Microfluidic Device for Washing Frozen/Thawed Erythrocytes

Over 15 million units of erythrocytes (red blood cells) are transfused every year in the United States. Cryopreservation of erythrocytes would help prevent shortages and provide a larger bank of rare blood types to draw from for transfusion. Unfortunately, cryopreservation is accompanied by the time consuming process of deglycerolizing erythrocytes. Glycerol is a common cryoprotectant used to preserve erythrocytes as they are frozen. Its ability to permeate cell membranes allows protection from osmotic damage that plagues cells as they are being cryopreserved. Unfortunately, glycerol is toxic to cells after an extended exposure, so it must be removed after thawing of the cells. This is traditionally done in batch process in which it takes and hour or more to deglycerolize a single unit of blood. With a three-step process, the deglycerolization time has been shown to be reduced to around three minutes. In the three-step process, a 12% saline solution is introduced to the erythrocytes, which draws the glycerol from the cells through osmotic pressures. A 3.4% glycerol solution is then used to dilute the 12% saline solution and finally a 0.2% glycerol solution is used to return the cells to isotonic conditions. The success of the deglycerolization process is dependent upon the amount of time the erythrocytes spend in the 12% saline solution. Too short a time spent in the 12% saline solution, and when the 3.4% saline solution is introduced, water enters the erythrocytes rapidly and they lyse; too long spent in the 12% saline solution, the cells shrivel and die. This three-step batch process, however, requires a large volume of solutions. A microfluidic device, such as the one used in this project, decreases the volume of saline solutions needed to preform the three-step deglycerolization process and is able to run the process continually. The residence time within the device corresponds to the amount of time the erythrocytes spend in the 12% saline solution, and 3.4% saline is added within the device to dilute the 12% saline. The 0.2% glycerol solution is added manually as the last step of the three-step process. The goal of the project is to reinforce the idea that rapid deglycerolization is possible with low hemolysis and that the microfluidic device can preform the same function as the batch process. Ultimately, as residence time that allows for under a 20% hemolysis to match the data found in the previous study is to be consistently achieved. So far, the general trend of the previous study’s hemolysis data has been replicated using the microfluidic device, but a hemolysis value under 20% has not been achieved. Working towards an ultimate goal of a 20% or less hemolysis value, more residence times must be evaluated and repeated trials to test consistency should be performed.