Development of optimal techniques for cryopreservation of human platelets

摘要

Using the current blood bank storage conditions at 22 deg C, the viability and function of human platelets can be maintained for only 5 days. This does not allow for the necessary and extensive banking of platelets need to treat patients afflicted with thrombocytopenia a side effect of many invasive surgeries such as cardiopulmonary bypass or bone marrow transplantation. The development of optimal techniques for long-term cryopreservation and banking of human platelets would provide the ability to greatly extend the viable life of the platelet and would fulfill an increasing and urgent need in many clinical applications. To determine the optimal techniques for platelet preservation, the expression of an activation marker, phosphatidylserine, on the platelet membrane during storage at 22 and 8 deg C as well as during the different freezing preservation processes was examined using flow cytometry and annexin V binding assay. Human platelets were identified by both CD41 and light scatter in flow cytometry. In crytopresexvation experiments, effects of the following factors on platelet activation were evaluated: (a) cryoprotective agents (CPAs) type: dimethyl sulfoxide (Me_2SO), ethylene glycol (EG), and propylene glycol (PG), (b) CPA concentration ranging from 0 to 3 M, and (c) ending temperatures of a slow cooling process at - 1 deg C/min. Our results demonstrated that (a) approximately 50% of platelets were activated on days 7 and 16 at 22 and 8 deg C, respectively; (b) platelets were not significantly activated after 30-min exposure to 1 M Me_2SO, EG, and PG at 22 deg C, respectively, and (c) there was a significant difference in cryoprotective efficacy among these three CPAs in preventing platelets from cryoinjury. After being cooled to - 10 deg C, 74% of the cryopreserved platelets survived (nonactivated) in 1 M Me_2SO solution, while in 1 MEG and 1 MPG solutions, 62 and 42% of the platelets survived, respectively. Using the information that Me_2SO consistently yields higher percentages of nonactivated platelets and does not seem to be cytotoxic to platelets for 30-min exposure time, this was found to be the optimal cryoprotective agent for platelets. In addition, significant Me_2SO toxicity to platelets was not noted until Me_2SO concentrations exceeded 2 M. Finally, a concentration of I M Me:SO proved to be the most effective at all cryopreservation ending temperatures tested (- 10, -30, -60, and - 196 deg C). In conclusion, under the present experimental conditions, a storage texture of 8 deg C appeared to be much better than 22 deg C. Although the potential chemical toxicity of 1 M Me_2SO, EG, or PG is negligible, 1 M Me_2SO was found to be optimum for cryopreservation of human platelets, lag has the least cryoprotective function for low-temperature platelet survival.