Dataset Information

Active volume regulation in adhered cells.

ABSTRACT: Recent experiments reveal that the volume of adhered cells is reduced as their basal area is increased. During spreading, the cell volume decreases by several thousand cubic micrometers, corresponding to large pressure changes of the order of megapascals. We show theoretically that the volume regulation of adhered cells is determined by two concurrent conditions: mechanical equilibrium with the extracellular environment and a generalization of Donnan (electrostatic) equilibrium that accounts for active ion transport. Spreading affects the structure and hence activity of ion channels and pumps, and indirectly changes the ionic content in the cell. We predict that more ions are released from the cell with increasing basal area, resulting in the observed volume-area dependence. Our theory is based on a minimal model and describes the experimental findings in terms of measurable, mesoscale quantities. We demonstrate that two independent experiments on adhered cells of different types fall on the same master volume-area curve. Our theory also captures the measured osmotic pressure of adhered cells, which is shown to depend on the number of proteins confined to the cell, their charge, and their volume, as well as the ionic content. This result can be used to predict the osmotic pressure of cells in suspension.

SUBMITTER: Adar RM

PROVIDER: S-EPMC7084104 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Active volume regulation in adhered cells.

Adar Ram M RM Safran Samuel A SA

Proceedings of the National Academy of Sciences of the United States of America 20200304 11

Recent experiments reveal that the volume of adhered cells is reduced as their basal area is increased. During spreading, the cell volume decreases by several thousand cubic micrometers, corresponding to large pressure changes of the order of megapascals. We show theoretically that the volume regulation of adhered cells is determined by two concurrent conditions: mechanical equilibrium with the extracellular environment and a generalization of Donnan (electrostatic) equilibrium that accounts for ...[more]

PMID: 32132211

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Project description:Adherent cultured cells are widely used biological tools for a variety of biochemical and biotechnology applications, including drug screening and gene expression analysis. One critical step in culturing adherent cells is the dissociation of cell monolayers into single-cell suspensions. Different enzymatic and non-enzymatic methods have been proposed for this purpose. Trypsinization, the most common enzymatic method for dislodging adhered cells, can be detrimental to cells, as it can damage cell membranes and ultimately cause cell death. Additionally, all available techniques require a prolonged treatment duration, typically on the order of minutes (5-10 min). Dissociation of cells becomes even more challenging in microfluidic devices, where, due to the nature of low Reynolds number flow and reduced mixing efficiency, multiple washing steps and prolonged trypsinization may be necessary to treat all cells. Here, we report a novel acoustofluidic method for the detachment of cells adhered onto a microchannel surface without exposing the cells to any enzymatic or non-enzymatic chemicals. This method enables a rapid (i.e., on the order of seconds), cost-effective, and easy-to-operate cell detachment strategy, yielding a detachment efficiency of ∼99% and cellular viability similar to that of the conventional trypsinization method. Also, as opposed to biochemical-based techniques (e.g., enzymatic), in our approach, cells are exposed to the dissociating agent (i.e., substrate-mediated acoustic excitation and microstreaming flow) only for as long as they remain attached to the substrate. After dissociation, the effect of acoustic excitation is reduced to microstreaming flow, therefore, minimizing unwanted effects of the dissociating agent on the cell phenotype. Additionally, our results suggest that cell excitation at acoustic powers lower than that required for complete cell detachment can potentially be employed for probing the adhesion strength of cell-substrate attachment. This novel approach can, therefore, be used for a wide range of lab-on-a-chip applications.

Project description:Sickle cell disease, a genetic disorder affecting a sizeable global demographic, manifests in sickle red blood cells (sRBCs) with altered shape and biomechanics. sRBCs show heightened adhesive interactions with inflamed endothelium, triggering painful vascular occlusion events. Numerous studies employ microfluidic-assay-based monitoring tools to quantify characteristics of adhered sRBCs from high resolution channel images. The current image analysis workflow relies on detailed morphological characterization and cell counting by a specially trained worker. This is time and labor intensive, and prone to user bias artifacts. Here we establish a morphology based classification scheme to identify two naturally arising sRBC subpopulations-deformable and non-deformable sRBCs-utilizing novel visual markers that link to underlying cell biomechanical properties and hold promise for clinically relevant insights. We then set up a standardized, reproducible, and fully automated image analysis workflow designed to carry out this classification. This relies on a two part deep neural network architecture that works in tandem for segmentation of channel images and classification of adhered cells into subtypes. Network training utilized an extensive data set of images generated by the SCD BioChip, a microfluidic assay which injects clinical whole blood samples into protein-functionalized microchannels, mimicking physiological conditions in the microvasculature. Here we carried out the assay with the sub-endothelial protein laminin. The machine learning approach segmented the resulting channel images with 99.1±0.3% mean IoU on the validation set across 5 k-folds, classified detected sRBCs with 96.0±0.3% mean accuracy on the validation set across 5 k-folds, and matched trained personnel in overall characterization of whole channel images with R2 = 0.992, 0.987 and 0.834 for total, deformable and non-deformable sRBC counts respectively. Average analysis time per channel image was also improved by two orders of magnitude (∼ 2 minutes vs ∼ 2-3 hours) over manual characterization. Finally, the network results show an order of magnitude less variance in counts on repeat trials than humans. This kind of standardization is a prerequisite for the viability of any diagnostic technology, making our system suitable for affordable and high throughput disease monitoring.

Dataset Information

Active volume regulation in adhered cells.

Publications

Active volume regulation in adhered cells.

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