perfusion cultures for clinical practices.