(142bh) Modeling of Cryoprotectant Transport Across Cell Membranes in a Micromixer

Cryopreservation of reproductive cells offers scientists a cost-effective alternative to maintaining a live strain of a genetically modified species.  Prior to cryopreservation, it is desirable to remove intracellular water to prevent ice crystal formation.  This is done by loading a cryoprotectant, such as glycerol, into the cell.  Microfluidic devices present a useful platform for this process because of their high-throughput nature, reproducible results, and small requisite sample volumes.  Despite these advantages, there have been very few studies investigating the use of a microfluidic device for such purposes.

In the present study, we numerically model the transport phenomena of glycerol across a zebrafish sperm cell membrane in a microchannel: namely, the cells and cryoprotectant moving throughout the micromixer, and the cryoprotectant transport across the sperm cell membrane.  The former is modeled using the finite volume method (in the computational fluid dynamics solver FLUENT) along with the discrete particle method to solve the three-dimensional Navier-Stokes equations, species continuity equation, and Newton’s Second Law.  The latter is simulated in MATLAB using the previous simulations’ data as inputs to the Kedem-Katchalsky equations for solute transport across a membrane.  Our simulations idealize sperm cells as spherical particles, and we use the fluid mixture of glycerol and water.  In experimental studies, data is usually collected at the end of the cryoprotectant loading process; the use of numerical methods allows us to simultaneously probe every cell as it moves throughout the micromixer.

Throughout the simulations, we focus on each cell’s internal cryoprotectant concentration and the cell’s size.  As the magnitude of cryoprotectant influx is different from the magnitude of water efflux, the cell’s size will change as a result (large enough changes being a cause of cell death during cryopreservation).  We characterize the effect of inlet flow rate, ranging from 0.05 μL/min to 2 μL/min.  Varying inlet flow rates will change the mixing performance of the device, which in turn affects the loading of cryoprotectant.  The two processes occur on different time scales, so if cells do not have a sufficient residence time, they do not reach equilibrium with the micromixer environment.  We also investigate the effect of inlet concentration over the range of 4.5 M glycerol to 9 M glycerol; more concentrated loading requires longer cellular residence times and cellular sizes show varying levels of the “shrink-swell” response as the inlet glycerol concentration changes.  These results lay the foundation for an optimal microfluidic cryopreservation protocol.