Electrochemical modulation enhances the selectivity of peripheral neurostimulation in vivo.

Electrical nerve stimulation serves an expanding list of clinical applications, but it faces persistent challenges in selectively activating bundled nerve fibers. In this study, we investigated electrochemical modulation with an ion-selective membrane (ISM) and whether it, used together with electrical stimulation, may provide an approach for selective control of peripheral nerves. Guided by theoretical transport modeling and direct concentration measurements, we developed an implantable, multimodal ISM cuff capable of simultaneous electrical stimulation and focused Ca2+ depletion. Acutely implanting it on the sciatic nerve of a rat in vivo, we demonstrated that Ca2+ depletion could increase the sensitivity of the nerve to electrical stimulation. Furthermore, we found evidence that the effect of ion modulation would selectively influence functional components of the nerve, allowing selective activation by electrical current. Our results raise possibilities for improving functional selectivity of new and existing bioelectronic therapies, such as vagus nerve stimulation.

Rapid and Low Cost Manufacturing of Cuff Electrodes.

For many peripheral neuro-modulation applications, the cuff electrode has become a preferred technology for delivering electrical current into targeted volumes of tissue. While basic cuffs with low spatial selectivity, having longitudinally arranged contacts, can be produced from relatively straightforward processes, the fabrication of more complex electrode configurations typically requires iterative design and clean-room fabrication with skilled technicians. Although facile methods for fabricating cuff electrodes exist, their inconsistent products have limited their adoption for rapid manufacturing. In this article, we report a fast, low-cost fabrication process for patterning of electrode contacts in an implantable peripheral nerve cuff. Using a laser cutter as we have prescribed, the designer can render precise contact geometries that are consistent between batches. This method is enabled by the use of silicone/carbon black (CB) composite electrodes, which integrate with the patterned surface of its substrate-tubular silicone insulation. The size and features of its products can be adapted to fit a wide range of nerve diameters and applications. In this study, we specifically documented the manufacturing and evaluation of circumpolar cuffs with radial arrays of three contacts for acute implantation on the rat sciatic nerve. As part of this method, we also detail protocols for verification-electrochemical characterization-and validation-electrophysiological evaluation-of implantable cuff electrodes. Applied to our circumpolar cuff electrode, we report favorable electrical characteristics. In addition, we report that it reproduces expected electrophysiological behaviors described in prior literature. No specialized equipment or fabrication experience was required in our production, and we encountered negligible costs relative to commercially available solutions. Since, as we demonstrate, this process generates consistent and precise electrode geometries, we propose that it has strong merits for use in rapid manufacturing.

High-throughput continuous-flow microfluidic electroporation of mRNA into primary human T cells for applications in cellular therapy manufacturing.

Implementation of gene editing technologies such as CRISPR/Cas9 in the manufacture of novel cell-based therapeutics has the potential to enable highly-targeted, stable, and persistent genome modifications without the use of viral vectors. Electroporation has emerged as a preferred method for delivering gene-editing machinery to target cells, but a major challenge remaining is that most commercial electroporation machines are built for research and process development rather than for large-scale, automated cellular therapy manufacturing. Here we present a microfluidic continuous-flow electrotransfection device designed for precise, consistent, and high-throughput genetic modification of target cells in cellular therapy manufacturing applications. We optimized our device for delivery of mRNA into primary human T cells and demonstrated up to 95% transfection efficiency with minimum impact on cell viability and expansion potential. We additionally demonstrated processing of samples comprising up to 500 million T cells at a rate of 20 million cells/min. We anticipate that our device will help to streamline the production of autologous therapies requiring on the order of 10[Formula: see text]-10[Formula: see text] cells, and that it is well-suited to scale for production of trillions of cells to support emerging allogeneic therapies.

Acoustophoretic rapid media exchange and continuous-flow electrotransfection of primary human T cells for applications in automated cellular therapy manufacturing.

Autologous cellular therapies based on modifying T cells to express chimeric antigen receptor genes have been highly successful in treating hematological cancers. Deployment of these therapies is limited by the complexity and costs associated with their manufacturing. Transitioning these processes from virus-based methods for gene delivery to a non-viral method, such as electroporation, has the potential to greatly reduce cost and manufacturing time while increasing safety and efficacy. Major challenges with electroporation are the negative impacts on cell health associated with exposure to high-magnitude electric fields, and that most commercial bulk electroporators are low-precision instruments designed for manually-operated, lower-throughput batch processing of cells. Negative effects on cell health can be mitigated by use of specialized electroporation medias, but this adds processing steps, and long-term exposure to these medias can reduce transfection efficiency and post-transfection viability. To enable automated, clinical-scale production of cellular therapies using electrotransfection in specialized medias, we developed a high-precision microfluidic platform that automatically and continuously transfers cells from culture media into electroporation media using acoustophoresis, and then immediately applies electric fields from integrated electrodes. This limits cell residence time in electroporation media to seconds, and enables high transfection efficiency with minimum impact on cell viability. We tested our system by transferring primary human T cells from a standard cell media to electroporation media, and then transfecting them with mRNA encoding an mCherry fluorescent protein. We achieved a media exchange efficiency of 86% and transfection efficiency of up to 60%, with less than a 5% reduction in viability.

Carbon fiber on polyimide ultra-microelectrodes.

OBJECTIVE: Most preparations for making neural recordings degrade over time and eventually fail due to insertion trauma and reactive tissue response. The magnitudes of these responses are thought to be related to the electrode size (specifically, the cross-sectional area), the relative stiffness of the electrode, and the degree of tissue tolerance for the material. Flexible carbon fiber ultra-microelectrodes have a much smaller cross-section than traditional electrodes and low tissue reactivity, and thus may enable improved longevity of neural recordings in the central and peripheral nervous systems. Only two carbon fiber array designs have been described previously, each with limited channel densities due to limitations of the fabrication processes or interconnect strategies. Here, we describe a method for assembling carbon fiber electrodes on a flexible polyimide substrate that is expected to facilitate the construction of high-density recording and stimulating arrays. APPROACH: Individual carbon fibers were aligned using an alignment tool that was 3D-printed with sub-micron resolution using direct laser writing. Indium deposition on the carbon fibers, followed by low-temperature microsoldering, provided a robust and reliable method of electrical connection to the polyimide interconnect. MAIN RESULTS: Spontaneous multiunit activity and stimulation-evoked compound responses with SNR >10 and >120, respectively, were recorded from a small (125 µm) peripheral nerve. We also improved the typically poor charge injection capacity of small diameter carbon fibers by electrodepositing 100 nm-thick iridium oxide films, making the carbon fiber arrays usable for electrical stimulation as well as recording. SIGNIFICANCE: Our innovations in fabrication technique pave the way for further miniaturization of carbon fiber ultra-microelectrode arrays. We believe these advances to be key steps to enable a shift from labor intensive, manual assembly to a more automated manufacturing process.

Directed assembly-based printing of homogeneous and hybrid nanorods using dielectrophoresis.

Printing nano and microscale three-dimensional (3D) structures using directed assembly of nanoparticles has many potential applications in electronics, photonics and biotechnology. This paper presents a reproducible and scalable 3D dielectrophoresis assembly process for printing homogeneous silica and hybrid silica/gold nanorods from silica and gold nanoparticles. The nanoparticles are assembled into patterned vias under a dielectrophoretic force generated by an alternating current (AC) field, and then completely fused in situ to form nanorods. The assembly process is governed by the applied AC voltage amplitude and frequency, pattern geometry, and assembly time. Here, we find out that complete assembly of nanorods is not possible without applying both dielectrophoresis and electrophoresis. Therefore, a direct current offset voltage is used to add an additional electrophoretic force to the assembly process. The assembly can be precisely controlled to print silica nanorods with diameters from 20-200 nm and spacing from 500 nm to 2 µm. The assembled nanorods have good uniformity in diameter and height over a millimeter scale. Besides homogeneous silica nanorods, hybrid silica/gold nanorods are also assembled by sequentially assembling silica and gold nanoparticles. The precision of the assembly process is further demonstrated by assembling a single particle on top of each nanorod to demonstrate an additional level of functionalization. The assembled hybrid silica/gold nanorods have potential to be used for metamaterial applications that require nanoscale structures as well as for plasmonic sensors for biosensing applications.

A micro-scale printable nanoclip for electrical stimulation and recording in small nerves.

OBJECTIVE: The vision of bioelectronic medicine is to treat disease by modulating the signaling of visceral nerves near various end organs. In small animal models, the nerves of interest can have small diameters and limited surgical access. New high-resolution methods for building nerve interfaces are desirable. In this study, we present a novel nerve interface and demonstrate its use for stimulation and recording in small nerves. APPROACH: We design and fabricate micro-scale electrode-laden nanoclips capable of interfacing with nerves as small as 50 µm in diameter. The nanoclips are fabricated using a direct laser writing technique with a resolution of 200 nm. The resolution of the printing process allows for incorporation of a number of innovations such as trapdoors to secure the device to the nerve, and quick-release mounts that facilitate keyhole surgery, obviating the need for forceps. The nanoclip can be built around various electrode materials; here we use carbon nanotube fibers for minimally invasive tethering. MAIN RESULTS: We present data from stimulation-evoked responses of the tracheal syringeal (hypoglossal) nerve of the zebra finch, as well as quantification of nerve functionality at various time points post implant, demonstrating that the nanoclip is compatible with healthy nerve activity over sub-chronic timescales. SIGNIFICANCE: Our nerve interface addresses key challenges in interfacing with small nerves in the peripheral nervous system. Its small size, ability to remain on the nerve over sub-chronic timescales, and ease of implantation, make it a promising tool for future use in the treatment of disease.