Continuous centrifugal microfluidics (CCM) isolates heterogeneous circulating tumor cells via full automation.

Understanding cancer heterogeneity is essential to finding diverse genetic mutations in metastatic cancers. Thus, it is critical to isolate all types of CTCs to identify accurate cancer information from patients. Moreover, full automation robustly capturing the full spectrum of CTCs is an urgent need for CTC diagnosis to be routine clinical practice. Methods: Here we report the full capture of heterogeneous CTC populations using fully automated, negative depletion-based continuous centrifugal microfluidics (CCM). Results: The CCM system demonstrated high performance (recovery rates exceeding 90% and WBC depletion rate of 99.9%) across a wide range of phenotypes (EpCAM(+), EpCAM(-), small-, large-sized, and cluster) and cancers (lung, breast, and bladder). Applied in 30 lung adenocarcinoma patients harboring epidermal growth factor receptor (EGFR) mutations, the system isolated diverse phenotypes of CTCs in marker expression and size, implying the importance of unbiased isolation. Genetic analyses of intra-patient samples comparing cell-free DNA with CCM-isolated CTCs yielded perfect concordance, and CTC enumeration using our technique was correlated with clinical progression as well as response to EGFR inhibitors. Conclusion: Our system also introduces technical advances which assure rapid, reliable, and reproducible results, thus enabling a more comprehensive application of robust CTC analysis in clinical practice.

Noble metal sensitized invasive porous bioelectrodes: advanced medical device for enhanced neuronal activity and chronic alcohol treatment.

Invasive bioelectrodes are widely used as an effective treatment for several acute and chronic diseases. In earlier work using high surface area invasive porous bioelectrodes evaluated in an animal model of alcoholism withdrawal, we demonstrated significantly improved electrophysiological and behavioral responses. In this study, we further modify the surface of these invasive porous bioelectrodes with noble metal (Ag, Au, Pt) nanoparticles. Compared to both conventional and porous bioelectrodes, noble metal sensitized invasive porous bioelectrodes show markedly increased low threshold (LT) and wide dynamic range (WDR) neuronal activity. In particular, Pt-sensitized invasive porous bioelectrodes show the highest WDR neuronal activity only upon insertion. In addition, Ag-sensitized invasive porous bioelectrodes, whose surface area is about 37 times greater than that of conventional bioelectrodes, show improved electrochemical properties with higher LT and WDR neuronal activity when stimulated. In an animal model of chronic alcoholism, using normal and alcohol-treated Sprague-Dawley (SD) rats evaluated with the elevated plus maze (EPM) test, the Ag-sensitized invasive porous bioelectrodes show about 20% higher open arms time. These results suggest that these noble metal-sensitized invasive bioelectrodes may offer improved therapeutic outcomes for the treatment of chronic alcoholism, and given these enhanced electrophysiological properties, for other conditions as well.

Cell Seeding Technology for Microarray-Based Quantitative Human Primary Skeletal Muscle Cell Analysis.

Pipetting techniques play a crucial role in obtaining reproducible and reliable results, especially when seeding cells on small target areas, such as on microarrays, biochips or microfabricated cell culture systems. For very rare cells, such as human primary skeletal muscle cells (skMCs), manual (freehand) cell seeding techniques invariably result in nonuniform cell spreading and heterogeneous cell densities, giving rise to undesirable variations in myogenesis and differentiation. To prevent such technique-dependent variation, we have designed and fabricated a simple, low-cost pipet guidance device (PGD), and holder that works with hand-held pipettes. This work validates the accuracy and reproducibility of the PGD platform and compares its effectiveness with manual and robotic seeding techniques. The PGD system ensures reproducibility of cell seeding, comparable to that of more expensive robotic dispensing systems, resulting in a high degree of cell uniformity and homogeneous cell densities, while also enabling cell community studies. As compared to freehand pipetting, PGD-assisted seeding of C2C12 mouse myoblasts showed 5.3 times more myotube formation and likewise myotubes derived from PGD-seeded human primary skMCs were 3.6 times thicker and 2.2 times longer. These results show that this novel, yet simple PGD-assisted pipetting technique provides precise cell seeding on small targets, ensuring reproducible and reliable high-throughput cell assays.

Method to purify and analyze heterogeneous senescent cell populations using a microfluidic filter with uniform fluidic profile.

To precisely purify and study aged (senescent) cells, we have designed, fabricated, and demonstrated a novel diamond-structure (DS) microfluidic filter. Nonuniform flow velocities within the microfilter channel can compromise microfluidic filter performance, but with this new diamond structure, further optimized via simulation, we achieve a uniform microfilter flow field, improving the throughput of size-based separation of senescent cells, as obtained by 39-passaged human dermal fibroblasts. After separating these aged cells into two groups, consisting of large- and small-sized cells, we assessed senescence by measuring lipofuscin accumulation and ß-galactosidase activity. Our results reveal that even though these senescent cells had been equivalently passaged in culture, a high degree of size distribution and senescent phenotype heterogeneity was observed. In particular, the smaller-sized cells tended to express a younger phenotype while the larger aged cells demonstrated an older phenotype. We suggest that size-based separation of senescent cells, subtyped into small- and large-sized cohorts, offers an alternative method to purify such aged cells, thereby enabling more precise study of the mechanisms of aging, autophagy impairment, and rejuvenation.

A trachea-inspired bifurcated microfilter capturing viable circulating tumor cells via altered biophysical properties as measured by atomic force microscopy.

Circulating tumor cells (CTCs), though exceedingly rare in the blood, are nonetheless becoming increasingly important in cancer diagnostics. Despite this keen interest and the growing number of potential clinical applications, there has been limited success in developing a CTC isolation platform that simultaneously optimizes recovery rates, purity, and cell compatibility. Herein, a novel tracheal carina-inspired bifurcated (TRAB) microfilter system is reported, which uses an optimal filter gap size satisfying both 100% theoretical recovery rate and purity, as determined by biomechanical analysis and fluid-structure interaction (FSI) simulations. Biomechanical properties are also used to clearly discriminate between cancer cells and leukocytes, whereby cancer cells are selectively bound to melamine microbeads, which increase the size and stiffness of these cells. Nanoindentation experiments are conducted to measure the stiffness of leukocytes as compared to the microbead-conjugated cancer cells, with these parameters then being used in FSI analyses to optimize the filter gap size. The simulation results show that given a flow rate of 100 µL min(-1), an 8 µm filter gap optimizes the recovery rate and purity. MCF-7 breast cancer cells with solid microbeads are spiked into 3 mL of whole blood and, by using this flow rate along with the optimized microfilter dimensions, the cell mixture passes through the TRAB filter, which achieves a recovery rate of 93% and purity of 59%. Regarding cell compatibility, it is verified that the isolation procedure does not adversely affect cell viability, thus also confirming that the re-collected cancer cells can be cultured for up to 8 days. This work demonstrates a CTC isolation technology platform that optimizes high recovery rates and cell purity while also providing a framework for functional cell studies, potentially enabling even more sensitive and specific cancer diagnostics.

SSA-MOA: a novel CTC isolation platform using selective size amplification (SSA) and a multi-obstacle architecture (MOA) filter.

Circulating tumor cells (CTCs) have gained increasing attention as physicians and scientists learn more about the role these extraordinarily rare cells play in metastatic cancer. In developing CTC technology, the critical criteria are high recovery rates and high purity. Current isolation methods suffer from an inherent trade-off between these two goals. Moreover, ensuring minimal cell stress and robust reproducibility is also important for the clinical application of CTCs. In this paper, we introduce a novel CTC isolation technology using selective size amplification (SSA) for target cells and a multi-obstacle architecture (MOA) filter to overcome this trade-off, improving both recovery rate and purity. We also demonstrate SSA-MOA's advantages in minimizing cell deformation during filter transit, resulting in more stable and robust CTC isolation. In this technique, polymer microbeads conjugated with anti-epithelial cell adhesion molecules (anti-EpCAM) were used to selectively size-amplify MCF-7 breast cancer cells, definitively differentiating from the white blood cells (WBCs) by avoiding the size overlap that compromises other size selection methods. 3 µm was determined to be the optimal microbead diameter, not only for size discrimination but also in maximizing CTC surface coverage. A multi-obstacle architecture filter was fabricated using silicon-on-glass (SOG) technology-a first such application of this fabrication technique-to create a precise microfilter structure with a high aspect ratio. The filter was designed to minimize cell deformation as simulation results predicted that cells captured via this MOA filter would experience 22% less moving force than with a single-obstacle architecture. This was verified by experiments, as we observed reliable cell capture and reduced cell deformation, with a 92% average recovery rate and 351 peripheral blood leukocytes (PBL) per millilitre (average). We expect the SSA-MOA platform to optimize CTC recovery rates, purity, and stability, increasing the sensitivity and reliability of such tests, thereby potentially expanding the utilization of CTC technologies in the clinic.

Diabetes: medical vs surgical disease?

The distinction between the medical and the surgical approach to disease has been a cornerstone of medical practice, and indeed with respect to the business and technology of medicine. It is common knowledge that diabetes is a medical disease-namely that drug therapy, whether it be via insulin or other medications, is the primary approach to therapy. This article argues that a reevaluation of the generalized (e.g., medication-based) approach to systemic blood sugar control may be in order. A consideration of the growing importance of interventional, device-based, or other surgical approaches to the primary management of diabetes has enormous implications for clinical practice as well as, of course, the business and technology of diabetes care.