We report a fully defined synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), which sustains long-term human embryonic stem (hES) cell growth in several different culture media, including commercially available defined media. The development of a standardized, controllable and sustainable culture matrix for hES cells is an essential step in elucidating mechanisms that control hES cell behavior and in optimizing conditions for biomedical applications of hES cells.
This paper describes a self-contained integrated microfluidic system that can separate motile sperm from small samples that are difficult to handle using conventional sperm-sorting techniques. The device isolates motile sperm from nonmotile sperm and other cellular debris, based on the ability of motile sperm to cross streamlines in a laminar fluid stream. The device is small, simple, and disposable yet is an integrated system complete with sample inlets, outlets, sorting channel, and a novel passively driven pumping system that provides a steady flow of liquid; it requires no external power source or controls. The device fulfills a need in clinical settings where small amounts of sperm need to be sorted. It also opens the way for convenient bioassays based on sperm motility including at-home motile sperm tests.
Immotile bovine caput epididymal sperm contain levels of protein phosphatase activity twofold higher than do mature motile caudal sperm. Comparison of the inhibition profiles of endogenous phosphatase activities detected by okadaic acid (OA) and calyculin A (CA) revealed a pattern consistent with the predominance of a type 1 protein phosphatase (PP1). Immunoblot analysis identified PP1 gamma 2 (the testis-specific isoform of PP1) as the only PP1 isoform in sperm and showed little protein phosphatase 2A (PP2A). In addition, of the known PP1 inhibitors, i.e., DARPP-32, inhibitor 1 (I1), and inhibitor 2 (I2), only I2-like activity was detected in sperm. Inhibition of PP1 by the heat-stable I2-like activity purified from sperm could be reversed with purified glycogen synthase kinase-3 (GSK-3). Furthermore, sperm extracts contain an inactive complex of PP1 and I2 (termed PP1I) that could also be activated by purified GSK-3. The presence of GSK-3 in sperm was demonstrated by activation of purified PP1I, and quantitation revealed that immotile caput sperm contained sixfold higher GSK-3 activity than motile caudal sperm. Immunoblot analysis confirmed the expression of GSK-3 in sperm and revealed the occurrence of both the alpha and beta isoforms. Our findings suggest that the higher PP1 activity measured in immotile sperm, presumably due to higher GSK-3 activity, is responsible for holding motility in check. This conclusion was supported by the observation that the phosphatase inhibitors OA and CA, at micromolar and nanomolar levels, respectively, were able to induce motility in completely immotile bovine caput epididymal sperm and to stimulate the kinetic activity of mature caudal sperm. The intrasperm levels of cAMP, pH, and calcium were unaltered by treatment with these inhibitors. The results suggest a biochemical basis for the development and regulation of sperm motility and a possible physiological role for the PP1/I2/GSK-3 system.
Evaporation is a critical problem when handling submicroliter volumes of fluids. This paper characterizes this problem as it applies to microfluidic cell culture in poly(dimethylsiloxane) (PDMS) devices and provides a practical solution. Evaporation-mediated osmolality shifts through PDMS membranes with varying thicknesses (10, 1, 0.2, or 0.1 mm) were measured over 96 h. Even in humidified cell culture incubators, evaporation through PDMS and associated shifts in the osmolality of culture media was significant and prevented mouse embryo and human endothelial cell growth and development. A simple diffusion model, where the measured diffusion coefficient for PDMS matches reported values of approximately 10-9 m2/s, accounts for these evaporation and osmolality shifts. To overcome this problem, a PDMS-parylene-PDMS hybrid membrane was developed that greatly suppresses evaporation and osmolality shifts, yet possesses thinness and the flexibility necessary to interface with deformation-based microfluidic actuation systems, maintains the clarity for optical microscopy, and enables the successful development of single-cell mouse embryos into blastocysts under static conditions and culture of human endothelial cells under dynamic recirculation of submicroliter volumes of media. These insights and methods demonstrated specifically for embryo and endothelial cell studies will be generally useful for understanding and overcoming evaporation-associated effects in microfluidic cell cultures.
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