Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
1994-03-31
2001-06-05
Martinell, James (Department: 1633)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C424S093100, C424S093210
Reexamination Certificate
active
06241984
ABSTRACT:
TECHNICAL FIELD
The field of this invention is growth and expansion of hematopoietic cells in culture.
BACKGROUND
Mammalian hematopoiesis has been studied in vitro through the use of various long-term marrow culture systems. Dexter and co-workers described a murine system from which CFU-S and CFU-GM could be assayed for several months, with erythroid and megakaryocytic precursors appearing for a more limited time. Maintenance of these cultures was dependent on the formation of an adherent stromal cell layer composed of endothelial cells, adipocytes, reticular cells, and macrophages. These methods were soon adapted for the study of human bone manrow. Human long-term culture systems were reported to generate assayable hematopoietic progenitor cells for 8 or 9 wks and, later, for up to 20 wks. Such cultures are again relying on the preestablishment of a stromal cell layer which is frequently reinoculated with a large, heterogeneous population of marrow cells. Hematopoietic stem cells have been shown to home and adhere to this adherent cell multilayer before generating and releasing more committed progenitor cells. Stromal cells are thought to provide not only a physical matrix on which stem cells reside, but also to produce membrane-contact signals and/or hematopoietic growth factors necessary for stem cell proliferation and differentiation. This heterogeneous mixture of cells comprising the adherent cell layer presents an inherently complex system from which the isolation of discrete variables affecting stem cell growth has proven difficult.
Recently, a study was conducted by McNiece and Langley which examined the stimulatory effect of recombinant human stem cell factor (MGF) on human bone marrow cells alone and in combination with recombinant human colony stimulating factors, GM-CSF, IL-3 and erythropoietin. The results showed that MGF stimulation of low density non-adherent, antibody depleted CD34+ cells suggests that MGF directly stimulates progenitor cells capable of myek)id and erythroid differentiation.
RELEVANT LITERATURE
Conditions which allow long term in vitro bone marrow culture are described in Dexter, et al. (1977) J. Cell. Physiol. 91:335-344; Gartner, et al. (1980) P.N.A.S. 77:4756-4759 and Hocking, et al. (1980) Blood 56:118-124. Survival of granulocytic progenitors is shown by Slovick, et al. (1984) Exp. Hematol. 12:327-338. Roberts, et al. (1987) J. Cell. Physiol. 132:203-214 describes the use of 3T3 cells in such cultures.
Cell surface antigen expression in hematopoiesis is discussed in Strauss, et al. (1983) Blood 61:1222-1231 and Sieff, et al. (1982) Blood 60:703-713. Descriptions of pluripotential hematopoietic cells; are found in McNiece, et al. (1989) Blood 74:609-612 and Moore, et al. (1979) Blood Cells 5:297-311. Characterization of a human hematopoietic progenitor cell capable of forming blast cell containing colonies in vitro is found in Gordon, et al. (1987) J. Cell. Physiol. 130:150-156 and Brandt, et al. (1988) J. Clin. Invest. 82:1017-1027.
Characterization of stromal cells is found in Tsai, et al. (1986) Blood 67:1418-1426 and Li, et al. (1985) Nature 316:633-636. The localization of progenitor cells in the adherent layer of cultures is discussed in Coulombel, et al. (1983) Blood 62:291-297 and Gordon, et al. (1985) Exp. Hematol. 13:937-940.
Eliason, et al. (1988) Exp. Hematol. 16:307-312 describes GM-CSF and IL-3 in hematopoiesis. The effect of growth factors in megakaryopoiesis is found in Brno, et al. (1988) Exp. Hematol. 16:371-377. McNiece, et al (1991) Exp. Hematol. 19:226-231 describes the use of stem cell factor in in vitro cultures.
SUMMARY OF THE INVENTION
In accordance with an aspect of the present invention there is provided a process for supporting mammalian bone marrow cells wherein such cells are maintained in a culture medium essentially free of stromal cells and which includes at least one cytokine effective for supporting such cells.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Preferred embodiments of this aspect of the present invention provide a process for supporting bone marrow cells which are hematopoietic stem cells, a process for supporting bone marrow cells which are hematopoietic progenitor cells and a process for supporting bone marrow cells which are CD34+ DR− CD15− cells.
In addition this invention provides that at least one cytokine be selected from the following cytokines: Interleukin (IL)-1, IL-3, IL-6, granulocyte/macrophage-colony stimulating factor (GM-CSF), human or murine stem cell factor, sometime referred to as human or murine mast cell growth factor (MGF) or Steel Factor (SL) and a fusion protein of GM-CSF/IL-3 (FP).
In accordance with another aspect of the present invention there is provided a process for supporting mammalian bone marrow cells wherein such cells are maintained in a culture medium containing a combination of cytokines effective for supporting such cells. Preferably, the bone marrow will be supported in a culture medium which is essentially free of stromal cells.
Additional preferred embodiments of this invention provide a process for supporting bone marrow cells which are he-matopoietic stem cells. A process for supporting bone marrow cells which are hematopoietic progenitor cells and a process for supporting bone marrow cells which are CD34+ DIR− CD15− cells.
Preferably, the culture medium will contain at least one of the following cytokine combinations IL-1/IL-3; IL-3/IL,-6; IL-3/MGF; IL-3/GM-CSF; MGF/FP. Applicant has found that such combinations provide for an improved rapid expansion of the cell population.
The term “supporting” with respect to stem cells and other progenitor cells means maintaining and/or expanding and/or promoting some differentiation of such cells.
The following are representative examples of cytokines which may be employed in the present invention. The cytokines may be human in origin, or may be derived from other mammalian species when active- on human cells. IL-1 is used in an amount effective to support the cells, generally such amount is at least 1 U/ml and need not exceed 10 U/ml, preferably 2.5 U/ml, where the specific activity is 10
8
CFU/mg protein. IL-6 is used in an amount effective to support the cells, generally such amount is at least 500 pg/ml and need not exceed 10 ng/ml, preferably 1 ng/ml. IL-3 is used in an amount effective to support the cells, generally such amount is at least 500 pg/ml and need not exceed 2 ng/ml, preferably 500 pg/ml. GM-CSF is used in an amount effective to support the cells, generally such amount is at least 100 pg/ml and need not exceed 1 ng/ml, preferably 200 pg/ml. c-kit ligand (MGF, steel factor, stem cell factor) may be human or murine in origin, is used in an amount effective to support the cells, generally such amount is at least 10 ng/ml and need not exceed 500 ng/ml, preferably 50 to 100 ng/ml. FP (fusion protein of IL-3 and GM-CSF as described in Broxmeyer, et al. [1990] Exp. Hematol. 18:615) is used in an amount effective to support the cells, generally such amount is at least 1 ng/ml and need not exceed 25 ng/ml, preferably 10 ng/ml.
The use of a cytokine in. the absence of stromal cells is particularly suitable for expanding the mammalian bone marrow stem cells and in particular progenitor cells. The cells which are supported in accordance with the present invention are preferably of human origin.
In accordance with a preferred aspect of the present invention, a cell population which is supported in accordance with the present invention is that which is positive for CD34 antigen and is negative for HLA-DR and is also negative for CD15.
Specifically this aspect of the present invention provides for a cell population of CD34+ DR− CD15− supported in accordance with the process described above, where the population has doubled in a period of time which does not exceed 15 days. Preferably, the population has doubled in 7 to 15 days.
In accordance with another aspect, the present invention provides for a cell population of bone marrow cells supported in
Brandt John
Hoffman Ronald
Martinell James
The Indiana University Foundation
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