Erythrocytic cells and method for preserving cells

Abstract

A dehydrated composition is provided that includes freeze-dried erythrocytic cells. Alcohol (e.g., sterol or cholesterol) is at least partially removed from erythrocytic cells including erythrocytic membranes. After removal of at least part of the alcohol, the erythrocytic cells have a low phase transition temperature range, an intermediate phase transition temperature range, and a high phase transition temperature range. The erythrocytic cells may be loaded with an oligosaccharide (e.g., trehalose) which preserves biological properties during freeze-drying and rehydration. A process for increasing cooperativity of a phase transition of an erythrocytic cell. A process for preserving and / or increasing the survival of dehydrated erythrocytic cells, including storing dehydrated erythrocytic cells having a residual water content equal to or less than about 0.30 gram of water per gram of dry weight erythrocytic cells.

Description

RELATED PATENT APPLICATIONS

[0001] This is a continuation-in-part patent application of copending patent application Ser. No. 09 / 927,760, filed Aug. 9, 2001. Patent application Ser. No. 09 / 927,760 is a continuation-in-part patent application of copending patent application Ser. No. 09 / 828,627, filed Apr. 5, 2001. Patent application Ser. No. 09 / 828,627 is a continuation patent application of patent application Ser. No. 09 / 501,773, filed Feb. 10, 2000. Benefit of all earlier filing dates is claimed with respect to all common subject matter.STATEMENT REGARDING FEDERAL SPONSORED RESEARCH AND DEVELOPMENT

[0002] Embodiments of this invention were made with Government support under Grant No. N66001-00-C-8048, awarded by the Department of Defense Advanced Research Projects Agency (DARPA). Further embodiments of this invention were made with Government support under Grant Nos. HL57810 and HL61204, awarded by the National Institutes of Health. The Government has certain rights to embodiments ...

Examples

example 1

[0176] Washing of Platelets. Platelet concentrations were obtained from the Sacramento blood center or from volunteers in our laboratory. Platelet rich plasma was centrifuged for 8 minutes at 320×g to remove erythrocytes and leukocytes. The supernatant was pelleted and washed two times (480×g for 22 minutes, 480×g for 15 minutes) in buffer A (100 mM NaCl, 10 mM KCl, 10 mM EGTA, 10 mM imidazole, pH 6.8). Platelet counts were obtained on a Coulter counter T890 (Coulter, Inc., Miami, Fla.).

[0177] Loading of Lucifer Yellow CH into Platelets. A fluorescent dye, lucifer yellow CH (LYCH), was used as a marker for penetration of the membrane by a solute. Washed platelets in a concentration of 1-2×109 platelets / ml were incubated at various temperatures in the presence of 1-20 mg / ml LYCH. Incubation temperatures and incubation times were chosen as indicated. After incubation the platelets suspensions were spun down for 20× at 14,000 RPM (table centrifuge), resuspended in buffer A, spun down ...

example 2

[0184] Washing Platelets. Platelets were obtained from volunteers in our laboratory. Platelet rich plasma was centrifuged for 8 minutes at 320×g to remove erythrocytes and leukocytes. The supernatant was pelleted and washed two times (480×g for 22 minutes, 480×g for 15 minutes) in buffer A (100 mM NaCl, 10 mM KCl, 10 mM EGTA, 10 mM imidazole, 10 μg / ml PGE1, pH 6.8). Platelet counts were obtained on a Coulter counter T890 (Coulter, Inc., Miami, Fla.).

[0185] Loading Platelets with Trehalose. Platelets were loaded with trehalose as described in Example 1. Washed platelets in a concentration of 1-2×109 platelets / ml were incubated at 37° C. in buffer A with 35 mM trehalose added. Incubation times were typically 4 hours. The samples were gently stirred for 1 minute every hour. After incubation the platelet solutions were pelleted (25 sec in a microfuge) and resuspended in drying buffer (9.5 mM HEPES, 142.5 mM NaCl, 4.8 mM KCl, 1 mM MgCl2, 30 mM Trehalose, 1% Human Serum Albumin, 10 μg / ml...

example 3

[0197] We view trehalose as the main lyoprotectant in the drying buffer. However, other components in the drying buffer, such as albumin, can improve the recovery. In the absence of external trehalose in drying buffer, the numerical recovery is only 35%. With 30 mM trehalose in the drying buffer the recovery is around 65%. A combination of 30 mM trehalose and 1% albumin gave a numerical recovery of 85%.

RELATED PATENT APPLICATIONS

[0001] This is a continuation-in-part patent application of copending patent application Ser. No. 09 / 927,760, filed Aug. 9, 2001. Patent application Ser. No. 09 / 927,760 is a continuation-in-part patent application of copending patent application Ser. No. 09 / 828,627, filed Apr. 5, 2001. Patent application Ser. No. 09 / 828,627 is a continuation patent application of patent application Ser. No. 09 / 501,773, filed Feb. 10, 2000. Benefit of all earlier filing dates is claimed with respect to all common subject matter.STATEMENT REGARDING FEDERAL SPONSORED RESEARCH AND DEVELOPMENT

[0002] Embodiments of this invention were made with Government support under Grant No. N66001-00-C-8048, awarded by the Department of Defense Advanced Research Projects Agency (DARPA). Further embodiments of this invention were made with Government support under Grant Nos. HL57810 and HL61204, awarded by the National Institutes of Health. The Government has certain rights to embodiments ...

Examples

example 1

[0176] Washing of Platelets. Platelet concentrations were obtained from the Sacramento blood center or from volunteers in our laboratory. Platelet rich plasma was centrifuged for 8 minutes at 320×g to remove erythrocytes and leukocytes. The supernatant was pelleted and washed two times (480×g for 22 minutes, 480×g for 15 minutes) in buffer A (100 mM NaCl, 10 mM KCl, 10 mM EGTA, 10 mM imidazole, pH 6.8). Platelet counts were obtained on a Coulter counter T890 (Coulter, Inc., Miami, Fla.).

[0177] Loading of Lucifer Yellow CH into Platelets. A fluorescent dye, lucifer yellow CH (LYCH), was used as a marker for penetration of the membrane by a solute. Washed platelets in a concentration of 1-2×109 platelets / ml were incubated at various temperatures in the presence of 1-20 mg / ml LYCH. Incubation temperatures and incubation times were chosen as indicated. After incubation the platelets suspensions were spun down for 20× at 14,000 RPM (table centrifuge), resuspended in buffer A, spun down ...

example 2

[0184] Washing Platelets. Platelets were obtained from volunteers in our laboratory. Platelet rich plasma was centrifuged for 8 minutes at 320×g to remove erythrocytes and leukocytes. The supernatant was pelleted and washed two times (480×g for 22 minutes, 480×g for 15 minutes) in buffer A (100 mM NaCl, 10 mM KCl, 10 mM EGTA, 10 mM imidazole, 10 μg / ml PGE1, pH 6.8). Platelet counts were obtained on a Coulter counter T890 (Coulter, Inc., Miami, Fla.).

[0185] Loading Platelets with Trehalose. Platelets were loaded with trehalose as described in Example 1. Washed platelets in a concentration of 1-2×109 platelets / ml were incubated at 37° C. in buffer A with 35 mM trehalose added. Incubation times were typically 4 hours. The samples were gently stirred for 1 minute every hour. After incubation the platelet solutions were pelleted (25 sec in a microfuge) and resuspended in drying buffer (9.5 mM HEPES, 142.5 mM NaCl, 4.8 mM KCl, 1 mM MgCl2, 30 mM Trehalose, 1% Human Serum Albumin, 10 μg / ml...

example 3

[0197] We view trehalose as the main lyoprotectant in the drying buffer. However, other components in the drying buffer, such as albumin, can improve the recovery. In the absence of external trehalose in drying buffer, the numerical recovery is only 35%. With 30 mM trehalose in the drying buffer the recovery is around 65%. A combination of 30 mM trehalose and 1% albumin gave a numerical recovery of 85%.

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