Rapid, Cost-Efficient, Enzyme-Free Passaging of Human Pluripotent Stem Cells on Feeder Cells by Ethylenediaminetetraacetic Acid-Mediated Dis-Adhesion.

Human pluripotent stem cells (human embryonic stem cells, hESCs, and human induced pluripotent stem cells, hiPSCs) were originally cultured on different types of feeder cells for maintenance in an undifferentiated state in long-term culture. This approach has been supplanted to a large extent by feeder-free culture protocols, but these involve more costly reagents and can promote a transition to a primed state, which restricts the cells' differentiation capacity. In both feeder and feeder-free conditions, the harvesting of hESC or hiPSC colonies for passaging is a necessary procedure for expanding the cultures. To provide an easy and high-yield procedure for passaging hESCs/hiPSCs cultured on feeder cells, we have established a harvesting method using dis-adhesion elicited by the calcium chelator ethylenediaminetetraacetic acid (EDTA). We have assessed the yield and quality of the resultant passaged cells by comparing this approach to the original mechanical harvesting approach, in which colonies are isolated with a scalpel under a microscope (mechanical harvesting was chosen as a comparator to avoid the reagent variability associated with enzymatic harvesting). In one set of experiments, two different hESC lines were maintained on a feeder cell layer of human foreskin fibroblasts. Each line was subjected to multiple passages using EDTA-based or mechanical harvesting and assessed for colony size and morphology, cell density, stemness marker expression, differentiation to the three germ layers in embryoid bodies, and genomic aberrations. In another set of experiments, we used EDTA-based harvesting on two different hiPSC lines and obtained similar results. EDTA-induced dis-adhesion saved time and gave a higher yield of colonies of a more favorable size and more uniform morphology compared to mechanical harvesting. It was also faster than enzymatic harvesting and not prone to enzyme batch variability. The EDTA-induced dis-adhesion method also facilitates the transfer of hESC/hiPSC lines from feeder cell-based culture to feeder-free conditions if desired for downstream use and analysis.

Design and characterization of targeted ultrasound microbubbles for diagnostic use.

Targeted ultrasound (US) contrast agents represent, because of their size (1 to 5 µm), a unique class of diagnostic imaging agents enabling true vascular imaging of conditions like inflammation and tumor angiogenesis. The objective of this study was to develop technology for preparing targeted microbubbles with binding and acoustic properties compatible with diagnostic use. Phosphatidylcholine (PC) was shown to represent the most favorable wall material. Various thiolated peptide binders were effectively conjugated to PC-based microbubbles containing maleimide functionalized lipids (95:5) without the need for biotin-streptavidin or antibody technology. By optimizing the technology, specific targeting of the inflammatory target E-selectin and the angiogenic target VEGFR2 in the presence of 100% serum was achieved. Increased phospholipid chain length from 18 carbons to 22 carbons improved the stability of the microbubbles during US exposure, without compromising binding or acoustic properties.