Brian Michael Davis

Nonthermal Atmospheric Plasma Rapidly Disinfects Multidrug-Resistant Microbes by Inducing Cell Surface Damage

ABSTRACT Plasma, a unique state of matter with properties similar to those of ionized gas, is an effective biological disinfectant. However, the mechanism through which nonthermal or “cold” plasma inactivates microbes on surfaces is poorly understood, due in part to challenges associated with processing and analyzing live cells on surfaces rather than in aqueous solution. Here, we employ membrane adsorption techniques to visualize the cellular effects of plasma on representative clinical isolates of drug-resistant microbes. Through direct fluorescent imaging, we demonstrate that plasma rapidly inactivates planktonic cultures, with >5 log10 kill in 30 s by damaging the cell surface in a time-dependent manner, resulting in a loss of membrane integrity, leakage of intracellular components (nucleic acid, protein, ATP), and ultimately focal dissolution of the cell surface with longer exposure time. This occurred with similar kinetic rates among methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Candida albicans. We observed no correlative evidence that plasma induced widespread genomic damage or oxidative protein modification prior to the onset of membrane damage. Consistent with the notion that plasma is superficial, plasma-mediated sterilization was dramatically reduced when microbial cells were enveloped in aqueous buffer prior to treatment. These results support the use of nonthermal plasmas for disinfecting multidrug-resistant microbes in environmental settings and substantiate ongoing clinical applications for plasma devices.

Single-Cell Mass Spectrometry of Subpopulations Selected by Fluorescence Microscopy

Specific subpopulations in a heterogeneous collection of cells, for example, cancer stem cells in a tumor, are often associated with biological or medical conditions. Fluorescence microscopy, based on biomarkers labeled with fluorescent probes, is a widely used technique for the visualization and selection of such cells. Phenotypic differences for these subpopulations at the molecular level can be identified by their untargeted analysis by single-cell mass spectrometry (MS). Here, we combine capillary microsampling MS with fluorescence microscopy for the analysis of metabolite and lipid levels in single cells to discern the heterogeneity of subpopulations corresponding to mitotic stages. The distributions of ATP, reduced glutathione (GSH), and UDP- N-acetylhexosamine (UDP-HexNAc) levels in mitosis reveal the presence of 2-3 underlying subpopulations. Cellular energy is found to be higher in metaphase compared to prometaphase and slightly declines in anaphase, telophase, and cytokinesis. The [GTP]/[GDP] ratio in cytokinesis is significantly higher than in prometaphase and anaphase. Pairwise correlations between metabolite levels show that some molecules within a group, including certain amino acids and nucleotide sugars, are strongly correlated throughout mitosis, but this is not related to their pathway distances. Correlations are observed between monophosphates (AMP and GMP), diphosphates (ADP and GDP), and triphosphates (ATP and GTP) of different nucleosides. In contrast, there is low correlation between diphosphates and triphosphates of the same nucleoside (ADP and ATP).

High-Capacity Redox Polymer Electrodes: Applications in Molecular and Cellular Processing

We present methods to fabricate high-capacity redox electrodes using thick membrane or fiber casting of conjugated polymer solutions. Unlike common solution casting or printing methods used in current organic electronics, the presented techniques enable production of PEDOT:PSS electrodes with high charge capacity and the capability to operate under applied voltages greater than 100 V without electrochemical overoxidation. The electrodes are shown integrated into several electrokinetic components commonly used in automated bioprocess or bioassay workflows, including electrophoretic DNA separation and extraction, cellular electroporation/lysis, and electroosmotic pumping. Unlike current metal electrodes used in these applications, the high-capacity polymer electrodes are shown to function without electrolysis of solvent (i.e., without production of excess H+, OH–, and H2O2 by-products). In addition, each component fabricated using the electrodes is shown to have superior capabilities compared with those fabricated with common metal electrodes. These innovations in electrokinetics include a low-voltage/high-pressure electroosmotic pump, and a “flow battery” (in which electrochemical discharge is used to generate electroosmotic flow in the absence of an applied potential). The novel electrodes (and electrokinetic demonstrations) enable new applications of organic electronics within the biology, health care, and pharmaceutical fields.

Automated Closed-System Expansion of Pluripotent Stem Cell Aggregates in a Rocking-Motion Bioreactor

Pluripotent stem cell suspension aggregates have proven to be an efficient and phenotypically stable means for expansion and directed differentiation. Bioreactor systems with automation of perfusion, fluidization, and gas exchange are essential for scaling up pluripotent stem cell cultures. Since stem cell pluripotency and differentiation are affected by both chemical and physical signals, we investigated a low-shear method for the expansion of cells in a rocking-motion bioreactor. The rocking motion drives continual mixing and aeration, and the single-use disposable bioreactors avoid issues around contamination during seeding, medium exchange, passage, and cell harvest. Serial passaging from a 150 mL to a 1 L scale was demonstrated, achieving cell densities of up to 4 million cells/mL. In an average of 13 experiments, pluripotent stem cell aggregates expanded 5.7-fold (with maximal 9.5-fold expansion) and maintained 97% viability over 4 days in a rocking bioreactor culture. In seven experiments with improved culture conditions, the average expansion was 6.8-fold. Maintenance of pluripotency was confirmed by differentiation to all three germ layers and surface marker expression, and the expanded aggregates maintained a stable normal karyotype. The automation associated with the rocking platform bioreactor required no user intervention during the 4-day culture, providing hands-off expansion of pluripotent stem cells.

Inferring Mechanism of Action of an Unknown Compound from Time Series Omics Data

Identifying the mechanism of action (MoA) of an unknown, possibly novel, substance (chemical, protein, or pathogen) is a significant challenge. Biologists typically spend years working out the MoA for known compounds. MoA determination is especially challenging if there is no prior knowledge and if there is an urgent need to understand the mechanism for rapid treatment and/or prevention of global health emergencies. In this paper, we describe a data analysis approach using Gaussian processes and machine learning techniques to infer components of the MoA of an unknown agent from time series transcriptomics, proteomics, and metabolomics data.

Enabling Technology in Cell-Based Therapies: Scale-Up, Scale-Out, or Program In-Place

Those of us working in clinical and medical technology and automation are most enthusiastic about our work when the instruments and techniques we are developing will directly affect patient well-being. The recent arrival of FDAapproved chimeric antigen receptor (CAR) T-cell therapies and the further expansion of T-cell and other cell-based therapies beyond oncology applications have reinvigorated discussions around the ways in which we harvest, culture, process, or directly alter therapeutic cells. However, the manufacturing process (i.e., selection of peripheral blood mononuclear cells from whole blood, activation of T cells, transduction with CAR viral vectors or transposons, and expansion in an appropriate bioreactor) for combination gene/cell therapies such as CAR T is complex, and there remain many opportunities for improvements to decrease the cost and improve the safety of these important new clinical tools. In this SLAS Technology special issue titled “Enabling Technology in Cell-Based Therapies: Scale-Up, Scale-Out, or Program In-Place,” we highlight technologies that are changing the ways in which researchers and clinicians process and use therapeutic cells. One of the major technology areas with the potential to simplify and decrease the costs associated with harvesting and processing therapeutic cells is the technique used to select and separate the cells with the greatest therapeutic potential from the complex mixtures of cells in harvested blood and expanded cultures. While the culture reactors that the cells are loaded into for expansion have become closed and automated systems, the separation techniques for selecting and monitoring those cells have remained dependent on traditional technologies, such as centrifugation, fluorescence-activated cell sorting, and magnetic-activated cell sorting. New techniques that utilize “smart” dynamic magnetic traps, microfluidic separators, and acoustic energy-based cell separation techniques provide new inline and closed-loop systems that may be directly integrated with the rest of the cell culture and processing machinery. In this issue, three groups present methods of cell separation that utilize unique microscale forces that were originally developed for research and diagnostic applications that are in the process of being scaled-up (i.e., developed for use with either higher cell concentrations or large sample volumes) for industrial applications. Each technology has unique challenges to successfully “scale-up,” and specific advantages within industrial processes, such as label-free, quantitative, or biophysical versus molecular separation mechanisms. The authors of each separation technology share progress in overcoming these challenges, while presenting new device packages and footprints that may be more easily integrated into inline and online cell processing equipment. Other processes in addition to cell separation will need to be adapted to provide the ability to continually alter the cell state during the expansion of cells within bioreactors to improve cell manufacturing, while reducing cost. In this issue, two research articles describe methods to better automate or package complex cell manipulations into closed bioreactor systems. One group describes a method to automatically control sizes of stem cell aggregates within automated culturing systems, while another describes unique polymeric electrode systems that may be built directly into processing systems for applications such as electrical cell lysis and electrokinetic molecular separations. In addition to modifying processes that alter cell state, improved cell manufacturing systems will need to maintain the capability to monitor cell product quality. In the final technical report within this issue, the authors present methods to rapidly sample and analyze cell culture media to detect the presence of unwanted microorganisms, demonstrating the first steps toward building inline sterility tests that may be implemented directly within new cell manufacturing bioreactor systems. Finally, this issue contains two review articles that detail the status of cell bioreactors in both stem cell and tissue/ organ engineering applications, thus providing the user with the tools to think about how the new processing technologies described may find their place in a variety of commercial cell therapy and manufacturing systems. 779764 JLAXXX10.1177/2472630318779764SLAS TECHNOLOGY: Translating Life Sciences InnovationPuleo et al. research-article2018

Pilot study assessing the impact of platelet activation electric stimulation protocols on hematopoietic and mesenchymal stem cell proliferation

Recent research has shown that pulsed electric fields can successfully activate platelets ex-vivo; activation means here the release of growth factors and clotting. Typically, platelets are in a complex biological matrix, such as platelet rich plasma (PRP), which contains a variety of cell types. While specific electric pulses can activate the platelets, the impact of electric simulation on other cell types is an open question. The pilot study presented here focuses on evaluating electric pulse effects on hematopoietic and mesenchymal stem cells when they are in a complex biological matrix also containing platelets. Experimental results indicate that stem cell proliferation at two weeks post treatment can be tuned as a function of electrical parameters. We demonstrate in this pilot study that stem cell proliferation can be either low (via conductive coupling, 8.5 kV/cm electric field amplitude) or high (via capacitive coupling, 2.5 kV/cm electric field amplitude) two weeks after stimulation, despite these two electric pulse delivery mechanisms inducing roughly similar growth factor release and immediate cell viability post treatment. These observations may open up additional ways of tuning electric pulse delivery for platelet activation and other biomedical applications.