Multiplexed Viability Assays for High-Throughput Screening of Spheroids of Multiple Sizes.

High-throughput (HT) drug screening is in high demand for successful drug discovery and personalized medicine. Spheroids act as a promising preclinical model for HT drug screening, which may decrease drug failures in clinical trials. Numerous spheroid-forming technological platforms are currently under development, which include synchronous, jumbo-sized, hanging drop, rotary, and nonadherent surface spheroid growth. Initial cell seeding concentration and time of culture play a vital role for spheroids to mimic the extracellular microenvironment of natural tissue, especially for HT preclinical evaluation. Hence microfluidic platforms become a potential technology to provide a confined space for the oxygen and nutrient gradients within the tissues while controlling the cell count and spheroid size in an HT manner. We describe here a microfluidic platform capable of generating spheroids of multiple sizes in a controlled manner with a predefined cell concentration for HT drug screening. Ovarian cancer spheroids grown on this microfluidic platform were evaluated for viability using a confocal microscope and flow cytometer. In addition, screening of the HT chemotherapeutic drug carboplatin was carried out on-chip to evaluate the impact of spheroid size on drug toxicity. This chapter summarizes a detailed protocol on microfluidic platform fabrication for spheroid growth, on-chip multi-sized spheroid analysis, and chemotherapeutic drug screening.

RF magnetron sputtering mediated NiTi/Ag coating on Ti-alloy substrate with enhanced biocompatibility and durability.

Mechanically robust, biocompatible and corrosion resistant Ag doped NiTi (NiTi/Ag) coatings were formed on implant grade commercially pure titanium substrates by R.F. magnetron sputtering. Five samples with varying silver content (0, 1, 3, 7, and 10 at.%) were prepared by controlling the power applied to Ag and NiTi targets. The intensity of X-ray photoelectron spectra peaks corresponding to Ni2p, Ti2p, Ag3d components were found proportional to respective coating compositions. The soft Ag crystallites were decreased the roughness and crystallinity of NiTi/Ag. Among all compositions, NiTi/Ag coating with 3 at.% Ag exhibited lowest friction coefficient (0.1) and wear rate (0.69 × 10-07 mm3/N ∗ mm). Electrochemical corrosion measurements indicated that Ag incorporation increased the corrosion resistance of NiTi. Increase in Ag content shifted Ecorr values in the anodic direction, and reduced the current density by one-order-of-magnitude. When cultured on NiTi/Ag coating with 3 at.% Ag, human dermal fibroblast neonatal cells demonstrated highest cell viability. The fluorescence micrographic image of the immunostained cells showed a well grown actin filament network. Overall, NiTi/Ag coated titanium substrates were found to be a promising orthopedic implant material.

Chitosanylated MoO3-Ruthenium(II) Nanocomposite as Biocompatible Probe for Bioimaging and Herbaceutical Detection.

Hybrid nanomaterials with inherent physicochemical properties and cytocompatibility are beneficial for healthcare utilities. This paper demonstrates the hybridization of transition-metal oxide (molybdenum trioxide, MoO3), optoelectrochemically active dye complex (Ru(II)), and biopolymer (chitosan, CS) into a single nanosystem. The asafetida-resin-mediated green synthesis of MoO3 nanoparticles (g-MoO3 NPs) enabled chemical adsorption of Ru(II) and CS. Optical imaging functionality of pristine g-MoO3, g-MoO3-Ru(II), and g-MoO3-Ru(II)/CS has been evaluated using Caenorhabditis elegans, as an in vivo animal model, at an excitation wavelength of 450 nm and observed emission of ∼600 nm. The localization of chitosan on the surface of g-MoO3-Ru(II) exhibits cytocompatibility promising for intracellular imaging. The intracellular antioxidant properties of the g-MoO3-Ru(II)/CS nanocomposite are more profound than pristine NPs as assessed by measuring reactive oxygen species and protein carbonyls against the standard drug resveratrol. The electrochemical transducing ability of the hybrid g-MoO3-Ru(II) nanocomposite has been tested using butein, as a model herbaceutical, with nanomolar precision (50-1250 nM). The triad composite of metal oxide, dye, and biopolymer enabled synergistic properties that are suitable for multifunctional application in intracellular imaging, antioxidant, and electrochemical sensor studies.

Methylene Blue-Fortified Molybdenum Trioxide Nanoparticles: Harnessing Radical Scavenging Property.

A hybrid nanosystem with impeccable cellular imaging and antioxidant functionality is demonstrated. The microwave irradiation-derived molybdenum trioxide nanoparticles (MoO3 NPs) were surface-functionalized with the cationic dye molecule, methylene blue (MB), which enables superior UV-visible absorbance and fluorescence emission wavelengths potential for bioimaging. The radical scavenging property of the pristine MoO3 NPs and MoO3-MB NPs were studied in vivo using Caenorhabditis elegans as the model system. Heat shock-induced oxidative stress in C. elegans was significantly resolved by the MoO3-MB NPs, in agreement with the in vitro radical scavenging study by electron paramagnetic resonance spectroscopy. Hybrid nanostructures of MoO3-MB demonstrate synergistic benefits in intracellular imaging with intrinsic biocompatibility and antioxidant behavior, which can facilitate application as advanced healthcare materials toward bioimaging and clinical therapeutics.

Pumpless steady-flow microfluidic chip for cell culture.

The current research engineered a pumpless energy-efficient microfluidic perfusion cell culture chip that works by modifying the basic gravity-driven siphon flow using an intravenous (IV) infusion set as a conventional, inexpensive, and sterile tool. The IV set was modified to control the constant hydrostatic head difference, thereby maintaining the steady flow rate medium perfusion. The micro-bioreactor chip demonstrated flexibility in controlling a wide range of flow rates from 0.1 to 10ml/min, among which 1- and 5-ml/min flow rates were examined as suitable shear flows for long-term dermal fibroblast cell culture, paving the way for artificial skin development.

Dual drug-loaded nanoparticles on self-integrated scaffold for controlled delivery.

Antioxidant (quercetin) and hypoglycemic (voglibose) drug-loaded poly-D,L-lactideco-glycolide nanoparticles were successfully synthesized using the solvent evaporation method. The dual drug-loaded nanoparticles were incorporated into a scaffold film using a solvent casting method, creating a controlled transdermal drug-delivery system. Key features of the film formulation were achieved utilizing several ratios of excipients, including polyvinyl alcohol, polyethylene glycol, hyaluronic acid, xylitol, and alginate. The scaffold film showed superior encapsulation capability and swelling properties, with various potential applications, eg, the treatment of diabetes-associated complications. Structural and light scattering characterization confirmed a spherical shape and a mean particle size distribution of 41.3 nm for nanoparticles in the scaffold film. Spectroscopy revealed a stable polymer structure before and after encapsulation. The thermoresponsive swelling properties of the film were evaluated according to temperature and pH. Scaffold films incorporating dual drug-loaded nanoparticles showed remarkably high thermoresponsivity, cell compatibility, and ex vivo drug-release behavior. In addition, the hybrid film formulation showed enhanced cell adhesion and proliferation. These dual drug-loaded nanoparticles incorporated into a scaffold film may be promising for development into a transdermal drug-delivery system.

Real-time electrical measurement of L929 cellular spontaneous and synchronous oscillation.

Nonexcitable cell types, fibroblasts of heart muscle or astrocytes, are well known for their spontaneous Ca(2+) oscillations. On the other hand, murine fibroblast (L929) cells are known to be deficient in cell-cell adhesive proteins and therefore lack gap junctions for cellular communication. However, these cells exhibit a unique property of collectively synchronized and spontaneous oscillation, as revealed by real-time monitoring of cells cultured on a 250-µm diameter microelectrode for more than 3 days using an electrical cell-substrate impedance-sensing system (ECIS). Live-cell imaging is a widely used technique for oscillation detection, but it has limitations relating to cellular physiological environment maintenance for microscopic analysis and for prolonged periods of study. The present research emphasizes an electrical-sensing technique (ECIS) capable of overcoming the most important issues inherent in live-cell imaging systems for the detection of L929 cellular spontaneous and synchronized oscillation in real-time for longer periods. Possible mechanisms involved in L929 oscillation were elucidated to be periodic extension/contraction of lamellipodia continued as blebbing, which is produced by signals from the actomyosin complex initiated by connexin hemichannel opening and adenosine triphosphate (ATP) release. By applying the connexin hemichannel inhibitor, flufenamic acid, the hindrance of ATP release and calcium transients were analyzed to elucidate this hypothesis.

A CMOS image sensor to recognize the cardiovascular disease markers troponin I and C-reactive protein.

A complementary metal oxide semiconductor (CMOS) image sensor was utilized to detect the interaction of cardiovascular disease markers, troponin I and C-reactive protein. Each marker with its respective antibodies was adsorbed to an indium nanoparticle (InNP)-coated glass substrate. Dielectric layers of antigens and antibodies bound onto and interacted on conducting InNPs. Normal room light passed through these protein-layer-bound substrates and hit the CMOS image sensor surface, and the number of photons was detected and converted into digital form. We tested this approach for real-time monitoring of cardiac disease markers based on photon count, demonstrating its low cost and its capacity to detect antigens with high sensitivity at picogram per milliliter concentration.

CMOS image sensor for detection of interferon gamma protein interaction as a point-of-care approach.

Complementary metal oxide semiconductor (CMOS)-based image sensors have received increased attention owing to the possibility of incorporating them into portable diagnostic devices. The present research examined the efficiency and sensitivity of a CMOS image sensor for the detection of antigen-antibody interactions involving interferon gamma protein without the aid of expensive instruments. The highest detection sensitivity of about 1 fg/ml primary antibody was achieved simply by a transmission mechanism. When photons are prevented from hitting the sensor surface, a reduction in digital output occurs in which the number of photons hitting the sensor surface is approximately proportional to the digital number. Nanoscale variation in substrate thickness after protein binding can be detected with high sensitivity by the CMOS image sensor. Therefore, this technique can be easily applied to smartphones or any clinical diagnostic devices for the detection of several biological entities, with high impact on the development of point-of-care applications.

Electrical cell-substrate impedance sensing as a non-invasive tool for cancer cell study.

Cell-substrate interactions are investigated in a number of studies for drug targets including angiogenesis, arteriosclerosis, chronic inflammatory diseases and carcinogenesis. One characteristic of malignant cancerous cells is their ability to invade tissue. Cell adhesion and cytoskeletal activity have served as valuable indicators for understanding the cancer cell behaviours, such as proliferation, migration and invasion. This review focuses on bio-impedance based measurement for monitoring the behaviours in real time and without using labels. Electric cell-substrate impedance sensing (ECIS) provides rich information about cell-substrate interactions, cell-cell communication and cell adhesion. High sensitivity of the ECIS method allows for observing events down to single-cell level and achieving nanoscale resolution of cell-substrate distances. Recently, its miniaturization and integration with fluorescent detection techniques have been highlighted as a new tool to deliver a high-content platform for anticancer drug development.

Complementary metal-oxide semiconductor (CMOS) image sensor: an insight as a point-of-care label-free immunosensor.

The present paper examines the efficiency of a complementary metal-oxide semiconductor (CMOS) using an indium nanoparticle (InNP) substrate for the high-sensitivity detection of antigen/antibody interactions at concentrations as low as 100 pg/ml under normal light. Metal NPs coated with antigen/antibody layers act as a dielectric layer on the conducting sphere, which enhances the number of photons hitting the sensor surface through a light-scattering effect. This photon number is proportional to the digital number observed with the CMOS sensor for detecting antigen/antibody interactions.