Unprecedented cell-selection using ultra-quick freezing combined with aquaporin expression.

Freezing is usually used for preservation and storage of biological samples; however, this process may have some adverse effects such as cell membrane damage. Aquaporin (AQP), a water channel protein, has been suggested to play some roles for cryopreservation although its molecular mechanism remains unclear. Here we show that membrane damage caused by ultra-quick freezing is rescued by the expression of AQP4. We next examine if the expression of AQP combined with ultra-quick freezing can be used to select cells efficiently under freezing conditions where most cells are died. CHO cells stably expressing AQP4 were exclusively selected from mixed cell cultures. Having identified the increased expression of AQP4 during ES cell differentiation into neuro-ectoderm using bioinformatics, we confirmed the improved survival of differentiated ES cells with AQP4 expression. Finally we show that CHO cells transiently transfected with Endothelin receptor A and Aqp4 were also selected and concentrated by multiple cycles of freezing/thawing, which was confirmed with calcium imaging in response to endothelin. Furthermore, we found that the expression of AQP enables a reduction in the amount of cryoprotectants for freezing, thereby decreasing osmotic stress and cellular toxicity. Taken together, we propose that this simple but efficient and safe method may be applicable to the selection of mammalian cells for applications in regenerative medicine as well as cell-based functional assays or drug screening protocols.

Influence of irrigated paddy fields on the fluorescence properties of fluvial dissolved organic matter.

This study investigated seasonal and geographical variation in the fluorescence properties of dissolved organic matter (DOM) in a river system that includes rice paddy fields in its watershed. The river system covers a northern area of the main island of Japan, and between 6 and 45% the watershed area of the tributaries is paddy fields. Water samples were collected monthly from eight tributaries for 1 yr, and the fluorescence properties of DOM were monitored by combined excitation-emission matrix (EEM) and parallel factor analysis. The EEM was statistically decomposed into six fluorescence components with different environmental dynamics. The relative proportion of these components varied with season and geographic location, the former having a greater effect than the latter. Seasonal variation is largely attributed to changes in the source of DOM associated with irrigated rice cultivation practice. The fluorescence component composition showed a stronger autochthonous signature, which varied over a wider range during off-cropping period (October-April) than cropping period (May-September). During the cropping period, a large amount of allochthonous DOM with relatively similar quality was transported into river water from flooded paddy fields and masked the variability in the quality of DOM among tributaries. Therefore, irrigated rice cropping practices are considered to be one of major factors that influence seasonal and geographical variation of fluvial DOM composition.