In the present study, we developed a microfluidic device that enables non-destructive isolation of chromosomes from single mammalian cells as well as real-time microscopic monitoring of the partial unfolding and stretching of individual chromosomes with increasing salt concentrations under a gentle flow. of the partial unfolding and stretching of individual chromosomes with increasing salt concentrations Lactose under a gentle flow. Using this device, we compared the folding stability of chromosomes between non-differentiated and differentiated cells and found that the salt concentration which induces the chromosome unfolding was lower (500?mM NaCl) for chromosomes derived from undifferentiated cells, suggesting that the chromatin folding stability of these cells is lower than that of differentiated cells. In addition, individual unfolded chromosomes, i.e., chromatin fibres, were stretched to 150C800?m non-destructively under 750?mM NaCl and showed distributions of highly/less folded regions along the fibres. Thus, our technique can provide insights into the aspects of chromatin folding that influence the epigenetic control of cell differentiation. Introduction In eukaryotic cells, genomic DNA bound to histones is folded and stored in the nucleus. Cellular activity is maintained by the expression of genes at the appropriate place and time, which requires the partial loosening of DNAChistone complexes. Since the control of gene expression involves chemical modifications of DNA bases and histones that alter the folding stability (loosening or tightening) of the chromatin at specific sites, gene expression profiles vary according to cell type and differentiation status1. Transcriptional activity differs among allogeneic cells2C4, and cancerous tissues harbour a mixed population of cells with distinct expression profiles5. As such, there is a need for a technique that enables epigenetic analyses at the single-cell level to evaluate the relationship between the distribution of chemical modifications of DNA or histones and the folding stability of chromatin as well as gene expression profiles. This information can provide insight into the mechanisms by which a state of differentiation is induced or maintained Lactose and how these mechanisms contribute to cancer development. Micrococcal nuclease sequencing, chromatin conformation capture sequencing, assay for transposase-accessible chromatin by high-throughput sequencing, and chromatin immunoprecipitation sequencing are analytical methods that can be used to identify DNA sites that lack or harbour loosely bound histones or that are bound by specific proteins at a single-base resolution6C10. However, since these approaches involve a DNA fragmentation step prior to sequencing and utilise short read sequences, it is difficult to obtain information about higher-order DNA structure and folding stability. In addition, whole-genome coverage is low when these methods are applied to single cells due to sample loss during preparation11. Immunofluorescence labelling of chromosomes is another epigenetic analysis technique12 that can be applied to single cells. In this method, chromosomes are spread out on a glass substrate near the source cells, which are seeded on the substrate with adequate spacing. However, this approach does not provide high-resolution Lactose information about the distribution of chemical modifications or folding stability along chromatin fibres. In addition, it is difficult to investigate changes in the higher-order folding structure resulting from alterations in the conditions of the surrounding solutionwhich alter the strength of interactions between DNA and DNA-binding proteinsdue to the adsorption of chromosomes onto the glass substrate. Consequently, a technique that allows for the examination of chromosomes isolated from single cells without fragmentation and adsorption onto a substrate is needed. Studies pioneering the use of single cell- and single chromosome-based techniques to investigate the properties of chromosomes have involved the extraction of mitotic chromosomes from mammalian/amphibian cells in an open cell culture dish under a microscope using micromanipulator-assisted micro-needles/-pipettes13,14. This approach has revealed the reversible condensation/decondensation of mitotic chromosomes by Mouse monoclonal to HK1 exposure to various cationic solutions in the open dish. However, this method has not been used to determine the correlation Lactose between the differentiation state of cells and the distribution of chromosome/chromatin folding stability. This lack of investigation may be attributed to practical challenges, e.g., sequential solution exchanges and the precise control of solution conditions in the open dish during the micromanipulation of cells/chromosomes. Recently, microfluidic devices have been utilised in single cell/molecule-level biochemical analyses/experiments15C18. A characteristic feature of microfluidic devices is their ability to precisely control solution conditions by introducing the solution of interest into microfluidic channels. Although such devices have been used for various types of bioanalysis, methods for investigating chromatin/chromosomes, i.e., the complex of DNA and proteins, in single cells are less developed than those used for single-cell genome-wide gene expression analyses in which the analyte is basically naked fragmented DNA. To date, nano-/microfluidic channel devices for chromosome/large genomic DNA analysis that have been developed employ off-chip-prepared chromosomes/genomic DNA and have not yet been used for single cell-based experiments19C21. We recently developed a method for isolating intact chromatin fibres from individual fission yeast cells that were.