WO1995002685A1 - Serum-free culture of progenitor cells - Google Patents

Serum-free culture of progenitor cells Download PDF

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Publication number
WO1995002685A1
WO1995002685A1 PCT/US1994/007896 US9407896W WO9502685A1 WO 1995002685 A1 WO1995002685 A1 WO 1995002685A1 US 9407896 W US9407896 W US 9407896W WO 9502685 A1 WO9502685 A1 WO 9502685A1
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cells
serum
population
cell
sba
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PCT/US1994/007896
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French (fr)
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Jane Lebkowski
Lisa R. Schain
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Applied Immune Sciences, Inc.
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Priority to AU73618/94A priority Critical patent/AU7361894A/en
Publication of WO1995002685A1 publication Critical patent/WO1995002685A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0634Cells from the blood or the immune system
    • C12N5/0647Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/90Serum-free medium, which may still contain naturally-sourced components
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/125Stem cell factor [SCF], c-kit ligand [KL]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20Cytokines; Chemokines
    • C12N2501/22Colony stimulating factors (G-CSF, GM-CSF)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20Cytokines; Chemokines
    • C12N2501/23Interleukins [IL]

Definitions

  • subject field concerns growth of cells with serum- free media.
  • subject field concerns serum-free culture of CD34 + cells.
  • Bone marrow is the source of all hematopoietic precursors. Extensive or complete damage to the marrow, whether as the result of disease within the cells of the marrow itself or as a consequence of therapeutic intervention to treat disease elsewhere in the patient, results in a situation that requires immediate action. Alternatively, even in a situation where the patient's marrow is not completely destroyed, there are instances when it is beneficial to provide a patient with supportive therapy consisting of periodic doses of progenitors (Leonard Sander, M.D. , unpublished data) .
  • GvHD graft- versus-host disease
  • Clinically important acute GvHD occurs in 30% to 50% of HLA identical transplants, and in approximately 75% of HLA non-identical transplants. Death results in 20% of all cases of acute or chronic GvHD.
  • the incidence of GvHD increases with the age of the host; many centers will not undertake an allogeneic transplant with patients over the age of 50 (Sullivan, Bone Marrow Transplantation p. 83 (Kluwer Academic Pub., Boston, 1990) ; Scientific American Medicine, section 5
  • GvHD the risk of contracting GvHD is reduced by performing HLA- atching of donor and host.
  • a successful match is sometimes made meaningless by the reluctance of many prospective marrow donors to undergo the potentially dangerous bone marrow explant procedure which usually requires general anesthesia and which is often accompanied by substantial discomfort.
  • large doses of immune system suppressing drugs must be given to the host to avoid the possibility of GvHD.
  • Immune suppressing drugs frequently have highly undesirable side-effects.
  • One means to avoid GvHD is with autologous circulatory system transplants. GvHD generally does not occur with an autologous transplant.
  • tumor purging often reduces the number of hematopoietic precursors.
  • tumor purging must be balanced against the need to return sufficient numbers of primitive hematopoietic cells to reasonably assure engraftment. Consequently, tumor purging has been used infrequently, and has been problematic when employed.
  • Hematopoietic and reconstitutive activity in primates is produced by cells bearing the CD34 antigen (Berenson et al . , "Antigen CD34 + Marrow Cells Engraft Lethally Irradiated Baboons," J. Clin. Invest. (1988) 81:951; Civin et al . , "A Hematopoietic Progenitor Cell Surface Antigen Defined by a Monoclonal Antibody Raised against KG-la Cells, " J. Immunol. (1984) 133:157; Watt et al .
  • CD34 + hematopoietic precursors as supportive therapy could significantly reduce cytopenic periods and consequently improve patient outcome.
  • Autologous bone marrow transplantation following high dose chemotherapy and/or radiation has become increasingly used as a treatment for human malignancies.
  • peripheral blood stem cells have been used exclusively, or in conjunction with bone marrow, to hasten patient engraftment.
  • a further advantage would be afforded to such therapy if CD34 + stem cells could be purified from the peripheral blood stem cell population, thereby significantly reducing the cell volume required for patient reinfusion. It would also be highly desirable if isolation of CD34 + cells could act to purge tumor cells from the engraftable fraction.
  • subsets of cells can have relatively narrow ranges of activities.
  • subsets can be specialized for response to a particular disease, such as neoplasia; infection, viral or bacterial, etc.; response to transplants; and the like. It is therefore of great interest to be able to identify and purify these subsets of cells, not only to understand their action, but also to use these cells for prophylactic and therapeutic purposes. In order to achieve the desired results, it is necessary that substantially pure populations of the desired subset or subsets of cells can be obtained.
  • the cells should be: (1) free of antibodies on their surface, (2) viable, (3) capable of fulfilling their normal function, and (4) responsive to activation by biologicals in the same manner as normal cells in their normal environment.
  • a method of manipulating circulatory system components such as cells from the lymphatic system, intrathecal fluid, peripheral blood, or bone marrow
  • An additional objective for an expansion method is the expansion of the precursor cells for banking and subsequent infusion.
  • the present invention sets forth a method for expanding progenitor cells. Accordingly, a population of progenitor cells is provided; these cells are provided from a cellular source such as the lymphatic system, intrathecal fluid, peripheral blood, or bone marrow. Thereafter, the progenitor cells are cultured in serum-free media. By use of serum-free media, an expanded population of progenitor cells results.
  • the expanded population of progenitor cells is of a population size sufficient for use in clinical applications.
  • the serum-free media can. comprise an array of growth factors.
  • the progenitor cells can be attached to a surface. The progenitor cells resulting from expansion of the provided progenitor cells can be attached to the surface or can be released into the serum-free media.
  • a population of cells prepared according to the method of the invention is also set forth.
  • a cellular composition is prepared by contacting circulatory system components with soybean agglutinin, wherein the soybean agglutinin (SBA) is bound to a plastic surface. Accordingly, SBA + cells become bound to the surface, and SBA" cells do not become bound to the surface. The SBA" circulatory system components are harvested. The SBA" circulatory system components are contacted with specific binding proteins, such as monoclonal antibodies, that are specific for the CD34 antigen; wherein the specific binding proteins are bound to a plastic surface. Accordingly, CD34 + cells become bound to the surface, and CD34 ⁇ do not become bound to the surface. Thereafter, the bound CD34 + cells are cultured with serum-free media, whereby progeny CD34 + cells are produced.
  • SBA soybean agglutinin
  • bound CD34 + cells are released from the surface.
  • the progeny CD34 + cells can become bound to the surface or can be released into the serum- free media. Should progeny cells be released into the serum-free media, CD34 + cells can be harvested from the media. Alternatively, should CD34 + cells be bound to the surface, they can be harvested by releasing them from the surface.
  • the specific binding proteins for the CD34 antigen are monoclonal antibodies
  • the monoclonal antibody can be from a group of monoclonal antibodies such as ICH3, My 10, 12.8, BEND10, or 8G12.
  • the released cells retain expression of the CD34 antigen, no lag time is involved in awaiting verification of CD34 + status.
  • Figure 1 Purification Scheme for CD34 + Cells.
  • FIG. 2 Two Color Flow Cytometric Analysis. Two color analysis was performed as described herein. A lymphoblastoid gate was used for the bone marrow mononuclear (BMMC) and SBA" cell fractions to eliminate the autofluorescense of high side scatter cells. A total cell gate was used for the CD34 + cell fraction.
  • BMMC bone marrow mononuclear
  • SBA SBA
  • Figure 3A-C Enrichment of Human Hematopoietic Progenitors. Aliquots of the indicated populations from bone marrow (A) , cytokine stimulated peripheral blood (B) , and unstimulated peripheral blood (C) were placed in methylcellulose culture as described in the Materials and Methods. The frequency of CFU-GM, BFU-E and CFU-GM were calculated in each population of cells. The fold enrichment of CFU-GM (solid bars) , BFU-E (hatched bars) and CFU-GEMM (dotted bars) in the SBA" and CD34 + cell populations was then calculated in reference to the mononuclear cell fraction. The fold enrichment in the mononuclear cell fraction was defined as 1. The results shown in these figures are from the same experiments detailed in Table 1.
  • Figure 4A-C Long-Term Bone Marrow Culture of CD34 + Cells. Bone marrow mononuclear (°) , SBA " (+) and CD34 + (*) cells were cultured in long term bone marrow culture as described herein. Two hundred thousand cells from each population were loaded into stromal layers and each population was tested in triplicate. Large-scale (624 cm 2 ) AIS Cellector®s CD34 were used to isolate the cells for this experiment. Panel A) , The number of cells in the nonadherent layer was counted at each time point and the % of the original number of seeded cells is plotted as a function of time.
  • Panel B The total number of CFU-GM produced at each time point during culture is plotted as a function of culture time.
  • Panel C) The cumulative production of CFU-GM during long term bone marrow culture is plotted as a function of time. To plot accumulation, the number of- CFU-GM at the start of the culture was defined as 0.
  • Figure 5 Cellular proliferation, over time, comparing serum-based ( ⁇ ) and serum-free (D) media for bone marrow derived cell population 497.
  • Figure 6 Cellular proliferation, over time, comparing use of serum-based ( ⁇ ) and serum-free (D) media, for bone marrow derived cell population 499.
  • Figure 7 Cellular proliferation, over time, comparing use of serum-based ( ⁇ ) and serum-free (O) media, for bone marrow derived cell population 500.
  • Figure 8 The percent of cells exhibiting CD34 antigen positivity, over time, for cell population 499, comparing serum-based ( ⁇ ) and serum-free (D) media.
  • Figure 9 The number of CD34 + cells produced, over time, for population 499, comparing use of serum-based ( ⁇ ) and serum-free (D) media.
  • Figure 10 The percent of CD34 + cell positivity in a cell culture, over time, comparing use of serum-based ( ⁇ ) and serum-free ( ⁇ ) media.
  • Figure 11 The total number of myeloid precursors (CFU-GM) in a culture of population 499, over time, comparing use of serum-based ( ⁇ ) and serum-free (D) media.
  • Figure 12 The total number of myeloid precursors (CFU-GM) in a culture of population 500, over time, comparing use of serum-based ( ⁇ ) and serum-free ( ⁇ ) media.
  • Figure 13 The total number of erythroid precursors (BFU-E) in a culture of population 499, over time, comparing use of serum-based ( ⁇ ) and serum-free (D) media.
  • Figure 14 The total number of erythroid precursors (BFU-E) in a culture of population 500, over time, comparing use of serum-based ( ⁇ ) and serum-free ( ⁇ ) media.
  • Figure 15 Cellular proliferation, over time, of peripheral blood stem cells using serum-free media comprising various combinations of growth factors: ( ⁇ ) IL-1, IL-3, and stem cell factor; (D) G-CSF, and SCF; ( ⁇ ) IL-3 and SCF; (0) IL-3, GM-CSF, and SCF; (A) IL-1, IL-3, GM-CSF, and SCF.
  • Figure 16 The total number of myeloid precursors (CFU-GM) in cultures of peripheral blood stem cells, over time, with use of serum-free media comprising various combinations of growth factors: ( ⁇ ) IL-1, IL-3, and stem cell factor; (D) G-CSF, and SCF; ( ⁇ ) IL-3 and SCF; ( 0) IL-3, GM-CSF, and SCF; (A) IL-1, IL-3, GM-CSF, and SCF.
  • Figure 17 The total number of erythroid precursors (BFU-E) for peripheral blood stem cells, over time, with use of media comprising various combinations of growth factors: () comprising IL-1, IL-3, and stem cell factor; (D) G-CSF, and SCF; ( ⁇ ) IL-3 and SCF; (0) IL-3, GM-CSF, and SCF; (A) IL-1, IL-3, GM-CSF, and SCF.
  • growth factors comprising IL-1, IL-3, and stem cell factor
  • D G-CSF, and SCF
  • IL-3 and SCF
  • A IL-1, IL-3, GM-CSF, and SCF.
  • CD34 + human hematopoietic stem/progenitor cells have numerous applications in basic research and human therapy.
  • isolated CD34 + cells can be used as a purged graft in autologous bone marrow transplantation, minimizing the reinfusion of tumor cells after high-dose chemotherapy or irradiation.
  • CD34 + cells can also be used as a T-cell depleted graft in allogeneic bone marrow transplantation, as a supportive care transfusion product post chemotherapy, or as a target cell in gene therapy protocols.
  • a subject device may take a wide variety of forms.
  • the device will be comprised of polystyrene surfaces, where the polystyrene is normally substantially free of cross-linking, less than about 0.5%, usually less than about 0.1%, preferably molded or extruded, so as to have a very smooth surface.
  • Polystyrene surfaces of this nature allow for substantial uniformity of derivatization, where the orientation of the receptor provides for a high level of accessibility of binding sites. It should be understood in referring to receptor, the term is entirely arbitrary. By receptor is intended a molecule which is able to specifically bind to a complementary molecule.
  • the surface will be derivatized by substitution of the benzene ring of the polystyrene with an electrophilic reagent, particularly by a Friedel-Crafts reaction in a solvent which does not soften or dissolve the polystyrene.
  • an electrophilic reagent particularly by a Friedel-Crafts reaction in a solvent which does not soften or dissolve the polystyrene.
  • sulfolane finds particular application.
  • Relatively mild conditions may be employed and the benzene may be derivatized with a variety of agents, such as nitro, which may be reduced to amino, halomethyl, which may be used to form an amino, hydroxy, or thiol group, or a substituted N- hydroxymethyl acetamide where the substituent is an active halogen or pseudohalogen.
  • a description of the reaction may be found in EPA 88-304516.3.
  • the derivatized polystyrene surface may then be reacted with the receptor. Under the conditions of derivatization, it is found that a high percentage of the benzenes at the surface are derivatized, so that one may obtain a high density of receptor at the surface. Depending upon the nature of the receptor, various reactions may be performed for bonding the receptor to the surface. Of particular interest is the bonding of proteins to the surface. Proteins can be bonded by contacting the proteins in an aqueous medium with the functionalized/derivatized surface, having active halogen, activated carboxy groups, e.g., esters, or the like, under mild conditions for sufficient time for complete reaction. Any remaining unreacted functional groups may be blocked by using an appropriate small molecule blocking agent.
  • active halogen may be blocked with aliphatic amines, thiols with maleimide, or the like. In some situations, there may be no need to block excess reactive groups, since they will not interfere with the subsequent steps in the process.
  • the surface may then be washed to remove the non-specifically bound receptor and evaluated to insure that appropriate receptor binding has occurred. Substantially homogeneous populations, greater than about 95%, usually 98%, of cells have been achieved by use of subject devices, where the cells may be in a quiescent or activated state.
  • cellular compositions may include any cellular population expressing a surface marker (ligand) recognized by the immobilized receptor.
  • compositions include cells bearing any of the recognized leukocyte antigens of the CD (cluster designation series) or others recognized by monoclonal antibodies to specific cell surface ligands.
  • Such compositions may include other blood cells, tumor cells, bacteria, viruses, or parasites similarly sharing a common surface marker. Virtually any cell population whose members share a surface ligand recognized by the immobilized receptor can constitute such a cellular composition.
  • the present method was used to process input samples as small as lxlO 7 cells, whereby the resulting population of expanded progenitor cells is expanded to a volume sufficient for use in a full-size clinical bone marrow graft.
  • CD34 + cells isolated using subject devices were up to 98% pure, had no detectable mouse immunoglobulin on their surface, and were functional in in vitro hematopoiesis assays.
  • the CD34 + purity of the cells was determined using phycoerythrin conjugated HPCA-2 anti-CD34 mAb, according to known methodologies.
  • isolated CD34 + cells can also be directly cultured in the subject devices yielding large expansions of progenitors.
  • CD34 + cells are provided, such as by being removed from a subject CD34 device, and the cells are placed in a cell culture bag comprising Teflon® (e.g., culture bags produced by American Fluoroseal, distributed by West Co., Lionville, PA). Thereafter, one or more growth factors and/or serum-free media are/is provided as described herein.
  • Teflon® e.g., culture bags produced by American Fluoroseal, distributed by West Co., Lionville, PA.
  • HMBA N-(H ⁇ droxymethyl) 2-bromoacetamide
  • BA-PS bromoacetamide polystyrene surface
  • the second step involves the generation of the bromoacetamide polystyrene surface (BA-PS) .
  • BA-PS bromoacetamide polystyrene surface
  • 2M triflic acid and 0.2M HMBA, both in tetramethylene sulfone (sulfolane) are mixed 1:1 in a volume sufficient to cover the inner surface of a polystyrene vessel being activated.
  • the reaction is allowed to proceed at 27 ⁇ C for 3 hours, the reaction solution is drained, the device washed with water, followed by ethanol, and the activated polystyrene chambers are air dried.
  • the resulting bromoacetamide polystyrene surface is stable in room air for six (6) months.
  • the next step is the receptor capture (the monoclonal antibody one wishes to covalently bind to the bromoacetamide-polystyrene surface) .
  • the monoclonal antibody of interest is diluted to approximately 0.01 - 0.05 mg/ml in phosphate buffered saline, pH 7.4.
  • the appropriate volume of diluted monoclonal antibody is introduced into the polystyrene chamber and the reaction is allowed to proceed for from about two to twenty, preferably about 2 to 4 hours, at 27°C with rotation.
  • the antibody remaining after the reaction is decanted and can be re-utilized up to 10 times in subsequent coating reactions.
  • the antibody bound device is then washed ten times with phosphate buffered saline (PBS), pH 7.4, and the surface is then stabilized by the addition of 2% sucrose/0.2% human serum albumin (HSA) , medical grade, to each device.
  • PBS phosphate buffered saline
  • HSA human serum albumin
  • the sucrose/albumin solution is allowed to coat the surface, after which the excess sucrose/HSA solution is decanted and the stabilized polystyrene chambers dried 24-96 hours in a vacuum ( ⁇ 0.10 Torr) at 25 ⁇ C. After drying, the vacuum is broken with dry nitrogen and the device is flushed with inert, dry gas and capped tightly. The device is sealed and then sterilized.
  • Sterilization is achieved by irradiation with, 2.7 ⁇ 0.2 megarads of electron beam or gamma irradiation. Sterility tests showed that the flasks were sterile after a 14 day in situ media incubation.
  • a variety of surface functionalization groups were employed and tested for the stability of binding of antibody to the surface.
  • the polystyrene was functionalized using N-(hydroxymethyl)2-haloacetamide, where the halo group was chloro, bromo or iodo; diazoniu and sulfonium. After monoclonal antibody attachment using these surfaces, the flasks were washed 10 times with PBS and once with 1% SDS at 55°C for 14 hours. The plastic surface was then assayed for radioactivity of the labeled monoclonal antibodies and the results expressed as surface density for monoclonal antibody in ng/cm 2 .
  • the bromoacetamide had a surface density of about 250 ng/cm 2 of antibody, more than 2.5 times that achieved by adsorption on an Immulon-2 11 ** (Dynatek) surface. While the bromoacetamide provided the highest surface density, the surface density for the other functionalities fell between 200 and about 240 ng/cm 2 . d. Stability of capture surface receptor.
  • the stability of the antibody binding was determined by coating the surface with 0.02 mg/ml of ( 35 S) human IgG. The flasks were washed five times with borate- carbonate buffer, once with borate-carbonate buffer for 8 hours and twice with borate-carbonate washes overnight. Aliquots of each wash were saved and assayed for radioactivity. After the second wash, there was no evidence of any antibody leaching. In a second study, using an ELISA assay for the antibody bound to the surface, the results observed showed that the amount of extractable antibody was less than the detection limit of the assay, (7.7 ng/ml) .
  • lymphokine release was tested with CD8 + cytotoxic T-cells captured from normal human peripheral blood according to the subject method.
  • the captured CD8 cells were shown to be free of surface-bound monoclonal antibody by flow cytometry analysis using fluoresceinated anti-mouse antibody. None of the released CD8 + cells were positive for surface mouse IgG.
  • the subject flasks can be re-used for cell capture, by washing in PBS containing 4M MgCl which regenerates the capture surface.
  • Such re-used flasks performed consistently for 4-6 cycles after which repeated washing reduced the bound antibody activity.
  • Detached cells recovering by decanting, were expanded numerically in standard tissue culture chambers supplemented with IL-2 and phytohemagglutinin.
  • Viability by Trypan blue exclusion was shown to be greater than 98% and the recovered, homogeneous cell population could be expanded by two orders of magnitude over a period of about 10 days.
  • a second method for cell recovery called ultrasonic release, utilized an ultrasonic bath (such as a Crest Ultrasonics model #H-4HT-1014-6) with an output of 40 to 90 kHz sonic output (main frequency at 40 kHz) evenly distributed through a water bath by means of the Crest Vibra-bar.
  • the power supply delivered 500 watts at 40 to 90 kHz.
  • the ultrasonic bath had an immersion tank of 10 x 14 inches, holding a volume of 6 gallons of fluid which contained one liter (0.5" from the tank bottom) for sonication in the subject studies.
  • a capture device containing the bound cells was immersed in the one liter of fluid in the ultrasonic bath and the power supply and power application time experimentally determined.
  • times and powers varied: For example, CD4 + T-cells: 78% max power, 17 sec; CD8 + T-cells: 30% max power, 20 sec; Leu 19 cells: 75% max power, 10 sec, etc., as is appreciated by one of ordinary skill in the art.
  • CD16 + NK-cells were recovered by sonication at maximum power for 15 to 20 seconds.
  • the cells recovered by sonication (1) were greater than 85% viable by Trypan blue exclusion, and (2) were extremely active in a lytic assay routinely utilized to quantitate NK-cell activity.
  • CD34 device e.g., AIS CellectorTM CD34, Applied Immune Sciences, Santa Clara, CA
  • CD34 + cells were washed from the device, and the adherent CD34 + cells collected by physical agitation.
  • Human CD34 + cells were isolated from a variety of sources by use of the subject devices. The process for utilizing these devices is outlined in Figure 1. Bone marrow or peripheral blood cells were depleted of red cells using ficoll/hypaque density gradients or gelatin sedimentation. The mononuclear cell preparation was then loaded into an SBA device to bind many differentiated cells such as red cells, B cells, fat cells, fibroblasts, endothelial cells, certain T-cells and tumor cells which normally bind to the soybean lectin.
  • the nonadherent SBA" fraction which was enriched in CD34 + cells, was collected; advantageously, the adherent SBA + cells were eluted from the devices by incubation at 37 ⁇ C with 200 mM N-acetylgalactosamine in RPMI for 0.5-3 hours.
  • the SBA" population was subsequently loaded into an CD34 device, for CD34 + cell isolation. Finally, the nonadherent CD34" cells were removed, and the CD34 + cells were harvested, advantageously by physical agitation of the device.
  • Bone marrow was collected from the iliac crest from consenting volunteers. Normal peripheral blood was collected as a leukopheresis product from consenting volunteers. Aliquots of cytokine-stimulated peripheral blood were collected from either normal volunteers who were stimulated 6 days with G-CSF, or from myeloma patients undergoing peripheral blood stem cell collections after chemotherapy along with G-CSF treatment, as a part of their normal course of therapy.
  • cells are obtained by use of methods such as counter-flow elutriation, flow cytometry, magnetic beads, affinity chromatography, cellular panning, or agglutination procedures.
  • Antigens used to identify progenitor cells include CD34 + , CD38", CD33 " , Thyl + , HLA-DR dull, rhodamine", HLA-DR " , and 7B7 " .
  • the foregoing antigens and cell types exemplify, but in no way limit, the antigens assessed and the cell types expanded by use of the invention.
  • the cell sample was diluted 1:5 - 1:10 using Dulbecco's Phosphate Buffered Saline (Ca/Mg free) containing 1 mM EDTA (DPBSE) . Forty mis of this suspension were underlayered with 10 ml of Histopaque
  • the mononuclear cell layer was collected, diluted 5-fold with DPBSE, and the cells were collected at 900 g for 20 minutes. To eliminate platelets, the cells were subsequently diluted with DPBSE and centrifuged at 450 g for 10 minutes. The cells were resuspended in DPBSE, counted, and used in later separation procedures.
  • gelatin sedimentation was used as an alternative procedure to eliminate red blood cells from the input samples.
  • the bone marrow or peripheral blood was directly mixed 1:1 (v:v) with 3% gelatin in DPBS. After a 20-40 minute incubation at room temperature, the white cell supernatant was extracted and diluted 3-5 fold with DPBSE. The white cells were collected at 900 g for 20 minutes, and were ready to use in accordance with the invention.
  • the devices comprised polystyrene vessels containing covalently immobilized protein (AIS).
  • the SBA devices had a variety of capacities; a 25 cm 2 and 150 cm 2 SBA device separated up to 2xl0 7 and 1.2X10 8 input cells, respectively.
  • a large-scale 3000 cm 2 SBA device processed up to 2xl0 9 cells.
  • the CD34 devices also processed a variety of cell loads.
  • a 25 cm 2 device accommodated 2xl0 7 cells, whereas a large-scale 624 cm 2 device processed up to 5xl0 8 cells.
  • the devices were primed/prepared by rehydrating the surface with four consecutive washes with DPBSE.
  • the device was shaken vigorously at each wash to remove a protective coating of the surface. (For information concerning the protective coating, please see U.S. Patent No. 5,283,034, issued 01 February 1994.)
  • the last wash was left on the device until use, to protect the immobilized lectin or antibody.
  • rbc-depleted cells were loaded into an SBA device. For this separation, the cells were incubated at 5xl0 6 cells/ml in 0.5% human IgG (Gamimune N, Cutter Labs) in DPBSE for 15-20 minutes at room temperature. The 25 cm 2 and 150 cm 2 SBA devices were then loaded with 4 ml and 25 ml of cells, respectively, containing at maximum 2xl0 7 and 1.2x10 s cells.
  • the large-scale 3000 cm 2 SBA device For the large-scale 3000 cm 2 SBA device, up to 2xl ⁇ 9 cells were incubated in 50 ml of 0.5% human IgG in DPBSE for 15-20 minutes at room temperature; after incubation, the cells were diluted to 265 ml with DPBSE and loaded directly into a primed 3000 cm 2 device. After loading, the SBA devices were incubated at room temperature for one hour. The large-scale 3000 cm 2 devices were incubated 30 minutes at room temperature on each of the two opposed, broad sides of the device. To remove the nonadherent SBA" cells, the devices were rocked gently, and the nonadherent cell suspension was collected.
  • the 25 and 150 cm 2 devices were then washed two times with DPBSE and the washes were pooled with the first nonadherent fraction.
  • the device was rocked steadily during the collection of the nonadherent cells. When this procedure was followed, subsequent washes of the device were not necessary.
  • the nonadherent cells from all devices were centrifuged at 900 g for 20 minutes, and subsequently resuspended at 5xl0 6 cells/ml in 0.5% human Ig in DPBSE for processing on CD34 devices.
  • CD34* Cell Selection and Collection on a CD34 Device After resuspension of the SBA " cells in the human IgG solution, 25 cm 2 and 624 a cm 2 CD34 devices were loaded with a maximum of 2xl0 7 and 5xl0 8 SBA" cells, respectively. The devices were then incubated at room temperature for 60 minutes. To remove the CD34" nonadherent cells, the devices were gently rocked and the fluid was drained. The devices were then washed 2-4 times using DPBSE containing 0.5% human serum albumin (HSA) .
  • HSA human serum albumin
  • the 25 cm 2 and 624 cm 2 devices were filled with 4 ml and 50 ml of DPBSE containing 0.5% HSA, respectively, and the devices were agitated vigorously in the plane of the binding surface, thereby dislodging the CD34 + cells.
  • the dislodged CD34 + cells were drained into centrifuge tubes pre-coated with HSA, and the devices were rinsed twice with DPBSE.
  • Progenitor cells were obtained according to methods known to those of ordinary skill in the art. For example, cells were obtained by use of the subject devices. Additionally, progenitor cells are obtained by use of methods such as counter-flow elutriation, flow cytometry, magnetic beads, affinity chromatography, cellular panning, or agglutination procedures. Use of the subject devices and methods is preferred as it provides a closed device for use in separating and culturing within a single vessel.
  • Antigens used.to identify progenitor cells include CD34 + , CD38", CD33 " , Thyl + , HLA-DR dull, rhodamine", HLA-DR " , and 7B7 ⁇ .
  • the foregoing antigens and cell types exemplify, but in no way limit, the antigens assessed and the cell types expanded by use of the procedure.
  • Presence of stem cells expressing the above-mentioned antigens are verified by long-term bone marrow culture assay as set forth herein; 4HC resistance assay (Brugger, et al., "J2x Vivo Expansion of Enriched Peripheral Blood CD34 + Progenitor Cells by Stem Cell Factor, Interleukin-IB (IL-1B) , IL-6, IL-3, Interferon- ⁇ , and Erythropoietin," Blood (1993) 81:2579-2584); anti-sense oligonucleotides (Hatzfeld, et al., "Release of Early Human Hematopoietic Progenitors from Quiescence by Anti-Sense Transforming Growth Factor Bl or Rb Oligonucleotides.” J.
  • progenitor cell types in addition to primitive stem cells, are also cultured and expanded by use of the present method. For example, to culture and expand progenitor cells of the myeloid, megakaryocyte cell lines, precursors expressing the antigens CD61 or CD41b are assayed, cultured and expanded in accordance with the invention.
  • progenitor cells by use of magnetic beads or by affinity chromatography has been used to scale-up cells to clinical size samples.
  • Such methods of obtaining progenitor cells have disadvantages known to those of ordinary skill in the art.
  • Other methods of obtaining progenitor cells are limited by the fact that the amount of cells obtained thereby is small. Any attempt to culture the cells to a clinical-size sample is generally not possible, since, during the expansion of the cells, they differentiate into non-progenitor cell lines.
  • 4-100 x 10 7 progenitor cells are supplied.
  • CD34 + cells were obtained using SBA and CD34 devices.
  • serum-free media for the expansion of purified CD34 + cells from bone marrow and peripheral blood was then evaluated.
  • serum-free is intended media that does not contain undefined serum components.
  • Hematopoietic Stem Cell-SFM media GibcoBRL, Grand Island, NY
  • CD34 + cells were captured on CD34 devices, and cultured directly in the devices at 37°C in 5% C0 2 with various combinations of growth factors.
  • growth factors IL-1, IL-3 and stem cell factor were used.
  • growth factors such as G-CSF and/or GM-CSF were also added.
  • IL-1, IL-3, GM-CSF and G-CSF were all at 10 ng/ml; SCF was at 50 ng/ml.
  • the cultures were incubated at 37°C at 5% C0 2 . Half media changes were made twice weekly until the termination of a culture.
  • FITC and phycoerythrin conjugated antibodies used for evaluation were: control mouse Ig; anti-CD3, clone SK7; anti-CD19, clone 467; anti-CD33, clone P676; anti-CD38, clone HB-7; anti-HLA- DR, clone L243; goat anti-mouse IgG (heavy and light chain) (Becton Dickinson) . These samples were analyzed on a FACScan flow cytometer (Becton Dickinson) . .
  • Table 1 shows the recovery of cells in the various SBA" and CD34 + populations from bone marrow, cytokine sti ulated peripheral blood stem cells and normal peripheral blood.
  • the mononuclear cell preparation was, on average, 2.4% CD34 + .
  • CD34 + cells Upon depletion of the SBA + population, there was a net enrichment of CD34 + cells to 8.1%.
  • 67.5% of the CD34+ cells were recovered in the SBA " fraction, as determined based on standard calculations using information contained in Tables 1 and 2.
  • the majority of those CD34 + cells not recovered in the SBA " fraction were CD34 + CD19 + pre B cells which normally bind SBA.
  • the final CD34 + cell fraction from bone marrow was an average of 74.2% pure, with a 30% overall recovery of CD34 + cells.
  • CD34 + cells were isolated from cytokine-stimulated (G-CSF) peripheral blood. Based on standard calculations, the input mononuclear cell fraction had a mean 3.1% frequency of CD34 + cells, which upon depletion of SBA binding cells was enriched to 10.9% with greater than 90% recovery of CD34 + cells. Again, based on standard calculations, the final CD34 + cell fraction from the cytokine-stimulated peripheral blood was highly purified, being 81.5% pure with a 22% overall recovery of the CD34 + cells.
  • the binding surface of a subject device provides high specificity of binding, even for extremely rare cell populations.
  • CD34 + cells from normal peripheral blood which were essentially nondetectable ( ⁇ 0.2%) in the input mononuclear cell fraction, were isolated to 43.4% purity on the devices, representing a greater than 200-fold purification of these CD34 + cells (Table 2) .
  • Figure 2 shows representative two color plots from the mononuclear cell, SBA", and CD34 + cell fractions from bone marrow.
  • CD3 + T cells which were present in the input cell fraction were eliminated from the final CD34 + fraction.
  • CD19 + B cells were almost totally eliminated after SBA depletion and were less than 2% of the final CD34 + cell fraction.
  • Particularly evident in the figure is the enrichment of CD34 + cells at both the SBA " and CD34 + steps of the purification process.
  • approximately 50% and 95% of the CD34 + cells expressed the CD33 and CD38 antigens respectively, suggesting that the purified population contained a mixture of committed and uncommitted CD34 + cells representative of those found in unfractionated bone marrow.
  • the final CD34 + cell population had no residual mouse immunoglobulin on its surface.
  • the CD34 + cell population after isolation on the subject devices, was phenotyped with many different anti-mouse immunoglobulin antibodies directed against the heavy, light, Fab and Fc portions of the murine immunoglobulin molecule.
  • no mouse immunoglobulin was found on the surface of the CD34 + cells.
  • Similar results were observed with the SBA" fraction, which was phenotyped with an anti-SBA polyclonal antibody. Therefore, the immobilized protein is an integral component of the AIS Cellector"' which is not released upon use.
  • FIG. 3A-C shows the enrichment of CFU-GM (solid bars) , BFU-E (hatched bars) and CFU-GEMM (dotted bars) activity in the SBA" and CD34 + cell fractions from bone marrow (3A) , cytokine stimulated peripheral blood (3B) , and normal unstimulated peripheral blood (3C) .
  • each of the three progenitor types were enriched approximately 3-fold after SBA + cell depletion.
  • the CD34 + cells from bone marrow and G-CSF stimulated peripheral blood were, on average, 25-30 fold enriched in CFU-GM and BFU-E progenitors.
  • the isolated CD34 + cells also showed enhanced activity in long-term bone marrow culture.
  • BMMC bone marrow mononuclear cells
  • SBA bone marrow mononuclear cells
  • CD34 + cells were seeded on irradiated allogeneic stroma and the nonadherent cells were assayed weekly for cell number and progenitor activity.
  • cultures seeded with CD34 + cells showed 2- to 50-fold more cells in the culture than did those cultures seeded with the unseparated mononuclear cells.
  • the CD34 device is a sterile culture vessel which supports the propagation of cells.
  • purified CD34 + cells were not dislodged from the device. Instead, cells were cultured directly in a device containing serum-free media with various combinations of growth factors such as IL-1, IL-3 and stem cell factor (SCF) , at 37°C in 5% C0 2 .
  • Half media changes were made twice weekly during the approximately 4-5 weeks of culture. On average, cell expansions were 15- to
  • Figures 5, 6 and 7 depict total number of cell proliferation over time, comparing use of serum- free and serum-based media.
  • Each of Figs. 5, 6 and 7 relate to a different donor population pool (pools 497, 499, and 500, respectively). These populations were derived from pooled bone marrow samples obtained in accordance with standard methodologies.
  • pools 497, 499, and 500 were derived from pooled bone marrow samples obtained in accordance with standard methodologies.
  • Figs. 5-7 superior expansion of cells by use of. serum- free media is noted.
  • the increase in expansion is on the order of 10- to 100-fold.
  • the expansions generally peaked at between 12 to 24 days.
  • the growth factors SCF, IL-1, and IL-3 were utilized in the cultures.
  • Figure 8 depicts the percent of cells from population 499, the population addressed in Fig. 6, as assessed for the percent of the cells which exhibit CD34 positivity over time. As noted in Figure 8, a higher percentage of cells grown in serum-free media expressed the CD34 antigen. The higher expression of CD34 antigen occurred during, at least, the first approximately two weeks of culture.
  • Figure 9 also relates to cellular population 499, the population addressed in Figures 6 and 8.
  • Figure 9 depicts the actual number of CD34 + cells relative to time in culture, as compared between serum-free and serum-based media.
  • CD34 + cells reached their highest number at approximately day 10, at which point there were approximately 1600% more CD34 + cells.
  • day 10 there were approximately 1.6 x 10 6 CD34 + cells.
  • the data in Figure 10 were obtained by use of 25 cm 2 CD34 devices.
  • CD34 + cell populations on the order of at least 4 x 10 7 are obtained.
  • CD34 + progenitor populations obtained by use of a 624 cm 2 device provide for clinical-size samples of progenitor cells from a single culture in a single culture vessel.
  • the increase in CD34 + cells between days 15 and 23 of culture in both serum- free and serum-based media corresponds to false positive readings, as is known to occur in differentiating cells.
  • Figure 10 depicts the percent of cells in a population expressing the CD33 antigen as compared to culture in serum-free and serum-based media.
  • serum-free and serum-based cultures began to express the CD33 antigen over time.
  • Expression of the CD33 antigen corresponds to commitment to a myeloid lineage.
  • the percent of cells expressing CD33 increased from approximately 50% at day 0 to approximately 85% at day 5. This data is notable in that it indicated that the serum-free culture provided expansion of cells in a manner consistent with that of cellular expansion in serum-based cultures. In other words, the serum-free culture had not produced an atypical cellular response.
  • FIGS 11 and 12 depict the total number of myeloid progenitors (CFU-GM's) in the serum-based and serum-free cultures for cellular population 499 (Fig. 11) and population 500 (Fig. 12) .
  • Myeloid progenitors were verified by use of known CFU-GM colony forming assays. As illustrated in each of Figures 11 and 12, there were a greater number of myeloid progenitors (CFU-GM) in cultures with serum-free media. In the culture for population 499 (Fig. 11) , there was an approximately 28-fold expansion at day 15, and an even higher expansion at day 25, approximately 36-fold. In the myeloid progenitor assay on population 500 (Fig.
  • Figures 13 and 14 depict the number of erythroid colonies (BFU-E) present in either serum-free or serum-based cultures, over time. Erythroid progenitors were verified by use of known BFU-E colony forming assays. Figure 13 corresponds to population 499, and Figure 14 corresponds to population 500. As depicted in Figs. 13 and 14, the number of erythroid progenitor colonies (BFU-E) peaked at approximately 7 days for each of the serum-free cultures. There was a substantially greater expansion of erythroid progenitors with the serum-free cultures. Especially notable regarding this data was the finding that the serum-free cultures consistently produced dramatic expansion of the erythroid progenitors.
  • Figures 15-17 illustrate the expansion of peripheral blood stem cells (PBSC) by use of serum-free media; in contrast, the results illustrated in Figs. 5- 14 corresponded to cells obtained from bone marrow.
  • the array of growth factors added to the serum-free media was varied.
  • the time course of cellular proliferation for peripheral blood stem cells corresponded to the time course of cellular proliferation in serum-free media for marrow-derived cells (as illustrated in Figs. 5-7) .
  • Fig. 16 depicts the total number of myeloid progenitor colonies in each peripheral blood stem cell-derived serum-free culture, over time.
  • the myeloid progenitor assay with peripheral blood stem cells corresponded to the myeloid progenitor assays (illustrated in Figs. 11 and 12) for marrow-derived cells. Accordingly, there was an increase in the total number of myeloid progenitors on the order of 10-fold occurring by a time in culture of about 7 to 14 days.
  • Figure 17 depicts the number of erythroid progenitor colonies over time for peripheral blood stem cell-derived serum-free cultures. There was an increase of approximately 100-fold in the number of erythroid progenitors by approximately day 7. This increase corresponded to the bone marrow-derived stem cells as assayed for erythroid progenitor activity (as illustrated in Figs. 13 and 14).
  • Figs. 15-17 indicated that the serum-free culture and expansion method of the invention produced corresponding culture values whether the cells were derived from peripheral blood or bone marrow.
  • the results from this study showed that the method and devices of the invention are used to isolate human CD34 + cells in large scale.
  • the covalent attachment chemistry provided surfaces densely coated with lectin or monoclonal antibody, allowing more efficient capture of target cells as compared to traditional "panning" techniques (Wysocki LJ, Sato VL, "'Panning 1 for Lymphocytes: A Method for Cell Separation," Proc Natl Acad Sci USA (1978) 75:2844- 2849) .
  • the isolated CD34 + cells were of high purity, and retained both short and long term culture hematopoietic activity.
  • methods of the invention relating to CD34 + cell purification take advantage of the binding properties of the soybean agglutinin lectin.
  • Soluble SBA has been utilized successfully in over 400 cell processing procedures to deplete T cells from bone marrow allografts (Reisner Y, et al., "Transplantation for Acute Leukemia with HLA A and B Nonidentical
  • SBA binds many differentiated cell types, yet CD34 + cells almost quantitatively remain in the unbound fraction. Only CD34 + , CD19 + pre B cells are bound by the immobilized lectin. Use of the SBA device improved the purity of the final CD34 + cell fraction 2- to 3-fold. During the use of the CD34 device, the device was vigorously agitated to remove the adherent CD34 + cells. Other methods can also be employed to remove the purified CD34 + cells. For the small 25 cm 2 devices, small magnetic propeller scrapers (post XCellerateTM) are easily used to recover the adherent fraction. In addition, CD34 + cells are removed by incubation of the devices with media without growth factors for 3 hours at 37 ⁇ C in a 5% C0 2 incubator. The adherent CD34 + cells then detach, and are collected by centrifugation.
  • a highly advantageous feature of devices of the invention is that the covalently immobilized protein is an integral component of the device, and is not removed upon the binding or release of the specific target cell population. This feature is particularly important when the clinical application of these purified cells is considered. Due to the antibody-free nature of these cells, repeated infusions of these cells into patients undergoing multiple rounds of chemotherapy is possible, while avoiding the risk of inducing an anti-mouse immunoglobulin antibody response by the human patient.
  • the CD34 device is also a sterile culture device; such devices were successfully used to propagate CD34 + progenitors ex vivo by use of serum-free media.
  • An objective of these stem cell expansions is to culture to a time point when maximal progenitor and CD34 + cell activity can be returned to the patient. Under selected circumstances cultured cells are not provided for the purpose of long-term engraftment, but to provide a bolus of progenitors that minimize or alleviate potentially life-threatening cytopenic periods due to repeated or intensive suppressive drug therapies.
  • a single set of peripheral blood stem cell collections provides enough CD34 + cells and progenitors to yield infusion products for multiple rounds of chemotherapy. This later application is especially important for use during later rounds of treatment, when hematopoietic rebound can be delayed. This system has been scaled-up for clinical application. The data established the feasibility of using this system for large-scale clinical expansions of progenitors, such as CD34 + cells, and demonstrated the superior performance of the serum-free culture conditions for the maintenance and expansion of hematopoietic progenitors.

Abstract

A method for serum-free culture of progenitor cells, whereby a single culture of progenitor cells is capable of producing an expanded population of progenitor cells. The expanded population of progenitor cells is, advantageously, of a size sufficient for use in human therapy. The cells can be obtained by culture and expansion of progenitor cells which are reversibly bound to a solid surface; the surface having covalently bound receptor.

Description

SERUM-FREE CULTURE OF PROGENITOR CELLS
TECHNICAL FIELD The subject field concerns growth of cells with serum- free media. In particular, subject field concerns serum-free culture of CD34+ cells.
BACKGROUND ART
Bone marrow is the source of all hematopoietic precursors. Extensive or complete damage to the marrow, whether as the result of disease within the cells of the marrow itself or as a consequence of therapeutic intervention to treat disease elsewhere in the patient, results in a situation that requires immediate action. Alternatively, even in a situation where the patient's marrow is not completely destroyed, there are instances when it is beneficial to provide a patient with supportive therapy consisting of periodic doses of progenitors (Leonard Sander, M.D. , unpublished data) . For example, regular boosters of cultured progenitor cells should reduce recovery time after courses of radiation and chemotherapy; this hematopoietic augmentation, or transfusion support, should also permit higher doses of cytotoxic therapies to be employed. Failure to respond will lead inevitably to death. Typically, a donor other than the patient provides marrow (an "allogeneic" transplant) in order to repopulate the host's marrow with hematopoietic progenitors. A major drawback to allogeneic transplants, except in the rare instances of identical twin host-donor pairs, is that some of the transplanted cells may mount an immune response against the recipient's cells and tissues, creating "graft-versus- host" disease. The clinical manifestations of "graft- versus-host" disease (GvHD) range from a mild skin irritation, to organ failure and death. Clinically important acute GvHD occurs in 30% to 50% of HLA identical transplants, and in approximately 75% of HLA non-identical transplants. Death results in 20% of all cases of acute or chronic GvHD. The incidence of GvHD increases with the age of the host; many centers will not undertake an allogeneic transplant with patients over the age of 50 (Sullivan, Bone Marrow Transplantation p. 83 (Kluwer Academic Pub., Boston, 1990) ; Scientific American Medicine, section 5
"Hematology," subsection VIII "The Leuke ias," p. 6 (Scientific American, 1993))
Thus, the risk of contracting GvHD is reduced by performing HLA- atching of donor and host. Unfortunately, a successful match is sometimes made meaningless by the reluctance of many prospective marrow donors to undergo the potentially dangerous bone marrow explant procedure which usually requires general anesthesia and which is often accompanied by substantial discomfort. Even where a graft is obtained that is well matched from an HLA perspective, large doses of immune system suppressing drugs must be given to the host to avoid the possibility of GvHD. Immune suppressing drugs frequently have highly undesirable side-effects. One means to avoid GvHD is with autologous circulatory system transplants. GvHD generally does not occur with an autologous transplant. However, the option of autologous transplantation is often unavailable because of tumor contamination of the marrow or the overall health status of the patient will not accommodate a suitable harvesting procedure. For tumor-contaminated marrow, a therapeutic goal is to remove all tumor cells from the marrow that is to be returned to the patient "tumor purging." Tumor purging often reduces the number of hematopoietic precursors. Thus, tumor purging must be balanced against the need to return sufficient numbers of primitive hematopoietic cells to reasonably assure engraftment. Consequently, tumor purging has been used infrequently, and has been problematic when employed.
Hematopoietic and reconstitutive activity in primates is produced by cells bearing the CD34 antigen (Berenson et al . , "Antigen CD34+ Marrow Cells Engraft Lethally Irradiated Baboons," J. Clin. Invest. (1988) 81:951; Civin et al . , "A Hematopoietic Progenitor Cell Surface Antigen Defined by a Monoclonal Antibody Raised Against KG-la Cells, " J. Immunol. (1984) 133:157; Watt et al . , "Distribution and Epitope Analysis of the Cell Membrane Glyco-protein (HPCA-1) Associated with Human Hematopoietic Progenitor Cells," Leukemia (1987) 1:417- 430) . These cells, upon cues from the microenvironment, proliferate to self renew and produce all mature lymphohematopoietic elements. Thus, isolation and expansion of CD34+ cells could have significant therapeutic value.
During many high dose and dose escalation chemotherapy regimens, significant hematopoietic cytopenia occurs, putting patients at risk for infections and other complications. The isolation, expansion and infusion of CD34+ hematopoietic precursors as supportive therapy could significantly reduce cytopenic periods and consequently improve patient outcome. Autologous bone marrow transplantation following high dose chemotherapy and/or radiation has become increasingly used as a treatment for human malignancies. More recently, peripheral blood stem cells have been used exclusively, or in conjunction with bone marrow, to hasten patient engraftment. A further advantage would be afforded to such therapy if CD34+ stem cells could be purified from the peripheral blood stem cell population, thereby significantly reducing the cell volume required for patient reinfusion. It would also be highly desirable if isolation of CD34+ cells could act to purge tumor cells from the engraftable fraction.
As the immune system becomes elucidated, it is increasingly evident that subsets of cells can have relatively narrow ranges of activities. Thus, subsets can be specialized for response to a particular disease, such as neoplasia; infection, viral or bacterial, etc.; response to transplants; and the like. It is therefore of great interest to be able to identify and purify these subsets of cells, not only to understand their action, but also to use these cells for prophylactic and therapeutic purposes. In order to achieve the desired results, it is necessary that substantially pure populations of the desired subset or subsets of cells can be obtained. Furthermore, the cells should be: (1) free of antibodies on their surface, (2) viable, (3) capable of fulfilling their normal function, and (4) responsive to activation by biologicals in the same manner as normal cells in their normal environment. Thus, a method of manipulating circulatory system components (such as cells from the lymphatic system, intrathecal fluid, peripheral blood, or bone marrow) is needed which provides for expansion of progenitor cells, while avoiding the differentiation of such cells. An additional objective for an expansion method is the expansion of the precursor cells for banking and subsequent infusion.
DISCLOSURE OF THE INVENTION The present invention sets forth a method for expanding progenitor cells. Accordingly, a population of progenitor cells is provided; these cells are provided from a cellular source such as the lymphatic system, intrathecal fluid, peripheral blood, or bone marrow. Thereafter, the progenitor cells are cultured in serum-free media. By use of serum-free media, an expanded population of progenitor cells results. Advantageously, the expanded population of progenitor cells is of a population size sufficient for use in clinical applications. The serum-free media can. comprise an array of growth factors. Additionally, the progenitor cells can be attached to a surface. The progenitor cells resulting from expansion of the provided progenitor cells can be attached to the surface or can be released into the serum-free media. A population of cells prepared according to the method of the invention is also set forth.
In another embodiment, a cellular composition is prepared by contacting circulatory system components with soybean agglutinin, wherein the soybean agglutinin (SBA) is bound to a plastic surface. Accordingly, SBA+ cells become bound to the surface, and SBA" cells do not become bound to the surface. The SBA" circulatory system components are harvested. The SBA" circulatory system components are contacted with specific binding proteins, such as monoclonal antibodies, that are specific for the CD34 antigen; wherein the specific binding proteins are bound to a plastic surface. Accordingly, CD34+ cells become bound to the surface, and CD34~ do not become bound to the surface. Thereafter, the bound CD34+ cells are cultured with serum-free media, whereby progeny CD34+ cells are produced. Optionally, bound CD34+ cells are released from the surface. The progeny CD34+ cells can become bound to the surface or can be released into the serum- free media. Should progeny cells be released into the serum-free media, CD34+ cells can be harvested from the media. Alternatively, should CD34+ cells be bound to the surface, they can be harvested by releasing them from the surface. Where the specific binding proteins for the CD34 antigen are monoclonal antibodies, the monoclonal antibody can be from a group of monoclonal antibodies such as ICH3, My 10, 12.8, BEND10, or 8G12. Should the CD34+ cells be released from the surface, they have substantially no specific binding proteins attached thereto. Furthermore, the released cells retain expression of the CD34 antigen, no lag time is involved in awaiting verification of CD34+ status.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 : Purification Scheme for CD34+ Cells.
Figure 2: Two Color Flow Cytometric Analysis. Two color analysis was performed as described herein. A lymphoblastoid gate was used for the bone marrow mononuclear (BMMC) and SBA" cell fractions to eliminate the autofluorescense of high side scatter cells. A total cell gate was used for the CD34+ cell fraction.
Figure 3A-C: Enrichment of Human Hematopoietic Progenitors. Aliquots of the indicated populations from bone marrow (A) , cytokine stimulated peripheral blood (B) , and unstimulated peripheral blood (C) were placed in methylcellulose culture as described in the Materials and Methods. The frequency of CFU-GM, BFU-E and CFU-GM were calculated in each population of cells. The fold enrichment of CFU-GM (solid bars) , BFU-E (hatched bars) and CFU-GEMM (dotted bars) in the SBA" and CD34+ cell populations was then calculated in reference to the mononuclear cell fraction. The fold enrichment in the mononuclear cell fraction was defined as 1. The results shown in these figures are from the same experiments detailed in Table 1.
Figure 4A-C: Long-Term Bone Marrow Culture of CD34+ Cells. Bone marrow mononuclear (°) , SBA" (+) and CD34+ (*) cells were cultured in long term bone marrow culture as described herein. Two hundred thousand cells from each population were loaded into stromal layers and each population was tested in triplicate. Large-scale (624 cm2) AIS Cellector®s CD34 were used to isolate the cells for this experiment. Panel A) , The number of cells in the nonadherent layer was counted at each time point and the % of the original number of seeded cells is plotted as a function of time. Panel B) , The total number of CFU-GM produced at each time point during culture is plotted as a function of culture time. Panel C) , The cumulative production of CFU-GM during long term bone marrow culture is plotted as a function of time. To plot accumulation, the number of- CFU-GM at the start of the culture was defined as 0.
Figure 5: Cellular proliferation, over time, comparing serum-based (■) and serum-free (D) media for bone marrow derived cell population 497.
Figure 6: Cellular proliferation, over time, comparing use of serum-based (■) and serum-free (D) media, for bone marrow derived cell population 499.
Figure 7: Cellular proliferation, over time, comparing use of serum-based (■) and serum-free (O) media, for bone marrow derived cell population 500.
Figure 8: The percent of cells exhibiting CD34 antigen positivity, over time, for cell population 499, comparing serum-based (■) and serum-free (D) media.
Figure 9: The number of CD34+ cells produced, over time, for population 499, comparing use of serum-based (■) and serum-free (D) media.
Figure 10: The percent of CD34+ cell positivity in a cell culture, over time, comparing use of serum-based (■) and serum-free (□) media.
Figure 11: The total number of myeloid precursors (CFU-GM) in a culture of population 499, over time, comparing use of serum-based (■) and serum-free (D) media. Figure 12: The total number of myeloid precursors (CFU-GM) in a culture of population 500, over time, comparing use of serum-based (■) and serum-free (□) media.
Figure 13: The total number of erythroid precursors (BFU-E) in a culture of population 499, over time, comparing use of serum-based (■) and serum-free (D) media.
Figure 14: The total number of erythroid precursors (BFU-E) in a culture of population 500, over time, comparing use of serum-based (■) and serum-free (□) media.
Figure 15: Cellular proliferation, over time, of peripheral blood stem cells using serum-free media comprising various combinations of growth factors: (■) IL-1, IL-3, and stem cell factor; (D) G-CSF, and SCF; (♦) IL-3 and SCF; (0) IL-3, GM-CSF, and SCF; (A) IL-1, IL-3, GM-CSF, and SCF.
Figure 16: The total number of myeloid precursors (CFU-GM) in cultures of peripheral blood stem cells, over time, with use of serum-free media comprising various combinations of growth factors: (■) IL-1, IL-3, and stem cell factor; (D) G-CSF, and SCF; (♦) IL-3 and SCF; ( 0) IL-3, GM-CSF, and SCF; (A) IL-1, IL-3, GM-CSF, and SCF.
Figure 17: The total number of erythroid precursors (BFU-E) for peripheral blood stem cells, over time, with use of media comprising various combinations of growth factors: () comprising IL-1, IL-3, and stem cell factor; (D) G-CSF, and SCF; (♦) IL-3 and SCF; (0) IL-3, GM-CSF, and SCF; (A) IL-1, IL-3, GM-CSF, and SCF. MODES FOR CARRYING OUT THE INVENTION.
Isolation and culture of large quantities of CD34+ human hematopoietic stem/progenitor cells have numerous applications in basic research and human therapy. For instance, isolated CD34+ cells can be used as a purged graft in autologous bone marrow transplantation, minimizing the reinfusion of tumor cells after high-dose chemotherapy or irradiation. CD34+ cells can also be used as a T-cell depleted graft in allogeneic bone marrow transplantation, as a supportive care transfusion product post chemotherapy, or as a target cell in gene therapy protocols. This study sets forth an elegant method used to isolate and culture human CD34+ cells in large-scale, from a variety of sources (such as bone marrow, peripheral blood, intrathecal fluid or the lymphatic system) using "subject devices" (AIS Cellectorws, Applied Immune Sciences, Santa Clara, CA) (for further discussion of the subject device please see copending USSN 07/785,222, filed October 28, 1991) comprising covalently immobilized proteins, for example, soybean agglutinin (SBA) or an anti-CD34 monoclonal antibody such as ICH3 (Watt S, et al. , "Distribution and Epitope Analysis of the Cell Membrane Glycoprotein (HPCA-1) Associated with Human Hematopoietic Progenitor Cells," Leukemia (1987) 1:417-430).
A subject device may take a wide variety of forms. For the most part, the device will be comprised of polystyrene surfaces, where the polystyrene is normally substantially free of cross-linking, less than about 0.5%, usually less than about 0.1%, preferably molded or extruded, so as to have a very smooth surface. Polystyrene surfaces of this nature allow for substantial uniformity of derivatization, where the orientation of the receptor provides for a high level of accessibility of binding sites. It should be understood in referring to receptor, the term is entirely arbitrary. By receptor is intended a molecule which is able to specifically bind to a complementary molecule.
The surface will be derivatized by substitution of the benzene ring of the polystyrene with an electrophilic reagent, particularly by a Friedel-Crafts reaction in a solvent which does not soften or dissolve the polystyrene. For this purpose, sulfolane finds particular application. Relatively mild conditions may be employed and the benzene may be derivatized with a variety of agents, such as nitro, which may be reduced to amino, halomethyl, which may be used to form an amino, hydroxy, or thiol group, or a substituted N- hydroxymethyl acetamide where the substituent is an active halogen or pseudohalogen. A description of the reaction may be found in EPA 88-304516.3.
The derivatized polystyrene surface may then be reacted with the receptor. Under the conditions of derivatization, it is found that a high percentage of the benzenes at the surface are derivatized, so that one may obtain a high density of receptor at the surface. Depending upon the nature of the receptor, various reactions may be performed for bonding the receptor to the surface. Of particular interest is the bonding of proteins to the surface. Proteins can be bonded by contacting the proteins in an aqueous medium with the functionalized/derivatized surface, having active halogen, activated carboxy groups, e.g., esters, or the like, under mild conditions for sufficient time for complete reaction. Any remaining unreacted functional groups may be blocked by using an appropriate small molecule blocking agent. For example, active halogen may be blocked with aliphatic amines, thiols with maleimide, or the like. In some situations, there may be no need to block excess reactive groups, since they will not interfere with the subsequent steps in the process. The surface may then be washed to remove the non-specifically bound receptor and evaluated to insure that appropriate receptor binding has occurred. Substantially homogeneous populations, greater than about 95%, usually 98%, of cells have been achieved by use of subject devices, where the cells may be in a quiescent or activated state. Moreover, cellular compositions may include any cellular population expressing a surface marker (ligand) recognized by the immobilized receptor. Such compositions include cells bearing any of the recognized leukocyte antigens of the CD (cluster designation series) or others recognized by monoclonal antibodies to specific cell surface ligands. Such compositions may include other blood cells, tumor cells, bacteria, viruses, or parasites similarly sharing a common surface marker. Virtually any cell population whose members share a surface ligand recognized by the immobilized receptor can constitute such a cellular composition.
The present method was used to process input samples as small as lxlO7 cells, whereby the resulting population of expanded progenitor cells is expanded to a volume sufficient for use in a full-size clinical bone marrow graft. Accordingly, CD34+ cells isolated using subject devices were up to 98% pure, had no detectable mouse immunoglobulin on their surface, and were functional in in vitro hematopoiesis assays. The CD34+ purity of the cells was determined using phycoerythrin conjugated HPCA-2 anti-CD34 mAb, according to known methodologies. Advantageously, since the subject devices are capable of serving as sterile culture vessels, isolated CD34+ cells can also be directly cultured in the subject devices yielding large expansions of progenitors.
In an alternate embodiment of the expansion of CD34+ cells, CD34+ cells are provided, such as by being removed from a subject CD34 device, and the cells are placed in a cell culture bag comprising Teflon® (e.g., culture bags produced by American Fluoroseal, distributed by West Co., Lionville, PA). Thereafter, one or more growth factors and/or serum-free media are/is provided as described herein.
1. DEVICE VALIDATION a. Synthesis of N-(Hγdroxymethyl) 2-bromoacetamide (HMBA) and generation of the bromoacetamide polystyrene surface (BA-PS) . HMBA is synthesized by conventional means (Leonard et al.. J. Pro. Chem. 50:2480 (1985)) from 2- bromoacetamide, available from commercial sources, in the presence of formalin at pH 10, which provides a 93% yield of the starting reactant, N-(hydroxymethyl) 2- bromoacetamide (HMBA) .
The second step involves the generation of the bromoacetamide polystyrene surface (BA-PS) . In this step, 2M triflic acid and 0.2M HMBA, both in tetramethylene sulfone (sulfolane) , are mixed 1:1 in a volume sufficient to cover the inner surface of a polystyrene vessel being activated. The reaction is allowed to proceed at 27βC for 3 hours, the reaction solution is drained, the device washed with water, followed by ethanol, and the activated polystyrene chambers are air dried. The resulting bromoacetamide polystyrene surface is stable in room air for six (6) months.
b. Cell capture surface preparation, stabilization and sterilization. The next step is the receptor capture (the monoclonal antibody one wishes to covalently bind to the bromoacetamide-polystyrene surface) . The monoclonal antibody of interest is diluted to approximately 0.01 - 0.05 mg/ml in phosphate buffered saline, pH 7.4. The appropriate volume of diluted monoclonal antibody is introduced into the polystyrene chamber and the reaction is allowed to proceed for from about two to twenty, preferably about 2 to 4 hours, at 27°C with rotation. The antibody remaining after the reaction is decanted and can be re-utilized up to 10 times in subsequent coating reactions.
The antibody bound device is then washed ten times with phosphate buffered saline (PBS), pH 7.4, and the surface is then stabilized by the addition of 2% sucrose/0.2% human serum albumin (HSA) , medical grade, to each device. The sucrose/albumin solution is allowed to coat the surface, after which the excess sucrose/HSA solution is decanted and the stabilized polystyrene chambers dried 24-96 hours in a vacuum (<0.10 Torr) at 25βC. After drying, the vacuum is broken with dry nitrogen and the device is flushed with inert, dry gas and capped tightly. The device is sealed and then sterilized. Sterilization is achieved by irradiation with, 2.7 ± 0.2 megarads of electron beam or gamma irradiation. Sterility tests showed that the flasks were sterile after a 14 day in situ media incubation.
c. Density of cell capture surface receptor
A variety of surface functionalization groups were employed and tested for the stability of binding of antibody to the surface. The polystyrene was functionalized using N-(hydroxymethyl)2-haloacetamide, where the halo group was chloro, bromo or iodo; diazoniu and sulfonium. After monoclonal antibody attachment using these surfaces, the flasks were washed 10 times with PBS and once with 1% SDS at 55°C for 14 hours. The plastic surface was then assayed for radioactivity of the labeled monoclonal antibodies and the results expressed as surface density for monoclonal antibody in ng/cm2. The bromoacetamide had a surface density of about 250 ng/cm2 of antibody, more than 2.5 times that achieved by adsorption on an Immulon-211** (Dynatek) surface. While the bromoacetamide provided the highest surface density, the surface density for the other functionalities fell between 200 and about 240 ng/cm2. d. Stability of capture surface receptor.
The stability of the antibody binding was determined by coating the surface with 0.02 mg/ml of (35S) human IgG. The flasks were washed five times with borate- carbonate buffer, once with borate-carbonate buffer for 8 hours and twice with borate-carbonate washes overnight. Aliquots of each wash were saved and assayed for radioactivity. After the second wash, there was no evidence of any antibody leaching. In a second study, using an ELISA assay for the antibody bound to the surface, the results observed showed that the amount of extractable antibody was less than the detection limit of the assay, (7.7 ng/ml) .
e. Cell recovery from a subject capture device and verification that covalently bound monoclonal antibody is retained in the device. Various techniques were employed to recover cells from subject devices. These cell recovery techniques provided quantitative cell recovery, good cellular viability, absence of monoclonal antibody on the surface of the recovered cells and full biological activity for both replication and function. For example, a recovery method termed lymphokine release, was tested with CD8+ cytotoxic T-cells captured from normal human peripheral blood according to the subject method. After decanting of the non-adherent cells and verifying confluent binding by microscopic observation, standard tissue culture media supplemented with recombinant IL-2 (300 units/ml) (usually between 20 and 1000 units/ml) and phytohemagglutinin (PHA:Gibco 0.1 mg/ml) (usually between 0.1 and 5.0 mg/ml) were added. After 48 to 72 hours of culture, the captured CD8+ cells spontaneously detached from the subject flask, leaving all the monoclonal antibody covalently attached to the polystyrene surface.
The captured CD8 cells were shown to be free of surface-bound monoclonal antibody by flow cytometry analysis using fluoresceinated anti-mouse antibody. None of the released CD8+ cells were positive for surface mouse IgG.
Further proof of retention of the antibody by the polystyrene surface was provided by in situ polystyrene blotting studies in which radiolabeled anti-mouse antibody was reacted with the derivatized polystyrene, washed vigorously and the surface either assayed by autoradiography or by direct scintillation counting. In both sets of experiments, the polystyrene surface was fully saturated with bound monoclonal antibody indicating retention of the antibody in the device.
Furthermore, the subject flasks can be re-used for cell capture, by washing in PBS containing 4M MgCl which regenerates the capture surface. Such re-used flasks performed consistently for 4-6 cycles after which repeated washing reduced the bound antibody activity.
Detached cells, recovering by decanting, were expanded numerically in standard tissue culture chambers supplemented with IL-2 and phytohemagglutinin.
Viability by Trypan blue exclusion was shown to be greater than 98% and the recovered, homogeneous cell population could be expanded by two orders of magnitude over a period of about 10 days. A second method for cell recovery, called ultrasonic release, utilized an ultrasonic bath (such as a Crest Ultrasonics model #H-4HT-1014-6) with an output of 40 to 90 kHz sonic output (main frequency at 40 kHz) evenly distributed through a water bath by means of the Crest Vibra-bar. The power supply delivered 500 watts at 40 to 90 kHz. The ultrasonic bath had an immersion tank of 10 x 14 inches, holding a volume of 6 gallons of fluid which contained one liter (0.5" from the tank bottom) for sonication in the subject studies. Immersion tanks of various sizes are commercially available. A capture device containing the bound cells was immersed in the one liter of fluid in the ultrasonic bath and the power supply and power application time experimentally determined. Depending upon the cell phenotype, times and powers varied: For example, CD4+ T-cells: 78% max power, 17 sec; CD8+ T-cells: 30% max power, 20 sec; Leu 19 cells: 75% max power, 10 sec, etc., as is appreciated by one of ordinary skill in the art.
The demonstrate that the cells recovered by sonication were still viable and retained their physiological activity, CD16+ NK-cells were recovered by sonication at maximum power for 15 to 20 seconds. The cells recovered by sonication (1) were greater than 85% viable by Trypan blue exclusion, and (2) were extremely active in a lytic assay routinely utilized to quantitate NK-cell activity.
Once again, to verify that the monoclonal antibodies covalently attached to a subject device were not released onto cells recovered from such devices, flow cytometry was employed . Using flow cytometric analysis, cells recovered by sonication were shown to be free of monoclonal antibody, as were cells recovered by the mitogen/lymphokine drive method described above. Thus, in cells recovered by various methods, the antibody remains behind when the cells are recovered, providing viable, homogeneous, fully functional cells free of monoclonal antibody.
2. ISOLATION OF CD34+ CELLS. a. Basic Scheme. Isolation of CD34+ cells from any human cell source using the subject devices (e.g., AIS Cellectorns, Applied Immune Sciences, Santa Clara, CA) typically involved three steps. In the first step, a sample was depleted of red blood cells (rbc) using either ficoll/hypaque density gradients or gelatin sedimentation. After rbc depletion, the majority of differentiated cells were depleted using an "SBA device" (e.g., AIS Cellector1**- SBA, Applied Immune Sciences, Santa Clara, CA) . The nonadherent, SBA" cells, which were enriched in CD34+ cells, were then loaded into a
"CD34 device" (e.g., AIS Cellector™ CD34, Applied Immune Sciences, Santa Clara, CA) for the purification of CD34+ cells. The nonadherent, CD34", cells were washed from the device, and the adherent CD34+ cells collected by physical agitation.
Human CD34+ cells were isolated from a variety of sources by use of the subject devices. The process for utilizing these devices is outlined in Figure 1. Bone marrow or peripheral blood cells were depleted of red cells using ficoll/hypaque density gradients or gelatin sedimentation. The mononuclear cell preparation was then loaded into an SBA device to bind many differentiated cells such as red cells, B cells, fat cells, fibroblasts, endothelial cells, certain T-cells and tumor cells which normally bind to the soybean lectin. The nonadherent SBA" fraction, which was enriched in CD34+cells, was collected; advantageously, the adherent SBA+ cells were eluted from the devices by incubation at 37βC with 200 mM N-acetylgalactosamine in RPMI for 0.5-3 hours.
After concentration, the SBA" population was subsequently loaded into an CD34 device, for CD34+ cell isolation. Finally, the nonadherent CD34" cells were removed, and the CD34+ cells were harvested, advantageously by physical agitation of the device.
b. Cell Sources. Bone marrow was collected from the iliac crest from consenting volunteers. Normal peripheral blood was collected as a leukopheresis product from consenting volunteers. Aliquots of cytokine-stimulated peripheral blood were collected from either normal volunteers who were stimulated 6 days with G-CSF, or from myeloma patients undergoing peripheral blood stem cell collections after chemotherapy along with G-CSF treatment, as a part of their normal course of therapy.
Alternatively, cells are obtained by use of methods such as counter-flow elutriation, flow cytometry, magnetic beads, affinity chromatography, cellular panning, or agglutination procedures. Antigens used to identify progenitor cells include CD34+, CD38", CD33", Thyl+, HLA-DR dull, rhodamine", HLA-DR", and 7B7". The foregoing antigens and cell types exemplify, but in no way limit, the antigens assessed and the cell types expanded by use of the invention.
c. Preparation of Bone Marrow or Peripheral Blood Mononuclear Cells. To prepare cells for CD34+ cell isolation, mononuclear cells were isolated from bone marrow, growth factor stimulated peripheral blood stem cell preparations, or normal peripheral blood cells using ficoll-hypaque density gradients.
Briefly, the cell sample was diluted 1:5 - 1:10 using Dulbecco's Phosphate Buffered Saline (Ca/Mg free) containing 1 mM EDTA (DPBSE) . Forty mis of this suspension were underlayered with 10 ml of Histopaque
(Sigma 1.077 g/ml) and centrifuged for 20 minutes at 900 g (no brake) . The mononuclear cell layer was collected, diluted 5-fold with DPBSE, and the cells were collected at 900 g for 20 minutes. To eliminate platelets, the cells were subsequently diluted with DPBSE and centrifuged at 450 g for 10 minutes. The cells were resuspended in DPBSE, counted, and used in later separation procedures.
As an alternative procedure to eliminate red blood cells from the input samples, gelatin sedimentation was used. For this procedure, the bone marrow or peripheral blood was directly mixed 1:1 (v:v) with 3% gelatin in DPBS. After a 20-40 minute incubation at room temperature, the white cell supernatant was extracted and diluted 3-5 fold with DPBSE. The white cells were collected at 900 g for 20 minutes, and were ready to use in accordance with the invention.
d. Preparation of the Devices. For the present study, the devices comprised polystyrene vessels containing covalently immobilized protein (AIS
Cellector™, Applied Immune Sciences, Santa Clara, CA) . The SBA devices had a variety of capacities; a 25 cm2 and 150 cm2 SBA device separated up to 2xl07 and 1.2X108 input cells, respectively. A large-scale 3000 cm2 SBA device processed up to 2xl09 cells. The CD34 devices also processed a variety of cell loads. A 25 cm2 device accommodated 2xl07 cells, whereas a large-scale 624 cm2 device processed up to 5xl08 cells.
The devices were primed/prepared by rehydrating the surface with four consecutive washes with DPBSE. The device was shaken vigorously at each wash to remove a protective coating of the surface. (For information concerning the protective coating, please see U.S. Patent No. 5,283,034, issued 01 February 1994.) The last wash was left on the device until use, to protect the immobilized lectin or antibody.
e. Depletion of Mature/Differentiated Cells Using an SBA Device. To remove most mature, differentiated elements, rbc-depleted cells were loaded into an SBA device. For this separation, the cells were incubated at 5xl06 cells/ml in 0.5% human IgG (Gamimune N, Cutter Labs) in DPBSE for 15-20 minutes at room temperature. The 25 cm2 and 150 cm2 SBA devices were then loaded with 4 ml and 25 ml of cells, respectively, containing at maximum 2xl07 and 1.2x10s cells. For the large-scale 3000 cm2 SBA device, up to 2xlθ9 cells were incubated in 50 ml of 0.5% human IgG in DPBSE for 15-20 minutes at room temperature; after incubation, the cells were diluted to 265 ml with DPBSE and loaded directly into a primed 3000 cm2 device. After loading, the SBA devices were incubated at room temperature for one hour. The large-scale 3000 cm2 devices were incubated 30 minutes at room temperature on each of the two opposed, broad sides of the device. To remove the nonadherent SBA" cells, the devices were rocked gently, and the nonadherent cell suspension was collected. The 25 and 150 cm2 devices were then washed two times with DPBSE and the washes were pooled with the first nonadherent fraction. For the 3000 cm2 SBA device, the device was rocked steadily during the collection of the nonadherent cells. When this procedure was followed, subsequent washes of the device were not necessary. The nonadherent cells from all devices were centrifuged at 900 g for 20 minutes, and subsequently resuspended at 5xl06 cells/ml in 0.5% human Ig in DPBSE for processing on CD34 devices.
f. CD34* Cell Selection and Collection on a CD34 Device. After resuspension of the SBA" cells in the human IgG solution, 25 cm2 and 624 a cm2 CD34 devices were loaded with a maximum of 2xl07 and 5xl08 SBA" cells, respectively. The devices were then incubated at room temperature for 60 minutes. To remove the CD34" nonadherent cells, the devices were gently rocked and the fluid was drained. The devices were then washed 2-4 times using DPBSE containing 0.5% human serum albumin (HSA) . After these washes, the 25 cm2 and 624 cm2 devices were filled with 4 ml and 50 ml of DPBSE containing 0.5% HSA, respectively, and the devices were agitated vigorously in the plane of the binding surface, thereby dislodging the CD34+ cells. The dislodged CD34+ cells were drained into centrifuge tubes pre-coated with HSA, and the devices were rinsed twice with DPBSE.
These washes were pooled with the dislodged CD34+ cells in the HSA coated tubes and the CD34+ cell fraction was collected at 900 g for 20 minutes.
3. Serum-Free Culture of Progenitor Cells. Progenitor cells were obtained according to methods known to those of ordinary skill in the art. For example, cells were obtained by use of the subject devices. Additionally, progenitor cells are obtained by use of methods such as counter-flow elutriation, flow cytometry, magnetic beads, affinity chromatography, cellular panning, or agglutination procedures. Use of the subject devices and methods is preferred as it provides a closed device for use in separating and culturing within a single vessel. Antigens used.to identify progenitor cells include CD34+, CD38", CD33", Thyl+, HLA-DR dull, rhodamine", HLA-DR", and 7B7~. The foregoing antigens and cell types exemplify, but in no way limit, the antigens assessed and the cell types expanded by use of the procedure.
Presence of stem cells expressing the above-mentioned antigens are verified by long-term bone marrow culture assay as set forth herein; 4HC resistance assay (Brugger, et al., "J2x Vivo Expansion of Enriched Peripheral Blood CD34+ Progenitor Cells by Stem Cell Factor, Interleukin-IB (IL-1B) , IL-6, IL-3, Interferon- γ, and Erythropoietin," Blood (1993) 81:2579-2584); anti-sense oligonucleotides (Hatzfeld, et al., "Release of Early Human Hematopoietic Progenitors from Quiescence by Anti-Sense Transforming Growth Factor Bl or Rb Oligonucleotides." J. EXP. Med. (1991) 174:925-929); HPPCFC analysis (Srour, et al., "Long-Term Generation and Expansion of Human Primitive Hematopoietic Progenitor Cells In Vitro." Blood (1993) 81:661-669); CFU-blast assay; or, by observing proliferation of cells with a specified, desired phenotype. Furthermore, progenitor cell types, in addition to primitive stem cells, are also cultured and expanded by use of the present method. For example, to culture and expand progenitor cells of the myeloid, megakaryocyte cell lines, precursors expressing the antigens CD61 or CD41b are assayed, cultured and expanded in accordance with the invention.
Obtaining progenitor cells by use of magnetic beads or by affinity chromatography has been used to scale-up cells to clinical size samples. However, such methods of obtaining progenitor cells have disadvantages known to those of ordinary skill in the art. Other methods of obtaining progenitor cells are limited by the fact that the amount of cells obtained thereby is small. Any attempt to culture the cells to a clinical-size sample is generally not possible, since, during the expansion of the cells, they differentiate into non-progenitor cell lines. Ideally, in order to have a sample size sufficient for clinical use, 4-100 x 107 progenitor cells are supplied.
In a preferred method to culture and expand progenitor cells, CD34+ cells were obtained using SBA and CD34 devices. Advantageously, the CD34+ cells adherent to the AIS Cellector1" CD34, were not removed from the device. The use of serum-free media for the expansion of purified CD34+ cells from bone marrow and peripheral blood was then evaluated. By "serum-free" is intended media that does not contain undefined serum components. Preferably, Hematopoietic Stem Cell-SFM media (GibcoBRL, Grand Island, NY) is used.
Accordingly, CD34+ cells were captured on CD34 devices, and cultured directly in the devices at 37°C in 5% C02 with various combinations of growth factors. Typically, growth factors IL-1, IL-3 and stem cell factor were used. For certain experiments, as indicated herein, growth factors such as G-CSF and/or GM-CSF were also added. For the experiments described herein, IL-1, IL-3, GM-CSF and G-CSF were all at 10 ng/ml; SCF was at 50 ng/ml. The cultures were incubated at 37°C at 5% C02. Half media changes were made twice weekly until the termination of a culture.
4. Assays and Determinations. a. Assays and Determinations by Flow Cvtometry. After purification, the CD34+ cells were held overnight at 4°C in DPBS containing 0.5% HSA. To assess final purity, the cells were stained using the FITC or phycoerythrin conjugated HPCA-2 (Becton Dickinson) anti-CD34 monoclonal antibody. FITC and phycoerythrin conjugated antibodies used for evaluation were: control mouse Ig; anti-CD3, clone SK7; anti-CD19, clone 467; anti-CD33, clone P676; anti-CD38, clone HB-7; anti-HLA- DR, clone L243; goat anti-mouse IgG (heavy and light chain) (Becton Dickinson) . These samples were analyzed on a FACScan flow cytometer (Becton Dickinson) . .
b. Assays and Determinations by Methylcellulose Progenitor Colony Assay. Defined numbers of the different cell populations were plated in 1.5 ml of Terry Fox methylcellulose containing PHA-stimulated leukocyte conditioned media. Additionally, for some experiments, this media was supplemented with 50 ng/ml SCF, which produced higher cloning efficiencies in the CD34+ cell fraction. The cells were plated in humidified 6-well culture dishes and incubated at 37°C in 5% C0 . After a number of days, CFU-GM, BFU-E and CFU-GEMM were counted on an inverted microscope.
c. Assays and Determinations bv Long-Term Bone Marrow Culture. To determine viability of long term bone marrow cultures, cultures are initiated on well- established human stromal cultures that were irradiated with 25 Gy according to the methods of Kaplan et al. (Gartner S, Kaplan HS, "Long-term Culture of Human Bone Marrow Cells," Proc Natl Acad Sci USA (1980) 77:4756- 4761) . Within 7 days after irradiation, 2-10xl05 cells are seeded into each irradiated stromal culture flask. The cultures are grown at 37°C in a 5-7% C02 atmosphere. Equal numbers of each different cell population are used to seed the stromal layers. Once per week for the following 6 weeks, nonadherent cells from each cell culture are collected, and an aliquot is plated in methylcellulose assay. The remaining cells from each population are distributed to the original flasks.
5. RESULTS a. Use of SBA Device.
Table 1 shows the recovery of cells in the various SBA" and CD34+ populations from bone marrow, cytokine sti ulated peripheral blood stem cells and normal peripheral blood.
Table 1. Cell Recoveries
% Recovery of Mononuclear Cells
SBA" Fraction CD34+ Fraction
Bone Marrow 20.4 +/- 12.0 0.9 +/- 0.6
(n=26)
Cytokine Stimulated Peripheral Blood 22.0 +/- 6.6 0.8 +/- 0.7 (n=5)
Nonmobilized Normal
Peripheral Blood 15.0 +/- 8.0 0.2 +/- 0.1 (n=3)
Table 1 Legend. The % recovery of mononuclear cells was determined from individual cell counts and was calculated as: # cells in output population// mononuclear cells x 100%. The results are expressed as the mean +/- standard deviation.
On average, 15.0, 22.0 and 20.4% of the mononuclear cells were recovered after depletion of SBA binding cells from normal peripheral blood, cytokine stimulated peripheral blood or bone marrow, respectively. These recoveries are equivalent to those observed in the SBA" fraction after soluble soybean agglutination (manuscript submitted) . After purification of CD34+ cells, on average, 0.9% of the bone marrow mononuclear cells were found in the final stem cell fraction. This recovery is expected, as CD34+ cells comprise approximately 0.5-3.0% of normal bone marrow (Civin, J. Immunol. (1984) 133-157) . From cytokine-sti ulated peripheral blood, on average, 0.8% of the mononuclear cells were collected in the CD34+ cell fraction, whereas normal unstimulated peripheral blood which usually has CD34+ cells levels below the detection limits of flow cytometry produced only 0.2% recoveries.
The CD34+ purities of the different cell fractions produced during the purification procedure were evaluated (Table 2) .
Table 2. Percentage CD34+ Cell Purity
Input
Mononuclear SBA" Cells CD34* Cells Bone Marrow 2.4 +/- 1.8 8.1 +/- 4.2 74.2 +/- 11.6
Cytokine Stimulated Peripheral Blood
Mononuclear
Cells 3.1 +/- 2.0 10.9 +/- 8.6 81.5 +/-
8.9
Normal Peripheral
Blood < 0.2* 0.4 +/- 0.4 43.4 +/-
16.8
Table 2 Legend. The purity was determined using a gate which excluded only debris. These values were collected from the same experiments described in Table 1.
For bone marrow, the mononuclear cell preparation was, on average, 2.4% CD34+. Upon depletion of the SBA+ population, there was a net enrichment of CD34+ cells to 8.1%. On average, 67.5% of the CD34+ cells were recovered in the SBA" fraction, as determined based on standard calculations using information contained in Tables 1 and 2. As was verified by flow cytometry, the majority of those CD34+ cells not recovered in the SBA" fraction were CD34+ CD19+ pre B cells which normally bind SBA. After purification, the final CD34+ cell fraction from bone marrow was an average of 74.2% pure, with a 30% overall recovery of CD34+ cells.
Similar purities were observed when CD34+ cells were isolated from cytokine-stimulated (G-CSF) peripheral blood. Based on standard calculations, the input mononuclear cell fraction had a mean 3.1% frequency of CD34+ cells, which upon depletion of SBA binding cells was enriched to 10.9% with greater than 90% recovery of CD34+ cells. Again, based on standard calculations, the final CD34+ cell fraction from the cytokine-stimulated peripheral blood was highly purified, being 81.5% pure with a 22% overall recovery of the CD34+ cells.
The binding surface of a subject device provides high specificity of binding, even for extremely rare cell populations. For instance, CD34+ cells from normal peripheral blood, which were essentially nondetectable (<0.2%) in the input mononuclear cell fraction, were isolated to 43.4% purity on the devices, representing a greater than 200-fold purification of these CD34+ cells (Table 2) .
To more fully characterize the cells at each step of the purification, two color flow cytometric analysis was performed according to standard methodologies. Figure 2 shows representative two color plots from the mononuclear cell, SBA", and CD34+ cell fractions from bone marrow. CD3+ T cells which were present in the input cell fraction were eliminated from the final CD34+ fraction. Likewise, CD19+ B cells were almost totally eliminated after SBA depletion and were less than 2% of the final CD34+ cell fraction. Particularly evident in the figure is the enrichment of CD34+ cells at both the SBA" and CD34+ steps of the purification process. In the final cell population, approximately 50% and 95% of the CD34+ cells expressed the CD33 and CD38 antigens respectively, suggesting that the purified population contained a mixture of committed and uncommitted CD34+ cells representative of those found in unfractionated bone marrow.
The final CD34+ cell population had no residual mouse immunoglobulin on its surface. In over 50 experiments, the CD34+ cell population, after isolation on the subject devices, was phenotyped with many different anti-mouse immunoglobulin antibodies directed against the heavy, light, Fab and Fc portions of the murine immunoglobulin molecule. In every test, no mouse immunoglobulin was found on the surface of the CD34+ cells. Similar results were observed with the SBA" fraction, which was phenotyped with an anti-SBA polyclonal antibody. Therefore, the immobilized protein is an integral component of the AIS Cellector"' which is not released upon use.
The isolated CD34+ cells not only have high purity, but retained functional hematopoietic activity. Figure 3A-C shows the enrichment of CFU-GM (solid bars) , BFU-E (hatched bars) and CFU-GEMM (dotted bars) activity in the SBA" and CD34+ cell fractions from bone marrow (3A) , cytokine stimulated peripheral blood (3B) , and normal unstimulated peripheral blood (3C) . On average, each of the three progenitor types were enriched approximately 3-fold after SBA+ cell depletion. In addition, the CD34+ cells from bone marrow and G-CSF stimulated peripheral blood were, on average, 25-30 fold enriched in CFU-GM and BFU-E progenitors. In general, 15-20 fold enrichments in CFU-GEMM were observed. It is important to note that in the assays, SCF was not added to the methylcellulose progenitor cultures. When SCF was added, higher cloning efficiencies, especially of the CD34+ cell fraction were observed, resulting in even higher progenitor enrichments (data not shown) .
Substantial hematopoietic activity was also found in the CD34+ cells isolated from nonmobilized (i.e., not stimulated with growth factors) normal peripheral blood. In these experiments, even higher enrichments of CFU-GM, BFU-E and CFU-GEMM were observed due to the very low level of these progenitors in the starting population. On average, 50-, 90- and 38-fold enrichments of CFU-GM, BFU-E, and CFU-GEMM, respectively, were observed after use of the subject process.
The isolated CD34+ cells also showed enhanced activity in long-term bone marrow culture. In these experiments, equal numbers of bone marrow mononuclear cells (BMMC) , SBA", cells or CD34+ cells were seeded on irradiated allogeneic stroma and the nonadherent cells were assayed weekly for cell number and progenitor activity. Figure 4A-C shows the results from one representative experiment (BMMC = 0; SBA" = +; CD34+ = *) . At each time point, cultures seeded with CD34+ cells showed 2- to 50-fold more cells in the culture than did those cultures seeded with the unseparated mononuclear cells. Moreover, those cultures seeded with CD34+ cells, in general, proliferated 2-5 weeks longer than did those cultures seeded with the BMMC population. Similarly, CFU-GM production was much higher in those cultures seeded with CD34+ cells, with the SBA" fraction showing intermediate levels of activity. At every time point, 10- to 1000-fold more colonies were produced by the CD34+ cells (Figure 4B) , yielding an overall 20- to 50-fold greater accumulation of CFU-GM. These results were consistent with the relative purity of the CD34+ cells in each cell population, and indicated that the purified CD34+ cells were viable, and retained complete functionality as measured by both short and long term in vitro hematopoietic assays.
b. Serum-Free Cell Expansion. Advantageously, the CD34 device is a sterile culture vessel which supports the propagation of cells. To study the proliferation of stem cells, purified CD34+ cells were not dislodged from the device. Instead, cells were cultured directly in a device containing serum-free media with various combinations of growth factors such as IL-1, IL-3 and stem cell factor (SCF) , at 37°C in 5% C02. Half media changes were made twice weekly during the approximately 4-5 weeks of culture. On average, cell expansions were 15- to
25-fold and 50- to 100-fold through the first and second weeks of culture, respectively, and were 5-fold higher than those in identical cultures containing fetal calf serum-based media. Cell numbers plateaued after 3-4 weeks in culture. Cultures maintained in serum-free conditions produced 7- to 10-fold expansions of CFU-GM, and 50- to 70-fold increases in BFU-E during the first week in culture. In contrast, only 3-fold expansions of CFU-GM, and no proliferation of BFU-E were observed with identical cultures containing fetal calf serum.
Additionally, the maintenance of CD34+ cells in these cultures was enhanced with the use of serum-free media; greater than 100% of the original CD34+ cells were present, even after 7-10 days in culture. The data represented by Figures 5-17 was derived from use of 25 cm2 CD34 devices (T-25 AIS Cellector™ CD34, Applied Immune Sciences, Santa Clara, CA) . For the experiments represented in these figures, the devices were not loaded to capacity with attached CD34+ cells. Accordingly, the ultimate numerical values of cell number obtainable in accordance with the invention are higher than the specific numbers supplied herein (data not shown) .
Figures 5, 6 and 7 depict total number of cell proliferation over time, comparing use of serum- free and serum-based media. Each of Figs. 5, 6 and 7 relate to a different donor population pool (pools 497, 499, and 500, respectively). These populations were derived from pooled bone marrow samples obtained in accordance with standard methodologies. In each of
Figs. 5-7, superior expansion of cells by use of. serum- free media is noted. The increase in expansion is on the order of 10- to 100-fold. The expansions generally peaked at between 12 to 24 days. As noted above, the growth factors SCF, IL-1, and IL-3 were utilized in the cultures.
Figure 8 depicts the percent of cells from population 499, the population addressed in Fig. 6, as assessed for the percent of the cells which exhibit CD34 positivity over time. As noted in Figure 8, a higher percentage of cells grown in serum-free media expressed the CD34 antigen. The higher expression of CD34 antigen occurred during, at least, the first approximately two weeks of culture.
Figure 9 also relates to cellular population 499, the population addressed in Figures 6 and 8. Figure 9 depicts the actual number of CD34+ cells relative to time in culture, as compared between serum-free and serum-based media. As a result of the increased percentage of cells in the serum-free culture that exhibit the CD34 antigen, and the rate of overall cellular expansion in the serum-free culture, the number of CD34+ cells was substantially greater in the serum- free culture. As noted in Figure 9, CD34+ cells reached their highest number at approximately day 10, at which point there were approximately 1600% more CD34+ cells. At day 10 there were approximately 1.6 x 106 CD34+ cells. The data in Figure 10 were obtained by use of 25 cm2 CD34 devices. By use of a 624 cm2 device, CD34+ cell populations on the order of at least 4 x 107 are obtained. Advantageously, CD34+ progenitor populations obtained by use of a 624 cm2 device provide for clinical-size samples of progenitor cells from a single culture in a single culture vessel.
As shown in Fig. 9, the increase in CD34+ cells between days 15 and 23 of culture in both serum- free and serum-based media corresponds to false positive readings, as is known to occur in differentiating cells.
Figure 10 depicts the percent of cells in a population expressing the CD33 antigen as compared to culture in serum-free and serum-based media. As depicted in Figure 10, serum-free and serum-based cultures began to express the CD33 antigen over time. Expression of the CD33 antigen corresponds to commitment to a myeloid lineage. Accordingly, in both serum-based and serum-free cultures, the percent of cells expressing CD33 increased from approximately 50% at day 0 to approximately 85% at day 5. This data is notable in that it indicated that the serum-free culture provided expansion of cells in a manner consistent with that of cellular expansion in serum-based cultures. In other words, the serum-free culture had not produced an atypical cellular response.
Figures 11 and 12 depict the total number of myeloid progenitors (CFU-GM's) in the serum-based and serum-free cultures for cellular population 499 (Fig. 11) and population 500 (Fig. 12) . Myeloid progenitors were verified by use of known CFU-GM colony forming assays. As illustrated in each of Figures 11 and 12, there were a greater number of myeloid progenitors (CFU-GM) in cultures with serum-free media. In the culture for population 499 (Fig. 11) , there was an approximately 28-fold expansion at day 15, and an even higher expansion at day 25, approximately 36-fold. In the myeloid progenitor assay on population 500 (Fig. 12) , the peak incidence in the number of myeloid progenitor cultures occurred at approximately day 6. In general, by use of serum-free media, increases in myeloid progenitors were on the order of 2- to 30-fold. In some experiments, CFU-GM colonies were increased up to 100-fold (data not illustrated) .
Figures 13 and 14 depict the number of erythroid colonies (BFU-E) present in either serum-free or serum-based cultures, over time. Erythroid progenitors were verified by use of known BFU-E colony forming assays. Figure 13 corresponds to population 499, and Figure 14 corresponds to population 500. As depicted in Figs. 13 and 14, the number of erythroid progenitor colonies (BFU-E) peaked at approximately 7 days for each of the serum-free cultures. There was a substantially greater expansion of erythroid progenitors with the serum-free cultures. Especially notable regarding this data was the finding that the serum-free cultures consistently produced dramatic expansion of the erythroid progenitors. Whereas it might be possible to obtain expansion of erythroid precursors with a serum- based culture, extensive, worker-intensive amounts of serum-based media screenings would need to take place. Advantageously, it was determined that the serum-free media produced erythroid progenitor increase on a consistent basis.
Figures 15-17 illustrate the expansion of peripheral blood stem cells (PBSC) by use of serum-free media; in contrast, the results illustrated in Figs. 5- 14 corresponded to cells obtained from bone marrow. In Figures 15-17, the array of growth factors added to the serum-free media was varied. Notably, as illustrated in Fig. 15, the time course of cellular proliferation for peripheral blood stem cells corresponded to the time course of cellular proliferation in serum-free media for marrow-derived cells (as illustrated in Figs. 5-7) .
Fig. 16 depicts the total number of myeloid progenitor colonies in each peripheral blood stem cell-derived serum-free culture, over time. Once again, the myeloid progenitor assay with peripheral blood stem cells corresponded to the myeloid progenitor assays (illustrated in Figs. 11 and 12) for marrow-derived cells. Accordingly, there was an increase in the total number of myeloid progenitors on the order of 10-fold occurring by a time in culture of about 7 to 14 days. Figure 17 depicts the number of erythroid progenitor colonies over time for peripheral blood stem cell-derived serum-free cultures. There was an increase of approximately 100-fold in the number of erythroid progenitors by approximately day 7. This increase corresponded to the bone marrow-derived stem cells as assayed for erythroid progenitor activity (as illustrated in Figs. 13 and 14).
Accordingly, the information presented in Figs. 15-17 indicated that the serum-free culture and expansion method of the invention produced corresponding culture values whether the cells were derived from peripheral blood or bone marrow.
5. DISCUSSION
The results from this study showed that the method and devices of the invention are used to isolate human CD34+ cells in large scale. The covalent attachment chemistry provided surfaces densely coated with lectin or monoclonal antibody, allowing more efficient capture of target cells as compared to traditional "panning" techniques (Wysocki LJ, Sato VL, "'Panning1 for Lymphocytes: A Method for Cell Separation," Proc Natl Acad Sci USA (1978) 75:2844- 2849) . The isolated CD34+ cells were of high purity, and retained both short and long term culture hematopoietic activity. In previous studies we have also demonstrated that the system can readily be utilized for tumor purging (Lebkowski, et al., "Rapid Isolation of Human CD34 Hematopoietic Stem Cells - Purging of Human Tumor Cells," Transplant (1992) 53:1011-1019) Use of an SBA device alone produced a one log depletion in tumor cells, whereas a purification procedure utilizing SBA and CD34 devices yielded a greater than 3 log reduction in tumor burden.
Advantageously, methods of the invention relating to CD34+ cell purification take advantage of the binding properties of the soybean agglutinin lectin. Soluble SBA has been utilized successfully in over 400 cell processing procedures to deplete T cells from bone marrow allografts (Reisner Y, et al., "Transplantation for Acute Leukemia with HLA A and B Nonidentical
Parental Marrow Cells," Lancet (1981) 2:327-340). SBA binds many differentiated cell types, yet CD34+ cells almost quantitatively remain in the unbound fraction. Only CD34+, CD19+ pre B cells are bound by the immobilized lectin. Use of the SBA device improved the purity of the final CD34+ cell fraction 2- to 3-fold. During the use of the CD34 device, the device was vigorously agitated to remove the adherent CD34+ cells. Other methods can also be employed to remove the purified CD34+ cells. For the small 25 cm2 devices, small magnetic propeller scrapers (post XCellerate™) are easily used to recover the adherent fraction. In addition, CD34+ cells are removed by incubation of the devices with media without growth factors for 3 hours at 37βC in a 5% C02 incubator. The adherent CD34+ cells then detach, and are collected by centrifugation.
A highly advantageous feature of devices of the invention is that the covalently immobilized protein is an integral component of the device, and is not removed upon the binding or release of the specific target cell population. This feature is particularly important when the clinical application of these purified cells is considered. Due to the antibody-free nature of these cells, repeated infusions of these cells into patients undergoing multiple rounds of chemotherapy is possible, while avoiding the risk of inducing an anti-mouse immunoglobulin antibody response by the human patient.
Advantageously, the CD34 device is also a sterile culture device; such devices were successfully used to propagate CD34+ progenitors ex vivo by use of serum-free media.
An objective of these stem cell expansions is to culture to a time point when maximal progenitor and CD34+ cell activity can be returned to the patient. Under selected circumstances cultured cells are not provided for the purpose of long-term engraftment, but to provide a bolus of progenitors that minimize or alleviate potentially life-threatening cytopenic periods due to repeated or intensive suppressive drug therapies. As appreciated by one of ordinary skill in the art, a single set of peripheral blood stem cell collections provides enough CD34+ cells and progenitors to yield infusion products for multiple rounds of chemotherapy. This later application is especially important for use during later rounds of treatment, when hematopoietic rebound can be delayed. This system has been scaled-up for clinical application. The data established the feasibility of using this system for large-scale clinical expansions of progenitors, such as CD34+ cells, and demonstrated the superior performance of the serum-free culture conditions for the maintenance and expansion of hematopoietic progenitors.
All publications and patent applications cited in this specification are incorporated by reference herein as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the invention. It is understood that the invention is solely limited by the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A method for expanding progenitor cells, said method comprising steps of: providing a starting population of progenitor cells obtained from a cellular source selected from the group consisting of peripheral blood, lymphatic system tissues, and bone marrow; culturing said cells in serum-free media, whereby an expanded population of progenitor cells results, the resulting population being of greater number than the starting population.
2. The method of claim 1 wherein the progenitor cells are CD34+ cells which have substantially no specific binding proteins attached thereto.
3. The method claim 1 wherein the culturing step comprises use of a growth factor.
4. The method of claim 3 wherein the growth factor comprises IL-3, stem cell factor, GM-CSF, or IL-1.
5. The method of claim 1, wherein the expanded population comprises sufficient progenitor cells for use in mammalian therapy.
6. The method of claim 1, wherein the resulting population comprises at least 3 x 107 progenitor cells.
7. The method of claim 1, further comprising a step of attaching the starting progenitor cells to a surface.
8. The method of claim 1 wherein the serum-free media is Hematopoietic Stem Cell-SFM.
9. A population of cells prepared according to the method of claim 1.
10. The cell population of claim 9 wherein the population comprises sufficient progenitor cells for use in mammalian therapy.
11. The cell population of claim 9, wherein the cell population comprises at least 3 x 107 progenitor cells.
12. A method for preparing a cellular composition, said method comprising: contacting circulatory system components, the circulatory system components comprising cells, with soybean agglutinin wherein said soybean agglutinin is covalently bound to a plastic surface, whereby SBA+ cells become bound to said surface and SBA" cells do not become bound to said surface; harvesting the SBA" circulatory system cells; contacting the SBA" circulatory system cells with specific binding proteins that are specific for CD34, wherein said specific binding proteins are covalently bound to a plastic surface, whereby CD34+ cells become bound to said surface and CD34" cells do not become bound to said surface; and culturing said bound CD34+ cells with serum-free media, whereby progeny CD34+ cells are produced.
13. The method of claim 12, further comprising a step of releasing the bound CD34+ cells from said surface, whereby the releasing step releases cells that have substantially no specific binding proteins attached thereto.
14. The method of claim 12 wherein progeny cells are released into the media.
15. The method of claim 14, further comprising a step of harvesting the CD34+ cells from the media.
16. The method of claim 12 wherein the specific binding proteins comprise monoclonal antibodies.
17. The method of claim 16 wherein the monoclonal antibodies are bound to the plastic surface by an acetamide linkage.
18. The method of claim 16 wherein the monoclonal antibody is specific for CD34.
19. The method of claim 18 wherein the monoclonal antibody is ICH3.
20. A substantially homogenous population of cells prepared according to the method of claim 12.
21. The method of claim 12 wherein the serum-free media is Hematopoietic Stem Cell-SFM.
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