Controlling water-in-water droplets and extracellular environments in microfluidics
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Many important processes in nature happen in water. In living cells, biological fluids, and soft materials, molecules do not always stay evenly mixed. Under certain conditions, one liquid can separate into two liquid phases. This process is called liquid–liquid phase separation. It can also occur in fully aqueous systems, where two water-rich phases form because they contain different polymers or salts. This leads to the formation of water-in-water droplets. Water-in-water droplets provide a gentle and biocompatible environment. Unlike water-in-oil droplets, they do not require an oil phase and are therefore attractive for biological studies. However, they are difficult to control because their interface is very weak. Their size, stability, composition, and surrounding environment are hard to regulate over time. This limits their use in both basic research and biological applications. The aim of this thesis was to develop a simple and reliable microfluidic platform to produce, trap, and control water-in-water droplets. Microfluidics allows liquids to be handled in very small channels with precise control. In this work, water-in-water droplets were formed directly inside dead-end chambers connected to a main channel. The main channel continuously supplied a controllable aqueous environment. As a result, droplets could form in place and remain stable without oil, surfactants, or complex flow control. This design has an important advantage. Because the droplets remain connected to the surrounding aqueous phase, their chemical environment can be changed in real time. For example, polymer concentration, salt concentration, or pH can be adjusted in the main channel and then influence the droplets inside the chambers. The platform was first tested using a PEG/dextran aqueous two-phase system. Droplet size and composition could be controlled in a stable and reproducible way. The same design was then applied to more complex phase-separating systems, including coacervate droplets and droplets with internal sub-compartments. After establishing droplet control, the platform was used for cell studies. Cells were guided into the dead-end chambers by a dextran concentration gradient. This gradient generated a physical driving force that moved cells into the chambers without direct mechanical pushing. By adjusting the gradient, the number of cells in each chamber could be controlled. This allowed reliable confinement of single cells or small cell groups. The method worked for both fission yeast and leukemia cells. Cells trapped in the chambers could then be enclosed inside PEG–dextran water-in-water droplets. This created a controllable three-dimensional extracellular environment around the cells. By changing the polymer concentration outside the droplets, physical properties such as density and viscosity could be tuned. In this way, the position and behavior of cells inside the droplets could also be regulated. The platform therefore combined cell capture, droplet formation, and extracellular-environment control in one system. The final part of the thesis studied how cells respond when the surrounding fluid becomes more viscous. This question is important because many biological environments are thick, crowded, and physically complex. Using fission yeast as a model organism, the platform allowed extracellular fluid viscosity to be changed under controlled conditions. The results showed that extracellular viscosity can strongly affect cell growth and division. Above a certain threshold, cells could no longer divide normally. Below this threshold, cells adapted by increasing the viscosity of their cytoplasm. This internal response was linked to preserved glucose-transporter activity and continued growth. Further experiments showed that glucan and trehalose are important for this adaptation. When their production was disrupted, cells became less tolerant to high extracellular viscosity. This thesis provides a new fully aqueous microfluidic platform for controlling water-in-water droplets and cell microenvironments. It also shows that cells can respond to physical changes outside the cell by tuning their own internal material properties.
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