Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
2000-01-21
2003-07-08
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S366000, C435S173500, C435S173600, C435S173700
Reexamination Certificate
active
06589786
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods and apparatuses for selecting specific cell types from cell suspensions, specifically those employing applied electric fields.
BACKGROUND OF THE INVENTION
The ability to isolate specific sub-populations of cells from cell suspensions is of critical importance to many applications in the biological sciences as well as to many therapies in clinical medicine. For example, the basis of many medical therapies for treating a variety of human diseases and for countering the effects of a variety of physiological injuries involves the isolation, manipulation, expansion, and/or alteration of specific biological cells. One particularly important example involves the reconstitution of the hematopoietic system via bone marrow or progenitor cell transplantation. More specific examples include: autologous, syngeneic, and allogenic stem cell transplants for immune system reconstitution following the myeloablative effects of severe high dose chemotherapy or therapeutic irradiation; severe exposure to certain chemical agents; or severe exposure to environmental radiation, for example from nuclear weapons or accidents involving nuclear power generators.
Intensive chemotherapy and/or irradiation for the treatment of a variety of cancers, including breast cancer, has become a commonly used approach in cancer care centers. Such treatments are associated with severe ablation of the bone marrow cells required for function of the blood and immune systems. Such bone marrow cells are derived from a small number of progenitor cells known as hematopoietic stem cells in the normal bone marrow. Therefore, patients receiving such therapies require life-saving transplants of stem cells in order to survive the effects of the treatment. Stem cell containing tissue for transplant may be derived from donor marrow (allogeneic transplant) or from the patient's own bone marrow or peripheral blood after mobilization (autologous transplant). In both instances, there is a need for effective cell separation methods to enrich the transplant tissue in stem cells and reduce the number of undesirable and deleterious cells (e.g. mature T cells for allogeneic transplants and residual cancerous cells for autologous transplants). For example, for autologous adjuvant stem cell transplant therapy following myeloablative cancer treatments, it is believed that reinfusion of residual tumor cells is a major cause of post therapy relapse. Clearly, removing such cells from transplanted tissue would be beneficial to the patient.
A number of cell isolation, cell separation, and cell purging strategies have been employed in the prior art for purifying or removing cells from a suspension. Prior art cell separation methods used to isolate cells or purge cell suspensions typically fall into one of three broad categories: physical separation methods typically exploit differences in a physical property between cell types, such as cell size or density (e.g. centrifugation or elutriation); chemical-based methods typically employ an agent that selectively kills or purges one or more undesirable cell types; and affinity-based methods typically exploit antibodies that bind selectively to marker molecules on a cell membrane surface of desired or undesired cell types, which antibodies may subsequently enable the cells to be isolated or removed from the suspension. While physical separation methods can be advantageous with regard to their ability to separate cells without causing undo damage to desired cells, current physical separation methods typically have relatively poor specificity and do not typically yield highly purified or highly purged cell suspensions. While many chemical and affinity methods have better selectivity than typical physical methods, they can often be expensive or time consuming to perform and can cause considerable damage to, or activation of, desired cells, for example stem cells, and/or can add undesirable agents to the purified or isolated cell suspensions (e.g. toxins, proliferation-inducing agents, and/or antibodies). An additional potential problem with antibody-based cell separation techniques typically employed for purification of stem cells, is that they select stem cells solely on the basis of cell surface markers (e.g., CD34) and will not select cells lacking such markers.
In addition to cancer therapy, there are a number of other important medical therapies which exist, or are under development, that are based on cells derived from a variety of different types of stem cells. Examples include pre-exposure prophylaxis or post-exposure therapies under development for a variety of biological exposures that may occur naturally (e.g., viral exposure for example with Ebola, etc.) or be inflicted by mankind (i.e., biological warfare agents). A variety of gene therapies involving genetically manipulated stem cells, are being contemplated or are under development for treating a variety of blood-related diseases (e.g., AIDS, leukemia, other cancers, etc.). Gene therapy techniques based on genetically manipulated stem and/or germ cells may also be useful in cloning organisms, such as animals. However, genetically manipulating stem cells using many current technologies is difficult, typically employing viruses or gene carriers that can be time consuming and expensive, or may be dangerous to perform and may not have high yields. Current research findings also suggest that the practical implementation of animal organ transplants into human recipients also may require procedures involving stem cells from both the donor and recipient. Many of these promising therapies would require cryopreservation and storage of donor specimens including human stem cells, for example, as derived from the stem cell-rich umbilical cord blood of newborns, which can provide such donors with a therapeutic basis for hematopoietic reconstitution or gene therapy should a health emergency occur later in life. If such storage demands are to be realistically met, the specimens will need to have minimal volume, and, therefore, successful implementation of such technologies may rest on the development and availability of effective methods for isolating trace numbers of stem cells from sources such as umbilical cord blood and the fetal liver. In order to achieve broad implementation of the therapies discussed above and others, rapid and cost effective methods are needed to isolate, with high purity, desired target cells from suspensions having a diverse mix of cell types and concentrations.
The use of applied electric fields to physically manipulate cells is known. Applied electric fields have been employed in the prior art for cell inactivation and sub-lethal cell membrane electroporation. For example, U.S. Pat. 5,048,404 to Bushnell discloses a system and method for sterilizing liquid foodstuffs by killing microorganisms with exposure to pulsed electric fields.
Sale and Hamilton (“Effects of High Electric Fields on Microorganisms I. Killing of Bacteria and Yeasts,”
Biochim et Biophys Acta,
148:781 (1967); and “Effects of High Electric Fields on Microorganisms II. Mechanism of Action of the Lethal Effect,”
Biochim et Biophys Acta,
148:789 (1967)) studied the effect of pulsed electric fields on suspensions of bacteria or suspensions of yeasts. Specifically, they investigated the effect on the degree of cell kill by the field as a function of field strength and exposure time. The effect of pulsed electric fields on the killing of bacteria was also studied by Hülsheger et al. (“Lethal Effects of High-Voltage Pulses on
E. Coli
K12,
” Radial Environ Biophys,
18:281(1980); and “Killing of Bacteria with Electric Pulses of High Field Strength,”
Radial Environ Biophys,
20:53(1981)). Hülsheger et al. studied the effects on bacterial cell death of a variety of experimental parameters and were able to demonstrate a 99.9% reduction in the number of living bacterial cells in suspensions after exposure to certain pulsed electric field parameters.
The lysis of erythrocytes in erythrocyte suspensions by pu
Eppich Henry M.
Mangano Joseph A.
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