Noninvasive and sensitive thermometry of a single cell during the normal physiological process is crucial for analyzing fundamental cellular metabolism and applications to cancer treatment. However, current thermometers generally sense the average temperature variation for many cells, thereby failing to obtain real-time and continuous data of an individual cell. In this study, we employed platinum (Pt) electrodes to construct an integrated microfluidic chip as a single-cell thermometer. The single-cell isolation unit in the microchip consisted of a main channel, which was connected to the inlet and outlet of a single-cell capture funnel. A single cell can be trapped in the funnel and the remaining cells can bypass and flow along the main channel to the outlet. The best capture ratio of a single MCF7 cell at a single-cell isolation unit was 90 % under optimal condition. The thermometer in the micro-chip had a temperature resolution of 0.007 °C and showed a good linear relationship in the range of 20-40 °C (R2 = 0.9999). Slight temperature increment of different single tumor cell (MCF7 cell, H1975 cell, and HepG2 cell) cultured on the chip was continuously recorded under normal physiological condition. In addition, the temperature variation of single MCF7 cell in-situ after exposure to a stimulus (4 % paraformaldehyde treatment) was also monitored, showing an amplitude of temperature fluctuations gradually decreased over time. Taken together, this integrated microchip is a practical tool for detecting the change in the temperature of a single cell in real-time, thereby offering valuable information for the drug screening, diagnosis, and treatment of cancer.

The enteric nervous system (ENS) is a complex neuronal network organized in ganglionated plexuses that extend along the entire length of the gastrointestinal tract. Largely independent of the central nervous system, the ENS coordinates motility and peristalsis of the digestive tract, regulates secretion and absorption, and is involved in immunological processes. Electrophysiological methods such as the patch-clamp technique are particularly suitable to study the function of neurons as well as the biophysical parameters of the underlying ion channels under both physiological and pathophysiological conditions. However, application of the patch-clamp method to ENS neurons remained difficult because they are embedded in substantial tissue layers that limit access to and targeted manipulation of these cells. Here, we present a robust step-by-step protocol that involves isolation of ENS neurons from adult mice, culturing of the cells, their transfection with plasmid DNA, and subsequent electrophysiological characterization of individual neurons in current-clamp and voltage-clamp recordings. With this protocol, ENS neurons can be prepared, transfected, and electrophysiologically characterized within 72 h. Using isolated ENS neurons, we demonstrate the feasibility of the approach by functional overexpression of recombinant voltage-gated NaV1.9 mutant channels associated with hereditary sensory and autonomic neuropathy type 7 (HSAN-7), a disorder characterized by congenital analgesia and severe constipation that can require parenteral nutrition. Although our focus is on the electrophysiological evaluation of isolated ENS neurons, the presented methodology is also useful to analyze molecules other than sodium channels or to apply alternative downstream assays including calcium imaging, proteomic and nucleic acid approaches, or immunochemistry.

Ovarian cancer remains the leading cause of death from gynecologic malignancy in the Western world. Tumors are comprised of heterogeneous populations of various cancer, immune, and stromal cells; it is hypothesized that rare cancer stem cells within these subpopulations lead to disease recurrence and treatment resistance. Technological advances now allow for the analysis of tumor genomes and transcriptomes at the single-cell level, which provides the resolution to potentially identify these rare cancer stem cells within the larger tumor.In this chapter, we review the evolution of next-generation RNA sequencing techniques, the methodology of single-cell isolation and sequencing, sequencing data analysis, and the potential applications in ovarian cancer. We also summarize the current published work using single-cell sequencing in ovarian cancer.By utilizing this novel technique to characterize the gene expression of rare subpopulations, new targets and treatment pathways may be identified in ovarian cancer to change treatment paradigms.

Single-cell RNA sequencing (scRNA-seq) of the bronchial epithelium enables examination of cellular subtypes and their responses to viral infections. Here, an optimized method for the isolation of virally infected primary bronchial epithelial cells using a commercially available microfluidic device is presented. Using this method single cells can be rapidly isolated with minimal equipment available in most laboratories. Isolation can be carried out inside biological safety cabinets, permitting the use of virally infected cells. Both cell-line and primary cells isolated using the device retained sufficient RNA integrity for the generation of short-read sequencing-compatible cDNA libraries to facilitate scRNA-seq.

Advances in human pluripotent stem cell (hPSC) techniques have led them to become a widely used and powerful tool for a vast array of applications, including disease modeling, developmental studies, drug discovery and testing, and emerging cell-based therapies. hPSC workflows that require clonal expansion from single cells, such as CRISPR/Cas9-mediated genome editing, face major challenges in terms of efficiency, cost, and precision. Classical sub-cloning approaches depend on limiting dilution and manual colony picking, which are both time-consuming and labor-intensive, and lack a real proof of clonality. Here we describe the application of three different automated cell isolation and dispensing devices that can enhance the single-cell cloning process for hPSCs. In combination with optimized cell culture conditions, these devices offer an attractive alternative compared to manual methods. We explore various aspects of each device system and define protocols for their practical application. Following the workflow described here, single cell-derived hPSC sub-clones from each system maintain pluripotency and genetic stability. Furthermore, the workflows can be applied to uncover karyotypic mosaicism prevalent in bulk hPSC cultures. Our robust automated workflow facilitates high-throughput hPSC clonal selection and expansion, urgently needed in the operational pipelines of hPSC applications. © 2020 The Authors. Basic Protocol: Efficient automated hPSC single cell seeding and clonal expansion using the iotaSciences IsoCell platform Alternate Protocol 1: hPSC single cell seeding and clonal expansion using the Cellenion CellenONE single-cell dispenser Alternate Protocol 2: hPSC single cell seeding and clonal expansion using the Cytena single-cell dispenser Support Protocol 1: Coating cell culture plates with Geltrex Support Protocol 2: hPSC maintenance in defined feeder-free conditions Support Protocol 3: hPSC passaging in clumps Support Protocol 4: Laminin 521 coating of IsoCell plates and 96-well/384-well plates Support Protocol 5: Preparation of medium containing anti-apoptotic small molecules Support Protocol 6: 96- and 384-well target plate preparation prior to single cell seeding Support Protocol 7: Single cell dissociation of hPSCs Support Protocol 8: IsoCell-, CellenONE-, and Cytena-derived hPSC clone subculture and expansion.

Single-cell isolation and transcriptomic analysis of a specific cell type or tissue offers the possibility of studying cell function and heterogeneity in time-dependent processes with remarkable resolution. The reduced tissue complexity and highly stereotyped development of Caenorhabditis elegans, combined with an extensive genetic toolbox and the ease of growing large tightly synchronized populations makes it an exceptional model organism for the application of such approaches. However, the difficulty to dissociate and isolate single cells from larval stages has been a major constraint to this kind of studies. Here, we describe an improved protocol for dissociation and preparation of single cell suspensions from developmentally synchronized populations of C. elegans L1 larvae. Our protocol has been empirically optimized to allow efficient FACS-based purification of large number of single cells from rare cell types, for subsequent extraction and sequencing of their mRNA.

Recent cell culture media for mammalian cells can be abundantly formulated with nutrients supporting production, but such media can be limited to use in host cell culture, transfection, cell cloning, and cell growth under the low cell density conditions. In many cases, appropriate platform media are used for cell line development, and then replaced with rich media for production. In this study, we demonstrate rich chemically defined media for Chinese hamster ovary (CHO) cells that are suitable as basal media both for cell line development and for final production of culture process. Set up for transfection, semi-solid media optimization, mini-pool screening, and single cell cloning media development were performed, and final clones were obtained with higher productivity in fed-batch culture mode using rich formulated media comparing with lean formulated media. Developed methods may remove the requirements for cell adaptation to production media after cell line development, and relieve the clonality issues associated with changing the culture media. Furthermore, established methods have advantages over traditional approaches, including saving resources and decreasing the time and the effort required to optimize the production process.

We present a novel technique for continuous label-free separation of particles based on their dielectrophoretic crossover frequencies. Our technique relies on our unique microfluidic geometry which performs hydrodynamic focusing, generates a stagnation flow with two outlets, and simultaneously produces an isomotive dielectrophoretic field via wall-situated electrodes. To perform particle separation, we hydrodynamically focus particles onto stagnation streamlines and use isomotive dielectrophoretic force to nudge the particles off these streamlines and direct them into appropriate outlets. Focusing particles onto stagnation streamlines obviates the need for large forces to be applied to the particles and therefore increases system throughput. The use of isomotive (spatially uniform) dielectrophoretic force increases system reliability. To guide designers, we develop and describe a simple scaling model for the particle separation dynamics of our technique. The model predicts the range of particle sizes that can be separated as well as the processing rate that can be achieved as a function of system design parameters: channel size, flow rate, and applied potential. Finally, as a proof-of-principle, we use this technique to separate polystyrene bead and cell mixtures of the same diameters as well as mixtures of both particles with varying diameters.

Brain endothelial cells (BECs) form the integral component of the blood-brain barrier (BBB) which separates the systemic milieu from the brain parenchyma and protects the brain from pathogens and circulating factors. In order to study BEC biology, it was of particular interest to establish a method that enables researchers to investigate and understand the underlying molecular mechanisms regulating their function during homeostasis, aging and disease. Furthermore, due to the heterogeneity of the cerebrovasculature and different vessel types that comprise the BBB, it is of particular interest to isolate primary BECs for single cell analysis from various subregions of the brain, such as the neurogenic and highly vascularized hippocampus and to enrich for specific vessel types. In the past, approaches to isolate endothelial cells were dependent on transgenic mice and often resulted in insufficiently pure cell populations and poor yield. This protocol describes a technique that allows single-cell isolation of highly pure brain endothelial cell populations using fluorescence activated cell sorting (FACS). Briefly, after perfusion and careful removal of the meninges, and dissection of the cortex/hippocampus, the brain tissue is mechanically homogenized and enzymatically digested resulting in a single cell suspension. Cells are stained with fluorochrome-conjugated antibodies identifying CD31+ brain endothelial cells, as well as CD45+CD11b+ myeloid cells for exclusion. Using flow cytometry, cell populations are separated and CD31+BECs are sorted in bulk into RNA later or as single cells directly into either RNA lysis buffer for single or bulk RNA-Seq analyses. The protocol does not require the expression of a transgene to label brain endothelial cells and thus, may be applied to any mouse model. In our hands, the protocol has been highly reproducible with an average yield of 1 × 105 cells isolated from an adult mouse cortex/hippocampus.

Tumors often show intra-tumor heterogeneity because of genotypic differences between all the cells that compose it and that derive from it. Recent studies have shown significant aspects of neuroblastoma heterogeneity that may affect the diagnostic-therapeutic strategy. Therefore, we developed a laboratory protocol, based on the combination of the advanced dielectrophoresis-based array technology and next-generation sequencing to identify and sort single cells individually and carry out their copy number variants analysis. The aim was to evaluate the cellular heterogeneity, avoiding overestimation or underestimation errors, due to a bulk analysis of the sample. We tested the above-mentioned protocol on two neuroblastoma cell lines, SK-N-BE(2)-C and IMR-32. The presence of several gain or loss chromosomal regions, in both cell lines, shows a high heterogeneity of the copy number variants status of the single tumor cells, even if they belong to an immortalized cell line. This finding confirms that each cell can potentially accumulate different alterations that can modulate its behavior. The laboratory protocol proposed herein provides a tool able to identify prevalent behaviors, and at the same time highlights the presence of particular clusters that deviate from them. Finally, it could be applicable to many other types of cancer.