Few-layered graphene increases the response of nociceptive neurons to irritant stimuli.

The unique properties of few-layered graphene (FLG) make it interesting for a variety of applications, including biomedical applications, such as tissue engineering and drug delivery. Although different studies focus on applications in the central nervous system, its interaction with the peripheral nervous system has been so far overlooked. Here, we investigated the effects of exposure to colloidal dispersions of FLG on the sensory neurons of the rat dorsal root ganglia (DRG). We found that the FLG flakes were actively internalized by sensory neurons, accumulated in large intracellular vesicles, and possibly degraded over time, without major toxicological concerns, as neuronal viability, morphology, protein content, and basic electrical properties of DRG neurons were preserved. Interestingly, in our electrophysiological investigation under noxious stimuli, we observed an increased functional response upon FLG treatment of the nociceptive subpopulation of DRG neurons in response to irritants specific for chemoreceptors TRPV1 and TRPA1. The observed effects of FLG on DRG neurons may open-up novel opportunities for applications of these materials in specific disease models.

Interactions of Graphene Oxide and Few-Layer Graphene with the Blood-Brain Barrier.

Thanks to their biocompatibility and high cargo capability, graphene-based materials (GRMs) might represent an ideal brain delivery system. The capability of GRMs to reach the brain has mainly been investigated in vivo and has highlighted some controversy. Herein, we employed two in vitro BBB models of increasing complexity to investigate the bionano interactions with graphene oxide (GO) and few-layer graphene (FLG): a 2D murine Transwell model, followed by a 3D human multicellular assembloid, to mimic the complexity of the in vivo architecture and intercellular crosstalk. We developed specific methodologies to assess the translocation of GO and FLG in a label-free fashion and a platform applicable to any nanomaterial. Overall, our results show good biocompatibility of the two GRMs, which did not impact the integrity and functionality of the barrier. Sufficiently dispersed subpopulations of GO and FLG were actively uptaken by endothelial cells; however, the translocation was identified as a rare event.

Improving reliability and reducing costs of cardiotoxicity assessments using laser-induced cell poration on microelectrode arrays.

Drug-induced cardiotoxicity is a major barrier to drug development and a main cause of withdrawal of marketed drugs. Drugs can strongly alter the spontaneous functioning of the heart by interacting with the cardiac membrane ion channels. If these effects only surface during in vivo preclinical tests, clinical trials or worse after commercialization, the societal and economic burden will be significant and seriously hinder the efficient drug development process. Hence, cardiac safety pharmacology requires in vitro electrophysiological screening assays of all drug candidates to predict cardiotoxic effects before clinical trials. In the past 10 years, microelectrode array (MEA) technology began to be considered a valuable approach in pharmaceutical applications. However, an effective tool for high-throughput intracellular measurements, compatible with pharmaceutical standards, is not yet available. Here, we propose laser-induced optoacoustic poration combined with CMOS-MEA technology as a reliable and effective platform to detect cardiotoxicity. This approach enables the acquisition of high-quality action potential recordings from large numbers of cardiomyocytes within the same culture well, providing reliable data using single-well MEA devices and single cardiac syncytia per each drug. Thus, this technology could be applied in drug safety screening platforms reducing times and costs of cardiotoxicity assessments, while simultaneously improving the data reliability.

Mirroring Action Potentials: Label-Free, Accurate, and Noninvasive Electrophysiological Recordings of Human-Derived Cardiomyocytes.

The electrophysiological recording of action potentials in human cells is a long-sought objective due to its pivotal importance in many disciplines. Among the developed techniques, invasiveness remains a common issue, causing cytotoxicity or altering unpredictably cell physiological response. In this work, a new approach for recording intracellular signals of outstanding quality and with noninvasiveness is introduced. By taking profit of the concept of mirror charge in classical electrodynamics, the new proposed device transduces cell ionic currents into mirror charges in a microfluidic chamber, thus realizing a virtual mirror cell. By monitoring mirror charge dynamics, it is possible to effectively record the action potentials fired by the cells. Since there is no need for accessing or interacting with the cells, the method is intrinsically noninvasive. In addition, being based on optical recording, it shows high spatial resolution and high parallelization. As shown through a set of experiments, the presented methodology is an ideal candidate for the next generation devices for the reliable assessment of cardiotoxicity on human-derived cardiomyocytes. More generally, it paves the way toward a new family of in vitro biodevices that will lay a new milestone in the field of electrophysiology.

Microfluidic Multielectrode Arrays for Spatially Localized Drug Delivery and Electrical Recordings of Primary Neuronal Cultures.

Neuropathological models and neurological disease progression and treatments have always been of great interest in biomedical research because of their impact on society. The application of in vitro microfluidic devices to neuroscience-related disciplines provided several advancements in therapeutics or neuronal modeling thanks to the ability to control the cellular microenvironment at spatiotemporal level. Recently, the introduction of three-dimensional nanostructures has allowed high performance in both in vitro recording of electrogenic cells and drug delivery using minimally invasive devices. Independently, both delivery and recording have let to pioneering solutions in neurobiology. However, their combination on a single chip would provide further fundamental improvements in drug screening systems and would offer comprehensive insights into pathologies and diseases progression. Therefore, it is crucial to develop platforms able to monitor progressive changes in electrophysiological behavior in the electrogenic cellular network, induced by spatially localized injection of biochemical agents. In this work, we show the application of a microfluidic multielectrode array (MEA) platform to record spontaneous and chemically stimulated activity in primary neuronal networks. By means of spatially localized caffeine injection via microfluidic nanochannels, the device demonstrated its capability of combined localized drug delivery and cell signaling recording. The platform could detect activity of the neural network at multiple sites while delivering molecules into just a few selected cells, thereby examining the effect of biochemical agents on the desired portion of cell culture.

On-Demand Intracellular Delivery of Single Particles in Single Cells by 3D Hollow Nanoelectrodes.

Delivery of molecules into intracellular compartments is one of the fundamental requirements in molecular biology. However, the possibility of delivering a precise number of nano-objects with single-particle resolution is still an open challenge. Here we present an electrophoretic platform based on 3D hollow nanoelectrodes to enable delivery of single nanoparticles into single selected cells and monitoring of the single-particle delivery by surface-enhanced Raman scattering (SERS). The gold-coated hollow nanoelectrode capable of confinement and enhancement of electromagnetic fields upon laser illumination can distinguish the SERS signals of a single nanoparticle flowing through the nanoelectrode. Tight wrapping of cell membranes around the nanoelectrodes allows effective membrane electroporation such that single gold nanorods are delivered on demand into a living cell by electrophoresis. The capability of the 3D hollow nanoelectrodes to porate cells and reveal single emitters from the background in continuous flow is promising for the analysis of both intracellular delivery and sampling.

Massively parallel sequencing of micro-manipulated cells targeting a comprehensive panel of disease-causing genes: A comparative evaluation of upstream whole-genome amplification methods.

Single Gene Disorders (SGD) are still routinely diagnosed using PCR-based assays that need to be developed and validated for each individual disease-specific gene fragment. The TruSight One sequencing panel currently covers 12 Mb of genomic content, including 4813 genes associated with a clinical phenotype. When only a limited number of cells are available, whole genome amplification (WGA) is required prior to DNA target capture techniques such as the TruSight One panel. In this study, we compared 4 different WGA methods in combination with the TruSight One sequencing panel to perform single nucleotide polymorphism (SNP) genotyping starting from 3 micro-manipulated cells. This setting simulates clinical settings such as day-5 blastocyst biopsy for Preimplantation Genetic Testing (PGT), liquid biopsy of circulating tumor cells (CTCs) and cancer-cell profiling. Bulk cell samples were processed alongside these WGA samples to serve as a performance reference. Target coverage, coverage uniformity and SNP calling accuracy obtained using any of the WGA, is inferior to the results obtained on bulk cell samples. However, results after REPLI-g come close. Compared to the other WGA methods, the method using REPLI-g WGA results in a better coverage of the targeted genomic regions with a more uniform read depth. Consequently, this method also results in a more accurate SNP calling and could be considered for clinical genotyping of a limited number of cells.

Short Tandem Repeat analysis after Whole Genome Amplification of single B-lymphoblastoid cells.

To allow multiple genetic analyses on a single cell, whole genome amplification (WGA) is required. Unfortunately, studies comparing different WGA methods for downstream human identification Short Tandem Repeat (STR) analysis remain absent. Therefore, the aim of this work was to assess the performance of four commercially available WGA kits for downstream human identification STR profiling on a B-lymphoblastoid cell line. The performance was assessed using an input of one or three micromanipulated cells. REPLI-g showed a very low dropout rate, as it was the only WGA method in this study that could provide a complete STR profile in some of its samples. Although Ampli1, DOPlify and PicoPLEX did not detect all selected STR markers, they seem suitable for genetic identification in single-cell applications.

Genome-Wide Copy Number Alteration Detection in Preimplantation Genetic Diagnosis.

Shallow whole genome sequencing has recently been introduced for genome-wide detection of chromosomal copy number alterations (CNAs) in preimplantation genetic diagnosis (PGD), using only 4-7 trophectoderm cells biopsied from day-5 embryos. This chapter describes the complete method, starting from whole genome amplification (WGA) on isolated blastomere(s), up to data analysis for CNA detection. The process is described generically and can also be used to perform CNA analysis on a limited number of cells (down to a single cell) in other applications. This unique description also includes some tips and tricks to increase the chance of success.

STR profiling and Copy Number Variation analysis on single, preserved cells using current Whole Genome Amplification methods.

The growing interest in liquid biopsies for cancer research and cell-based non-invasive prenatal testing (NIPT) invigorates the need for improved single cell analysis. In these applications, target cells are extremely rare and fragile in peripheral circulation, which makes the genetic analysis very challenging. To overcome these challenges, cell stabilization and unbiased whole genome amplification are required. This study investigates the performance of four WGA methods on single or a limited number of cells after 24 hour of Streck Cell-Free DNA BCT preservation. The suitability of the DNA, amplified with Ampli1, DOPlify, PicoPLEX and REPLI-g, was assessed for both short tandem repeat (STR) profiling and copy number variant (CNV) analysis after shallow whole genome massively parallel sequencing (MPS). Results demonstrate that Ampli1, DOPlify and PicoPLEX perform well for both applications, with some differences between the methods. Samples amplified with REPLI-g did not result in suitable STR or CNV profiles, indicating that this WGA method is not able to generate high quality DNA after Streck Cell-Free DNA BCT stabilization of the cells.

Performance of four modern whole genome amplification methods for copy number variant detection in single cells.

Whole genome amplification (WGA) has become an invaluable tool to perform copy number variation (CNV) detection in single, or a limited number of cells. Unfortunately, current WGA methods introduce representation bias that limits the detection of small CNVs. New WGA methods have been introduced that might have the potential to reduce this bias. We compared the performance of PicoPLEX DNA-Seq (Picoseq), DOPlify, REPLI-g and Ampli-1 WGA for aneuploidy screening and copy number analysis using shallow whole genome massively parallel sequencing (MPS), starting from single or a limited number of cells. Although the four WGA methods perform differently, they are all suited for this application.

Forensic SNP Genotyping using Nanopore MinION Sequencing.

One of the latest developments in next generation sequencing is the Oxford Nanopore Technologies' (ONT) MinION nanopore sequencer. We studied the applicability of this system to perform forensic genotyping of the forensic female DNA standard 9947 A using the 52 SNP-plex assay developed by the SNPforID consortium. All but one of the loci were correctly genotyped. Several SNP loci were identified as problematic for correct and robust genotyping using nanopore sequencing. All these loci contained homopolymers in the sequence flanking the forensic SNP and most of them were already reported as problematic in studies using other sequencing technologies. When these problematic loci are avoided, correct forensic genotyping using nanopore sequencing is technically feasible.

Performance of a TthPrimPol-based whole genome amplification kit for copy number alteration detection using massively parallel sequencing.

Starting from only a few cells, current whole genome amplification (WGA) methods provide enough DNA to perform massively parallel sequencing (MPS). Unfortunately, all current WGA methods introduce representation bias which limits detection of copy number aberrations (CNAs) smaller than 3 Mb. A recent WGA method, called TruePrime single cell WGA, uses a recently discovered DNA primase, TthPrimPol, instead of artificial primers to initiate DNA amplification. This method could lead to a lower representation bias, and consequently to a better detection of CNAs. The enzyme requires no complementarity and thus should generate random primers, equally distributed across the genome. The performance of TruePrime WGA was assessed for aneuploidy screening and CNA analysis after MPS, starting from 1, 3 or 5 cells. Although the method looks promising, the single cell TruePrime WGA kit v1 is not suited for high resolution CNA detection after MPS because too much representation bias is introduced.

Shallow whole genome sequencing is well suited for the detection of chromosomal aberrations in human blastocysts.

OBJECTIVE: To add evidence that massive parallel sequencing (MPS) is a valuable substitute for array comparative genomic hybridization (arrayCGH) with a resolution that is more appropriate for preimplantation genetic diagnosis (PGD) in translocation carriers. DESIGN: Study of diagnostic accuracy. SETTING: University hospital. PATIENT(S): Fifteen patients with a balanced structural rearrangement were included in the study: eight reciprocal translocations, four Robertsonian translocations, two inversions, and one insertional translocation. INTERVENTION(S): Trophectoderm biopsy was performed on 47 blastocysts. MAIN OUTCOME MEASURE(S): In the current study, shallow whole genome MPS on a NextSeq500 (Illumina) and Ion Proton (Life Technologies) instrument was performed in parallel on 47 whole genome amplified trophectoderm samples. Data analyses were performed using the QDNAseq algorithm implemented in Vivar. RESULT(S): In total, 5 normal and 42 abnormal embryos were analyzed. All aberrations previously detected with arrayCGH could be readily detected in the MPS data using both technologies and were correctly identified. The smallest detected abnormality was a ∼ 4.5 Mb deletion/duplication. CONCLUSION(S): This study demonstrates that shallow whole genome sequencing can be applied efficiently for the detection of numerical and structural chromosomal aberrations in embryos, equaling or even exceeding the resolution of the routinely used microarrays.

Whole genome amplification with SurePlex results in better copy number alteration detection using sequencing data compared to the MALBAC method.

Current whole genome amplification (WGA) methods lead to amplification bias resulting in over- and under-represented regions in the genome. Nevertheless, certain WGA methods, such as SurePlex and subsequent arrayCGH analysis, make it possible to detect copy number alterations (CNAs) at a 10 Mb resolution. A more uniform WGA combined with massive parallel sequencing (MPS), however, could allow detection at higher resolution and lower cost. Recently, MALBAC, a new WGA method, claims unparalleled performance. Here, we compared the well-established SurePlex and MALBAC WGA for their ability to detect CNAs in MPS generated data and, in addition, compared PCR-free MPS library preparation with the standard enrichment PCR library preparation. Results showed that SurePlex amplification led to more uniformity across the genome, allowing for a better CNA detection with less false positives compared to MALBAC amplified samples. An even more uniform coverage was observed in samples following a PCR-free library preparation. In general, the combination of SurePlex and MPS led to the same chromosomal profile compared to a reference arrayCGH from unamplified genomic DNA, underlining the large potential of MPS techniques in CNA detection from a limited number of DNA material.