USA As Represented by Secretary of the Navy

Thomas R Groel

Alan S Rudolph

Barry J Spargo

Irina B Bakaltcheva

Samuel B Leslie

Byeong S Chang

Richard G Emler

One aspect of the present invention is a method for storing tissues and cells (typically erythrocytes) having the step of (1) stabilizing the cells with a reversible stabilizing agent. This method typically will have the additional steps of (2) loading the cells with a cryoprotectant, and typically (3) storing the cells in liquid, frozen, or dry state. This method will also typically have the additional step of (4) prior to use, reversing the stabilization reaction. Preferably, the erythrocytes are pre-treated with CO to complex the hemoglobin with CO. It is anticipated that a practical method according to the invention will include reoxygenation of the eryathrocytes, and also washing out reagents prior to in vivo use. Another aspect of the present invention is an erythrocyte that has had its shape stabilized by the reversible crosslinking of proteins in the erythrocyte, such as the structural proteins of the cytoskeleton. Another aspect of the invention is a population of such reversibly cross-linked erythrocytes. Another aspect of the invention is the in vivo use of such erythrocytes, after the reversal of the crosslinking reaction. The use of more gentle, reversible cross-linking as described below is desirable to result in the recovery of erythrocyte deformability and extended post-transfusion survival.

Shape stabilized erythrocytes

One aspect of the present invention is a method for storing tissues and cells (typically erythrocytes) having the step of (1) stabilizing the cells with a reversible stabilizing agent. This method typically will have the additional steps of (2) loading the cells with a cryoprotectant, and typically (3) storing the cells in liquid, frozen, or dry state. This method will also typically have the additional step of (4) prior to use, reversing the stabilization reaction. Preferably, the erythrocytes are pre-treated with CO to complex the hemoglobin with CO. It is anticipated that a practical method according to the invention will include reoxygenation of the erythrocytes, and also washing out reagents prior to in vivo use. Another aspect of the present invention is an erythrocyte that has had its shape stabilized by the reversible crosslinking of proteins in the erythrocyte, such as the structural proteins of the cytoskeleton. Another aspect of the invention is a population of such reversibly crosslinked erythrocytes. Another aspect of the invention is the in vivo use of such erythrocytes, after the reversal of the crosslinking reaction. The use of more gentle, reversible cross-linking as described below is desirable to result in the recovery of erythrocyte deformability and extended post-transfusion survival.

Applications of reversible crosslinking and co-treatment in stabilization and viral inactivations of erythrocytes

A process and medium are disclosed for the lyophilization of platelets. During lyophilization, carbohydrate-load platelets are supercooled while suspended in a buffer solution including a biocompatible polymer that serves to preserve the structure of the platelets. The supercooled platelets are then frozen at a temperature below the glass transition temperature of the suspension. A vacuum is placed on the frozen suspension to remove most of the water therefrom. Then, the temperature of the platelets is increased to the supercooled temperature while the vacuum is maintained. After being sealed under vacuum, the lyophilized platelets may be reconstituted to form viable, transfusable platelets. The reconstituted platelets have a high aggregation index, retain normal agglutination and degranulation capability, and are able to participate in clot formation.

Method of lyophilizing platelets by incubation with high carbohydrate

A process and medium are disclosed for the lyophilization of cells, specifically red blood cells and platelets, and cell-like matter, which comprises the use of solution including a carbohydrate, and biocompatible polymers to permit reconstitution of transfusably useful cells which are viable by the measure of ATP and 2,3 DPG.

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Manassas, VA, United States of America

Thomas R Groel