In isotonic conditions, approximately 70% of the cell’s volume is water. However, it would be wrong to think that the intracellular solutes and macromolecules bathe in a dilute solution. It has long been known that most, if not all, of the intracellular water exhibits physical properties unlike those in the bulk. This is attributed to the presence of high concentrations of proteins (200-300 g/l), ions, amino acids, fatty acids, sugars and other small solutes in the cytoplasm enmeshed in a network of cytoskeletal macromolecules (actin filaments, microtubules and intermediate filaments). In individual organelles (such as mitochondria) the protein concentration may be even higher. Within the cytoplasm, at any given time, water molecules are either a part of a tight cluster (bulk water) or in the close vicinity (vicinal water) of a surface (cell or organelle membrane) or a solute (a macromolecule, ion or amino acid). There is not a consensus in the literature on the relative populations of vicinal and bulk water within the cytosol. Crowding inside a cell influences the translational diffusivity of molecules in the cytosol as a function of their size (Figure 6). Translational diffusivity, on the other hand, determines the rates of diffusion-limited biochemical reactions and therefore the activity of the organism. The goal of biopreservation is to reversibly arrest the biological activity by slowing down the biochemical reactions (and by slowing down diffusion). We are therefore interested in devising methods to quantify the molecular mobility in live mammalian cells under various conditions of osmotic stress, environmental conditions (such as temperature, pressure), different biopreservation solutions and various preservation processes (such as cryopreservation, lyopilization and desiccation).

During freezing/thawing or desiccation/re-hydration, the water content inside a cell changes significantly, depending on the rate of cooling/warming and desiccation/re-hydration imposed. The time-scales of biochemical reactions and the preservation conditions applied to the organism play crucial roles in determining the success of preservation. For example, the ratio of the time-scale of water diffusion, tD, across the cell membrane where r, Lp and are the cell radius, membrane permeability and osmotic pressure differential, respectively) to the time scale of cooling the cell experiences, where cp2, p1, q” and are the specific heat, mass density, heat flux and temperature differential, respectively) determines the fate of a cell during freezing such that (Figure 7):

causes excessive dehydration of the cell,

establishes an intra/extracellular equilibrium such that the intracellular water transported across the membrane balances the extracellular osmotic increase induced by freezing (the solute-concentration effect) minimizing the amount of intracellular free water, results in rapid cooling (faster than the cell can reach equilibrium with its surroundings) inducing Intracellular Ice Formation (IIF) known to be lethal to most cells, and theoretically, yields to ultra-fast cooling without ice crystallization (if as an additional constraint where, is the time-scale of structural relaxations) enabling vitrification of the extracellular medium, and more importantly the cytosol.

In order to determine the cytosolic mobility change during desiccation in the presence of membrane permeable biopreservation solutions, we are utilizing microfluidic systems that enable us to do real-time measurements inside living mammalian cell exposed to changing environmental conditions. In the movies below (Figure 8), an H35 hepatoma cell placed in a microchannel is being desiccated in a Ci=30% w/w glycerol solution while the cytosolic viscosity increase is being measured using a fluorescent dye. Using the microfluidic device designed specifically for this research, the variation of intracellular mobility could be determined in real-time as a function of the desiccation protocol utilized (to be published soon).