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Title:  Ex vivo generation of functional leukemia cells in a three-dimensional bioreactor
United States Patent: 
7,087,431
Issued: 
August 8, 2006

Inventors: 
Wu; J. H. David (Pittsford, NY), Mantalaris; Athanassios (Middlesex, GB), Panoskaltsis; Nicki (Middlesex, GB)
Assignee: 
University of Rochester (Rochester, NY)
Appl. No.:  09/796,830
Filed: 
March 1, 2001


 

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Abstract

The present invention provides cultured leukemia cells. The method comprises isolating mononuclear cells, which contain leukemia cells, culturing the leukemia cells in a chamber having a scaffolding covered or surrounded with culture medium, where the scaffolding allows for leukemia cells to have cell to cell contacts in three dimensions. The subject leukemia cells are useful for screening compounds which inhibit or stimulate leukemia cell function or formation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of culturing leukemia cells. The method comprises isolating mononuclear cells, which contain leukemia cells and culturing such cells in a chamber having a scaffolding covered or surrounded in culture medium where the scaffolding allows for the leukemia cells to have cell to cell contacts in three dimensions.

The present invention also provides a method for screening for compounds which effect leukemia cell formation. The method comprises isolating mononuclear cells which contain leukemia cells, culturing the cells in a container having a scaffolding covered or surrounded with culture medium where the scaffolding allows for leukemia cells to have cell to cell contacts in three dimensions, adding a test compound to the container, removing cultured cells, and determining the ability of a test compound to effect leukemia cell formation.

The present invention also provides a method for diagnosis or prognosis of leukemia. The method comprises isolating mononuclear cells which possibly contain leukemia cells, culturing the cells in a container having a scaffolding covered or surrounded with culture medium where the scaffolding allows for cells to have cell to cell contacts in three dimensions and leukemia cell formation, and determining the type and the stage of leukemia.

As used herein, the term "leukemia cells" means abnormal cells of the bone marrow and lymphoid tissues and may include, e.g., acute myelocytic leukemia cells, chronic lymphocytic leukemia cells. Acute myelocytic leukemia cells are abnormal cells of the bone marrow of the myeloid lineage arrested at an early stage of maturation. Chronic lymphocytic leukemia cells are abnormal cells of the bone marrow and lymphoid tissues of the lymphocyte lineage arrested at a mature stage of development. The mononuclear cells may be isolated from many different sources such as bone marrow aspirate or biopsy, chloroma, or spleen, peripheral blood, infiltrated organs, tissues, and body fluids, cord blood, fetal liver, and cell lines. The leukemia cells are preferably mammalian leukemia cells. In a more preferred embodiment, the mammalian leukemia cells are human leukemia cells.

In accordance with the present invention, a bioreactor system and method for generating functional leukemia cells is provided. As used herein, "functional leukemia cells" mean leukemia cells which function to sustain the leukemic state (i.e. they do not mature in culture but remain viable and behave as they would in the human host). The bioreactor of the present invention provides a three-dimensional structure which mimics the natural extracellular matrix and ample surface area of the leukemia cells and allows cell to cell interaction at a tissue-like cell density. It is understood that the bioreactor of the present invention may have many different configurations so long as it provides a three-dimensional structure. With respect to the bioreactor, the term "three-dimensional structure" is used interchangeably with the term "scaffolding".

The bioreactor for use in generating functional leukemia cells comprises a container or vessel having at least one chamber or section with scaffolding located therein. The scaffolding for use in the chamber or container may consist of tangled fibers, porous particles, or a sponge-like material. The scaffolding may be formed from a material selected from the group consisting of a synthetic polymer, a natural substance, and a semisynthetic material and may be degradable or non-degradable. Culture media is placed over or around the porous or fibrous substrate.

FIG. 1A (see Original Patent) illustrates one possible configuration of a bioreactor which may be used to generate functional leukemia cells. In FIG. 1 (see Original Patent), the porous or fibrous scaffolding is located in a lower, culture chamber. It is understood that the bioreactor of the present invention may have any number of configurations so long as it provides a three dimensional structure (scaffolding).

The walls of the container or vessel may comprise any number of materials such as glass, ceramic, plastic, polycarbonate, vinyl, polyvinyl chloride (PVC), metal, etc. Culture medium which will support the growth and differentiation of hemopoietic and/or accessory cells into functional leukemia cells is placed over and/or around the porous or fibrous material.

Many different porous or fibrous materials may be used as scaffolding in the bioreactor such as, e.g., tangled fibers, porous particles, sponge, or sponge-like material. The porous or fibrous scaffolding allows leukemia cells to lodge onto, proliferate and, if needed, differentiate. For purposes of example only and not limitation, suitable scaffolding substrates may be prepared using a wide variety of materials including natural polymers such as polysaccharides and fibrous proteins, synthetic polymers such as polyamides (nylon), polyesters, polyurethanes and minerals including ceramics and metals, coral, gelatin, polyacrylamide, cotton, glass fiber, corrageenans, and dextrans. Examples of tangled fibers include glass wool, steel wool, and wire or fibrous mesh.

Examples of porous particles include, e.g., beads (glass, plastic, or the like) cellulose, agar, hydroxyapatite, treated or untreated bone, collagen, gels such as Sephacryl, Sephadex, Sepharose, agarose or polyacrylamide. "Treated" bone may be subjected to different chemicals such as, acid or alkali solutions. Such treatment alters the porosity of bone. If desired, the substrate may be coated with an extracellular matrix or matrices, such as, collagen, matrigel, fibronectin, heparin sulfate, hyalumonic and chondroitin sulfate, laminin, hemonectin, or proteoglycans.

The fibrous or porous material used as scaffolding in the bioreactor forms openings or pores into which leukemia cells enter. Once entered, the cells become entrapped or adhered to the fibrous or porous material and colonize and/or aggregate thereon. Cell attachment and colonization can occur merely by inoculating the cells into the culture medium which overlays and/or surrounds the porous or fibrous substrate. Cell attachment and colonization may also occur by inoculating the cells directly onto the porous or fibrous substrates.

In accordance with the present invention, leukemia cells must be able to enter the openings (pores) of the fibrous or porous material. The skilled artisan is cognizant of the different sizes of leukemia cells and, therefore, the pore size needed to accommodate such cells. Generally speaking, a pore size in the range of from about 15 microns to about 1000 microns may be used. Preferably, a pore size in the range of from about 100 microns to about 300 microns is used.

In a preferred embodiment, a membrane is placed in the bioreactor in order to facilitate gas exchange. The membrane is gas permeable and may have a thickness in the range of from about 10 to about 100 .mu.m. In a more preferred embodiment, the membrane has a thickness of about 50 .mu.m. The membrane is placed over an opening in the bottom or side of the chamber or container. In order to prevent excessive leakage of media and cells from the bioreactor, a gasket may be placed around the opening and/or a solid plate placed under or alongside the opening and the assembly fastened.

The cell medium used in the bioreactor may be any of the widely known media used to support growth and differentiation of bone marrow cells and, in particular, growth and differentiation of hemopoietic and/or accessory cells into functional leukemia cells. For example, the following classical media may be used and supplemented, if desired, with vitamin and amino acid solutions, serum, and/or antibiotics: Fisher's medium (Gibco), Basal Media Eagle (BME), Dulbecco's Modified Eagle Media (D-MEM), Iscoves's Modified Dulbecco's Media, Minimum Essential Media (MEM), McCoy's 5A Media, and RPMI Media.

Specialized media may also be used such as, MyeloCult.TM. (Stem Cell Technologies) and Opti-Cell.TM. (ICN Biomedicals). If desired, serum free media may be used such as StemSpan SFEM.TM. (StemCell Technologies), StemPro 34 SFM (Life Technologies), and Marrow-Gro (Quality Biological Inc.).

In a preferred embodiment, McCoy's 5A medium (Gibco) is used at about 70% v/v, supplemented with vitamin and amino acid solutions. In an even more preferred embodiment, the culture medium comprises approximately 70% (v/v) McCoy's 5A medium (Gibco), approximately 1.times.10.sup.-6 M hydrocortisone, approximately 50 .mu.g/ml penicillin, approximately 50 mg/ml streptomycin, approximately 0.2 mM L-glutamine, approximately 0.45% sodium bicarbonate, approximately 1.times. MEM sodium pyruvate, approximately 1.times. MEM vitamin solution, approximately 0.4.times.MEM amino acid solution, approximately 12.5% (v/v) heat inactivated horse serum and approximately 12.5% heat inactivated FBS, or autologous serum. The medium chamber may be continuously perfused if desired.

The bioreactor is inoculated with leukemia cells by gently adding e.g., pipetting, into the three-dimensional scaffolding portion of the bioreactor. Alternatively, the leukemia cells may be added to the culture covering and/or surrounding the three dimensional scaffolding. Cells will settle or migrate into the porous or fibrous material making up the scaffolding. The number of cells added to the bioreactor depends on the total area of the three-dimensional scaffolding and volume of culture media. Preferably, leukemia cells isolated from any of the sources discussed extensively herein, are centrifuged through a gradient such as a Ficol/Plaque to remove mature red blood cells, yielding mononuclear cells.

For a bioreactor having a culture chamber of about 3/16'' height by about 5/16'' width by about 5/16'' length and packed with about 0.01 g of a porous or fibrous substrate, the number of mononuclear cells added to the bioreactor may be anywhere in the range of from about 10.sup.4 to 10.sup.9 mononuclear cells. Preferably, 4 6.times.10.sup.6 cells may be used to inoculate the bioreactor. Using these guidelines, one skilled in the art is able to adjust the number of cells used to inoculate the bioreactor depending on the total area of the three-dimensional scaffolding, volume of culture media, type of three-dimensional scaffolding, and source of leukemia cells.

Preferably, the culture is fed every second day with the culture medium with or without exogenous growth factors. Various other ingredients may be added to the culture media in order to further stimulate leukemia cells growth and differentiation. Thus, for example, cytokines, such as, granulocyte colony stimulating factor, granulocyte monocyte colony stimulating factor, IL3, or IL2 may be added to the culture medium.

The cell culture is allowed to grow anywhere from about a few days to a few weeks. Preferably, the cultures are harvested after about three weeks.

Cells may be harvested in any number of well known methods. The chamber may be treated with any suitable agent, such as collagenase, to release the adhering cells. Non-adhering cells may be collected as they release into the medium. Cells may also be removed from the substrate by physical means such as shaking, agitation, etc. Thereafter, the cells are collected using any known procedure in the art such as, pipetting or centrifugation. Preferably, non-adherent cells are released by gentle stirring and mixing the bed of porous or fibrous material and then collected by centrifugation or sedimentation.

If desired, the cell samples collected from the bioreactor may be further enriched for leukemia cells using well known methods of positive selection. Thus, for example, a solid support (such as beads) having an antibody that binds leukemia cells conjugated thereto, may be mixed with the cell sample. Antibody conjugated beads with leukemia cells bound thereto are then collected by gravity or other means such as a magnet, in the case of magnetic beads.

Negative selection may also be used as a means of enriching the leukemia cells population in the cell sample removed from the bioreactor. With a negative selection scheme, a solid support (such as beads) having conjugated thereto one or more antibodies which react with cells other than leukemia cells may be mixed with the cell sample. Antibody conjugated beads with cells other than leukemia cells bound thereto are then collected by gravity or other means such as a magnet, in the case of magnetic beads.

In either positive or negative selection, the leukemia cells may be further isolated by filtration based on size. In accordance with the present invention, however, the cell samples removed from the bioreactor comprise functional leukemia cells which may be used in many different clinical and drug screening settings without being further enriched.

Leukemia cells may be identified using any of the well known indicia such as abhorrent surface marker staining which is unique to each leukemia type.

The cultured leukemia cells of the present invention have a myriad of uses in the therapeutic and pharmaceutical industries. For example, the subject leukemia cells may be used to screen for drugs and therapeutics (including immunotherapies) which either inhibit or stimulate leukemia cell formation or function.

It is now known that leukemia is associated with overproduction of leukemia cells. For example, leukemia cell formation is a condition whereby a disturbance in the DNA of a previously normal cell of the bone marrow or lymphoid tissues makes cells more prone to proliferation. Thus, inhibitors of leukemia cell formation identified by the assays of the present invention are useful for the treatment of leukemia.

Thus in accordance with the present invention, there are provided methods of screening for drugs which affect leukemia cell formation. As used herein, "drug" or "test compound" encompasses any element, molecule, chemical compound, hormone, growth factor, nucleotide sequence (including oligonucleotides), protein (including peptides), cells, irradiation, and reagents which have the ability to inhibit or stimulate leukemia cell formation and function.

In a typical screening assay for a drug which affects leukemia cell production, cultured leukemia cells are removed from the bioreactor and placed in a petri dish, flask, microscope slide, microtiter dish or the like with enough culture medium or buffered solution to keep the leukemia cells alive. The liquid medium should preferably mimic the blood environment of the body since this is ultimately where the drug which inhibits function will be acting. Preferably, a pH of approximately 7.2, and a temperature of about 37.degree. C. is maintained. The number of leukemia cells which may be used in a screening assay is empirical. Typically, a sample containing 1.times.10.sup.6 total cells may be used, depending upon the number of leukemia cells in the cell sample.

The number of leukemia cells in a cell sample relative to other cells may be determined microscopically by counting morphologically leukemic cells or blasts. Immunohistochemical staining, flow cytometry, or a combination thereof may also be performed. Methods of cell counting are well known in the art. The concentration of the test compound--i.e., the drug to be screened as a potential inhibitor of leukemia cell activity is empirical. One skilled in the art is familiar with methods of adjusting concentrations of different compositions in order to best identify the effects of a test compound in the screening assay. Typically, a range of concentrations is used and those portions of the range which exhibit serious deleterious effects on leukemia cell viability are eliminated from further study. Those portions of the range having less deleterious effects on leukemia cell viability are identified and used for further study of inhibitory or stimulatory effects on leukemia formation, leukemia activity, or leukemia cells functionality.

The mixture of leukemia cells and test compound is incubated for a time and under conditions sufficient for the inhibition or stimulation of leukemia cell activity to be carried out. As defined herein, a sufficient time can be anywhere from about five minutes to several hours or more. When leukemia cells are tested in a petri dish, flask, microscope slide, microtiter dish, or the like, a sufficient time may be several minutes to several hours. Of course, the test time may be extended if needed in order to see effects on the cells. The skilled artisan is able to determine the optimal time for running the screening assay by removing samples and examining cells microscopically for viability.

A preferred buffer for use in the reactions is Phenol red-free MEM supplemented with 1.times. nonessential amino acids, 1.times. L-glutamine, 10% FBS, 50 U/ml penicillin, and 50 .mu.g/ml streptomycin. In a preferred embodiment, the test reaction volume is between about 0.5 and about 2 ml. In a more preferred embodiment, the reaction volume is about 1 ml. In a preferred embodiment, the incubation temperature is approximately 37.degree. C.

In an alternative embodiment, there is provided a method for screening for drugs which either inhibit or stimulate leukemia cell formation. In this embodiment, a test compound is added directly to the bioreactor. The test compound may be added to the culture medium or into the three dimensional scaffolding. The time at which the test compound is added is empirical but is relatively early. Typically, control runs are performed in which no test compounds are added to the bioreactor.

The ability of a test compound to inhibit leukemia cell formation may be determined by leukemia cell count, immunohistochemical staining, flow cytometry, or a combination thereof. Methods of cell counting are well known in the art. Cell counts are compared between experimental and control assays. Increased numbers of leukemia cells compared to control runs correlate with the identification of a stimulator of leukemia cell formation. Decreased numbers of leukemia cells compared to control runs correlate with the identification of an inhibitor of leukemia cell formation.

As described above, however, any available test compound may be used to screen for effective inhibitors of leukemia cell formation, activity, or functionality.

In one aspect of the present invention the leukemia cells are isolated from a leukemia patient and, then, subjected to the cell culturing process of the present invention. The test compound is then used to treat such cultured cells in order to determine which compound is particularly effective in treating the patient's leukemia. Based on this test procedure, the most effective test compound is administered to the patient to inhibit growth of leukemia cells in the leukemia patient.

The present invention provides the means of sustaining a patient's leukemic cells in culture in order to provide leukemic antigens for immunotherapy purposes. Examples of this include donor lymphocyte infusions and dendritic cells therapies.

The present invention also provides a method for identifying genes which are related to in leukemia cell formation or function. In this aspect of the invention, various parameters of the culture conditions may be changed (e.g., nutrient ingredients (including leukemia cell agents, temperature, oxygen concentration, CO.sub.2 concentration, and nutrient composition), cytokine environment, cellular content, and inhibition of receptors, signaling, or adhesion molecules. After altering one or more parameters, leukemia cell number and function is determined. The leukemia cell number may be determined by morphological, immunohistochemical, or flow cytometric techniques. If changes in leukemia cell number and function occur in a test sample when compared to a control sample, then the system may be used to further screen for the gene or genes accountable for the change. Differential gene display, its modified versions such as RNA arbitrarily primed (RAP)-PCR technique or gene microarray analysis, may be used to further identify and characterize the genes involved. These methods are well known in the art. Alternatively, comparison with a 2-dimensional Dexter culture which does not support leukemia cells can be used to screen for genes. Furthermore, the genes associated with the leukemia cells of the test sample may be identified by cloning the genes expressed by the purified or enriched subject leukemia cells.
 


Claim 1 of 33 Claims

1. A method of culturing leukemia cells, comprising: isolating mononuclear cells, which contain leukemia cells, from a subject and culturing the cells in a chamber having a scaffolding covered or surrounded with culture medium, wherein said scaffolding allows for leukemia cells to have cell to cell contacts in three dimensions, and the leukemia cells are cultured for a duration of at least four weeks.
 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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