Kavita M. Jeerage

Neurite outgrowth and differentiation of rat cortex progenitor cells are sensitive to lithium chloride at non-cytotoxic exposures.

Neuron-specific in vitro screening strategies have the potential to accelerate the evaluation of chemicals for neurotoxicity. We examined neurite outgrowth as a measure of neuronal response with a commercially available rat cortex progenitor cell model, where cells were exposed to a chemical during a period of cell differentiation. In control cultures, the fraction of beta-III-tubulin positive neurons and their neurite length increased significantly with time, indicating differentiation of the progenitor cells. Expression of glial fibrillary acidic protein, an astrocyte marker, also increased significantly with time. By seeding progenitor cells at varying densities, we demonstrated that neurite length was influenced by cell-cell spacing. After ten days, cultures seeded at densities of 1000 cells/mm(2) or lower had significantly shorter neurites than cultures seeded at densities of 1250 cells/mm(2) or higher. Progenitor cells were exposed to lithium, a neuroactive chemical with diverse modes of action. Cultures exposed to 30 mmol/L or 10 mmol/L lithium chloride (LiCl) had significantly lower metabolic activity than control cultures, as reported by adenosine triphosphate content, and no neurons were observed after ten days of exposure. Cultures exposed to 3 mmol/L, 1 mmol/L, or 0.3 mmol/L LiCl, which encompass lithiums therapeutic range, had metabolic activity similar to control cultures. These cultures exhibited concentration-dependent decreases in neurite outgrowth after ten days of LiCl exposure. Neurite outgrowth results were relatively robust, regardless of the evaluation methodology. This work demonstrates that measurement of neurite outgrowth in differentiating progenitor cell cultures can be a sensitive endpoint for neuronal response under non-cytotoxic exposure conditions.

Gold Nanoparticle Quantitation by Whole Cell Tomography

Many proposed biomedical applications for engineered gold nanoparticles require their incorporation by mammalian cells in specific numbers and locations. Here, the number of gold nanoparticles inside of individual mammalian stem cells was characterized using fast focused ion beam-scanning electron microscopy based tomography. Enhanced optical microscopy was used to provide a multiscale map of the in vitro sample, which allows cells of interest to be identified within their local environment. Cells were then serially sectioned using a gallium ion beam and imaged using a scanning electron beam. To confirm the accuracy of single cross sections, nanoparticles in similar cross sections were imaged using transmission electron microscopy and scanning helium ion microscopy. Complete tomographic series were then used to count the nanoparticles inside of each cell and measure their spatial distribution. We investigated the influence of slice thickness on counting single particles and clusters as well as nanoparticle packing within clusters. For 60 nm citrate stabilized particles, the nanoparticle cluster packing volume is 2.15 ± 0.20 times the volume of the bare gold nanoparticles.

Three-dimensional hydrogel constructs for exposing cells to nanoparticles

Abstract In evaluating nanoparticle risks to human health, there is often a disconnect between results obtained from in vitro toxicology studies and those from in vivo activity, prompting the need for improved methods to rapidly assess the hazards of engineered nanomaterials. In vitro studies of nanoparticle toxicology often rely on high doses and short exposure periods due to the difficulty of maintaining monolayer cell cultures over extended time periods as well as the difficulty of maintaining nanoparticle dispersions within the culture environment. In this work, tissue-engineered constructs are investigated as a platform for providing doses of nanoparticles over different exposure periods to cells within a three-dimensional environment that can be tuned to mimic in vivo conditions. Uptake of quantum dots (QDs) by model neural cells was first investigated in a high-dose exposure scenario, resulting in a strong concentration-dependent uptake of carboxyl-functionalised QDs. Poly(ethylene glycol) hydrogel scaffolds with varying mesh sizes were then investigated for their ability to support cell survival and proliferation. Cells were co-encapsulated with carboxyl-functionalised poly(ethylene glycol)-coated QDs at a lower dose than is typical for monolayer cultures. Although the QDs leach from the hydrogel within 24 h, they are also incorporated by cells within the scaffold, enabling the use of these constructs in future studies of cell behaviour and function.

Direct nuclear magnetic resonance observation of odorant binding to mouse odorant receptor MOR244-3.

Mammals are able to perceive and differentiate a great number of structurally diverse odorants through the odorants interaction with odorant receptors (ORs), proteins found within the cell membrane of olfactory sensory neurons. The natural gas industry has used human olfactory sensitivity to sulfur compounds (thiols, sulfides, etc.) to increase the safety of fuel gas transport, storage, and use through the odorization of this product. In the United States, mixtures of sulfur compounds are used, but the major constituent of odorant packages is 2-methylpropane-2-thiol, also known as tert-butyl mercaptan. It has been fundamentally challenging to understand olfaction and odorization due to the low affinity of odorous ligands to the ORs and the difficulty in expressing a sufficient number of OR proteins. Here, we directly observed the binding of tert-butyl mercaptan and another odiferous compound, cis-cyclooctene, to mouse OR MOR244-3 on living cells by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. This effort lays the groundwork for resolving molecular mechanisms responsible for ligand binding and resulting signaling, which in turn will lead to a clearer understanding of odorant recognition and competition.

Correlating Multiscale Measurements of Nanoparticles in Primary Cells

Nanoparticles are emerging as invaluable tools in disease diagnosis, disease treatment and imaging contrast enhancement agents [1]. The interactions of nanoparticles with host organisms are complex and affect biological systems over length scales that vary from the size of molecules to that of full organisms. In order to understand these interactions between nanoparticles and organisms, a variety of imaging and measurement techniques are required. We present imaging and analysis methods to statistically catalog cellular development on the macroscale, to identify the location of nanoparticles in cellular cultures on the microscale, and to identify the interaction of cellular organelles and nanoparticles on the nanoscale.

Single Cell Viability Measured by Scanning Electrochemical Microscopy and Live/Dead TM Staining

Metabolic activity is an unambiguous indicator of cell viability and health. A widely utilized probe molecule for cell cultures is resazurin, which is reduced to resorufin by living cells. Scanning electrochemical microscopy (SECM) can probe the metabolic activity of single cells via O2 reduction [1]; respiration causes a decrease in the O2 concentration above living cells. However, based on its standard reduction potential, oxidized ferrocenemethanol (FcCH2OH) may act as an alternate electron acceptor. By locally oxidizing FcCH2OH at a microelectrode, we probe metabolic activity using feedback measurements, which have higher resolution than passive measurements of O2 diffusion profiles. If FcCH2OH is regenerated by the cell, the current will increase when the microelectrode is positioned above a cell. This is known as positive feedback and has been reported for HeLa cells [2]. However if FcCH2OH diffusion is blocked by the cell, the current will decrease when the microelectrode is positioned above a cell. This is known as negative feedback and has been reported for COS-7 cells [3].

Rapid Synthesis and Correlative Measurements of Electrocatalytic Nickel/Iron Oxide Nanoparticles

Electrocatalytic core-shell nanoparticles, such as nickel/iron oxides for the oxygen evolution reaction (OER) in alkaline electrolytes, require rapid synthesis and measurement for practical use. To meet this challenge, we investigated a novel process of adding Ni(II) species to Fe nanoparticles immediately after synthesis, which we expected to yield Ni-rich shells around Fe-rich cores. Cyclic voltammetry showed that the overpotential decreased as the molar ratio of Ni to Fe in the synthesis vessel increased from 0.2 mol Ni:1 mol Fe to 1.5 mol Ni:1 mol Fe, consistent with an increase of Ni composition. Unexpectedly, the overpotential increased abruptly at 2.0 mol Ni:1 mol Fe. X-ray photoelectron spectroscopy revealed that this synthesis ratio resulted in less Ni at the nanoparticle surfaces than lower synthesis ratios. These results demonstrate the sensitivity of rapid electrochemical measurements to surface composition, and the limits of Ni(II) adsorption and reduction to rapidly form Ni-rich shells around Fe-rich cores. Cyclic voltammetry also showed that the onset of the methanol oxidation reaction (MOR) correlates with the oxidation of Ni(OH)2 to NiOOH. Therefore, tuning materials to improve performance as OER catalysts also improves their performance as MOR catalysts.

Localization and Number of Au Nanoparticles in Optically Indexed Cells by FIB Tomography

Gold nanoparticles (GNPs) are gaining importance as therapeutic chemical delivery vehicles, medical diagnostic tools, and phototherapeutic and contrast enhancement agents. GNPs are uniquely suited for these biological uses because of their chemical stability, novel optical properties, and broad potential for functionalization. Additionally, each of these beneficial properties is further enhanced by the ability to manufacture GNPs in an almost endless combination of sizes and shapes. This versatility has allowed researchers to access and modify biological processes inside of a large variety of cells [1] and the observation of innocuous uptake of citrate stabilized GNPs [2]. To describe the effect of GNPs, characterization of affected cells and tissues is required from the macroscopic to nanoscopic level. In particular, the location of cells in the tissue or culture of interest and then the mapping of the number and spatial distribution of the GNPs inside of those cells is required, and frequently requires multiple imaging techniques [3]. We achieve the large scale mapping of mammalian stem cells using reflection optical microscopy and then explore the location and number of the nanoparticles inside these cells after exposure to 60 nm GNPs using focused ion beam – scanning electron (FIB-SEM) based tomography.