113 Continuous removal of glycerol from frozen-thawed blood in a microscale membrane device

The technique of freezing red blood cells (RBCs) in the presence of 40% w/v glycerol extends the shelf life to 10 years as opposed to only 6 weeks for conventional refrigerated blood. Despite the clear logistical advantages, frozen blood is not preferred in clinical therapy because of the prohibitively time consuming post-thaw process needed to remove the glycerol. A rapid glycerol removal method is necessary to facilitate more widespread use of frozen blood. In a previous study, we used mathematical predictions of cell membrane transport to guide the design of a procedure that varies the extracellular solution composition with time to provide the necessary concentration driving force for rapid glycerol removal while keeping the cell volume within osmotic tolerance limits to minimize cell damage. This batch procedure removed the intracellular glycerol in approximately three minutes with a hemolysis of less than 20%. In this study, the feasibility of adapting this batch procedure for use in a continuous device is examined. The current device consists of a membrane sandwiched between two PEEK sheets with a microchannel (15 cm long, 200–400 mm wide, 100 mm deep) etched into each sheet. Pins fashioned in the PEEK sheets facilitate proper alignment when the device is assembled, enabling mass transfer through the membrane. In this study, the membrane used was the AN69 membrane, a biocompatible copolymer membrane commonly used for hemodialysis. In the first experiment, fresh human RBCs glycerolized to 10% w/v were flowed counter-current to isotonic saline. The solution concentration in this experiment was specifically chosen because it is expected to yield gradual mass transfer without causing excessive osmotic damage. This experiment resulted in a glycerol mass composition reduction up to 50% for a single pass, with a low hemolysis average of about 3.4% for all flow rates tested. In the second experiment, 40% w/v glycerolized frozen-thawed RBCs were flowed counter-current to 40% w/v glycerol solution to assess the fragility of frozen-thawed cells. Because the osmolality of both streams are equal, no mass transfer will occur and any damage is expected to only be due to mechanical stresses. In this experiment, the level of hemolysis only increased by 2% due to mechanical stresses incurred during transit through the device. In the third experiment, 40% w/v glycerolized frozen-thawed RBCs were flowed counter-current to 3.4% w/v saline. This experiment resulted in a glycerol mass composition reduction of 55.0 ± 0.3% and hemolysis of 15.1 ±0.7% for the slowest flow rate tested, which is comparable to the removal achieved in the first step of the batch procedure. The ability to vary the flow rate and the channel dimensions, as well as the solution used as a dialysate enable us to control the mass transfer inside the channel. Furthermore, the ability to link multiple channels in series allows us to potentially match the concentration changes from the batch procedure in a continuous application. Ultimately, this method has the potential for rapid continuous removal of glycerol, which will make frozen blood a more attractive option for blood banking. Source of funding: National Science Foundation Grant #1150861. Conflict of interest: None declared. adam.higgins@oregonstate.edu