TECHNOLOGY

The liver performs many key functions, the most prominent of which is serving as the metabolic hub of the body. For this reason, the liver is the focal point of many investigations aimed at understanding an organism's toxicological response to endogenous and exogenous challenges. Because so many drug failures have involved direct liver toxicity or other organ toxicity from liver generated metabolites, the pharmaceutical industry has constantly sought superior, predictive in-vitro models that can more quickly and efficiently identify problematic drug candidates before they incur major development costs, and certainly before they are released to the public. In this broad review, we present a survey and critical comparison of in-vitro liver technologies along a broad spectrum, but focus on the current renewed push to develop "organs-on-a-chip". One prominent set of conclusions from this review is that while a large body of recent work has steered the field towards an ever more comprehensive understanding of what is needed, the field remains in great need of several key advances, including establishment of standard characterization methods, enhanced technologies that mimic the in-vivo cellular environment, and better computational approaches to bridge the gap between the in-vitro and in-vivo results.

Dyes such as hematoxylin and eosin (H&E) and immunohistochemical stains have been increasingly used to visualize tissue composition in research and clinical practice. We present an alternative approach to obtain the same information using stain-free chemical imaging. Relying on Fourier transform infrared (FT-IR) spectroscopic imaging and computation, stainless computed histopathology can enable a rapid, digital, quantitative and non-perturbing visualization of morphology and multiple molecular epitopes simultaneously in a variety of research and clinical pathology applications.

Microbial density control is a major challenge for food preservation especially in the low-income countries, where 850 million undernourished people live. In this work, we report on non-thermal, chemical-free microbial cell density control by intermittently delivered pulsed electric fields (IDPEF). We show that IDPEF allows for precise control of L. monocytogenes density in contaminated milk, an essential product for small farmers in low-income countries. Using L. monocytogenes growth kinetics, we designed an IDPEF protocol that consists of 2 sequences of 10 square wave pulses, 50 μ s duration, 12.5 kV cm^-1 electric field strength, delivered at 0.5 Hz and 1 min pause between the sequences applied every 1.5 hours. In a 12 hours experiment at 32 °C, L. monocytogenes density of untreated samples reached (9.1 ± 0.6) ⋅ 10⁷ CFU ml^-1 and (7.1 ± 0.3) ⋅ 10⁸ CFU ml^-1 for initial load of (1.4 ± 0.2) ⋅ 10³ CFU ml^-1 and (3.1 ± 0.3) ⋅ 10⁶ CFU ml^-1 respectively; the final density in the IDPEF treated samples, however, was 120 ± 44 CFU ml^-1 and (1.1 ± 0.3) ⋅ 10⁵ CFU ml^-1. The energy required for IDPEF storage of milk in the low-income countries could be generated by a small-scale 2kW solar energy system operating 5.5 hours per day in combination with small-scale energy storage system. We believe that IDPEF storage technology can empower millions of small farmers in the low-income countries by providing them a simple and energy efficient technology for milk preservation.

The isolation of CD4 positive T lymphocyte (CD4+) from peripheral blood is important for monitoring patients after HIV infection. Here, we demonstrate a fast isolation strategy for CD4+ cells that involves mixing blood and glass microbubbles. After the specific binding of target cells to the microbubbles carrying specific antibodies on their surface, target cells will spontaneously float to the top of the blood vial and can be quickly separated. Using this strategy, we demonstrate that the isolation of CD4+ cells in less than 5 minutes and with better than 90% efficiency. This strategy for cell isolation based on buoyancy and glass microbubbles is quick and inexpensive, minimizes blood handling, does not require magnetic fields, or centrifugation equipment, and could lead to new, efficient strategies for AIDS diagnosis in resource-limited areas.

Electrolytic ablation is a minimally invasive tissue ablation technique that operates by delivering low magnitude direct current to the target region over long periods of time, generating electrolytic products that destroy cells. Our study seeks to examine the hypothesis that permeabilizing the cell membrane with reversible electroporation will reduce the electrolytic dose required for tissue ablation, by exposing the interior of the cell to the electrolytic products. The hypothesis is examined by evaluating the extent of tissue damage when electrolytic and reversible electroporation sequences are delivered separately and in combination, it in vivo, to rat liver tissue. The study shows that combining reversible electroporation with electrolysis produces a substantial increase in the extent of tissue ablation compared to that achieved by electrolysis alone.

None of the ABCG2 inhibitors are effective clinically against multidrug resistant tumors overexpressing ABCG2. New in vitro models are needed to characterize inhibitors and discover new ones. We report a 3D spheroid model and image-based method to quantify ABCG2 action. Nonadhesive micro-molds were used to self-assemble spheroids overexpressing ABCG2; these spheroids were then incubated with the transporter substrate Hoechst 33342. Time-lapse fluorescent microscopy was used to determine the transporter-dependent efflux of Hoechst 33342 and dose response of three inhibitors (Ko143, Iressa, Elacridar). This 3D microtissue model was also used to determine the time to maximal effect as well as duration of effect after inhibitor removal. All acted within one hour and Elacridar had a surprisingly long duration of effect, active 5 hours after removal. This model can be used with multiple cell types, provides new insight into the pharmacokinetics of inhibitors, and can be adapted to high throughput analyses.

Long-term preservation of live cells is critical for a broad range of clinical and research applications. With the increasing diversity of cells that need to be preserved (e.g. oocytes, stem and other primary cells, genetically modified cells), careful optimization of preservation protocols becomes tedious and poses significant limitations for all but the most expert users. To address the challenge of long-term storage of critical, heterogeneous cell types, we propose a universal protocol for cell vitrification that is independent of cell phenotype and uses only low concentrations of cryoprotectant (1.5 M PROH and 0.5 M trehalose). We employed industrial grade microcapillaries made of highly conductive fused silica, which are commonly used for analytical chemistry applications. The minimal mass and thermal inertia of the microcapillaries enabled us to achieve ultrafast cooling rates up to 4,000 K/s. Using the same low, non-toxic concentration of cryoprotectant, we demonstrate high recovery and viability rates after vitrification for human mammary epithelial cells, rat hepatocytes, tumor cells from pleural effusions, and multiple cancer cell lines.

Proteins bearing short sequence tags (ExoTags) can be specifically modified in a reaction catalyzed by the ADP-ribosyltransferase of Pseudomonas ExoT. ExoT tolerates a wide range of substituents at the adenosine N6 position, allowing the convenient installation of chemical diversity at the site of the tag. The ExoTag can be placed at either the N- or the C-terminus of the protein of interest or internally. The minimum tag is Arg-Leu-Ser-Arg (RLSR). Tandem copies of the minimal ExoTag enhance the efficiency of modification and allow modification at multiple sites within the tag. ExoT can be used to conjugate NAD-RNA to ExoTag bearing proteins, providing a convenient method for introducing nucleic acid bar codes for highly sensitive detection of proteins by polymerase chain reaction.