WO1997028253A1 - Method for culturing cells having angiogenic potential - Google Patents

Method for culturing cells having angiogenic potential Download PDF

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Publication number
WO1997028253A1
WO1997028253A1 PCT/FR1997/000186 FR9700186W WO9728253A1 WO 1997028253 A1 WO1997028253 A1 WO 1997028253A1 FR 9700186 W FR9700186 W FR 9700186W WO 9728253 A1 WO9728253 A1 WO 9728253A1
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cells
collagen
angiogenic potential
vascular
type iii
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PCT/FR1997/000186
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French (fr)
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Claude Souvignet
Béatrice PLUMAS-MARTY
Jean-Louis Tayot
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Saduc (S.A.)
Laboratoire Hemeris (S.A.R.L.)
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Publication of WO1997028253A1 publication Critical patent/WO1997028253A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/069—Vascular Endothelial cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates to the field of cell culture and more particularly to a method of culturing cells with angiogenic potential to induce the vascular morphogenesis of these cells.
  • Vascular morphogenesis is defined as the set of cellular and tissue events allowing the formation of blood vessels (Risau, W. "Dif ferentiation of the endothelium”. Faseb. J .. 1995, 6-933):
  • Vasculogenesis is essentially an embryonic phenomenon whereas angiogenesis appears not only during embryogenesis, but also during adulthood. This late angiogenesis responds to physiological processes, such as tissue healing or endometrial regeneration, as well as to pathological phenomena, for example during degenerative complications of insulin-dependent diabetes, chronic inflammatory rheumatism, tumor progression or endometriosis.
  • the inhibition of angiogenesis is therefore a therapeutic objective in these pathologies, whereas on the contrary, induction of angiogenesis is sought in tissue healing, skin grafting or post-ischemic re-vascularization (Battegay , EJ "Angiogenesi ⁇ : Mechanistic insights, neovascular diseases, and therapeutic prospects ". J. Mol. Med., 1995, 333-346).
  • Animal models allow the exploration of both direct and indirect mechanisms of angiogenesis.
  • Such models are for example the following:
  • vi tro models use the ability of cultured cells to form tubular structures centered on a luminal space representative of physiological light from the vessels of the circulatory system.
  • Such models are for example:
  • EHS matrix gel It is an extracellular matrix which is secreted by a mouse EHS tumor and the composition of which was initially described by HK Kleinman (Kleinman, HK, ML McGarvey, LA Liotta, PG Robey, K. Tryggvason, GR Martin, "Isolation and characterization of type IV procollagen, laminin and heparin sulfate proteoglycan from EHS sarcoma ". Bi ochemi stry, 1982, 21, 6188-6193).
  • This matrix is purified by a non-denaturing process to serve as a three-dimensional support for cell culture.
  • vascular embryonic bodies The cells of the early stroma of the mouse embryo, designated in the literature by ES cells, form embryonic bodies after cultivation in vitro under appropriate conditions. These embryonic bodies are capable of forming full cysts, the cells of the outer shell of which express certain angiogenic differentiation characteristics of the vascular type, designated vascular embryonic bodies (Wang, R., R. Clark., VL Bautch, "Embryonnic stem cell- derived cystic embryo ⁇ d bodies form vascular channels: an in vitro model of blood vessel development ", Development 1992, 114, 303- 316). This model explores the very initial phases of embryonic vasculogenesis. Indeed, although the priming of vascular channels exists in this model, these structures remain inside the embryonic body and do not continue their development. It is therefore exclusively a phenomenon of vasculogenesis.
  • the aim of the present invention is precisely to provide a model covering the two main phases of vascular morphogenesis, vasculogenesis, that is to say the formation of neo-vessels from undifferentiated cells, and angiogenesis, this ie vascular extension from pre-formed neo-vessels.
  • An additional object of the present invention which is not achieved by any of the models of the prior art, is to be able to easily distinguish, cell multiplication, migration with invasion of the surrounding tissue, tubing with formation of an internal lumen , budding with the formation of secondary structures.
  • the collagen gel used in step (b) consists of types I and III collagen; according to a preferred embodiment, it comprises between approximately 15 and 100%, and advantageously between 30 and 100%, of type III collagen.
  • the cells with angiogenic potential envisaged in the context of the invention are more particularly chosen from cells of a line of embryonic mammalian stem cells, teratocarcinoma cells, tumor cells, or any other type of cells having angiogenic potential. as cells that have acquired this angiogenic potential by genetic modification.
  • a mammalian embryonic stem cell line mention may be made of mouse ES cells, such as the D3 line derived from mouse blastocysts (ATCC CRL 1934).
  • step (b) Independently of the culture medium adapted to the cells cultivated in step (b) and which is a conventional medium which the skilled person is able to determine according to the nature of the cells used, the Applicant has observed that the addition in said step (b) of one or more growth factors, for example incorporated into the collagen gel, and more particularly of FGF (fibroblast growth factor) and especially of VEGF
  • vascular endothlial growth factor or a mixture of FGF and VEGF, makes it possible to approximately double the number of bodies embryonic, to promote and accelerate the formation of tubular structures.
  • the method of the invention defined above makes it possible to induce in cells with angiogenic potential a surprising cellular effect, in fact, if the vascular potential of the embryonic bodies was known, the revelation of the expression in vi tro of this potential of differentiation is remarkable.
  • This process provides a new model of vascular morphogenesis ensuring separate exploration during step (c) during the same experiment of the two phases representative of vascular morphogenesis:
  • the invention therefore also relates to an in vi tro model of vascular morphogenesis, characterized in that it comprises cells with angiogenic potential included in a collagen gel comprising type III collagen or consisting exclusively of type III collagen, in a culture medium adapted to said cells.
  • This collagen gel is advantageously made up of collagen types I and III of placental origin, and according to a preferred embodiment, it comprises between approximately 5 and 100% of collagen type III.
  • the model of the invention also comprises one or more growth factors.
  • Factors of preferred growths are FGF but especially VEGF, or a mixture of FGF and VEGF.
  • the collagen gel consists of collagen types I and III, and in this case comprises between approximately 15 and 100%, and advantageously between 30 and 100 %, type III collagen.
  • the cells with angiogenic potential are chosen from cells of a line of embryonic mammalian stem cells, teratocarcinoma cells, tumor cells, or any other type of cells presenting an angiogenic potential such as cells having acquired by genetic modification this potential angiogenic.
  • a mammalian embryonic stem cell line mention may be made of mouse ES cells, such as the line D3 derived from mouse blastocysts (ATCC CRL 1934).
  • the method of the invention and the model associated with it, described above are particularly suitable for industrial screening of medicaments on the basis of their pro cu anti vascular properties.
  • they give the possibility of studying vascular development, but also vascular dysfunctions, using for example neutralizing antibodies or antisense oligonucleotide probes.
  • the implementation of the process of the invention with molecules, in particular whose activity is unknown makes it possible to select those which would have a property of modulation of the phenomena of vasculogenesis or angiogenesis.
  • Such molecules would have a major pharmacological interest in multiple pathologies, both human and animal, involving vascular dysfunction, such as degenerative complications of diabetes, inflammatory joint rheumatism, tumor and metastatic progression.
  • the invention also relates to a method for screening for substances capable of promoting or inhibiting vascular morphogenesis using a method for culturing cells with angiogenic potential described above, characterized in that the test substance to be tested is introduced. at least one of steps (a) to (c) of this process, and in that the presence or absence of vascular morphogenesis is measured by any suitable means.
  • the implementation of this screening method can be advantageously carried out using a kit comprising at least one protected compartment containing a model of the invention described above, or one or more series of protected compartments, each series comprising a compartment containing a gel collagen and at least one of the following compartments:
  • the application of the method of the invention to drug screening that finds applications in the field of diagnosis, in humans or animals, of the metastatic potential of a tumor and of pathologies with a vascular component.
  • the vascular supply of a tumor is often correlated with its metastatic potential.
  • the measurement of the angiogenic potential according to the method of the invention makes it possible to predict the severity of the tumor.
  • establishing a prognosis for the severity of a solid malignant tumor could then be improved by taking into account the angiogenic power of the tumor.
  • cytological tests are carried out to measure the development of the intra-tumor vascular network but no functional test exists.
  • the invention therefore also relates to a method for diagnosing the metastatic potential of a tumor in a patient using a method for culturing cells with angiogenic potential described above, characterized in that the cells with angiogenic potential included in the collagen gel are tumor cells taken from said patient.
  • kits comprising one or more series of protected compartments, each series comprising a compartment containing a collagen gel and at least one of the following compartments:
  • Pathologies with a vascular component such as diabetes, rheumatism, endometriosis and cancers cause the presence in biological fluids of angiogenic factors, such as FGF or VEGF, which may constitute markers of the development of the disease.
  • the invention therefore relates another method for diagnosing a pathology with a vascular component in a patient using a method for culturing cells with angiogenic potential described above, characterized in that in at least one of steps (a) to (c) , a biological sample taken from said patient and likely to contain angiogenic factors capable of promoting vascular morphogenesis is added.
  • the biological samples on which this diagnosis can be performed are in particular serum, urine, cerebrospinal fluid, plasma, etc.
  • kits comprising at least one protected compartment containing a model of the invention described above, or one or more series of protected compartments, each series comprising a compartment containing a collagen gel, and at least one of the following compartments: - a compartment containing cells with angiogenic potential,
  • the method of culturing cells with angiogenic potential of the invention makes it possible to prepare a collagen-based biomaterial in which are included, depending on the stage of step (c), cells, either with angiogenic potential, or having more or less already induced vascular differentiation.
  • the invention therefore also relates to a cellularized biomaterial consisting of a collagen gel comprising type III collagen or consisting exclusively of type III collagen, in which cells with angiogenic potential are included or cells which have more or less induced vascular differentiation.
  • the above biomaterial can also comprise or be impregnated with a culture medium suitable for the cells incorporated in the collagen gel.
  • the collagen gel used in the composition of this biomaterial consists of collagen types I and III, and according to a preferred embodiment, it comprises between approximately 15 and 100% and advantageously between 30 and 100%, collagen type III. It can be human or animal collagens, obtained from placenta.
  • the cells used in the composition of this biomaterial may, moreover, have been, prior to their inclusion in the collagen gel, genetically modified to produce therapeutic active principles, such as growth factors.
  • This biomaterial can also, according to the method of 1 : invention, be impregnated with at least one growth factor, such as VEGF and / or FGF.
  • at least one growth factor such as VEGF and / or FGF.
  • a biomaterial in accordance with the invention is useful as a cellular implant to replace or compensate for the defective vascularization of an organ, or even in a cell therapy strategy, to allow the in vi vo production of a therapeutic active principle.
  • An application of the invention close to the previous one, consists in using in a body, as an implant colonizable by cells, a biomaterial consisting of a collagen gel comprising type III collagen or consisting exclusively of type III collagen, impregnated with at least one growth factor, preferably chosen from FGF and VEGF.
  • a biomaterial consisting of a collagen gel comprising type III collagen or consisting exclusively of type III collagen, impregnated with at least one growth factor, preferably chosen from FGF and VEGF.
  • the biomaterial previously, it comprises, according to a preferred embodiment, between approximately 15 and 100% and advantageously between 30 and 100%, of type III collagen.
  • It can be human or animal collagens, obtained from placenta.
  • This biomaterial is particularly useful as an implant that can be colonized in vi vo, in the organism of a human or animal subject, by cells of said organism.
  • This biomaterial can then comprise, in addition to the collagen gel and the growth factor (s), a suitable, or even selective, medium, promoting its colonization
  • FIGS. 1 and 2 represent photos, in inverted microscopy, of vascularized embryonic bodies obtained with the process of the invention.
  • ES cells of the D3 line derived from mouse blastocysts are cultured in 100 mm culture dishes (Costar, Cambridge, Ma, USA, ref. 3100) coated with 0.2% gelatin (Sigma, Saint
  • the culture medium consists of “Dubelco Modified Eagle's Medium (DMEM) High Glucose (Gibco 41965-039) medium supplemented with 15% fetal calf serum (FCS, Gibco, Grand Island, NY, USA), sodium pyruvate 1 mM (Gibco 1136-039), non-essential amino acids 0.1 mM (Gibco 11140-035), L-Glutamine 2 mM (Gibco 25030-032), 150 mM mono-thio-glycerol (Sigma, Saint Louis, Mi, USA M17353) and a mixture of penicillin-streptomycin 100 ⁇ g / ml (Gibco 15140-030).
  • DMEM Dubelco Modified Eagle's Medium
  • FCS Gibco, Grand Island, NY, USA
  • FCS fetal calf serum
  • FCS Gibco, Grand Island, NY, USA
  • FCS fetal calf serum
  • FCS Gibco, Grand Island,
  • the cells are maintained in undifferentiated form by adding human recombinant LIF (leukemia inhibiting factor) 1000 u / ml (Schmitt, R., M. Bruyns, HR Snodgrass, "Hematopoetic development of embryonic stem cells in vitro: cytokin and receptor gene expression ". Genes Dev., 1991, 5, 728-740).
  • LIF leukemia inhibiting factor
  • the culture medium supplemented with LIF is renewed every day. Under these conditions, 95% of the population of ES cells is in an undifferentiated state clearly visible in phase contrast microscopy. A stock of several dozen cryotubes containing 5 million ES cells from the same passage was prepared in advance.
  • a D3 cell ampoule is thawed and then cultured as indicated above. After two days of culture, the cells are subconfluent. They are then peeled off with a 0.25% trypsin solution (Gibco) in saline buffer (PBS) containing 1 mM EDTA, then seeded at the rate of 5 million cells per dish for 24 hours. Three hours before the induction of differentiation, the medium containing LIF is renewed. After trypsination, the differentiation is carried out in bacteriological culture dishes (35/10 mm) (Greiner, 627102). Are mixed by box:
  • differentiation inductions are carried out by replacing the collagen solution with 1.1% methyl cellulose (Methocel high viscosity, Fluka A. G., Buchs, Switzerland) according to the same technique.
  • the collagen concentration must of course be adapted in order to have a gel which is neither too liquid nor too solid;
  • the amount of collagen can be between 0.5 mg / ml and 20 mg / ml.
  • the proportions of cells relative to the amount of collagen are likely to admit variations compared to the diagram reported above. Conclusive results have been obtained with mixtures comprising between 500 to 1500 cells for approximately 1.5 mg of collagen.
  • the gels are dried on a slide according to the method initially described (Lanotte, M., "Terminal dif f erentiation of hematopoietic cell clones cultured in tridimesional collagen matrix: in situ cell morphology and enzyme histochemistry analysis”. Biol. Cell., 1984 , 50, 107-120), then colored with May Grunewald Giemsa (Biolyon, Unipath, Dardilly, France 09262 and 09766) according to the supplier's recommendations.
  • the dried gels can also be fixed with a 3% paraformaldehyde solution prepared in phosphate buffered saline (PBS) in order to allow the performance of an indirect immunofluorescence test.
  • PBS phosphate buffered saline
  • the slides are then brought into contact at 37 ° C. for one hour with a rat monoclonal antibody directed against a vascular cell marker molecule (PECAM for Platelet Endothelial Cell Adhesion Molecule, or von Willebrand Factor) (Risau, W., "Di ff erentiation of the endothelium ". Faseb J., 1995, 6-933).
  • PECAM Platelet Endothelial Cell Adhesion Molecule, or von Willebrand Factor
  • the slides are incubated for 30 minutes with a solution containing fragments F (ab ') 2 of goat serum directed against rat immunoglobulins G diluted to 1/100 (Jackson ImmunoResearch, Immunotech, France) in PBS. Last two washes in PBS allow observation of the slides under a fluorescence microscope.
  • EHS embryonic bodies
  • Methyl cellulose allows the formation of CE in a few days. After three days, cell clusters are observed. These clumps grow until they are visible to the naked eye after five days.
  • the CE thus obtained can have different morphologies depending on whether or not they are surrounded by a cell shell. However, no external vascular structure is observed with this culture support.
  • the method of the invention using a collagen I + III gel support allows the formation of CEs having radiant tubular structures.
  • the undifferentiated ES cells incorporated into the collagen I + III gel containing the SD solution have the same appearance as in the presence of methyl cellulose during the first days of culture. But, between D5 and D8, the CEs trained (at least thirty per box) see their size increase. During the following days, some of them present radiant tubular structures whose length is mostly less than the diameter of the CE in question. The percentage of vascularization can nevertheless reach 50%. These tubular structures are not revealed in immunofluorescence by monoclonal antibodies directed at the center of the marker molecules of cells of vascular origin.
  • VEGF vascular endothelial growth factor
  • FIG. 1 represents, in reverse microscopy, different types of embryonic bodies obtained on D10 in a collagen gel supplemented with VEGF and FGF.
  • VEGF vascular endothelial growth factor
  • FGF FGF
  • the photo of Figure 2 represents, in inverted microscopy, a highly vascularized CE

Abstract

A method for culturing cells having angiogenic potential in order to induce vascular morphogenesis in said cells is disclosed. According to the method, (a) a cell preparation containing cells having angiogenic potential is prepared; (b) the cells are contacted with a collagen gel containing type III collagen or consisting entirely of type III collagen, in the presence of a suitable culture medium for said cells; and (c) the cells are allowed to develop in the collagen gel for a sufficient time to enable vascular morphogenesis. In vitro vascular morphogenesis models, and the use thereof, in particular for drug screening and for diagnosing diseases with a vascular component in humans and animals, are also disclosed.

Description

PROCEDE DE CULTURE DE CELLULES A POTENTIEL ANGIOGENIQUE METHOD FOR CULTURING CELLS WITH ANGIOGENIC POTENTIAL
La présente invention concerne le domaine de la culture cellulaire et plus particulièrement un procédé de culture de cellules à potentiel angiogénique pour induire la morphogénèse vasculaire de ces cellules. La morphogénèse vasculaire est définie comme l'ensemble des événements cellulaires et tissulaires permettant la formation des vaisseaux sanguins (Risau, W. "Dif f erentiation of the endothelium" . Faseb. J.. 1995, 6- 933) :The present invention relates to the field of cell culture and more particularly to a method of culturing cells with angiogenic potential to induce the vascular morphogenesis of these cells. Vascular morphogenesis is defined as the set of cellular and tissue events allowing the formation of blood vessels (Risau, W. "Dif ferentiation of the endothelium". Faseb. J .. 1995, 6-933):
- soit à partir de cellules indifférenciées (angioblastes) , il s'agit alors de la vasculogénèse,- either from undifferentiated cells (angioblasts), this is then vasculogenesis,
- soit à partir de vaisseaux préformés, et il s'agit alors de 1 ' angiogénèse . La vasculogénèse est essentiellement un phénomène embryonnaire alors que 1 ' angiogénèse apparaît non seulement durant l'embryogenèse, mais également durant la vie adulte. Cette angiogénèse tardive répond à des processus physiologiques, tels que la cicatrisation tissulaire ou la régénération endométriale, ainsi qu'à des phénomènes pathologiques, lors par exemple de complications dégénérativeε du diabète insulino-dépendant , de rhumatismes inflammatoires chroniques, d'une progression tumorale ou d'une endometriose. L'inhibition de 1 ' angiogénèse est donc un objectif thérapeutique dans ces pathologies, alors qu'au contraire, l'induction de 1 ' angiogénèse est recherchée dans la cicatrisation tissulaire, la greffe de peau ou la re-vascularisation post-ischémique (Battegay, E. J. " Angiogenesiε : Mechanistic insights, neovascular diseases, and therapeutic prospects". J. Mol. Med. , 1995, 333-346) .- either from preformed vessels, and this is then angiogenesis. Vasculogenesis is essentially an embryonic phenomenon whereas angiogenesis appears not only during embryogenesis, but also during adulthood. This late angiogenesis responds to physiological processes, such as tissue healing or endometrial regeneration, as well as to pathological phenomena, for example during degenerative complications of insulin-dependent diabetes, chronic inflammatory rheumatism, tumor progression or endometriosis. The inhibition of angiogenesis is therefore a therapeutic objective in these pathologies, whereas on the contrary, induction of angiogenesis is sought in tissue healing, skin grafting or post-ischemic re-vascularization (Battegay , EJ "Angiogenesiε: Mechanistic insights, neovascular diseases, and therapeutic prospects ". J. Mol. Med., 1995, 333-346).
Ces perspectives thérapeutiques nécessitent de disposer de modèles in vivo ou in vitro permettant le criblage de substances capables d'inhiber ou, au contraire, d'induire 1 ' angiogénèse. On connait dans l'art antérieur de tels modèles, parmi lesquels on peut distinguer les modèles animaux et les modèles in vitro.These therapeutic perspectives require the availability of in vivo or in vitro models allowing the screening of substances capable of inhibiting or, on the contrary, inducing angiogenesis. In the prior art, such models are known, among which animal models and in vitro models can be distinguished.
Les modèles animaux permettent l'exploration à la fois des mécanismes directs et indirects de 1 ' angiogénèse . De tels modèles sont par exemple les suivants :Animal models allow the exploration of both direct and indirect mechanisms of angiogenesis. Such models are for example the following:
- La néovascularisation de la cornée d'oeil de lapin (Hendkin, P. "Ocular neovascularization . The Kril mémorial lecture". Am. J. Ophtalmol . , 1978, 85, 287-301) . Dans ce modèle, la cornée est normalement vascularisée et un traumatisme chimique ou physique induit la formation de néo-vaisseaux capillaires. Ce modèle animal est limité par les complexités de manipulation, notamment la standardisation du traumatisme, le coût et les difficultés de l'expérience puisque un lapin est nécessaire pour chaque test. En outre, son emploi est soumis aux réglementations sur l'expérimentation animale. - La membrane d'oeuf de poule embryonné- The neovascularization of the rabbit eye cornea (Hendkin, P. "Ocular neovascularization. The Kril memorial reading". Am. J. Ophtalmol., 1978, 85, 287-301). In this model, the cornea is normally vascularized and a chemical or physical trauma induces the formation of capillary neo-vessels. This animal model is limited by the complexities of handling, in particular the standardization of the trauma, the cost and the difficulties of the experiment since a rabbit is necessary for each test. In addition, its use is subject to regulations on animal testing. - The embryonated chicken egg membrane
(Sorgente, N. , K. E. Kuetter, L. W. Soble, "The résistance of certain tissues to invasion". Lab. Invest . , 1975, 32, 217-222) . Il s'agit d'un modèle traditionnel de l'embryologie selon lequel la formation rapide d'un réseau vasculaire durant la phase initiale de l'embryogenèse permet l'analyse des effets inhibiteurs de 1 ' angiogénèse . Bien que le matériel mis en oeuvre dans ce modèle soit peu coûteux et non soumis à une réglementation contraignante, il est de manipulation délicate et non automatisable. En outre, les informations recueillies grâce à ce modèle aviaire ne sont pas directement transposables à l'homme.(Sorgente, N., KE Kuetter, LW Soble, "The resistance of certain tissues to invasion". Lab. Invest., 1975, 32, 217-222). It is a traditional model of embryology according to which the rapid formation of a vascular network during the initial phase of embryogenesis allows the analysis of the inhibitory effects of angiogenesis. Although the equipment used in this model is inexpensive and not subject to restrictive regulations, it is delicate to handle and cannot be automated. In addition, the information collected using this avian model cannot be directly transposed to humans.
L'utilisation de modèles animaux est rendue difficile par la variabilité inter-individus limitant les possibilités de standardisation, et par la mise en oeuvre de manipulations lourdes peu automatisables. En outre, la question de la spécificité d'espèce des effets observés est toujours soulevée, dès que le modèle s'éloigne du modèle mammifère. Enfin, la mise en place de réglementations visant à limiter l'usage d'animaux dans l'expérimentation cosmétique et pharmaceutique, n'est pas favorable aux développements de ce type de modèle.The use of animal models is made difficult by the inter-individual variability limiting the possibilities of standardization, and by the implementation of heavy manipulations which are not very automated. In addition, the question of the species specificity of the effects observed is always raised, as soon as the model moves away from the mammalian model. Finally, the implementation of regulations aimed at limiting the use of animals in cosmetic and pharmaceutical experimentation is not favorable to the development of this type of model.
Les modèles in vi tro utilisent la capacité de cellules en culture de former des structures tubulaires centrées sur un espace luminal représentatif de la lumière physiologique des vaisseaux de l'appareil circulatoire. De tels modèles sont par exemple :In vi tro models use the ability of cultured cells to form tubular structures centered on a luminal space representative of physiological light from the vessels of the circulatory system. Such models are for example:
- Les gels tridimensionnels de collagène I (Montesano, R. , L. Orci, "Phorbol ester induces angiogenesis in vi tro from large vessel endothelial cell". J . Cel l . Physiol . , 1987, 139, 284-291) (Montesano, R. , L. Orci, "Tumor-promoting phorbol ester induces angiogenesis in vi tro " , Cel l , 1985, 42, 469-477) . Ce modèle permet de cultiver des cellules d'une lignée aortique de boeuf à la surface d'un gel formé de collagène purifié de type I en présence d'effecteurs de différenciation cellulaire tel que le phorbol myristyl acétate (PMA) . Ces cellules induisent la formation de cordes cellulaires qui parfois envahissent le gel sous-jacent pour former de véritables tubules dans l'épaisseur du gel. Ce modèle est limité à l'utilisation de quelques lignées cellulaires très particulières ayant le potentiel de former des structures tubulaires différenciées. L'inducteur de différenciation le plus démonstratif dans ce modèle est un ester de phorbol, le PMA, qui est un promoteur tumoral non physiologique, alors que les inducteurs physiologiques, comme le FGF (fibroblast growth factor) , ont des effets dont la reproductibilité interlaboratoire n'est pas satisfaisante. La notion de structures tubulaires tri¬ dimensionnelles, qui est un élément crucial des phénomènes de morphogénèse vasculaire, est toujours discutable dans ce modèle où l'envahissement du gel par les tubules néo¬ formés reste très peu profond, et où seul un expérimentateur averti peut clairement différencier les cordes cellulaires formées à la surface du gel, des tubules très légèrement sous-jacents. En conséquence, une simple photographie de la culture en microscopie inversée ne permet pas d'affirmer acec certitude l'envahissement du gel, et seule la technique lourde d'analyse histologique du gel en coupes sériées peut apporter la preuve de l'envahissement du gel par les formations tubulaires.- Three-dimensional collagen I gels (Montesano, R., L. Orci, "Phorbol ester induces angiogenesis in vi tro from large vessel endothelial cell". J. Cel l. Physiol., 1987, 139, 284-291) (Montesano , R., L. Orci, "Tumor-promoting phorbol ester induces angiogenesis in vi tro", Cel l, 1985, 42, 469-477). This model makes it possible to cultivate cells of a beef aortic line on the surface of a gel formed from purified collagen type I in the presence of cell differentiation effectors such as phorbol myristyl acetate (PMA). These cells induce the formation of cellular cords which sometimes invade the underlying gel to form real tubules in the thickness of the gel. This model is limited to the use of a few very specific cell lines with the potential to form differentiated tubular structures. The most demonstrative inducer of differentiation in this model is an ester of phorbol, PMA, which is a non-physiological tumor promoter, while physiological inducers, such as FGF (fibroblast growth factor), have effects whose interlaboratory reproducibility is not satisfactory. The notion of three-dimensional tubular structures, which is a crucial element of the phenomena of vascular morphogenesis, is always debatable in this model where the invasion of the gel by the neo¬ formed tubules remains very shallow, and where only a knowledgeable experimenter clearly differentiate the cellular strings formed on the surface of the gel, very slightly underlying tubules. Consequently, a simple photograph of the culture in inverted microscopy does not make it possible to confirm with certainty the invasion of the gel, and only the heavy technique of histological analysis of the gel in serial sections can provide evidence of the invasion of the gel. by tubular formations.
- Le gel de matrice EHS . Il s'agit de matrice extracellulaire qui est sécrétée par une tumeur EHS de souris et dont la composition a été initialement décrite par H. K. Kleinman (Kleinman, H. K. , M. L. McGarvey, L. A. Liotta, P. G. Robey, K. Tryggvason, G. R. Martin, "Isolation and characterization of type IV procollagen, laminin and heparin sulfate proteoglycan from EHS sarcoma". Bi ochemi stry, 1982, 21, 6188-6193) . Cette matrice est purifiée par un procédé non dénaturant pour servir de support tri-dimensionnel de culture cellulaire. Les cellules à potentiel vasculaire forment en quelques heures à la surface de ce gel des structures en réseau assimilées à des structures vasculaires. La rapidité des phénomènes fait admettre aux spécialistes du domaine qu'il s'agit plus d'un phénomène de migration et d'organisation cellulaire, que d'une véritable différenciation fonctionnelle. De plus, des cellules non programmées pour former in vi vo des néo-capillaires, donnent dans ce modèle, des structures en réseau comparables à celles obtenues avec des cellules vasculaires. La spécificité vasculaire des phénomènes observés dans ce modèle n'est donc pas admise de façon générale. Ce produit est d'ailleurs commercialisé dans diverses applications de recherche, autres que l'étude des phénomènes angiogéniques .- EHS matrix gel. It is an extracellular matrix which is secreted by a mouse EHS tumor and the composition of which was initially described by HK Kleinman (Kleinman, HK, ML McGarvey, LA Liotta, PG Robey, K. Tryggvason, GR Martin, "Isolation and characterization of type IV procollagen, laminin and heparin sulfate proteoglycan from EHS sarcoma ". Bi ochemi stry, 1982, 21, 6188-6193). This matrix is purified by a non-denaturing process to serve as a three-dimensional support for cell culture. Cells with vascular potential form in a few hours on the surface of this gel network structures assimilated to vascular structures. The rapidity of the phenomena makes specialists in the field admit that it is more a phenomenon of migration and cellular organization, than a real functional differentiation. In addition, cells not programmed to form neo-capillaries in vi vo, give in this model, network structures comparable to those obtained with vascular cells. The vascular specificity of the phenomena observed in this model is therefore not generally accepted. This product is also marketed in various research applications, other than the study of angiogenic phenomena.
- Les lignées de cellules embryonnaires de souris. Les cellules du stroma précoce de l'embryon de souris, désignées dans la littérature par cellules ES, forment des corps embryonnaires après culture in vitro dans des conditions appropriées. Ces corps embryonnaires sont capables de former des kystes pleins dont les cellules de la coque externe expriment certains caractères de différenciation angiogénique de type vasculaire, désignés corps embryonnaires vasculaires (Wang, R. , R. Clark., V. L. Bautch, "Embryonnic stem cell-derived cystic embryoïd bodies form vascular channels : an in vitro model of blood vessel development " , Development 1992, 114, 303- 316) . Ce modèle explore les phases très initiales de la vasculogénèse embryonnaire. En effet, bien que l'amorce de canaux vasculaires existe dans ce modèle, ces structures restent à 1 ' intérieur du corps embryonnaire et ne poursuivent pas leur développement. Il s'agit donc exclusivement d'un phénomène de vasculogénèse.- Mouse embryonic cell lines. The cells of the early stroma of the mouse embryo, designated in the literature by ES cells, form embryonic bodies after cultivation in vitro under appropriate conditions. These embryonic bodies are capable of forming full cysts, the cells of the outer shell of which express certain angiogenic differentiation characteristics of the vascular type, designated vascular embryonic bodies (Wang, R., R. Clark., VL Bautch, "Embryonnic stem cell- derived cystic embryoïd bodies form vascular channels: an in vitro model of blood vessel development ", Development 1992, 114, 303- 316). This model explores the very initial phases of embryonic vasculogenesis. Indeed, although the priming of vascular channels exists in this model, these structures remain inside the embryonic body and do not continue their development. It is therefore exclusively a phenomenon of vasculogenesis.
La multiplication des modèles in vitro témoigne de la difficulté de définir un modèle général, simple d'emploi et reproductible. En effet, comme indiqué pour chacun des exemples de l'art antérieur rappelés ci- dessus, l'organisation tri-dimensionnelle des structures vasculaires néo-formées est difficile à démontrer. En outre, les phénomènes observés avec les modèles existants ne sont représentatifs que de certaines étapes du processus complexe qui s'enchaînent selon une chronologie précise pour permettre la formation d'un néo-capillaire. Ainsi, l'intérêt de la diversité des modèles existants repose sur leur complémentarité, chaque modèle étant adapté à 1 ' étude de certains aspects seulement de 1 'angiogénèse; or cette diversité n'est pas compatible avec une utilisation industrielle facile.The proliferation of in vitro models testifies to the difficulty of defining a general model, easy to use and reproducible. Indeed, as indicated for each of the examples of the prior art mentioned above, the three-dimensional organization of the neo-formed vascular structures is difficult to demonstrate. In addition, the phenomena observed with existing models are only representative of certain stages of the complex process which are linked according to a precise chronology to allow the formation of a neo-capillary. Thus, the interest of the diversity of existing models rests on their complementarity, each model being adapted to the study of certain aspects only of angiogenesis; this diversity is not compatible with easy industrial use.
Le but de la présente invention est précisément d'offir un modèle couvrant les deux grandes phases de la morphogénèse vasculaire, la vasculogénèse, c'est à dire la formation de néo-vaisseaux à partir de cellules indifférenciées, et 1 'angiogénèse, c'est à dire l'extension vasculaire à partir de néo-vaisseaux pré¬ formés. Un but supplémentaire de la présente invention, qui n'est réalisé par aucun des modèles de l'art antérieur, est de pouvoir distinguer aisément, la multiplication cellulaire, la migration avec envahissement du tissu avoisinant, la tubulisation avec formation d'une lumière interne, le bourgeonnement avec formation de structures secondaires.The aim of the present invention is precisely to provide a model covering the two main phases of vascular morphogenesis, vasculogenesis, that is to say the formation of neo-vessels from undifferentiated cells, and angiogenesis, this ie vascular extension from pre-formed neo-vessels. An additional object of the present invention, which is not achieved by any of the models of the prior art, is to be able to easily distinguish, cell multiplication, migration with invasion of the surrounding tissue, tubing with formation of an internal lumen , budding with the formation of secondary structures.
Ces buts sont atteints grâce à un procédé de culture de cellules à potentiel angiogénique, général, simple d'emploi et reproductible, permettant d'induire la morphogénèse vasculaire desdites cellules, comprenant les étapes consistant à : - (a) obtenir une préparation cellulaire contenant des cellules à potentiel angiogénique;These aims are achieved by a method of culturing cells with angiogenic potential, general, simple to use and reproducible, making it possible to induce the vascular morphogenesis of said cells, comprising the steps consisting in: - (a) obtaining a cell preparation containing cells with angiogenic potential;
- (b) mettre en contact ces cellules avec un gel de collagène comprenant du collagène de type III ou constitué exclusivement de collagène de type III, en présence d'un milieu de culture adapté auxdites cellules;- (b) bringing these cells into contact with a collagen gel comprising type III collagen or consisting exclusively of type III collagen, in the presence of a culture medium suitable for said cells;
(c) laisser les cellules se développer dans le gel de collagène pendant une durée suffisante pour permettre la morphogénèse vasculaire.(c) allowing the cells to grow in the collagen gel for a time sufficient to allow vascular morphogenesis.
Le gel de collagène mis en oeuvre à l'étape (b) est constitué de collagène des types I et III; selon une forme de réalisation préférée, il comprend entre environ 15 et 100 %, et avantageusement entre 30 et 100 %, de collagène de type III.The collagen gel used in step (b) consists of types I and III collagen; according to a preferred embodiment, it comprises between approximately 15 and 100%, and advantageously between 30 and 100%, of type III collagen.
La préparation de gels de collagène de ces types sont notamment décrit dans les demandes de brevet européen publiées sous les numéros 214 035, 242 270, 667The preparation of collagen gels of these types is described in particular in the European patent applications published under the numbers 214 035, 242 270, 667
332. D'autres collagènes de ces types sont disponibles commercialement.332. Other collagens of these types are commercially available.
Il peut s'agir de collagènes humains ou d'origine animale, obtenus de placenta.It can be human or animal collagens, obtained from placenta.
Les cellules à potentiel angiogénique envisagées dans le cadre de 1 ' invention sont plus particulièrement choisies parmi des cellules d'une lignée de cellules souches embryonnaires de mammifère, des cellules de tératocarcinome, des cellules tumorales, ou tout autre type de cellules présentant un potentiel angiogénique comme des cellules ayant acquis par modification génétique ce potentiel angiogénique. A titre d'exemple spécifique de cellules d'une lignée de cellules souches embryonnaires de mammifère, on peut citer des cellules ES de souris, telle que la lignée D3 dérivée de blastocystes de souris (ATCC CRL 1934) .The cells with angiogenic potential envisaged in the context of the invention are more particularly chosen from cells of a line of embryonic mammalian stem cells, teratocarcinoma cells, tumor cells, or any other type of cells having angiogenic potential. as cells that have acquired this angiogenic potential by genetic modification. By way of specific example of cells of a mammalian embryonic stem cell line, mention may be made of mouse ES cells, such as the D3 line derived from mouse blastocysts (ATCC CRL 1934).
Indépendamment du milieu de culture adapté aux cellules cultivée de l'étape (b) et qui est un milieu classique que l'homme du métier est à même de déterminer selon la nature des cellules mises en oeuvre, la Demanderesse a observé que l'adjonction à ladite étape (b) d'un ou plusieurs facteurs de croissance, par exemple incorporés au gel de collagène, et plus particulièrement de FGF (fibroblast growth factor) et surtout de VEGFIndependently of the culture medium adapted to the cells cultivated in step (b) and which is a conventional medium which the skilled person is able to determine according to the nature of the cells used, the Applicant has observed that the addition in said step (b) of one or more growth factors, for example incorporated into the collagen gel, and more particularly of FGF (fibroblast growth factor) and especially of VEGF
(vascular endothlial growth factor) ou d'un mélange de FGF et de VEGF, permet de doubler environ le nombre de corps embryonnaires, de favoriser et d'accélérer la formation des structures tubulaires.(vascular endothlial growth factor) or a mixture of FGF and VEGF, makes it possible to approximately double the number of bodies embryonic, to promote and accelerate the formation of tubular structures.
Le procédé de l'invention défini ci-dessus permet d'induire chez des cellules à potentiel angiogénique un effet cellulaire surprenant, en effet, si le potentiel vasculaire des corps embryonnaires était connu, la révélation de l'expression in vi tro de ce potentiel de différenciation est remarquable. Ce procédé permet de disposer d'un nouveau modèle de morphogénèse vasculaire assurant l'exploration distincte au cours de l'étape (c) lors d'une même expérimentation des deux phases représentatives de la morphogénèse vasculaire :The method of the invention defined above makes it possible to induce in cells with angiogenic potential a surprising cellular effect, in fact, if the vascular potential of the embryonic bodies was known, the revelation of the expression in vi tro of this potential of differentiation is remarkable. This process provides a new model of vascular morphogenesis ensuring separate exploration during step (c) during the same experiment of the two phases representative of vascular morphogenesis:
- la formation de troncs initiaux à partir de corps embryonnaires, laquelle est le reflet de la vasculogénèse, la formation de tubules secondaires au niveau des fourches de division des tubules primaires, laquelle est le reflet d'un bourgeonnement caractéristique de 1 'angiogénèse.- The formation of initial trunks from embryonic bodies, which is a reflection of vasculogenesis, the formation of secondary tubules at the forks of division of the primary tubules, which is a reflection of budding characteristic of angiogenesis.
L'invention concerne donc aussi un modèle in vi tro de morphogénèse vasculaire, caractérisé en ce qu'il comprend des cellules à potentiel angiogénique incluses dans un gel de collagène comprenant du collagène de type III ou constitué exclusivement de collagène de type III, dans un milieu de culture adapté auxdites cellules. Ce gel de collagène est avantageusement constitué de collagène des types I et III d'origine placentaire, et selon une forme de réalisation préférée, il comprend entre environ 5 et 100 % de collagène de type III.The invention therefore also relates to an in vi tro model of vascular morphogenesis, characterized in that it comprises cells with angiogenic potential included in a collagen gel comprising type III collagen or consisting exclusively of type III collagen, in a culture medium adapted to said cells. This collagen gel is advantageously made up of collagen types I and III of placental origin, and according to a preferred embodiment, it comprises between approximately 5 and 100% of collagen type III.
Selon une forme de réalisation particulière, et indépendamment du milieu de culture adapté aux cellules, le modèle de l'invention comprend également un ou plusieurs facteurs de croissance. Des facteurs de croissances préférés sont le FGF mais surtout le VEGF, ou un mélange de FGF et de VEGF.According to a particular embodiment, and independently of the culture medium adapted to the cells, the model of the invention also comprises one or more growth factors. Factors of preferred growths are FGF but especially VEGF, or a mixture of FGF and VEGF.
Conformément au procédé défini précédemment, on préfère, dans le modèle de l'invention, que le gel de collagène soit constitué de collagène des types I et III, et dans ce cas comprenne entre environ 15 et 100 %, et avantageusement entre 30 et 100%, de collagène de type III. les cellules à potentiel angiogénique soient choisies parmi des cellules d'une lignée de cellules souches embryonaires de mammifère, des cellules de tératocarcinome, des cellules tumorales, ou tout autre type de cellules présentant un potentiel angiogénique comme des cellules ayant acquis par modification génétique ce potentiel angiogénique. A titre d'exemple spécifique de cellules d'une lignée de cellules souches embryonaires de mammifère, on peut citer des cellules ES de souris, telle que la lignée D3 dérivée de blastocystes de souris (ATCC CRL 1934) .According to the method defined above, it is preferred, in the model of the invention, that the collagen gel consists of collagen types I and III, and in this case comprises between approximately 15 and 100%, and advantageously between 30 and 100 %, type III collagen. the cells with angiogenic potential are chosen from cells of a line of embryonic mammalian stem cells, teratocarcinoma cells, tumor cells, or any other type of cells presenting an angiogenic potential such as cells having acquired by genetic modification this potential angiogenic. As a specific example of cells of a mammalian embryonic stem cell line, mention may be made of mouse ES cells, such as the line D3 derived from mouse blastocysts (ATCC CRL 1934).
En raison de leur simplicité, le procédé de l'invention et le modèle qui y est associé, précédemment décrits, sont particulièrement adaptés au criblage industriel de médicament sur la base de leurs propriétés pro cu anti vasculaires. En outre, ils donnent la possibilité d'étudier le développement vasculaire, mais également les dysfonctionnements vasculaires, en utilisant par exemple des anticorps neutralisants ou des sondes oligonucléotidiques anti-sens. En effet, la mise en oeuvre du procédé de l'invention avec des molécules, notamment dont l'activité est inconnue, permet de sélectionner celles qui présenteraient une propriété de modulation des phénomènes de vasculogénèse ou d'angiogénèse. De telles molécules auraient un intérêt pharmacologique majeur dans de multiples pathologies tant humaines que animales impliquant un dysfonctionnement vasculaire, comme des complications dégénérâtives du diabète, des rhumatismes inflammatoires articulaires, la progression tumorale et métastatique.Because of their simplicity, the method of the invention and the model associated with it, described above, are particularly suitable for industrial screening of medicaments on the basis of their pro cu anti vascular properties. In addition, they give the possibility of studying vascular development, but also vascular dysfunctions, using for example neutralizing antibodies or antisense oligonucleotide probes. Indeed, the implementation of the process of the invention with molecules, in particular whose activity is unknown, makes it possible to select those which would have a property of modulation of the phenomena of vasculogenesis or angiogenesis. Such molecules would have a major pharmacological interest in multiple pathologies, both human and animal, involving vascular dysfunction, such as degenerative complications of diabetes, inflammatory joint rheumatism, tumor and metastatic progression.
En conséquence, l'invention concerne aussi un procédé de criblage de substances susceptibles de favoriser ou inhiber la morphogénèse vasculaire mettant en oeuvre un procédé de culture de cellules à potentiel angiogénique décrit précédemment, caractérisé en ce que l'on introduit la substance à tester à l'une au moins des étapes (a) à (c) de ce procédé, et en ce que l'on mesure par tout moyen approprié la présence ou l'absence de morphogénèse vasculaire. La mise en oeuvre de ce procédé de criblage peut être avantageusement réalisée grâce à une trousse comprenant au moins un compartiment protégé renfermant un modèle de l'invention décrit précédemment, ou une ou plusieurs séries de compartiments protégés, chaque série comprenant un compartiment renfermant un gel de collagène et au moins un des compartiments suivants :Consequently, the invention also relates to a method for screening for substances capable of promoting or inhibiting vascular morphogenesis using a method for culturing cells with angiogenic potential described above, characterized in that the test substance to be tested is introduced. at least one of steps (a) to (c) of this process, and in that the presence or absence of vascular morphogenesis is measured by any suitable means. The implementation of this screening method can be advantageously carried out using a kit comprising at least one protected compartment containing a model of the invention described above, or one or more series of protected compartments, each series comprising a compartment containing a gel collagen and at least one of the following compartments:
- un compartiment renfermant des cellules à potentiel angiogénique,- a compartment containing cells with angiogenic potential,
- un compartiment renfermant un milieu de culture adaptée auxdites cellules;- A compartment containing a culture medium adapted to said cells;
- un compartiment renfermant un ou plusieurs facteurs de croissance.- a compartment containing one or more growth factors.
Outre, l'application du procédé de l'invention au criblage de drogues, celui trouve des applications dans le domaine du diagnostic, chez l'homme ou l'animal, du potentiel métastatique d'une tumeur et de pathologies à composante vasculaire.In addition, the application of the method of the invention to drug screening, that finds applications in the field of diagnosis, in humans or animals, of the metastatic potential of a tumor and of pathologies with a vascular component.
En effet, la vascularisation d'une tumeur est souvent corrélée à son potentiel metastasique. En conséquence, la mesure du potentiel angiogénique selon le procédé de 1 ' invention permet de pronostiquer la gravité de la tumeur. En cancérologie clinique, l'établissement d'un pronostic de gravité d'une tumeur maligne solide pourrait être alors amélioré par la prise en compte du pouvoir angiogénique de la tumeur. Actuellement des tests cytologiques sont réalisés pour mesurer le développement du réseau vasculaire intra-tumoral mais aucun test fonctionnel n'existe. L'invention concerne donc aussi un procédé de diagnostic du potentiel métastatique d'une tumeur chez un patient mettant en oeuvre un procédé de culture de cellules à potentiel angiogénique décrit précédemment, caractérisé en ce que les cellules à potentiel angiogénique incluses dans le gel de collagène sont des cellules tumorales prélevées chez ledit patient.In fact, the vascular supply of a tumor is often correlated with its metastatic potential. In Consequently, the measurement of the angiogenic potential according to the method of the invention makes it possible to predict the severity of the tumor. In clinical oncology, establishing a prognosis for the severity of a solid malignant tumor could then be improved by taking into account the angiogenic power of the tumor. Currently cytological tests are carried out to measure the development of the intra-tumor vascular network but no functional test exists. The invention therefore also relates to a method for diagnosing the metastatic potential of a tumor in a patient using a method for culturing cells with angiogenic potential described above, characterized in that the cells with angiogenic potential included in the collagen gel are tumor cells taken from said patient.
La mise en oeuvre de ce procédé diagnostic du potentiel metastasique d'une tumeur peut être avantageusement réalisée grâce à une trousse comprenant une ou plusieurs séries de compartiments protégés, chaque série comprenant un compartiment renfermant un gel de collagène et au moins un des compartiments suivants :The implementation of this method for diagnosing the metastatic potential of a tumor can be advantageously carried out using a kit comprising one or more series of protected compartments, each series comprising a compartment containing a collagen gel and at least one of the following compartments:
- un compartiment renfermant un milieu de culture adaptée auxdites cellules; - un compartiment renfermant un ou plusieurs facteurs de croissance;- A compartment containing a culture medium adapted to said cells; - a compartment containing one or more growth factors;
- un compartiment dans lequel seront placées des cellules tumorales prélevées chez ledit patient.- a compartment in which tumor cells taken from said patient will be placed.
Les pathologies à composante vasculaire, telles que des diabètes, des rhumatismes, 1 'endometriose et des cancers engendrent la présence dans les fluides biologiques de facteurs angiogéniques, comme le FGF ou le VEGF, susceptibles de constituer des marqueurs du développement de la maladie. L'invention concerne donc encore un procédé de diagnostic d'une pathologie à composante vasculaire chez un patient mettant en oeuvre un procédé de culture de cellules à potentiel angiogénique décrit précédemment, caractérisé en ce qu'à l'une au moins des étapes (a) à (c) , on ajoute un échantillon biologique prélevé dudit patient et susceptible de contenir des facteurs angiogéniques capables de favoriser la morphogénèse vasculaire. Les échantillons biologiques sur lesquels peuvent être pratiqués ce diagnostic sont notamment le sérum, l'urine, le liquide céphalo-rachidien, le plasma, etc...Pathologies with a vascular component, such as diabetes, rheumatism, endometriosis and cancers cause the presence in biological fluids of angiogenic factors, such as FGF or VEGF, which may constitute markers of the development of the disease. The invention therefore relates another method for diagnosing a pathology with a vascular component in a patient using a method for culturing cells with angiogenic potential described above, characterized in that in at least one of steps (a) to (c) , a biological sample taken from said patient and likely to contain angiogenic factors capable of promoting vascular morphogenesis is added. The biological samples on which this diagnosis can be performed are in particular serum, urine, cerebrospinal fluid, plasma, etc.
La mise en oeuvre de ce procédé de diagnostic de pathologies à composante vasculaire chez un patient peut être avantageusement réalisée grâce à une trousse comprenant au moins un compartiment protégé renfermant un modèle de l'invention décrit précédemment, ou une ou plusieurs séries de compartiments protégés, chaque série comprenant un compartiment renfermant un gel de collagène, et au moins un des compartiments suivants : - un compartiment renfermant des cellules à potentiel angiogénique,The implementation of this method of diagnosing pathologies with a vascular component in a patient can be advantageously carried out using a kit comprising at least one protected compartment containing a model of the invention described above, or one or more series of protected compartments, each series comprising a compartment containing a collagen gel, and at least one of the following compartments: - a compartment containing cells with angiogenic potential,
- un compartiment renfermant un milieu de culture adaptée auxdites cellules;- A compartment containing a culture medium adapted to said cells;
- un compartiment dans lequel sera placée un échantillon biologique prélevé dudit patient.- a compartment in which a biological sample taken from said patient will be placed.
Outre des applications au criblage de drogue et au diagnostic, le procédé de culture de cellules à potentiel angiogénique de l'invention, permet de préparer un biomatériau à base de collagène dans lequel sont incluses, selon le stade de l'étape (c) , des cellules, soit à potentiel angiogénique, soit ayant plus ou moins déjà induit une différenciation vasculaire. L'invention se rapporte donc aussi à un biomatériau cellularisé constitué d'un gel de collagène comprenant du collagène de type III ou constitué exclusivement de collagène de type III, dans lequel sont incluses des cellules à potentiel angiogénique ou des cellules ayant plus ou moins induit une différenciation vasculaire. Le biomatériau précédent peut en outre comprendre ou être imprégné d'un milieu de culture adapté aux cellules incorporées dans le gel de collagène. Avantageusement, le gel de collagène entrant dans la composition de ce biomatériau, est constitué de collagène des types I et III, et selon une forme de réalisation préférée, il comprend entre environ 15 et 100 % et avantageusement entre 30 et 100 %, de collagène de type III. Il peut s'agir de collagènes humains ou d'origine animale, obtenus de placenta.In addition to applications in drug screening and in diagnosis, the method of culturing cells with angiogenic potential of the invention makes it possible to prepare a collagen-based biomaterial in which are included, depending on the stage of step (c), cells, either with angiogenic potential, or having more or less already induced vascular differentiation. The invention therefore also relates to a cellularized biomaterial consisting of a collagen gel comprising type III collagen or consisting exclusively of type III collagen, in which cells with angiogenic potential are included or cells which have more or less induced vascular differentiation. The above biomaterial can also comprise or be impregnated with a culture medium suitable for the cells incorporated in the collagen gel. Advantageously, the collagen gel used in the composition of this biomaterial, consists of collagen types I and III, and according to a preferred embodiment, it comprises between approximately 15 and 100% and advantageously between 30 and 100%, collagen type III. It can be human or animal collagens, obtained from placenta.
Les cellules entrant dans la composition de ce biomatériau peuvent, en outre, avoir été, préalablement à leur inclusion dans le gel de collagène, génétiquement modifiées pour produire des principes actifs thérapeutiques, comme des facteurs de croissance.The cells used in the composition of this biomaterial may, moreover, have been, prior to their inclusion in the collagen gel, genetically modified to produce therapeutic active principles, such as growth factors.
Ce biomatériau peut encore conformément au procédé de 1 : invention être imprégné d'au moins un facteur de croissance, tel que le VEGF et/ou le FGF..This biomaterial can also, according to the method of 1 : invention, be impregnated with at least one growth factor, such as VEGF and / or FGF.
Un biomatériau conforme à 1 ' invention est utile comme implant cellularisé pour remplacer ou compenser la vascularisation défectieuse d'un organe, ou encore dans une stratégie de thérapie cellulaire, pour permettre la production in vi vo d'un principe actif thérapeutique.A biomaterial in accordance with the invention is useful as a cellular implant to replace or compensate for the defective vascularization of an organ, or even in a cell therapy strategy, to allow the in vi vo production of a therapeutic active principle.
Une application de l'invention, proche de la précédente, consiste à mettre en oeuvre dans un organisme, à titre d'implant colonisable par des cellules, un biomatériau constitué d'un gel de collagène comprenant du collagène de type III ou constitué exclusivement de collagène de type III, imprégné d'au moins un facteur de croissance, de préférence choisi parmi le FGF et le VEGF. Comme pour le biomatériau précédemment, il comprend, selon une forme de réalisation préférée, entre environ 15 et 100 % et avantageusement entre 30 et 100 %, de collagène de type III. Il peut s'agir de collagènes humains ou d'origine animale, obtenus de placenta. Ce biomatériau est particulièrement utile comme implant colonisable in vi vo , dans l'organisme d'un sujet humain ou animal, par des cellules dudit organisme. Ce biomatériau peut alors comprendre outre le gel de collagène et le(s) facteur (s) de croissance, un milieu adapté, voir sélectif, favorisant sa colonisation par un ou plusieur types cellulaires de l'organisme.An application of the invention, close to the previous one, consists in using in a body, as an implant colonizable by cells, a biomaterial consisting of a collagen gel comprising type III collagen or consisting exclusively of type III collagen, impregnated with at least one growth factor, preferably chosen from FGF and VEGF. As for the biomaterial previously, it comprises, according to a preferred embodiment, between approximately 15 and 100% and advantageously between 30 and 100%, of type III collagen. It can be human or animal collagens, obtained from placenta. This biomaterial is particularly useful as an implant that can be colonized in vi vo, in the organism of a human or animal subject, by cells of said organism. This biomaterial can then comprise, in addition to the collagen gel and the growth factor (s), a suitable, or even selective, medium, promoting its colonization by one or more cell types of the organism.
D'autres avantages et caractéristiques de 1 ' invention apparaîtront à la lecture des exemples qui suivent donnés à titre non limitatifs et se référant aux dessins en annexe, dans lesquels les figures 1 et 2 représentent des photos, en microscopie inversée, de corps embryonnaires vascularisés obtenus avec le procédé de l'invention.Other advantages and characteristics of the invention will appear on reading the examples which follow, given without limitation and with reference to the appended drawings, in which FIGS. 1 and 2 represent photos, in inverted microscopy, of vascularized embryonic bodies obtained with the process of the invention.
I - Culture de cellules ES indifférenciées.I - Culture of undifferentiated ES cells.
Les cellules ES de la lignée D3 dérivée de blastocystes de souris (ATCC CRL 1934) sont cultivées dans des boîtes de culture 100 mm (Costar, Cambridge, Ma, USA, réf. 3100) recouvertes de gélatine 0,2 % (Sigma, SaintES cells of the D3 line derived from mouse blastocysts (ATCC CRL 1934) are cultured in 100 mm culture dishes (Costar, Cambridge, Ma, USA, ref. 3100) coated with 0.2% gelatin (Sigma, Saint
Louis, Mi, USA M17353)Louis, Mi, USA M17353)
Le milieu de culture est constitué de milieu "Dubelco Modified Eagle's Médium (DMEM) High Glucose (Gibco 41965-039) additionné de 15 % de sérum de veau foetal (FCS, Gibco, Grand Island, NY, USA) , de pyruvate de sodium 1 mM (Gibco 1136-039) , d'acides aminés non essentiels 0,1 mM (Gibco 11140-035) , de L-Glutamine 2 mM (Gibco 25030-032) , de mono-thio-glycérol 150 mM (Sigma, Saint Louis, Mi, USA M17353) et d'un mélange de pénicilline-streptomycine 100 μg/ml (Gibco 15140-030) .The culture medium consists of “Dubelco Modified Eagle's Medium (DMEM) High Glucose (Gibco 41965-039) medium supplemented with 15% fetal calf serum (FCS, Gibco, Grand Island, NY, USA), sodium pyruvate 1 mM (Gibco 1136-039), non-essential amino acids 0.1 mM (Gibco 11140-035), L-Glutamine 2 mM (Gibco 25030-032), 150 mM mono-thio-glycerol (Sigma, Saint Louis, Mi, USA M17353) and a mixture of penicillin-streptomycin 100 μg / ml (Gibco 15140-030).
Les cellules sont maintenues sous forme indifférenciées par ajout de LIF recombinant humain (leukemia inhibiting factor) 1000 u/ml (Schmitt, R. , M. Bruyns , H. R. Snodgrass, "Hematopoetic development of embryonic stem cells in vitro : cytokin and receptor gène expression". Gènes Dev. , 1991, 5, 728-740) . Le milieu de culture additionné de LIF est renouvelé tous les jours. Sous ces conditions, 95% de la population de cellules ES est dans un état indifférencié clairement visible en microscopie à contraste de phase. Un stock de plusieurs dizaines de cryotubes contenant 5 millions de cellules ES provenant du même passage a été préparé à 1 ' avance.The cells are maintained in undifferentiated form by adding human recombinant LIF (leukemia inhibiting factor) 1000 u / ml (Schmitt, R., M. Bruyns, HR Snodgrass, "Hematopoetic development of embryonic stem cells in vitro: cytokin and receptor gene expression ". Genes Dev., 1991, 5, 728-740). The culture medium supplemented with LIF is renewed every day. Under these conditions, 95% of the population of ES cells is in an undifferentiated state clearly visible in phase contrast microscopy. A stock of several dozen cryotubes containing 5 million ES cells from the same passage was prepared in advance.
Ce protocole expérimental permet bien entendu un grand nombre de variantes à la portée de l 'homme du métier à partir de l'enseignement de cet exemple. Il est possible notamment de remplacer le LIF par tout autre inhibiteur de la différenciation, comme l'Oncostatin M.This experimental protocol obviously allows a large number of variants within the reach of the skilled person from the teaching of this example. It is possible in particular to replace the LIF with any other inhibitor of differentiation, such as Oncostatin M.
II - Expériences de différenciation.II - Differentiation experiences.
Une ampoule de cellule D3 est décongelée puis mise en culture comme indiquée ci-dessus. Après deux jours de culture, les cellules sont subconfluentes . Elles sont alors décollées par une solution de trypsine 0,25 % (Gibco) dans du tampon salin (PBS) contenant 1 mM d'EDTA, puis ensemencées à raison de 5 milions de cellules par boîte durant 24 heures. Trois heures avant l'induction de la différenciation, le milieu contenant du LIF est renouvelé. Après trypsination, la différenciation est réalisée dans des boîtes de culture bactériologiques (35/10 mm) (Greiner, 627102) . Sont mélangés par boîte :A D3 cell ampoule is thawed and then cultured as indicated above. After two days of culture, the cells are subconfluent. They are then peeled off with a 0.25% trypsin solution (Gibco) in saline buffer (PBS) containing 1 mM EDTA, then seeded at the rate of 5 million cells per dish for 24 hours. Three hours before the induction of differentiation, the medium containing LIF is renewed. After trypsination, the differentiation is carried out in bacteriological culture dishes (35/10 mm) (Greiner, 627102). Are mixed by box:
- 500 μl d'une solution de collagène I+III préparé à partir de placenta humain (50 % de chacun des deux types de collagène) ,- 500 μl of a collagen I + III solution prepared from human placenta (50% of each of the two types of collagen),
- 250 μl de milieu Iscove Médium Dulbecco- 250 μl of Iscove Medium Dulbecco medium
Modified (IMDM) deux fois concentré (Gibco 42200-014) ,Modified (IMDM) twice concentrated (Gibco 42200-014),
- 750 μl d'une solution de différenciation deux fois concentré (SD) contenant de la transferrine 300 mg (Boehringer Mannheim 652202) , de l 'insuline 10 mg/ml (Boehringer 977420) , du mono-thio-glycérol 450 mM, 15 % de sérum de veau foetal (FCS, Gibco, Grand Island, NY, USA) dans de 1 ' ISCOVE Glutamax (Gibco 31980- 022)- 750 μl of a twice concentrated differentiation solution (SD) containing transferrin 300 mg (Boehringer Mannheim 652202), insulin 10 mg / ml (Boehringer 977420), mono-thio-glycerol 450 mM, 15 % of fetal calf serum (FCS, Gibco, Grand Island, NY, USA) in ISCOVE Glutamax (Gibco 31980-022)
- 50 μl de suspension contenant au moins 500 cellules .- 50 μl of suspension containing at least 500 cells.
En contrôle, des inductions de différenciation sont réalisées en remplaçant la solution de collagène par de la méthyl-cellulose 1,1 % (Methocel high viscosity, Fluka A. G., Buchs, Suisse) selon la même technique.In control, differentiation inductions are carried out by replacing the collagen solution with 1.1% methyl cellulose (Methocel high viscosity, Fluka A. G., Buchs, Switzerland) according to the same technique.
Enfin, des essais sont également réalisés en utilisant comme support de culture, une solution de matrice Matrigel (EHS) (Collaborative Biomédical Product,Finally, tests are also carried out using as a culture support, a Matrigel matrix solution (EHS) (Collaborative Biomedical Product,
Becton Dickinson) utilisée diluée au 1/2, additionnée du milieu SD et d'une solution d ' IMDM tous deux concentrés deux fois . Pour certains essais, les cytokines seules ou en mélange suivantes ont été incorporées au milieu de culture :Becton Dickinson) used diluted to 1/2, supplemented with SD medium and a solution of IMDM, both concentrated twice. For certain tests, the following cytokines alone or as a mixture were incorporated into the culture medium:
VEGF recombinant humain à raison deHuman recombinant VEGF due to
50 ng/ml ( Pre protech 100-20 ) , bFGF recombinant humain à raison de 100 ng/ml (R&D 233-FB-025) .50 ng / ml (Pre protech 100-20), human recombinant bFGF at a rate of 100 ng / ml (R&D 233-FB-025).
Toutes les expériences rapportées ci-dessus ont été réalisées selon le même schéma, lequel constitue un exemple acceptant de nombreuses variantes à la portée de l'homme du métier à partir de l'enseignement de ce schéma. Notamment, des essais favorables ont été réalisés avec un gel de collagène constitué de collagène I+III comprenant 10 % de collagène III pour 90 % de collagène I. Le collagène préféré est un collagène placentaire humain non dénaturé de type 1+111 préparé en solution à 3 mg/ml et dilué à 1 mg/ml en présence du milieu de culture qui élève son pH et le gélifie. Le gel ainsi obtenu en quelques heures est un gel semi-solide qui est facilement démoulable et séchable sur lame. La concentration en collagène doit bien entendu être adaptée pour disposer d'un gel ni trop liquide, ni trop solide ; à titre indicatif, la quantité de collagène peut être comprise entre 0,5 mg/ml et 20 mg/ml. Les proportions de cellules par rapport à la quantité de collagène sont susceptibles d'admettre des variations par rapport au schéma rapporté ci-dessus. Des résultats concluant ont été obtenus avec des mélanges comprenant entre 500 à 1500 cellules pour environ 1,5 mg de collagène.All the experiments reported above were carried out according to the same scheme, which constitutes an example accepting many variants within the reach of the skilled person from the teaching of this scheme. In particular, favorable tests have been carried out with a collagen gel consisting of collagen I + III comprising 10% of collagen III for 90% of collagen I. The preferred collagen is an undenatured type 1 + 111 human placental collagen prepared in solution at 3 mg / ml and diluted to 1 mg / ml in the presence of the culture medium which raises its pH and gels it. The gel thus obtained in a few hours is a semi-solid gel which is easily demouldable and dryable on a slide. The collagen concentration must of course be adapted in order to have a gel which is neither too liquid nor too solid; As an indication, the amount of collagen can be between 0.5 mg / ml and 20 mg / ml. The proportions of cells relative to the amount of collagen are likely to admit variations compared to the diagram reported above. Conclusive results have been obtained with mixtures comprising between 500 to 1500 cells for approximately 1.5 mg of collagen.
III - Quantification du phénomène observé.III - Quantification of the phenomenon observed.
Les boîtes sont observées quotidiennement au microscope inversé et photographiées. Des comptages peuvent être réalisés afin d'évaluer le nombre de corps embryonnaires :The boxes are observed daily under an inverted microscope and photographed. Counts can be made to assess the number of embryonic bodies:
- ne présentant pas de structure vasculaire (CO) , - présentant une ou plusieurs structures tubulaires inférieures à son diamètre (C+) , présentant un ensemble de tubules de longueur supérieure ou égale à son diamètre (C++) . Un pourcentage de vascular isation est ensuite obtenu en calculant le rapport : (C+)+(C++) / nombre total de corps embryonnaires par boîte.- not having a vascular structure (CO), - having one or more tubular structures smaller than its diameter (C +), having a set of tubules of length greater than or equal to its diameter (C ++). A percentage of vascular isation is then obtained by calculating the ratio: (C +) + (C ++) / total number of embryonic bodies per dish.
IV - Coloration et immunof luorescence indirecte.IV - Staining and indirect immunofluorescence.
Occasionnellement, les gels sont séchés sur lame selon la méthode initialement décrite (Lanotte, M. , "Terminal dif f erentiation of hematopoietic cell clones cultured in tridimesional collagen matrix : in situ cell morphology and enzyme histochemistry analysis". Biol . Cell. , 1984, 50, 107-120) , puis colorés au May Grunewald Giemsa (Biolyon, Unipath, Dardilly, France 09262 et 09766) selon les recommandations du fournisseur. Les gels séchés peuvent également être fixés par une solution de paraformaldehyde 3 % préparée dans du tampon phosphate salin (PBS) afin de permettre la réalisation d'un test d ' immunof luorescence indirecte. Les lames sont ensuite mises en contact à 37 °C durant une heure avec un anticorps monoclonal de rat dirigé contre une molécule marqueur des cellules vasculaires (PECAM pour Platelet Endothelial Cell Adhésion Molécule, ou Facteur von Willebrand) (Risau, W. , "Di ff erentiation of the endothelium" . Faseb J., 1995, 6-933) . Après plusieurs lavages effectués en PBS, les lames sont incubées 30 minutes avec une solution contenant des fragments F(ab')2 de sérum de chèvre dirigé contre les immunoglobulines G de rat diluée au 1/100 (Jackson ImmunoResearch, Immunotech, France) dans du PBS. Deux derniers lavages en PBS permettent l'observation des lames au microscope à fluorescence .Occasionally, the gels are dried on a slide according to the method initially described (Lanotte, M., "Terminal dif f erentiation of hematopoietic cell clones cultured in tridimesional collagen matrix: in situ cell morphology and enzyme histochemistry analysis". Biol. Cell., 1984 , 50, 107-120), then colored with May Grunewald Giemsa (Biolyon, Unipath, Dardilly, France 09262 and 09766) according to the supplier's recommendations. The dried gels can also be fixed with a 3% paraformaldehyde solution prepared in phosphate buffered saline (PBS) in order to allow the performance of an indirect immunofluorescence test. The slides are then brought into contact at 37 ° C. for one hour with a rat monoclonal antibody directed against a vascular cell marker molecule (PECAM for Platelet Endothelial Cell Adhesion Molecule, or von Willebrand Factor) (Risau, W., "Di ff erentiation of the endothelium ". Faseb J., 1995, 6-933). After several washes carried out in PBS, the slides are incubated for 30 minutes with a solution containing fragments F (ab ') 2 of goat serum directed against rat immunoglobulins G diluted to 1/100 (Jackson ImmunoResearch, Immunotech, France) in PBS. Last two washes in PBS allow observation of the slides under a fluorescence microscope.
V - Résultats.V - Results.
La matrice Matrigel (EHS) additionnée ou non de facteurs de croissance ne permet pas l'obtention de corps embryonnaires (CE) . En effet, les cellules ES ne s'associent pas en amas constitués de quelques cellules et ne forment pas de structures plus importantes. Elles ne peuvent pas être observées au microscope et doivent certainement mourir très rapidement .The Matrigel matrix (EHS) with or without the addition of growth factors does not allow the production of embryonic bodies (EC). In fact, ES cells do not associate in clusters made up of a few cells and do not form larger structures. They cannot be observed under a microscope and must certainly die very quickly.
La méthyl-cellulose additionnée ou non de facteurs de croissance permet la formation de CE en quelques jours. AU bout de trois jours, des amas cellulaires sont observés. Ces amas grossissent jusqu'à être visibles à l'oeil nu au bout de cinq jours. Les CE ainsi obtenus peuvent présenter des morphologies différentes selon qu'ils sont ou non entourés d'une coque cellulaire. Mais aucune structure vasculaire extérieure n'est observée avec ce support de culture. Ces résultats sont à rapprocher de plusieurs travaux antérieurs décrivant des canaux vasculaires confinés à 1 ' intérieur des CE (Wang. R. , R. Clark. , V. L. Bautch, "Embryonnic stem cell-derived cystic embryoïd bodies from vascular channels : an in vitro model of blood vessel development " , Development 1992, 114, 303-316 ; Doetschman, T. , A. Kier, J. D. Coffin, "Embryonic stem cells model Systems for vasculogenesis and cardiac disorders". Hypertension, 1993,Methyl cellulose, with or without added growth factors, allows the formation of CE in a few days. After three days, cell clusters are observed. These clumps grow until they are visible to the naked eye after five days. The CE thus obtained can have different morphologies depending on whether or not they are surrounded by a cell shell. However, no external vascular structure is observed with this culture support. These results are to be compared with several previous works describing vascular channels confined within the EC (Wang. R., R. Clark., VL Bautch, "Embryonnic stem cell-derived cystic embryoid bodies from vascular channels: an in vitro model of blood vessel development ", Development 1992, 114, 303-316; Doetschman, T., A. Kier, JD Coffin," Embryonic stem cells model Systems for vasculogenesis and cardiac disorders ". Hypertension, 1993,
22, 618-629) . L'adjonction de facteurs de croissance permet de doubler le nombre de CE obtenus à partir de 1500 cellules ES indifférenciées (obtention de 50 CE au minimum par boîte en présence de facteurs de croissance) . De manière remarquable, le procédé de 1 ' invention mettant en oeuvre un support de gel de collagène I+III permet la formation de CE présentant des structures tubulaires rayonnantes. Les cellules ES indifférenciées incorporées à 1 ' intérieur du gel de collagène I+III contenant la solution SD présentent le même aspect qu'en présence de méthyl cellulose durant les premiers jours de la culture. Mais, entre J5 et J8 , les CE formés (une trentaine par boîte au minimum) voient leur taille augmenter. Au cours des jours suivants, certains d'entre eux présentent des structures tubulaires rayonnantes dont la longueur est la plupart du temps inférieure au diamètre du CE considéré. Le pourcentage de vascularisation peut néanmoins atteindre 50 %. Ces structures tubulaires ne sont pas révélées en immunofluorescence par des anticorps monoclonaux dirigés centre des molécules marqueurs des cellules d'origine vasculaire.22, 618-629). The addition of growth factors makes it possible to double the number of CEs obtained from 1500 undifferentiated ES cells (obtaining at least 50 CEs per dish in the presence of growth factors). Remarkably, the method of the invention using a collagen I + III gel support allows the formation of CEs having radiant tubular structures. The undifferentiated ES cells incorporated into the collagen I + III gel containing the SD solution have the same appearance as in the presence of methyl cellulose during the first days of culture. But, between D5 and D8, the CEs trained (at least thirty per box) see their size increase. During the following days, some of them present radiant tubular structures whose length is mostly less than the diameter of the CE in question. The percentage of vascularization can nevertheless reach 50%. These tubular structures are not revealed in immunofluorescence by monoclonal antibodies directed at the center of the marker molecules of cells of vascular origin.
L'addition de VEGF et de FGF favorise et accélère la différenciation des cellules ES en cellules endothéliales . Ainsi, lorsque du FGF, du VEGF ou un mélange de ceux-ci est incorporé au gel de collagène, des structures tubulaires commencent à se former dès j6. Le calcul du pourcentage de vascularisation a permis de montrer :The addition of VEGF and FGF promotes and accelerates the differentiation of ES cells into endothelial cells. Thus, when FGF, VEGF or a mixture of these is incorporated into the collagen gel, tubular structures begin to form from day 6. The calculation of the percentage of vascularization made it possible to show:
- qu'il existe bien un décalage d'apparition des structures tubulaires sans facteur de croissance par rapport aux conditions avec des facteurs de croissance,- that there is a lag in the appearance of tubular structures without growth factor compared to the conditions with growth factors,
- que le FGF et surtout le VEGF introduits individuellement sont suffisants à induire une tubulisation,- that the FGF and especially the VEGF introduced individually are sufficient to induce tubulation,
- que le FGF et le VEGF agissent en synergie puisque la condition réunissant ces deux facteurs donne les meilleurs résultats (75 % de vascularisation à J10) . Les expériences d ' immunof luorescence ont clairement démontré le caractère vasculaire des structures tubulaires obtenues en présence de VEGF. Ce facteur semble particulièrement efficace pour induire la différenciation des cellules ES en cellules endothéliales exprimant les molécules PECAM mais également le facteur von Willebrand qui est un marqueur tardif de la différenciation endothéliale.- that FGF and VEGF act in synergy since the condition combining these two factors gives the best results (75% of vascularization on D10). Immunofluorescence experiments clearly demonstrated the vascular character of the tubular structures obtained in the presence of VEGF. This factor seems particularly effective for inducing the differentiation of ES cells into endothelial cells expressing PECAM molecules but also the von Willebrand factor which is a late marker of endothelial differentiation.
La photo de la figure 1 représente, en microscopie inversée, différents types de corps embryonnaires obtenus à J10 dans un gel de collagène supplémenté par du VEGF et du FGF. On observe sur cette photo, de la gauche vers le droite : un CE non vascularisé, un CE entouré de cellules ayant migré dans le gel, enfin un CE fortement vascularisé, c'est à dire, dont la longueur des tubules est supérieure au diamètre du corps et présentant des branchements secondaires, témoignant du processus d ' angiogénèse.The photo of FIG. 1 represents, in reverse microscopy, different types of embryonic bodies obtained on D10 in a collagen gel supplemented with VEGF and FGF. We can see in this photo, from left to right: a non-vascularized CE, a CE surrounded by cells that have migrated into the gel, finally a highly vascularized CE, that is to say, whose tubule length is greater than the diameter of the body and presenting secondary branches, testifying to the process of angiogenesis.
La photo de la figure 2 représente, en microscopie inversée, un CE fortement vasculariséThe photo of Figure 2 represents, in inverted microscopy, a highly vascularized CE
(branchements tertaires) avec présence de nombreuses cellules intra- tubulaires probablement d'origine hématopoïétique . (tertiary connections) with the presence of numerous intra-tubular cells probably of hematopoietic origin.

Claims

REVENDICATIONS
1) Procédé de culture de cellules à potentiel angiogénique pour induire la morphogénèse vasculaire desdites cellules, caractérisé en ce qu'il comprend les étapes consistant à :1) Method for culturing cells with angiogenic potential to induce the vascular morphogenesis of said cells, characterized in that it comprises the steps consisting in:
(a) obtenir une préparation cellulaire contenant des cellules à potentiel angiogénique;(a) obtaining a cell preparation containing cells with angiogenic potential;
- (b) mettre en contact ces cellules avec un gel de collagène comprenant du collagène de type III ou constitué exclusivement de collagène de type III, en présence d'un milieu de culture adapté auxdites cellules;- (b) bringing these cells into contact with a collagen gel comprising type III collagen or consisting exclusively of type III collagen, in the presence of a culture medium suitable for said cells;
(c) laisser les cellules se développer dans le gel de collagène pendant une durée suffisante pour permettre la morphogénèse vasculaire.(c) allowing the cells to grow in the collagen gel for a time sufficient to allow vascular morphogenesis.
2) Procédé selon la revendication 1, caractérisé en ce que le gel de collagène est constitué de collagène des types I et III.2) Method according to claim 1, characterized in that the collagen gel consists of collagen types I and III.
3) Procédé selon l'une des revendications 1 ou 2, caractérisé en ce que le gel de collagène comprend entre environ 15 et 100 %, et avantageusement entre 30 et 100 %, de collagène de type III.3) Method according to one of claims 1 or 2, characterized in that the collagen gel comprises between about 15 and 100%, and advantageously between 30 and 100%, of type III collagen.
4) Procédé selon l'une quelconque des revendications précédentes, caractérisé en que le collagène est d'origine placentaire.4) Method according to any one of the preceding claims, characterized in that the collagen is of placental origin.
5) Procédé de culture de cellules à potentiel angiogénique selon l'une quelconque des revendications 1 à 4, caractérisé en ce que lesdites cellules sont choisies parmi :5) Method for culturing cells with angiogenic potential according to any one of claims 1 to 4, characterized in that said cells are chosen from:
- des cellules d'une lignée de cellules souches embryonnaires de mammifère; des cellules de tératocarcinome à potentiel angiogénique; des cellules tumorales à potentiel angiogénique; - des cellules ayant acquis par modification génétique un potentiel angiogénique.- cells of a mammalian embryonic stem cell line; angiogenic potential teratocarcinoma cells; tumor cells with angiogenic potential; - cells that have acquired angiogenic potential by genetic modification.
6) Procédé selon l'une quelconque des revendications précédentes, caractérisé en ce qu'à l'étape (b) on ajoute un ou plusieur facteurs de croissance.6) Method according to any one of the preceding claims, characterized in that in step (b) one or more growth factors are added.
7) Procédé selon la revendication 6, caractérisé en ce que les facteurs de croissance ajoutés à l 'étape (b) sont le bFGF ou le VEGF, ou un mélange de ceux-ci .7) Method according to claim 6, characterized in that the growth factors added in step (b) are bFGF or VEGF, or a mixture of these.
8) Modèle i n vi t ro de morphogénèse vasculaire, caractérisé en ce qu'il comprend des cellules à potentiel angiogénique incluses dans un gel de collagène comprenant du collagène de type III ou constitué exclusivement de collagène de type III, dans un milieu de culture adapté auxdites cellules, éventuellement additionné d'un ou plusieur facteurs de croissance.8) In vi t ro model of vascular morphogenesis, characterized in that it comprises cells with angiogenic potential included in a collagen gel comprising type III collagen or consisting exclusively of type III collagen, in a suitable culture medium to said cells, optionally supplemented with one or more growth factors.
9) Modèle selon la revendication 8, caractérisé en ce que le gel de collagène est constitué de collagène des types I et III.9) Model according to claim 8, characterized in that the collagen gel consists of collagen types I and III.
10) Modèle selon l'une des revendications 8 ou 9, caractérisé en ce que le gel de collagène comprend entre 5 et 100 % de collagène de type III.10) Model according to one of claims 8 or 9, characterized in that the collagen gel comprises between 5 and 100% type III collagen.
11) Modèle selon l'une quelconque des revendications 8 à 10, caractérisé en ce que les cellules à potentiel angiogénique sont choisies parmi : - des cellules d'une lignée de cellules souches embryonaires de mammifère; des cellules de tératocarcinome à potentiel angiogénique; - des cellules tumorales à potentiel angiogénique;11) Model according to any one of claims 8 to 10, characterized in that the cells with angiogenic potential are chosen from: - cells of a mammalian embryonic stem cell line; angiogenic potential teratocarcinoma cells; - tumor cells with angiogenic potential;
- des cellules ayant acquis par modification génétique un potentiel angiogénique.- cells that have acquired angiogenic potential by genetic modification.
12) Procédé de criblage de substances susceptibles de favoriser ou inhiber la morphogénèse vasculaire mettant en oeuvre un procédé selon l'une quelconque des revendications 1 à 7, caractérisé en ce que l'on introduit la substance à tester à l'une au moins des (a) à (c) , et en ce que l'on mesure par tout moyen approprié la présence ou l'absence de morphogénèse vasculaire.12) A method of screening substances capable of promoting or inhibiting vascular morphogenesis using a method according to any one of claims 1 to 7, characterized in that the test substance is introduced into at least one of the (a) to (c), and in that the presence or absence of vascular morphogenesis is measured by any suitable means.
13) Trousse de criblage de substances susceptibles de favoriser ou inhiber la morphogénèse vasculaire, pour la mise en oeuvre d'un procédé selon la revendication 12, caractérisée en ce qu'elle comprend au moins un compartiment protégé renfermant un modèle selon l'une quelconque des revendications 8 à 11, ou une ou plusieurs séries de compartiments protégés, chaque série comprenant un compartiment renfermant un gel de collagène et au moins un des compartiments suivants :13) Screening kit for substances capable of promoting or inhibiting vascular morphogenesis, for the implementation of a method according to claim 12, characterized in that it comprises at least one protected compartment containing a model according to any one of claims 8 to 11, or one or more series of protected compartments, each series comprising a compartment containing a collagen gel and at least one of the following compartments:
- un compartiment renfermant des cellules à potentiel angiogénique, - un compartiment renfermant un milieu de culture adapté auxdites cellules;- a compartment containing cells with angiogenic potential, - a compartment containing a culture medium adapted to said cells;
- un compartiment renfermant un ou plusieurs facteurs de croissance. 14) Procédé de diagnostic du potentiel metastasique d'une tumeur chez un patient mettant en oeuvre un procédé selon l'une quelconque des revendications 1 à 7, caractérisé en ce que les cellules à potentiel angiogénique incluses dans le gel de collagène sont des cellules tumorales prélevées chez ledit patient.- a compartment containing one or more growth factors. 14) Method for diagnosing the metastatic potential of a tumor in a patient using a method according to any one of claims 1 to 7, characterized in that the cells with angiogenic potential included in the collagen gel are tumor cells taken from said patient.
15) Trousse de diagnostic du potentiel metastasique d'une tumeur chez un patient pour la mise en oeuvre d'un procédé selon la revendication 14, caractérisée en ce qu'elle comprend une ou plusieurs séries de compartiments protégés, chaque série comprenant un compartiment renfermant un gel de collagène et au moins un des compartiments suivants : - un compartiment renfermant un milieu de culture adapté auxdites cellules;15) Kit for diagnosing the metastatic potential of a tumor in a patient for the implementation of a method according to claim 14, characterized in that it comprises one or more series of protected compartments, each series comprising a compartment containing a collagen gel and at least one of the following compartments: - a compartment containing a culture medium adapted to said cells;
- un compartiment renfermant un ou plusieurs facteurs de croissance;- a compartment containing one or more growth factors;
- un compartiment dans lequel seront placées des cellules tumorales prélevées chez ledit patient.- a compartment in which tumor cells taken from said patient will be placed.
16) Procédé de diagnostic d'une pathologie à composante vasculaire chez un patient mettant en oeuvre un procédé selon l'une quelconque des revendications 1 à 5, caractérisé en ce qu'à l'une au moins des étapes (a) à (cj , on ajoute un échantillon biologique prélevé dudit patient et susceptible de contenir des facteurs angiogéniques capables de favoriser la morphogénèse vasculaire.16) A method of diagnosing a pathology with a vascular component in a patient using a method according to any one of claims 1 to 5, characterized in that in at least one of steps (a) to (cj , a biological sample taken from said patient and likely to contain angiogenic factors capable of promoting vascular morphogenesis is added.
17) Trousse de diagnostic d'une pathologie à composante vasculaire chez un patient pour la mise en oeuvre d'un procédé selon la revendication 16, caractérisée en ce qu'elle comprend au moins un compartiment protégé renfermant un modèle selon l'une quelconque des revendications 8 à 11, ou une ou plusieurs séries de compartiments protégés, chaque série comprenant un compartiment renfermant, un gel de collagène et au moins un des compartiments suivants : - un compartiment renfermant des cellules à potentiel angiogénique,17) Kit for diagnosing a pathology with a vascular component in a patient for the implementation of a method according to claim 16, characterized in that it comprises at least one protected compartment containing a model according to one any one of claims 8 to 11, or one or more series of protected compartments, each series comprising a compartment containing, a collagen gel and at least one of the following compartments: - a compartment containing cells with angiogenic potential,
- un compartiment renfermant un milieu de culture adapté auxdites cellules;- A compartment containing a culture medium adapted to said cells;
- un compartiment dans lequel sera placée un échantillon biologique prélevé dudit patient.- a compartment in which a biological sample taken from said patient will be placed.
18) Biomatériau pour la réalisation d' implants colonisables in vi vo par les cellules d'un organisme, caractérisé en ce qu'il est constitué d'un gel de collagène comprenant du collagène de type III ou constitué exclusivement de collagène de type III, ledit gel étant imprégné d'au moins un facteur de croissance, de préférence choisi parmi le VEGF et le FGF.18) Biomaterial for producing implants that can be colonized in vi vo by the cells of an organism, characterized in that it consists of a collagen gel comprising type III collagen or consisting exclusively of type III collagen, said gel being impregnated with at least one growth factor, preferably chosen from VEGF and FGF.
19) Biomatériau selon la revendication 18, caractérisé en ce que ledit gel de collagène comprend entre environ 15 et 100 %, et avantageusement entre 30 et 100 %, de collagène de type III.19) Biomaterial according to claim 18, characterized in that said collagen gel comprises between approximately 15 and 100%, and advantageously between 30 and 100%, of type III collagen.
20) Biomatériau pour la réalisation d'implants, caractérisé en ce qu'il est constitué d'un gel de collagène comprenant du collagène de type III ou constitué exclusivement de collagène de type III dans lequel est inclus des cellules à potentiel angiogénique ou des cellules ayant plus ou moins induit une différenciation vasculaire.20) Biomaterial for producing implants, characterized in that it consists of a collagen gel comprising type III collagen or consisting exclusively of type III collagen in which cells with angiogenic potential or cells are included having more or less induced vascular differentiation.
21) Biomatériau selon la revendication 20, caractérisé en ce que les cellules à potentiel angiogénique ou les cellules ayant plus ou moins induit 97/28253 27 PC17FR97/0018621) Biomaterial according to claim 20, characterized in that the cells with angiogenic potential or the cells having more or less induced 97/28253 27 PC17FR97 / 00186
une différenciation vasculaire, sont génétiquement modifiées pour produire in vivo un principe actif thérapeutique .vascular differentiation, are genetically modified to produce in vivo a therapeutic active ingredient.
22) Biomatériau selon l'une quelconque des revendications 20 et 21, caractérisé en ce qu'il est imprégné d'au moins un facteurs de croissance, de préférence choisi parmi le VEGF et le FGF. 22) Biomaterial according to any one of claims 20 and 21, characterized in that it is impregnated with at least one growth factor, preferably chosen from VEGF and FGF.
PCT/FR1997/000186 1996-01-30 1997-01-30 Method for culturing cells having angiogenic potential WO1997028253A1 (en)

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FR9601056A FR2744133A1 (en) 1996-01-30 1996-01-30 METHOD FOR CULTURING CELLS WITH ANGIOGENIC POTENTIAL TO INDUCE VASCULAR MORPHOGENESIS OF SAID CELLS, IN VITRO MODELS OF VASCULAR MORPHOGENESE THUS OBTAINED, AND THEIR APPLICATIONS, IN PARTICULAR TO DRUG SCREENING
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