Method for eliminating fragile cells from stored cells
A method for reducing hemolysis in cells including washing cells in a solute solution having the capabilities of reducing cell hemolysis by at least about 0.50% for each 100 mOsm increase in osmolarity of the solute solution. A cell produced by the method for reducing hemolysis. The method permits removal of osmotically fragile cells from the population.
1 . A method for reducing hemolysis in cells comprising
washing cells in a solute solution having the capabilities of reducing cell hemolysis by at least about 0.50% for each 100 mOsm increase in osmolarity of the solute solution.
2 . The method of claim 1 wherein said solute solution reduces cell hemolysis from about 0.50% to about 8.0% for each 100 mOsm increase in osmolarity of the solute solution.
3 . The method of claim 1 wherein said solute solution reduces cell hemolysis from about 1.0% to about 4.0% for each 100 mOsm increase in osmolarity of the solute solution.
4 . The method of claim 1 wherein said solute solution reduces cell hemolysis from about 1.0% to about 2.0% for each 100 mOsm increase in osmolarity of the solute solution.
5 . The method of claim 1 wherein said solute solution comprises an osmolarity ranging from about 100 mOsm to about 1500 mOsm.
6 . The method of claim 1 wherein said solute solution comprises an osmolarity ranging from about 200 mOsm to about 1000 mOsm.
7 . The method of claim 1 wherein said solute solution comprises an osmolarity ranging from about 300 mOsm to about 600 mOsm.
8 . The method of claim 4 wherein said solute solution comprises an osmolarity ranging from about 300 mOsm to about 600 mOsm.
9 . The method of claim 1 wherein said solute solution comprising a salt solution having a phosphate buffered saline (PBS) solution including NaCl, Na 2 HPO 4 , and KH 2 PO 4 .
10 . The method of claim 1 wherein said solute solution comprises a PBS buffer having 154 mM NaCl, 5.6 mM Na 2 HPO 4 , 1.06 mM KH 2 PO 4 , and a pH 7.2.
11 . The method of claim 1 additionally comprising removing damaged cells from the washed cells.
12 . The method of claim 11 wherein removing damaged cells comprises centrifuging the washed cells.
13 . The method of claim 11 additionally comprising suspending the cells in the solute solution.
14 . The method of claim 1 additionally comprising loading a solute into the cells prior to washing the cells.
15 . The method of claim 14 wherein said loading of the cells comprises disposing the cells in a solution having a solute concentration of sufficient magnitude to produce hyperosmotic pressure on the cells for transferring a solute from the solution into the cells.
16 . The method of claim 15 wherein said solute concentration includes an extracellular cellular solute concentration for elevating extracellular osmolarity within the solution to a value which is greater than a value of the intracellular osmolarity of the cells.
17 . The method of claim 15 wherein said transferring a solute is by fluid phase endocytosis.
18 . The method of claim 15 wherein said solute comprises trehalose and said cells comprise erythrocytic cells.
19 . The method of claim 18 wherein said transferring of trehalose from the solution into the erythrocytic-cells is without degradation of the trehalose.
20 . The method of claim 18 wherein a gradient of trehalose concentration (M) within the erythrocytic cells to extracellular trehalose concentration (M) within the solution ranges from about 0.130 to about 0.200.
21 . The method of claim 18 wherein a gradient of trehalose concentration (M) within the erythrocytic cell to extracellular trehalose concentration (M) within the solution ranges from about 0.04 to about 0.12.
22 . The method of claim 18 wherein said solute solution has a trehalose concentration ranging from about 320 mM to about 4000 mM.
23 . A cell produced in accordance with the method of claim 1 .
24 . The method of claim 18 wherein loading trehalose into erythrocytic cells comprises disposing the erythrocytic cells in a trehalose solution having a trehalose concentration of at least about 25% greater than the intracellular osmolarity of the erythrocytic cells for loading the trehalose into the erythrocytic cells.
25 . The method of claim 14 additionally comprising
preventing a decrease in a loading efficiency gradient in the loading of the solute into the cells.
26 . The method of claim 25 wherein said solute comprises an oligosaccharide and said preventing a decrease in a loading efficiency gradient in the loading of the oligosaccharide into the cells comprises maintaining a concentration of the oligosaccharide in the oligosaccharide solution below a concentration ranging from about 35 mM to about 65 mM.
27 . The method of claim 25 wherein said solute comprises an oligosaccharide and said preventing a decrease in a loading efficiency gradient in the loading of the oligosaccharide into the cells comprises maintaining a positive gradient of loading efficiency to concentration of the oligosaccharide in the oligosaccharide solution.
28 . The method of claim 1 additionally comprising retaining the solute in the cells during the washing.
29 . The method of claim 28 wherein said washing is with a washing buffer, and retention of the solute in the cells increases from about 25% to about 175% when a buffer concentration increases from about 50% to about 400%.
30 . The method of claim 28 additionally comprising washing the cells with a washing buffer wherein a ratio of an extracellular buffer concentration (mOsm) to an intracellular solute concentration (mM) ranges from about 14.0 to about 4.0.
31 . A method for removing fragile cells from cells comprising:
washing cells in a solute solution having the capabilities of reducing cell hemolysis to produce washed cells including fragile cells; and
removing the fragile cells from the washed cells.
32 . The method of claim 31 wherein said solute solution has the capabilities of reducing hemolysis by at least about 0.50% for each 100 mOsm increase in osmolarity of the solute solution.