A scalable label-free approach to separate human pluripotent cells from differentiated derivatives.

The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the device's utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 10(6)-10(7) cells per min and up to 50% removal of specific cell types per single pass. The level of enrichment and depletion of soft, pluripotent hESC in the respective channels was found to rise with increasing stiffness of the differentiating cells, suggesting CEM can serve as a major discriminator. Our results demonstrate the principle of a scalable, label-free, solution for separation of heterogeneous cell populations deriving from human pluripotent stem cells.

Experimental measurement of particle size distribution and voidage in an expanded bed adsorption system.

This paper presents an experimental analysis of matrix bead size distribution and voidage variations with axial height in an expanded bed adsorption system. Use of a specially constructed expanded bed with side ports has enabled sampling from within the expanded bed along the vertical axis. Particles removed from within the bed were measured for their size distributions. Residence time distribution studies were used to estimate bed voidage. Measurements of axial and radial particle size distributions and axial voidage distribution have been made at different flow rates. Particle size was found to be radially constant, indicating constant stratification in the column. The particle size was found to decrease with increasing axial height. Voidage increased with axial height from a settled bed value of 0.39 to approaching unity for high liquid velocities and increased at a constant axial position with increased flowrate. This information provides key insight into bed stability and data for the improved modeling of this important unit operation.

Immobilised metal ion affinity chromatography purification of alcohol dehydrogenase from baker's yeast using an expanded bed adsorption system.

Alcohol dehydrogenase (ADH) from solutions of homogenised packed bakers' yeast has been successfully purified using immobilised metal-ion affinity chromatography in an expanded bed. Method scouting carried out using pure ADH solutions loaded onto 5-ml HiTrap columns charged with Zn2+, Ni2+ and Cu2+ and eluted using 0-50 mM EDTA gradient found that charging with Zn2+ gave the highest recovery and the lowest EDTA concentration required for elution. These results were used to develop a protocol for the expanded bed system and further tested using clarified yeast homogenate loaded onto XK16/20 packed beds (approximately 30 ml) packed with Chelating Sepharose FastFlow matrix in order to determine the optimum elution conditions using EDTA. The ADH was found to elute at 5 mM EDTA and the dynamic and total binding capacities of Streamline chelating for ADH were found to be 235 U/ml and 1075 U/ml matrix, respectively. Expanded bed work based on a step EDTA elution protocol demonstrated that ADH could be successfully eluted from unclarified homogenised bakers' yeast diluted to 10 mg/ml total protein content with a recovery of 80-100% that was maintained over five consecutive runs with a vigorous clean-in-place procedure between each run.