120 Deglycerolization and separation of human red blood cells in a microfluidic focusing channel

In current practice, removal of glycerol from frozen-thawed red blood cells (RBCs) is completed through a batch process that requires approximately 45 min per unit of blood. This process demands that the need for frozen RBCs be anticipated, and any unused deglycerolized units of blood be disposed of after a short post-thaw shelf life. Previous research in our lab demonstrated that rapid glycerol removal is possible using a multistep process involving exposure to solutions with decreasing concentrations of non-permeating solutes. This process maintains RBCs within their osmotic tolerance limits to minimize cell damage while enhancing the speed of glycerol removal. The ultimate goal is to perform the aforementioned rapid deglycerolization procedure in a continuous flow microfluidic device. The current research focuses on the first step of a proposed three-step deglycerolization process to demonstrate the ability to remove glycerol from a 40% w/v glycerolized human RBCs in a microfocusing device and to separate wash solution from cells at the outlet. The glycerolized cell stream is flowed between two 3270 mOsm saline wash streams allowing for a high contact area between the wash and cells for rapid diffusion of glycerol. The device separates RBCs from the wash solution at the exit of the device by taking advantage of the Fahraeus–Lindqvist effect in which blood cells tend to occupy the center of a microchannel creating a cell-free boundary layer. The device is able to skim off the cell-free layer with minimal collection of RBCs, creating an all in one passive deglycerolization device. In order to facilitate rapid prototyping and allow for cleaning and sterilization between runs, a device was fabricated by compressing a polyimide shim between two pieces of 21 cm× 5 cm× 0.95 cm acrylic with CNC milled inlet holes and through holes for compression screws. The polyimide shim was micromachined with a 200 μm W × 127 μm H × 14 cm L main channel from a CAD drawing, using a UV laser cutting tool. The optically transparent device allows for visualization of the flow for the entire length of the microchannel via microscopy and high speed imaging. Blood and wash solutions are flowed through the device and collected from their respective outlets for analysis. Syringe pumps feed the device with 3:1 ratio of wash to glycerolized blood. At the outlet, approximately 92% of the red blood cells exited the device in the expected blood collection channel, with the remaining 8% leaving with the wash streams. The wash stream represented 46% of the total volume out of the device. The difference in glycerol mass percent between the deglycerolized blood and wash output was approximately 30%, indicating substantial mass transfer into the wash solution during the short (∼12 s) transit time through the device. Further work is being done to improve the separation of wash solution from deglycerolized cells with additional separation branches as well as running multiple devices in series to carry out a complete multistep RBC deglycerolization process. Source of funding: National Science Foundation Grant#1150861. Conflict of interest: None declared. adam.higgins@oregonstate.edu