Analysis of cellular water content in T cells reveals a switch from slow metabolic water gain to rapid water influx prior to cell division.

Cell growth is driven by the acquisition and synthesis of both dry biomass and water mass. In this study, we examine the increase of water mass in T cell during cell growth. We found that T-cell growth is characterized by an initial phase of slow increase in cellular water, followed by a second phase of rapid increase in water content. To study the origin of the water gain, we developed a novel methodology we call cold aqua trap-isotope ratio mass spectrometry, which allows analysis of the isotope composition of intracellular water. Applying cold aqua trap-isotope ratio mass spectrometry, we discovered that glycolysis-coupled metabolism of water accounts on average for 11 fl out of the 20 fl of water gained per cell during the initial slow phase. In addition, we show that at the end of the rapid phase before initiation of cell division, a water influx occurs, increasing the cellular water mass by threefold. Thus, we conclude that activated T cells switch from metabolizing water to rapidly taking up water from the extracellular medium prior to cell division. Our work provides a method to analyze cell water content as well as insights into the ways cells regulate their water mass.