Lincoln C. Young

A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis

We have developed a prototype three-channel microfluidic chip that is capable of generating a linear concentration gradient within a microfluidic channel and is useful in the study of bacterial chemotaxis. The linear chemical gradient is established by diffusing a chemical through a porous membrane located in the side wall of the channel and can be established without through-flow in the channel where cells reside. As a result, movement of the cells in the center channel is caused solely by the cells chemotactic response and not by variations in fluid flow. The advantages of this microfluidic chemical linear gradient generator are (i) its ability to produce a static chemical gradient, (ii) its rapid implementation, and (iii) its potential for highly parallel sample processing. Using this device, wildtype Escherichia coli strain RP437 was observed to move towards an attractant (e.g., l-asparate) and away from a repellent (e.g., glycerol) while derivatives of RP437 that were incapable of motility or chemotaxis showed no bias of the bacterias distribution. Additionally, the degree of chemotaxis could be easily quantified using this assay in conjunction with fluorescence imaging techniques, allowing for estimation of the chemotactic partition coefficient (CPC) and the chemotactic migration coefficient (CMC). Finally, using this approach we demonstrate that E. coli deficient in autoinducer-2-mediated quorum sensing respond to the chemoattractant l-aspartate in a manner that is indistinguishable from wildtype cells suggesting that chemotaxis is insulated from this mode of cell-cell communication.

Batch, fed‐batch, and microcarrier cultures with CHO cell lines in a pressure‐cycle driven miniaturized bioreactor

Miniaturized bioreactors for suspension cultures of animal cells, such as Chinese Hamster Ovary (CHO) cells, could improve bioprocess development through the ability to cheaply explore a wide range of bioprocess operating conditions. A miniaturized pressure‐cycled bioreactor for animal cell cultures, described previously (Diao et al., 2008 ), was tested with a suspension CHO cell line producing commercially relevant quantities of human IgG. Results from the suspended CHO cell line showed that the cell growth was comparable to conventional flask controls and the target protein production was enhanced in the minibioreactor, which may be due to the relatively high oxygen transfer rate and the moderate shear stress, measured and simulated previously. Microcarrier culture using an anchorage‐dependent CHO cell line and Cytodex 3 also showed a similar result: comparable growth and enhanced production of a model protein (secreted alkaline phosphatase or SEAP). Various fed‐batch schemes were applied to the CHO cells producing human IgG, yielding cell numbers (1.1 × 107/mL) at day 8 and titers of human IgG (2.3 g/L) at day 14 that are typical industrial values for CHO cell fed‐batch cultures. The alteration of the volumetric oxygen transfer coefficient is a key parameter for viability of the CHO cell line producing human IgG. We conclude that the minibioreactor can provide favorable cell culture environments; oxygen transfer coefficient and mixing time can be altered to mimic values in a larger scale system allowing for potential prediction of response during scale‐up. Biotechnol. Bioeng. 2012;109: 137–145.

Determination of Genotypes Using a Fully Automated Molecular Detection System

CONTEXT Although the value of pharmacogenomics to improve patient outcomes has become increasingly clear, adoption in medical practice has been slow, which can be attributed to several factors, including complicated and expensive testing procedures and required equipment, lack of training by private practice physicians, and reluctance of both private and commercial payers to reimburse for such testing. OBJECTIVES To evaluate a fully automated molecular detection system for human genotyping assays, starting with anticoagulated whole blood samples, and to perform all sample preparation, assay, and analysis steps automatically with actionable results reported by the systems software. DESIGN The genotypes of 254 random individuals were determined by performing bidirectional DNA sequencing, and that information was used to statistically train the imaging software of the automated molecular detection system to distinguish the 3 possible genotypes (ie, homozygous wild type, heterozygous, and homozygous mutant) at each of 3 different loci (CYP2C9*2, CYP2C9*3, and VKORC1). RESULTS The resulting software algorithm was able to correctly identify the genotypes of all 254 individuals (100%) evaluated without any further user analysis. CONCLUSIONS The EncompassMDx workstation (Rheonix, Inc, Ithaca, New York) is a molecular detection system that can automatically determine the genotypes of individuals in an unattended manner. Considerably less technical expertise was required to achieve results identical to those obtained using more complex, time-consuming, and expensive bidirectional DNA sequencing. This optimized system may dramatically simplify and reduce the costs of pharmacogenomics testing, thus leading to more-widespread use.