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 ...

AI Technical Summary

Benefits of technology

[0019] Practice of the present invention permits the manipulation or modification of platelets while maintaining, or preserving, biological properties, such as a response to thrombin. Further, use of the method to preserve platelets can be practiced on a large, commercially feasible scale, and avoids platelet activation. The inventive freeze-dried platelets, and hemostasis aids including the freeze-dried platelets, are substantially shelf stable at ambient temperatures when packaged in moisture barrier materials.

[0020] Embodiments of the present invention also provide a process for preserving and / or increasing the survival of dehydrated eukaryotic cells after storage comprising providing eukaryotic cells from a mammalian species (e.g., a human); loading the eukaryotic cells with a preservative (e.g., an oligosaccharide, such as trehalose); dehydrating the eukaryotic cells while maintaining a residual water content in the eukaryotic cells greater than about 0.15 (e.g., from about 0.20 to about 0.75) gram of water per gram of dry weight eukaryotic cells to increase eukaryotic cell survival, preferably to greater than about 80%, upon rehydrating after storage; storing the dehydrated eukaryotic cells having the residual water content greater than about 0.15 gram of water per gram of dry weight eukaryotic cells; and rehydrating the stored dehydrated eukaryotic cells with the stored dehydrated eukaryotic cells having an increase in survival following dehydration and storage. In a preferred embodiment, more than about 80% of the stored dehydrated cells survive the dehydration and storage.

[0023] Embodiments of the present invention yet also further provide a generally dehydrated composition comprising freeze-dried eukaryotic cells selected from a mammalian species (e.g., a human) and being effectively loaded internally (e.g., incubating the eukaryotic cells at a temperature from about 30° C. to less than about 50° C. so as to uptake external trehalose via fluid phase endocytosis) with at least about 10 mM trehalose therein to preserve biological properties during freeze-drying and rehydration. The amount of trehalose loaded inside the freeze-dried eukaryotic cells is preferably from about 10 mM to about 50 mM. The freeze-dried eukaryotic cells comprise at least about 0.15 (e.g., from about 0.20 to about 0.75) gram of residual water per gram of dry weight eukaryotic cells to increase eukaryotic cell survival upon rehydrating.

[0025] Further aspects of embodiments of the present invention include a process for increasing the loading efficiency of an oligosaccharide into eukaryotic cells. The process comprises providing eukaryotic cells having a first phase transition temperature range and a second phase transition temperature range (e.g., a temperature greater than about 25° C., such as from about 30° C. to less than about 50° C.) which is greater than the first phase transition temperature range; disposing the eukaryotic cells in an oligosaccharide solution for loading an oligosaccharide (e.g., trehalose) into the eukaryotic cells; and heating the oligosaccharide solution to the second phase transition temperature range to increase the loading efficiency of the oligosaccharide into the eukaryotic cells. The process additionally comprises taking up external oligosaccharide via fluid phase endocytosis from the oligosaccharide solution.

[0031] Additional features of the present invention include a solution for loading erythrocytic cells, an erythrocytic cell composition, and a generally dehydrated composition. The solution for loading erythrocytic cells comprises reduced-alcohol (e.g. reduced-sterol) erythrocytic cells having three phase transition temperature ranges, and an oligosaccharide solution containing the reduced-alcohol erythrocytic cells for loading oligosaccharide from the oligosaccharide solution into the reduced-alcohol erythrocytic cells. External oligosaccharide is taken up via lipid phase endocytosis from the oligosaccharide solution at a temperature in a range of temperatures approximating one of the three phase transition temperature ranges. The erythrocytic cell composition comprises reduced-alcohol erythrocytic cells loaded internally with an oligosaccharide from an oligosaccharide solution. Preferably, the oligosaccharide is loaded from the oligosaccharide solution at a temperature in a range of temperatures selected from the group consisting of a low phase transition temperature range, an intermediate phase transition temperature range, and a high phase transition temperature range. The generally dehydrated composition comprises freeze-dried reduced-alcohol erythrocytic cells effectively loaded internally with at least about 10 mM of the oligosaccharide (e.g., trehalose) therein to preserve biological properties during freeze-drying and rehydration. The amount of the oligosaccharide loaded inside the freeze-dried reduced-alcohol erythrocytic cells may be from about 10 mM to about 200 mM. The freeze-dried reduced-alcohol erythrocytic cells may comprise less than about 0.30 gram of residual water per gram of dry weight erythrocytic cells to increase erythrocytic cell survival upon rehydrating.

Problems solved by technology

Blood transfusion centers are under considerable pressure to produce platelet concentrates for transfusion.

Today platelet rich plasma concentrates are stored in bloodbags at 22° C.; however, the shelf life under these conditions is limited to five days.

The rapid loss of platelet function during storage and risk of bacterial contamination complicates distribution and availability of platelet concentrates.

When activated they are substantially useless for an application such as transfusion therapy.

However, a considerable fraction of these cells are partly lysed after thawing and have the shape of a balloon.

Proper functioning of lyophilized platelets that have been fixed by such fixative agents in hemostasis is questionable.

However, electroporation is very damaging to the cell membranes and is believed to activate the platelets.

Activated platelets have dubious clinical value.

Images