[go: up one dir, main page]

WO1995006112A1 - CROISSANCE Si(IN VITRO) DE PRECURSEURS DE NEUTROPHILES ET DE MEGACARYOCYTES DANS DES MILIEUX EXEMPTS DE SERUM - Google Patents

CROISSANCE Si(IN VITRO) DE PRECURSEURS DE NEUTROPHILES ET DE MEGACARYOCYTES DANS DES MILIEUX EXEMPTS DE SERUM Download PDF

Info

Publication number
WO1995006112A1
WO1995006112A1 PCT/US1994/009622 US9409622W WO9506112A1 WO 1995006112 A1 WO1995006112 A1 WO 1995006112A1 US 9409622 W US9409622 W US 9409622W WO 9506112 A1 WO9506112 A1 WO 9506112A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
serum
free
megakaryocyte
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1994/009622
Other languages
English (en)
Inventor
Stephen L. Smith
Xiaoying Qiao
Susan M. Maciukas
James G. Bender
Dennis E. Van Epps
Maureen F. Loudovaris
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baxter International Inc
Original Assignee
Baxter International Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baxter International Inc filed Critical Baxter International Inc
Priority to JP7507759A priority Critical patent/JPH09505462A/ja
Priority to EP94927235A priority patent/EP0719326A1/fr
Priority to AU76744/94A priority patent/AU702871B2/en
Publication of WO1995006112A1 publication Critical patent/WO1995006112A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0644—Platelets; Megakaryocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/98—Xeno-free medium and culture conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the invention relates generally to human hematopoietic cells cultured in serum-free media. More specifically, the invention relates to serum-free cell suspensions enriched for neutrophil and megakaryocyte precursors useful for treating neutropenia and thrombocytopeni .
  • Cancer treatments such as high-dose chemotherapy and radiation destroy hematopoietic cells in the bone marrow, leaving the patients severely depleted of neutrophils and platelets. After such treatments, patients often spend several weeks in intensive care due to infections and fever resulting from neutropenia. Thrombocytopenia leads to reduced clotting and bleeding disorders requiring platelet transfusions. Lack of neutrophils and platelets is the leading cause of morbidity and mortality following these cancer treatments, and contributes to the high cost of cancer therapy.
  • Hematopoietic growth factors are administered after therapy to stimulate remaining stem cells to proliferate and differentiate into mature infection fighting cells. Although hematopoietic growth factors can shorten the total period of neutropenia, there remains a critical 10 - 15 day period immediately following therapy when the patient is severely neutropenic and thus infection prone. Even with growth factor stimulation, 10 to 15 days are required for the patient's stem cells to proliferate and progress through the various stages of differentiation leading to mature neutrophils. Megakaryocyte and platelet recovery requires an even longer time, generally greater than 15 days. Thus growth factor treatment leaves a gap during which the patient is deficient in infection fighting cells and blood clotting ability.
  • Post-therapy bone marrow transplantation can also ameliorate neutropenia after about 10 - 15 days. Since allogenic bone marrow transplantation is often complicated by Graft versus Host Disease, autologous bone marrow transplantation is preferred whenever practical. Bone marrow is harvested from the patient prior to therapy, frozen, and then thawed and transplanted back into the patient after therapy. Autologous bone marrow transplantation carries the risk that the transplanted bone marrow may harbor tumor cells. In any event, bone marrow does not contain sufficient mature neutrophils or their immediate precursors to restore the patient's immunity during the critical 10 -15 day period after therapy.
  • Mobilization occurs as a .result of either chemotherapy, or administration of hematopoietic growth factors, or both. It is believed that hematopoietic stem cells in the bone marrow are "mobilized” into the peripheral blood stream as a natural result of the recovery of myelosuppressed bone marrow or in response to relatively large doses of hematopoietic growth factors.
  • Growth factors used for mobilization include interleukin-3 (IL-3), granulocyte colony stimulating factor (G-CSF) , granulocyte-macrophage colony stimulating factor (GM-CSF) , stem-cell factor (SCF) and a recombinant fusion protein having the active moieties of both IL-3 and GM-CSF (Brandt, SJ, et al., N En ⁇ J Med 318:169, 1988; Crawford, J, et al., N Eng J Med 325:164, 1991; Neidhart, J, et al., J Clin Oncol 7:1685, 1989).
  • IL-3 interleukin-3
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • SCF stem-cell factor
  • Mobilized peripheral blood stem cells are harvested after chemotherapy or growth factor treatment, and then reinfused into the patient following the next round of high dose chemotherapy or radiation. The reinfused stem cells then proliferate and differentiate in vivo, eventually to restore the patient's neutrophil and platelet population.
  • differentiated hematopoietic cells were generated in vitro in the laboratory of the present inventors (Smith, S.L., et al.. Experimental Hematoloqy 21:870-877, 1993). Committed precursors of neutrophils were successfully generated in vitro using fetal bovine and horse serum- containing media. However, the potential for therapeutic use of the cells would be greatly enhanced if the cells were grown without animal sera or animal proteins in the culture medium.
  • animal serum supplementation was relied upon as a source for protein and growth factors in culture media formulations.
  • researchers have long sought media formulations free of animal proteins for the growth of therapeutic cells because of the potential for life-threatening immune reactions raised by infusion of foreign proteins.
  • animal sera, and in particular fetal bovine sera contain many unknown growth factors, certain factors being more or less important for each cell type. Even if every factor in fetal bovine serum were identified and chemically synthesized, it would still be a matter of trial and error and much experimentation to discover which factors promote the proliferation and differentiation of each cell type. In spite of the difficulty, researchers have succeeded in formulating serum- free media in which certain hematopoietic cells may be grown.
  • Serum-free media formulations containing bovine serum albumin and various hematopoietic growth factors were shown to promote the proliferation of murine bone marrow cells (Ponting, I.L.O., et al., Growth Factors 4:165-173, 1991; Merchauv, S., et al., Internatl J Cell Cloning 2:356-367, 1984;).
  • the invention provides serum-free, animal protein-free media formulations to be used in conjunction with hematopoietic growth factors for the in vitro growth of human neutrophil and megakaryocyte precursors.
  • the medium is comprised of a base medium, corticosteroid, transferrin, insulin, cholesterol, ethanolamine, and human albumin.
  • the invention also provides methods for preparing serum-free, animal protein-free suspensions of human hematopoietic precursor cells wherein the cellular component comprises at least about 16% neutrophil precursors and at least about 1% megakaryocyte precursors.
  • Serum-free, animal protein-free suspensions of human hematopoietic cells are provided wherein the cellular component comprises at least about 30%, preferably greater than 60% neutrophil precursors.
  • the neutrophil precursors are comprised of blast cells, promyelocytes, neutrophilic myelocytes, and neutrophilic metamyelocytes.
  • serum-free, animal protein-free cell suspensions wherein the cellular component comprises at least about 3%, preferably greater than 8% megakaryocyte precursors.
  • serum-free, animal-protein free cell suspensions wherein the cellular component comprises colony- forming units and cluster-forming units.
  • Figure 1 depicts the differential cell count of cultures grown in serum-free medium (295-1), in serum-containing medium (HLTM) , and cultures which were grown for 10 days in 295-1 and then transferred to HLTM for an additional 10 days.
  • Figure 2 shows blast cells (solid arrows) in a 10 day serum- free culture.
  • Figure 3 shows promyelocytes (solid arrows) and a neutrophilic metamyelocyte (hollow arrow) in a 10-day serum-free culture.
  • Figure 4 shows neutrophilic myelocytes (solid arrows) and neutrophilic metamyelocytes (hollow arrows) in a 10 day serum- free culture.
  • Figure 5 shows the cell phenotypes as determined by flow cytometry.
  • Figure 6 shows a typical field of colonies (solid arrows) and clusters (hollow arrow) .
  • Figure 7 shows megakaryocyte precursors labeled by i munocytochemistry (red) in a field of myelocytic cells (blue) .
  • Figure 8 shows forward light scatter of the cells in flow cytometry; (a) control (b) anti-CD41a.
  • Figure 9 shows a typical megakaryocyte burst developed in a fibrin clot assay.
  • Figure 10 shows the kinetics of megakaryocyte cell growth in serum-free cultures compared to normal media.
  • Figure 11 shows the effects of serum-free culture media compared to normal media or megakaryocyte growth from isolated CD34 + cultures.
  • Figure 12 shows the growth comparisons of megakaryocytes in different cultures.
  • Figure 13 shows the effects of various cytokine combinations on megakaryocyte growth.
  • the invention provides serum-free media formulations for the in vitro growth of human neutrophil and megakaryocyte precursors.
  • the resulting serum-free, animal protein-free cell suspensions are suitable for infusion into a patient for the treatment of neutropenia and thrombocytopenia.
  • the absence of animal proteins in the cell suspensions is especially advantageous since animal proteins are known to cause immune reactions in humans.
  • a high proportion of the cells in the suspensions are at suitably advanced stages of differentiation so that soon after infusion they are expected to differentiate within the patient to form mature neutrophils, megakaryocytes and platelets.
  • Essential reagents in the serum-free media formulations are the base medium, corticosteroid, transferrin, insulin, cholesterol, ethanolamine, and human albumin.
  • the base medium may be any standard cell culture medium containing inorganic salts, vitamins, amino acids, and at least one energy source such as glucose or pyruvate.
  • the base medium contains a bicarbonate buffer and a pH indicator such as phenol red.
  • the pH of the final medium is maintained at 6.8 - 7.4, preferably at about 7.2, by means of a controlled 0 2 /C0 2 gas atmosphere.
  • the osmolarity of the basal medium is preferably in the range of 260-290 mOsm/Kg.
  • Hematopoietic growth factors are also necessary. Growth factors are selected from the following: interleukin-3 (IL-3), granulocyte-colony stimulating factor (G-CSF), granulo ⁇ yte/macrophage-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-11 (IL-11), stem cell factor (SCF), interleukin-1 (IL-1), thrombopoietin and gamma interferon.
  • Whole growth factors may be replaced by chimeric or fusion proteins having the active moieties of one or more factors. For instance, in place of IL-3 and GM-CSF, a single fusion protein having the active moieties of both IL-3 and GM- CSF may be used.
  • the growth factors are produced recombinantly in microorganisms or mammalian single-cell hosts, and have amino acid sequences either identical to or highly homologous with the active moieties of corresponding human growth factors.
  • the factor is highly purified from any host proteins associated with it such that, when added to the herein provided media formulations, no host protein is detectable.
  • growth factor combinations and concentrations may be manipulated to yield various results.
  • G-CSF and GM-CSF will enhance the production of neutrophils whereas thrombopoietin will enhance the production of megakaryocytes.
  • the serum free media formulations provided herein are expected to further enable the identification of specific growth factor functions due to the absence of confounding factors from serum.
  • serum-free refers to a medium formulation which does not include any form of whole serum, neither animal nor human.
  • a protein which has been purified from other serum components is considered serum-free.
  • Human serum albumin provides a source of protein in the media. It is generally believed that protein is required to provide a viscosity similar to that of blood so that hematopoietic cells may thrive. Moreover, protein acts as a substrate for proteases which might otherwise digest cell membrane proteins. Albumin is thought to act as a carrier for trace elements and essential fatty acids. HSA is greatly advantageous over protein derived from animals such as bovine serum albumin (BSA) due to the reduced immunogenic potential of HSA.
  • BSA bovine serum albumin
  • the HSA may be derived from pooled human plasma fractions, or may be recombinantly produced in such hosts as bacteria and yeast, or in vegetable cells such as potato and tomato.
  • the HSA used in the present formulations is free of pyrogens and viruses, and is approved by regulatory agencies for infusion into human patients.
  • the HSA may be deionized using resin beads prior to use in the media.
  • Transferrin and insulin used in these serum-free media formulations may be derived from animal sera, but it is most preferable to use products which are recombinantly synthesized. It is understood that when transferrin and insulin are derived from an animal source, they are purified to remove other animal proteins, and thus are at least 99% pure. Transferrin is used in these formulations at only 25 - 125 ⁇ g/ml, while insulin is used at only 1 - 25 ⁇ g/ml. Therefore, any trace of animal protein other than insulin or transferrin in the medium is non-detectable using standard techniques such as HPLC and gel electrophoresis.
  • the term "essentially free of animal proteins” refers to a medium formulation or a cell suspension in which no animal proteins other than transferrin or insulin are detectable.
  • the term "animal” is understood to exclude microorganisms and humans.
  • the transferrin and insulin are genetically engineered proteins produced by microorganisms such as bacteria and yeast.
  • the amino acid sequences of the recombinant transferrin and insulin are identical to or highly homologous with those of humans.
  • the most preferable serum-free media formulations herein contain no animal-derived proteins and do not have even a non-detectable presence of animal protein.
  • the corticosteroid component may be any naturally occurring or synthetic glucocorticoid hormone, preferably hydrocortisone at a concentration of 1 - 10 ⁇ M.
  • the corticosteroid may also be dexamethasone, methylprednisone, or other glucocorticoids approved for clinial use.
  • Cholesterol may be chemically synthesized or purified from human serum. Cholesterol is used in the present media formulations at a concentration of 0.01 - 0.1 mg/ml, most preferably at 0.05 mg/ml.
  • ethanolamine added to serum free media in the range of 50 - 200 ⁇ M, preferably 100 ⁇ M greatly increased the proliferation and development of neutrophil and megakaryocyte precursors.
  • Ethanolamine also known as ⁇ -aminoethyl alcohol, is a viscous, hygroscopic liquid which is commonly used as a surfactant in the manufacture of pharmaceuticals.
  • the exact function of ethanolamine in the present media formulations is not known, however this does not diminish the importance of the discovery of its beneficial effects, especially within the context of the specific media formulations provided herein.
  • serum-free media are made fresh each day and continuously refreshed in the cultures.
  • reducing agents such as ⁇ -monothioglycerol and ⁇ - mercaptoethanol, which are thought to diminish free-radical formation, may be added to the serum-free media formulations to enhance stablility during storage times of up to 20 days or longer. Reducing agents may also be helpful when the media formulations are used in static culture and refreshed only after several days of use.
  • Antibiotics such as gentimicin may also be added to the media as a precaution against bacterial infection of the cultures.
  • lipids containing the above reagents, known herein as media "295-1", were found to be optimal for the development of megakaryocyte precursors within a population of neutrophil precursors, as will be described below. However, it was discovered that the addition of other lipids to the above essential reagents could enhance the proliferative potential of neutrophil precursors.
  • triglycerides and/or phospholipids are included as additional sources of lipid.
  • a preferable source of lipid is a sterile, non-pyrogenic fat emulsion prepared from soybean oil and egg yolk phospholipids, typified by Intralipid® (Kabi Pharmacia) .
  • Such a preparation preferably contains a mixture of neutral triglycerides of predominantly unsaturated fatty acids such as linoleic, oleic, palmitic, linolenic, and stearic acid.
  • Such a preparation may also contain phosphatidylethanolamine and phosphatidylcholine from egg yolk.
  • Another source of lipid is a human plasma fraction precipitated by ethanol and preferably rendered virus-free by pasteurization.
  • a growth-enhancing media supplement, Nutrimax*" (Baxter Hyland)
  • Nutrimax* is derived from Cohn's fraction IV 4 and contains triglycerides as well as cholesterol.
  • a cholesterol-containing lipid preparation it may substitute for the cholesterol in the 295-1 formulation above provided the lipid preparation supplies cholesterol at a final concentration of at least about 30 ⁇ g/ml.
  • the cells originate either from bone marrow, from cord blood, or from peripheral blood.
  • Bone marrow samples may be obtained either from normal donors or from patients.
  • Umbilical cord blood is obtained after normal gestations.
  • Peripheral blood is obtained either from normal donors or from cancer patients.
  • cancer patients are treated with hematopoietic growth factors to "mobilize” or stimulate their stem cells to move from bone marrow to their peripheral blood stream, thus greatly increasing the number of stem/progenitor cells in their peripheral blood samples.
  • the term “stem/progenitor cell” refers to a hematopoietic cell having the CD34+ cell surface antigen (stem cells and colony-forming units).
  • the number of CD34+ cells comprises only about 0.1% of total leukocytes. Mobilization brings the number of CD34+ cells up to about 1 - 4% of total leukocytes.
  • CD34+ cells comprise about 0.1 - 1% of total leukocytes.
  • Normal bone marrow typically contains only 1 - 2% CD34+ cells.
  • White blood cells are first separated from the samples of bone marrow or cord or peripheral blood by standard methods such as centrifugation through a gradient.
  • a leukocyte population from bone marrow generally contains only about 10 - 15% myeloblasts and promyelocytes (Geigy Scientific Tables, Vol 3, C. Lentner, ed. Ciba-Geigy, Basel, Switzerland, 1984) .
  • Mature megakaryocytes in bone marrow, as recognized by Wright-Giemsa staining, comprise only about 0.05% of the leukocyte population, while immunostaining specific for megakaryocyte lineage cells labels up to about 0.2%. Since, in a healthy individual, neutrophil and megakaryocyte precursors differentiate fully in the bone marrow, a precursor is only very rarely discovered in normal blood.
  • leukocytes may be cultured directly in serum-free medium.
  • stem/progenitor cells are separated from the leukocyte population by positive selection. Positive selection of stem/progenitor cells may be based on their binding to an antibody specific for CD34+, followed by separation of antibody-bound stem/progenitor cells using paramagnetic beads (Hardwick, RA, et al., J Hematother 1:379-386, 1992; Smith, SL, et al., supra).
  • Positively selected stem/progenitor cells are placed in culture at densities ranging from 5,000 to 100,000 cells/ml, preferably at 10,000 cells/ml.
  • Any standard tissue culture flasks or bags may be used in either a static or a perfusion culture system (Roller, MR, et al., BIO/TECHNOLOGY 11:358-363; Emerson, SG, et al., PCT W092/11355).
  • a static culture system When a static culture system is used, the cells are fed at intervals of 5 to 7 days to replenish nutrients and remove wastes.
  • Cells are cultured in serum-free medium for 7 - 14 days, more preferably 9 - 12 days, at which time the cell suspension contains a suitable population of neutrophil and megakaryocyte precursors for use in the treatment of neutropenia and thrombocytopenia.
  • serum-free medium formulation 295-1 the cell population contains at least about 16% neutrophil precursors and at least about 1% megakaryocyte precursors.
  • the cell population contains over 30% neutrophil precursors, more preferably over 60% neutrophil precursors.
  • the neutrophil precursor population is composed of blast cells, promyelocytes, neutrophilic myelocytes, and neutrophilic metamyelocytes, which are the precursors of mature banded and segmented neutrophils (Marmont, AM., et al., IN "Neutrophils”, Atlas of Blood Cells, Function and Pathology,Eds. Zucker-Franklin, D., et al., 2nd Edition, pp. 159-190; Jandl, JH 1987 Blood. Textbook of Hematology. Little, Brown & Co., Boston/Toronto, pp.441-480).
  • Flow cytometric analysis of the 10-day serum free cultures was performed to determine the cells' phenotypes based on labeling with cell surface antigens.
  • Cells which are positive for the CD15 antigen and negative for the CDllb antigen (CDl5+/CDllb-) have been shown by morphological analysis to be predominantly myelocytes and promyelocytes (Smith, SL, et al., supra) .
  • Cells which are CD15+/CDllb+ have been shown to be predominantly mature segmented neutrophils.
  • the 10-day serum- free cultures of the present invention were shown to contain 20 - 60% CDl5+/CDllb- cells. When the cultures were transferred at day 10 from serum-free to serum containing medium, the phenotypes were seen to progress to predominantly mature forms as identified by CD15+/CDllb+ labeling ( Figure 5c) .
  • megakaryocyte precursor refers to a nucleated cell which expresses the platelet/megakaryocyte specific glycoprotein Ilb/IIIa, also known as "CD41a", as identified by immunostaining and/or flow cytometry.
  • the megakaryocyte precursor population is composed mainly of promegakaryoblasts and megakaryoblasts (Long MW, Stem Cells 11:33-44, 1993; Paulus, JM, "Platelet kinetics", in: Williams, WJ, et al., (eds), Hematology, McGraw-Hill, Ner York: 1251-1260, 1990).
  • the megakaryocyte precursor component of the serum-free 9 - 12 day cultures comprises about 1% of the total cells, more preferably at least about 3% of the total cells, most preferably greater than 8% of the total cells.
  • serum-free medium 295-1 were superior to those obtained in the control, serum-containing medium.
  • Culture in serum-free medium yielded 3 - 22 times the number of megakaryocte precursors as compared with control, which suggested that the serum component may have an inhibitory effect on megakaryocyte precursor growth.
  • a cell suspension greatly enriched for megakaryocyte precursors enables the effective treatment of various types of thrombocytopenia in addition to those caused by cancer therapy.
  • the herein provided cell suspensions may replace platelet infusions in the treatment of thrombocytopenia.
  • the co-existence of a high proportion of megakaryocyte precursors with neutrophil precursors renders these serum-free cell suspensions ideal for the co-treatment of thrombocytopenia and neutropenia.
  • the administered cells will further differentiate in vivo to form mature neutrophils and megakaryocytes, which ultimately form platelets.
  • This expectation is based upon the discovery that when this cell population is switched to serum-containing medium for a further 10 days of culture, the population advances to more mature neutrophilic forms ( Figure 1).
  • the megakaryocyte precursors were observed to form mature megakaryocytes and release platelets. The results from these assays suggest that cells from the serum-free cultures can undergo further maturation after they are returned to in vivo conditions.
  • One of the primary advantages of the 7 - 14 day serum-free cell culture lies in its population of more mature neutrophil and megakaryocyte precursors, as described above.
  • another advantage of this cell culture lies in its small population of earlier cell types, i.e. "progenitors", which are capable of a number of successive rounds of proliferation and differentiation.
  • the daughter cells of these progenitors are expected to follow the precursors in maturation to provide a later population of neutrophils and megakaryocytes, thereby extending the duration of effective treatment possible from an infusion of the original cell suspension.
  • CFU colony-forming units
  • clFU cluster-forming units
  • CFU is herein defined as a single cell which, when plated in a serum-containing methylcellulose culture for 14 days, proliferates to form a closely associated group of 50 or more cells (a "colony”). Under the same conditions, a clFU forms a group of fewer than 50 cells (a "cluster"). Colonies are further defined by the cell type(s) they contain, as identified by Wright-Giemsa staining (Zucker-Franklin, et al., supra) .
  • the colonies which are expected to ultimately form mature neutrophils and mono ⁇ ytes are designated "granulocyte/macrophage" or GM.
  • the clusters are composed of early granulocyte precursors and macrophages, with the granulocytic types predominating.
  • this cell population preferably contains about 0.5 to 3.0% CFU and clFU combined.
  • GM-CFU preferably comprise about 5 - 50% of the total CFU/clFU.
  • the cells within the clusters are more differentiated than the cells within the GM colonies.
  • the clFU within the population are expected to provide a bridge of committed precursors before the GM-CFU have gone through a sufficient number of divisions and stages of differentiation to replenish the precursor population.
  • the clFU comprise about 40 - 60% of the total CFU/clFU.
  • a colony of cells of the megakaryocyte lineage may contain fewer cells due to their unique form of differentiation.
  • a megakaryocyte burst forming unit MK- BFU
  • MK- BFU megakaryocyte burst forming unit
  • a megakaryocyte colony forming cell MK-CFC
  • MK-CFC megakaryocyte colony forming cell
  • the single megakaryocyte forming unit can only undergo endomitosis (DNA duplication without cell division) and form a single polyploid cell in the colony culture.
  • the S-MK is thus regarded as more mature than the MK-CFC.
  • MK-BFU represent 30 - 57% of the total cells in 5 - 7 day cultures capable of megakaryocyte burst/colony formation.
  • MK-CFC By day 12 in serum-free culture, all the MK colony forming units have progressed to MK-CFC or S-MK.
  • the serum-free cell suspensions provided herein may be administered to a patient in an amount sufficient to abrogate neutropenia and thrombocytopenia following ablative cancer therapy.
  • the enrichment of neutrophil and megakaryocyte precursors in these cell suspensions allows the administration of an effective therapeutic number of desired cells with fewer total cells returned to the patient.
  • Persons skilled in the art will be able to determine the optimal quantity and conditions for administration of the cell suspensions based on their own clinical experience and using guidelines from literature on stem cell infusions (Spitzer, G., et al.. Blood 55:317-323; Douay, et al., EXP Hematol 14:358-365, 1986).
  • Serum free media were formulated as follows. Optimum concentrations were determined by separate titrations based on cell proliferation.
  • Lipid derived from a human plasma fraction (Pentex® Ex-Cyte®, Miles Inc., Kankakee, IL; cholesterol, 4.96 mg/ml; triglycerides, 1.67 mg/ml) was used at dilutions from 1:50 - 1:500, optimum 1:100.
  • Lipids derived from soybean oil and egg yolk (Intralipid®, Kabi Pharmacia, Clayton, NC; neutral triglycerides with fatty acid residues comprising 50% linoleic, 26% oleic, 10% palmitic, 9% linolenic, and 3.5% stearic; phospholipids comprising primarily phosphatidylcholine and phosphatidylethanolamine), at 0.03 - 2%, optimum 1%.
  • Stock solutions of each lipid were prepared in ethanol at a dilution of 1:10.
  • Intralipid® was sonicated in a test tube for 30 minutes.
  • Control serum-containing medium was composed of the above base media, standard supplements and growth factors, plus 12.5% fetal bovine serum and 12.5% horse serum (Sigma).
  • Mononuclear cells were obtained from samples of bone marrow, cord blood, or peripheral blood. Normal individuals provided the bone marrow and cord blood.
  • the peripheral blood cells were obtained from cancer patients whose hematopoietic cells had been "mobilized” into their peripheral blood from their bone marrow by the administration of hematopoietic growth factors (Brandt, SJ, et al., supra; Crawford, J., et al., supra: Neidhart, J., et al., supra) .
  • the cells in the samples were separated by centrifigation through Histopaque® (Ficoll®Hypaque®, Sigma) and the mononuclear cells from the interface band were collected (Smith, S.L., et al., Exp Hematol 21:870-877, 1993).
  • the suspensions of mononuclear cells were enriched for stem/progenitor cells by positive selection of CD34+ cells using magnetic beads as follows.
  • the mononuclear cells were first sensitized for 30 minutes on ice with 0.5 ⁇ g of anti- CD34 antibody per IO 6 cells [anti-CD34 #9069 prepared by serum-free cell culture (Baxter Hyland, Hayward, CA) and purified by protein G affinity chromatography (Baxter Immuntherapy Division)].
  • the cells were then washed 3 times by centrifugation in IMDM to remove unbound antibody and mixed with sheep antimouse IgG ⁇ Fc coated magnetic beads at a bead- to-cell ratio of 1:1.
  • the mixture of beads and cells was then rotated for 30 minutes at 4°C.
  • CD34+ cells were released from the beads by adding 50 U/ml of Chymodiactin® (Bootes Pharmaceutical, Lincolnshire, IL) in RPMI 1640 (Sigma) and incubating for 15 minutes in a 37°C water bath.
  • Chymodiactin® Bootes Pharmaceutical, Lincolnshire, IL
  • the cells released from the beads were then evaluated for CD34+ purity by staining with the FITC-8G12 monoclonal antibody to CD34 (Baxter Immunotherapy Division, Irvine, CA) for 15 minutes on ice and quantitated with a FACSan® flow cytometer (Becton Dickenson, San Jose, CA) as described (Smith, S.L., et al, supra ) .
  • EXAMPLE 3 Culture of hematopoietic cells in serum-free media. Enriched preparations of CD34+ cells were cultured in serum- free media at initial concentrations of 5xl0 3 - lxlO 5 cells/ml in either polystyrene flasks or plastic bags. Tissue-culture- treated flasks, as well as non-treated flasks of 25, 75 and 150 cm 2 (Corning, Corning, NY) were used. The ethylvinyl acetate (EVA) gas-permeable plastic bags were type PL269 of 250, 500, and 1000 ml capacity (Baxter Fenwal, Deerfield, IL) .
  • EVA ethylvinyl acetate
  • the 250 ml bags were filled with 20-60 ml, the 500 ml bags were filled with 60-100 ml, and the 1000 ml bags were filled with 100-400 ml of cell suspension.
  • the cultures were placed in a 5% C0 2 /5% 0 2 , 37°C high humidity incubator for the times indicated in the examples below.
  • the cultures were fed at intervals of 5 - 7 days, with dilutions of 1:2 or 1:4.
  • the concentration of nonadherent cells in the cultures was determined by diluting 0.5 ml from the culture in 9.5 ml 10% cetrimide (Sigma) and counting on a Coulter ZBI (Coulter Electronics, Hialeah, FL) .
  • EXAMPLE 4 Assessment of growth of neutrophil precursors in serum-free medium.
  • the cell cultures of Example 3 were sampled at indicated days in culture, stained by the Wright-Giemsa method, and differentially counted according to morphological characteristics (Marmont, A.M., et al. "Neutrophils", in Atlas of Blood Cells. Eds. Zucker-Franklin, D., et al., 2nd Edition, pp.159-190; Jandl, J.H. (1987) Blood, Textbook of Hematology. Little, Brown & Co., Boston/Toronto, pp.441-480).
  • Figure 1 depicts the differential cell count of a typical cell culture grown in serum free medium formulation 295-1 (example 1) compared with growth in serum-containing medium ("HLTM").
  • Hematopoietic growth factors were added to both media as follows: IL-3 at 1000 U/ml, G-CSF at 500 U/ml, GM-CSF at 500 U/ml, and SCF at 20 ng/ml.
  • IL-3 1000 U/ml
  • G-CSF at 500 U/ml
  • GM-CSF GM-CSF at 500 U/ml
  • SCF serum-containing medium
  • FIG. 1 Blast cells (solid arrows).
  • Figure 3. Promyelocytes (solid arrows) and neutrophilic metamyelocyte (hollow arrow).
  • Band forms and segmented neutrophils (“Bands/Segs", Fig. 1), the most mature neutrophilic cells, were readily recognized according to standard criteria (Marmont, A.M., et al., supra) .
  • the category designated "Other” in Figure 1 includes monocytes, macrophages, and megakaryocyte precursors which are not identifiable by Wright-Giemsa staining. Monocytes and macrophages were identified as described in the Atlas of Blood Cells (supra. Johnston, RB, Jr., The mononuclear phagocyte system, pp.321-377) .
  • Megakaryocyte precursors were identified by immunocytochemical staining as described below. Mast cells, which can be identified by toluidine blue staining of their heparin granules, represented less than 0.01% of the total cell population.
  • EXAMPLE 5 Neutrophil precursors grown in serum-free medium; phenotype identified by flow cytometry. At 10 and 20 days of culture, aliquots of l-2xl0 5 cells were removed from the culture vessels and washed 1 - 2 times in phosphate buffered saline containing 0.05% bovine serum albumin and 0.1% azide (PAB) . The cells were then labeled with CD15 (LeuMl) FITC-conjugated and CDllb (Leul5) PE- conjugated monoclonal antibodies (Becton Dickenson) for 10 minutes on ice. Following 1 additional wash in PAB, the cells were suspended in 1 ml PAB and analyzed by flow cytometry for coexpression of CD15 and CDllb.
  • CD15 LeuMl
  • CDllb Leul5
  • PE- conjugated monoclonal antibodies Becton Dickenson
  • the CDl5+CDllb- phenotype comprised 20 - 60% of the cells in serum-free cultures, which was similar to the day 10 profile of serum-containing cultures (Fig. 5a, lower right quadrant) .
  • cultures which had been maintained in serum-free media still contained predominantly the CD15+CDllb- phenotype (Fig. 5b).
  • cultures which had been transferred from serum-free to serum-containing media at day 10 contained 70 - 100% differentiated cells of the CD15+llb+ phenotype, similar to the phenotypic profile of cultures which had been grown in serum-containing media for the entire 20 days (Fig. 5c).
  • Cells which were CDl5+/CDllb- were sorted by FACS cell sorting and identified morphologically as neutrophilic precursors in the promyelocyte and myelocyte stages of differentiation (Smith, SL, et al., supra) .
  • Cells which were CDl5+/CDllb+ were sorted and identified as the more mature forms, i.e. metamyelocytes, band forms, and segmented neutrophils.
  • Colony forming units and cluster forming units in serum free cultures Colony forming units and cluster forming units in serum free cultures.
  • Cells were grown in various media formulations using the following growth factors: IL-3 at 300 U/ml, G-CSF at 300 U/ml, GM-CSF at 300 U/ml, and SCF at 20 ng/ml.
  • colony assays were set up in methyl-cellulose containing IMDM, 30% FBS (Sigma), 7% Leptalb • 7 (Armour Pharmaceuticals, Kankakee, IL) and recombinant growth factors at 150 U/ml rIL-3, 200 U/ml rGM- CSF, 150 U/ml rG-CSF, 160 U/ml rIL-6 and 10 U/ml erythropoietin (Amgen, Thousand Oaks, CA) .
  • Colony assays were set up in triplicate at 5 to lOxlO 3 cells/ml in 35 ml dishes (Nunc) and scored following 14-day incubation in 5% C0 2 /5% 0 2 at 37°C. Colonies were defined as groups of greater than 50 cells, and clusters were defined as groups of fewer than 50 cells (see Figure 6).
  • the colonies were identified macroscopically as either CFU-GM, macrophage (CFU-M) , burst- forming unit erythroid (BFU-E) or mixed (CFU-Mix) (myeloid and erythroid) with periodic morphological verification of plucked colonies using Wright-Giemsa staining. Results are shown in Tables 1 - 4 below.
  • P.I. proliferation index (total cells at day 10/total cells at day 0)
  • GM I. granulocyte/macrophage-colony forming index (GM colonies formed from 10 day culture/GM colonies formed from day 0 suspension) .
  • the number of CFU-GM in day 10 cultures increased 5 - 20 fold over day 0.
  • the number of CFU-Mac in day 10 cultures increased 2 - 10 fold over day 0.
  • the number of cluster forming units in day 10 serum-free cultures increased 10 -600 fold over day 0.
  • GM colonies comprised about 10-50% of total colonies/clusters
  • Mac colonies comprised about 5-20% of total colonies/clusters
  • clusters comprised about 10-60% of total colonies/clusters.
  • CFU colony-forming units
  • clFU cluster-forming units
  • the absolute number of cluster-forming units per 10,000 cells ranged from 16 to 136.
  • Cluster-forming units are the individual cells which proliferate directly into more mature forms of myelocytes such as band forms and segmented neutrophils.
  • cells were cyto-centrifuged onto microscopic slides, fixed with absolute acetone, and incubated with an anti-CD41a monoclonal antibody designated P2 (AMAC, estbrook, ME) .
  • the antibody recognizes platelet/megakaryocyte specific glycoprotein Ilb/IIIa. Glycoprotein Ilb/IIIa is present in megakaryocyte precursors, mature megakaryocytes, and platelets, but not in other blood cells.
  • a secondary antibody biotinylated goat anti-mouse IgG, Kirkegaard & Perry, Gaithersburg, MD was added and bound to the primary antibody.
  • BM Macroph bone marrow macrophages (98% of the cells in thi ⁇ culture were morphologically macrophages) ; ND, not done.
  • Cytokine combination (1) SCF , IL-3 & IL-6.
  • Cytokine combination (2 ) SCF , IL-3 , GM-CSF & G-CSF.
  • Figure 8 shows the flow cytometric dot-plot of a culture sample which was grown in the serum-free medium for 10 days.
  • the X axis represents the forward light scatter (FSC) which measures the cell size. Those cells which plotted further to the right are higher in FSC (hFSC) and larger in cell size.
  • the Y axis represent the green fluorescence intensity which measures the FITC conjugated antibody staining.
  • the isotype control staining of the sample using FITC conjugated anti-mouse IgG is presented in Figure 8a, showing no non-specific antibody binding to the cells.
  • EXAMPLE 8 Megakaryocyte colony formation analyzed by fibrin clot assay. At selected days in culture, cells were plated in a serum-free fibrin-clot colony culture (MK-CFC assay) and evaluated for their ability to form megakaryocyte colonies. Cells (2 x lOV l) were suspended in a semisolid fibrin-clot culture medium containing 1% BSA (Sigma), 0.02 % fibrinogen (KABI, Sweden) and 0.02 U/ml thrombin (Sigma) in IMDM (Zauli, G, et al., EXP Hematol 20:850-854, 1992).
  • MK-CFC assay serum-free fibrin-clot colony culture
  • Cytokines including SCF, IL-3, IL-6, and/or IL-11 were also utilized as indicated.
  • a 0.5 ml sample of the cell/medium suspension was placed on an ordinary microscope slide. The slide was placed in a humidified 150 mm Petri dish and cultured at 37°C, 5% C0 2 for 14 days. During this time, megakaryocyte precursors in the original culture proliferated to form megakaryocyte bursts and colonies, or differentiated into more mature single megakaryocytes. The fibrin-clot cultures were then fixed and i munostained as described in Example 7.
  • the above described MK-CFC assay offers several improvements and advantages over previously described assays.
  • the Petri dish has been replaced by the microscope slide as a substrate for the megakaryocyte colony cultures, which offers these advantages: (1)
  • the slide enables the use of absolute acetone, the best fixative to preserve the protein antigen for the immunocytochemical detection.
  • the colony cultures were grown in plastic Petri dishes, which would dissolve in acetone.
  • the slide facilitates the immunostaining by decreasing the amount of antibody used for each culture and diminishing the artifact caused by antibody dry out during staining.
  • the slide permits clear examination of the in situ colonies by using high power microscopic objectives.
  • the traditional evaluation methods included morphological recognition wherein megakaryocyte colonies were described as clusters of big, highly refractile cells under the inverted microscope without any staining (Sonoda, Y., et al., Blood 81:624-630, 1993) and immunfluorescent microscopy where megakaryocyte colonies were distinguished from other colonies as bright green vs. dull green (background) (Bruno, E., et al., Blood 73:671-677, 1989; Zauli, G. , et al., Exp. Hematol. 20:850-854, 1992). These methods yielded ambiguous results because it was difficult to distinguish between true megakaryocyte colonies and non-megakaryocyte lineage colonies, which also grow in fibrin clots.
  • the present method provides more accurate counting of megakaryocyte colonies because they are identified by a specific red label, within blue-labelled non-megakaryocytes, under the light microscope ( Figure 9). Results are summarized in Table 8 below. Table 8
  • PB CD34 + cells were grown in the pre-cultured conditions for the indicated times and then plated into fibrin-clot cultures for a 14-day of further growing.
  • Cul culture; Cond, conditions; MK, megakaryocyte; BFU-MK, megakaryocyte burst-forming cell, the colony containing 40- 100 megakaryocytes; CFC-MK, megakaryocyte colony-forming cell, the colony containing 2-39 megakaryocytes; S-MK, single megakaryocyte found in the colony culture.
  • BFU- MK early megakaryocyte progenitors
  • HLTM serum-containing
  • S-MK single megakaryocytes
  • BM bone marrow
  • PB peripheral blood
  • apheresis products of PB cells and plasma were simultaneously collected using a CS3000TM blood cell separator (Fenwal Division, Baxter Healthcare, Deerfield, IL) from cancer patients 4-15 days after G-CSF and/or chemotherapy mobilization.
  • Cell samples were collected in citrate dextrose formula A (ACDS) and diluted 1:1 with Iscove's modified Dulbecco's medium (IMDM) (Gibco, Grand Island, NY) containing 2% fetal bovine serum (FBS) (Sigma, St. Louis, MO).
  • ACDS citrate dextrose formula A
  • IMDM Iscove's modified Dulbecco's medium
  • FBS fetal bovine serum
  • Low- density MNC were obtained after density centrifugation over HistopaqueTM ficoll-hypaque (1.077 g/ml) for 20 min at 300g.
  • the plasma was spun for 10 min at 200g to remove platelets, passed through a 0.45 ⁇ m filter and stored at -20°C.
  • Human aplastic serum was obtained from thrombocytopenic patients after BM transplantation as described by Debili et al. (Blood 80:3022 (1992)).
  • Aplastic serum and plasma (AS) that demonstrated megakaryocyte stimulatory activities in pre- screened studies were utilized in these experiments at a final concentration of about 10%.
  • CD34 positive cells were selected as previously described in Example 2 with slight modifications. Briefly,. adherent cells were depleted by mixing the MNC with uncoated polystyrene Dynal paramagnetic beads (Fenwal Division, Baxter Healthcare, Deerfield, IL) . Non-adherent cells were incubated with the anti-CD34 monoclonal antibody (mAB) 9C5 (Immunotherapy Division, Baxter Healthcare, Santa Ana, CA) and then mixed with magnetic beads coated with sheep anti-mouse IgG. (Fenwal Division, Baxter Healthcare, Deerfield, IL) at the ratio of 0.5 - 1 bead to 1 cell.
  • mAB anti-CD34 monoclonal antibody
  • Bead-cell rosettes were collected after the positive selection using a Dynal MPC-1 Magnet (Immunotherapy Division, Baxter Healthcare, Santa Ana, CA) or IsolexTM 50 magnetic cell separator (Immunotherapy Division, Baxter Healthcare, Santa Ana, CA) .
  • Cells were released from beads by overnight incubation with 200 U/ml IL-3 at 37°C, 5% C0 2 .
  • For quantitation aliquots of selected cells were stained with another mAB against CD34, fluorescein (FITC) conjugated 8G12 (Immunotherapy Division, Baxter Healthcare, Santa Ana, CA) .
  • FITC fluorescein conjugated 8G12
  • BM mononuclear cell (MNC) cultures were initiated at 10 s cells/ml while CD34 cell cultures were initiated at 10 4 cells/ml.
  • Cultures were grown in the human long term culture medium (HLTM), the serum-free medium (Immunotherapy Division, Baxter Healthcare) or the human serum or plasma-supplemented medium. Immunocytochemistry and flow cytometry was performed as described in Example 7.
  • HLTM CD34 cell cultures generally contained less blast cells (9.2 ⁇ 3.2%, ranged 1 -
  • CD41a + cells were observed in day 10-14 in serum-free CD34 cultures (Fig. 9). Under serum-free conditions, the percent of CD41a + megakaryocyte progenitors was substantially higher than that observed under serum containing conditions. Similarly, the fold expansion of these cells as compared to the starting cell population was 51 fold in serum-free media versus 14.1 fold in serum containing media (Table 9). These cells showed cytoplasmic CD41a immunoreactivity, containing a single, non-lobulated nucleus and non-granular cytoplasm. Their size was similar or slightly bigger than the surrounding granulocytes. Without the antibody staining, they could not be recognized by light microscopy.
  • PB CD34 + cell cultures were established to compare the ability of different culture media to support megakaryocyte growth.
  • PB CD34 + cells from individual donors were grown in serum-free and HLTM cultures. Cultures were supplemented with the identical cytokine combination and refed with the same regimen. Results from 6 experiments are presented in Figure 11. Experiments 1-3 were supplemented with SCF, IL-3, GM-CSF & G-CSF; while experiments 4-6 were supplemented with SCF, IL- 3 & IL-6. Cell viability in paired cultures was similar. Cell count was generally lower in serum-free cultures than in HLTM cultures with the exception of experiment 2 ( Figure 11).
  • Megakaryocyte frequency in serum-free cultures was 7.6 ⁇ 3.1 fold higher (ranged 3-22) than their paired HLTM cultures.
  • the absolute megakaryocyte number in serum-free cultures was 17.4 ⁇ 7.1 fold higher (ranged 2-45) than their comparative HLTM cultures ( Figure 11, megakaryocyte cellularity) .
  • IMDM BM MNC cultures were further supplemented with AS.
  • HLTM cultures constantly yielded 2-3% megakaryocyte, no matter if they were derived from purified CD34 + cells or un-purified BM MNC.
  • serum-free PB CD34 cell cultures grew more megakaryocyte (16.1 ⁇ 4.3%) and expanded more (22.0 ⁇ 6.0 fold) than serum-free BM CD34 cell cultures (9.3 ⁇ 1.6% megakaryocyte, 8.9 ⁇ 1.3 fold expansion) (Figure 12). Cell viability in both cultures was similar (80.0 ⁇ 6.0% vs. 86.3 ⁇ 1.3%).
  • the serum-free medium appeared to support megakaryocyte growth better in PB CD34 than in BM CD34 cell cultures, or there may have been more megakaryocyte precursors to begin with.
  • the serum-free medium compared unfavorably with HLTM BM MNC cultures ( Figure 12).
  • the cell count increased in HLTM BM MNC (4-10 fold) but not in serum-free BM MNC cultures.
  • the growth of megakaryocytes from BM MNC appeared to depend on the presence of stromal foci, which were formed in IMDM (containing human AS) and HLTM (containing animal serum) but not in serum-free cultures.
  • the addition of AS to the serum-free medium on day 0 rendered the medium supportive of megakaryocyte growth from BM MNC in both HLTM and IMDM media ( Figure 12).
  • paired PB CD34 + cell cultures were set up and assayed for their ability to support megakaryocyte growth. Results from a typical experiment are presented in Figure 13.
  • the only effective cytokine combination was SCF + IL-3 + IL-6.
  • SCF + IL-6 or SCF + GM-CSF combinations supported baseline megakaryocyte growth.
  • the addition of IL-3 to either of these combinations increased megakaryocyte proliferation ( Figure 13).
  • GM-CSF stimulated more cell proliferation than IL-6 when combined with SCF & IL-3, without changing the megakaryocyte frequency.
  • Serum-free cultures supplemented with GM-CSF + SCF + IL-3 thus contained higher total cell count and megakaryocyte number than cultures supplemented with IL-6 + SCF + IL-3 ( Figure 13, megakaryocyte cellularity) .
  • the same was true when comparing serum-free cultures supplemented with SCF, IL-3, GM-CSF & G-CSF (Experiment 1-3 in Figure 11) with those supplemented with SCF, IL-3 & IL-6 (Experiment 4-6 in Figure 11).
  • the addition of 200 U/ml IL-11 to the SCF + IL-3 + GM-CSF combination did not stimulate further megakaryocyte proliferation (Figure 13).
  • IL-11 was titrated at concentrations between 50 and 800 U/ml with the SCF + IL-3 + IL-6 combination in PB CD34 + cell cultures. Although high dose IL-11 (400 U ⁇ ml) might enhance cell proliferation, no obvious synergistic effects of IL-11 with this cytokine combination on megakaryocyte growth were observed.
  • PB CD34 + cells showed a higher megakaryocyte potential than BM CD34 + cells when they were grown in the serum-free medium.
  • the majority of megakaryocyte derived from serum-free cultures expressed the GP Ilb/IIIa antigen, and were morphologically unrecognizable as mature megakaryocytes. They could undergo further differentiation and acquired megakaryocyte morphology upon further stimulation by aplastic serum.
  • This example shows the preparation and growth of serum-free suspensions of human hematopoietic cells in media other than the 295-1 media described previously.
  • CD34 + cells were selected from the apheresis blood products with the IsolexTM 50 (Baxter Healthcare, Corp., Fenwal Division) or the IsolexTM 300 (Baxter Healthcare, Corp., Immunotherapy Division, Irvine, CA) Magnetic Cell Separator. These systems are designed to select CD34 + cells from apheresis blood using an anti-CD34 monoclonal antibody (9C5) and retrieval using Dynal IgG-FC, ST magnetic beads.
  • the 9C5 anti-CD34 monoclonal antibody was added to the apheresis blood product at 0.5 ug (stock vial at 1 ug/ul) per 1x10* cells in the IsolexTM 50 or IsolexTM 300 column. The cells were then rotated for 30 minutes on the IsolexTM rotator. The cells were washed twice, in order to remove the unbound 9C5 monoclonal antibody, in RPMI 1640 containing 1% human serum albumin (HSA) .
  • HSA human serum albumin
  • the cells were then diluted in the column to l-2xl0 7 cells/ml with RPMI 1640 containing 10 mg/ml (1%) HSA and 1 mg/ml (0.1%) Gammagard R (Baxter Healthcare Corp., Hyland Division, Glendale, CA) .
  • Dynal IgG j FC, ST magnetic beads (Dynal, Oslo, Norway) were added to the cell suspension at 0.5 bead to cell ratio and rotated for 30 minutes on the IsolexTM rotator.
  • the CD34 cell:bead complexes were then removed using a magnet.
  • the CD34 + cell:bead complexes were resuspended to 3xl0 5 cells/ml in X-VIVO 10 (BioWhittaker, Inc., Walkersville, MD) containing 1% HSA and PIXY 321 at 100 ng/ml (Complete Media).
  • PIXY is a fusion protein containing the active domains of both IL-3 and GM-CSF.
  • the suspension was then placed in a gas- permeable culture bag (Baxter, Roundlake, IL) and incubated for 48 hours in a cell culture incubator set at 5% carbon dioxide and 37°C.
  • the starting culture medium was 20-50 mis in volume. These containers were referred to as the primary culture containers.
  • the released cells were retrieved from the culture by removal of the magnetic beads using the MaxSepTM Cell Separator (Baxter Healthcare, Corp., Irvine, CA) (Day 9 of culture). The released cells were then transferred into a secondary culture container and cultured as described in Example 3. At day 5, the cultures were analyzed for cell number and cell viability using a hemacytometer and trypan blue viability staining. The cell concentration was then readjusted to lxlO 5 cells/ml with Complete Media and cultured for an additional 5 days. Flow cytometry, colony assays and morphology evaluations were performed as described in Examples 5-7.
  • Table 10 summarizes the results of 5 experiments for cell proliferation at day 5 and 10 in which the viable proliferation index (P.I.) was calculated by dividing the total number of cells at day 5 or 10 by the total number of cells at day 0 and then multiplying by the % viable.
  • P.I. viable proliferation index
  • the increases in cell number, total CFU, clusters, CFU-GM, CD15 + cells, and early granulocytes at day 10 of culture were also determined.
  • the increases in total CFU, clusters, and CFU-GM were calculated from the change in the numbers present at day 0 to the final numbers at day 10 relative to the P.I. (Table 11).
  • the increase in CD15 + cells and early granulocytes at day 10 of culture takes into account that 80 ⁇ 17% (mean purity) of the cells were CD34 + and defined as blasts at day 0.
  • CD34 + cells were cultured in X-VIVO 10 media containing 1% HSA, 300 U/ml IL-3 and 20 ng/ml SCF with or without the addition of 40 ng/ml IL-6. At day 11 of culture, cell counts were performed and analyzed for the percentage of
  • CD41a + cells by immunocytochemistry. The results from 5 independent experiments are shown below in Tables 15-17. The data indicate that 1) CD41a + cells can be generated in this serum-free media under these conditions, and 2) that there was no significant difference seen on the proliferation index of
  • CD34 + cells percentage of CD41a + cells and the total number of megakaryocytes in the presence or absence of IL-6.
  • the human hematopoietic cell compositions described herein can be cultured in serum-free media containing, for example, just IL-3 and SCF.
  • EXAMPLE 11 COMPARISONS OF SERUM-FREE SUSPENSIONS OF HUMAN HEMATOPOIETIC CELLS This example compares the proliferation of human hematopoietic stem/progenitor cells in various serum-free media and characterizes the precursor cells produced from the different cultures.
  • CD34 + hematopoietic cells from peripheral blood (PB) was performed by methods similar to those described in Example 9. Apheresis products from five patients who received G-CSF during recovery from cyclophosphamide chemotherapy were used for the comparison of proliferation and precursor production. Briefly, following isolation of CD34 + cells, cultures were seeded on day 0 with the selected cells at a density of l-5xl0 4 cells/ml in either X-vivo 10, X-vivo 15 or X-vivo 20 (BioWhittaker) .
  • the media was supplemented with 300 U/ml IL-3, 40ng/ml IL-6 and 20 ng/ml SCF and the cultures were incubated at 37°C, 5% C0 2 , 5% 0 2 and high humidity for 8-12 days.
  • the percentage of CD34 + cells was determined as described in Example 9.
  • procedures for flow cytometry, cell morphology determination and immunochemistry were performed as described in Example 9.
  • CD34 + cells were determined by flow cytometry at day 0 of culture. The results showed no difference in purity with CD34 + cells comprising between 92% and 99% of the cultures in each of the medias. Similarly, no difference was seen in the cell viability or morphology following release in either the X-vivo 10, X-vivo 15 or X-vivo 20 medias.
  • total number of cells at day 10/number of cells at day 0 (total number of cells at day 10/number of cells at day 0).
  • results determined from all five cultures showed no significant difference between the serum-free medias with the mean proliferation indexes being 95.8 ⁇ 22.0, 112.0 ⁇ 7.4 and 170.2+39.5 for X-vivo 10, 15 and 20, respectively.
  • the percentage of early neutrophil precursors (CDl5 + /CDllb") was also determined in each of the serum-free medias. The results obtained again showed no significant difference in any of the medias. Specifically, the mean percentage of neutrophil precursors for each of the five cultures was 20.76 ⁇ 5.98, 17.20 ⁇ 6.63 and 17.63 ⁇ 6.20 for X-vivo 10, X-vivo 15 and X-vivo 20, respectively. Conversely, the percentage of more mature neutrophils was less then 5% in all cultures in the 10 day time period.
  • FACS percentage CD41a + cells determined by flow cytometry ICC: percentage CD41a + cells determined by immunochemistry
  • the total number of neutrophil precursors and megakaryocyte cells expanded in each of the three serum-free medias was also assessed.
  • the cell expansion for each lineage was determined by multiplying the initial number of CD34 + cells (IO 6 ) by the proliferation index at day 10 and by the percentage of cells within the particular lineage. For both lineages, the results indicate that there was no significant difference between each of the medias.
  • the fold expansion of neutrophil precursors was 20.9 ⁇ 4.7, 17.20+2.98 and 17.63 ⁇ 2.77 in X-vivo 10, 15 and 20, respectively.
  • Fold expansion for megakaryocytes was determined to be 9.77 ⁇ 3.1, 6.6 ⁇ 1.9 and 12.2 ⁇ 4.7 in X-vivo 10, 15 and 20, respectively.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Control Of Transmission Device (AREA)

Abstract

On décrit des compositions de milieux exempts de sérum et de protéines animales, devant être utilisées conjointement avec des facteurs de croissance hématopoiétique pour la croissance in vitro de précurseurs de neutrophiles et de mégacaryocytes humains. Le milieu est composé d'un milieu de base, d'un corticostéroïde, de transferrine, d'insuline, de cholestérol, d'éthanolamine et d'albumine humaine. L'invention concerne également des procédés de préparation de suspensions, exemptes de sérum et de protéines, animales, de cellules précurseurs hématopoiétiques humaines, dans lesquelles le composant cellulaire comprend au moins 16 % environ de précurseurs de neutrophiles et au moins 1 % environ de précurseurs de mégacaryocytes. On décrit des suspensions exemptes de sérum et de protéines, animales, dans lesquelles le composant cellulaire comprend au moins 30 % environ, et de préférence plus de 60 % de précurseurs de neutrophiles. Les précurseurs de neutrophiles comprennent des cellules blastiques, des promyélocytes, des myélocytes neutrophiles et des métamyélocytes neutrophiles. On décrit également des suspensions de cellules exemptes de sérum et de protéines, animales, dans lesquelles le composant cellulaire comprend au moins 3 % environ et de préférence plus de 8 % de précurseurs de mégacaryocytes, ainsi que des suspensions de cellules exemptes de sérum et de protéines animales, dans lesquelles le composant cellulaire comprend des unités formant des colonies et des unités formant des grappes.
PCT/US1994/009622 1993-08-23 1994-08-23 CROISSANCE Si(IN VITRO) DE PRECURSEURS DE NEUTROPHILES ET DE MEGACARYOCYTES DANS DES MILIEUX EXEMPTS DE SERUM Ceased WO1995006112A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP7507759A JPH09505462A (ja) 1993-08-23 1994-08-23 無血清培地内での好中球前駆細胞及び巨核球前駆細胞の試験管内増殖
EP94927235A EP0719326A1 (fr) 1993-08-23 1994-08-23 CROISSANCE Si(IN VITRO) DE PRECURSEURS DE NEUTROPHILES ET DE MEGACARYOCYTES DANS DES MILIEUX EXEMPTS DE SERUM
AU76744/94A AU702871B2 (en) 1993-08-23 1994-08-23 (In vitro) growth of neutrophil and megakaryocyte precursors in serum-free media

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11027793A 1993-08-23 1993-08-23
US08/110,277 1993-08-23

Publications (1)

Publication Number Publication Date
WO1995006112A1 true WO1995006112A1 (fr) 1995-03-02

Family

ID=22332160

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/009622 Ceased WO1995006112A1 (fr) 1993-08-23 1994-08-23 CROISSANCE Si(IN VITRO) DE PRECURSEURS DE NEUTROPHILES ET DE MEGACARYOCYTES DANS DES MILIEUX EXEMPTS DE SERUM

Country Status (7)

Country Link
EP (1) EP0719326A1 (fr)
JP (1) JPH09505462A (fr)
AU (1) AU702871B2 (fr)
CA (1) CA2168091A1 (fr)
IL (1) IL110755A (fr)
SG (1) SG52619A1 (fr)
WO (1) WO1995006112A1 (fr)

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040866A1 (fr) * 1995-06-07 1996-12-19 Novartis Ag Milieux sans serum pour des cellules hematopoietiques primaires et procedes d'utilisation
US5945337A (en) * 1996-10-18 1999-08-31 Quality Biological, Inc. Method for culturing CD34+ cells in a serum-free medium
US6207454B1 (en) 1989-10-16 2001-03-27 Amgen Inc. Method for enhancing the effciency of gene transfer with stem cell factor (SCF) polypeptide
US6811776B2 (en) 2000-12-27 2004-11-02 The Regents Of The University Of Michigan Process for ex vivo formation of mammalian bone and uses thereof
US6841147B2 (en) 1989-10-16 2005-01-11 Amgen, Inc. Stem cell factor compositions
US6852313B1 (en) 1989-10-16 2005-02-08 Amgen Inc. Method of stimulating growth of melanocyte cells by administering stem cell factor
US7144731B2 (en) 1989-10-16 2006-12-05 Amgen Inc. SCF antibody compositions and methods of using the same
US7534434B2 (en) 2004-12-28 2009-05-19 The Rockefeller University Glycolipids and analogues thereof as antigens for NK T cells
US7837990B2 (en) 2005-03-28 2010-11-23 The Rockefeller University In vivo expanded NKT cells and methods of use thereof
US20100297090A1 (en) * 1996-03-12 2010-11-25 Life Technologies Corporation Hematopoietic cell culture nutrient supplement
US7923245B2 (en) 2003-12-26 2011-04-12 Miho Furue Medium for ES culturing
US7923013B2 (en) 2004-12-28 2011-04-12 The Rockefeller University Glycolipids and analogues thereof as antigens for NKT cells
EP2423303A1 (fr) * 2006-03-24 2012-02-29 Children's Medical Center Corporation Procédé de modulation de croissance de cellules souches hématopoïétiques
US8168429B2 (en) 2004-01-21 2012-05-01 Mitsubishi Tanabe Pharma Corporation Method of amplifying hematopoietic stem cell and hematopoietic progenitor cell
US8198084B2 (en) 1996-08-30 2012-06-12 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof
WO2013184809A1 (fr) * 2012-06-05 2013-12-12 The Regents Of The University Of California Procédés et compositions pour la production rapide de cellules épithéliales pigmentées de la rétine à partir de cellules multipotentes
AU2013202353B2 (en) * 2006-03-24 2015-05-07 Children's Medical Center Corporation Method to modulate hematopoietic stem cell growth
US9051548B2 (en) 2009-02-03 2015-06-09 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US9056085B2 (en) 2009-02-03 2015-06-16 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US9068164B1 (en) * 1995-02-02 2015-06-30 Tristem Trading (Cyprus) Limited Method of preparing an undifferentiated cell
US9402852B2 (en) 2006-10-20 2016-08-02 Children's Medical Center Corporation Method to enhance tissue regeneration
US9452186B2 (en) 2011-12-02 2016-09-27 Fate Therapeutics, Inc. Enhanced stem cell composition
AU2015207905B2 (en) * 2006-03-24 2017-04-27 Children's Medical Center Corporation Method to modulate hematopoietic stem cell growth
US10111907B2 (en) 2011-12-02 2018-10-30 Fate Therapeutics, Inc. Methods of treating ischemia
US10851412B2 (en) 2013-03-15 2020-12-01 Fate Therapeutics, Inc. Cell potency assay for therapeutic potential
FR3128874A1 (fr) * 2021-11-09 2023-05-12 Crossject Solution pharmaceutique d’hydrocortisone pour dispositif d’injection

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013006806A1 (fr) * 2011-07-06 2013-01-10 Cellerant Therapeutics, Inc. Cellules progénitrices mégacaryocytaires pour la production de plaquettes
JP7521738B2 (ja) * 2020-05-18 2024-07-24 キヤノン株式会社 目的細胞の生産方法、目的細胞による生産物の生産方法、および無血清培地
CA3192530A1 (fr) * 2020-09-21 2022-03-24 Xiaoming Chen Formulation pharmaceutique aqueuse de phosphate de sodium d'hydrocortisone et de monothioglycerol
WO2025018336A1 (fr) * 2023-07-20 2025-01-23 学校法人近畿大学 Milieu sans sérum

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992006178A1 (fr) * 1990-10-05 1992-04-16 Systemix, Inc. Croissance, production et composition des megacaryocytes et des plaquettes

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816401A (en) * 1985-09-09 1989-03-28 University Of Rochester Serum free cell culture medium
US5004681B1 (en) * 1987-11-12 2000-04-11 Biocyte Corp Preservation of fetal and neonatal hematopoietic stem and progenitor cells of the blood

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992006178A1 (fr) * 1990-10-05 1992-04-16 Systemix, Inc. Croissance, production et composition des megacaryocytes et des plaquettes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ACTA HAEMATOL. JPN., vol. 49, no. 1, 1986, pages 27 - 33 *
BIOLOGICAL ABSTRACTS, vol. 82, 1986, Philadelphia, PA, US; abstract no. 20424, K. KUBOTA ET AL.: "MURINE GRANULOCYTE/MACROPHAGE AND MEGAKARYOCYTE COLONY FORMATION IN SERUM-FREE CULTURES." page AB-94; *
M. TERAMURA ET AL.: "CLONAL GROWTH OF HUMAN MEGAKARYOCYTE PROGENITORS IN SERUM-FREE CULTURES: EFFECT OF RECOMBINANT HUMAN INTERLEUKIN 3.", EXPERIMENTAL HEMATOLOGY, vol. 16, no. 10, 1988, LAWRENCE, KANS., US, pages 843 - 848 *
S.L. SMITH ET AL.: "EXPANSION OF NEUTROPHIL PRECURSORS AND PROGENITORS IN SUSPENSION CULTURES OF CD34+ CELLS ENRICHED FROM HUMAN BONE MARROW.", EXPERIMENTAL HEMATOLOGY, vol. 21, no. 7, July 1993 (1993-07-01), LAWRENCE, KANS., US, pages 870 - 877 *

Cited By (54)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6967029B1 (en) 1989-10-16 2005-11-22 Amgen Inc. Method for increasing hematopoietic progenitor cells by stem cell factor
US6207454B1 (en) 1989-10-16 2001-03-27 Amgen Inc. Method for enhancing the effciency of gene transfer with stem cell factor (SCF) polypeptide
US7144731B2 (en) 1989-10-16 2006-12-05 Amgen Inc. SCF antibody compositions and methods of using the same
US6841147B2 (en) 1989-10-16 2005-01-11 Amgen, Inc. Stem cell factor compositions
US6852313B1 (en) 1989-10-16 2005-02-08 Amgen Inc. Method of stimulating growth of melanocyte cells by administering stem cell factor
US9068164B1 (en) * 1995-02-02 2015-06-30 Tristem Trading (Cyprus) Limited Method of preparing an undifferentiated cell
WO1996040866A1 (fr) * 1995-06-07 1996-12-19 Novartis Ag Milieux sans serum pour des cellules hematopoietiques primaires et procedes d'utilisation
US20100297090A1 (en) * 1996-03-12 2010-11-25 Life Technologies Corporation Hematopoietic cell culture nutrient supplement
US9321996B2 (en) 1996-08-30 2016-04-26 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof
US8455246B2 (en) 1996-08-30 2013-06-04 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof
US8198084B2 (en) 1996-08-30 2012-06-12 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof
US8815573B2 (en) 1996-08-30 2014-08-26 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof
US8785194B2 (en) 1996-08-30 2014-07-22 Life Technologies Corporation Serum-free mammalian cell culture medium, and uses thereof
US6372210B2 (en) 1996-10-18 2002-04-16 Quality Biological, Inc. Method for repopulating human bone marrow comprising culturing CD34+ cells in a serum free medium
US6224860B1 (en) 1996-10-18 2001-05-01 Quality Biological, Inc. Method for repopulating human bone marrow comprising culturing CD34+ cells in a serum free medium
US5945337A (en) * 1996-10-18 1999-08-31 Quality Biological, Inc. Method for culturing CD34+ cells in a serum-free medium
US7229826B2 (en) 1999-12-28 2007-06-12 The Regents Of The University Of Michigan Process for ex vivo formation of mammalian bone and uses thereof
US7371575B2 (en) 1999-12-28 2008-05-13 The Regents Of The University Of Michigan Process for ex vivo formation of mammalian bone and uses thereof
US6811776B2 (en) 2000-12-27 2004-11-02 The Regents Of The University Of Michigan Process for ex vivo formation of mammalian bone and uses thereof
US7923245B2 (en) 2003-12-26 2011-04-12 Miho Furue Medium for ES culturing
US8168429B2 (en) 2004-01-21 2012-05-01 Mitsubishi Tanabe Pharma Corporation Method of amplifying hematopoietic stem cell and hematopoietic progenitor cell
US8586051B2 (en) 2004-12-28 2013-11-19 The Rockefeller University Glycolipids and analogues thereof as antigens for NKT cells
US7923013B2 (en) 2004-12-28 2011-04-12 The Rockefeller University Glycolipids and analogues thereof as antigens for NKT cells
US7534434B2 (en) 2004-12-28 2009-05-19 The Rockefeller University Glycolipids and analogues thereof as antigens for NK T cells
US8163290B2 (en) 2004-12-28 2012-04-24 The Rockefeller University Glycolipids and analogues thereof as antigens for NKT cells
US7837990B2 (en) 2005-03-28 2010-11-23 The Rockefeller University In vivo expanded NKT cells and methods of use thereof
AU2015207905B2 (en) * 2006-03-24 2017-04-27 Children's Medical Center Corporation Method to modulate hematopoietic stem cell growth
EP3424507A1 (fr) * 2006-03-24 2019-01-09 Children's Medical Center Corporation Procédé de modulation de croissance de cellules souches hématopoïétiques
US10278990B2 (en) 2006-03-24 2019-05-07 Children's Medical Center Corporation Methods for promoting hematopoietic reconstitution
AU2013202353B2 (en) * 2006-03-24 2015-05-07 Children's Medical Center Corporation Method to modulate hematopoietic stem cell growth
US9028811B2 (en) 2006-03-24 2015-05-12 Children's Medical Center Corporation Methods for promoting HSC self-renewal
US10272110B2 (en) 2006-03-24 2019-04-30 Children's Medical Center Corporation Methods for promoting HSC engraftment
EP2423303A1 (fr) * 2006-03-24 2012-02-29 Children's Medical Center Corporation Procédé de modulation de croissance de cellules souches hématopoïétiques
US8563310B2 (en) 2006-03-24 2013-10-22 Children's Medical Center Corporation Methods for promoting hematopoietic reconstitution
US8551782B2 (en) 2006-03-24 2013-10-08 Children's Medical Center Corporation Methods for promoting HSC engraftment
AU2017202149B2 (en) * 2006-03-24 2018-12-06 Children's Medical Center Corporation Method to modulate hematopoietic stem cell growth
CN102626517A (zh) * 2006-03-24 2012-08-08 儿童医疗中心有限公司 调节造血干细胞生长的方法
US10736906B2 (en) 2006-10-20 2020-08-11 Children's Medical Center Corporation Method to enhance tissue regeneration
US9402852B2 (en) 2006-10-20 2016-08-02 Children's Medical Center Corporation Method to enhance tissue regeneration
US9056085B2 (en) 2009-02-03 2015-06-16 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US9737567B2 (en) 2009-02-03 2017-08-22 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US10092599B2 (en) 2009-02-03 2018-10-09 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US9051548B2 (en) 2009-02-03 2015-06-09 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US10159697B2 (en) 2009-02-03 2018-12-25 Children's Medical Center Corporation Methods for enhancing hematopoietic stem/progenitor cell engraftment
US10111907B2 (en) 2011-12-02 2018-10-30 Fate Therapeutics, Inc. Methods of treating ischemia
US10172888B2 (en) 2011-12-02 2019-01-08 Fate Therapeutics, Inc. Enhanced stem cell composition
US9452186B2 (en) 2011-12-02 2016-09-27 Fate Therapeutics, Inc. Enhanced stem cell composition
US10980838B2 (en) 2011-12-02 2021-04-20 Fate Therapeutics, Inc. Methods of treating ischemia
US11052118B2 (en) 2011-12-02 2021-07-06 Fate Therapeutics, Inc. Enhanced stem cell composition
US9458428B2 (en) 2012-06-05 2016-10-04 The Regents Of The University Of California Methods and compositions for the rapid production of retinal pigmented epithelial cells from pluripotent cells
WO2013184809A1 (fr) * 2012-06-05 2013-12-12 The Regents Of The University Of California Procédés et compositions pour la production rapide de cellules épithéliales pigmentées de la rétine à partir de cellules multipotentes
US10851412B2 (en) 2013-03-15 2020-12-01 Fate Therapeutics, Inc. Cell potency assay for therapeutic potential
FR3128874A1 (fr) * 2021-11-09 2023-05-12 Crossject Solution pharmaceutique d’hydrocortisone pour dispositif d’injection
WO2023083825A1 (fr) * 2021-11-09 2023-05-19 Crossject Solution pharmaceutique d'hydrocortisone pour dispositif d'injection

Also Published As

Publication number Publication date
IL110755A (en) 2000-11-21
IL110755A0 (en) 1994-11-11
AU7674494A (en) 1995-03-21
JPH09505462A (ja) 1997-06-03
CA2168091A1 (fr) 1995-03-02
SG52619A1 (en) 1998-09-28
EP0719326A1 (fr) 1996-07-03
AU702871B2 (en) 1999-03-11

Similar Documents

Publication Publication Date Title
US6037174A (en) Preparation of serum-free suspensions of human hematopoietic cells or precursor cells
AU702871B2 (en) (In vitro) growth of neutrophil and megakaryocyte precursors in serum-free media
US6241984B1 (en) Human hematopoietic progenitor cell preparations and their expansion in a liquid medium
US5744361A (en) Expansion of human hematopoietic progenitor cells in a liquid medium
Sandstrom et al. Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells
Debili et al. The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors
US6667034B2 (en) Methods for regulating the specific lineages of cells produced in a human hematopoietic cell culture, methods for assaying the effect of substances on lineage-specific cell production, and cell compositions produced by these cultures
US6733746B2 (en) Hematopoietic cell culture nutrient supplement
WO1996040866A1 (fr) Milieux sans serum pour des cellules hematopoietiques primaires et procedes d'utilisation
US5955357A (en) In-vitro-derived human neutrophil precursor cells
WO1994025571A1 (fr) Population de cellules souches erythroides humaines
Mazur et al. Isolation of large numbers of enriched human megakaryocytes from liquid cultures of normal peripheral blood progenitor cells
US5846529A (en) Infusion of neutrophil precursors for treatment of neutropenia
US5888499A (en) Method for increasing neutrophil populations using in vitro-derived human neutrophil precursor cells
Smith et al. Neutrophil maturation of CD34+ cells from peripheral blood and bone marrow in serum-free culture medium with PIXY321 and granulocyte-colony stimulating factor (G-CSF)
Hirao et al. Synergism of interleukin 12, interleukin 3 and serum factor on primitive human hematopoietic progenitor cells
Sandstrom Ex vivo expansion of human hematopoietic cells using better defined culture conditions
WO1999000486A1 (fr) Compositions et procedes permettant d'induire la croissance et la differenciation de cellules souches hematopietiques
US20040043009A1 (en) Method of repairing primate mammalian tissue
Dai et al. Purification and culture of erythroid progenitor cells
Zandstra Cytokine-dependent regulation of human hematopoietic cell self-renewal and differentiation in suspension cultures

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2168091

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 1994927235

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1994927235

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1994927235

Country of ref document: EP