WO1992006178A1 - Croissance, production et composition des megacaryocytes et des plaquettes - Google Patents

Croissance, production et composition des megacaryocytes et des plaquettes Download PDF

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WO1992006178A1
WO1992006178A1 PCT/US1991/007159 US9107159W WO9206178A1 WO 1992006178 A1 WO1992006178 A1 WO 1992006178A1 US 9107159 W US9107159 W US 9107159W WO 9206178 A1 WO9206178 A1 WO 9206178A1
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cells
megakaryopoiesis
factor
megakaryocyte
growth
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George Tidmarsh
Judy C. Young
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Systemix Inc
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Systemix Inc
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Priority to FI922586A priority Critical patent/FI922586A7/fi
Priority to CA002069879A priority patent/CA2069879A1/fr
Priority to KR1019920701338A priority patent/KR927003795A/ko
Publication of WO1992006178A1 publication Critical patent/WO1992006178A1/fr
Priority to NO92922213A priority patent/NO922213L/no
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    • 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
    • C07K14/475—Growth factors; Growth regulators
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/91—Heparin

Definitions

  • the field of this invention is growth and production of hematopoietic cells, more particularly megakaryocytes and platelets.
  • Thrombopoiesis is the differentiation and maturation of megakaryocytes and platelets. Despite enormous efforts that have been made to understand the process of thrombopoiesis and the factors involved with thrombopoiesis, there remains an extraordinary amount of confusion and uncertainty about the process. Factors that appear to have effects in vitro seem to have little or no effect in vivo. An activity called thrombopoietin has been elusive and has yet to be isolated in pure form and characterized. Efforts to provide for long-term culture of megakaryocyte progenitors providing for their maturation and formation of platelets has also been problematical. Thro bopoiesis is an extremely important process for the health of individuals.
  • Platelets play a vital role in a number of protective processes, providing various factors, participating in clotting, and the like. There is, therefore, substantial interest in providing procedures which will aid in the understanding of thrombopoiesis, as well as providing for long-term production of megakaryocytes and platelets which may find application as therapeutic procedures.
  • Methods are provided for growing megakaryocytes and platelets in culture for extended periods of time, by employing media which encourage megakaryocyte progenitor and megakaryocyte growth.
  • Various sources of megakaryocyte progenitor cells are employed.
  • a megakaryocyte growth factor is provided for enhancing megakaryocyte progenitor and megakaryocyte growth.
  • Assay systems are provided for assaying for the effect of factors on the proliferation and maturation of megakaryocyte progenitors and megakaryocytes, as well as the formation of platelets.
  • the megakaryocytes and platelets find use in the treatment of thrombo ⁇ ytopenia, identification of megakaryopoiesis activity of compounds or compositions, and for an understanding of the mechanism of megakaryopoiesis.
  • Megakaryocytes can be grown in the presence of heparin and a megakaryo ⁇ ytic growth factor or human plasma. In culture, the megakaryocytes may be maintained for extended periods of time without replenishment of the progenitor cells from an exogenous source.
  • Bioassays are provided for monitoring megakaryopoiesis and factors influencing megakaryopoiesis.
  • the megakaryocyte progenitors, megakaryocytes and platelets can be produced by growing in culture in an appropriate nutrient medium human progenitor cells under conditions which are not supportive, preferably are inhibitory, cf fibroblast growth, while inactivating, as appropriate, inhibitors of megakaryopoiesis, particularly those produced by platelets.
  • the source of progenitor cells may be any convenient source, including bone marrow, fetal liver, non-adherent cells from bone marrow, fractionated cells, leukocytes, e.g., buff coat free of erythrocytes, CD34+ fraction from bone marrow, low density cells.
  • Class II HLA positive cells, or the like so long as early progenitors are present which are capable of maintaining production of megakaryocytes.
  • the cell composition which is employed will desirably include hematopoietic stem cells.
  • the cellular composition may be fetal, neonate, or adult. While the subject methodology may be used with any primate hematopoietic cellular composition comprising stem cells, the methodology desirably finds use with human cell compositions.
  • megakaryocytes For many purposes, the growth of megakaryocytes, megakaryocyte progenitors, and platelets is desirable.
  • having a system whereby one can grow cells of the megakaryocytic lineage is extremely important in providing a source of megakaryocytic cells and platelets.
  • containers which are used for the growth of cultures are coated to enhance fibroblast formation.
  • containers are employed which remain uncoated or have been treated to avoid encouraging fibroblast formation.
  • Containers can be obtained where the containers are free of coating, either by being commercially available or by requesting such containers from sources such as Falcon, Corning, or the like.
  • the containers may include multiple well plates, roller bottles, fermentors, Petri dishes or the like.
  • the medium which is employed may be any convenient medium providing the required salts, minerals and nutritional supplements, such as amino acids, glucose and vitamins. Also, a small amount of a physiologically acceptable reductant is used such as 2-mercaptoethanol.
  • the mercaptoethanol will generally be present in from about 10 " - 10 "6 M, preferably about 10 '5 M.
  • Various additives may be employed to discourage or inhibit fibroblast formation and proliferation or preferentially encourage megakaryopoiesis.
  • Additives which may be employed to discourage fibroblast formation in the presence of fetal calf serum include heparin, citrate, or other additives that prevent aggregation and degranulation of platelets.
  • the amount of heparin will be at least about 50 ⁇ g/ml, preferably at least about 100 ⁇ g/ml and usually not exceed 4 mg/ml, more usually not exceed 2 mg/ml, preferably being in the range of about 0.5 to 1.5 mg/ml.
  • the amount of citrate will vary, when present, generally in the range of about 0.1 to 1%.
  • Other additives will be used in accordance with their activity and any adverse effects on the system.
  • the medium generally will also have a low level of plasma or serum, human plasma or serum, fetal calf serum or fetal bovine serum, preferably being about 10% or less, more preferably being about 5%, there generally being at least about 1% in the absence of specific cytokines in a defined medium.
  • the amount of serum, as well as the other additives may be optimized, where cells of the megakaryocytic lineage are encouraged to grow, while fibroblast growth and proliferation may be inhibited. Any complex eukaryotic growth media may be used, Which includes MCDB107, IMDM, RPMI, EX-CEL, etc., normally enhanced with a low level of serum.
  • PF-4 platelet factor 4
  • thrombospondin thrombospondin
  • heparin is able to bind to PF-4 and diminish its inhibitory activity against megakaryopoiesis.
  • the heparin prevents the PF-4 and its degradation product from exerting their inhibitory influence.
  • other methods may be used for minimizing the level of PF-4.
  • the calcium level should be in the range of about 0 to lOmM, while the phosphate concentration will be in the range of about 0 to lOmM.
  • the level of ADP may also be controlled by providing for a system to convert ADP to ATP.
  • a convenient system includes creatinine phosphate and creatinine phosphate kinase, although other systems may find use.
  • the concentration of the creatinine kinase and creatinine kinase phosphate will be maintained in a range to provide the desired ADP/ATP levels. These concentrations are not critical and are intended to be primarily of convenience, enhancing the productivity of the system. Platelet preservation may also be achieved with dextrose acid citrate.
  • Megakaryocytic cell cultures may also be grown in the substantial absence of serum ( ⁇ 1%) by employing at least one growth factor in combination with the subject megakaryocytic growth factor.
  • Growth factors include the interleukins IL-1, -3, -6 and erythropoietin, particularly IL-6 and erythropoietin at relatively high concentrations, usually greater than about 25 ng/ml, preferably greater than about 50 ng/ml, and may be 100 ng/ml or higher.
  • a rich defined medium should be employed, having high levels of cof ctors, e.g., vitamins, and other components, e.g., ⁇ -carotene, insulin, etc., such as X-VIVO and EX-CEL (J.R. Scientific) .
  • cof ctors e.g., vitamins, and other components, e.g., ⁇ -carotene, insulin, etc., such as X-VIVO and EX-CEL (J.R. Scientific) .
  • the cell composition obtained in the medium is highly enriched for megakaryocyte progenitors, megakaryocytes and platelets, greater than 10% by number, usually greater than 50%, of the liquid culture medium being in the megakaryocyte/platelet dedicated lineage.
  • Cell compositions in liquid culture can be obtained having at least 90 weight percent of megakaryoblastoid cells and megakaryocytes (excluding platelets in the weight count) , particularly megakaryocytes.
  • a culture comprising cells committed to the megakaryocyte/platelet lineage, an adherent population of cells which may provide factors and a conditioned medium supportive of megakaryopoiesis by virtue of comprising the appropriate growth factors, agent(s) for stabilizing platelets and, as appropriate, agent(s) for inhibiting fibroblast growth and proliferation.
  • the cells of interest may be isolated from the culture medium or bone marrow. Cells of interest may also be available from buffy coat isolated from blood by using separation techniques associated with surface membrane protein markers. Cells of interest will fractionate as CD34+, -41+, -14-, and usually -33+.
  • megakaryocyte progenitors will be diploid, as distinguished from the polyploid mature megakaryocytes.
  • a highly enriched cellular-composition of megakaryocyte progenitors by separation using magnetic beads, employing positive and negative selection, FACS, affinity columns, and the like.
  • FACS fluorescence-activated cell sorting
  • Factor(s) present in the conditioned medium associated with megakaryopoiesis are found to be stable for greater than two weeks at 41 ⁇ C, usually at ambient temperatures or higher.
  • human bone marrow cells per ml will be seeded, preferably lxlO 6 cells per ml, or a unit of leukocytes (buffy coat) from an adult, containing about 2 x 10 8 white cells and freed of erythrocytes.
  • leukocytes a unit of leukocytes (buffy coat) from an adult, containing about 2 x 10 8 white cells and freed of erythrocytes.
  • Conventional physiologic conditions will be employed for growing human cells in culture, namely about 37°C, in a humidified chamber, with air having about 5% carbon dioxide, where the oxygen will be at least about 5% usually at least about 20%. Since higher oxygen levels are reported to inhibit fibroblast growth, high oxygen levels, usually not exceeding 90% may be employed, depending on the other conditions used for the growth of the cells.
  • wells of 6 - 10 cm or 96 well plates may be employed, which allows for the growth of the adherent cells to the surface.
  • megakaryocytic cells can be observed and the culture may be continued for six months or more.
  • the media is changed on a regular basis, generally at least about twice weekly, preferably at least about every 2 days.
  • adherent cells set down, which differentiate to mature megakaryocytes, observed as multi-lobular cells.
  • An adherent layer is observed which is a combination of megakaryocytes and adherent (stromal) cells.
  • Islands of cells appear on the adherent layer which have the characteristic CD41 marker for megakaryocytes but are mononucleated, supporting the cells being megakaryocyte progenitors (megakaryoblasts) .
  • the progenitor cells will also carry the CD34 marker.
  • the megakaryocyte cells are shown to have the surface protein markers CD41 (Ilb/IIIa)
  • Megakaryocytic cells may be further distinguished by staining with Megacolor (Cytocolor, Inc., Hinckley, OH) .
  • Megacolor Cytocolor, Inc., Hinckley, OH
  • the cytoplasm stains intensely purple. Also, as evidenced by scanning electron microscope, a majority of the cells in the culture are actively budding platelets.
  • the level of src protein expression and its kinase activity may be used as a measure of megakaryocytic cell growth, measuring megakaryocyte progenitors, megakaryocytes and platelets.
  • the assay can be carried out with cellular lysates of relatively homogeneous or heterogeneous phenotype or populations selected by one or more markers, isolating the src kinase, e.g. by means of specific antibodies, and then measuring src kinase activity under conditions supportive of src kinase catalyzed phosphorylation. To remove non-specific proteins from the isolated src kinase, one or more washes may be employed.
  • nu ⁇ leotide triphosphate e.g. radioisotope labeled
  • Western blotting or SRC ELISA can be used to quantitate src protein.
  • concentration of platelets As the cultures mature, the concentration of platelets continues to increase, so that after about one week, the concentration of platelets is ⁇ 10 6 platelets per ml, and may be maintained at about 10 6 -10 7 platelates/ml.
  • the level of platelet production may be maintained by the continuous growth of the culture and maintenance of the medium at the appropriate component composition, and may be increased by concentration of the platelets using various techniques for removing fluid.
  • the cells may be harvested by separating the adherent layer mechanically, using chelating agents, e.g., EDTA, sonication, enzymes, e.g., proteases, such as trypsin, collagenase, plasmin, etc., or the like.
  • chelating agents e.g., EDTA, sonication
  • enzymes e.g., proteases, such as trypsin, collagenase, plasmin, etc., or the like.
  • the particular manner employed is not critical to this invention.
  • the megakaryoblasts and megakaryocytes may then be isolated using various techniques, such as cytospin, FACS, affinity separation, density separation, magnetic bead separation, and the like.
  • a substantially pure cellular composition can be obtained of cells dedicated to megakaryopoiesis, namely dedicated megakaryocyte progenitors, megakaryocytes and platelets.
  • composition will be free of components of peripheral blood, which might be associated with isolation of such cellular composition from bone marrow or blood.
  • each of the types of cells may be isolated in substantially pure form, namely, megakaryoblasts, megakaryocytes and platelets, generally comprising at least 75% by number of the same cellular type, usually at least about 90% of the same cellular type and up to and including about 100%.
  • the conditioned medium, or human plasma may be used for isolation of the factors associated with megakaryopoiesis. Fractionating ammonium sulfate precipitation is used to enrich protein fractions for growth factors. Further enrichment is achieved using gel permeation to separate different size fractions, followed by assaying for the enriched fractions. The enriched fractions are then further enriched using rp- HPLC, eluting with an aqueous acidic medium against n- propanol or acetonitrile,. e.g., 0.1% trifluoroacetic acid, or 1M acetic acid.
  • Purification of the megakaryocyte activity may be further achieved by HPLC using a cationic column, where the column buffer is at a Ph of about 7.5 to 9, and the sample is eluted with a high ionic strength eluent, e.g. 2 M inorganic salt, e.g. NaCl, using a linear gradient in the column buffer.
  • a high ionic strength eluent e.g. 2 M inorganic salt, e.g. NaCl
  • the activity is observed in the fractions of the tail end of the highest peak when using a linear gradient, generally eluting in the fractions of about 1.25 to 1.6 M salt.
  • fractions are bioassayed and enriched fractions may be further purified by gel electrophoresis or other separation technique.
  • a megakaryocytic growth factor can be obtained by fractionating medium from the culture or human plasma through a heparin column.
  • the factor is characterized by binding to heparin, capable of being transferred from heparin-Sepharose to heparin in a conventional complex eukaryotic growth media, with heparin at a concentration of at least about 1 mg/ml, preferably at least about 3 mg/ml, and at 4 ⁇ C.
  • the ratio of heparin-Sepharose beads to medium will generally be at least about 1 g:2 ml, preferably 5 ml, and not more than about 1 g:10 ml.
  • the factor is further characterized by supporting cell growth and proliferation of cells of the megakaryocytic lineage in the culture system as described above, while its absence results in the substantial absence of proliferation and maturation of cells of the megakaryocytic lineage.
  • the molecular weight of the factor appears to be greater than about 40kD. It also is stable at 56° C. in whole plasma. It also appears to be species specific, not being effective with mouse cells under comparable conditions to being effective with human cells. Finally, it does not bind Affi-gel blue CM.
  • the subject factor may be further purified by conventional techniques as described above to provide for 1,000-fold or greater enhancement in concentration and specific activity.
  • the factors may be evaluated using a standard semi-solid medium assay composition, e.g., methylcellulose assay composition comprising factors which support megakaryocyte growth.
  • a standard semi-solid medium assay composition e.g., methylcellulose assay composition comprising factors which support megakaryocyte growth.
  • the methylcellulose which is employed will generally be at least about 1.5% and not more than about 3% v/v in water.
  • the cellular source may be the same as that used for the culture. Also included in the culture will be at least about 15% fetal calf serum, fetal bovine serum, or human plasma, preferably about 20%.
  • a convenient volume for carrying out the assay is to employ about 200 ⁇ l in a 24-well plate.
  • cytokines IL-1, -3 and -6 as well as erythropoietin are included, generally at least at about 0.5 times saturation or higher.
  • Other factors may be included such as si or steel factor at comparable concentration. By saturation it is intended that a further increase in concentration does not have a significant effect on the number of colonies of cells which are produced. Conveniently, about 100 ng/ml of each of the factors may be employed.
  • the cells which are seeded will include CD41+, CD34+ cells, which cells serve as megakaryocyte progenitors.
  • the cells may be whole bone marrow or an enriched fraction comprising CD41+, CD34+ cells.
  • the mature cells in the colonies which form will, for the most part, be free of the CD34 marker and will normally have the CD33 marker, as well as having the morphology of the mature megakaryocyte.
  • a factor or composition of interest may be studied by adding such factor or composition to the medium with all of the above-indicated factors or where one or more of the above-indicated factors are lacking.
  • one may determine the inherent capability of a composition in conjunction with fetal calf serum to stimulate megakaryocyte growth or its ability to interact with other factors or cytokines to provide for megakaryocyte growth.
  • the assay is carried out for at least about 5, usually at least 7 days, usually not more than about 21 days, preferably from about 7 to 14 days.
  • compositions or factors By employing various dosages of a composition or factor, one can determine the activity of such composition or factor. In this manner, rapid screening can be carried out to determine whether a particular composition or factor is useful for the growth of megakaryocytes or the production of platelets.
  • the megakaryocyte progenitors and/or megakaryocytes may be used for the treatment of thrombocytopenia by themselves or in conjunction with the infusion of platelets.
  • autologous megakaryocytic cells may be expanded in culture and returned to the host to minimize bleeding or other effect of the treatment which megakaryocytes and/or platelets may address.
  • the cells may be infused in a physiologically acceptable medium, e.g., saline, generally being present at a concentration of at least about 10 5 cells/ml, usually in the range of about 10 6 - 10 8 cells/ml.
  • the dosage will generally vary in the range of about 10 6 - 10 8 cells/kg of host.
  • the composition will be at least 25%, more usually at least 50% and frequently at least 90% by weight of megakaryocyte progenitors and megakaryocytes.
  • One or more infusions may be necessary, as required.
  • Platelets may be harvested and administered in accordance with conventional ways.
  • the megakaryocytic cells may be used for gene therapy, where the cells may serve to maintain function under conditions where normal megakaryocytic cells may be adversely affected or to provide a function normally unavailable from megakaryocytic cells.
  • the multiple drug resistance (mdr) phenotype may be introduced into the cells, which would make them resistant to chemotherapeutic agents, allowing for the continued normal megakaryocytic response during the chemotherapy treatment.
  • Genes encoding growth factors may be introduced, which would provide an enhanced source of growth factors in the bone marrow, where a deficiency might exist during a particular episode or one wished to enhance the growth of megakaryocytes or other hematopoietic cells.
  • Various methods are known for providing transformation of cells, providing for stable extrachromosomal elements or homologous or random integration. Viral vectors incapable of replication have been described in the literature, which can be used for infection, transfection and integration. By being able to grow megakaryocytic cells in culture, one has the opportunity to transform such cells and expand and clone desirable transformants.
  • Heparin Sigma 178 U/ng, stock solution 200 mg/ml. Plate: 100 mm Falcon plate. Plate 1: No added heparin.
  • Plate 2 1000 ⁇ g/ml heparin sigma concentration. Both plates were tended to by removal of 10 ml medium and addition of media plus heparin approximately every day, occasionally every other day. By day 10 a noticeable difference in the predominant cell morphology was evident. Photomicrographs were taken evidencing the morphology. Plate 1 had sheets of fibroblast-like cells which would form large balls of cells often dislodging from the plate. Plate 2 had confluent layers of rounder cells with a different morphology. No sheets of fibroblasts were observed.
  • Bones K266 and K267 samples were stripped of protein by cutting along the long axis in one half, incubating at 37 ⁇ C with collagenase or trypsin and every 0.5 hr, mixing for 3 hours followed by three washes with media. Cell concentration was 2xl0 6 per ml.
  • Example 3 The effect of different plates on megakaryocyte and fibroblast growth.
  • the Corning control plate showed a rapid growth of adherent cells with a fibroblast morphology. Within 7 to 10 days, the culture was overgrown in areas and the cells peeled away from the plate. Often, balls of these cells were found floating in the medium. The medium became yellow rapidly despite frequent changes. In the Corning heparin-containing medium, a mixed pattern of growth was observed with both fibroblast-like cells and cells with the "egg-shaped" morphology. The growth of the fibroblast-like cells was very significantly reduced and the overall cellularity of the culture was reduced compared to the control plate. At about 2-3 weeks, the fibroblast-like cells predominated in the culture and the number of egg-shaped cells was reduced. Overall, the non-adherent cell number was minimal.
  • Example 4 Fluorescence-activated cell sorter scan.
  • Cells from the K266 and K267 cultures were collected by gentle pipetting, containing about 10 5 cells/ml.
  • the antibodies used were anti-CD7 and anti- CD42 in 0.1 PBS plus 2% FCS.
  • the cells were incubated for 20 min on ice with 20 ⁇ l of antibody and then washed through FCS.
  • FITC labeled goat anti-mouse Ig diluted 1:100 (20 ⁇ l/sample) was incubated on ice with the mixture described above for 20 min, followed by washing the cells through FCS.
  • the cells were then analyzed on a FACScan.
  • the scan was ungated, standard threshold 52 (arbitrary unites) and the amplification was 1.
  • Example 5 Methylcellulose assay.
  • a methylcellulose assay was carried out with conventional conditions, employing 20% fetal calf serum and 100 ng/ml each of IL-1, -3 and -6 and erythropoietin.
  • Whole bone marrow was employed at different seeding levels as well as selected cells based on CD34 and CD41.
  • the CD34+ CD41+ is between 0.5 and 0.9% of the whole bone marrow population. The following table indicates the results:
  • the colonies comprised of morphologically identifiable megakaryocytes. Thus, colonies were observed at the lower concentration from the CD34+ CD41+ cells which matured to megakaryocytes. In addition, when whole bone marrow was employed at a seeding of 10 5 per ml of cells, a significant number of colonies was observed which provided megakaryocytes. In addition, the CD34+ CD41+ population was found to be sensitive to the indicated factors, so that by removing one factor and replacing such factor with a test composition, one can determine the effect of such test factor on the formation of megakaryocytes,
  • Example 6 Growth of megakaryocytes from buffy coat.
  • One unit of leukocytes (buffy coat) from an adult human contains about 2 x 10 8 white cells.
  • the red cells in the unit are lysed by a 10 min incubation at 37 ⁇ C in a solution of ammonium chloride buffered with Tris-HCl.
  • the leukocytes were then plated under standard heparin culture conditions as described in Example 1 at 10 7 cells/10 cm plate, 5 x 10 6 cells/6 cm plate, or 10 5 cells/well in a microtiter plate in a volume of 10 ml, 5 ml, or 100 ⁇ l, approximately 20 10 cm plates can be made.
  • aliquots of 1-2 x 10 7 fresh leukocytes can be frozen in liquid nitrogen in 10% DMSO and 20% FCS. These vials can be thawed at a later time for use in heparin cultures as described above.
  • Example 7 Megakaryocyte assay by measuring src protein by ELISA.
  • Megakaryocyte cultures are established with 8x10* cells in 96 well plates. Each growth factor source is tested in quadruplicate (4 wells) . After 10 days of growth the medium is removed from the wells and the cultures are air dried overnight. The src ELISA is performed by rehydrating the cells in PBS for 5 minutes. The src antibody (2-17) (mouse monoclonal) is diluted 1:800 in PBS + 1% BSA. 40 ⁇ l are added to each well and incubated 30 minutes. The wells are washed x 3 with PBS. The second antibody, goat ⁇ mouse Ig (alkaline phosphatase conjugated) (Caltag) is diluted 1:250 in PBS + 1% BSA.
  • Example 8 Isolation of megakaryocyte growth factor. 5 g of heparin-Sepharose CL-6B (Pharmacia) were swollen in PBS to a final volume of 25 ml, followed by washing in 1 liter of PBS. Fresh human plasma (25 ml) pooled from three donors was added to the swollen beads in a 50 ml tube and mixed at 4 ⁇ C overnight. The plasma was removed and filtered (referred to as "depleted”) . Beads were then mixed for 5 h with 25 ml of IMDM containing 3 mg/ml heparin at 4 ⁇ C. The medium was decanted and filtered (referred to as "eluate”) .
  • Heparin cultures were then set up as described in Examples 1 and 6 with either bone marrow or buffy coat, where the various forms of plasma: (1) plasma (10%) used without treatment; (2) depleted plasma; or (3) depleted plasma plus the eluate (30% final volume) , were used in place of the fetal calf serum described in Example 1.
  • Src kinase assay was performed on cultures grown 2 wks. Total protein in the dish was measured: CPM in src band was determined after excising band from the gel and scintillation counting. The results are as follows:
  • Sepharose column (eluate) Expressed as percentage of culture #3.
  • Example 9 Purification of megakaryocyte growth factor. Human plasma protein (0.65g) was placed on a 50mm x
  • 150mm DEAE-cellofine column prepared with buffer A (lOmM Tris-HCl pH 8.0, 0.1 mM EDTA) and eluted with a linear gradient of buffer A and 2 M NaCl in buffer A.
  • the flow rate was 2ml/min and 5ml fractions were collected.
  • the bioactivity was found to be in the higher salt concentration or downside of the major peak during elution with the salt gradient, the major portion of the activity being in the fractions from about 1.25 to 1.6 M NaCl.
  • the fractions may be assayed in a 96 titer well plate, with 4 replicates.
  • the fractions are dialyzed against PBS.
  • the medium comprising 5-10% of the fractions to be assayed in X-Vivo (Whittaker) , lmg/ l heparin, are seeded with 8 x 10 A cells/well and fed every three days. After two weeks the cells are inspected for megakaryocyte morphology and staining with Megacolor. It is evident from the above results, that an efficient economic procedure has been provided for producing human megakaryocytes in culture.
  • a continuous source of platelets is provided for treatment and study of the function of platelets, as well as their formation.
  • megakaryopoiesis may be studied.
  • a factor associated with megakaryopoiesis is provided and other factors may be similarly isolated.

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Abstract

On produit des mégacaryocytes et des plaquettes en culture en employant des conditions encourageant la mégacaryopoïèse tout en décourageant la formation de fibroblastes. En particulier on constate que du plasma humain ou de l'héparine avec du sérum de foetus de veau favorisent la formation de mégacaryocytes dans des récipients dépourvus de revêtements de support de fibroblastes. Un facteur associé à la mégacaryopoïèse est décrit.
PCT/US1991/007159 1990-10-05 1991-10-04 Croissance, production et composition des megacaryocytes et des plaquettes Ceased WO1992006178A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
FI922586A FI922586A7 (fi) 1990-10-05 1991-10-04 Megakaryosyyttien ja verihiutaleiden kasvu, tuotanto ja koostumus
CA002069879A CA2069879A1 (fr) 1990-10-05 1991-10-04 Croissance, production et composition de megakaryocytes et de plaquettes
KR1019920701338A KR927003795A (ko) 1990-10-05 1991-10-04 거핵세포 및 혈소판 성장, 제조방법 및 이것들을 포함하는 조성물
NO92922213A NO922213L (no) 1990-10-05 1992-06-04 Megakaryocyt- og blodplatevekst, fremstilling og blanding

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US59368890A 1990-10-05 1990-10-05
US593,688 1990-10-05
US68266691A 1991-04-09 1991-04-09
US682,666 1991-04-09

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PCT/US1991/007159 Ceased WO1992006178A1 (fr) 1990-10-05 1991-10-04 Croissance, production et composition des megacaryocytes et des plaquettes

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EP (1) EP0504361A4 (fr)
JP (1) JPH05502385A (fr)
KR (1) KR927003795A (fr)
AU (1) AU639378B2 (fr)
CA (1) CA2069879A1 (fr)
WO (1) WO1992006178A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
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WO1993023059A1 (fr) * 1992-05-15 1993-11-25 Institut Des Vaisseaux Et Du Sang Utilisation de glycosaminoglycanes exogenes ou derives dans le traitement des thrombopenies
WO1995006112A1 (fr) * 1993-08-23 1995-03-02 Baxter International Inc. CROISSANCE Si(IN VITRO) DE PRECURSEURS DE NEUTROPHILES ET DE MEGACARYOCYTES DANS DES MILIEUX EXEMPTS DE SERUM
EP0675726A4 (fr) * 1992-12-11 1996-09-25 Systemix Inc Croissance, production et composition des megacaryocytes et des thrombocytes.
US5766581A (en) * 1994-03-31 1998-06-16 Amgen Inc. Method for treating mammals with monopegylated proteins that stimulates megakaryocyte growth and differentiation
US5795569A (en) * 1994-03-31 1998-08-18 Amgen Inc. Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation
US5846529A (en) * 1993-08-23 1998-12-08 Nexell Therapeutics, Inc. Infusion of neutrophil precursors for treatment of neutropenia
US6037174A (en) * 1993-08-23 2000-03-14 Nexell Therapeutics, Inc. Preparation of serum-free suspensions of human hematopoietic cells or precursor cells

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WO2006109367A1 (fr) * 2005-04-05 2006-10-19 Obihiro University Of Agriculture And Veterinary Medicine Méthode de libération cellulaire, solution de libération cellulaire, méthode de culture cellulaire, milieu de culture cellulaire, solution cellulaire, préparation de solution cellulaire, méthode de colonisation cellulaire et solution de colonisation cellulaire
JP2008109866A (ja) * 2006-10-30 2008-05-15 Jms Co Ltd 培地添加剤、この培地添加剤を含む培地、及びこの培地を用いた細胞の培養方法
PL2539441T3 (pl) * 2010-02-22 2017-06-30 Université Pierre Et Marie Curie (Paris 6) Podłoże do hodowli komórkowej do wzrostu i różnicowania komórek linii hematopoetycznych
KR102873857B1 (ko) 2023-04-17 2025-10-24 오알에스코리아 유한회사 드레싱 장치

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See also references of EP0504361A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993023059A1 (fr) * 1992-05-15 1993-11-25 Institut Des Vaisseaux Et Du Sang Utilisation de glycosaminoglycanes exogenes ou derives dans le traitement des thrombopenies
EP0675726A4 (fr) * 1992-12-11 1996-09-25 Systemix Inc Croissance, production et composition des megacaryocytes et des thrombocytes.
WO1995006112A1 (fr) * 1993-08-23 1995-03-02 Baxter International Inc. CROISSANCE Si(IN VITRO) DE PRECURSEURS DE NEUTROPHILES ET DE MEGACARYOCYTES DANS DES MILIEUX EXEMPTS DE SERUM
US5846529A (en) * 1993-08-23 1998-12-08 Nexell Therapeutics, Inc. Infusion of neutrophil precursors for treatment of neutropenia
AU702871B2 (en) * 1993-08-23 1999-03-11 Nexell Therapeutics Inc. (In vitro) growth of neutrophil and megakaryocyte precursors in serum-free media
US6010697A (en) * 1993-08-23 2000-01-04 Nexell Therapeutics, Inc. Infusion of neutrophil precursors for treatment of neutropenia
US6037174A (en) * 1993-08-23 2000-03-14 Nexell Therapeutics, Inc. Preparation of serum-free suspensions of human hematopoietic cells or precursor cells
US5766581A (en) * 1994-03-31 1998-06-16 Amgen Inc. Method for treating mammals with monopegylated proteins that stimulates megakaryocyte growth and differentiation
US5795569A (en) * 1994-03-31 1998-08-18 Amgen Inc. Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation

Also Published As

Publication number Publication date
EP0504361A1 (fr) 1992-09-23
EP0504361A4 (en) 1994-08-17
AU639378B2 (en) 1993-07-22
AU8714491A (en) 1992-04-28
KR927003795A (ko) 1992-12-18
JPH05502385A (ja) 1993-04-28
CA2069879A1 (fr) 1992-04-06

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