scholarly journals Analysis of in vivo single cell behavior by high throughput, human-in-the-loop segmentation of three-dimensional images

2015 ◽  
Vol 16 (1) ◽  
Author(s):  
Michael Chiang ◽  
Sam Hallman ◽  
Amanda Cinquin ◽  
Nabora Reyes de Mochel ◽  
Adrian Paz ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Three Dimensional ◽  
Cell Behavior ◽  
Human In The Loop ◽  
Three Dimensional Images

scholarly journals Systematic comparison of high-throughput single-cell RNA-seq methods for immune cell profiling

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Tracy M. Yamawaki ◽  
Daniel R. Lu ◽  
Daniel C. Ellwanger ◽  
Dev Bhatt ◽  
Paolo Manzanillo ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Immune Cell ◽  
Cell Types ◽  
Data Interpretation ◽  
Detection Sensitivity ◽  
Rna Seq ◽  
Cell Recovery

Abstract Background Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. Results Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5′ v1 and 3′ v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. Conclusion Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

On-chip hydrogel arrays individually encapsulating acoustic formed multicellular aggregates for high throughput drug testing

Lab on a Chip ◽  
2020 ◽  
Vol 20 (12) ◽  
pp. 2228-2236 ◽  
Author(s):  
Xuejia Hu ◽  
Shukun Zhao ◽  
Ziyi Luo ◽  
Yunfeng Zuo ◽  
Fang Wang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
High Throughput ◽  
Solid Tumor ◽  
Drug Testing ◽  
Three Dimensional ◽  
Multicellular Aggregates ◽  
3D Environments ◽  
On Chip ◽  
Insight Into

Multicellular aggregates in three-dimensional (3D) environments provide novel solid tumor models that can provide insight into in vivo drug resistance.

scholarly journals Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Hilary A. Kenny ◽  
Madhu Lal-Nag ◽  
Erin A. White ◽  
Min Shen ◽  
Chun-Yi Chiang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Organotypic Culture ◽  
Three Dimensional ◽  
In Vivo Efficacy

scholarly journals Systematic Comparison of High-Throughput Single-Cell RNAseq Methods for Immune Cell Profiling

2020 ◽  
Author(s):  
Chi-Ming Kevin Li ◽  
Tracy M Yamawaki ◽  
Daniel R Lu ◽  
Daniel C Ellwanger ◽  
Dev Bhatt ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Immune Cell ◽  
Cell Types ◽  
Drop Out ◽  
Detection Sensitivity ◽  
Rna Seq ◽  
Cell Recovery

Abstract Background: Elucidation of immune populations with single-cell RNA-seq has greatly benefited the fieldof immunology by deepening the characterization of immune heterogeneity and leading to thediscovery of new subtypes. However, single-cell methods inherently suffer from limitations in therecovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropoutevents. This issue is often compounded by limited sample availability and limited prior knowledge ofheterogeneity, which can confound data interpretation.Results: Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. Weprepared 21 libraries under identical conditions of a defined mixture of two human and two murinelymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluatemethods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expressionsignatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5’v1 and 3’ v3 methods. We demonstrate that these methods have fewer drop-out events whichfacilitates the identification of differentially-expressed genes and improves the concordance of singlecellprofiles to immune bulk RNA-seq signatures.Conclusion: Overall, our characterization of immune cell mixtures provides useful metrics, which canguide selection of a high-throughput single-cell RNA-seq method for profiling more complex immunecellheterogeneity usually found in vivo.

Patient-derived organoids for personalized drug screening in intrahepatic cholangiocarcinoma.

2020 ◽  
Vol 38 (4_suppl) ◽  
pp. 581-581
Author(s):  
Ricardo J. Antonia ◽  
Kan Toriguchi ◽  
Eveliina Karelehto ◽  
Dania Annuar ◽  
Luika Timmerman ◽  
...  
Keyword(s):  
High Throughput ◽  
Drug Screening ◽  
Intrahepatic Cholangiocarcinoma ◽  
Three Dimensional ◽  
Small Samples ◽  
Patient Specific ◽  
Mtor Inhibition ◽  
Drug Responses

581 Background: Despite standard treatment with gemcitabine and cisplatin, median survival for unresectable Intrahepatic Cholangiocarcinoma (ICC) is < 1 year. Clearly, novel therapeutic strategies are urgently needed. The paucity of targetable mutations in ICC and the as yet unproven benefit of genetically targeted drugs led us to ask whether a reliable clinical benefit may be revealed by patient-specific therapeutic testing in novel models of ICC. Here we describe our ability to establish patient-derived three-dimensional organoid cultures (PDO) that enable individualized identification of active single agents or drug combinations in surrogate models of ICC. Methods: To model patient-specific drug responses, we used the freshly resected ICCs from small samples of single patient tumors to generate PDXs and PDOs, small spheroidal clusters of tumor cells grown in vitro. We have employed a high-throughput drug screening platform using AI-enhanced robotics (Yamaha Motor Corporation) to identify and distribute single, uniformly sized PDOs into 384-well ultra-low adherent plates. This is coupled with a TECAN D300e drug dispenser that rapidly delivers nanoliter volumes of a 34-drug panel, thereby facilitating rapid, reliable drug response analyses. Results: Our data show that PDOs retain characteristic genomic and histological features of the patients’ tumors. Drug responses were specific to each patient tumor, but PDOs from all patients responded to a greater or lesser degree to mTOR inhibition, suggesting that this pathway is important in ICC. The responses of PDO to the mTOR inhibitor Sapanisertib (INK128), was recapitulated in the same patient’s PDX. Further, INK128 was synergistic with gemcitabine in patient 970 PDOs as well as in vivo in PDX also from patient 970. Conclusions: As it is believed that PDX can predict patient responses to drugs, our results suggest that PDO may also predict patient drug responses. The establishment of PDO may allow economical patient-specific, high throughput drug screens that could ultimately inform clinical practice. [Table: see text]

scholarly journals Functional In vivo Single-cell Transcriptome (FIST) Analysis Reveals Molecular Properties of Light-Sensitive Neurons in Mouse V1

2018 ◽  
Author(s):  
Jianwei Liu ◽  
Na Pan ◽  
Le Sun ◽  
Mengdi Wang ◽  
Junjing Zhang ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Three Dimensional ◽  
Whole Cell ◽  
Mrna Sequencing ◽  
Layer 2 ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome ◽  
Live Mouse

ABSTRACTVision formation is classically based on projections from the retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single neurons with specific functions in V1 still remain unknown. Some neurons in mouse V1 are tuned to light stimulus. To determine the molecular properties of light-stimulated neurons in layer 2/3 of V1, we developed a method of functional in vivo single-cell transcriptome (FIST) analysis that integrates sensory evoked calcium imaging, whole-cell electrophysiological patch-clamp recordings, single-cell mRNA sequencing and three-dimensional morphological characterization in a live mouse, based on a two-photon microscope system. In our study, 58 individual cells from layer 2/3 of V1 were identified as either light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of every single cell after individual functional tests were aspirated through the patch-clamp pipette for mRNA sequencing. Furthermore, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in the live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with high expression of genes related to transmission regulation, such as Rtn4r, Nr4a1, and genes involved in membrane transport, such as Na+/K+ ATPase, NMDA-type glutamatergic receptor, preferentially respond to light stimulation. Our findings demonstrate the ability of FIST analysis to characterize the functional, morphological and transcriptomic properties of a single cell in alive animal, thereby providing precise neuronal information and predicting its network contribution in the brain.

scholarly journals Single-cell transcriptomics elucidates in vitro reprogramming of human intestinal epithelium cultured in a physiodynamic gut-on-a-chip

2021 ◽  
Author(s):  
Woojung Shin ◽  
Zhe Su ◽  
S. Stephen Yi ◽  
Hyun Jung Kim
Keyword(s):  
Single Cell ◽  
Intestinal Epithelium ◽  
Drug Transport ◽  
Three Dimensional ◽  
Cancer Cell Line ◽  
Static Condition ◽  
On Chip ◽  
Epithelial Layers

The microphysiological human gut-on-a-chip has demonstrated in vivo-relevant cellular fidelity of intestinal epithelium compared to its cultures in a static condition. Microfluidic control of morphogen gradients and mechanical cues robustly induced morphological histogenesis with villi-like three-dimensional (3D) microarchitecture, lineage-associated cytodifferentiation, and physiological functions of a human intestinal Caco-2 epithelium. However, transcriptomic dynamics that orchestrates morphological and functional reprogramming of the epithelium in a microphysiological culture remains elusive. Single-cell transcriptomic analysis revealed that a gut-on-a-chip culture that offers physiological motions and flow drives three distinctive subclusters that offer distinct gene expression and unique spatial representation in 3D epithelial layers. The pseudotemporal trajectory of individual cells visualized the evolutionary transition from ancestral genotypes in static cultures into more heterogeneous phenotypes in physiodynamic cultures on cell cycles, differentiation, and intestinal functions including digestion, absorption, drug transport, and metabolism of xenobiotics. Furthermore, the inversed transcriptomic signature of oncogenes and tumor-suppressor genes of Caco-2 cells verified that a gut-on-a-chip culture drives a postmitotic reprogramming of cancer-associated phenotypes. Thus, we discovered that a physiodynamic on-chip culture is necessary and sufficient for a cancer cell line to be reprogrammed to elicit in vivo-relevant heterogeneous cell populations with restored normal physiological signatures.

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