A Single‐Cell Assay for Time Lapse Studies of Exosome Secretion and Cell Behaviors

Abstract

To understand the inhomogeneity of cells in biological systems, there is a growing demand on the capability of characterizing the properties of individual single cells. Since single cell studies require continuous monitoring of the cell behaviors instead of a snapshot test at a single time point, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and, for appropriate cell types, proliferation of single cells and convenient, non-invasive tests of single cell behaviors from molecular markers. In this paper we present a highly versatile single-cell assay that can accommodate different cellular types, enable easy and efficient single cell loading and culturing, and be suitable for the study of effects of in-vitro environmental factors in combination with drug screening. One salient feature of the assay is the non-invasive collection and surveying of single cell secretions at different time points, producing unprecedented insight of single cell behaviors based on the biomarker signals from individual cells under given perturbations. Above all, the acquired information is quantitative, for example, measured by the number of exosomes each single cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single cell properties.

Bone Marrow-Derived Mesenchymal Stem Cells-Derived Exosomes Promote Survival of Retinal Ganglion Cells Through miRNA-Dependent Mechanisms

“…While few studies have monitored exosome secretion rate, our control numbers corroborate those seen in cell lines [44]. For example, a recent study reported secretion rate of 4 3 10 8 exosomes per 100,000 cells per 24 hours in MCF10A cell lines [45]. Flow cytometry of exosomes revealed two important conclusions.…”

Microfluidic Single‐Cell Omics Analysis

“…To enrich the secretions, Chiu et al resorted to a bioprinting method, in which glass slides were modified with capture antibodies and then placed on microwells. The exosomes secreted from corresponding single cells below could be collected and subsequently measured by streptavidin quantum dots …”

“…Thus each protein could also gain unique fluorescent barcoding as the corresponding microbead, rendering this strategy to reach exponential growth for protein targets . Fluorescence readout exhibits a simple and rapid approach of protein measurement, and is especially suitable for secretions detection, but still suffers from limited complexity and low sensitivity when detecting low‐copy proteins.…”

Quantitative Analysis of Exosome Secretion Rates of Single Cells

“…A chip size between 5 x 5 mm 2 and 12 x 12 mm 2 is recommended. A cell loading chip made of PDMS is also recommended (Chiu et al ., 2016). …”

“…After each collection, anti-body conjugated quantum dots are used to label exosomes to allow counting and analysis of exosome surface proteins. Detailed studies of exosome properties related to cell behaviors such as responses to drugs and stress at single cell resolution can be found in the publication (Chiu et al ., 2016). …”