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Exosomes are believed to be secreted from multivesicular endosomes and containing proteins and nucleic acids, including mRNA and microRNAs, which have been implicated to play a role in neurodegenerative diseases. Neuron-derived exosomes at the circulation provide a unique potential as biomarkers towards assessment of Alzheimer's disease (AD), even at the pre-clinical stage. This review briefly discusses their biogenesis and transport, exosomal protein verses soluble protein, evidence for their role in AD, isolation of exosomes, and challenges and future directions to realize reliable blood-based biomarkers to meet phenomenal unmet clinical and pre-clinical need of AD.

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.

Non-planar microstructure-based tissue culture devices have emerged as powerful tools to mimic in vivo physiological microenvironments in a wide range of medical applications. Here we report a spontaneous aqueous molding approach - inspired by Stenocara gracilipes beetles - to rapidly fabricate non-planar microstructure devices for facilitating tissue-based bioassays. The device fabrication is determined from the self-assembled liquid morphology, which is induced by condensation or guided by surface tension. Through experiments and modeling, we reveal that the molding mainly comprises two typical circular and striped domains, highlighting versatile applications for bioengineering. In addition, the molding characteristic is dependent on the geometry of the patterned wetting surfaces, the working volume of the liquid, and the interaction between the liquid and the substrate. The theoretical model, based on the geometry of the patterned liquid, is highly consistent with experimental data. We also demonstrate that our approach can facilitate the culturing of tumor spheroids incorporated with biomimic nano-cilia, rapid high-throughput drug screening, tumor spheroid migration assay, and in vitro modeling of blood vessels. Remarkably, the delivery of multiple concentrations of drugs and their associate mixtures (a total of 25 test spots in one device) can be carried out simultaneously within seconds. Taken together, these insights may offer new opportunities to tailor non-planar microstructures, and our proposed methodology can be applicable for the emerging needs in tumor cell biology and tissue engineering.

We present a microarray chip integrated with novel round bottom μ-well arrays and biomimetic nano-cilia, which can, for the first time, not only recapitulate in vivo-like tumor microenvironment but also be achieved in situ for hallmarks of tumor-based bioassays.

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.

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

Engineered materials have been employed as versatile tools to explore fundamental processes in cell biology/drug development as well as an approach towards intelligent devices, thereby becoming key components in modern technology. Herein, a ParaStamp technique is revealed to possess applications for cell patterning, drug screening, and rewritable functional patterning. The ParaStamp includes a micropatterned polydimethylsiloxane master and a liquid-phased paraffin oil generated at high temperature, which can transfer the patterned paramembrane onto varied material surfaces such as glass, polystyrene, and flexible foil. This technique is simple and cost-effective to meet the high-throughput requirement for industries. Taken together, the findings herein should provide general insights in cell biology, biodetection, and the development of smart hydrophobic surfaces.

Communication between cells and extracellular environments is of interest because of its critical roles in cell development and differentiation. Particularly, this signal transduction is commonly believed to rely on the contact and binding of the participating molecules/proteins, suggesting that the binding distance needed is less than a few nanometers. However, it is difficult to precisely match the rapidly binding interaction which depends on the probability of molecular collision in living systems, raising a hypothesis that another mechanism exists, could promote this signal communication, and remains unknown. Here we report that a long-range signal delivery over 10-μm and 20-μm polydimethylsiloxane(PDMS) barriers can be observed in microfluidically tetracycline (Tet) inducible expression systems. Results show that a significant increment of the long-range induced green fluorescent protein in human embryonic kidney 293T (HEK 293T) cells by the stimulation of Tet is demonstrated, and that such a signal induction is not dominated by Tet diffusion and displays a specific bindingless property. In addition, our experimental results, combined with theoretical modeling, suggest that this communication exhibits a bump-shaped characteristic depending on barrier thickness, materially structural property, surface roughness, and agonist concentration. It strongly relies on the PDMS barrier to delivery signal; therefore, we call such a mechanism as “wired” cell communication instead of wireless. These results could ignite interests in the novel and “wired” cell communication, which we call it X-signal, and in the use of such systems for the study of cellular biology and development of new drug.