Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of storing cells in a viable state
Reexamination Certificate
2002-01-16
2004-08-03
Chen, Shin-Lin (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of storing cells in a viable state
C435S002000, C435S100000, C435S325000, C435S372000
Reexamination Certificate
active
06770478
ABSTRACT:
FIELD OF THE INVENTION
Embodiments of the present invention generally broadly relate to living mammalian cells. More specifically, embodiments of the present invention generally provide for the preservation and survival of human cells, especially eukaryotic cells and erythrocytic cells.
Embodiments of the present invention also generally broadly relate to the therapeutic uses of blood platelets, eukaryotic cells, and erythrocytic cells; and more particularly to manipulations or modifications of platelets, eukaryotic cells, and erythrocytic cells, such as in preparing freeze-dried compositions that can be rehydrated at the time of application. When freeze-dried platelets are rehydrated, they have a normal response to thrombin and other agonists with respect to that of fresh platelets. When eukaryotic cells and erythrocytic cells are rehydrated, they are immediately restored to viability.
The inventive compositions and methods for embodiments of the present invention are useful in many applications, such as in medicine, pharmaceuticals, biotechnology, and agriculture, and including transfusion therapy, as hemostasis aids and for drug delivery.
BACKGROUND OF THE INVENTION
Blood transfusion centers are under considerable pressure to produce platelet concentrates for transfusion. The enormous quest for platelets necessitates storage of this blood component, since platelets are important contributors to hemostasis. Platelets are generally oval to spherical in shape and have a diameter of 2-4 &mgr;m. 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. Platelets tend to become activated at low temperatures. When activated they are substantially useless for an application such as transfusion therapy. Therefore the development of preservation methods that will increase platelet lifespan is desirable.
Several techniques for preservation of platelets have been developed over the past few decades. Cryopreservation of platelets using various agents, such as glycerol (Valeri et al.,
Blood,
43, 131-136, 1974) or dimethyl sulfoxide, “DMSO” (Bock et al.,
Transfusion,
35, 921-924, 1995), as the cryoprotectant have been done with some success. The best results have been obtained with DMSO. However, a considerable fraction of these cells are partly lysed after thawing and have the shape of a balloon. These balloon cells are not responsive to various agonists, so that overall responsiveness of frozen thawed platelets to various agonists is reduced to less than 35% compared with fresh platelets. The shelf life of cryopreserved DMSO platelets at −80° C. is reported to be one year, but requires extensive washing and processing to remove cryoprotective agents, and even then the final product has a severe reduction in ability to form a clot.
Attempts to dry platelets by lyophilization have been described with paraformaldehyde fixed platelets (Read et al.,
Proc. Natl. Acad. Sci. USA,
92, 397401, 1995). U.S. Pat. No. 5,902,608, issued May 11, 1999, inventors Read et al. describe and claim a surgical aid comprising a substrate on which fixed, dried blood platelets are carried. These dried blood platelets are fixed by contacting the platelets to a fixative such as formaldehyde, paraformaldehyde, gutaraldehyde, or permanganate. Proper functioning of lyophilized platelets that have been fixed by such fixative agents in hemostasis is questionable.
Spargo et al., U.S. Pat. No. 5,736,313, issued Apr. 7, 1998, have described a method in which platelets are loaded overnight with an agent, preferably glucose, and subsequently lyophilized. The platelets are preincubated in a preincubation buffer and then are loaded with carbohydrate, preferably glucose, having a concentration in the range of about 100 mM to about 1.5 M. The incubation is taught to be conducted at about 10° C. to about 37° C., most preferably about 25° C.
U.S. Pat. No. 5,827,741, Beattie et al., issued Oct. 27, 1998, discloses cryoprotectants for human platelets, such as dimethylsulfoxide and trehalose. The platelets may be suspended, for example, in a solution containing a cryoprotectant at a temperature of about 22° C. and then cooled to below 15° C. This incorporates some cryoprotectant into the cells.
Trehalose is a disaccharide found at high concentrations in a wide variety of organisms that are capable of surviving almost complete dehydration (Crowe et al.,
Anhydrobiosis. Annul. Rev. Physiol.,
54, 579-599, 1992). Trehalose has been shown to stabilize certain cells during freezing and drying (Leslie et al.,
Biochim. Biophys. Acta,
1192, 7-13, 1994; Beattie et al.,
Diabetes,
46, 519-523, 1997).
Other workers have sought to load platelets with trehalose through use of electroporation before drying under vacuum. However, electroporation is very damaging to the cell membranes and is believed to activate the platelets. Activated platelets have dubious clinical value.
Platelets have also been suggested for drug delivery applications in the treatment of various diseases, as is discussed by U.S. Pat. No. 5,759,542, issued Jun. 2, 1998, inventor Gurewich. This patent discloses the preparation of a complex formed from a fusion drug including an A-chain of a urokinase-type plasminogen activator that is bound to an outer membrane of a platelet.
Accordingly, a need exists for the effective and efficient preservation of platelets such that they maintain, or preserve, their biological properties, particularly their response to platelet agonists such as thrombin, and which can be practiced on a large, commercially feasible scale. Further, it would also be useful to expand the types of present vehicles that are useful for encapsulating drugs and used for drug delivery to targeted sites. Accordingly further, a need also exists for the effective and efficient preservation of eukaryotic cells and erythrocytic cells, such that these cells respectively maintain their biological properties and may readily become viable after storage.
SUMMARY OF THE INVENTION
In one aspect of the present invention, a dehydrated composition is provided comprising freeze-dried platelets that are effectively loaded with trehalose to preserve biological properties during freeze-drying and rehydration. These platelets are rehydratable so as to have a normal response to at least one agonist, such as thrombin. For example, substantially all freeze-dried platelets of the invention when rehydrated and mixed with thrombin (1 U/ml) form a clot within three minutes at 37° C. The dehydrated composition can include one or more other agents, such as antibiotics, antifungals, growth factors, or the like, depending upon the desired therapeutic application.
In another aspect of the present invention, a hemostasis aid is provided where the above-described freeze-dried platelets are carried on or by a biocompatible surface. A further component of the hemostasis aid may be a therapeutic agent, such as an antibiotic, an antifungal, or a growth factor. The biocompatible surface may be a bandage or a thrombic surface, such as freeze-dried collagen. Such a hemostasis aid can be rehydrated just before the time of application, such as by hydrating the surface on or by which the platelets are carried, or, in case of an emergency, the dry hemostasis treatment aid could be applied directly to the wound or burn and hydrated in situ.
Methods of making and using inventive embodiments are also described. One such method is a process of preparing a dehydrated composition comprising providing a source of platelets, effectively loading the platelets with trehalose to preserve biological properties, cooling the trehalose loaded platelets to below their freezing point, and lyophilizing the cooled platelets. The trehalose loading includes incubating the platelets at a temperature from greater than about 25° C. to less than about 40° C. with a trehalose solution
Crowe John H.
Crowe Lois M.
Oliver Ann F.
Tablin Fern
Tsvetkova Nelly M.
Carpenter John W.
Carpenter & Kulas LLP
Chen Shin-Lin
The Regents of the University of California
LandOfFree
Erythrocytic cells and method for preserving cells does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Erythrocytic cells and method for preserving cells, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Erythrocytic cells and method for preserving cells will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3351376