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High-content adhesion assay to address limited cell samples.

ABSTRACT: Cell adhesion is a broad topic in cell biology that involves physical interactions between cells and other cells or the surrounding extracellular matrix, and is implicated in major research areas including cancer, development, tissue engineering, and regenerative medicine. While current methods have contributed significantly to our understanding of cell adhesion, these methods are unsuitable for tackling many biological questions requiring intermediate numbers of cells (10(2)-10(5)), including small animal biopsies, clinical samples, and rare cell isolates. To overcome this fundamental limitation, we developed a new assay to quantify the adhesion of ~10(2)-10(3) cells at a time on engineered substrates, and examined the adhesion strength and population heterogeneity via distribution-based modeling. We validated the platform by testing adhesion strength of cancer cells from three different cancer types (breast, prostate, and multiple myeloma) on both IL-1? activated and non-activated endothelial monolayers, and observed significantly increased adhesion for each cancer cell type upon endothelial activation, while identifying and quantifying distinct subpopulations of cell-substrate interactions. We then applied the assay to characterize adhesion of primary bone marrow stromal cells to different cardiac fibroblast-derived matrix substrates to demonstrate the ability to study limited cell populations in the context of cardiac cell-based therapies. Overall, these results demonstrate the sensitivity and robustness of the assay as well as its ability to enable extraction of high content, functional data from limited and potentially rare primary samples. We anticipate this method will enable a new class of biological studies with potential impact in basic and translational research.

SUBMITTER: Warrick JW

PROVIDER: S-EPMC3832292 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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High-content adhesion assay to address limited cell samples.

Warrick Jay W JW Young Edmond W K EW Schmuck Eric G EG Saupe Kurt W KW Beebe David J DJ

Integrative biology : quantitative biosciences from nano to macro 20130221 4

Cell adhesion is a broad topic in cell biology that involves physical interactions between cells and other cells or the surrounding extracellular matrix, and is implicated in major research areas including cancer, development, tissue engineering, and regenerative medicine. While current methods have contributed significantly to our understanding of cell adhesion, these methods are unsuitable for tackling many biological questions requiring intermediate numbers of cells (10(2)-10(5)), including s ...[more]

PMID: 23426645

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Project description:Cigarette smoke (CS) has been reported to affect the adhesion of monocytes to endothelial cells, a critical step in atherogenesis. Using an in vitro adhesion assay together with innovative computational systems biology approaches to analyze omics data, our study aimed at investigating CS-induced mechanisms by which monocyte-endothelial cell adhesion is promoted. Disease-relevant primary human coronary artery endothelial cells (HCAECs) were treated for 4 hours with (1) conditioned media of human monocytic Mono Mac-6 (MM6) cells preincubated with low or high concentrations of aqueous CS extract (sbPBS) from reference cigarette 3R4F for 2 hours (indirect treatment, I), (2) unconditioned media similarly prepared, however without MM6 cells (direct treatment, D), or (3) freshly generated sbPBS (fresh direct treatment, FD). sbPBS promoted MM6 cells-HCAECs adhesion following I and FD, but not D. In I, the effect was mediated at a low concentration through activation of vascular inflammation processes promoted in HCAECs by a paracrine effect of the soluble mediators secreted by sbPBS-treated MM6 cells. Tumor necrosis factor alpha (TNF-alpha major inducer, was actually shed by unstable CS compound-activated TNFalpha-converting enzyme. In FD, the effect was triggered at a high concentration that also induced some toxicity. This effect was mediated through an yet unknown mechanism associated with a stress damage response promoted in HCAECs by unstable CS compounds present in freshly generated sbPBS, which had decayed in D unconditioned media. In conclusion, aqueous CS extract directly and indirectly promotes monocytic cell-endothelial cell adhesion in vitro via distinct concentration-dependent mechanisms. Samples from E-MTAB-3644 dataset correspond to 3R4F sbPBS15 and PBS15 (vehicle control: 15% v/v). In the E-MTAB-3923 dataset, samples correspond to THS2.2 abPBS15, THS2.2 abPBS75 and PBS75 (75% v/v). All THS2.2 abPBS15 from E-MTAB-3923 must be compared with their respective control, PBS15 from E-MTAB-3644; while THS2.2 abPBS75 from E-MTAB-3923 must be compared with their respective control, PBS75 from E-MTAB-3923.

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High-content adhesion assay to address limited cell samples.

Publications

High-content adhesion assay to address limited cell samples.

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