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WO1993012805A1 - Procede de regulation des lignages cellulaires humains hematopoeïtiques - Google Patents

Procede de regulation des lignages cellulaires humains hematopoeïtiques Download PDF

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WO1993012805A1
WO1993012805A1 PCT/US1992/011228 US9211228W WO9312805A1 WO 1993012805 A1 WO1993012805 A1 WO 1993012805A1 US 9211228 W US9211228 W US 9211228W WO 9312805 A1 WO9312805 A1 WO 9312805A1
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cells
human
medium
cell
culture
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Bernhard O. Palsson
R. Douglas Armstrong
Michael F. Clarke
Stephen G. Emerson
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Regents Of The University Of Michigan
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N5/0641—Erythrocytes
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/14—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1394—Bone marrow stromal cells; whole marrow

Definitions

  • the present invention relates to methods and compositions for the growth of mammalian cells in culture, particularly the growth of hematopoietic cell cultures.
  • the present invention also relates to a functioning in vitro human tissue system, which may serve as a model for hematopoiesis.
  • the present invention further relates to a method for assaying the effect of a substance and/or physical condition on a human
  • the present invention also relates to a method for controlling the lineage development in an in vitro human tissue system and cultures of cells in which the population of a particular cell type has been enhanced relative to the total cell population in the culture or depleted.
  • the present invention also relates to a method for controlling the lineage development in an in vitro human tissue system and cultures of cells in which the population of a particular cell type has been enhanced relative to the total cell population in the culture or depleted.
  • invention relates to a method of bone marrow transplantation, in which the tissue implanted into the donee has been cultured by the present method.
  • progenitors which are assayed by their development into contiguous colonies of mature blood cells in 1-3 week cultures in semisolid media such as methylcellulose or agar.
  • Stem cells themselves derive from a class of progenitor cells called stem cells. Stem cells have the capacity, upon division, for both self-renewal and
  • stem cells differentiate into progenitors.
  • stem cells also may give rise to osteoblasts and osteoclasts, and perhaps cells of other tissues as well.
  • compositions which permit, for the first time, the successful in vitro culture of human hematopoietic stem cells, which results in their proliferation and differentiation into progenitor cells and more mature blood cells of a specific lineage.
  • fibroblasts and endothelial cells within adhering layers as central cellular stromal elements. These cells both provide sites of attachment for developing hematopoietic cells and can be induced to secrete
  • hematopoietic growth factors which stimulate progenitor cell proliferation and differentiation. These hematopoietic growth factors include granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin 6 (IL-6).
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • IL-6 interleukin 6
  • HGFs hematopoietic growth factors
  • Bone cell and progenitor cell expansion for bone marrow transplantation is a potential application of human long-term bone marrow cultures.
  • Human autologous and allogeneic bone marrow transplantation are currently used as therapies for diseases such as leukemia, lymphoma and other life-threatening disorders. For these procedures however, a large amount of donor bone marrow must be removed to insure that there is enough cells for engraftment.
  • stem cells and progenitor cells would reduce the need for large bone marrow donation and would make possible obtaining a small marrow donation and then expanding the number of stem cells and progenitor cells in vitro before infusion into the recipient. Also, it is known that a small number of stem cells and progenitor cells circulate in the blood stream. If these stem cells and progenitor cells could be collected by phoresis and expanded, then it would be possible to obtain the required number of stem cells and progenitor cells for transplantation from peripheral blood and eliminate the need for bone marrow donation.
  • Bone marrow transplantation requires that approximately 1 x 10 8 to 2 x 10 8 bone marrow mononuclear cells per kilogram of patient weight be infused for engraftment. This requires the bone marrow donation of the same number of cells which is on the order of 70 ml of marrow for a 70 kg donor. While 70 ml is a small fraction of the donors marrow, it requires an intensive donation and significant loss of blood in the donation process. If stem cells and progenitor cells could be expanded ten-fold, the donation procedure would be greatly reduced and possibly involve only collection of stem cells and progenitor cells from peripheral blood and expansion of these stem cells and progenitor cells.
  • Progenitor cell expansion would also be useful as a supplemental treatment to chemotherapy and is another
  • Bone marrow is one of the most prolific tissues in the body and is therefore often the organ that is initially damaged by chemotherapy drugs.
  • the result is that blood cell production is rapidly destroyed during chemotherapy treatment and chemotherapy must be terminated to allow the hematopoietic system to replenish the blood cell supply before a patient is retreated with chemotherapy. It may take a month or more for the once quiescent stem cells to raise up the white blood cell count to acceptable levels to resume chemotherapy during which case the drop in blood cell count is repeated.
  • the cancer has time to grow and possibly become more resistant to the chemotherapy drugs due to natural selection.
  • the hematopoietic cells required for progenitor cell expansion may come from either bone marrow withdrawal or peripheral blood collection. Bone marrow harvests would result in collection of approximately 4 x 10 5 CFU-GM
  • progenitor cells Phoresis of 5 liters of peripheral blood would collect approximately 10 5 CFU-GM although this number could be increased to 10 6 CFU-GM by prior treatment of the donor with GM-CSF. Rapid recovery of a patient would require transfusion of approximately 1 x 10 8 to 5 x 10 8 CFU-GM.
  • Gene therapy is a rapidly growing field in medicine which is also of inestimable clinical potential. Gene therapy is, by definition, the insertion of genes into cells for the purpose of medicinal therapy. Research in gene therapy has been on-going for several years in several types of cells in vitro and in animal studies, and has recently entered the first human clinical trials. Gene therapy has many potential uses in treating disease and has been reviewed extensively. See, e.g., Boggs, Int. J. Cell Cloning. (1990) 8:80-96, Kohn et al, Cancer Invest. (1989) 7 (2): 179-192, Lehn. Bone Marrow Transp. (1990) 5:287-293, and Verma, Scientific Amer. (1990) pp. 68-84.
  • the human hematopoietic system is an ideal choice for gene therapy in that hematopoietic stem cells are readily accessible for treatment (bone marrow or peripheral blood harvest), they are believed to possess unlimited self-renewal capabilities (inferring lifetime therapy), and upon
  • reinfusion can expand and repopulate the marrow.
  • hematopoietic system Several disorders of the hematopoietic system include thalassemia, sickle cell anemia, Falconi's anemia, acquired immune deficiency syndrome (AIDS) and SCIDS (ADA, adenosine deaminase deficiency). These candidates include both diseases that are inherited such as hemoglobinopathies and virally caused diseases of the hematopoietic system such as AIDS.
  • a salient problem which remain to be addressed for successful human gene therapy is the ability to insert the desired therapeutic gene into the chosen cells in a quantity such that it will be beneficial to the patient. To date, no method for doing this is available.
  • red cells transfused are used in the treatment of anemia following elective surgery, in cases of traumatic blood loss, and in the supportive care of, e.g., cancer patients.
  • controlled production of platelets would permit the in vitro production of platelets for platelet transfusion therapy, for example for cancer patients in whom thrombocytopenia is caused by chemotherapy.
  • red cells and platelets current volunteer donor pools are accompanied by the risk of
  • Controlled, selective depletion of a particular lineage of cells from within a hematopoietic cell population can similarly confer important advantages. For example,
  • T-cell depleted bone marrow sample could then be used to "rescue" the patient following hematolymphoid ablation and autologous bone marrow transplantation.
  • T-cell depletion is the prevention of graft-versus-host disease (GVHD) in allogeneic bone marrow transplantation.
  • GVHD graft-versus-host disease
  • Depletion of T-cells from a stem/progenitor cell population prior to allogeneic transplant would directly reduce the incidence and severity of GVHD. This depletion in turn would greatly decrease the morbidity and mortality of allogeneic bone marrow transplantation.
  • GVHD graft-versus-host disease
  • a further application of selective cell removal is the purging of malignant cells from bone marrow cultures for autologous bone marrow transplantation of cancer patients in which the cancer has metastasized. If it were possible to maintain a viable and productive human hematopoietic in vitro culture under conditions, which would lead to the depletion and extinction of malignant cells, then one could utilize such a culture for an autologous bone marrow transplant after a bout of chemotherapy, without the consequence of reintroducing metastasized malignant cells to the patient via the bone marrow transplant.
  • the present invention is based on the inventors'
  • the above medium replacement rate is used in conjunction with the addition of hematopoietic growth factors to the rapidly exchanged culture medium.
  • the inventors have discovered that the increased medium exchange rate used in accordance with the present invention, with the optional addition of hematopoietic growth factors to the rapidly exchanged culture medium, surprisingly (1) supports cultures in which human stem cells proliferate over extended periods of time of at least 5 months, (2) supports cultures in which human hematopoietic progenitor cells are produced by division and differentiation of human stem cells through extended culture periods of at least 5 months, (3) stimulates the increased metabolism of and growth factor, including GM-CSF, secretion from human stromal cells,
  • hematopoietic mononuclear cell population (5) provides a method for assaying the affect of a substance or substances or physical conditions on a human hematopoietic cell population, (6) provides for the depletion of malignant cells from a human hematopoietic cell population, and (7) supports cultures in which human stem cells continue to divide over long periods of time and, thus, may be genetically transformed with a suitable vector such as a retrovirus.
  • the present invention provides, for the first time, human stem cell survival and proliferation in culture.
  • the present invention provides a functioning human in vitro tissue system which may serve as a model for a human hematopoietic cell mass or the process of hematopoiesis.
  • the advantages of the present invention may be observed whenever the present invention is applied to any standard system for liquid human hematopoietic culture.
  • the rapid medium exchange rates used in accordance with the present invention with the optional addition of supplementary hematopoietic growth factors to the culture, the inventors have surprisingly discovered that one is able to make standard systems for liquid human hematopoietic cultures, which comprise cultures performed in the presence or absence of animal sera or plasmas, including horse, calf, fetal calf, or human serum, perform in a qualitatively superior manner.
  • Human liquid hematopoietic cultures which may be used in accordance with the invention can be performed at cell densities of from 10 4 to 5x10 8 cells per ml of culture, using standard known medium components such as, for example, IMDM, MEM, DMEM, RPMI 1640, Alpha Medium or McCoy's Medium, which can use combinations of serum albumin, cholesterol and/or insulin, transferrin, lecithin, selenium and inorganic salts. As known, these cultures may be supplemented with
  • corticosteroids such as hydrocortisone at a concentration of 10 -4 to 10 -7 M, or other corticosteroids at equal potent dose such as cortisone, dexamethasone or Solu-Medrol® (Upjohn).
  • the medium is kept at an oxygen concentration that corresponds to an oxygen- containing atmosphere which contains from 1 to 20 vol. percent oxygen, preferably 3 to 12 vol. percent oxygen.
  • the preferred range of O 2 concentration refers to the concentration of O 2 near the cells, not necessarily at the point of O 2
  • the cell mass used may be enriched, by any desired amount, such as by up to 10 3 fold or more, either for stem cell content or for hematopoietic progenitor cell content.
  • Different known methods may be used to achieve this enrichment, corresponding either to a negative selection method or a positive selection method. For example, in accordance with the negative
  • mature cells are removed using immunological techniques, e.g., labelling non-progenitor, non-stem cells with a panel of mouse anti-human monoclonal antibodies, then removing the mouse antibody-coated cells by adherence to rabbit-anti-mouse Ig-coated plastic dishes.
  • immunological techniques e.g., labelling non-progenitor, non-stem cells with a panel of mouse anti-human monoclonal antibodies, then removing the mouse antibody-coated cells by adherence to rabbit-anti-mouse Ig-coated plastic dishes.
  • stem cells and progenitor cells may be used to be selected cells.
  • the present invention relies on a fundamental alteration of the conditions of liquid human bone marrow cultures under any of the above conditions; rapid replacement of the nutrient medium.
  • Standard culture schedules call for medium and serum to be exchanged weekly, either as a single exchange performed weekly or a one-half medium and serum exchange performed twice weekly.
  • nutrient medium of the culture is replaced, preferably
  • the present medium replacement rate may be expressed as 1 ml of medium per 10 7 cells per about 24 to about 48 hour period.
  • the medium exchange rate may be increased proportionality to achieve a constant medium and serum flux per cell per unit time.
  • Replacement of the nutrient medium in accordance with the invention may be carried out in any manner which will achieve the result of replacing the medium, e.g., by removing an aliquot of spent culture medium and replacing it with a fresh aliquot.
  • the flow of the aliquot being added may be by
  • the new medium is added to the culture in a manner such that it contacts the cell mass. Most preferably, it is added to the culture in a manner mimicking in vivo perfusion, i.e., it is perfused through at least part of the cell mass and up to the whole cell mass .
  • Another, optional but important, embodiment of the present invention resides in the addition of hematopoietic growth factors to the rapidly exchanged cultures.
  • the rapidly exchanged cultures a particularly preferred aspect of this embodiment, the
  • cytokines IL-3 and GM-CSF are both added, together, to the medium at a rate of from 0.1 to 100 ng/ml/day, preferably about 0.5 to 10 ng/ml/day, most preferably 1 to 2 ng/ml/day.
  • Epo may be added to the nutrient medium in an amount of from 0.001 to 10 U/ml/day, preferably 0.05 to 0.15 U/ml/day.
  • Mast cell growth factor MMF, c-kit ligand. Steel factor
  • MMF c-kit ligand. Steel factor
  • IL-1 ( ⁇ or ⁇ ) may also be added in an amount of from 10 to 100 units/ml per 3 to 5 day period. Additionally, IL-6, G-CSF, basic fibroblast growth factor, IL-7, IL-8, IL-9, IL-10, IL-11, PDGF, or EGF may be added, at a rate of from 1 to 100 ng/ml/day.
  • the metabolic product level in the medium is normally maintained within a particular range.
  • Glucose concentration is usually maintained in the range of about 5 to 20 mM.
  • Lactate concentration is usually maintained below 35 mM.
  • Glutamine concentration is generally maintained in the range of from about 1 to 3 mM. Ammonium concentration is usually maintained below about 2.4 mM. These concentrations can be monitored by either periodic off line or on line continuous measurements using known methods. See, e.g., Caldwell et al. J. Cell Physiol. (1991) 147:344-353.
  • the cells which may be cultured in accordance with the present invention may be human peripheral blood mononuclear cells, human bone marrow cells, human fetal liver cells, human cord blood cells and/or human spleen cells. Each of these cell masses contains human stem cells and human hematopoietic progenitor cells.
  • the cell culture may be enriched to augment the human stem cell content of the cell mass.
  • enrichment may achieved as described above, and, when used in accordance with the invention, provides the first useful means for genetic therapy via gene transfer into human bone marrow stem cells.
  • human stem cells cultured in accordance with the invention is added to human stem cells cultured in accordance with the invention to obtain transformed human bone marrow stem cells.
  • Such genetic transformation of human stem cells may be carried out as described in U.S. Patent application serial no. 07/740,590, which is incorporated herein by
  • the present invention provides increased levels of stem cell and human hematopoietic progenitor cell replication, whereas, by contrast, prior cultures provided only for human hematopoietic progenitor cell replication at a decreasing rate (i.e., decaying cultures).
  • the present culture system provides increased levels of stem cell and human hematopoietic progenitor cell replication, whereas, by contrast, prior cultures provided only for human hematopoietic progenitor cell replication at a decreasing rate (i.e., decaying cultures).
  • hematopoietic growth factors provides a very effective means for obtaining stem cell infection in vitro.
  • Bone marrow stomal cells may or may not be present in the cultures of the invention.
  • stromal cells are present in the cell culture in an amount of approximately 10 -3 to 10 -1 (stromal cells/total cells).
  • the cultures of the invention surprisingly provide increased metabolism and GM-CSF and IL-6 secretion from human bone marrow stromal cells. Whereas no GM-CSF is detected in human bone marrow stromal cells supernatant, rapid medium exchange in accordance with the invention stimulates human bone marrow stromal cells to secrete 300
  • the medium used in accordance with the invention may comprise three basic components.
  • the first component is a media component comprised of IMDM, MEM, DMEM, RPMI 1640, Alpha Medium or McCoy's Medium, or an equivalent known culture medium component.
  • the second is a serum component which comprises at least horse serum or human serum and may optionally further comprise fetal calf serum, newborn calf serum, and/or calf serum.
  • the third component is a corticosteroid, such as hydrocortisone, cortisone,
  • the serum component can be replaced in whole or in part with any standard serum
  • compositional make up of various media which can be used are set forth below.
  • McCoy's 5A Medium Formulated with Hanks' and Suspension Salts is a GIBCO modincation and is not cited in references 1-3 a. HCl form listed by the Tissue Culture Standards Committee. In Vitro (1974) 9:6.
  • the serum component may be present in the culture in an amount of at least 1% (v/v) to 50% (v/v).
  • the preferred range will depend on whether or not serum is being used alone or is, at least in part, replaced by a serum replacement. When using no serum replacement, the serum concentration may be
  • the third component, corticosteroid may be present in an amount of from 10 -7 M to 10 -4 M, and is preferably present in an amount of from 5 x 10 -6 to 5 x 10 -5 M.
  • the serum may be present in an amount of from 10 -7 M to 10 -4 M, and is preferably present in an amount of from 5 x 10 -6 to 5 x 10 -5 M.
  • the serum may be present in an amount of from 10 -7 M to 10 -4 M, and is preferably present in an amount of from 5 x 10 -6 to 5 x 10 -5 M.
  • component can be replaced by any of several standard serum replacement mixtures which typically include insulin, albumin, and transferrin, lecithin, selenium or cholesterol. See,
  • T and B lymphocytes are lost from these cells
  • T and B lymphocytes are lost over time.
  • T-cell-derived diseases there are several T-cell-derived diseases and
  • the autoimmune deficiency diseases e.g., AIDS
  • the autoimmune deficiency diseases result because of abnormal T-cell function caused by direct viral infection. Since this order results as a direct infection of the mature T-cell, and is not derived from defective hematopoietic stem or progenitor cells, selective eradication of the mature T-cells has notable potential therapeutic benefit.
  • T-cell depletion has other applications as well.
  • a limiting factor to the improved success of allogeneic bone marrow transplant is T-cell mediated. Depletion of T-cells from a stem/progenitor cell population prior to allogeneic transplant would enhance the reingraftment success by reducing the T-cell mediated graft versus host-rejection response.
  • the inventors have discovered that the present methods, including the present culture media conditions, which allow for the in vitro replication and differentiation of human stem and hematopoietic progenitor cells do not allow for
  • hematopoietic progenitor cells are capable of in vitro
  • T-cells and B-cells a major class of peripheral blood cells, do not proliferate or maintain viability in these in vitro culture conditions.
  • T-cells require, among other factors, the growth factor interleukin-2 (IL-2) to remain viable.
  • IL-2 growth factor interleukin-2
  • T- cells grown without such proper support die in approximately 3 to 4 days in a medium substantially free of IL-2.
  • progenitor cells are maintained in accordance with the
  • hematopoietic mononuclear cell population die, if the medium is substantially free of IL-2.
  • a mixed human hematopoietic mononuclear cell fraction can be effectively depleted of T-cells and B-cells, under conditions which allow for expansion of stem and progenitor cells.
  • T-cell- depleted hematopoietic stem and/or progenitor cells can then be used for reingraftment of patient bone marrow.
  • the reestablished marrow will then produce anew, normal T-cell population.
  • this procedure itself would not serve to necessarily eradicate the HIV from the patient, and reinfection of the newly developed T-cell in vivo is likely. Accordingly, this therapy would be considered as supportive, but, if used with other virus-eradication
  • this procedure is operative for curative treatment protocols as well.
  • Allogeneic bone marrow transplant whereby a human hematopoietic stem/progenitor cell population is depleted of viable T-cells and then used to reestablish the hematopoietic system in a recipient individual. The depletion of the T-cell population will increase the prospect of successful
  • progenitor cells see, e.g., U.S. Patent No. 5,061,620 as representative.
  • the reported methods do not provide a method for the long term culture of viable and replicating stem cells, while the present method affords just such a result.
  • the present method for controlling the lineage development in a human hematopoietic tissue system may be practiced in conjunction with genetic transformation of at least a portion of the stem cells in the hematopoietic tissue system.
  • the present invention provides a method for assaying the effect of a substance or substances and/or physical condition on a hematopoietic cell culture or the process of hematopoiesis.
  • one may add to a cell culture, carried out in accordance with the invention, at least one substance
  • Compounds or substances which may be tested include those which are expected to exert some effect on the hematopoietic system. Such compounds include, for example, hematopoietic growth factors, drugs, hormones, etc.
  • endogenously produced substance examples include monoclonal antibodies, which bind to and neutralize endogenously produced growth factors, and antagonists, which bind to and block growth factor receptors on the surface of cells.
  • the present assay may also be used to ascertain the effect of physical conditions on the hematopoietic system or hematopoietic process.
  • Such conditions include, for example, temperature, pressure, light intensity, gravity, etc.
  • the effect of temperature, pressure, and light intensity may be determined by varying these parameters using conventional techniques and apparatus, such as heaters, refrigerators, pressurized or reduced-pressure chambers, and light sources.
  • the effect of gravity may be determined by, e.g., carrying out the present culture in a zero-gravity environment, such as the space shuttle.
  • the present assay may also be used to
  • the present assay is not limited to determining the effect of a single chemical substance or physical condition but may be used to detect the effect of the combined action of any number of chemical substances and/or physical conditions.
  • the parameters which can be monitored in carrying out the present assay include the cell population profile of the hematopoietic cell culture, the total cell population, the relative population of any particular type of cell, the presence, absence, or concentration of any other substance in the medium being removed from the culture, the consumption of nutrients, the morphology of any or all of the particular cell types present in the culture, the lifetime and duration of the culture, and the kinetics of hematopoiesis.
  • the present invention provides a functioning in vitro human hematopoietic tissue system which may serve as a model for the study of the naturally occurring in vivo hematopoietic system and the process of hematopoiesis.
  • the effect of any chemical substance and/or physical condition on the hematopoietic system or the process of hematopoiesis may be determined by the present assay. It should be recognized that the present assay exhibits a significant advantage in that the effects of (i) slow acting substances and/or conditions or (ii) substances and/or conditions for which the effect exhibits a lag phase may be readily determined, because the present system provides a long term functioning hematopoietic tissue system.
  • the present assay may be carried out when at least a portion of the stem cells in the human hematopoietic tissue system have been genetically transformed. Such genetic transformation may be used to introduce genetic markers useful for the subsequent
  • stem cells are identified from the transformed stem cells.
  • such genetic transformation may also serve as a method for introducing into the medium the chemical substance to be studied.
  • the production of the substance by either the stem cell or a cell derived therefrom will provide a constant source of the substance.
  • the present invention provides an improved method of bone marrow transplantation.
  • culturing bone marrow tissue according to the present method may of the drawbacks attendant to conventional bone marrow transplants may be avoided.
  • the present method of bone marrow transplantation may be advantageously applied in situations in which the bone marrow tissue has been previously removed from a patient, before the patient is subjected to either
  • the present method also exhibits distinct advantages.
  • culturing according to the present method using medium substantially free of IL-2, results in a bone marrow culture substantially free of T-cells and B-cells and since such cells are principally involved in graft versus host disease, implantation of allogeneic bone marrow tissue
  • cultured according to the present method poses a reduced risk of graft versus host disease.
  • hematopoietic tissue system containing viable stem cells.
  • the present method of bone marrow transplantation may be carried out as follows: removing a tissue sample from a donor; culturing said tissue sample according to the present method; and implanting said cultured tissue in a donee.
  • the donor and donee may be the same or different.
  • the tissue sample may be obtained from the donor
  • tissue sample After the tissue sample has been obtained, it is then cultured according to the present method, for a time
  • the cultured tissue may then be implanted in the donee, again, according to conventional techniques.
  • EXAMPLE 1 MEDIUM REPLACEMENT.
  • Human bone marrow cells were obtained from heparinized aspirates from the iliac crest of informed and consenting individuals. The bone marrow was separated by a Ficoll-Paque (Pharmacia, No. 17-0840-02) density gradient centrifugation and the low density cells ( ⁇ 1.077 gm/cm 3 ) were collected and washed 3 times with Iscove' s Modified Duibecco's Medium (IMDM). The cells were counted between the second and third washes. The cells were then seeded onto 24-well tissue culture plates (Costar No. 3524) in duplicate or triplicate at 1, 2, and 5 ⁇ 10 6 cells/ml at 322 ⁇ l/well.
  • IMDM Iscove' s Modified Duibecco's Medium
  • Methylcellulose and morphologic assays Once every other week, the non-adherent cells removed for cell counts were plated in methylcellulose in the presence of erythropoietin, GM-CSF, and IL-3, and the Granulocyte Macrophage-Colony
  • CFU-GM Forming Units
  • Nonadherent cell production was examined both as a function of inoculum cell density (over the range 1-5 ⁇ 10 6 cells/ml) and medium exchange rate.
  • the medium exchange rate was varied from one medium volume exchange per week, the traditional Dexter
  • the medium exchange rate in contrast, strongly
  • the cell production under the 3.5/wk and 1/wk protocols can be directly compared by plotting the cell production under the 3.5/wk exchange rate as a percentage of the production of the cultures with an exchange rate of 1/wk. This comparison shows that during the initial decay phase the cell production under the two protocols is similar. However, between weeks 3.5 and 18, the cell production under the 3.5/wk exchange rate is consistently higher.
  • the proliferative potential of the cultures can be measured by their ability to produce cells following the initial decay.
  • the normalized cumulative cell production following week 3 was independent of the cell inoculation density for the medium exchange rates of 7/wk, 3.5/wk. Cell production data from the cultures at similar medium exchange rates were qualitatively and statistically similar, and were therefore density averaged and combined to obtain a larger statistical sample.
  • the density averaged cumulative cell production between weeks 3.5 and 20 was: 0.22 for the 7/wk; 0.40 for the 3.5/wk; and 0.15 for the 1/wk cultures.
  • the increase in the medium exchange rate from 1/wk to 7/wk thus increased the cell production about 60% over the typical
  • Granulocyte-macrophage progenitor cell production is a major component of Granulocyte-macrophage progenitor cell production
  • Granulocyte-macrophage progenitor cell assays were performed from replicates of a given medium perfusion schedule and inoculum density (Table 1). The medium perfusion rate had a pronounced effect on the number of granulocyte-macrophage progenitor cells produced. The 3.5/wk medium exchange
  • progenitor cell production showed the greatest longevity in terms of progenitor cell production. These cultures produced progenitors at a stable rate between weeks 4 and 18. The optimum conditions in terms of progenitor cell production are the cultures exchanged 3.5 times per week and inoculated at 5 • 10 6 cells/ml. These cultures produced a significant number of progenitor cells until week 20. Statistical analysis, using a paired t-test, showed that the optimum medium exchange rate cultures of
  • Progenitor cells can only be present after several weeks in culture by differentiation from an earlier cell, presumably a stem cell, which is still present in culture. Thus, these data suggest that more physiologic, rapid medium/serum exchange rate and higher cell densities may have provided conditions that supported some degree of stem cell renewal for five months.
  • Nonadherent cell morphology To determine whether the prolonged hematopoiesis supported by the 3.5/wk cultures was qualitatively different from the other cultures, the non- adherent cells collected between weeks 10 and 19 were stained and typed morphologically. At the exchange rates of 1/wk and 7/wk, the cells produced were mostly macrophages by week 15 and thereafter (Table 2), which is similar to results from studies in other laboratories. In contrast, the cultures perfused at a rate of 3.5 medium volumes per week and seeded at 5 • 10 6 cells/ml produced granulocytes as well as
  • Table 1 The average number of nonadherent progenitor cells removed from long term bone marrow cultures (LTBMCs) as a function of the medium perfusion rate and inoculum density.
  • EXAMPLE 2 MEDIUM REPLACEMENT COMBINED WITH SUPPLEMENTATION OF MEDIUM WITH HEMATOPOIETIC GROWTH FACTORS.
  • Human bone marrow cells were obtained following informed consent from heparinized aspirates of the iliac crest bone marrow, under a protocol approved by the University of Michigan Human Investigation Committee. The bone marrow was separated by a Ficoll-Paque (Pharmacia) density gradient centrifugation and the low density cells ( ⁇ 1.077gm/cm 3 ) were collected and washed 3 times with IMDM. The cells were counted between the second and third washes. The cells were then seeded onto 6-well tissue culture plates (Costar No.
  • Hematopoietic growth factors Due to the frequent culture supplementation via rapid medium exchange, hematopoietic growth factors were added daily to the medium at
  • concentrations used were 1 ng/ml of IL-3, 1 ng/ml of GM-CSF (Amgen
  • hematopoietic cells removed from culture were counted and plated at 1-10 5 cells/ml or fewer cells in methylcellulose.
  • GM-CSF and Epo were added to the methylcellulose at 20 ng/ml and 2 U/ml, respectively.
  • the cells were plated in 24 well plates at 0.25 ml/well and incubated at 37°C for 14 days. The colonies were then counted under an inverted microscope and colonies greater than 50 cells were scored as GM-colony forming units (CFU-GM), erythroid burst-forming unit (BFU-E), or granulocyte erythroid megakaryocyte macrophage-colony forming unit (CFU-GEMM).
  • CFU-GM GM-colony forming units
  • BFU-E erythroid burst-forming unit
  • CFU-GEMM granulocyte erythroid megakaryocyte macrophage-colony forming unit
  • LTBMC conditions The cultures were incubated at 37°C in a humidified 5% CO 2 /95% air atmosphere and perfused (medium exchanged) at a rate of 50% daily medium exchange. During the first week in culture, all cells removed during the daily medium exchange were centrifuged and returned to the original wells. After the first week in culture, 50% of the total nonadherent cells were removed from the cultures on a biweekly basis during the medium exchange, mononucleated cells counted, and fresh medium returned to the wells. The remaining five days per week when the cells were not counted, 50% of the medium was removed from each of the culture wells and replaced with fresh medium, the removed medium was centrifuged, the medium decanted from the cell pellet, and the cells returned to their original wells.
  • IL-3+GM-CSF produced an average 3.5-fold increase in nonadherent cells as compared to controls through week 8.
  • adding neither IL-6 nor G-CSF to the combination of IL-3+GM-CSF+Epo improved the nonadherent cell production rate, but instead resulted in cell production rates indistinguishable from the cultures
  • the combination of IL-3+GM-CSF+Epo induced cumulative cell production that was more than 3-fold greater than the number of cells inoculated.
  • the cell production rate was the highest during the first 6 weeks in culture during which time the culture produced approximately as many cells as were inoculated every two weeks. This maximum cell production rate was 15% of the estimated in vivo bone marrow cell production rate where 50% of the myeloid cell mass is generated daily.
  • the combination of IL-3+GM-CSF resulted in more than a 2-fold expansion in cell number and at rates comparable to the combination of IL3+GM-CSF+Epo during weeks 3-7 in culture.
  • Morphologic analysis of nonadherent cells The addition of multiple HGFs also increased the variety of myeloid cells produced in the cultures.
  • the control cultures produced nonadherent cells that were predominately macrophages after week 3 in the culture. Production of erythroid cells
  • Epo Epo alone, IL-3+Epo, and IL-3+GM-CSF+Epo
  • nonadherent cells being erythroid through week 3.
  • IL-3+Epo ⁇ GM-CSF was present, the cultures continued to produce erythroid cells throughout the 16 weeks in culture with about 5-15% of the nonadherent cells being typed as erythroid.
  • CFU-GM granulocyte macrophage colony forming units
  • IL-3+Epo ⁇ GM-CSF cultures was approximately 10-fold higher than controls during weeks 3 to 5.
  • Erythroid burst forming unit (BFU-E) production in human LTBMC has been reported to be low and cease quickly (Coutinho et al, Blood (1990) 75(11): 2118-2129). The rapidly
  • IL-3 alone induced a mild short-lived stimulation of BFU-E production in weeks 3-5.
  • IL-3 plus either Epo or GM-CSF induced a 10 to
  • IL-3+Epo resulted in sustained red cell production not previously observed in any human LTBMC.
  • IL-3+ GM-CSF supplementation resulted in sustained granulocyte production.
  • Supplementation with different HGFs may likewise lead to preferential production of different blood cells, such as platelets, B lymphocytes or T lymphocytes.
  • the critical feature of the present invention is that the combination of rapid medium exchange conditions combined with hematopoietic growth factor supplementation results in essentially
  • hematopoietic growth factors by stromal cells in vitro.
  • HGFs Increased medium perfusion and addition of HGFs may therefore also induce other HGF production (e.g. kit-ligand) by
  • LTMBCs stimulating hematopoietic or accessory cells in the cultures. Therefore, increasing the medium perfusion rate may provide LTMBCs benefits other than just increasing metabolite and decreasing waste product levels.
  • EXAMPLE 3 USE OF RAPID EXCHANGE CULTURE SYSTEM TO DEPLETE A BONE MARROW CELL POPULATION OF LYMPHOID CELLS.
  • the present methods including the present culture media conditions, which allow for the in vitro replication and differentiation of human stem and hematopoietic progenitor cells can simultaneously promote the disappearance of specified cells present in the original composition, notably lymphoid cells such as T-cells and B- cells.
  • lymphoid cells such as T-cells and B- cells.
  • hematopoietic progenitor cells human T-cells and B-cells do not proliferate or maintain viability in these in vitro culture conditions.
  • a mixed human hematopoietic mononuclear cell fraction can be effectively depleted of T-cells and/or
  • B-cells T-cells. and early Hematopoietic Cells: At weekly intervals, cells were removed from the cultures and analyzed by flow cytometry on a FACS Scan (Becton-Dickinson) Cell Analyzer. B-cells were enumerated with anti-CD19, T- cells with anti-CD3, and early hematopoietic progenitor cells with anti-CD34, using fluoresceinated normal mouse Ig as a background control (all antibodies were from Becton-Dickinson, Inc).
  • T-cells and B-cells were rapidly lost in these cultures, while primitive hematopoietic progenitor (CD34+) cells were maintained: Table 3. Selective Depletion of T and B Lymphocytes from within a Proliferating Human Bone Marrow Culture.
  • the described culture conditions support the survival and proliferation of hematopoietic progenitor cells, while simultaneously depleting the culture of T and B
  • Human hematopoietic cell populations prepared in this way are directly applicable to allogeneic bone marrow transplantation, and also to depletion of virally infected T- and/or B-cells from a hematopoietic cell population for auto- or allo-transplantation.
  • EXAMPLE 4 USE OF RAPIDLY EXCHANGED/PERFUSED CULTURE SYSTEM TO DEPLETE A HEMATOPOIETIC CELL POPULATION OF TUMOR CELLS.
  • cancer cells including T- and B-cell leukemia and lymphoma cells, and chronic myelogenous leukemia cells, can be depleted from within a hematopoietic cell population by
  • the essential feature of the culture system that it allows the survival and proliferation of hematopoietic stem and progenitor cells while not supporting the survival and proliferation of other cell types, results in the loss of cancer cells which are
  • CML chronic myelogenous leukemia cells
  • % of cultured cells that belong to the CML clone can decrease from 90% or more to under 5%, and in some cases less than 1%.
  • the presently described culture system can be directly applicable to the purging of cancer cells from within any hematopoietic cell mass (bone marrow, peripheral blood, cord blood, fetal blood) for autologous or allogeneic bone marrow transplantation, and can be applied directly to the treatment of cancer patients.
  • the exact time of culture required to achieve substantial depletion of the malignant cells may depend on the exact type and the number of malignant cells present. However, the time required to achieve a culture substantially free of any particular type of malignant cell may be easily determined by one of skill in the art by using the present method in
  • EXAMPLE 5 USE OF RAPIDLY EXCHANGED/PERFUSED SYSTEM TO ASSAY THE INFLUENCE OF SPECIFIED MOLECULES ON HEMATOPOIESIS
  • this system provides a method for assaying the affect of a substance or substances on a hematopoietic cell culture.
  • one adds to a cell culture carried out in accordance with the invention at least one substance suspected of having an affect, which may be either beneficial or detrimental, on the cell culture.
  • One compares the cell culture profile obtained in the absence of the substance being tested to the cell culture profile obtained in the presence of the substance.
  • This embodiment may be used in accordance with the various embodiments used in the present invention to ascertain the particular affect of a suspected substance on a human stem or hematopoietic cell system. As examples.
  • Interleukin 6 IL-6
  • G-CSF Granulocyte colony stimulating factor
  • Hematopoietic growth factors IL-3, 1 ng/ml; GM-CSF, 1 ng/ml of GM-CSF; Epo, 0.1 U/ml; IL-6, 10 U/ml (Collaborative Research Inc.), and G-CSF, 0.1 ng/ml (Amgen Biologicals).
  • Table 4 Cumulative cell production by cell lineage in growth factor supplemented LTBMCs over the first 6.5 weeks of culture.
  • Progenitor cell production Progenitor cell production was dramatically influenced by addition of HGFs to LTBMCs.
  • Granulocyte macrophage progenitor cell (CFU-GM) production was also affected by HGF supplementation of the LTBMCs.
  • IL-3 or GM-CSF produced only a slight increase in total CFU-GM (Table 5) whereas IL-3+GM-CSF+Epo induced approximately a 1.7-fold increase in total CFU-GM production.
  • IL-3+GM-CSF+Epo with IL-6 or G-CSF induced a 2.4 and 3.1-fold increase in the number of CFU-GM removed from culture,
  • CFU-GEMM production was significantly influenced by HGF supplementation. Individual supplementation of the LTBMCs with either IL-3 or GM-CSF alone, increased the number of CFU-GEMM removed from culture 1.8 and 1.6 fold, respectively. However, combinations of CSFs induced a much larger increase in CFU-GEMM production than did either IL-3 or GM-CSF alone. Supplementation with IL-3+GM-CSF and IL-3+GM-CSF+Epo induced a 3.5 and 10.3-fold increase in the total number of CFU-GEMM removed, suggesting that Epo plays either a direct or indirect role in early hematopoietic events.
  • progenitor cell production is an indirect measure of primitive stem cell activity.
  • the four process rates that determine the progenitor cell pool size are:
  • V 1 The rate of production of progenitor cells by more undifferentiated stem cells
  • V 2 The rate of loss of progenitor cells through the act of sampling of nonadherent cells from the culture
  • V 3 The rate of loss of progenitor cells by their differentiation to mature cells of a particular lineage
  • V 4 The rate of death of progenitor cells.
  • the initial number, C 0 of each progenitor cell species can be determined by assay at culture initiation.
  • V 1 is V 1 greater than zero?
  • V 4 cannot be assessed.
  • V 2 can be measured by assaying the nonadherent cells removed from culture, and V 3 can be estimated from nonadherent cell production data.
  • a conservative estimate of the rate of progenitor cell differentiation, V 3 can be determined by assuming that each progenitor cell has 10 divisions remaining to become a terminally differentiated cell. Therefore, each progenitor cell is equivalent to 2 10 (1024) mature cells.
  • V 1 is greater than zero.
  • Table 6 The cumulative production of BFU-E removed and differentiated in growth factor supplemented rapidly perfused human long-term bone marrow cultures.
  • this example demonstrates how the rapid medium exchange/perfusion hematopoietic cultures allow for the precise analysis of the hematopoietic activity of added molecules, particularly in regard to their effects on
  • This system can be used to analyze the hematopoietic activity of any desired soluble substance or substances.
  • Table 7 The cumulative production of CFU-GM removed and differentiated in growth factor supplemented rapidly perfused human long-term bone marrow cultures.
  • the liquid medium may be pumped with a syringe pump that may be located in a refrigerator adjacent to an incubator, maintained at a temperature sufficient to sustain hematopoiesis.
  • the fresh medium may be kept at 4°C preventing decay of chemically unstable medium components such as glutamine and growth factors.
  • the medium may be fed through a PharMed tubing
  • This tubing may have a "slack" so that the syringe pump can be moved to a laminar flow hood where the syringes can be replaced in a sterile environment.
  • the extra tubing may be kept in the refrigerator so that only a short tube segment is at room or at incubator temperature. This arrangement is important since liquid residence time in the tube can be on the order of days (depending of the flow rate used).
  • the gas may come from either a cylinder containing premixed gases (a mixture of O 2 , N 2 and CO 2 ) or may be simply taken from the inside of the incubator (a mixture of air and CO 2 ).
  • the flowrate and composition of the gas stream may thus be easily controlled.
  • the gas may be pumped with an aquarium pump through a sandstone in a 100 ml medium flask to give relative humidities as close to 100% as possible.
  • the gas line may contain a sterile filter.
  • the spent medium may be collected in a 100 ml medium bottle. Samples may be taken from it for analysis of medium components.
  • the sets of chambers may be kept in an CO 2 incubator with a humidification system.
  • the perfusion chamber may be made from two specially machined pieces of a polycarbonate slab, the chamber top and bottom. Between the top and bottom piece two identical 1/4 inch silicone rubber gaskets may be placed.
  • a membrane When the chamber is assembled, a membrane may be placed between the two silicone gaskets which in turn may be placed between the top and bottom piece and four bolts may be used to hold the chamber together.
  • the difference between the top and bottom piece is the number of ports provided.
  • the bottom piece may have two ports for gas inlet and outlet, whereas the top piece may have three ports, a medium inlet and outlet and a sample port, placed in the middle of the top piece.
  • the outlet port may be constructed so that an angle is formed relative to the horizontal position to provide gravity induced settling for any non-adherent cells that might be floating about the chamber.
  • the geometry of the hole in the silicone gasket may be circular, but an elliptical shape with the inlet and outlet ports placed in focal points of the ellipse may provide a better fluid distribution.
  • the lower membrane may be a gas exchange membrane, such as silicone, teflon, mylar, etc.
  • the membrane is preferably hydrophobic to prevent loss of water and is permeable to gases.
  • the gas exchange membrane can provide a mechanical support for the second membrane.
  • the second membrane is for cell attachment and growth and may be, e.g., an inorganic ceramic based membrane. It can be coated with extra-cellular protein.
  • PepTite-2000® RGD based adhesion protein a product of Telios
  • a highly desirable property of a ceramic inorganic membrane is that it becomes transparent upon
  • the second membrane may serve as a surface for the attachment of the adherent cells.
  • chambers have a cracked inorganic membrane after autoclaving.
  • the tubing for medium and gases may be connected to the top and bottom pieces using polypropylene fittings and Luer Locks rings. By employing silicone O-rings, good seals are formed and no leakage problems have occurred.
  • the perfusion chamber can be assembled in other
  • the syringe pump should carry up to ten syringes (for a set of ten parallel chambers) of the appropriate size. Currently we are using a flowrate of 2 ml/day and 10 ml syringes, requiring syringe change every 5 days. The pump should be accurate, reliable, and be able to operate at 4°C over extended periods of time. We have used a Harvard '22' syringe pump with a multiple syringe holder.
  • the air may be pumped with an aquarium pump.
  • Medium bottles may be used for the spent medium and for the
  • the cells are treated prior to inoculation in the same fashion as they are prepared for Dexter cultures. After aspiration from a donor, mononuclear cells are separated on a discontinuous density gradient (Ficoll) and then washed several times in the culture medium. This procedure typically takes about half a day.
  • Ficoll discontinuous density gradient
  • the medium used is the standard Dexter medium, 10% horse serum, 10% fetal calf serum, 10 -6 M hydrocortisone and IMDM.
  • hematopoietic growth factors are added. Typically, Il-3, GM-CSF and Epo are used as previously described. The search continues for the optimal combination of added hematopoietic factors.
  • Perfusion Chambers The preparation of the perfusion chambers starts one day prior to inoculation. Assembly of a set of 6-10 perfusion chambers takes about 6 to 8 hours. This involves sizing/cutting tubing, putting fittings into the chamber, preparing the medium bottles, etc. At the end of the day the full chamber assembly (less the tubing and attachments for the gas exchange) is autoclaved without medium (all components are autoclaved). The set of chambers is then typically stored in a hood overnight. The following day the full set of components is assembled in the hood, the medium introduced, any adhesion protein applied, cells inoculated, the chambers placed in the incubator, the syringes loaded into the pump and stored in the refrigerator. If cell preparation is included, these steps take another day. Thus, the chambers are running at the end of the second say. The perfusion typically begins after the cells have settled in the chamber for 12 to 24 hours. Running the Perfusion Chambers:
  • the chambers are set up they are easy to maintain; the syringes need to be replaced at a fixed interval and non- adherent cells collected.
  • the syringes are typically replaced on a fixed schedule. For instance, during the initial runs with the chambers, 10 ml syringes were used at a flow rate of 2 mis per day. Syringes thus were replaced every 5th day.
  • the syringe pump is moved from the incubator to the hood where the syringes are replaced in a sterile environment. This transfer of the pump is allowed by the "slack" in the medium inlet line as described above. We have yet to experience contamination problems during this procedure.
  • the inorganic membrane becomes transparent once it is hydrated and thus during operation one can observe the cells in the chamber through a microscope. To do so one needs a long distance objective.
  • Sampling cells can be achieved using one of two methods. Firstly, we let cells settle by gravity inversion for two hours and then we replace 2 mis in the chamber by pushing liquid through the inlet port and collecting it from the outlet line. Secondly, we have pulled directly through the sampling port 2 mis leaving air space in the chamber that then disappears within a day due to the incoming medium. The second method is more invasive and yields a higher number of cells (approximately two to four fold).
  • the cell sampling takes place in the laminar flow hood.
  • the set of chambers is moved from the incubator to the hood and the length of the inlet medium line allows for this transfer. Since the solubility of oxygen is higher at lower temperatures, the cooling to room temperature often results in the formation of bubbles in the chambers (the bubbles are often located between the two membranes) once they are
  • the adherent cells can be removed in a similar fashion after treatment with trypsinization.

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Abstract

On décrit des procédés, y compris des conditions de milieux de culture, qui produisent la division des cellules-souches humaines in vitro et/ou l'optimisation des cultures des cellules-souches hématopoéïtiques humaines, et/ou l'accroissement du métabolisme ou de la sécrétion GM-CSF ou IL-6 ou des cellules stromales humaines, et/ou un procédé pour déterminer l'effet d'une substance ou l'état d'une population cellulaire hématopoéïtique humaine, et/ou pour provoquer la déplétion des cellules malignes ou le contenu des cellules T ou des cellules B d'une population cellulaire hématopoéïtique humaine. Les procédés s'appuient sur la culture des cellules-souches humaines et/ou des cellules-souches hématopoéïtiques humaines, et/ou des cellules stromales humaines dans un milieu de culture liquide qui est remplacé, de préférence irrigué, soit continuellement, soit périodiquement, à un taux de 1 ml de milieu par ml de culture par période d'environ 24 à environ 48 heures, et sur l'élimination de produits métaboliques et la recharge de produits nutritifs appauvris tout en maintenant la culture dans des conditions physiologiquement acceptables. Eventuellement, on ajoute des facteurs de croissance au milieu de culture. Les conditions de culture décrites permettent d'obtenir des procédés améliorés de transplantation de la moëlle osseuse.
PCT/US1992/011228 1992-01-02 1992-12-31 Procede de regulation des lignages cellulaires humains hematopoeïtiques WO1993012805A1 (fr)

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