An automated system for high-throughput single cell-based breeding.

When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.

Hybrid dynamic coating with n-dodecyl beta-D-maltoside and methyl cellulose for high-performance carbohydrate analysis on poly(methyl methacrylate) chips.

Hybrid dynamic coating using n-dodecyl beta-d-maltoside (DDM) and methyl cellulose (MC) has been developed for suppression of analyte adsorption and electroosmotic flow (EOF) in a poly(methyl methacrylate) (PMMA) channel. The adsorption of APTS-labeled sugars in a PMMA channel was obviously suppressed with DDM dynamic coating; however, EOF was reduced only by a factor of approximately 25%, resulting in irreproducible separations. In contrast, both analyte adsorption and EOF in a PMMA channel were efficiently minimized with MC coating; however, concentrated MC above 0.3% was required to achieve high-performance separations, which greatly increased viscosity of the solution and caused difficulties during buffer loading and rinsing. In addition, n-dodecyltrimethylammonium chloride did not show observable effects on reducing analyte adsorption, although it has the same hydrophobic alkyl chain as DDM. These results strongly indicated that the polysaccharide moiety of surface modifiers has a specific affinity to surface charges and is crucial to achieving efficient and stable dynamic coating on the PMMA surface. Hybrid dynamic coating with 0.25% DDM and 0.03% MC was found to minimize both analyte adsorption and EOF in a PMMA channel to a negligible level, while still keeping a low viscosity of the solution. High-speed and high-throughput profiling of the N-linked glycans derived from alpha1-acid glycoprotein, fetuin, and ribonuclease B was demonstrated in both single-channel and 10-channel PMMA chips using DDM-MC hybrid coating. We propose that DDM-MC hybrid coating might be a general method for suppressing analyte adsorption and EOF in polymer MCE devices. The current MCE-based method might be a promising alternative for high-throughput screening of carbohydrate alterations in glycoproteins.

High-performance genetic analysis on microfabricated capillary array electrophoresis plastic chips fabricated by injection molding.

We have developed a novel technique for mass production of microfabricated capillary array electrophoresis (mu-CAE) plastic chips for high-speed, high-throughput genetic analysis. The mu-CAE chips, containing 10 individual separation channels of 50-microm width, 50-microm depth, and a 100-microm lane-to-lane spacing at the detection region and a sacrificial channel network, were fabricated on a poly(methyl methacrylate) substrate by injection molding and then bonded manually using a pressure-sensitive sealing tape within several seconds at room temperature. The conditions for injection molding and bonding were carefully characterized to yield mu-CAE chips with well-defined channel and injection structures. A CCD camera equipped with an image intensifier was used to monitor simultaneously the separation in a 10-channel array with laser-induced fluorescence detection. High-performance electrophoretic separations of phiX174 HaeIII DNA restriction fragments and PCR products related to the human beta-globin gene and SP-B gene (the surfactant protein B) have been demonstrated on mu-CAE plastic chips using a methylcellulose sieving matrix in individual channels. The current work demonstrated greatly simplified the fabrication process as well as a detection scheme for mu-CAE chips and will bring the low-cost mass production and application of mu-CAE plastic chips for genetic analysis.