title: An agar gel membrane-PDMS hybrid microfluidic device for long term single cell dynamic study.
abstract: Significance of single cell measurements stems from the substantial temporal fluctuations and cell-cell variability possessed by individual cells. A major difficulty in monitoring surface non-adherent cells such as bacteria and yeast is that these cells tend to aggregate into clumps during growth, obstructing the tracking or identification of single-cells over long time periods. Here, we developed a microfluidic platform for long term single-cell tracking and cultivation with continuous media refreshing and dynamic chemical perturbation capability. The design highlights a simple device-assembly process between PDMS microchannel and agar membrane through conformal contact, and can be easily adapted by microbiologists for their routine laboratory use. The device confines cell growth in monolayer between an agar membrane and a glass surface. Efficient nutrient diffusion through the membrane and reliable temperature maintenance provide optimal growth condition for the cells, which exhibited fast exponential growth and constant distribution of cell sizes. More than 24 h of single-cell tracking was demonstrated on a transcription-metabolism integrated synthetic biological model, the gene-metabolic oscillator. Single cell morphology study under alcohol toxicity allowed us to discover and characterize cell filamentation exhibited by different E. coli isobutanol tolerant strains. We believe this novel device will bring new capabilities to quantitative microbiology, providing a versatile platform for single cell dynamic studies.
authors.affiliation: Department of Mechanical and Aerospace Engineering, University of California, Los Angeles, CA 90095, USA.
authors.forename: Ieong
authors.initials: I
authors.lastname: Wong
authors.forename: Shota
authors.initials: S
authors.lastname: Atsumi
authors.forename: Wei-Chih
authors.initials: WC
authors.lastname: Huang
authors.forename: Tung-Yun
authors.initials: TY
authors.lastname: Wu
authors.forename: Taizo
authors.initials: T
authors.lastname: Hanai
authors.forename: Miu-Ling
authors.initials: ML
authors.lastname: Lam
authors.forename: Ping
authors.initials: P
authors.lastname: Tang
authors.forename: Jian
authors.initials: J
authors.lastname: Yang
authors.forename: James C
authors.initials: JC
authors.lastname: Liao
authors.forename: Chih-Ming
authors.initials: CM
authors.lastname: Ho
mesh_terms: D000362:Agar / Q000737:chemistry*; D018929:Cell Culture Techniques / Q000295:instrumentation*; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation*; D005782:Gels / Q000737:chemistry; D008567:Membranes, Artificial; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D009757:Nylons / Q000737:chemistry*
chemical_list: D004129:Dimethylpolysiloxanes; D005782:Gels; D008567:Membranes, Artificial; D009757:Nylons; C085707:poly(dimethylsiloxane)-polyamide copolymer; D000362:Agar