Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential.

Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

Continuous and quantitative delivery of molecules into individual cells with a diffusional microburet.

Direct delivery of molecules into the cytosol of live cells is required in many areas of biology and clinical research. Molecules of interest include indicator dyes, biomolecules, and pharmacological agents. In this work we describe continuous delivery of molecules into single cells using a diffusional microburet, DMB. The DMB is a pulled glass micropipette with a fine tip that contains a microscopic plug made of a hydrogel such as agar or polyacrylamide. This plug prevents flow but allows diffusive delivery of the molecule of interest from the DMB body into the cytosol, driven by its concentration gradient. This leads to a scheme of sustained intracellular dosing that is highly reproducible and quantifiable yet does not require the addition of solution volume to the cell. Potential loss of biomolecules from the cytosol through the plug of the DMB can be greatly reduced by proper choice of the pore size and tortuosity of the hydrogel in the DMB tip. The intracellular concentration of fluorescent molecules during delivery can be obtained calibration free. In this work we demonstrate dosing of Lucifer Yellow CH, LY, a charged fluorescent dye, into individual a7r5 vascular smooth muscle cells with a DMB. New types of quantitative analytical experiments on single live cells that the DMB technology enables are titration of intracellular ions and ligands, binding sites, and efflux pathways such as those that are involved in drug resistance.