Stimulation of the hepatoportal nerve plexus with focused ultrasound restores glucose homoeostasis in diabetic mice, rats and swine.

Peripheral neurons that sense glucose relay signals of glucose availability to integrative clusters of neurons in the brain. However, the roles of such signalling pathways in the maintenance of glucose homoeostasis and their contribution to disease are unknown. Here we show that the selective activation of the nerve plexus of the hepatic portal system via peripheral focused ultrasound stimulation (pFUS) improves glucose homoeostasis in mice and rats with insulin-resistant diabetes and in swine subject to hyperinsulinemic-euglycaemic clamps. pFUS modulated the activity of sensory projections to the hypothalamus, altered the concentrations of metabolism-regulating neurotransmitters, and enhanced glucose tolerance and utilization in the three species, whereas physical transection or chemical blocking of the liver-brain nerve pathway abolished the effect of pFUS on glucose tolerance. Longitudinal multi-omic profiling of metabolic tissues from the treated animals confirmed pFUS-induced modifications of key metabolic functions in liver, pancreas, muscle, adipose, kidney and intestinal tissues. Non-invasive ultrasound activation of afferent autonomic nerves may represent a non-pharmacologic therapy for the restoration of glucose homoeostasis in type-2 diabetes and other metabolic diseases.

Peripheral Focused Ultrasound Neuromodulation (pFUS).

BACKGROUND: A fundamental limit to the study of the peripheral nervous system and its effect on organ function is the lack of tools to selectively target and stimulate specific neurons. Traditional implant and electrode-based systems remain too large and invasive for use at the organ or sub-organ level (without stimulating or effecting neighboring organs and tissues). Recent progress in optical and genetic tools (such as optogenetics) has provided a new level of molecular specificity and selectivity to the neurons that are stimulated by bioelectronic devices. However, the modified neurons that result from use of these tools (that can be selectively activated based on expression of light, heat, or stimuli sensitive ion channels) often still require stimulation by implantable devices and face difficult scientific, technical, and regulatory hurdles for clinical translation. NEW METHOD: Herein, we present a new tool for selective activation of neuronal pathways using anatomical site-specific, peripheral focused ultrasound neuromodulation (pFUS). RESULTS: We utilize three experimental models to expand upon and further characterize pFUS beyond data outlined to our initial report (Cotero et al., 2019a), and further demonstrate its importance as a new investigative and translational tool. First, we utilized an interconnected microporous gel scaffold to culture isolated dorsal root ganglion (DRG) neurons in an interconnected, three-dimensional in vitro culture. (Griffin et al., 2015, Tay et al., 2018) Using this system, we directly applied ultrasound (US) stimuli and confirmed US activation of peripheral neurons at pressures consistent with recent in vivo observations. (Cotero et al., 2019a, Zachs, 2019, Gigliotti et al., 2013) Next, we tested the capability of pFUS to activate previously reported nerve pathways at multiple locations within the neural circuit, including primary sensory ganglia (i.e. inferior ganglion of the vagus nerve), peripheral ganglia (i.e. sacral ganglia), and within target end-organs. In addition, we compared selective activation of multiple anatomically overlapping neural pathways (i.e. activation of the cholinergic anti-inflammatory pathway (Tracey, 2009, Pavlov and Tracey, 2012) vs. metabolic sensory pathways (O'Hare and Zsombok, 2015, Roh et al., 2016, Pocai et al., 2005) after stimulation of each separate target site. Finally, we utilized an established model of metabolic dysfunction (the LPS-induced inflammation/hyperglycemia model) to demonstrate pFUS capability to stimulate and assess alternative therapeutic stimulation sites (i.e. liver, pancreas, and intestines) in a simple and clinically relevant manner. This is demonstrated by ultrasound induced attenuation of LPS-induced hyperglycemia by stimulation at all three anatomical targets, and mapping of the effect to a specific molecular product of excitable cell types within each stimulus site. COMPARISON WITH EXISTING METHODS: The ease-of-use and non-invasive nature of pFUS provides a solution to many of the challenges facing traditional toolsets, such as implantable electrodes and genetic/optogenetic nerve stimulation strategies. CONCLUSIONS: The pFUS tool described herein provides a fundamental technology for the future study and manipulation of the peripheral nervous and neuroendocrine systems.

Novel DNA-based T-Cell Activator Promotes Rapid T-Cell Activation and Expansion.

Autologous chimeric antigen receptor engineered T-cell therapies are beginning to dramatically change the outlook for patients with several hematological malignancies. Yet methods to activate and expand these cells are limited, often pose challenges to automation, and have biological limitations impacting the output of the injectable dose. This study describes the development of a novel, highly flexible, soluble DNA-based T-cell activation and expansion platform which alleviates the limitations of current technologies and provides rapid T-cell activation and expansion.

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.

Correction: Unsupervised capture and profiling of rare immune cells using multi-directional magnetic ratcheting.

Correction for 'Unsupervised capture and profiling of rare immune cells using multi-directional magnetic ratcheting' by Coleman Murray et al., Lab Chip, 2018, 18, 2396-2409.

Unsupervised capture and profiling of rare immune cells using multi-directional magnetic ratcheting.

Immunotherapies (IT) require induction, expansion, and maintenance of specific changes to a patient's immune cell repertoire which yield a therapeutic benefit. Recently, mechanistic understanding of these changes at the cellular level has revealed that IT results in complex phenotypic transitions in target cells, and that therapeutic effectiveness may be predicted by monitoring these transitions during therapy. However, monitoring will require unique tools that enable capture, manipulation, and profiling of rare immune cell populations. In this study, we introduce a method of automated and unsupervised separation and processing of rare immune cells, using high-force and multidimensional magnetic ratcheting (MR). We demonstrate capture of target immune cells using samples with up to 1 : 10 000 target cell to background cell ratios from input volumes as small as 25 microliters (i.e. a low volume and low cell frequency sample sparing assay interface). Cell capture is shown to achieve up to 90% capture efficiency and purity, and captured cell analysis is shown using both on-chip culture/activity assays and off-chip ejection and nucleic acid analysis. These results demonstrate that multi-directional magnetic ratcheting offers a unique separation system for dealing with blood cell samples that contain either rare cells or significantly small volumes, and the "sample sparing" capability leads to an expanded spectrum of parameters that can be measured. These tools will be paramount to advancing techniques for immune monitoring under conditions in which both the sample volume and number of antigen-specific target cells are often exceedingly small, including during IT and treatment of allergy, asthma, autoimmunity, immunodeficiency, cell based therapy, transplantation, and infection.

Plasma membrane temperature gradients and multiple cell permeabilization induced by low peak power density femtosecond lasers.

Calculations indicate that selectively heating the extracellular media induces membrane temperature gradients that combine with electric fields and a temperature-induced reduction in the electropermeabilization threshold to potentially facilitate exogenous molecular delivery. Experiments by a wide-field, pulsed femtosecond laser with peak power density far below typical single cell optical delivery systems confirmed this hypothesis. Operating this laser in continuous wave mode at the same average power permeabilized many fewer cells, suggesting that bulk heating alone is insufficient and temperature gradients are crucial for permeabilization. This work suggests promising opportunities for a high throughput, low cost, contactless method for laser mediated exogenous molecule delivery without the complex optics of typical single cell optoinjection, for potential integration into microscope imaging and microfluidic systems.