Erythrocytic cells and method for loading solutes
A dehydrated composition is provided that includes freeze-dried erythrocytic cells. A method 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 the cell. A method for retaining a solute in a cell.
1 . A method 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 the cell.
2 . The method of claim 1 wherein said solute concentration includes an extracellular cellular solute concentration for elevating extracelluar osmolarity within the solution to a value which is greater than a value of the intracellular osmolarity of the cell.
3 . The method of claim 1 wherein said transferring a solute is by fluid phase endocytosis.
4 . The method of claim 1 wherein said solute comprises trehalose and said cell comprises an erythrocytic cell.
5 . The method of claim 4 wherein said transferring of trehalose from the solution into the erythrocytic cell is without degradation of the trehalose.
6 . The method of claim 4 wherein a gradient of trehalose concentration (mM) within the erythrocytic cell to extracellular trehalose concentration (mM) within the solution ranges from about 0.130 to about 0.200.
7 . The method of claim 4 wherein a gradient of trehalose concentration (mM) within the erythrocytic cell to extracellular trehalose concentration (mM) within the solution ranges from about 0.04 to about 0.12.
8 . The method of claim 4 wherein a gradient of trehalose concentration (mM) within the erythrocytic cell to extracellular trehalose concentration (mM) within the solution ranges from about 0.08 to about 0.12.
9 . The method of claim 4 wherein said solute solution has a trehalose concentration ranging from about 320 mM to about 4000 mM.
10 . The method of claim 4 wherein said solute solution has a trehalose concentration ranging from about 320 mM to about 2000 mM.
11 . The method of claim 4 wherein said solute solution has a trehalose concentration ranging from about 500 mM to about 1000 mM.
12 . A cell produced in accordance with the method of claim 1 .
13 . An erythrocytic cell produced in accordance with the method of claim 11 .
14 . A method for loading trehalose into an erythrocytic cell comprising disposing an erythrocytic cell in a trehalose solution having a trehalose concentration of at least about 25% greater than the intracellular osmolarity of the erythrocytic cell for loading the trehalose into the erythrocytic cell.
15 . The method of claim 14 wherein said loading the trehalose into the erythrocytic cell is by fluid phase endocytosis.
16 . The method of claim 14 wherein said loading of the trehalose from the trehalose solution into the erythrocytic cell is without degradation of the trehalose.
17 . The method of claim 14 said loading of the trehalose produces a loaded erythrocytic cell having a gradient of loaded trehalose concentration (mM) within the erythrocytic cell to extracellular trehalose concentration (mM) within the trehalose solution ranging from about 0.130 to about 0.200.
18 . The method of claim 14 wherein said loading of the trehalose produces a loaded erythrocytic cell having a gradient of loaded trehalose concentration (mM) within the erythrocytic cell to extracellular trehalose concentration (mM) within the trehalose solution ranging from about 0.04 to about 0.08.
19 . The method of claim 14 wherein said loading of the trehalose produces a loaded erythrocytic cell having a gradient of loaded trehalose concentration (mM) within the erythrocytic cell to extracellular trehalose concentration (mM) within the trehalose solution ranging from about 0.04 to about 0.12.
20 . The method of claim 14 wherein said trehalose solution has a trehalose concentration of at least about 50% greater than the intracellular osmolarity of the erythrocytic cell.
21 . The method of claim 14 wherein said trehalose solution has a trehalose concentration ranging from about 25% to at least about 1000% greater than the intracellular osmolarity of the erythrocytic cell.
22 . An erythrocytic cell produced in accordance with the method of claim 14 .
23 . The method of claim 1 additionally comprising
preventing a decrease in a loading efficiency gradient in the loading of the solute into the cell.
24 . The method of claim 23 wherein said solute comprises an oligosaccharide and said preventing a decrease in a loading efficiency gradient in the loading of the oligosaccharide into the cell comprises maintaining a concentration of the oligosaccharide in the oligosaccharide solution below a concentration ranging from about 35 mM to about 65 mM.
25 . The method of claim 23 wherein said loading comprises loading by fluid phase endocytosis.
26 . The method of claim 24 wherein said loading comprises loading by fluid phase endocytosis.
27 . The method of claim 23 wherein said solute comprises an oligosaccharide and said preventing a decrease in a loading efficiency gradient in the loading of the oligosaccharide into the cell comprises maintaining a positive gradient of loading efficiency to concentration of the oligosaccharide in the oligosaccharide solution.
28 . The method of claim 23 wherein said solute comprises an oligosaccharide and said preventing a decrease in a loading efficiency gradient in the loading of the oligosaccharide into the cell comprises maintaining a positive gradient of loading efficiency (%) to concentration (mM) of the oligosaccharide in the oligosaccharide solution.
29 . The method of claim 27 wherein said oligosaccharide comprises trehalose.
30 . The method of claim 28 wherein said oligosaccharide comprises trehalose.
31 . A method for loading trehalose into cells comprising:
disposing cells in a trehalose solution having a trehalose concentration of at least about 25% greater than the intracellular osmolarity of the cells for loading trehalose into the cells; and
preventing a decrease in a loading efficiency gradient in the loading of the trehalose into the cells.
32 . The method of claim 31 wherein said preventing a decrease in a loading efficiency gradient in the loading of the trehalose into the cells comprises maintaining a concentration of the trehalose in the trehalose solution below a concentration ranging from about 35 mM to about 65 mM.
33 . The method of claim 31 wherein said loading comprises loading by fluid phase endocytosis.
34 . The method of claim 32 wherein said loading comprises loading by fluid phase endocytosis.
35 . The method of claim 31 wherein said preventing a decrease in a loading efficiency gradient in the loading of the trehalose into the cells comprises maintaining a positive gradient of loading efficiency to concentration of the trehalose in the trehalose solution.
36 . The method of claim 31 wherein said preventing a decrease in a loading efficiency gradient in the loading of the trehalose into the cells comprises maintaining a positive gradient of loading efficiency (%) to concentration (mM) of the trehalose in the trehalose solution.
37 . The method of claim 31 wherein said cells comprise erythrocytic cells.
38 . The method of claim 36 wherein said cells comprise erythrocytic cells.
39 . A method for loading an oligosaccharide into cells comprising:
disposing cells in an oligosaccharide solution having an oligosaccharide concentration of at least about 25% greater than the intracellular osmolarity of the cells for loading oligosaccharide into the cells; and
preventing a decrease in a loading gradient in the loading of the oligosaccharide into the cells.
40 . The method of claim 39 wherein said preventing a decrease in a loading 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.
41 . The method of claim 39 wherein said loading comprises loading by fluid phase endocytosis.
42 . The method of claim 40 wherein said loading comprises loading by fluid phase endocytosis.
43 . The method of claim 39 wherein said preventing a decrease in a loading gradient in the loading of the oligosaccharide into the cells comprises maintaining a positive gradient of concentration of oligosaccharide loaded into the cells to concentration of the oligosaccharide in the oligosaccharide solution.
44 . The method of claim 43 wherein said oligosaccharide comprises trehalose.
45 . The method of claim 39 wherein said cells comprise erythrocytic cells.
46 . The method of claim 1 additionally comprising retaining the solute in the cell.
47 . The method of claim 1 additionally comprising washing the cell, and retaining the solute in the cell during the washing.
48 . The method of claim 47 wherein said washing is with a washing buffer, and retention of the solute in the cell increases from about 25% to about 175% when a buffer concentration increases from about 50% to about 400%.
49 . The method of claim 47 wherein said washing is with a washing buffer, and retention of the solute in the cell increases from about 50% to about 150% when a buffer concentration increases from about 100% to about 300%.
50 . The method of claim 47 wherein said washing is with a washing buffer, and retention of the solute in the cell increases from about 75% to about 125% when a buffer concentration increases from about 150% to about 250%.
51 . The method of claim 47 wherein said washing is with a washing buffer, and retention of the solute in the cell increases about 100% when a buffer concentration increases about 200%.
52 . The method of claim 1 additionally comprising washing the cell 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.
53 . The method of claim 1 additionally comprising washing the cell with a washing buffer wherein a ratio of an extracellular buffer concentration (mOsm) to an intracellular solute concentration (mM) ranges from about 12.0 to about 5.0.
54 . The method of claim 1 additionally comprising washing the cell with a washing buffer wherein a ratio of an extracellular buffer concentration (mOsm) to an intracellular solute concentration (mM) ranges from about 9.0 to about 6.0.
55 . The method of claim 1 additionally comprising washing the cell with a washing buffer wherein a ratio of an extracellular buffer concentration (mOsm) to an intracellular solute concentration (mM) ranges from about 8.0 to about 7.0.
56 . The method of claim 14 additionally comprising retaining the trehalose in the erythrocytic cell.
57 . The method of claim 14 additionally comprising washing the erythrocytic cell, and retaining the trehalose in the erythrocytic cell during the washing.
58 . The method of claim 57 wherein said washing is with a washing buffer, and retention of the trehalose in the erythrocytic cell increases from about 25% to about 175% when a buffer concentration increases from about 50% to about 400%.
59 . The method of claim 47 wherein said washing is with a washing buffer, and retention of the trehalose in the erythrocytic cell increases from about 50% to about 150% when a buffer concentration increases from about 100% to about 300%.
60 . The method of claim 57 wherein said washing is with a washing buffer, and retention of the trehalose in the erythrocytic cell increases from about 75% to about 125% when a buffer concentration increases from about 150% to about 250%.
61 . The method of claim 57 wherein said washing is with a washing buffer, and retention of the trehalose in the erythrocytic cell increases about 100% when a buffer concentration increases about 200%.
62 . The method of claim 14 additionally comprising washing the erythrocytic cell with a washing buffer wherein a ratio of an extracellular buffer concentration (mOsm) to an intracellular trehalose concentration (mM) ranges from about 14.0 to about 4.0.
63 . The method of claim 14 additionally comprising washing the erythrocytic cell with a washing buffer wherein a ratio of an extracellular buffer concentration (mOsm) to an intracellular trehalose concentration (mM) ranges from about 12.0 to about 5.0.
64 . The method of claim 14 additionally comprising washing the erythrocytic cell with a washing buffer wherein a ratio of an extracellular buffer concentration (mOsm) to an intracellular trehalose concentration (mM) ranges from about 9.0 to about 6.0.
65 . The method of claim 14 additionally comprising washing the erythrocytic cell with a washing buffer wherein a ratio of an extracellular buffer concentration (mOsm) to an intracellular trehalose concentration (mM) ranges from about 8.0 to about 7.0.
66 . A method for retaining a solute in a cell comprising disposing a cell containing a solute in a solution wherein a ratio of an extracellular buffer concentration (mOsm) to an intracellular solute concentration (mM) ranges from about 14.0 to about 4.0.