Large-scale arrays of picolitre chambers for single-cell analysis of large cell populations

Abstract

We present a new method to analyze the cytoplasmic contents of single cells in large cell populations. This new method consists of an array of microchambers in which individual cells are collected, enclosed, and lysed to create a reaction mixture of the cytoplasm with extracellular detection agents. This approach was tested for the analysis of red blood cells in 10,000 microchambers in parallel. Single cells were routinely collected in more than 60% of microchambers, the collected cells were robustly (up to 99%) lysed by electric fields, and the cytoplasm enclosed in each microchamber was analyzed with fluorescence microscopy. Using a heterogeneous cell mixture, we verified that the new method could distinguish individual cells by cytoplasmic composition and the analysis compared well with conventional flow-cytometric evaluation of mixed cell populations. In contrast to flow-cytometry, the new method monitored single cells over time, thus characterizing the distributions of caspase activities of 5000 individual cells. This approach should be interesting for a variety of applications that would benefit from the ability to measure the distribution of cytoplasmic compounds in complex cell populations, including hematology, oncology, and immunology.

Biomechanical properties of red blood cells in health and disease towards microfluidics

“…Thus, because microscopy analysis is considered as the most important step in the diagnosis of hereditary disorders, a novel system such as a microfluidic device that combines RBC image analysis and the quantification of membrane properties such as the three fundamental membrane moduli (the shear elastic modulus, the area expansion modulus, and the bending modulus) and the relaxation time constant could be a novel tool in the diagnosis of hereditary disorders and could help to establish the relationship between membrane defects and the clinical status of the patient. The initial steps have been conducted towards this goal through the application of a microfluidic-based method, the SICMA 86,87 technology, that is able to measure the Elongation Index of individual cells in a heterogeneous population, allowing the recognition of subpopulations of pathological cells by the comparison with the healthy ones. A 30% reduction in RBC deformability has been observed in spherocytosis by using SICMA technology.…”

“…The cell can be osmotically swelled before the experiments, in order to obtain a spherical shape, that makes small deformations easier to detect 67 Microfluidic devices, coupled with automated image analysis, are suitable for point-of-care applications, 77 allowing to test a large number of cells by using microcirculation-mimicking patterns, 78 transparent substrata such as polydimethylsiloxane and glass, and video microscopy. Examples include visualizations of cell deformation as a function of pressure drop, in which the classical parachute-like shape observed in vivo was observed in in vitro experiments as well, 66,[79][80][81][82][83][84] estimations of cell membrane viscoelastic properties such as RBC shear elastic modulus and surface viscosity by using diverging channels, 65 measurements of the RBC time recovery constant in start-up experiments, 35 cell characterization by electric impedance microflow cytometry, 85 and single-cell microchamber array (SiCMA) technology 86,87 (Figures 3(D1) and 3(D2)). The latter applies a dielectrophoretic force to deform RBCs and used image analysis to analyse RBCs shape changes, allowing the evaluation the deformability of single RBCs in terms of Elongation Index %, defined as (x À y)/(x þ y) Â 100, where x and y are RBC major and minor axes, respectively.…”

Frequency-Dependent Impedance Analysis for Discriminating Cancer and Normal Cells Using Microchamber Array

“…Therefore, the width of a single chamber was 1.57 times longer than the average diameter of the cells, which is in the range of the optimal condition for singlecell loading. (17) Figure 4 shows the number of chambers occupied with respect to the cell concentrations. The number of chambers occupied by single cells shows an increasing tendency as cell concentration increases.…”

An electroactive microwell array for trapping and lysing single-bacterial cells

“…[2][3][4][5] Especially, microwell arrays enable large scale arraying and high-throughput single-cell measurements of cellular responses. 6 Previous microwell array methods used gravity as a simple mechanism to trap single cells; [6][7][8][9] however, this method remains in the applications to mammalian cells for which typical volume is in the range of picoliters. To the best of our knowledge, no single-cell arraying method for bacteria such as Escherichia coli ͑E.…”