Methods of harvesting rare cells from blood products

Details Methods of harvesting rare cells from blood products Methods of harvesting rare cells from blood products

1999-09-30

2003-04-08

Smith, Lynette R. F. (Department: 1645)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S455000, C435S325000, C435S002000, C435S378000, C436S519000, C436S520000, C436S536000, C436S537000, C204S166000, C210S806000, C210S793000, C210S678000, C424S093700

Reexamination Certificate

active

06544751

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to harvesting rare cells from blood products and/or obtaining products of the rare cells.
BACKGROUND OF THE INVENTION
Blood is a complex biological tissue comprising a solution, suspended particulate macromolecules, and a wide variety of cell types. Blood is routinely collected from donors and typically separated into component fractions (e.g., packed red cells (PRC), platelet concentrate (PC), and plasma), and fractions such as plasma can be further processed. The component fractions are typically used to produce transfusion products.
Donated blood or component fractions can be filtered in order to separate leukocytes from the blood product, since transfusion of leukocytes can cause undesirable effects in the recipient. For example, certain leukocytes (e.g., granulocytes) which mediate immune response by distinguishing between “self” and “non-self” matter are also responsible for “graft versus host” (GVH) disease in which transfused leukocytes attack the host, often causing substantial damage to various host tissues. As the onset of GVH greatly complicates blood transfusions even between related individuals, efficient means for filtering such cells have been developed in order to minimize this risk.
As a cell fraction, leukocytes represent a disparate grouping of rare cell types. Granulocytes (e.g., neutrophils, basophils, eosinophils, etc.), monocytes, macrophages, and the like are generally phagocytotic and capable, at varying degrees, of amoeboid movement. These cell types represent the majority of rare cells (about 60-80%). These amoeboid cells can be activated by the presence of certain hormones (e.g., bacterial toxins, cytokines (notably interleukin IL-2), products of the complement complex (notably C3a, C4a, and C5a), hormone activating factors, etc.), and these cells exhibit chemotaxic movement along concentration gradients of activating factors. Thus, while blood-borne, these amoeboid cells are often localized in the sites of inflammation, infection, or injury outside of blood vessels, having left the blood vessels by the processes of diapedesis and migration in response to chemotaxic stimuli. Granulocytes isolate and/or destroy “foreign” matter, and granulocytes also present antigens to lymphocytes. Other rare cells, (e.g., lymphocytes, dendritic cells, and stem cells) are distinguishable from the amoeboid leukocytes such as granulocytes.
Donated blood represents a potential source of rare cells for clinical use and further study. However, methods for routinely and economically harvesting rare cells from blood products are generally lacking or are problematic. For example, rare cells are often isolated indiscriminately or with low efficiency. Furthermore, methods for preferentially isolating a given population of cells primarily confined to one given population (e.g., filtration leukapheresis of granulocytes) are often undesirable in certain applications. The inability to preferentially isolate some types of lymphocytes (e.g., cells that produce interferon, lymphokines, hormones, and other factors) is a particular problem, since recombinantly produced factors are often inferior in quality, or difficult and/or expensive to purify.
In view of the foregoing problems, there exists a need for an improved method for harvesting rare cells from blood products. The present invention provides such a method for harvesting rare cells from blood products. The present inventive method further provides for the isolation or separation of sub-populations of rare cells, and it affords the ability to culture a population of rare cells in order to obtain products of rare cells.
SUMMARY OF THE INVENTION
The present invention provides a method of harvesting rare cells from blood products. The method involves first contacting a blood product containing rare cells with a porous medium and selectively retaining rare cells of interest on the porous medium. Subsequently, the porous medium is contacted with an elution fluid to elute a population of the rare cells from the porous medium. In some embodiments, the method involves selectively eluting a first population of rare cells and retaining a second population of rare cells on the porous medium.
Embodiments of the method include obtaining products of rare cells selectively retained on the porous medium by contacting the porous medium containing the rare cells of interest with a nutrient-rich culture solution such that a population of the rare cells is cultured on the porous medium. The products of the rare cells thereafter can be isolated from the culture solution.
The present invention provides rare cells (or any sub-population thereof), or the products produced by such rare cells, for a variety of uses, e.g., for further study, or for clinical or academic uses. Embodiments of the present invention further provide, develop, or facilitate means of assaying activity, number, cell type, concentration, viability, filterability, secretory or metabolic activity, or other parameters relating to rare cells. Additionally, embodiments of the present inventive method provide for developing methods for testing the efficiency of leukodepletion of blood products or depletion of sub-populations of rare cells. These and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
SPECIFIC DESCRIPTION OF THE INVENTION
Definitions
As used herein, including the claims appended hereto, the following terms are defined as follows:
Rare cells are cells in blood products; generally rare cells are blood cells other than erythrocytes or platelets, e.g., leukocytes. Rare cells can be unspecialized cells such as stem cells, or specialized cells, e.g., types of leukocytes such as lymphocytes (including classes of lymphocytes such as T cells and B cells) and dendritic cells.
Blood product is a composition, any component of which (and preferably all of which) is derived from blood. Thus, blood product can be whole blood; however, the blood product also can be any fraction thereof (e.g., plasma, packed red cells, buffy coat, a concentrated suspension of cells in a solution, especially a product including rare cells).
A particulate is retained on a porous medium if the porous medium acts as a substrate for the particulate; thus, a particulate is retained either on the surface of the porous medium or within the pores of the porous medium.
A class of particulates is selectively retained on a substrate if it is retained with greater affinity in comparison with another class of particulates. A class of particulates is not selectively retained if it is retained with the same or lesser affinity than another class of particulates, even if the class of particulates is retained on the substrate with some affinity.
A class of particulates is selectively eluted from the substrate if it is eluted from the substrate to a greater extent than another class of particulates, even if that other class of particulates is eluted from the substrate to some extent.
For ease of reference, the upstream surface of a porous medium is the surface initially contacted with the blood product, while the downstream surface is the other side, e.g., the side through which permeate is initially discharged.
The present invention provides a method of harvesting rare cells from blood products. The method involves first contacting a blood product containing rare cells with a porous medium wherein rare cells of interest are selectively retained on the porous medium. Subsequently, the porous medium is contacted with an elution fluid wherein a population of the rare cells of interest is eluted from the porous medium. Embodiments of the method include separating rare cells into distinct populations of cells.
The present inventive method involves first contacting a blood product containing rare cells with a porous medium. An appropriate blood product is any blood product having rare cells; suitable blood products can comprise other components as well (such as

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