Identification of rare progenitor cells from human periosteal tissue using droplet microfluidics

Abstract

The isolation and characterisation of single cells from a heterogeneous population are important processes in cell biology, immunology, stem cell research, and cancer research. In the development of novel cell-based therapies, there is a considerable need to target specific cell types to allow for further analysis and amplification ex vivo. We introduce, herein, the use of droplet-based microfluidics as a platform technology for the identification and quantification of distinct cell phenotypes. Using molecular labelling of specific cell populations by antibodies and fluorescent dyes, detection of single cells encapsulated within picolitre-sized aqueous droplets can be performed using high-sensitivity confocal fluorescence detection. Specifically, rare progenitor cells were immunodetected within a heterogeneous population of cells isolated from human periosteal tissue. Using this model human cell population, the accuracy and reproducibility of the droplet system were tested and the results were verified using conventional flow cytometry. It was found that the quantitation of phenotypic subpopulations measured using both techniques is directly comparable. Accordingly, this study demonstrates the biological capacity of droplet-based microfluidics for cellular analysis and provides a necessary first step towards the development of a novel cell sorting technology.

Stem cells in microfluidics

“…[106][107][108][109][110][111] For instance, a direct cell analysis approach has been reported using microfluidics, which was based on analysis of interphoton burst recurrence times from a single particle for discrimination between different cell populations, allowing species discrimination in a realtime fashion with the potential for cell counting and single-cell sizing. 106 Besides, a microfluidic stochastic confinement approach utilizing wells and droplets to separate rare single cells from a bulk mixture was reported.…”

“…110 Furthermore, similar techniques have even been adopted for investigation of stem cells. 111 Cells labeled with antibodies and fluorescent dyes have been explored by using droplet-based microfluidic systems to physically encapsulate and simultaneously characterize single cells, which may be useful for the enrichment of stem cells. Those methods provide a direct way to investigate the cellular behavior simultaneously and have potential for further medical applications.…”

“…Differentiation of ESCs is often initiated by the generation of embryoid bodies ͑EBs͒, which are three-dimensional cell aggregates formed by culturing ESCs on a nonadherent substrate. 113,114 In this section, a variety of methods for studying the response of ESCs in microfluidic systems are introduced and reviewed, including microwells, [115][116][117][118][119][120][121] membrane fabrication, [122][123][124] feederlayer coating, [125][126][127] arrayed microreactors with microwells, [128][129][130][131] and physical and chemical stimulation 111,[132][133][134][135][136][137][138][139] to study the morphology of ESCs.…”

“…109 Furthermore, droplet-based microfluidic systems have also been used for cell analysis. 110,111 For instance, simultaneous determination of gene expression and enzymatic activity in individual bacterial cells has been performed in microdroplets utilizing a microfluidic device capable of storing picoliter droplets containing single bacteria with constant volumes. 110 Furthermore, similar techniques have even been adopted for investigation of stem cells.…”

Analytical techniques for single-cell metabolomics: state of the art and trends

“…Although current studies involving the application of fluorescent probes to metabolites make use of fluorescence microscopy, implementation of such assays in combination with flow cytometry or fluorescenceactivated cell sorting [34] could enable studies of intracellular metabolites in large populations of cells. Since cytometry can already be realized in microchip format [35], such microscale cell handling and signal readout could be applied to record signals from metabolite probes in cell populations. In fact, a number of cytometric assays probing cell viability, metabolic functions, and redox states or determining the content of lipids and certain secondary metabolites (e.g., photosynthetic dyes) in single cells, with (or, in some cases, without) staining, are available (for more information, see the review by Davey and Kell [34]).…”