A DIALYSIS-WASHING SYSTEM FOR REMOVAL OF CRYOPROTECTIVE AGENT FROM CRYOPRESERVED HUMAN RED BLOOD CELLS

摘要

In cryopreservation of human red blood cells (RBCs), a cryoprotective agent glycerol (40% w/v) must be used to prevent cells from cryoinjury. However, the cryoprotectant needs to be removed from the cells after thawing because of the toxicity of glycerol to the cells at room or body temperatures. Further more,when the cells are transfused into a patient’s body if glycerol is not removed, the high osmolality (5000+mOsm) will cause great osmotic shock to the cells in the body. Currently, glycerol is removed using a procedure involving centrifugation, which introduces mechanical forces and osmotic stress causing cell packing/clumping and significant cell loss. Besides, the currently used method is an open system, which may cause bacteria contamination. To prevent cell clumping, cell loss, and osmotic injury, a novel diffusion-washing device (without centrifugation) is developed to remove glycerol. Similar to hemodialysis process, the perfusion-washing system is composed of a mini-module dialyzer, blood side tubings and dialysate side tubings. The mechanism is that, the cryopreserved cell suspension, red blood cells with glycerol run through hollow, porous membrane fibers inside the cylindrical jacket (blood side) of the mini-module dialyzer. Isotonic medium is pumped into the dialyzer and run in the opposite direction in the gaps among the hollow fibers (dialysate side). Using this counter-flow dialysis approach, glycerol diffuses from cell suspension into the perfusion medium across the fiber membranes, while the RBCs can not go across the membrane because of its larger size. The washed cell suspension is re-circulated by automated pumps and runs through the mini-module dialyzer continuously (closed system) until almost all of the glycerol is removed from the cell suspension (Osmolality less than 420mOsm). The amount of the residual glycerol is measured using a freezing point osmometer. Results show that the dialysis-washing method can remove the cryoprotective agent in about 30 minutes. The count recovery and hemoglobin recovery of the RBCs is about 90% and 85%.