title: Isolation of cell nuclei in microchannels by short-term chemical treatment via two-step carrier medium exchange.
abstract: Separation/purification of nuclei from cells is a critical process required for medical and biochemical research applications. Here, we report a flow-through microfluidic device for isolating cell nuclei by selectively digesting the cell membrane by using the concept of hydrodynamic filtration (HDF). When a cell suspension is continuously introduced into a microchannel (main channel) possessing multiple side channels, cells flow through the main channel, whereas the carrier medium of the cells is drained through the side channels. Introductions of a cell treatment solution containing a surfactant and a washing buffer enable the two-step exchange of the carrier-medium and the cell treatment by the surfactant for a short span of time. The precise control of the treatment time by changing the flow rate and/or the size of the microchannel enables the selective digestion of cell membranes, resulting in the isolation of cell nuclei after separation from membrane debris and cytoplasmic components according to size. We examined several surfactant molecules and demonstrated that Triton X-100 exhibited high efficiency regarding nucleus isolation for both adherent (HeLa) and nonadherent (JM) cells, with a recovery ratio of ~80 %. In addition, the isolation efficiency was evaluated by western blotting. The presented flow-through microfluidic cell-nucleus separator may be a useful tool for general biological applications, because of its simplicity in operation, high reproducibility, and accuracy.
authors.affiliation: Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, Chiba University, Inage-ku, Chiba, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Toyama
authors.forename: Masumi
authors.initials: M
authors.lastname: Yamada
authors.forename: Minoru
authors.initials: M
authors.lastname: Seki
mesh_terms: D002458:Cell Fractionation / Q000295:instrumentation*; D002467:Cell Nucleus / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D017830:Octoxynol / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D013997:Time Factors
chemical_list: D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D017830:Octoxynol