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US 5,690,963 AGranted
Freeze dried red blood cells
AssigneeThe United States of America as represented by the Secretary of the Navy
InventorsBarry J. Spargo, Alan S. Rudolph, Byeong S. Chang, Thomas R. Groel, II
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Abstract
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.
Claims4
A process for treating erythrocytes for storage comprising in order the steps of
- (a) resuspending erythrocytes in a first phosphate-citrate incubation buffer which comprises glucose in a concentration range of 10-1500 mM, sodium citrate in a concentration range of 1-50 mM, sodium diphosphate dibasic in a concentration range of 1-50 mM, sodium phosphate monobasic in a concentration range of 1-15 mM, ammonium phosphate in a concentration range of 1-100 mM and adenine in a concentration range of 0-5 mM;
- (b) incubating the resuspended erythrocytes in said phosphate-citrate incubation buffer;
- (c) removing said first phosphate-citrate incubation buffer;
- (d) resuspending the incubated erythrocytes in a second phosphate-citrate incubation buffer comprising the phosphate-citrate incubation buffer of step (a) and an additional carbohydrate in a concentration of from about 500 to about 1000 mM;
- (e) incubating the resuspended erythrocytes in the second phosphate-citrate incubation buffer;
- (f) removing the second phosphate-citrate incubation buffer;
- (g) resuspending the incubated erythrocytes in a phosphate-citrate lyophilization buffer comprising the phosphate-citrate incubation buffer of step (d) and a polymer having a molecular weight of from about 100 kD to about 500 kD in a concentration of about 10-30% w/v;
- (h) lyophilizing the resuspended erythrocytes of step (g) by the steps of
- (1) slowly cooling the resuspended erythrocytes to -15.degree. C.;
- (2) incubating said cooled erythrocytes at -10.degree. to -15.degree. C.;
- (3) primary drying the erythrocytes of step (2) by further cooling said erythrocytes of step (2) to -50.degree. C. and placing said primary-dried erythrocytes under a vacuum;
- (4) further drying the primary-dried erythrocytes of step (3) by keeping the primary-dried erythrocytes under vacuum while elevating the temperature of said primary-dried erythrocytes to -15.degree. C., until the residual water content of the lyophilized erythrocytes is less than 10% by weight;
- (i) sealing the lyophilized erythrocytes under vacuum for storage;
- and
- (j) storing the sealed erythrocytes;
- wherein the step (b) of incubating the erythrocytes in the first phosphate-citrate incubation buffer is carried out for a sufficient time such that when the stored erythrocytes of step (j) are subsequently rehydrated, the rehydrated erythrocytes have an ATP level of at least 100% and a 2,3 DPG level of at least 150-200% that of freshly drawn packed erythrocytes.
13 more elements
A process for treating erythrocytes for storage comprising in order the steps of
- (a) resuspending erythrocytes in a first phosphate-citrate incubation buffer which comprises glucose in a concentration range of 10-1500 mM, sodium citrate in a concentration range of 1-50 mM, sodium diphosphate dibasic in a concentration range of 1-50 mM, sodium phosphate monobasic in a concentration range of 1-15 mM, ammonium phosphate in a concentration range of 1-100 mM and adenine in a concentration range of 0-5 mM;
- (b) incubating the resuspended erythrocytes in said phosphate-citrate incubation buffer;
- (c) removing said first phosphate-citrate incubation buffer;
- (d) resuspending the incubated erythrocytes in a second phosphate-citrate incubation buffer comprising the phosphate-citrate incubation buffer of step (a) and an additional carbohydrate in a concentration of from about 500 to about 1000 mM;
- (e) incubating the resuspended erythrocytes in the second phosphate-citrate incubation buffer;
- (f) removing the second phosphate-citrate incubation buffer;
- (g) resuspending the incubated erythrocytes in a phosphate-citrate lyophilization buffer comprising the phosphate-citrate incubation buffer of step (d) and a polymer having a molecular weight of from about 100 kD to about 500 kD in a concentration of about 10-30% w/v;
- (h) lyophilizing the resuspended erythrocytes of step (g) by the steps of
- (1) slowly cooling the resuspended erythrocytes to -15.degree. C.;
- (2) incubating said cooled erythrocytes at -10.degree. to -15.degree. C.;
- (3) primary drying the erythrocytes of step (2) by further cooling said erythrocytes of step (2) to -50.degree. C. and placing said primary-dried erythrocytes under a vacuum;
- (4) further drying the primary-dried erythrocytes of step (3) by keeping the primary-dried erythrocytes under vacuum while elevating the temperature of said primary-dried erythrocytes to -15.degree. C., until the residual water content of the lyophilized erythrocytes is less than 10% by weight;
- (i) sealing the lyophilized erythrocytes under vacuum for storage;
- and
- (j) storing the sealed erythrocytes;
- (k) rehydrating the stored erythrocytes of step (j) by suspending said erythrocytes in a phosphate-citrate reconstitution buffer comprising a polymer having a molecular weight of from about 100 kD to about 500 kD in a concentration of about 10-30% w/v at a temperature in the range of 15.degree.-50.degree. C.;
- wherein the step (b) of incubating the erythrocytes in the first phosphate-citrate incubation buffer is carried out for a sufficient time such that the rehydrated erythrocytes of step (k) have an ATP level of at least 100% and a 2,3 DPG level of at least 150-200% that of freshly drawn packed erythrocytes.
14 more elements
Dependencies are inferred from the claim text.