Shape stabilized erythrocytes

Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se

Reexamination Certificate

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C435S366000

Reexamination Certificate

active

06436705

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to the physicochemical modification of cells and tissues, in particular erythrocytes, by reversible crosslinking agents, to increase storage stability in liquid, frozen, and dry form.
2. Description of the Related Art
There is a continuing need in the art for methods to improve the shelf-life of tissues and cells, including especially erythrocytes (red blood cells or RBCs). Typically, erythrocytes are stored under refrigeration as packed cells. As refrigerated packed cells, erythrocytes have a shelf life of six weeks. It is a goal of the art to extend this shelf life.
Other methods for storing erythrocytes under consideration include freezing and lyophilization (freeze-drying). However, these methods put a great deal of stress on erythrocytes, leading to excessive hemoglobin loss.
For a method of storing erythrocytes, whether by refrigeration, freezing, or lyophilization, for subsequent mammalian (especially human) transfusion to be satisfactory, it is desired to satisfy certain criteria. These criteria include the following: (a) avoiding erythrocyte cell membrane rupture, and consequent hemoglobin loss; (b) maintaining the ability of erythrocytes to take up and release oxygen, which will include avoiding the oxidation of hemoglobin to methemoglobin (which does not take up oxygen); (c) avoiding the loss of cell deformability, so that erythrocytes may circulate through capillaries; and (d) maintaining the viability of these erythrocytes.
The field of cryobiology describes two fundamental strategies for freezing and freeze-drying of mammalian cells: the use of cryoprotectant solutes and cryofixatives. The earliest attempt to apply these strategies to the lyophilization of erythrocytes was explored by Maryman in the early 1960's. In this work human and rat erythrocytes were lyophilized using the polymer PVP as a cryoprotectant. These experiments resulted in little success and the effort was abandoned as no cellular recovery and hemoglobin droplet formation was reported. Almost 25 years later, a group of investigators led by Crowe and colleagues used cryoprotectant carbohydrates to stabilize membranes in the dry state toward the stabilization of erythrocytes. This method employed cryoprotectant carbohydrates as water-replacement molecules with polymers such as PVP to result in red cell stabilization to freeze-drying. Thus, the development of lyophilization media is based on mixtures of stabilizing carbohydrates and matrix stabilizing polymers. Early application of such mixtures to lyophilization of red cells by Goodrich et al. showed only limited success (Goodrich Jr et al., U.S. Pat. No. 4,874,690; Goodrich Jr and Williams C. M., U.S. Pat. No. 5,171,661; Goodrich Jr et al., U.S. Pat. No. 5,178,884). Erythrocytes lyophilized in concentrated glucose and 40% PVP showed osmotic fragility and upon reconstitution and washing the cells swelled to spherocytes and lysed.
A second strategy for the stabilization of biological structures for freeze-drying is the use of fixatives. Bode A and Read M (1995) have shown that platelets lightly treated with paraforn-aldehyde retain structural integrity and some hemostatic functionality after lyophilization and rehydration. The stabilization of platelets by this irreversible crosslinking agent also results in viral inactivation. Issues that remain to be addressed in the clinical development of these preparations is the preclinical efficacy in animal models of homeostasis, and the potential for toxicities associated with trace paraformaldehyde, which can increase membrane rigidity and change the rheological properties of the cells. The loss of red cell deformability by fixation could cause significant problems in the circulatory system due to their size and shear forces encountered upon transit through the microcirculation.
U.S. Pat. No. 4,711,852 teaches a method for preparing a blood gas-hemoglobin analysis control by stabilizing red blood cells with the crosslinking agent dimethyladipimidate (DMA). Higher degree of stability was achieved with the imidoester DMA as compared to other protein cross-linking agents (formaldehyde, sodium tetrathionate, diamide, diethyl oxydiformate and dimethyl suberimidate). However, these red blood cells could not be used for transfusion.
It is desirable to add cryoprotectants to erythrocytes prior to freezing, to protect them during freezing. Unfortunately, erythrocyte membranes have little or no permeability to many cryoprotectants, including sugars, including monosaccharides (e.g., glucose) and disaccharides (e.g., sucrose). Moreover, if erythrocyte membranes were made more permeable to such cryoprotectants, such permeability would likely be deleterious to erythrocyte viability in vivo.
In short, a method for treating erythrocytes for long term (>6 weeks) storage and subsequent transfusion should satisfy the following criteria: (a) the method should maintain the ability of the erythrocytes, at the time of transfusion, to take up and release oxygen, as part of the normal respiration process; (b) the method should maintain the ability of the erythrocytes, at the time of transfusion, to pass through the circulatory system, including the capillaries, by maintaining the ability of the erythrocytes to deform; (c) the method should not rupture the cell membrane of the erythrocytes; (d) the method should preserve, at the time of transfusion. the ability of the erythrocytes to metabolize sufficiently to maintain viability for some time after transfusion.
SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to improve the storage of tissues and cells.
It is a further object of this invention to improve the storage of erythrocytes.
It is a further object of this invention to improve the liquid storage of erythrocytes under refrigeration.
It is a further object of this invention to improve the storage of erythrocytes by freezing.
It is a further object of this invention to improve the storage of erythrocytes by lyophilization.
It is a further object of this invention to improve the ability to load cryoprotectants into erythrocytes.
It is a further object of this invention to protect the ability of erythrocytes to take up and release oxygen during long term storage.
It is a further object of this invention to protect the integrity of cell membranes during long term storage.
It is a further object of this invention to protect the metabolic viability of cells after long term storage.
It is a further object of this invention to protect the physical properties of cells (e.g., deformability) after long term storage.
It is a further object of this invention to achieve all of the foregoing objects in a manner that is consistent with viability and in vivo use of cells and tissues, including erythrocytes (e.g., for human and other mammalian transfusion).
These and additional objects of the invention are accomplished by the structures and processes hereinafter described.
One aspect of the present invention is a method for storing tissues and cells (typically eiythrocytes) having the step of (1) stabilizing the cells with a reversible stabilizing agent. This method typically will have the additional steps of (2) loading the cells with a cryoprotectant, and typically (3) storing the cells in liquid, frozen, or dry state. This method will also typically have the additional step of (4) prior to use, reversing the stabilization reaction. Preferably, the erythrocytes are pre-treated with CO to complex the hemoglobin with CO.
It is anticipated that a practical method according to the invention will include reoxygenation of the erythrocytes, and also washing out reagents prior to in vivo use.
Another aspect of the present invention is an erythrocyte that has had its shape stabilized by the reversible crosslinking of proteins in the erythrocyte, such as the structural proteins of the cytoskeleton.
Another aspect of the invention is a population of such reversibly cross-linked erythrocytes.
Another aspect of the invention is the in vi

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