Erythrocytic cells and method for loading solutes
A dehydrated composition is provided that includes freeze-dried erythrocytic cells. The invention further provides methods for loading a solute into a cell comprising disposing a cell in a solution having a solute concentration of sufficient magnitude to produce hyperosmotic pressure on the cell for transferring a solute from the solution into cell.
1 . A method for loading a solute into an erythrocytic cell, comprising:
disposing an erythrocytic cell in a solution having a solute concentration of sufficient magnitude to produce hyperosmotic pressure on the cell, thereby transferring a solute from the solution into the cell.
2 . The method of claim 1 wherein said solute is present in said solution in a concentration of between 700 and 1000 mM.
3 . The method of claim 1 , wherein said solute is a disaccharide.
4 . The method of claim 3 , wherein said disaccharide is trehalose.
5 . The method of claim 1 , wherein said solution further comprises a potassium salt.
6 . The method of claim 5 , wherein said potassium salt is potassium phosphate.
7 . The method of claim 1 , wherein said solution further comprises α-crystallin.
8 . The method of claim 1 , wherein said solution further comprises a strong reducing agent.
9 . The method of claim 8 , wherein said strong reducing agent is ascorbic acid.
10 . The method of claim 1 , wherein said solution comprises a disaccharide, a-crystallin, ascorbic acid, and a potassium salt.
11 . A method of claim 1 , further wherein the loading is conducted at a temperature of between 25 and 40° C.
12 . A method of claim 11 , further wherein the loading is conducted at a temperature of between 30 and 40° C.
13 . A method of claim 11 , further wherein the loading is conducted at a temperature of about 37° C.
14 . An erythrocyte loaded with from 10 mM to 50 mM trehalose.
15 . An erythrocyte of claim 11 , further comprising ascorbic acid.
16 . An erythrocyte of claim 11 , further comprising α-crystallin.
17 . A method for separating fragile or damaged cells from a population of erythrocytes, said method comprising
contacting said population with a first solution which is hyperosmotic with respect to a solute,
loading a solute into said erythrocytes,
removing said erythrocytes from said hyperosmotic solution,
contacting said erythrocytes with a second solution which is mildly hypoosmotic in comparison to said hyperosmotic solution, thereby lysing fragile or damaged cells, and
separating said fragile or damaged cells from said population.
18 . A method of claim 14 , wherein said separation is by centrifugation.
19 . A method for freeze-drying erythrocytes comprising lowering the hematocrit of said erythrocytes to between 2 and 5%.
20 . A method for freeze-drying erythrocytes, comprising drying said erythrocytes in the presence of liposomes.
21 . A method of claim 18 , wherein said liposomes are composed primarily of unsaturated lipids.
22 . A method for freeze-drying erythrocytes, comprising freeze-drying said erythrocytes in the presence of 200-300 mOsm of potassium salts.
23 . A method of claim 19 , wherein said erythrocytes are present in a hematocrit of up to 15%.
24 . A buffer for drying erythrocytes, said buffer comprising liposomes.
25 . A buffer of claim 21 , wherein said liposomes are composed primarily of unsaturated lipids.
26 . A buffer for drying erythrocytes, said buffer comprising ascorbic acid.
27 . A buffer for rehydrating dried erythrocytes, said buffer comprising methylene blue.
28 . A buffer for rehydrating dried erythrocytes, said buffer comprising transition metal ions.
29 . A buffer of claim 24 , wherein said transition metal ions are selected from the group consisting of zinc, copper, magnesium, and nickel.
30 . A solution for rehydrating dried erythrocytes, said solution comprising ascorbic acid.
31 . A solution for rehydrating dried erythrocytes, said solution comprising methylene blue, ascorbic acid, and transition metal ions.
32 . A method for rehydrating dried erythrocytes, said method comprising contacting said dried erythrocytes with a solution comprising methylene blue.
33 . A method for rehydrating dried erythrocytes, said method comprising contacting said dried erythrocytes with a solution comprising transition metal ions.
34 . A method for rehydrating dried erythrocytes, said method comprising contacting said dried erythrocytes with a solution comprising ascorbic acid.
35 . A method for rehydrating dried erythrocytes, said method comprising contacting said dried erythrocytes with a solution comprising methylene blue, and transition metal ions.