Platelet cryopreservation using a trehalose and phosphate formulation

Abstract

Long-term storage of platelets is infeasible due to platelet activation at low temperatures. In an effort to address this problem, we evaluated the effectiveness of a formulation combining trehalose and phosphate in protecting platelet structure and function following cryopreservation. An annexin V binding assay was used to quantify the efficacy of the trehalose and phosphate formulation in suppressing platelet activation during cryopreservation. Of the platelets cryopreserved with the trehalose plus phosphate formulation, 23% +/- 1.2% were nonactivated, compared with 9.8% +/- 0.26% nonactivated following cryopreservation with only trehalose. The presence of both trehalose and phosphate in the cryopreservation medium is critical for cell survival and preincubation in trehalose plus phosphate solutions further enhances viability. The effectiveness of trehalose plus phosphate in preserving platelets in a nonactivated state is comparable to 6% dimethyl sulfoxide (Me(2)SO). Measurements of platelet metabolic activity using an alamarBlue assay also established that trehalose plus phosphate is superior to trehalose alone. Finally, platelets protected by the trehalose plus phosphate formulation exhibit similar aggregation response upon thrombin addition as fresh platelets, but an increase of cytosolic calcium concentration upon thrombin addition was not observed in the cryopreserved platelets. These results suggest that trehalose and phosphate protect several aspects of platelet structure and function during cryopreservation, including an intact plasma membrane, metabolic activity, and aggregation in response to thrombin, but not intracellular calcium release in response to thrombin.

Cryopreservation of primary human hepatocytes: The benefit of trehalose as an additional cryoprotective agent

“…This was not only confirmed by the trypan blue test: the same results were achieved by measurement of plating efficiency and total protein and in a cell proliferation assay 26. In the few available publications, the viability of the other types of cells was also enhanced after thawing, if trehalose was used as cryoprotective additive 13, 27, 28. In cryopreservation experiments, the function of the cells is often assayed only during the short culture period of a few days or even just in suspension directly after thawing 4, 11, 26.…”

Trehalose: Current Use and Future Applications

“…of liposomes 64 and platelets. 65 In all of these applications, the ionic species are thought to increase the strength of the interactions present within the glassy matrix, through enhanced hydrogen bonding network, and demonstrated increased T g . In another application, the addition of glucose at 1:10 weight ratio improved the storage stability of glucose-6-phosphate dehydrogenase 66 ; glucose incorporation was thought to lower the free volume by filling in the void in the trehalose-protein glass, thus reducing molecular mobility, and consequently, improved stability.…”

Bacterial Infections: Bacterial Contamination, Testing, and Post-Transfusion Complications

“…Time and temperature of storage may influence bacterial growth, extent of phagocytosis, and the occurrence of complement-mediated killing. 128 Investigators have also found that the proliferation of Y. enterocolitica in RBCs may be decreased by storage at 0°C. [124][125][126] Lowering Component Storage Temperature Platelet storage at room temperature maintains platelet hemostatic efficacy, but this temperature provides a hospitable culture medium for many bacteria.…”