WO2009146746A1 - Utilisation de fxa, de fviia, de tf ou d'une combinaison de ceux-ci en tant qu'agents anti-apoptotiques - Google Patents

Utilisation de fxa, de fviia, de tf ou d'une combinaison de ceux-ci en tant qu'agents anti-apoptotiques Download PDF

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WO2009146746A1
WO2009146746A1 PCT/EP2008/056994 EP2008056994W WO2009146746A1 WO 2009146746 A1 WO2009146746 A1 WO 2009146746A1 EP 2008056994 W EP2008056994 W EP 2008056994W WO 2009146746 A1 WO2009146746 A1 WO 2009146746A1
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cells
cell
factor
neurones
astrocytes
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Valéry DAUBIE
Roland Pochet
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Universite Libre de Bruxelles ULB
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    • 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
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals
    • 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
    • C12N2501/70—Enzymes

Definitions

  • the invention lies in the technical field of in vitro cell culturing tools and more particularly in the field of cell culture medium additives for the prevention of apoptosis, the culturing of osteoblasts or osteoblast-like cells, neurones or neurones-like cells, astrocytes or astrocytes- like cells, mesenchymal stem cells, hematopoietic stem cells, endothelial cell progenitors and their use in clinical settings.
  • Bone diseases are widely spread throughout the world, affecting women, men, and children of all ages.
  • One example of a bone disease includes osteonecrosis (ON), referring to the death of bone which can occur by hypoxia from a decrease or loss in blood supply to specific parts of bones or by some other means. Eventually the dead section of bone weakens and collapses.
  • ON can be post-traumatic (fractures and joint dislocations, where e.g. the arterial blood vessels to a part of the bone have been interrupted or physically damaged) and non- traumatic.
  • ON is called non traumatic when ON occurs without any history of trauma.
  • Another well known bone disease is osteoporosis which is characterized by a decreased bone density, with a loss of biomechanical strength, resulting in a high prevalence and incidence of fractures.
  • Osteoporosis and related fractures are particularly common in postmenopausal women; and patients treated with corticosteroids.
  • fractures -so called non- union fractures or pseudarthrosis- a lack of, or a delayed bone healing may occur.
  • the origin hereof could be at least partly due to a lack of proliferation of the osteoblastic cells.
  • Typical medical treatment of these diseases includes, bed rest, orthotics, non weight bearing and the administration of medication.
  • surgery may sometimes be required.
  • Surgical techniques include decompression of the necrotic area by taking of a plug of bone out of a diseased area and may involve creating a hole or bore in the bone or the joint or bone grafting, which involves surgically removal of the dead bone and filling the empty space with bone graft that is either taken from the patient or from the bone bank.
  • Cellular therapy is a further alternative treatment, involving the use of cultured or otherwise obtained living cells which may be injected in cavities or bores applied in bone tissues or joints. Cellular material applied to the bone is used to replace, repair or ameliorate the biological function of the bone or joint tissue.
  • Osteoblasts are specialized bone cells that form new bone material. The building of bone by osteoblasts is balanced by the action of other bone cells called osteoclasts that break down bone material. In the bones of healthy adults, bone mass remains relatively stable because osteoblasts and osteoclasts are working at about the same rate. This allows bones to continuously remodel and to heal when damaged.
  • osteoblasts can be affected by various drugs and key nutrients and can sometimes be impaired in certain disease states such as osteoporosis and osteopenia, e.g., primary, postmenopausal, senile, corticoid-induced, any secondary, mono- or multisite osteonecrosis, any type of fracture, e.g., non-union, mal-union, delayed union fractures or compression, conditions requiring bone fusion (e.g., spinal fusions and rebuilding), maxillo-facial fractures, bone reconstruction, e.g., after traumatic injury or cancer surgery, cranio-facial bone reconstruction, osteogenesis imperfecta, osteolytic bone cancer, Paget's Disease, osteopetrosis, endocrinological disorders, hypophosphatemia, hypocalcemia, renal osteodystrophy, osteomalacia, adynamic bone disease, rheumatoid arthritis, hyperparathyroidism, primary hyperparathyroidism, secondary hyperparathyroidism, periodontal disease,
  • Neurones and astrocytes grafting can be useful for curative treatment of neurodegenerative disease such as Alzheimer's disease, Parkinson disease, and various sclerosis (multiple sclerosis, amyotrophic lateral sclerosis,).
  • Stem cells are seen to be used for tissue engineering or tissue repair in various fields (cardiac , vascular, tissue, bone, skin, nervous system). As there are not enough stem cells available in human body, a proliferation step is necessary to increase the available amount of such cells.
  • the use of the medium of the invention avoids serum-free medium- induced apoptosis.
  • Hematopoietic stem cells and endothelial cell progenitors find use in clinical settings regarding the treatment of blood-related disorders such as haemophilia, leukaemia, neutropenia.
  • blood progenitor cells are Bone marrow transplantation (BMT) and peripheral biood stem eel! transplantation (PBSCT).
  • BMT and PBSCT are most commonly used in the treatment of leukemia and lymphoma. They are most effective when the leukemia or lymphoma is in remission (the signs and symptoms of cancer have disappeared).
  • BMT and PBSCT are also used to treat other cancers such as neuroblastoma (cancer that arises in immature nerve cells and affects mostly infants and children) and multiple myeloma.
  • researchers are evaluating BMT and PBSCT in clinical trials (research studies) for the treatment of various types of cancer.
  • osteoblasts neurones or neurones-like cells, astrocytes or astrocytes-like cells, mesenchymal stem cells, hematopoietic stem cells, or endothelial cell progenitors, suitable for clinical administration is not so easy.
  • osteoblasts need a specific culturing medium in order to be able to survive.
  • fetal calf serum or some other sort of bovine serum is provided in the culturing medium, in order to provide the cells with the necessary growth factors and signaling molecules keeping the cells alive.
  • Fetal or bovine calf serum may be contaminated with pathogens such as viruses, bacteria, prions etc, providing a high risk in administering cultured cells to a patient.
  • pathogens such as viruses, bacteria, prions etc.
  • the present invention provides new means and methods for culturing clinical grade osteoblasts, neurones or neurones-like cells, astrocytes or astrocytes-like cells, mesenchymal stem cells, hematopoietic stem cells, or endothelial cell progenitors, without the drawbacks of the prior art methods.
  • the present invention provides means and methods to culture osteoblasts or osteoblast-like cells, neurones or neurones-like cells, astrocytes or astrocytes-like cells, mesenchymal stem cells, hematopoietic stem cells, endothelial cell progenitors, in serum-free medium in such a way that spontaneous apoptosis of said cells is significantly reduced or avoided.
  • the invention provides a culture medium for cell-lines or primary cells comprising: a) serum-free medium, b) 1-30% autologous or heterologous platelet-rich- plasma or platelet-poor-plasma and c) a factor or a combination of factors selected from the group consisting of Tissue Factor (TF), Factor Vila and Factor Xa.
  • the plasma is autologous.
  • the plasma is platelet-rich- plasma.
  • the plasma may optionally also be defibrinated or not.
  • additionally CaCI2 is added to the medium.
  • the added factors are in purified or recombinant forms.
  • Tissue Factor TF
  • Factor Vila Factor Xa
  • compound c of the medium of the invention.
  • the cell-lines or primary cells are preferably selected from the group consisting of osteoblasts or osteoblast-like cells, neurones or neurones-like cells, astrocytes or astrocytes- like cells, mesenchymal stem cells, hematopoietic stem cells, endothelial cell progenitors, In the most preferred embodiment, the cells are of the osteoblast-like phenotype, osteoblast cell-lines or are isolated primary osteoblasts.
  • the method further provides a method of growing cell-lines or primary cells in vitro comprising the steps of adding to the culture a medium according to the invention.
  • the cell- lines or primary cells are preferably selected from the group consisting of osteoblasts or osteoblast-like cells, neurones or neurones-like cells, astrocytes or astrocytes-like cells, mesenchymal stem cells, hematopoietic stem cells, endothelial cell progenitors, and can be derived from an immortalised cell-line or are primary cells isolated from a subject.
  • the invention further provides a method of inhibiting apoptosis of cell-lines or primary cells in an in vitro culture comprising adding to the culture medium a factor or a combination of factors selected from the group consisting of Tissue Factor (TF), Factor Vila and Factor Xa.
  • TF Tissue Factor
  • Factor Vila Factor Xa
  • the combination of Tissue Factor (TF), Factor Vila and Factor Xa is used.
  • the cell-lines or primary cells are preferably selected from the group consisting of osteoblasts or osteoblast-like cells, neurones or neurones-like cells, astrocytes or astrocytes- like cells, mesenchymal stem cells, hematopoietic stem cells, endothelial cell progenitors, derived from an immortalised cell-line or are primary osteoblast cells isolated from a subject.
  • the invention further provides for the use of any one of Factor Vila, Tissue Factor and/or Factor Xa, or any combination thereof in the prevention of apoptosis of cell-lines or primary cells in vitro.
  • the cell-lines or primary cells are preferably selected from the group consisting of osteoblasts or osteoblast-like cells, neurones or neurones-like cells, astrocytes or astrocytes-like cells, mesenchymal stem cells, hematopoietic stem cells, endothelial cell progenitors.
  • the invention also provides for cell-lines or primary cells or cell populations and cultures comprising such, which are obtainable or directly obtained using the methods of the invention.
  • the invention further provides for the use of cell-lines or primary cells or cell populations and cultures comprising such in therapy of disorders wherein administration of said cells is needed.
  • the invention also provides a method for preventing and/or treating a disease wherein administration of cells is needed, comprising:
  • said cells are osteoblasts or osteoblast-like cells
  • disease is selected from the group of local or systemic disorders, such as, any type of osteoporosis or osteopenia, e.g., primary, postmenopausal, senile, corticoid-induced, any secondary, mono- or multisite osteonecrosis, any type of fracture, e.g., non-union, mal-union, delayed union fractures or compression, conditions requiring bone fusion (e.g., spinal fusions and rebuilding), maxillo-facial fractures, bone reconstruction, e.g., after traumatic injury or cancer surgery, cranio-facial bone reconstruction, osteogenesis imperfecta, osteolytic bone cancer, Paget's Disease, osteopetrosis, endocrinological disorders, hypophosphatemia, hypocalcemia, renal osteodystrophy, osteomalacia, adynamic bone disease, rheumatoid arthritis, hyperparathyroidism, primary hyperparathyroidism, secondary hyperparathyroidism, periodontal disease,
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising cell-lines or cells obtainable or directly obtained by the methods of the invention, or comprising an isolated cell population comprising said cells suitable for administration at a site where said cells are needed in the subject.
  • said cells are osteoblasts or have an osteoblast-like phenotype.
  • the advantage of the means and methods of the invention lies in the ability to reduce apoptotic events in an in vitro culturing system, without the need of adding serum components which may be unsuitable for use in clinical settings due to the accompanied risks of e.g. contamination with pathogens.
  • the invention provides a simple improvement of the in vitro culturing of highly needed progenitor or stem cells.
  • FIG. 1 Free FXa induces cell viability
  • A SaOS-2 cell viability (36 h incubation) in serum- starved medium, or stimulated with FXa (50 nM), FXa (50 nM) with DEGR (1 ⁇ M), FVIIa (50 nM) and sTF-FVIIa (50 nM).
  • B SaOS-2 cell viability in serum-starved medium, or stimulated with FXa (50 nM), with or without FVIIa (50 nM) or sTF-FVIIa complex (50 nM).
  • C SaOS-2 cell viability in serum-starved medium, or stimulated with TRAP-6 (10 ⁇ M) and SLIGRL (10 IM). Proteases and agonists were diluted in serum-starved medium. 100% are from 15% serum-containing medium. Average value of 24 wells from three independent experiments.
  • FIG.2 Results of apoptosis measurement ( Figure 2a) and measurement of dividing cells ( Figure 2b) in serum free culture medium wherein either FXa, FIIa or Trap-6 or FBS was added (Trap-6 is a PAR-1 agonist, downstream of Factor Xa). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the present invention provides new methods and means for inhibiting apoptosis of cells in an in vitro culture system.
  • the inventors have unexpectedly found that especially activated platelet-rich plasma or platelet-poor plasma can be used as an anti-apoptotic agent in in vitro cell culture.
  • FBS fetal bovine serum
  • the inventors have investigated whether it is possible to grow and culture human osteoblasts or osteoblast-like cells, in serum-free medium, without having the problem of excessive apoptosis of said cells.
  • the inventors have now surprisingly found that apoptosis of osteoblast-like cells in serum-free medium can be inhibited or reduced by adding the following compositions to the medium:
  • PRP Platelet-rich plasma
  • PPP platelet-poor plasma
  • the invention therefore provides for methods to culture osteoblast or osteoblast-like cells comprising the steps of:
  • Either newly isolated cells, or primary cells already in culture or cell lines will be put in culture boxes (or other) at a concentration varying between 1000 cells/ml to 100000 cells/ml, except for stem cells which can be seeded at a lower concentration, with medium described here below. 5. Depending on cell type, cells will be re-seeded from every 2 days to every 7 days.
  • the invention further provides for a culture medium for osteoblasts or osteoblast-like cells comprising: 1. DMEM or McCoy 5A (or other medium known for culturing such cells)
  • PRP Platelet-rich plasma
  • PPP Platelet-rich plasma
  • Platelet Rich Plasma is a component of whole blood that contains concentrated amounts of platelets.
  • Platelet-rich plasma PRP
  • PRP Platelet-rich plasma
  • the two main devices are: AngelTM (Sorin Group, Italy) and MagellanTM (Medtronic Inc, USA) Tissue factor (TF):
  • Tissue factor is a 47kDa transmembrane glycoprotein consisting of 263 amino acids: 219 amino acids constitutes the extracellular domain, 23 amino acids the transmembrane domain, and the cytoplasmic tail is maid of 21 amino acids.
  • tissue factor molecule Four types are known. Three of them can be obtained by genetic engineering:
  • tissue factor comprising 206 amino acids.
  • Amino acids 1 to 166 are identical to the extracellular domain of complete tissue factor, while residues 167-206 constitute a unique C terminal.
  • Tissue factor is the trigger of the coagulation cascade.
  • TF forms a ternary complex with factor Vila (FVIIa) and zymogen factor X (FX), which is then activated to factor Xa (FXa).
  • FVIIa factor Vila
  • FX zymogen factor X
  • TF also presents signaling effects by binding to both FVIIa and FXa. Both TF-FVIIa and TF- FVIIa-FXa complexes activate either G-protein coupled receptor PAR-2 (TF-FVIIa) or PAR-1 and PAR-2 (TF-FVIIa-FXa). This allows complexes to modulate various cellular processes.
  • TF-FVIIa G-protein coupled receptor PAR-2
  • TF-FVIIa-FXa PAR-1 and PAR-2
  • Zymogen factor VII is a serine protease of 406 amino acids, with a molecular weight of 45kDa.
  • Activated factor VII is made of two polypeptidic chains of 2OkDa and 3OkDa. Light chain is linked to heavy chain through a disulfide bridge.
  • Zymogen factor X is a 58kDa serine protease made of 2 chains, with 139 and 306 amino acids. Cleaving of peptide bond Arg194-lle195 makes activation of FX into FXa. Isolated factor Xa is produced by American Diagnostica (USA) for research purposes only.
  • Osteoblasts can be used in clinical settings to cure or reduce the effects of bone related disorders, wherein the term "bone-related disorder” as used herein refers to any type of bone disease, the treatment of which may benefit from the administration of osteogenic lineage cells, e.g., osteoprogenitors, osteoblasts or osteoblast phenotype cells to a subject having the disorder.
  • osteogenic lineage cells e.g., osteoprogenitors, osteoblasts or osteoblast phenotype cells
  • such disorders may be characterised, e.g., by decreased bone formation or excessive bone resorption, by decreased number, viability or function of osteoblasts or osteocytes present in the bone, decreased bone mass in a subject, thinning of bone, compromised bone strength or elasticity, etc.
  • bone-related disorders which can benefit from administration of osteoblasts or osteoblast phenotype cells of the present invention may include local or systemic disorders, such as, any type of osteoporosis or osteopenia, e.g., primary, postmenopausal, senile, corticoid-induced, any secondary, mono- or multisite osteonecrosis, any type of fracture, e.g., non-union, mal-union, delayed union fractures or compression, conditions requiring bone fusion (e.g., spinal fusions and rebuilding), maxillofacial fractures, bone reconstruction, e.g., after traumatic injury or cancer surgery, cranio- facial bone reconstruction, osteogenesis imperfecta, osteolytic bone cancer, Paget's Disease, osteopetrosis, endocrinological disorders, hypophosphatemia, hypocalcemia, renal osteodystrophy, osteomalacia, adynamic bone disease, rheumatoid arthritis, hyperparathyroidism, primary hyperpara
  • subject refers preferably to animals, more preferably warm-blooded animals, yet more preferably vertebrates, and even more preferably mammals specifically including humans and non-human mammals, that have been the object of treatment, observation or experiment.
  • mamal includes any animal classified as such, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, pet animals, companion animals and experimental animals, such as, for example, mice, rats, hamsters, rabbits, dogs, cats, guinea pigs, cattle, cows, sheep, horses, pigs and primates, e.g., monkeys and apes. Particularly preferred are human subjects, including both genders and all age categories thereof.
  • the present treatments are particularly to be given to subjects in need thereof, which phrase includes subjects that would benefit from treatment of a given condition, such as a disease or condition associated with immunodeficiency or immunosuppression.
  • a given condition such as a disease or condition associated with immunodeficiency or immunosuppression.
  • Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and/or those in whom said condition is to be prevented.
  • treat or “treatment” encompass both the therapeutic treatment of an already developed disorder, such as the therapy of an already developed disease or condition associated with immunodeficiency or immunosuppression, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent the chances of contraction and progression of a disease or condition associated with immunodeficiency or immunosuppression.
  • Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • prophylactically effective amount refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician.
  • therapeutically effective amount refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or disorder being treated. Methods are known in the art for determining therapeutically and prophylactically effective doses.
  • the treatment may employ autologous (i.e., cells derived from the subject to be treated), allogeneic (i.e., cells derived from subject(s) other than the subject to be treated, but belonging to the same species) or xenogenic (i.e., cells derived from subject(s) belonging to species other than the subject to be treated) bone-forming cells and cell populations as defined herein.
  • autologous i.e., cells derived from the subject to be treated
  • allogeneic i.e., cells derived from subject(s) other than the subject to be treated, but belonging to the same species
  • xenogenic i.e., cells derived from subject(s) belonging to species other than the subject to be treated
  • Envisaged are in particular treatments of human subjects using human autologous or allogeneic osteoblast bone-forming cells or cell populations as defined herein.
  • the herein defined bone-forming cells and cell populations may be formulated into and administered as pharmaceutical compositions.
  • the cells or cell populations of the invention may be introduced alone or in admixture with further components useful in the repair of bone wounds and defects.
  • Such compositions include, but are not limited to bone morphogenetic proteins, hydroxyapatite/tricalcium phosphate particles (HA/TCP), gelatin, poly-lactic acid, poly-lactic glycolic acid, hyaluronic acid, chitosan, poly-L-lysine, and collagen.
  • osteoblasts may be combined with
  • Demineralized Bone Matrix or other matrices to make the composite osteogenic (bone forming in it own right) as well as osteo-inductive. Similar methods using autologous bone marrow cells with allogeneic DBM have yielded good results (Connolly et al. 1995. Clin
  • the composition may contain further components ensuring the viability of such cells, e.g., osteoblasts or osteoblast phenotype cells therein.
  • the cells or cell populations can be supplied in the form of a pharmaceutical composition, comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration.
  • a pharmaceutical composition comprising an isotonic excipient prepared under sufficiently sterile conditions for human administration.
  • the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P.
  • the composition may comprise a suitable buffer system to suitable pH, e.g., near neutral pH (e.g., phosphate or carbonate buffer system), and may comprise sufficient salt to ensure iso-osmotic conditions for the cells or cell populations, i.e., preventing osmotic stress.
  • suitable solution for these purposes may be phosphate-buffered saline (PBS) as known in the art.
  • the composition may comprise a carrier protein, e.g., albumin, which may increase the viability of the cells.
  • the albumin may be of human origin (e.g., isolated from human material or produced recombinantly). Suitable concentrations of albumin are generally known.
  • the cells or cell populations can be administered in a manner that permits them to graft or migrate to the intended tissue site and reconstitute or regenerate the functionally deficient area. Administration of the composition will depend on the musculoskeletal site being repaired. For example, osteogenesis can be facilitated in concordance with a surgical procedure remodel tissue or insert a split, or a prosthetic device such as a hip replacement. In other circumstances, invasive surgery will not be required, and the composition can be administered by injection or (for repair of the vertebral column) using a guidable endoscope.
  • the cell preparation can further include or be co-administered with a complementary bioactive factor such as a bone morphogenic protein, such as BMP-2 or BMP-4, or any other growth factor.
  • a complementary bioactive factor such as a bone morphogenic protein, such as BMP-2 or BMP-4, or any other growth factor.
  • Other potential accompanying components include inorganic sources of calcium or phosphate suitable for assisting bone regeneration (WO 00/07639).
  • cell preparation can be administered on a carrier matrix or material to provide improved tissue regeneration.
  • the material can be a granular ceramic, or a biopolymer such as gelatin, collagen, osteonectin, fibrinogen, or osteocalcin.
  • Porous matrices can be synthesized according to standard techniques (e.g., Mikos et al., Biomaterials 14:323, 1993; Mikos et al., Polymer 35:1068, 1994; Cook et al., J. Biomed. Mater. Res. 35:513, 1997).
  • the cell preparation as define above may be administered in a form of liquid composition.
  • the cells or cell populations of the invention may be transferred to and/or cultured on suitable substrate to provide for implants.
  • the substrate on which the cells can be applied and cultured can be a metal, such as titanium, cobalt/chromium alloy or stainless steel, a bioactive surface such as a calcium phosphate, polymer surfaces such as polyethylene, and the like.
  • a metal such as titanium, cobalt/chromium alloy or stainless steel
  • a bioactive surface such as a calcium phosphate
  • polymer surfaces such as polyethylene, and the like.
  • siliceous material such as glass ceramics
  • the substrate may be porous or non-porous.
  • cells that have proliferated, or that are being differentiated in culture dishes can be transferred onto three-dimensional solid supports in order to cause them to multiply and/or continue the differentiation process by incubating the solid support in a liquid nutrient medium of the invention, if necessary.
  • Cells can be transferred onto a three-dimensional solid support, e.g. by impregnating said support with a liquid suspension containing said cells.
  • the impregnated supports obtained in this way can be implanted in a human subject.
  • Such impregnated supports can also be re-cultured by immersing them in a liquid culture medium, prior to being finally implanted.
  • the three-dimensional solid support must be biocompatible so as to enable it to be implanted in a human. It can be of any suitable shape such as a cylinder, a sphere, a plate, or a part of arbitrary shape.
  • materials suitable for the biocompatible three-dimensional solid support particular mention can be made of calcium carbonate, and in particular aragonite, specifically in the form of coral skeleton, porous ceramics based on alumina, on zirconia, on tricalcium phosphate, and/or hydroxyapatite, imitation coral skeleton obtained by hydrothermal exchange enabling calcium carbonate to be transformed into hydroxyapatite, or else apatite-wollastonite glass ceramics, bioactive glass ceramics such as Bioglass(TM) glasses or biodegradable polymer scaffolds.
  • Autologous or heterologous plasma is used for serum-free culturing of the human osteoblasts or osteoblast-like cells.
  • This plasma can be isolated from the same patient to which the osteoblast cells have to be administered (autologous) or can be derived from one or more other subjects (heterologous).
  • the isolated plasma or serum Prior to use, can be heat inactivated. Heat inactivation is used in the art mainly to remove the complement. Where the present method employs plasma or serum autologous to the cells cultured in the presence thereof, it may be unnecessary to heat inactivate the plasma or serum. Where the plasma or serum is heterologous, heat inactivation may be advantageous.
  • Heat inactivation typically involves incubating the plasma or serum at 56°C for 30 to 60min, e.g., 30min, with steady mixing, after which the plasma or serum is allowed to gradually cool to ambient temperature.
  • the plasma or serum may also be sterilised prior to use by e.g., filtration through one or more filters with pore size smaller than 1 ⁇ m, preferably smaller than 0.5 ⁇ m, e.g., smaller than 0.45 ⁇ m, 0.40 ⁇ m, 0.35 ⁇ m, 0.30 ⁇ m or 0.25 ⁇ m, more preferably 0.2 ⁇ m or smaller, e.g., 0.15 ⁇ m or smaller, 0.10 ⁇ m or smaller.
  • the method may employ a mixture of autologous and heterologous plasma as defined above.
  • human plasma or serum may be included in the media at a proportion (volume of serum / volume of medium) between 0.5% and 30%, preferably between 1 % and 20%, more preferably between 2% and 10%, such as between 5% and 10%, e.g., at about 5%, 6%, 7%, 8%, 9% or 10%.
  • Any medium capable of supporting the growth of fibroblasts in cell culture may be used in the present method of culturing osteoblasts or osteoblast-like cells.
  • Media formulations that will support the growth of fibroblasts include, but are not limited to, Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), BGJb, F-12 Nutrient Mixture (Ham), and the like, which are commercially available (e.g., Invitrogen, Carlsbad, California).
  • a particularly suitable medium for use in the present method may be alpha-MEM, IMDM, X- Vivo-10, X-Vivo 20 serum free medium (clinical grade), available from Invitrogen or Cambrex (New Jersey).
  • Such liquid culture media contain ingredients necessary for mammal cell development, which are known per se.
  • these ingredients include inorganic salts (in particular Na, K, Mg, Ca, and possibly Cu, Fe, and Zn), amino acids, vitamins, and sources of carbon (e.g. glucose), etc.
  • the media may further contain one or more compounds of interest, including, but not limited to, sodium bicarbonate, antibiotic and/or antimycotic components, such as, penicillin, streptomycin and/or amphotericin, etc.
  • the osteoblast-like cells are not contacted with any component obtained from a non-human animal, in particular non-human mammal. If osteoblasts or osteoblast phenotype cells are to be administered to a human subject, the absence of contact between the BMSC and components obtained from non-human animals ensures optimal acceptance of the cells by the subject and avoids accidental transmission of infectious agents thereto. The latter concern becomes increasingly important due to the appearance of prion diseases, e.g., BSE, which can be transmitted from animals to humans.
  • prion diseases e.g., BSE
  • osteoblast-like cells are not contacted with any serum component derived from a non-human animal.
  • FCS or BCS addition of, e.g., FCS or BCS to cell culture media is commonplace in the art to sustain cell culture growth, the method of the invention avoids this use.
  • a cell refers to one or more than one cell.
  • the terms “comprising”, “comprises” and “comprised of as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.
  • Example 1 culturing of SaOS-2 cell-line in serum-free medium
  • the method of the invention comprises the following steps: the addition to the serum-free culture medium of purified factor Xa in a concentration of 5OnM or; the addition to the serum-free culture medium of a complex of human recombinant tissue factor, recombinant factor Vila and purified factor Xa, all in a concentration of 5OnM resulting in a reduction of apoptosis after 36h of culturing in an incubator (37°C, 5% CO2).
  • the values of apoptosis are respectively (mean ⁇ SEM) :

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Abstract

La présente invention porte sur de nouveaux moyens et procédés pour cultiver des ostéoblastes, des neurones ou cellules de type neurones, des astrocytes ou des cellules de type astrocytes, des cellules souches mésenchymateuses, des cellules souches hématopoïétiques ou des progéniteurs de cellules endothéliales de qualité clinique, sans les inconvénients des procédés de l'état antérieur de la technique. En particulier, l'invention porte sur des moyens et sur des procédés pour cultiver des ostéoblastes ou des cellules de type ostéoblastes, des neurones ou des cellules de type neurones, des astrocytes ou des cellules de type astrocytes, des cellules souches mésenchymateuses, des cellules souches hématopoïétiques, des progéniteurs de cellules endothéliales, dans un milieu exempt de sérum d'une façon telle, à savoir par addition de TF, de FVIIa, de FXa ou d'un complexe de ceux-ci, qu'une apoptose spontanée desdites cellules est significativement réduite ou évitée.
PCT/EP2008/056994 2008-06-05 2008-06-05 Utilisation de fxa, de fviia, de tf ou d'une combinaison de ceux-ci en tant qu'agents anti-apoptotiques Ceased WO2009146746A1 (fr)

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WO2010089379A1 (fr) * 2009-02-05 2010-08-12 Pierre Philippart Procédé et moyen de production de tissus et tissus obtenus
CN113101303A (zh) * 2021-04-15 2021-07-13 四川大学 自发性凋亡的间充质干细胞的制备方法及用途

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010089379A1 (fr) * 2009-02-05 2010-08-12 Pierre Philippart Procédé et moyen de production de tissus et tissus obtenus
US9259442B2 (en) 2009-02-05 2016-02-16 Pierre Philippart Method and means for producing tissues and tissues obtained
CN113101303A (zh) * 2021-04-15 2021-07-13 四川大学 自发性凋亡的间充质干细胞的制备方法及用途

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