Studying 3D cell cultures in a microfluidic droplet array under multiple time-resolved conditions

The relevance of traditional cell cultures to cellular behavior in vivo is limited, since the two-dimensional (2D) format does not appropriately reproduce the microenvironment that regulates cell functions. In this context, spheroids are an appealing 3D cell culture format to complement standard techniques, by combining a high level of biological relevance with simple production protocols. However the methods for spheroid manipulation are still labor intensive, which severely limits the complexity of operations that can be performed on statistically relevant numbers of individual spheroids. Here we show how to apply hundreds of different conditions on spheroids in a single microfluidic chip, where each spheroid is produced and immobilized in an anchored droplet. By using asymmetric anchor shapes, a second drop can be merged with the spheroid-containing drop at a later time. This time-delayed merging uniquely enables two classes of applications that we demonstrate: (1) the initiation of cell-cell interactions on demand, either for building micro-tissues within the device or for observing antagonistic cell-cell interactions with applications in immuno-therapy or host-pathogen interactions, (2) a detailed dose-response curve obtained by exposing an array of hepatocyte-like spheroids to droplets containing a wide range of acetaminophen concentrations. The integrated microfluidic format allows time-resolved measurements of the response of hundreds of spheroids with a single-cell resolution. The data shows an internally regulated evolution of each spheroid, in addition to a heterogeneity of the responses to the drug that the single-cell analysis correlates with the initial presence and location of dead cells within each spheroid.

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The effect of quercetin on endothelial cells is modified by heterocellular interactions

Food & Function ◽

10.1039/d0fo00141d ◽

2020 ◽

Vol 11 (5) ◽

pp. 3916-3925

Author(s):

Sarka Tumova ◽

Michael J. Houghton ◽

Gary Williamson

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Single Cell ◽

Cell Function ◽

Cell Interactions ◽

Cell Type ◽

Endothelial Cell Function ◽

Single Cell Type ◽

Cell Cell

Single cell-type models are useful for determining mechanisms, but in vivo, cell–cell interactions are important, and neighbouring cells can impact endothelial cell function.

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TAMI-35. DETECTING SINGLE-CELL INTERACTIONS IN ORGANOTYPIC CULTURES OF GLIOBLASTOMA USING BARCODED RABIES VIRUS

Neuro-Oncology ◽

10.1093/neuonc/noab196.819 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi205-vi205

Author(s):

Brian Andersen ◽

Michael Wheeler ◽

Zhaorong Li ◽

E Antonio Chiocca ◽

David Reardon ◽

...

Keyword(s):

Single Cell ◽

Rabies Virus ◽

Single Cell Analysis ◽

Cell Interactions ◽

Cellular Interactions ◽

Cell Analysis ◽

Viral Glycoprotein ◽

Organotypic Cultures ◽

Interaction Detection ◽

Cell Cell

Abstract Cell-cell interactions are thought to drive tumor-promoting signals in the microenvironment of glioblastoma, but standard approaches for single cell analysis do not directly identify cell interactions and the mechanisms that mediate them. We recently developed a novel method to analyze cell-cell interactions—rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNAseq). RABID-seq was first implemented in transgenic mice to investigate the interactions of astrocytes with other cells in the CNS enabling the study of astrocyte connectome perturbations and candidate therapeutic targets in multiple sclerosis and its pre-clinical model, experimental autoimmune encephalomyelitis (EAE). Here, we report the first use of RABID-seq in human tissues in organotypic cultures established from three IDH-wildtype glioblastoma (GBM) patients. In organotypic GBM cultures, initial infection by pseudotyped barcoded rabies virus deficient for viral glycoprotein was achieved after previous culture transduction with a lentivirus containing the avian TVA receptor and rabies glycoprotein under the human EF1a promoter. We employed this system to initially infect approximately 1,000 malignant or non malignant cells in the tumor microenvironment. After five days, infected cells were isolated from cultures and processed for single cell analysis using SMART seq. We were able to capture at least 6,000 interacting cells per tumor specimen, from which barcodes were recovered and cDNA was sent for sequencing. Here we present connectomic data from our initial cohort of three glioblastoma patients as an introduction to RABID-seq, with a focus on astrocyte-tumor interactions. Candidate mechanisms of cellular interactions will undergo functional validation in murine models of glioblastoma.

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Faculty Opinions recommendation of A high-throughput single-cell analysis of human CD8⁺ T cell functions reveals discordance for cytokine secretion and cytolysis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13709958.15123059 ◽

2012 ◽

Author(s):

Bart Roep ◽

Menno van Lummel

Keyword(s):

T Cell ◽

Single Cell ◽

High Throughput ◽

Single Cell Analysis ◽

Cd8 T Cell ◽

Cytokine Secretion ◽

Cell Analysis ◽

Cell Functions

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Single Cell Analysis Reveals That IL-4 Receptor/Stat6 Signaling Is Not Required for the In Vivo or In Vitro Development of CD4+Lymphocytes with a Th2 Cytokine Profile

The Journal of Immunology ◽

10.4049/jimmunol.164.6.3047 ◽

2000 ◽

Vol 164 (6) ◽

pp. 3047-3055 ◽

Cited By ~ 193

Author(s):

Dragana Jankovic ◽

Marika C. Kullberg ◽

Nancy Noben-Trauth ◽

Patricia Caspar ◽

William E. Paul ◽

...

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Cytokine Profile ◽

Cell Analysis ◽

In Vitro Development ◽

Th2 Cytokine ◽

Vitro Development ◽

Cd4 Lymphocytes

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P-165 Cytometry-Based Single Cell Analysis of Intact Epithelial Signaling Reveals MAPK Activation Divergent from TNF-α-Induced Apoptosis in Vivo

Inflammatory Bowel Diseases ◽

10.1097/01.mib.0000480283.46656.d8 ◽

2016 ◽

Vol 22 ◽

pp. S59-S60

Author(s):

Alan Simmons ◽

Amrita Banerjee ◽

Eliot McKinley ◽

Cherieʼ Scurrah ◽

Jeffrey Franklin ◽

...

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Cell Analysis ◽

Mapk Activation ◽

Tnf Α ◽

Induced Apoptosis

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Single cell analysis in Vivo

Optics in the Life Sciences ◽

10.1364/boda.2015.bw1a.1 ◽

2015 ◽

Author(s):

Charles P. Lin

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Cell Analysis

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Inositol phosphates: health implications, methods of analysis, and occurrence in plant foods

Journal of Food Bioactives ◽

10.31665/jfb.2018.1126 ◽

2018 ◽

Vol 1 ◽

Cited By ~ 1

Author(s):

Quynh H. Duong ◽

Karen G. Lapsley ◽

Ronald B. Pegg

Keyword(s):

Inositol Phosphates ◽

Negative Effects ◽

Plant Foods ◽

Plant Seeds ◽

Cell Functions ◽

Wide Range ◽

Multiple Cell ◽

The Relationship

Inositol phosphates (InsPs), especially myo-inositol hexakisphosphate (InsP6), are important binders of phosphorus and minerals in plant seeds. However, they have long been considered as anti-nutritional components of plant foods due to their possible negative effects on the absorption of minerals and proteins in mammals. On the other hand, recent findings have found InsPs to be ubiquitous in eukaryote cells and actively participating in multiple cell functions. In vivo and in vitro studies have also documented the preventive potential of these compounds against the development of a wide range of diseases. In light of these findings, interest in the relationship between these compounds and human health has been renewed. It is suggested that the interactions of InsPs with other nutrients in the gut are complex, that the absorption of dietary InsPs might be implied but is not certain, and that the disease fighting capabilities of InsPs hold both promises and limitations. At the same time, the analysis of these compounds in foods and biological samples still faces many challenges, calling for more advanced modification and developments in the future.

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Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617961114 ◽

2017 ◽

Vol 114 (18) ◽

pp. E3659-E3668 ◽

Cited By ~ 70

Author(s):

Ann Wiegand ◽

Jonathan Spindler ◽

Feiyu F. Hong ◽

Wei Shao ◽

Joshua C. Cyktor ◽

...

Keyword(s):

Single Cell ◽

Mononuclear Cells ◽

Single Cells ◽

Constitutive Expression ◽

Multiple Time ◽

Hiv Rna ◽

Infected Cells ◽

The Impact ◽

Hiv 1

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2–18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

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