High-throughput Cell Research Articles

Skyline Computation for Low-Latency Image-Activated Cell Identification

High-throughput label-free single cell screening technology has been studied for noninvasive analysis of various kinds of cells. We tackle the cell identification task in the cell sorting system as a continuous skyline computation. Skyline Computation is a method for extracting interesting entries from a large population with multiple attributes. Jointed rooted-tree (JR-tree) is continuous skyline computation algorithm that manages entries using a rooted-tree structure. JR-tree delays extend the tree to deeper levels to accelerate tree construction and traversal. In this study, we proposed the JR-tree-based parallel skyline computation accelerator. We implemented it on a field-programmable gate array (FPGA). We evaluated our proposed software and hardware algorithms against an existing software algorithm using synthetic and real-world datasets.

Optically Guided Single Cell Mass Spectrometry of Rat Dorsal Root Ganglia to Profile Lipids, Peptides and Proteins.

The mammalian dorsal root ganglia (DRG) are located on the dorsal roots of the spinal nerves and contain cell bodies of primary sensory neurons. DRG cells have been classified into subpopulations based on their size, morphology, intracellular markers, response to stimuli, and neuropeptides. To understand the connections between DRG chemical heterogeneity and cellular function, we performed optically guided, high-throughput single cell profiling using sequential matrix-assisted laser desorption/ionization mass spectrometry (MS) to detect lipids, peptides, and several proteins in individual DRG cells. Statistical analysis of the resulting mass spectra allows stratification of the DRG population according to cellular morphology and, presumably, major cell types. A subpopulation of small cells contained myelin proteins, which are abundant in Schwann cells, and mass spectra of several larger cells contained peaks matching neurofilament, vimentin, myelin basic protein S, and thymosin beta proteins. Of the over 1000 cells analyzed, approximately 78 % produced putative peptide-rich spectra, allowing the population to be classified into three distinct cell types. Two signals with m/z 4404 and 5487 were exclusively observed in a cell type, but could not be matched to results of our previous liquid chromatography-MS analyses.

High-throughput refractive index-based microphotonic sensor for enhanced cellular discrimination

This paper presents a novel microphotonic sensor based on silicon technologies for high-throughput single cell measurements. It employs a highly sensitive Fabry-Pérot resonant cavity to extract cellular refractive index information. The integrated large cross-section rib waveguides provide a single-mode like behavior important for resonant cell sensing. Differentiated myeloid cells derived from a promyelocytic leukemia cell line were injected in a microchannel, sheathlessly focused using inertial forces and analyzed while flowing through the resonant cavity volume. Results were compared against a commercial flow cytometer and showed a substantial improvement on cellular discrimination. Thus, this sensor has the ability to discriminate cell populations, usually identified using fluorescent parameters, without any dyes and can reach measurement rate as high as 2000 cells per second. By harnessing the cell's effective volume refractive index, our device offers complementary measurements readily improving actual technologies and thus providing crucial information for research and clinical professionals.

Age-Correlated Phenotypic Alterations in Cells Isolated From Human Degenerated Intervertebral Discs With Contained Hernias

Human intervertebral disc (hIVD) cells were isolated from 41 surgically excised samples and assessed for their phenotypic alterations with age. Toward the design of novel anti-aging strategies to overcome degenerative disc disease (DDD), we investigated age-correlated phenotypic alterations that occur on primary hIVD cells. Although regenerative medicine holds great hope, much is still to be unveiled on IVD cell biology and its intrinsic signaling pathways, which can lead the way to successful therapies for IDD. A greater focus on age-related phenotypic changes at the cell level would contribute to establish more effective anti-aging/degeneration targets. The study was subdivided in four main steps: i) optimization of primary cells isolation technique; ii) high-throughput cell morphology analysis, by imaging flow cytometry (FC) and subsequent validation by histological analysis; iii) analysis of progenitor cell surface markers expression, by conventional FC; and iv) statistical analysis and correlation of cells morphology and phenotype with donor age. Three subsets of cells were identified on the basis of their diameter: small cell (SC), large cell (LC), and super LC (SLC). The frequency of SCs decreased nearly 50% with age, whereas that of LCs increased nearly 30%. Interestingly, the increased cells size was due to an enlargement of the pericellular matrix (PCM). Moreover, the expression pattern for CD90 and CD73 was a reflexion of age, where older individuals show reduced frequencies of positive cells for those markers. Nevertheless, the elevated percentages of primary positive cells for the mesenchymal stem cells (MSCs) marker CD146 found, even in some older donors, refreshed hope for the hypothetical activation of the self-renewal potential of the IVD. These findings highlight the remarkable morphological alterations that occur on hIVD cells with aging and degeneration, while reinforcing previous reports on the gradual disappearance of an endogenous progenitor cell population. N/A.

Automated Microfluidic Instrument for Label-Free and High-Throughput Cell Separation.

Microfluidic technologies for cell separation were reported frequently in recent years. However, a compact microfluidic instrument enabling thoroughly automated cell separation is still rarely reported until today due to the difficult hybrid between the macrosized fluidic control system and the microsized microfluidic device. In this work, we propose a novel and automated microfluidic instrument to realize size-based separation of cancer cells in a label-free and high-throughput manner. Briefly, the instrument is equipped with a fully integrated microfluidic device and a set of robust fluid-driven and control units, and the instrument functions of precise fluid infusion and high-throughput cell separation are guaranteed by a flow regulatory chip and two cell separation chips which are the key components of the microfluidic device. With optimized control programs, the instrument is successfully applied to automatically sort human breast adenocarcinoma cell line MCF-7 from 5 mL of diluted human blood with a high recovery ratio of ∼85% within a rapid processing time of ∼23 min. We envision that our microfluidic instrument will be potentially useful in many biomedical applications, especially cell separation, enrichment, and concentration for the purpose of cell culture and analysis.

A novel in vitro model for the assessment of postnatal myonuclear accretion

BackgroundDue to the post-mitotic nature of myonuclei, postnatal myogenesis is essential for skeletal muscle growth, repair, and regeneration. This process is facilitated by satellite cells through proliferation, differentiation, and subsequent fusion with a pre-existing muscle fiber (i.e., myonuclear accretion). Current knowledge of myogenesis is primarily based on the in vitro formation of syncytia from myoblasts, which represents aspects of developmental myogenesis, but may incompletely portray postnatal myogenesis. Therefore, we aimed to develop an in vitro model that better reflects postnatal myogenesis, to study the cell intrinsic and extrinsic processes and signaling involved in the regulation of postnatal myogenesis.MethodsProliferating C2C12 myoblasts were trypsinized and co-cultured for 3 days with 5 days differentiated C2C12 myotubes. Postnatal myonuclear accretion was visually assessed by live cell time-lapse imaging and cell tracing by cell labeling with Vybrant® DiD and DiO. Furthermore, a Cre/LoxP-based cell system was developed to semi-quantitatively assess in vitro postnatal myonuclear accretion by the conditional expression of luciferase upon myoblast–myotube fusion. Luciferase activity was assessed luminometrically and corrected for total protein content.ResultsLive cell time-lapse imaging, staining-based cell tracing, and recombination-dependent luciferase activity, showed the occurrence of postnatal myonuclear accretion in vitro. Treatment of co-cultures with the myogenic factor IGF-I (p < 0.001) and the cytokines IL-13 (p < 0.05) and IL-4 (p < 0.001) increased postnatal myonuclear accretion, while the myogenic inhibitors cytochalasin D (p < 0.001), myostatin (p < 0.05), and TNFα (p < 0.001) decreased postnatal myonuclear accretion. Furthermore, postnatal myonuclear accretion was increased upon recovery from electrical pulse stimulation-induced fiber damage (p < 0.001) and LY29004-induced atrophy (p < 0.001). Moreover, cell type-specific siRNA-mediated knockdown of myomaker in myoblasts (p < 0.001), but not in myotubes, decreased postnatal myonuclear accretion.ConclusionsWe developed a physiologically relevant, sensitive, high-throughput cell system for semi-quantitative assessment of in vitro postnatal myonuclear accretion, which can be used to mimic physiological myogenesis triggers, and can distinguish the cell type-specific roles of signals and responses in the regulation of postnatal myogenesis. As such, this method is suitable for both basal and translational research on the regulation of postnatal myogenesis, and will improve our understanding of muscle pathologies that result from impaired satellite cell number or function.

Abstract P4-05-03: Heat shock protein 27 (HSP27) and HER2 positively correlate in breast cancer and effect cell responsiveness to neratinib and cMET inhibitor

Abstract Background: Upregulation of heat shock protein-27 (HSP27), a key member of the HSP family, has been shown to confer resistance to chemotherapy and radiotherapy in solid tumours including breast cancer. Evidence is also emerging that HSP27 is linked to the metastatic spread of breast cancer and key cellular traits including cellular migration. Neratinib is an orally available tyrosine kinase inhibitor that irreversibly binds and inhibits EGFR, HER2 and HER4 receptor tyrosine kinases. cMET, the receptor for hepatocyte growth factor and target for cancer therapies, has been shown to be trans-phosphorylated by EGFR. In the current study, we evaluated HSP27 expression within a breast cancer cohort and its implications in cellular responsiveness to cMET inhibition and neratinib. Methods: HSP27 transcript expression was analysed in our chemotherapy naïve breast cancer cohort (n=124) using quantitative PCR (qPCR) and compared to clinic-pathological information including long-term survival over a ten year follow up period. In addition, the correlation between HSP27 and HER2 expression was explored using Spearman Rank order. High-throughput cell migration analysis was performed using ECIS, on MCF-7 control and HSP27 siRNA knockdown cells in conjunction with neratinib and PHA 665752, a small molecule cMET inhibitor. Results: In our primary breast cancer cohort, there were no significant associations between HSP27 transcript expression levels and tumour grade, TNM or estrogen receptor (ER) status. Combined survival expression analysis indicated that the worst patient prognosis was associated with high levels of both HER2 and HSP27 and high HER2 and low HSP27 whereas best patient prognosis was associated with low HER2 and low HSP27 expression. Knockdown of HSP27 in MCF7 cells brought about a reduction in cellular migration compared to the control. Additionally, this reduction was enhanced by the addition of neratinib, in a concentration dependent manner, and also cMET inhibition when individually treated. Furthermore, the greatest inhibitory effects on MCF-7 migration were seen following HSP27 knockdown and combined treatment with neratinib and PHA 665752. Conclusions: Our current data suggests that HSP27 confers low sensitivity to drugs such as neratinib and PHA 665752, particularly in relation to cellular migration and hence potentially metastasis. Therefore, the targeting of HSP27, HER2 and cMET appear to act syngeristically to regulate cellular migration in vitro. Furthermore, clinically expression of HER2 and HSP27 may serve as a prognostic marker for breast cancer survival. Hence, combination therapies that target both HSP and HER2 pathways may provide new clinical opportunities for preventing breast cancer progression. Citation Format: Owen S, Sanders AJ, Ruge F, Lalani AS, Avogadri-Connors F, Bryce RP, Jiang WG. Heat shock protein 27 (HSP27) and HER2 positively correlate in breast cancer and effect cell responsiveness to neratinib and cMET inhibitor [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-05-03.

Leaf-templated, microwell-integrated microfluidic chips for high-throughput cell experiments

As an alternative to conventional cell culture and animal testing, an organ-on-a-chip is applied to study the biological phenomena of organ development and disease, as well as the interactions between human tissues and external stimuli such as chemicals, forces and electricity. The pattern design of a microfluidic channel is one of the key approaches to regulate cell growth and differentiation, because these channels work as a crucial vasculature system to control the fluidic flow throughout the organ-on-a-chip device. In this study, we introduce a novel leaf-templated, microwell-integrated microfluidic chip for high-throughput cell experiments, consisting of a leaf-venation layer for fluent fluid flow, and a microwell-array layer for cell to reside. Computational fluid dynamics analysis was carried out to study the fluidic flow within leaf-venation network, which was further used to optimize the design of microwell arrays. A simple leaf-venation-mold-based microreplication method was developed to transfer the intact native leaf venation network into leaf-venation layer and 3D printing technology was used to fabricate the microwell-array layer. The layers were then assembled and used for perfusion culture, showing that leaf-templated microfluidic channels provided a sufficient culture medium for cells within each microwell. These results indicate a novel and effective strategy to generate a biomimetic microfluidic chip with an effective vascular transport system for high-throughput cell experiments.

High-Throughput Cell Deformability Screening to Identify Novel Anti-Cancer Compounds

High-Throughput Cell Deformability Screening to Identify Novel Anti-Cancer Compounds

Linking in vitro estrogenicity to adverse effects in the inland silverside (Menidia beryllina).

High-throughput cell assays that detect and integrate the response of multiple chemicals acting via a common mode of action have the potential to enhance current environmental monitoring practices. Establishing the linkage between in vitro and in vivo responses is key to demonstrating that in vitro cell assays can be predictive of ecologically relevant outcomes. The present study investigated the potency of 17β-estradiol (E2), estrone (E1), nonylphenol (NP), and treated wastewater effluent using the readily available GeneBLAzer® estrogen receptor transactivation assay and 2 life stages of the inland silverside (Menidia beryllina). In vitro estrogenic potencies were ranked as follows: E2 > E1 >> NP. All 3 model estrogens induced vitellogenin and choriogenin expression in a dose-dependent manner in larvae and juveniles. However, apical effects were only found for E2 and E1 exposures of juveniles, which resulted in female-skewed sex ratios. Wastewater effluent samples exhibiting low in vitro estrogenicity (below the 10% effective concentration [EC10]), did not cause significant changes in M. beryllina. Significant induction of estrogen-responsive genes was observed at concentrations 6 to 26 times higher than in vitro responses. Gonadal feminization occurred at concentrations at least 19 to 26 times higher than the in vitro responses. These findings indicated that in vitro cell assays were more sensitive than the fish assays, making it possible to develop in vitro effect thresholds protective of aquatic organisms. Environ Toxicol Chem 2018;37:884-892. © 2017 SETAC.

Microfluidic Templated Multicompartment Microgels for 3D Encapsulation and Pairing of Single Cells.

Controlled encapsulation and pairing of single cells within a confined 3D matrix can enable the replication of the highly ordered cellular structure of human tissues. Microgels with independently controlled compartments that can encapsulate cells within separately confined hydrogel matrices would provide precise control over the route of pairing single cells. Here, a one-step microfluidic method is presented to generate monodisperse multicompartment microgels that can be used as a 3D matrix to pair single cells in a highly biocompatible manner. A method is presented to induce microgels formation on chip, followed by direct extraction of the microgels from oil phase, thereby avoiding prolonged exposure of the microgels to the oil. It is further demonstrated that by entrapping stem cells with niche cells within separate but adjacent compartments of the microgels, it can create complex stem cell niche microenvironments in a controlled manner, which can serve as a useful tool for the study of cell-cell interactions. This microfluidic technique represents a significant step toward high-throughput single cells encapsulation and pairing for the study of intercellular communications at single cell level, which is of significant importance for cell biology, stem cell therapy, and tissue engineering.

Field of view scanning based quantitative interferometric microscopic cytometers for cellular imaging and analysis.

Microimaging is of great significance in the biological and medical fields, since it can realize observations acting as important references for cellular research and disease diagnosis. However, traditional microscopy only offers qualitative sample contours; moreover, it is difficult to reach large-amount sample observations limited by the fixed field of view (FoV). To realize massive cellular measurements quantitatively, three designed quantitative interferometric microscopic cytometers based on the FoV scanning are introduced and compared in details in this article. These devices not only retrieve the quantitative sample phase distributions in the extended FoV, but also provide the detailed information of massive cells, such as cellular volume, area, and roundness. Considering their capabilities as quantitative imaging and large-amount sampling, it is believed that these quantitative interferometric microscopic cytometers (QIMCs) can be potentially adopted in high-throughput cell imaging and statistical analysis for both the biological and medical applications.

CDKN1C (P57): one of the determinants of human endometrial stromal cell decidualization.

Decidualization is regulated by crosstalk of progesterone and the cAMP pathway. It involves extensive reprogramming of gene expression and includes a wide range of functions. To investigate how cell cycle regulatory genes drive the human endometrial stromal cell (ESC) exit cell cycle and enter differentiation, primary cultured ESC was treated with 8-Br-cAMP and MPA and cell cycle distribution was investigated by flow cytometry. High-throughput cell cycle regulatory gene expression was also studied by microarray. To validate the results of microarray chip, immunohistochemistry and semi-quantitative method of optical density were used to analyze the expression of cell cycle regulator proteins in proliferative phase of endometrium (n=6) and early pregnancy decidua (n=6). In addition, we selected cyclin-dependent kinase inhibitor 1c (CDKN1C, also known as P57) and cyclin-dependent kinase inhibitor 2b (CDKN2B, also known as P15) in order to study their role in the process of decidualization by the RNAi method. ESC was arrested at G0/G1 checkpoints during decidualization. Cell cycle regulatory genes P57 and P15 were upregulated, while cyclin D1 (CCND1), cyclin-dependent kinase 2 (CDK2), and cell division cycle protein 2 homolog (CDC2) were downregulated during ESC differentiation both in vitro and vivo. P57 siRNA impaired ESC decidualization and caused different morphological and ultrastructural changes as well as a relatively low secretion of prolactin, but P15 siRNA had no effects. We concluded that P15, CCND1, CDK2, and CDC2 may participate in ESC withdraw from the cell cycle and go into differentiation both in vitro and in vivo. P57 is one of the key determinants of ESC differentiation due to its effect on the cell cycle distribution, but its association with the decidua-specific transcription factor needs further investigation.

High-throughput impedance-based cell adhesion and migration assay with single-cell resolution using CMOS microelectrode array

High-throughput impedance-based cell adhesion and migration assay with single-cell resolution using CMOS microelectrode array

Single-Cell Genomics of Microbial Dark Matter.

Single-cell genomics allows bypassing the culturing step and to directly access environmental microbes one cell at a time. The method has been successfully applied to explore archaeal and bacterial candidate phyla, referred to as microbial dark matter. Here I summarize the single-cell genomics workflow, including sample preparation and preservation, high-throughput fluorescence-activated cell sorting, cell lysis and amplification of environmental samples. Furthermore I describe phylogenetic screening based on 16S rRNA genes and suggest a suitable library preparation and sequencing approach.

A fluorescent microbead-based microfluidic immunoassay chip for immune cell cytokine secretion quantification.

Quantitative and dynamic analyses of immune cell secretory cytokines are essential for precise determination and characterization of the "immune phenotype" of patients for clinical diagnosis and treatment of immune-related diseases. Although multiple methods including the enzyme-linked immunosorbent assay (ELISA) have been applied for cytokine detection, such measurements remain very challenging in real-time, high-throughput, and high-sensitivity immune cell analysis. In this paper, we report a highly integrated microfluidic device that allows for on-chip isolation, culture, and stimulation, as well as sensitive and dynamic cytokine profiling of immune cells. Such a microfluidic sensing chip is integrated with cytometric fluorescent microbeads for real-time and multiplexed monitoring of immune cell cytokine secretion dynamics, consuming a relatively small extracted sample volume (160 nl) without interrupting the immune cell culture. Furthermore, it is integrated with a Taylor dispersion-based mixing unit in each detection chamber that shortens the immunoassay period down to less than 30 minutes. We demonstrate the profiling of multiple pro-inflammatory cytokine secretions (e.g. interleukin-6, interleukin-8, and tumor necrosis factors) of human peripheral blood mononuclear cells (PBMCs) with a sensitivity of 20 pg ml-1 and a sample volume of 160 nl per detection. Further applications of this automated, rapid, and high-throughput microfluidic immunophenotyping platform can help unleash the mechanisms of systemic immune responses, and enable efficient assessments of the pathologic immune status for clinical diagnosis and immune therapy.

Spatiotemporal Developmental Trajectories in the Arabidopsis Root Revealed Using High-Throughput Single Cell RNA Sequencing

High-throughput single cell RNA-sequencing (scRNA-seq) is becoming a cornerstone of developmental research, providing unprecedented power in understanding dynamic processes. Here, we present a high-resolution scRNA-seq expression atlas of the Arabidopsis root, composed of thousands of independently profiled cells. This atlas provides precise spatiotemporal information, identifying defining expression features for all major cell types, including the scarce cells of the quiescent centre. These reveal new developmental regulators and downstream genes that translate cell fate into distinctive cell shapes and functions. Developmental trajectories derived from pseudotime analysis depict a finely resolved cascade of cell progressions from the niche through differentiation that are supported by mirroring expression waves of highly interconnected transcription factors. This study demonstrates the power of applying scRNA-seq to plants, and provides a unique spatiotemporal perspective of root cell differentiation.

Intelligent Cell Search Engine

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a real-time machine intelligence technology based on a radically new architecture that realizes real-time image-based intelligent cell search and sorting at an unprecedented rate. The technology integrates high-throughput cell imaging, cell focusing, and cell sorting on a hybrid software-hardware data management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision making, and actuation. Specifically, we use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. Our technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

Profiling the circulating miRnome reveals a temporal regulation of the bone injury response.

Bone injury healing is an orchestrated process that starts with an inflammatory phase followed by repair and remodelling of the bone defect. The initial inflammation is characterized by local changes in immune cell populations and molecular mediators, including microRNAs (miRNAs). However, the systemic response to bone injury remains largely uncharacterized. Thus, this study aimed to profile the changes in the plasma miRnome after bone injury and determine its biological implications.Methods: A rat model of femoral bone defect was used, and animals were evaluated at days 3 and 14 after injury. Non-operated (NO) and sham operated animals were used as controls. Blood and spleen were collected and peripheral blood mononuclear cells (PBMC) and plasma were separated. Plasma miRnome was determined by RT-qPCR array and bioinformatics Ingenuity pathway analysis (IPA) was performed. Proliferation of bone marrow mesenchymal stem/stromal cells (MSC) was evaluated by Ki67 staining and high-throughput cell imaging. Candidate miRNAs were evaluated in splenocytes by RT-qPCR, and proteins found in the IPA analysis were analysed in splenocytes and PBMC by Western blot.Results: Bone injury resulted in timely controlled changes to the miRNA expression profile in plasma. At day 3 there was a major down-regulation of miRNA levels, which was partially recovered by day 14 post-injury. Interestingly, bone injury led to a significant up-regulation of let-7a, let-7d and miR-21 in plasma and splenocytes at day 14 relative to day 3 after bone injury, but not in sham operated animals. IPA predicted that most miRNAs temporally affected were involved in cellular development, proliferation and movement. MSC proliferation was analysed and found significantly increased in response to plasma of animals days 3 and 14 post-injury, but not from NO animals. Moreover, IPA predicted that miRNA processing proteins Ago2 and Dicer were specifically inhibited at day 3 post-injury, with Ago2 becoming activated at day 14. Protein levels of Ago2 and Dicer in splenocytes were increased at day 14 relative to day 3 post-bone injury and NO animals, while in PBMC, levels were reduced at day 3 (albeit Dicer was not significant) and remained low at day 14. Ephrin receptor B6 followed the same tendency as Ago2 and Dicer, while Smad2/3 was significantly decreased in splenocytes from day 14 relative to NO and day 3 post-bone injury animals.Conclusion: Results show a systemic miRNA response to bone injury that is regulated in time and is related to inflammation resolution and the start of bone repair/regeneration, unravelling candidate miRNAs to be used as biomarkers in the monitoring of healthy bone healing and as therapeutic targets for the development of improved bone regeneration therapies.

Sequencing of Genomes from Environmental Single Cells.

Sequencing of single bacterial and archaeal cells is an important methodology that provides access to the genetic makeup of uncultivated microorganisms. We here describe the high-throughput fluorescence-activated cell sorting-based isolation of single cells from the environment, their lysis and strand displacement-mediated whole genome amplification. We further outline 16S rRNA gene sequence-based screening of single-cell amplification products, their preparation for Illumina sequencing libraries, and finally propose computational methods for read and contig level quality control of the resulting sequence data.