Progress 07/01/02 to 06/30/05
Outputs
These DARPA funded studies were designed to develop a method to freeze-dried human red blood cells for long term storage. Human red blood cells (RBCs) were diluted in physiological saline to a hematocrit of between five and fifteen percent. These dilute RBCs were then incubated with liposomes, and the mixture subsequently incubated with high concentrations of trehalose (up to five hundred millimolar trehalose). Trehalose loaded cells were then washed and freeze-dried in a mixture of trehalose and albumin (five percent). Freeze-dried RBCs were rehydrated either in buffer or in a mixture of buffer and liposomes. Less than 15 percent of the total cells survived under either condition. The surviving RBCs had increased catalase and glutathione peroxidase activity suggesting that there had been oxidative damage to the cells. Freeze-dried rehydrated cells were extremely fragile and could only be washed in buffer if fifty percent polyethylene glycol (PEG) was present. PEG
stabilized RBCs could be stored for several days in the cold.
Impacts
Freeze-dried red blood cells will improve blood bank stocks in case of emergencies. They will also provide critically needed blood on the battlefield.
Publications
- Satpathy, G.R., Torok,Z., Bali, R., Dwyer, D.M., Little, E., Walker, N.J., Tablin, F., Crowe, J.H., and N.M. Tsvetkova. Loading red blood cells with trehalose: a step towards biostabilization. Cryobiology 2004 49:123-136.
- Torok, Z., Satpathy, G.R., Banerjee, M., Bali, R., Little, E., Novaes, R., Ly, H.V., Dwyre, D.M., Kheirolomoon, A., Tablin, F., Crowe, J.H., and N.M. Tsvetkova. Preservation of trehalose-loaded red blood cells by lyophilization. Cell Preservation Technology 2005 3:96-111
- Kheirolomoon, A., Satpathy, G.R., Torok, Z., Banerjee, M., Bali, R., Novaes, R.C., Little, E., Manning D.M., Dwyre, D.M., Tablin, F., Crowe, J.H. and N.M. Tsvetkova. Phospholipid vesicles increase the survival of freeze-dried human red blood cells. Cryobiology 2005 51:290-305.
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Progress 01/01/04 to 12/31/04
Outputs
Studies from the Center for Biostabilization funded by this DARPA grant are designed to improve stability of red blood cells and platelets in the dry state. To this end we have successfully dried human platelets which have been loaded with trehalose and freeze-dried in a mixture of trehalose and albumin. When rehydrated, these cells are responsive to platelet agonists, thrombin, ADP, and collagen by formation of a clot in the presence of plasma. Further, the cells are able to accommodate osmotic stress in a fashion virtually identical to fresh human platelets. Red blood cell studies are continuing to improve the formulation required for freeze-drying. Currently trehalose loaded human red blood cells are freeze-dried in a mixture of trehalose, albumin and hydroxyethyl starch. While these cells are intact and viable upon rehydration they are very fragile, and efforts are underway to improve the rehydration conditions for further assessment. Rehydrated red blood cells
have a low hemolytic index, and relatively low methemoglobin (7-10%). Our studies on both of these cellular systems are continuing and we anticipate further funding from DARPA for this work.
Impacts
Freeze-dried red blood cells will improve blood bank stocks in case of emergencies. They will also provide critically needed blood on the battlefield.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
We have made great progress in understanding the requirements and biophysical characteristics of red blood cells necessary for achieving a successful freeze-dried rbc. We have determined that normal human red blood cells have 40% cholesterol in their cellular membranes, which leads to extensive raft formation, but limited cellular permeability. In addition, we have characterized the membrane phase transitions of red blood cells in an effort to determine if we can successfully use these phase transitions to introduce trehalose into the red blood cells. Current studies are focused on understanding the metabolic parameters necessary for stabilization of red blood cells as well as ways to increase the intracellular trehalose concentration in order to optimize stabilization of both the red cell membranes and proteins.
Impacts
Freeze-dried red blood cells will improve blood bank stocks in case of emergencies. They will also provide critically needed blood on the battlefield.
Publications
- Wolkers, W.F., L.M. Crowe, N.M. Tsvetkova, F. Tablin and J.H. Crowe. 2002 In situ assessment of erythrocyte membrane properties during cold storage. Molecular Membrane Biology 19:59-65.
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