WO1998055597A1 - Procede de mise en culture de cellules - Google Patents

Procede de mise en culture de cellules Download PDF

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Publication number
WO1998055597A1
WO1998055597A1 PCT/AU1998/000428 AU9800428W WO9855597A1 WO 1998055597 A1 WO1998055597 A1 WO 1998055597A1 AU 9800428 W AU9800428 W AU 9800428W WO 9855597 A1 WO9855597 A1 WO 9855597A1
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cells
haemopoietic
cell
mammalian
stromal
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PCT/AU1998/000428
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English (en)
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Helen Christine O'neill
Keping Ni
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The Australian National University
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Priority to AU77513/98A priority Critical patent/AU7751398A/en
Publication of WO1998055597A1 publication Critical patent/WO1998055597A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1394—Bone marrow stromal cells; whole marrow

Definitions

  • the present invention relates generally to a cell culture process and more particularly to a method of developing dendritic cells. Even more particularly the present invention provides a method of developing precursor dendritic cells.
  • the dendritic cells ofthe present invention are useful ter alia as targets for gene therapy or immunotherapy of cancer, infectious disease, autoimmunity and in tumour therapy.
  • HSC haemopoietic stem cells
  • the HSC's can generate all blood cell components including erythroid, myeloid, lymphoid, granulocytic and dendritic cell lineages (Spangrude G et al. 1988). Heterogeneity amongst HSC is consistent with the existence of lineage restricted progenitor cells (Flemming W. et al. 1993). Definition of these progenitors is fundamentally important to the planning of lineage-specific immunotherapy or gene therapy.
  • DC represents an apparent "lineage” of reticular, interdigitating cells with a common “scavenger” and “stimulatory” function in the immune system. They express a common phenotype, morphology and antigen presenting capacity As “professional” APC, they are located at sites of antigen exposure (Stingl, G etal. 1995 and Steinman, R 1991) where they very efficiently internalise, process and present soluble antigen They express and use the mannose receptor for antigen uptake and also take up soluble molecules through macropinocytosis (Sallusto, F et al. 1995). They can present endogenous and autologous antigens to T cells (Stockinger, B. et al.
  • Dendritic cells are derived from HSC's but the exact process of their development from precursors and the distinction between dendritic cells at different stages of development is poorly understood (Reid et al, 1990) Mature dendritic cells do not divide in vitro (Bowers & Berkowitz, 1986) Dendritic cells precursors have been isolated from blood (Reid et al, 1990; Inaba et al, 1992a), bone marrow (Reid et al, 1990; Inaba et al, 1993b), afferent lymph (Pugh et al, 1983, Fossum, 1989), thymus (Wu et al, 1991) and liver (Lu et al, 1994)
  • Dendritic cells represent only a trace cell population in tissues and have been shown to be short- lived in vivo (Fossum, 1989) Studies on the properties and functional maturation of dendritic cells have been limited by the scarcity of cells and by the failure of dendritic cells to survive beyond a few days in culture (Steinman & Cohn, 1974) They do not replicate in vitro (Fossum, 1989) .
  • the dendritic cell lineage has not been well characterised because of the difficulty in isolating pure populations and sufficient numbers of these rare cells to perform functional and developmental studies Accordingly, there is considerable controversy with respect to the relationship, if any, between dendritic cells located in various tissue sites.
  • the inventors used in vitro long term stromal culture, in the absence of exogenous growth factors, to support the proliferation and differentiation of stem cells to cells with dendritic cell properties.
  • Said cells have been characterised as typical dendritic cells sharing characteristics of morphology, phenotype and a potent capacity of T cell stimulation.
  • one aspect of the present invention contemplates a method of supporting the viability, proliferation and/or differentiation of mammalian dendritic cells, said method comprising cultuiing a mammalian haemopoietic cell population wherein said haemopoietic cell population forms a stromal cell layer which supports dendritic cells
  • a stromal cell layer which "supports" a specific cell type (e g a dendritic cell or a stem cell) should be understood to describe the development of said specific cell type from an adherent or non-adherent haemopoietic cell wherein the viability, proliferation and/or differentiation of said specific cell type is maintained and/or induced by growth factors or other molecules produced by said stromal cell layer and/or by cell/cell contact between said specific cell type and said stromal cell layer.
  • a specific cell type e g a dendritic cell or a stem cell
  • dendritic cells should be read as including reference to dendritic cells at any differentiative stage of development, cells exhibiting dendritic cell morphology, phenotype or functional activity and mutants or variants thereof
  • Variants include, but are not limited to cells exhibiting some but not all of the morphological or phenotypic features or functional activities of dendritic cells at any differentiative stage of development
  • Mutants include but are not limited to dendritic cells which are transgenic wherein said transgenic cells are engineered to express one or more genes such as genes encoding antigens, immune modulating agents, cytokines or receptors.
  • the morphological features of dendritic cells may include, but are not limited to, long cytoplasmic processes, dendrites, irregularly shaped membrane (although round cells are also observed) or pseudopodia
  • Phenotypic characteristics may include, but are not limited to expression of CD1 lc, MHC Class II, NLDC-145, N418 or 33D1.
  • Functional activity includes, but is not limited to, the capacity to internalise antigens, re-expressing peptides of said antigens in association with MHC Class I and II molecules and presenting said peptides to T cells.
  • the expression of particular morphological, phenotypic and functional features will vary according to the differentiative state ofthe dendritic cells.
  • dendritic cell precursors are a most effective antigen presenting cell
  • Expression of particular morphological, phenotypic and functional features may also vary between different populations of dendritic cells, such as dendritic cells arising from different cell lineages.
  • dendritic cells such as dendritic cells arising from different cell lineages.
  • lymphoid-like dendritic cells vary from myeloid-like dendritic cells.
  • the present invention contemplates a method of supporting the viability, proliferation and/or differentiation of mammalian precursor dendritic cells, said method comprising cultuiing a mammalian haemopoietic cell population wherein said haemopoietic cell population forms a stromal cell layer which supports precursor dendritic cells
  • haemopoietic cell population refers to a population of cells which is derived from haematolymphoid tissue or which comprises multipotential, haemopoietic, stromal, myeloid or lymphoid precursor cells. Examples include but are not limited to cells isolated from the bone marrow, spleen, lymph node, thymus or blood. Some or all ofthe haemopoietic cell population may also be transgenic in that said cells may be engineered to express one or more genes such as genes encoding antigens, immune modulating agents, cytokines or receptors.
  • mammal is the adjectival form of "mammal” and includes humans, primates, livestock animals (e.g. horses, cattle, sheep, pigs, donkeys), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs), companion animals (e g. dogs, cats) and captive wild animals (e g kangaroos, deer, foxes).
  • livestock animals e.g. horses, cattle, sheep, pigs, donkeys
  • laboratory test animals e.g. mice, rats, rabbits, guinea pigs
  • companion animals e g. dogs, cats
  • captive wild animals e g kangaroos, deer, foxes.
  • the mammal is a human or laboratory test animal. Even more preferably the mammal is a human.
  • cultures can be established from disassociated whole tissues, using medium supplemented with 10% v/v FCS and 2-mercaptoethanol.
  • the stromal layer is a mixture of cell types which, depending on the cell source, may include one or more of fibroblasts, endothelial cells, macrophages, scattered-adherent cells or epithelial cells. For example, cultures established from spleen cells achieve the growth of a stromal layer within four weeks of initial culture. Mature cells die off during the first two weeks of culture.
  • the stromal layer present in spleen cultures is a monolayer of cell types including fibroblasts, endothelial cells, isolated fixed macrophages and dendritic cells.
  • the initial stages in the development ofthe stromal monolayer are characterised by slowly dividing fibroblasts and endothelial cells. These cells form a connecting network within about two weeks of initiation of culture and the stromal layer first becomes confluent within about three weeks. Scattered macrophages are also present at this time and very small stem cells are obvious.
  • the present invention contemplates a method of supporting the viability, proliferation and/or differentiation of mammalian dendritic cells, said method comprising culturing mammalian spleen cells wherein said spleen cells form a stromal cell layer which supports dendritic cells.
  • the dendritic cells are precursor dendritic cells
  • the present invention contemplates a method of supporting the viability, proliferation and/or differentiation of mammalian precursor dendritic cells, said method comprising culturing mammalian spleen cells wherein said spleen cells form a stromal layer which supports precursor dendritic cells.
  • Stem cells may be multipotential or committed.
  • a multipotential stem cell is one which can commit to any cell lineage while a committed stem cell is one which has committed to developing as a cell of a particular linage or as a particular cell type.
  • a stem cell committed to developing as a cell of a particular lineage may be committed to developing as a lymphoid or myeloid cell while a stem cell committed to developing as a specific cell type may be committed to developing for example as a monocyte, dendritic cell, T cell or B cell
  • the presence of stem cells can be confirmed utilising, for example, colony forming assays, which confirm colony forming capacity, or utilising animals to confirm long term re-constituting capacity
  • the present invention contemplates a method of supporting the viability and/or proliferation of mammalian stem cells, said method comprising culturing a mammalian haemopoietic cell population wherein said haemopoietic cell population forms a stromal cell layer which supports stem cells.
  • the haemopoietic cell population is spleen or bone marrow and even more preferably spleen.
  • the present invention contemplates a method of supporting the viability and/or proliferation of mammalian stem cells, said method comprising culturing mammalian spleen cells wherein said spleen cells form a stromal cell layer which supports stem cells.
  • a related aspect of the present invention contemplates a method of producing a mammalian stromal cell culture said method comprising culturing a mammalian haemopoietic cell population in the presence of serum proteins for a time and under conditions sufficient to support the viability, proliferation and/or differentiation of dendritic cells and/or stem cells from said haemopoietic cell population.
  • serum proteins includes, but is not limited to, fractionated, unfractionated, purified and unpurified serum derived proteins or functional derivatives or chemical equivalents thereof.
  • serum proteins are foetal calf serum
  • the present invention also encompasses a method of producing a stromal cell culture comprising culturing a haemopoietic cell population in the presence of serum proteins and one or more additives or functional derivatives or chemical equivalents thereof
  • Said additives include, but are not limited to vitamins, amino acids and sugars
  • said additives may be glucose, folic acid, L-asparagine and L-arginine
  • Additives may also include cytokines such as GM- CSF, G-CSF, M-CSF, IL-3, TNF- ⁇ , IL-1, IL-6, IL-7, Flt3-L and SCF
  • Said factors may be added to the cultures alone or in combination to expand the number of dendritic cells produced at any time during culture or to expand a population of cells with the characteristics including those of stem cells or myeloid cells The same factors or combinations can be added to the non- adherent cells collected from these cultures to modify their function or number.
  • Dendritic cells, stem cells and stromal cells can be generated from haemopoietic cells which have been overlayed onto a growth supporting stromal cell layer Said stromal cell layer provides a matrix which supports the development of dendritic cells, stem cells and stromal cells.
  • another aspect ofthe present invention contemplates a method of supporting the viability, proliferation and /or differentiation of mammalian dendritic cells and/or stromal cells said method comprising co-culturing a mammalian haemopoietic cell population with a mammalian stromal cell layer wherein said dendritic cells and/or stromal cells develop from said haemopoietic cell population.
  • co-culture encompasses the co-culturing of two or more non-adherent populations of cells, adherent and non-adherent populations of cells or two or more adherent populations of cells.
  • said co-culture is of a non-adherent haemopoietic stem cell population and an adherent stromal cell layer.
  • the present invention contemplates a method of supporting the viability, proliferation and/or differentiation of mammalian dendritic cells and/or stromal cells said method comprising co-culturing a non-adherent mammalian haemopoietic cell population with an adherent mammalian stromal cell layer wherein said dendritic cells and/or stromal cells develop from said haemopoietic cell population
  • Said stromal cell layer is constituted by one or more phenotypes of stromal cells. These include, but are not limited to, fibroblasts, endothelial cells, macrophages, and epithelial cells. Examples of sources of stromal cells include but are not limited to haematolymphoid tissue such as spleen, thymus, blood, bone marrow or lymph node or stroma derived from primary long term cultures and mutants thereof, which have been depleted of non-adherent cells "Mutants" include but are not limited to stromal cells which are transgenic wherein said transgenic cells are engineered to express one or more genes such as genes encoding antigens, immune modulating agents, cytokines and receptors. Preferably said stromal cells are spleen derived
  • haemopoietic cell population is spleen or bone marrow derived and most preferably bone marrow derived.
  • the present invention contemplates a method of supporting the viability, proliferation and/or differentiation of mammalian dendritic cells and/or stromal cells said method comprising co-culturing a non-adherent mammalian bone marrow population with an adherent mammalian spleen cell layer wherein said dendritic cells and/or stromal cells develop from said bone marrow cell population
  • the present invention contemplates a method of supporting the viability and/or proliferation of mammalian stem cells said method comprising co-culturing a mammalian haemopoietic cell population with a stromal cell layer wherein said stem cells develop from said haemopoietic cell population.
  • said co-culture is of a non-adherent haemopoietic cell population and an adherent stromal cell layer.
  • said stromal cell are spleen derived.
  • haemopoietic cell population is spleen or bone marrow derived and most preferably bone marrow derived
  • the present invention contemplates a method of supporting the viability and/or differentiation of mammalian stem cells said method comprising co-culturing a non-adherent mammalian bone marrow population with an adherent mammalian spleen cell layer wherein said stem cells develop from said haemopoietic cell population.
  • the process of the present invention may be "syngeneic", “allogeneic” or “xenogeneic” with respect to the individuals within an animal species from which haemopoietic cells and stromal cells are isolated
  • a “syngeneic” process means that the individual from which the haemopoietic cells are derived has the same MHC genotype as the origin ofthe stromal cells.
  • murine haemopoietic cells are overlayed onto a murine stromal cell layer
  • An "allogeneic" process is where the haemopoietic cells are from an MHC-incompatible individual from which the stromal cells are derived
  • Balb/C derived haemopoietic cells are overlayed onto a CBA derived stromal cell layer.
  • a "xenogeneic” process is where the haemopoietic cells are from a different species to that from which the stromal cells are derived.
  • human derived haemopoietic cells are overlayed onto a murine derived stromal layer
  • Another aspect of the present invention contemplates a method of producing a haemopoietic cell/stromal cell co-culture said method comprising depleting said stromal cells of non-adherent cells, irradiating said stromal cells, overlaying onto said stromal cells a single cell suspension of haemopoietic cells and culturing said cells in the presence of serum proteins for a time and under conditions sufficient for dendritic cells, stem cells and stromal cells to develop from said haemopoietic cell population.
  • said serum proteins are foetal calf serum.
  • the present invention also encompasses a method of producing a haemopoietic cell/stromal cell co-culture comprising co-culturing said haemopoietic cell population and stromal cells in the presence of serum proteins and additives or functional derivatives or chemical equivalents thereof.
  • Said additives includes, but are not limited to vitamins, amino acids and sugars.
  • said additives may be glucose, folic acid, L-asparagine and L-arginine
  • the stromal cell layer ofthe present invention secretes multiple factors Culturing ofthe stromal cell conditioned medium with a haemopoietic cell population supports the development of dendritic cells and stem cells.
  • another aspect of the present invention contemplates a cell culture supernatant comprising tissue culture supernatant harvested from a stromal cell layer cultured in accordance with the methods of the present invention wherein said cell culture supernatant supports the viability, proliferation and/or differentiation of mammalian dendritic cells, stem cells and/or stromal cells
  • cell culture supernatant should be read as including reference to cell culture supernatant derived from cultures of adherent and/or non-adherent haematolymphoid tissue and includes functional derivatives and chemical equivalents thereof.
  • “Functional derivatives” include, but are not limited to fractions, homologs, analogs, mutants and variants having the functional activity of supporting the viability, proliferation and/or differentiation of dendritic cells, stem cells and/or stromal cells. This includes functional derivatives from natural or recombinant sources "Chemical equivalents" can act as a functional analog of said cell culture supernatant.
  • Said chemical equivalents may be chemically synthesised or may be detected following, for example, natural product screening
  • Said cell culture supernatant comprises a combination of proteinaceous and non-proteinaceous molecules which exhibit haemopoietic functional activity, for example, IL-3 and IL-6.
  • Another aspect of the present invention contemplates cell culture molecules comprising molecules harvested from stromal cell layers cultured in accordance with the methods of the present invention wherein said cell culture molecules support the viability, proliferation and/or differentiation of mammalian dendritic cells, stem cells and/or stromal cells
  • cell culture molecules should be read as including reference to molecules derived from cultures of adherent and/or non-adherent haematolymphoid tissue and includes functional derivatives and chemical equivalents thereof
  • “Functional derivatives” include, but are not limited to fractions, homologs, analogs, mutants and variants having the functional activity of supporting the viability, proliferation and/or differentiation of dendritic cells, stem cells and/or stromal cells This includes functional derivatives from natural or recombinant sources
  • Chemical equivalents can act as a functional analog of said cell culture molecules.
  • Said chemical equivalents may be chemically synthesised or may be detected following, for example, natural product screening
  • Said cell culture molecules may be proteinaceous or non-proteinaceous molecules, for example, cell surface receptors, cell surface adhesion molecules or extra cellular matrix molecules
  • Said molecules are useful for inducing differentiation or proliferation or maintaining viability of stem cells, dendritic cells or stromal cells
  • Another aspect of the present invention contemplates a method of producing dendritic cells and/or stem cells, said method comprising culturing a haemopoietic cell population for a time and under conditions sufficient to produce dendritic cells and/or stem cells Said "conditions" include culturing said haemopoietic cells either in the presence of stromal cells or only in the presence of growth factors or other molecules produced by said stromal cells.
  • a further aspect ofthe present invention contemplates the use of a mammalian haemopoietic cell population in the manufacture of a stromal cell culture which is capable of producing mammalian dendritic cells and/or stem cells.
  • Another further aspect of the present invention contemplates the use of a haemopoietic cell population and a stromal cell layer in the manufacture of a stromal cell co-culture which is capable of producing dendritic cells, stem cells and/or stromal cells
  • the present invention contemplates the use of dendritic cells, stem cells and/or stromal cells in the manufacture of a medicament for the treatment of a mammal.
  • the methods and compositions ofthe present invention are useful for generating dendritic cells, stem cells and stromal cells for use in a range of therapeutic and diagnostic procedures.
  • the cells produced by the methods ofthe present invention can be used as immunogens for the production of monoclonal antibodies specific for new cell surface markers which define these rare and poorly defined cell types.
  • Dendritic cells can be used for in vitro testing ofthe immunogenicity of vaccines
  • Dendritic cells can also be transplanted to various sites in an animal for effective migration or modulation of immune responses
  • Dendritic cells of the present invention can also be engineered to express foreign genes for effective gene therapy after transplantation and represent a long lived stem cell population which can colonise a host after transplantation, producing long term reconstituting cells which can replicate and continue to express new genes
  • Said dendritic cells may also be used in immunotherapy to induce either tolerance or immunity to foreign antigens
  • both the dendritic cells and the stromal cells ofthe present invention can be used to isolate genes and proteins expressed specifically by these cell types
  • the present invention is further described by the following non-limiting Figures and Examples. In the Figures:
  • Figure 1 is a photographic representation of scanning electron microscopy of non-adherent cells produced in early long term spleen culture.
  • Figure la Heterogeneity of non-adherent cells generated in spleen LTC. Spleen cells were cultured in sDMEM for 15 days Magnification (clockwise from top left): 3400x, 3400x, 4200x; 5000x.
  • Figure lb Scanning electron microscopy of dendritiform cells present in culture of spleen (15 days). Magnification 6700x.
  • Figure 2 is a photographic representation of transmission electron microscopy showing characteristic features of cells produced in spleen culture (15 days) under various magnifications. Clockwise from top left: 5800x, 700x, 3000x, 2900x, 2900x, 6500x
  • Figure 3 is a photographic representation of the characteristic pattern of cell growth in established LTC. Microscopy under different magnification shows (A) proliferation of small (early) cells in clusters; (B) foci of stroma with attached proliferating cells, (C) high power view of a focus, (D) non-adherent cells with dendritic-like morphology, (E) stroma-free region of culture showing heterogeneity amongst non-adherent cells, (F) higher power view of E Magnification • 240x(A,C,D,F), 150x(B,E)
  • Figure 4 is a photographic representation ofthe morphology of non-adherent cells produced in LTC. Modified Wright's Giemsa staining of cytospin preparations reveals cell heterogeneity in early (32 days) spleen LTC (A, 500x), in comparison with non-adherent cells produced by a continuous culture of spleen LTC-X1 maintained for >12 months (B, 800x)
  • Figure 5 is a photographic representation ofthe stroma present in LTC Stroma derived from different organs including spleen (A, 200x), BM (B, 320x), thymus (C, 200x), thymus (D, 320x) at various stages of development after removal of non-adherent cells by washing.
  • Figure 6 is a photographic representation of fluorescent antibody staining of endothelial cells using antibody against Factor VUJ-related antigen.
  • A Control human HUVEC endothelial cells.
  • B Splenic stroma X3.
  • C Thymic stroma Tl. Magnification 750x.
  • Figure 7 is a graphical representation ofthe expression of cell surface antigens on DC generated in spleen LTC.
  • Non-adherent cells were collected from LTC XI and X3 at various times from 100 days after LTC establishment.
  • Control cells included spleen cells, bone marrow (BM), LPS activated lymph node B cells and peritoneal exudate cells (PEC).
  • the solid line gives background staining of FITC-conjugated secondary antibody NIL to cells absorbed with isotype control antibodies in place of specific antibodies.
  • Figure 8(a) is a graphical representation ofthe cell surface antigen expression on non-adherent cells produced in spleen LTC.
  • Non-adherent cells from established LTC (>21 days) were screened for the presence of markers representing different cell types using a range of specific antibodies. Binding of antibody to cells was measured in an indirect assay using FACS analysis Specific staining was calculated by subtraction of binding in the presence of an isotype control antibody. Data represent mean+SE of positive cells for cultures which have >10% cells binding antibody. The % positive cultures (number analysed) is also shown for each antibody Spleen and BM cells from syngeneic mice were used as a control for all screening experiments to confirm antibody activity.
  • Figure 8(b) is a graphical representation of CD4 and CD8 ⁇ expression on non-adherent cells produced by LTC Cells were first incubated with an Fc-receptor blocking antibody and stained in a two colour assay using FITC-anti-CD8 and PE-anti-CD4 Cells were counted on a FACSort counting 50,000 events Controls were incubated with single or no antibodies, and thymocytes.
  • Figure 9 is a graphical representation of antigen presenting capacity of dendritic-like cells a-b:
  • Figure 10 is a photographical representation of growth factors present in the supernatant of LTC from spleen and thymus support the development of bone marrow cultures,
  • A spleen LTC
  • B KT2 (thymic LTC)
  • C medium alone
  • r-IL-3 50U/ml
  • FIG 11 is a graphical representation of FACS analysis showing phenotypic changes in bone marrow cells cultured in various growth factor sources
  • CSN were collected from splenic stroma XI (CSN XI) and thymic stroma KT-2 (CSN KT2) 2 x 10 3 cells were cultured in 50% CSN for 6 days with medium changes every two days r-IL-3 (50 U/ml) was used as a control growth factor.
  • Bone marrow was used as a source of control cells for antibody staining
  • Figure 12 is a photographic representation of stromal support of BM cell growth and differentiation.
  • BM from B10.A(2R) mice was cultured for 4 days on irradiated syngeneic splenic stroma XI (1.2) (A) and XI (8.3) (B) (Magnification lOOx)
  • Control cultures included BM alone (C) and irradiated stroma XI (8 3) alone (D) (lOOx)
  • Non-adherent cells from (B) are shown at higher magnification, 320x (E)
  • Figure 13 is a photographic representation of the electron microscopy of cells produced in secondary LTC.
  • B10.A(2R) BM was cultured on syngeneic XI splenic stroma for 45 days. Magnification. A, 1740x; B, 2300x; C, 3300x, D, 2400x
  • Figure 14 is a photographic representation of established secondary LTC of lymphoid cells growing on stroma derived from spleen.
  • B10 A(2R) spleen has been cultured for 20 days on syngeneic irradiated splenic stroma XI. (Magnification, 400x).
  • BIO A(2R) BM has been cultured for 9 days on syngeneic splenic stroma XI (8.3) (200x).
  • Figure 15 is a graphical representation of cells generated in secondary LTC being potent stimulators of an MLR to both allogeneic DBA/lj (A) and syngeneic B10.A(2R) (B) spleen responder cells.
  • Figure 16 is a graphical representation of the capacity of cells produced in secondary LTC to present conalbumin and induce a proliferative response in the D10.G4.1 Th2 cell line
  • Nonadherent cells generated in secondary culture of B10.A(2R) BM on splenic stroma XI for 70 days (A) and X1-3H for 150 days (B) were compared with normal BIO A(2R) spleen cells as control APC APC were pulsed with conalbumin at 100 ⁇ g/ml Antigen or APC alone gave no significant proliferation
  • Figure 17 is a graphical representation of dose-dependency of antigen- specific responses
  • Figure 19 is a graphical representation of non-adherent cells produced in secondary LTC presenting antigen to naive T cells.
  • Non-adherent cells were collected for use as APC from a culture of B10.A(2R) BM on the XI spleen stroma, maintained in 1% NMS for 60 days.
  • A(2R) mice were cultured with or without 1000 APC in the presence of various concentrations a protein mixture containing equal concentrations of conalbumin, ovalbumin and bovine serum albumin, as antigen.
  • the proliferation of spleen MNC stimulated by APC in the absence of exogenous antigen was 570 cpm
  • Figure 20 is a photographical representation of dendritic cell colonies in agar developing from non-adherent cells produced by LTC-XI. Cells were cultured in agar supplemented with CSN from LTC-XI Figure 20 shows a cluster of mature dendritic cells with long membrane extensions stained with Wright-Giemsa stain and photographed under light microscopy (x320)
  • mice were bred under specific pathogen free conditions at the John Curtin School of Medical Research, Canberra, Australia, and used when 4 to 10 weeks of age.
  • Controls also included cells cultured in the absence of stroma Dulbecco's modified Eagle's medium was used for cell culture following supplementation to give a final concentration of 10% v/v FCS, 5 10 "5 M 2-mercaptoethanol, lOmM Hepes, 100 U/ml penicillin, 100 mg/ml streptomycin, 5g/L glucose, 6 mg/L folic acid, 36 mg/L L-asparagine, 116 mg/L L-arginine HC 1 (sDMEM) Cells were cultured for one week before any medium changes were made Half medium was replenished every 3-5 days thereafter.
  • sDMEM sDMEM
  • Antibody binding was assessed in an indirect assay using an appropriate FITC-conjugated second stage antibody or with phycoerythrin-conjugated avidin to detect the binding of biotinylated antibody This staining procedure has been described previously (O'Neill & Ni, 1993) Briefly,
  • non-adherent cells (10 - 10 ) were collected from LTC, and washed twice with sDMEM/3mM NaN by centrifugation at 200g for 5 min Specific antibody was absorbed to cells in a 50 ⁇ l volume in the wells of a microtitre plate. After incubation for 30 min on ice, cells were washed thrice by centrifugation (350g for 1 min). The same procedure was repeated for absorption of the labelled second stage reagent. Fluorescence was measured by log analysis using a FACScan
  • CTLA4-Ig specific for B7(CD80/CD86) (Linsley et al, 1991), CD40-Hgand (CD40L) conjugated with CD8 ⁇ which binds CD40 (Lane et al, 1993)
  • CD40L CD40-Hgand conjugated with CD8 ⁇ which binds CD40
  • Factor VHI-related antigen expressed by endothelial cells was examined by indirect immunofluorescence microscopy using the method described (Wagner et al, 1982).
  • the primary antibody was a rabbit anti-human Factor VHI-related antigen (Dako Ltd , Botany, Australia), and the secondary antibody was a FITC-conjugated goat anti-rabbit IgG
  • HUVEC human umbilical vein endothelial cell line, HUVEC (kindly provided by X Liang, JCSMR, Canberra, Australia) was used as a control cell line for Factor VIII staining It was grown on 0 1% w/v gelatine-coated flasks with sDMEM and passaged weekly. The stromal cell culture was trypsinised, shaken gently to produce a single cell suspension, then washed twice to remove enzyme before passage.
  • a Diff-Quik staining kit (Lab Aids, Narrabeen, Australia ) was used for Wright's Giesma staining of cells, by following the manufacturer's instructions. Briefly, cytospin preparations of cells or blood smears were air dried, fixed in methyl alcohol, stained with methylene dye mixture (1.25 g/L), rinsed with deionized water, air dried and examined by microscopy under oil immersion.
  • Astra blue was used for staining of mast cells as described previously (Stoble et al, 1981) Briefly, cytospin preparations or blood smears were fixed in Carnoy's fixative (60% v/v absolute ethanol, 30% v/v chloroform, 10% v/v glacial acetic acid) for 45 min at room temperature and then air-dried. Slides were stained in 1% w/v Astra blue in HCl(pH 0 3) for 30 min, washed for 10 min in 0.7M-HC1, counterstained in 0 5% Safranin-O/0 125M HC1 for 20s, then washed in running tap water and air-dried.
  • Non-adherent cells from LTC were collected from culture supernatant after gentle shaking ofthe flask. Cells were washed twice in sDMEM before use as stimulators They were irradiated (20 Greys, ⁇ -irradiation, Co source) and applied in graded numbers to 2 x 10 allogeneic spleen responder cells in a final volume of 200 ⁇ l in 96 well U-bottom microtitre plates. Responder cells were prepared from spleen or lymph node after centrifugation through an Isopaque-Ficoll gradient.
  • responders used were enriched for T cells by treatment with anti- Class II antibody (N22) and rabbit complement (Low-Tox M, Cedarlane, Canada) to remove any potential antigen presenting cells (APC) in the responder population Proliferation was measured
  • k D10.G4J (D10) is an H-2 restricted, conalbumin-specific T helper type 2 (Th2) clone (Kaye etal, 1983) It was purchased from ATCC (Rockville, Pike, MD) and maintained in sDMEM
  • D10 cells were routinely restimulated fortnightly with irradiated C57BL/6J spleen cells or with conalbumin (100 ⁇ g/ml) presented by AKR J or B10 A(2R) spleen cells D10 cells were used at
  • CSN Culture supernatant
  • D10 cells were used to titrate IL-1 activity, HT-2 cells for IL-2/IL-4 activity; BCL1 cells were used to detect IL-4, IL-5 and GM-CSF; B9 cells were used in IL-6 assays; IL-3 and GM-CSF were detected using FDC-Pl cells, IL-3 was titrated on
  • Tumor necrosis factor- (TNF- ) activity was detected using WEHI- 164 cells.
  • r-IL-3 and r-IL-5 were produced and kindly provided by I Young (JCSMR, Australia,
  • Mouse r-IL-6 was supplied by P Hodgkin (DNAX, Palo Alto, CA); Mouse r-GM- CSF was provided by N. Nicola (WEHI, Melbourne, Australia); Mouse r-TNF- ⁇ was purchased from Genzyme (Cambridge, MA).
  • EXAMPLE 10 ESTABLISHMENT OF LONG TERM CULTURE (I)
  • Spleen stromal cultures began to produce small, non-adherent cells arising from the monolayer within 2 weeks. These small cells were first detectable at a time when splenic lymphocytes had clearly died off and were no longer present in culture Within a further 2 to 3 weeks, larger, immature lymphoid-like cells appeared, some with obvious pseudopodia. These cells were produced continually in LTC and some of them migrated to stroma free areas. About
  • lymph node cells produced immature mononuclear cells with pseudopodia but cells were present in fewer numbers than in LTC of spleen. Spleen cultures could be maintained continuously as LTC producing non-adherent cells. Some have been maintained for up to 4 years Typically the number of immature, lymphoblastic
  • non-adherent cells produced in LTC were generally very large and round with fewer and shorter processes or veils
  • the vast majority of cells had the typical, irregularly-shaped DC membrane with processes and some had developed vacuoles/endosomes.
  • This population has been shown to contain APC When cells were passaged by transfer into a new flask, a stromal layer of multiple cell types reformed confluently within 3 to 4 weeks and non-adherent cells with antigen presenting capacity were further released.
  • Spleen stromal cultures began to produce small, non-adherent cells arising from the monolayer within 2 weeks (Fig 3 -A) These small cells were first detectable at a time when splenic lymphocytes had clearly died off and were no longer present in culture Within a further 2 to 3 weeks, there was an increase in cell number and larger, immature "lymphoid"-like cells appeared, some with obvious pseudopodia (Figs 3-D, E & F) These cells were produced continually in LTC and some of them moved to stroma free areas (Figs 3-E & F) BM cultures were less successful and only 60% produced non-adherent cells.
  • lymph node cells produced immature mononuclear cells with pseudopodia but cells were present in far fewer numbers than in LTC of spleen.
  • Spleen cultures could be maintained continuously as LTC producing non-adherent cells. Some have been maintained for up to 30 months. Generally, spleen cultures are optimal producers until about 4 months. After this time, the stromal layer becomes overgrown and the majority of cultures deteriorate, producing less cells. Removing stroma at medium change can result in maintenance ofthe culture for a long period of time.
  • Spleen LTC have been routinely generated from 6 week old mice, by establishing cultures at 2-5 x 10 lymphoid cells per ml. No obvious strain differences have been noted and cultures have been successfully established from B10.A(2R), B10.A(5R), CBA/H, C57BL/6J, C57BL/KaThyl.l, AKR/J and DBA/lj strains of mice These cultures have been easy to maintain in sDMEM medium. However, some cultures have also been successfully established in RPMI medium supplemented with 10% FCS, antibiotics and 2-mercaptoethanol (5 x 10 M). LTC continued to produce non-adherent cells until the monolayer became confluent.
  • Haematopoietic cells display lineage-specific enzyme activities which are useful in the dissection of cell lineage. Histochemical staining was carried out to assess cell types present in the nonadherent cell population of LTC. Methods used include Astra blue and Alcian blue staining for mast cells, myeloperoxidase staining for granulocytes and monocytes/M ⁇ and non-specific esterase staining for myeloid cells and lymphoid cells.
  • Astra blue staining cells were only detected in 6 out of 23 spleen LTC which were all early LTC within 4 weeks of culture initiation (Table 2). For these, the mean percent positive cells was only 1.9 ⁇ 1.7%. There were no positively stained cells in the non-adherent cell populations collected from lymph node, bone marrow and thymus LTC. Control bone marrow and peritoneal exudate cells contained -7% Astra blue positive or mast cells. Very few positive staining cells were found in control blood populations ( ⁇ 0J%). The intensity of stain in cells from spleen LTC was weak and less granular in comparison with staining of positive control cells.
  • Alcian blue staining was also used to detect the presence of mast cells in the non- adherent cells produced in LTC. An even lower percentage of positive staining was observed in control cells of bone marrow and peritoneal exudate cells and no positive cells were detected in 7 samples of non-adherent cells produced by LTC.
  • Myeloperoxidase has been reported as absent in human blood DC but present in monocytes and peritoneal exudate M ⁇ (Van Voorhis et al, 1982) Myeloperoxidase activity was measured amongst the non-adherent cells produced by LTC in order to determine lineage characteristics of cells present. Endogenous myeloperoxidase activity was not detectable in the non-adherent cell population generated in LTC from spleen, BM or lymph node (Table 2). In comparison, control cell populations from bone marrow, spleen and peritoneal exudate cells contained measurable numbers of positive cells (Table 2).
  • non-adherent cells produced in LTC were generally very large and round with processes or veils Before this time, a mixture of various lymphoid, granular and myeloid cells could be detected in the supernatant of LTC (Fig 4).
  • Foci contained cells with characteristic pseudopodia resembling DC which were being released into the medium. These were common in well established LTC such as LTC X3 which has been in culture for >12 months (Fig. 4) When cultures were passaged by transfer into a new flask, a stromal layer of multiple cell types reformed confluently within 3 to 4 weeks and non-adherent cells were further released.
  • the stromal layer present in spleen LTC is a mix of cell types, including fibroblasts, endothelial cells (EC), with isolated fixed M ⁇ and DC. Cultures established with a cell number of greater than 5 x 10 cells per ml were generally unsuccessful and produced few non-adherent cells This could be due to the overgrowth of fibroblasts which inhibits the growth of other cells.
  • Fibroblasts have been reported to inhibit DC growth in granulocyte-macrophage colony stimulating factor (GM-CSF) supported BM cultures (Inaba et al, 1992b). Similarly, at the time of re-establishment of cultures, either from frozen stocks or after passaging, any overgrowth of fibroblasts leads to unproductive cultures. Cultures which showed evidence of adipose cell growth also produced no non-adherent cells.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • Stable cultures producing non-adherent cells maintain a stromal layer comprising foci or outgrowths of EC well connected to a fibroblast network and to M ⁇ . These can be clearly seen at the time of cell passage into a new flask. Foci of EC were evident by the clustering of nonadherent cells on top of them (Figs 3-B & C).
  • One notable feature of LTC was the appearance of small, loosely attached stem cells randomly scattered across the whole stromal layer, representing 10-20% ofthe non-adherent cell population They never clustered, nor increased in number.
  • foci had a base of confluent EC interconnected with fibroblasts and a few scattered M ⁇ . Large, immature mononuclear cells were continuously shed from these foci into the medium (Figs 3-E & F).
  • the yield of cells from productive cultures was typically 5 x 10 to 10 cells every 3 to 4 days in a 75cm flask. In cultures where foci did not form, no cells were released into the culture medium.
  • the stromal layer in spleen LTC is a mixture of cell types, including fibroblasts and EC, with isolated fixed M ⁇ (Table 4).
  • a well developed stromal network of spleen LTC XI is shown in Fig 4-A.
  • long term bone marrow cultures formed a loosely connected network comprising fibroblasts, M ⁇ and some scattered semi-adherent cells (Fig 5-B).
  • Thymic stroma once established, comprised mainly epithelial cells, with few other cell types, forming a mesh-like network, including M ⁇ and fibroblasts (Figs 5-C & D).
  • M ⁇ present in spleen stroma were morphologically heterogeneous and dominated different regions of the culture flask. Three types of M ⁇ were observed. Polygonal M ⁇ with few membrane extensions were usually found in cultures which were poor producers of non-adherent cells. A second type was characterised as adherent round M ⁇ with smoother edges. These M ⁇ were usually present in productive LTC, intermingled with EC. The third type of M ⁇ resembled stellate cells, scattered mainly in non-confluent areas ofthe culture. This type of M ⁇ was smaller, usually present with fibroblasts and spindle-shaped cells and was easily overtaken by other cells.
  • Fibroblasts were a major cell type present in stromal cultures of spleen, BM and lymph node. Fibroblasts were also present in some thymic LTC They usually emerged in cultures later than M ⁇ and EC. However, they spread more quickly than did other cells, and easily formed dense cell sheets excluding the growth of other cells. Fibroblast/spindle cells may be precursors of adipose cells (Wilkins & Jones, 1995) Adipose cells developed in a few long term BM cultures which did not produce floating cells (Zipori et al, 1984; de Wynter et al, 1993). Stromal cultures of spleen were very heterogeneous, comprising endothelial cells, fibroblasts, stellate cells, M ⁇ , spindle-shaped cells and small round scattered cells.
  • EC were a component ofthe stromal layer of cultures which continued to produce haemopoietic cells. They formed extensive cell sheets with scattered stem cells on top of clustered EC. There was a continuing presence of small cells resembling stem cells in cultures producing non-adherent cells. These were a minority of cells and never grew to become more than 5-10% ofthe nonadherent population ofthe culture. The existence of EC was confirmed by detection of Factor Vlll-related antigen using specific antibody staining. This marker is expressed on human EC (Jaffe et al, 1973), and rabbit anti-human Factor- Vffl-associated antigen was used to detect antigen expression on mouse EC (Obeso et al, 1990).
  • the 33D1 antibody bound to a smaller or different subset of CD1 lc + or MHC Class IIJ cells.
  • the NLDC-145 antibody also had a different staining specificity than did antibodies N418 or 33D1 (Fig 7 & Table 5).
  • CD1 lc + cells have been shown to be enriched in the high FSC/high SSC subset of non-adherent cells generated in LTC. This confirms the finding of large-sized irregular-shaped DC in the non-adherent cell population produced in LTC and detectable by electron microscopy (Fig 1).
  • LTC XI and LTC X3 Some established LTC were further characterized phenotypically and representative profiles of antibody staining on LTC XI and LTC X3 are shown in Fig 7.
  • the c-kit marker expressed by bone marrow cells and the ThB marker expressed by early lymphoid cells and some isolates of DC were also expressed by LTC XI and LTC X3 cells.
  • the myeloid cell markers CD1 lb, F4/80 and Gr-1 were expressed by non-adherent cells produced in LTC XI and X3, although Gr-1 was expressed at much lower levels than on granulocytes in bone marrow (Fig 7).
  • Low affinity Fc receptors detected by antibody 2 4G2 and MHC Class II were also expressed by cells from LTC XI and X3, but cells did not express the B cell markers B220 or CD40.
  • Cells generated in spleen LTC were negative for a range of T cell markers detected by antibodies MR5.2, AT83, GK1.5, 53-6.7, H57-579.
  • Non-adherent cells obtained from many LTC of both spleen and bone marrow were collected and analysed for expression of cell surface markers to identify haemopoietic cell types at different times after establishment of cultures and to investigate the possibility that cells produced were in fact DC.
  • a range of different antibodies was used to screen for the presence of different haemopoietic cell types and to check for consistency in the production of cells in LTC.
  • Data collected from multiple cultures at many time points including 49 spleen LTC and 9 BM LTC is summarised in Fig. 8a.
  • the number of non-adherent cells produced in LTC increased rapidly between 2 and 3 weeks of culture, with the formation of a confluent stromal layer.
  • LTC became more visibly stable after 3-4 weeks and cells released into the supernatant at this stage were screened for expression of lineage specific markers. Multiple staining tests were performed on many individual LTC using small numbers of non-adherent cells collected at various times after establishment of culture. For the purpose of summarising data cultures were scored as positive if they contained >10% cells binding specific antibody.
  • T cell activation Greater than 60% of cultures contained cells which bound antibodies specific for myeloid cell markers including CD1 lb, low affinity Fc receptors and the F4/80 marker. The average size of these populations was 35% for CD1 lb, 78% for FcR and 48% for F4/80. Less than 50% of cultures stained weakly with antibody specific for the Gr-1 marker on granulocytes, but level of expression was low, 24% of cells. B cells detected by B220 expression were infrequent in cultures after 3 weeks of establishment Only 1 in 9 cultures had a level of 12% B cells. Markers specific for T cells were not detectable on spleen LTC after 3 weeks, and cells expressing many T cell markers, including Thy 1.2, CD4, TCR- ⁇ or CD40L were absent. CD8 + cells have been detected in low number in well established LTC, consistent with production of different subsets or types of dendritic cells. A small subset (1-1.5%) is consistent with the production of lymphoid-like dendritic cells ( Figure 8b)
  • HSA Heat stable antigen
  • non-adherent cells from LTC were screened for MLR- stimulating capacity at multiple time points. These tests involved allogeneic spleen responder cells from at least two strains of mice While the capacity of cells from different LTC varied, about 20% of LTC produced cells with high APC capacity These were up to 500-fold stronger than spleen cell populations in inducing an MLR response. LTC producing cells with a strong MLR stimulating capacity were shown to maintain that capacity over long periods of culture and could be maintained after passaging Fig 9a-b shows the allogeneic MLR response induced by non- adherent cells from LTC XI derived from BIO. A(2R) spleen, which indicates that cells produced in this LTC have 200 and 500-fold greater capacity to induce an allogeneic response than syngeneic spleen cells.
  • Non-adherent cells generated in LTC were also tested for capacity to stimulate the I-A restricted conalbumin-specific T helper cell clone, D10. Capacity to stimulate an antigen-specific response is an important indication that the proliferation induced by these cells represents their capacity to act as APC and is not due to some non-specific process.
  • non-adherent cells generated in LTC were 25 to 125 fold more efficient at presenting antigen to D10 cells than were control spleen cells from B10.A(2R) mice (Fig 9c) Cells produced by some cultures have stronger antigen presenting capacity than others and these patterns have remained relatively constant for individual cultures
  • Non-adherent cells removed from LTC did not survive if cultured in medium alone and the addition of CSN from LTC to non-adherent cells in culture, postponed but did not prevent cell death.
  • CSN from stromal cell cultures collected 48 h after a previous medium change was therefore tested for capacity to support haematopoiesis in BM cells
  • CSN from both B10 A(2R) splenic stroma XI (CSN XI) and from C57BL/KaThyl 1 thymic stroma KT-2 (CSN KT-2) supported B10.A(2R) BM cell growth. 50 U/ml r-IL-3 was used as a control growth factor.
  • stromal CSN displayed haematopoietic ability distinct from that of IL-3, CSN were screened for the presence of various cytokine activities relevant to DC proliferation.
  • CSN derived from splenic and thymic stroma from 3 different strains of mice were assessed for capacity to support growth factor-dependent cell proliferation (Table 6).
  • Table 6 There was some consistency in the profile of cytokine production across different stromal cell lines. All stroma produced no detectable levels of EL-1, no measurable IL-2/EL-4, nor did they produce TNF- ⁇ .
  • IL-3 and/or GM-CSF production was predicted because CSN from all cultures supported the proliferation of FDC-Pl, an IL-3/GM-CSF dependent cell line.
  • IL-3 production was confirmed since CSN from all lines supported proliferation of the IL-3 -dependent cell line, 32Dcl23. Uniform production of IL-6 was detected by proliferation of factor-dependent B9 cells. This activity was equivalent to -20 ⁇ g/ml of r-IL-6. No CSN induced measurable proliferation of BCL1 cells, which are dependent on IL-4, IL-5 and GM-CSF for proliferation Data presented in Table 6 are consistent with the production of at least IL-3 and IL-6 by stromal cells
  • BM and lymphoid cell populations would not readily form a stromal cell layer capable of supporting continuous production of non-adherent cells ( Whitlock et al, 1984; Ni and O'Neil, 1997)
  • the preformed stromal layer is acting as the support medium for cell proliferation from precursors present in the overlay cell populations with little or no contribution from the overlaid cells.
  • secondary LTC producing non-adherent cells can be quickly established using both syngeneic and allogeneic lymphoid cells cocultured as an overlay on spleen stroma with an overall success rate of 90% (Table 7).
  • Fig 13A-B The majority of non-adherent cells generated in established secondary BM LTC using syngeneic spleen stroma were rounded in shape, with cytoplasmic processes and a low nuclear/cytoplasmic ratio (Fig 13A-B). These cells contained endosome-like bodies with a clear membrane bilayer (Fig 13B) and cells also contained many electron dense, lysosome-like bodies in the cytoplasm
  • Figs 13 A-D Most were dendritic cells with - 10% small immature cells present in the population (Fig 13 A).
  • Fig 13 A The existence of vacuoles, mitochondria, well developed endoplasm and Golgi-complexes in immature cells indicates that cells were metabolically active
  • Fig 14 Maintenance of cell production depends on a healthy stromal
  • BM cells differentiated when cultured on stroma Table 8 shows phenotypic changes in B10.A(2R) BM cells cultured on syngeneic spleen stroma for three weeks.
  • BM cells which express the stem cell factor receptor (c-kit, CD1 17) dropped from a control value of 46 4% to
  • the B cell marker, B220 expressed on 28% of control BM cells, decreased to very low levels during the first week of culture and disappeared by 3 weeks.
  • the expression ofthe Gr-1 antigen on granulocytes reduced by more than 50% during the first week, then expanded to include -70% of the population during the
  • the cells produced in LTC were collected free of stroma and tested for binding of antibodies specific for cell surface markers used to delineate DC
  • a range of antibodies specific for haematolymphoid cells was used to detect different cell types present in LTC and to screen for consistent production of cells. This included a range of antibodies specific for T & B cells, myeloid, granulocyte, DC and stem cells.
  • the number of cells staining with DC specific antibodies N418 and 33D1 increased rapidly during the first 4 weeks of BM culture on syngeneic spleen stroma XI and X3 (Table 9). Cells binding 33D1 expanded from nil to -50% of cells by 4 weeks and the number of cells staining with N418 increased to similar levels.
  • lymphoid DC has been described in thymus and spleen which expresses the CD8 ⁇ molecule (Vremec et al, 1992).
  • Cultured BM cells also show changes in Forward light scatter (FSC) and Side (90°) light scatter (SSC) .
  • FSC Forward light scatter
  • SSC Side (90°) light scatter
  • Table 3 shows marker expression on B10.A(2R) spleen and thymus cell populations at 21 days after culture on syngeneic spleen stroma X3
  • Both the cultured spleen cells and thymocytes bound the DC specific antibodies N418 (37% and 70%, respectively) and 33D1 (14% and 35%, respectively). This represented a large change in cell composition since control spleen and thymus cell populations contain almost no cells which bind these DC-specific antibodies.
  • BM, spleen cells and thymocytes cultured on splenic stroma gave rise to cell populations enriched for cells which express DC/myeloid cell markers
  • Capacity of cells to act as APC is another method for determining presence of dendritic-like cells in LTC than is cell surface antigen expression.
  • Non-adherent cells generated in secondary culture resembling DC by marker expression and morphology were assessed at various time points for functional characteristics typical of DC.
  • MLR employed both allogeneic and syngeneic spleen cells as responders.
  • BM cells cultured on syngeneic spleen stroma XI and X3 for 8 days developed strong capacity to stimulate both syngeneic B10.A(2R) and allogeneic DBA/lj cell proliferation in an MLR assay.
  • Table 10 is a summary of MLR data of responses induced by BM cells cultured on syngeneic spleen stroma for different periods of time.
  • MLR-stimulating capacity of secondary cultured BM can be detected as early as day 8 and maintained for as long as 160 days (Table 10) MLR stimulating capacity was verified using at least 3 strains of mice, as responders from both syngeneic and allogeneic strains Similar experiments have confirmed that non-adherent cells produced in secondary LTC of spleen and thymus on spleen stroma also produce cells with potent APC capacity
  • D10G4J D10 conalbumin-specific Th2 clone.
  • Strong D10 cell proliferation was induced by BM cells cultured on XI or X1-3H stroma for various periods of time (Fig 16).
  • Antigen or APC alone did not induce any significant D10 cell proliferation.
  • APC produced in secondary LTC between 10 & 30 times more syngeneic B10.A(2R) spleen cells were needed to induce equivalent levels of D10 cell proliferation
  • D10 cell proliferation induced by APC was antigen-specific and dose dependent D10 cells did not proliferate in the absence of antigen and cell proliferation increased with antigen dose over the concentration range tested (Fig 17)
  • Two different doses of 2R spleen were used as APC controls in this experiment.
  • Spleen cells in a lower number 10,000 cells
  • Spleen cells at a higher dose (250,000 cells) were very efficient at presenting even lower concentrations of antigen
  • CTLA4-Ig completely inhibited the allogeneic and syngeneic spleen proliferative response induced by APC produced in secondary culture of B10 A(2R) BM on syngeneic spleen stroma (Fig 18).
  • An isotype human IgG control antibody used at the same concentrations did not inhibit the response.
  • a role for CD80/CD86 was indicated only for the MLR response since CTLA-4Ig did not inhibit the D10 antigen-specific proliferative response D10 cells do not require a second signal from CD80/CD86 for stimulation
  • Non-adherent cells generated in primary LTC have been shown to be capable of presenting soluble protein antigen to unprimed spleen cells (Ni and O'Neill, 1997)
  • Cells generated in secondary LTC were tested for similar capacity
  • B 10 A(2R) BM cells cultured on syngeneic spleen stroma and maintained in medium containing 1% v/v NMS for 60 days were tested for capacity to present soluble protein antigen to unprimed syngeneic spleen cells.
  • Soluble protein antigen was a mixture of equal concentrations of conalbumin, ovalbumin and bovine serum albumin.
  • Proliferation of B10.A(2R) spleen in the presence of proteins was at least three times greater than proliferation without antigen (Fig 19).
  • the response to the protein mixture was antigen dose dependent over the range 100-1000 mg/ml for both the syngeneic response to the protein mixture (Fig 19), and allogeneic responses
  • ⁇ Percent positive staining cells mean ⁇ SD (number of cultures)
  • Tl ND not determined, BM, bone marrow, LN, lymph node, PEC, peritoneal exudate cells
  • # Data represents mean percent positive cells ⁇ SD for only those cultures which contain positive staining cells
  • Non- Adherent cells Stem cells Very small, round cells forming loose colonies randomly over the stromal layer, never clustering or increasing in number
  • Mononuclear cells Heterogeneous dend ⁇ tic-like cells 10 to 30 ⁇ m in diameter present as individual floating cells or shedding from a cluster of cells as part of foci Cells vary in shape from round to irregular, all with processes Table 5. Staining of cell surface markers from LTC which contain DC.
  • Non-adherent cells from LTC were collected at various times and tested for staining of antibodies specific for DC Data shows only cell populations which stain positively (>40%) with N418
  • ⁇ Data represents staining of a subset of large granular cells, gated by FACS for high forward and 90° scatter profile
  • Stromal layers originated from spleen LTC which had ceased production of non-adherent cells.
  • Cells from various lymphoid organs derived from syngeneic or allogeneic mice were overlaid on stroma and cultures were examined for non-adherent cell production.
  • Hi or “lo” refers to relative cell yield; Hi > 5xl0 4 c/ml after 3 days after medium change , lo ⁇ 5x10 4 c/ml
  • the isotype (rat IgG2b) control antibodies were GKl 5 and 53-6 7 t t Secondary culture of syngeneic B10 A(2R) BM cells on spleen stroma (XI) to
  • B10 A(2R) BM cells were used in secondary LTC on different syngeneic splenic stioma (Xl-8 3, X1-1 2, XI , X3, and XI -3H) for various lengths of time f
  • 10" spleen mononuclear cells were cultured with vanous numbers of non-adherent cells collected from LTC for 72 hrs Response was assessed by " ⁇ T incorporation over the f nal 6 hrs of culture Controls included nonadherent cells alone and splenic responders alone APC background was always below 200 cpm
  • Data represents number of cells required to give five-fold increase in ⁇ T incorporation over background of allogeneic and syngeneic responders alone ⁇ 80 cells ( M M I ), 80 - 400 (++++), 400 - 2,000 (+++), 2,000 - 10,000 (++), 10.000 - 50,000 (+) > 50,000 (+/-)

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Abstract

L'invention concerne d'une manière générale un procédé de mise en culture de cellules et notamment un procédé de formation de cellules dendritiques. L'invention se rapporte, plus particulièrement, à un procédé de formation de cellules dendritiques précurseurs. Les cellules dendritiques de l'invention sont utiles, entre autres, en tant que cibles pour la thérapie génique ou l'immunothérapie contre le cancer, les maladies infectieuses, l'auto-immunité et pour le traitement des tumeurs.
PCT/AU1998/000428 1997-06-06 1998-06-05 Procede de mise en culture de cellules WO1998055597A1 (fr)

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AU77513/98A AU7751398A (en) 1997-06-06 1998-06-05 A method for culturing cells

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US6440735B1 (en) 1998-03-31 2002-08-27 Geron Corporation Dendritic cell vaccine containing telomerase reverse transcriptase for the treament of cancer
US7402307B2 (en) 1998-03-31 2008-07-22 Geron Corporation Method for identifying and killing cancer cells
CN114480277A (zh) * 2022-04-01 2022-05-13 首都医科大学附属北京佑安医院 一种刺激树突状细胞成熟的方法及培养基

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6440735B1 (en) 1998-03-31 2002-08-27 Geron Corporation Dendritic cell vaccine containing telomerase reverse transcriptase for the treament of cancer
US7402307B2 (en) 1998-03-31 2008-07-22 Geron Corporation Method for identifying and killing cancer cells
US7824849B2 (en) 1998-03-31 2010-11-02 Geron Corporation Cellular telomerase vaccine and its use for treating cancer
CN114480277A (zh) * 2022-04-01 2022-05-13 首都医科大学附属北京佑安医院 一种刺激树突状细胞成熟的方法及培养基
CN114480277B (zh) * 2022-04-01 2022-07-22 首都医科大学附属北京佑安医院 一种刺激树突状细胞成熟的方法及培养基

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