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WO1993011225A1 - Culture de cellules osseuses - Google Patents

Culture de cellules osseuses Download PDF

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Publication number
WO1993011225A1
WO1993011225A1 PCT/GB1992/002185 GB9202185W WO9311225A1 WO 1993011225 A1 WO1993011225 A1 WO 1993011225A1 GB 9202185 W GB9202185 W GB 9202185W WO 9311225 A1 WO9311225 A1 WO 9311225A1
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Prior art keywords
cells
bone
asc
culture
primary
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PCT/GB1992/002185
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English (en)
Inventor
Martin James Ogilvie Francis
Jon Nicholas Beresford
Stephen Ellis Graves
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Isis Innovation Limited
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Publication of WO1993011225A1 publication Critical patent/WO1993011225A1/fr

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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
    • A61L27/3821—Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor 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
    • A61L27/3895—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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones

Definitions

  • the present invention is concerned with the culturing of bone cells.
  • Bone is a specialised connective tissue. It provides support and protection for other tissues of the body, allows movement and functions as an organ in mineral and haemopoietic homeostasis. It contains a large number of cell types and a unique intercellular matrix. Some of the cells are specific to bone and are responsible for its development, maintenance and repair.
  • the matrix contains both organic and inorganic components.
  • the organic component consists chiefly of collagen, but also contains a large number of other organic molecules.
  • the inorganic (or mineral) component is a poorly crystalline carbonate-containing analogue of hydroxyapatite. There is an intimate relationship between cells, organic matrix production and the deposition of mineral that results in the formation of bone.
  • Ascorbic acid (Vitamin C) is one factor known to be important in the normal function of human bone forming cells (osteoblasts) . Unlike most other mammals humans do not synthesise ascorbic acid but are entirely dependent on dietary ascorbate to supply their requirements. In scurvy, which is due to a deficiency of ascorbate, bone formation completely ceases.
  • ascorbic acid was shown to increase collagen, but not non-collagenous protein synthesis. Mineralisation was also not affected, but was deposited as a calcified cartilage core (Chen and Raisz, 1975). Ascorbic acid is known to be essential for mineralised matrix production by bone-derived cells in vitro (Anderson et al. 1984; Aronow et al. 1990; Bellows et al. 1986; Nefussi et al, 1985; Ecarot- Charrier et al. 1983; Tenenbaum and Heersche, 1982).
  • bone cell tissue culture systems have been developed. The majority of these systems have used bone cells obtained from animals however their relevance to human bone cell physiology is not known. Studies involving human cells have been much less frequent. This is in part due to the difficulty of obtaining sufficient growth of suitably differentiated cells using conventional techniques when compared to the animal models.
  • autologous bone is the best alternative but it has three main problems. The first is that there is a limited supply so it can only be used in treating small areas of bone loss. The second is its failure to provide structural stability. This is because only small fragments of mainly trabecular bone are obtained when harvesting and in many situations it is not possible to achieve the structural stability using these fragments. The third is that there is considerable morbidity to the patient when bone is taken from other areas of the body. Allografts on the other hand have been particularly useful because they provide the structural stability not possible to achieve with autologous grafting. Whole bones or large segments of bone can be completely replaced using allografts. Unfortunately the bone is dead and there is no inherent bone forming property within it.
  • Na ascorbate is the usual form of ascorbate used in these situations. In cell culture conditions it is unstable, having a half life of 7-10 hours. As culture medium is changed every 2-3 days the addition of Na ascorbate results in a pulsed treatment of the cells. Under normal in vivo circumstances there is a constant level of ascorbate, therefore the use of Na ascorbate cannot be regarded as physiological.
  • L- Ascorbic Acid 2-Phosphate L- Ascorbic Acid 2-Phosphate
  • ASC-2P The structure of ASC-2P is shown in Figure 1.
  • Primary culture The earliest stage in culturing bone and bone-forming cells, that is, when the cells and/or bone explants are first placed in culture, is known as primary culture.
  • First passage cells from bone are the cells derived from bone explants or other bone cell primary cultures typically after four to six weeks in primary culture.
  • Bone and bone-forming cells include marrow stromal osteoprogenitors and may come from the bone marrow, bone and periosteum (the fibrous layer covering the outer surface of the bone) .
  • the invention is of primary interest for human bone and bone-forming cells, it also extends to the cells of other animals.
  • a long- acting source of ascorbate is one which provides a continuing supply of ascorbate in a form usable by the cells, at a practicable frequency of culture medium change.
  • the preferred long-acting source of ascorbate is L-ascorbic acid 2-phosphate (ASC 2P) , a commercially available compound.
  • the nature of the culture medium is not material to the invention, and conventional media can be used.
  • the ascorbate concentration in the fresh culture medium as added should preferably be in the range 2-2,000 ⁇ M, and the ascorbate concentration in contact with the cells should preferably not fall below 2 ⁇ M preferably 20 ⁇ M.
  • adipocytic differentiation develops only in the cells from explants cultured in ASC 2P.
  • Adipocytic differentiation of cells derived from human bone derived cells has not been previously reported.
  • the populations of cells derived from explants are more proliferative, produce more matrix and have a greater capacity to react to factors influencing cellular differentiation and function.
  • cell culture conditions inducing cell proliferation can be achieved using other substances influencing cells such as growth factors, the resulting cells are of a poorer quality and the cost of such methods can be substantial.
  • the resulting first passage cells may be used in various ways. They may be further cultured in vitro, preferably in the presence of a long-acting source of ascorbate such as ASC 2P, under conditions which are not material to the invention and which may be conventional.
  • the cells, either at the first passage stage or after further culturing may be implanted into a patient, or used in other ways.
  • the invention further provides a method of culturing bone and bone-forming cells wherein the cells are cultured in vitro with a long-acting source of ascorbate from the primary culture stage, and are subsequently implanted into the recipient as an allograft or an autograft or a xenograft.
  • the cells can be obtained by taking either a small amount of bone or alternatively bone marrow which is also a source of osteogenic cells. In the presence of a long-acting source of ascorbate the cells can be rapidly expanded over a 4-6 week period and then reimplanted. To ensure the bone-derived cells are retained in the appropriate area it may be necessary to implant them in a biocompatible (i.e. supports bone formation) matrix. This has the advantage of being able to shape the graft to the exact dimensions needed. If structural stability is required then the cells can be implanted into a structurally stable biocompatible material such as ceramic. A number of materials already developed would be suitable such as A-W glass ceramic. The rate of bone formation after implantation is likely to be rapid i.e. measured in weeks.
  • the advantage of the invention in this embodiment is that using a long-acting source of ascorbate allows culturing of osteogenic cells to be much faster and they appear to be of much better "quality" than cells cultured using conventional techniques.
  • the cells produce a large amount of matrix.
  • Bone matrix has the ability to induce non-osteogenic cells to differentiate into bone cells. So there is the two fold advantage of being able to implant large numbers of the patients own healthy bone cells with a significant amount of matrix which may promote recruitment of further bone cells from surrounding non- osteogenic tissue.
  • Bone substitutes derived from animal bone are currently commercially available.
  • the invention provides a method in which one or more materials are added to the cells cultured as above and the effect of the material or materials on the growth and condition of the cells is assessed.
  • A-W glass ceramic a material that has already been used for replacing bone in humans, is known to show useful properties as a biomaterial in vivo. Its effects in vitro on bone cells have now been investigated. Using conventional culture techniques the material is detrimental to human bone-derived cells. However using cells that have been cultured in ASC 2P from the explant stage there is promotion of osteoblastic differentiation and matrix production by the A-W glass ceramic. Another material that has now been tested is the bioactive material that is the subject of our U.K. patent application GB 91 22 329.7. This is also shown to promote osteoblastic differentiation and matrix production when bone cells are provided with ASC 2P. The methods used to assess these materials are distinguished only by the fact that the cells involved are cultured in ASC 2P from the explant stage. The cells therefore have the ability to react to osteoblastic stimuli in a much better manner than cells obtained using conventional techniques and are rendered more appropriate for use is assessing effectiveness of biomaterials.
  • the pH was adjusted to 7.35 at 37 * C by the addition of 10M NaOH. 10% v/v fetal calf serum was added before use.
  • Human bone derived cells were obtained by outgrowth from explants of normal human trabecular bone using a modification of the method described by Beresford et al. (1983)
  • the bone was cut into small fragments 3-5 mm in diameter, washed by vigorous vortexing in calcium and magnesium free phosphate buffered saline (PBS) to remove blood and marrow. This wash was repeated three times.
  • Eight to ten explants were then placed into T80 flasks containing 10 mis of culture medium and incubated at 31 ' C in an atmosphere of 5% C0 2 and 95% humidity. The medium was changed completely at 7 and 14 days. Thereafter the medium was completely changed three times a week (Mon, Wed and Fri) .
  • the culture medium in half of the flasks was supplemented with L- Ascorbic Acid 2-Phosphate at 100 ⁇ M concentration throughout. The cells were maintained for four weeks after which time they were passaged.
  • the cells were then cultured for a further seven days and the media changed every two days.
  • the parameters tested were: cell proliferation assessed by cell counts, total DNA and by thymidine incorporation, total protein content, alkaline phosphatase activity, osteocalcin release, collagen and non-collagenous protein synthesis.
  • Cells were incubated for 24 hours in one ml of medium containing 5 ⁇ Ci [methyl- ⁇ H]thymidine (5 Ci/mmol) . The incubation was terminated by removing the labelled medium and washing the cell layer three times with one ml of PBS' containing 1 mM non- radioactive thymidine. The cells were then detached by incubating for 30 mins at 37'C in one ml of trypsin/EDTA solution, supplemented with 1 mM non- radioactive thymidine. The cell suspension was transferred to 4.5 ml polypropylene tubes. The wells were then washed twice with 0.5 ml ultra-pure water containing 1 mg/ml of bovine serum albumin.
  • Total cell layer protein was assessed by colourimetric assay using Co ⁇ massie Brilliant Blue
  • Protein content was then determined by adding a 50 ⁇ l aliquot to 2.5 mis of the protein assay solution.
  • This solution contained 0.01% (w/v) Coomassie Brilliant Blue G-250, 4.7% (v/v) ethanol and 8.5% (w/v) phosphoric acid.
  • the result was determined spectrophotometrically (system 2600 Guilford Instrument Lab. Inc., Ohio, USA) at 595 nm wavelength.
  • the values obtained were compared to a range of standards (1-25 ⁇ g) prepared from bovine serum albumin in 0.2% NP-40. The final result for one well was the mean of three replicates from that well.
  • Alkaline Phosphatase Activity was determined by measuring the release of p-nitrophenol from p- nitrophenyl phosphate as described in Sigma technical bulletin No.104 (Sigma Ltd, Dorset, UK). The samples used to , determine the total cell layer protein content were also used to measure alkaline phosphatase activity. An aliquot of 100 ⁇ l was added to 900 ⁇ l of assay buffer.
  • the assay buffer was prepared by dissolving 40 mg of p-nitrophenyl phosphate disodium in 10 mis of 221-alkaline buffer solution (2-amino-2 methyl-1 propanol buffer 1.5 mol/1 pH 10.3 at 25'C) and adding 20 mis of ultra-pure water. The samples were then incubated for 30 mins at 37 * C and the reaction terminated by the addition of one ml of 1 M NaOH (final concentration of 0.5 M) . The amount of p-nitrophenol released was determined using a spectrophotometer
  • Osteocalcin release into the medium was determined by radioimmunoassay.
  • the cells were cultured for 24 hrs in one ml of medium which contained 2% FCS as well as the additives being tested.
  • the media were further supplemented with 10 ⁇ 8 M 1,25(OH) 2 Vitamin D 3 and 10 ⁇ 8 M Vitamin K.
  • 10 ⁇ 8 M 1,25(OH) 2 Vitamin D 3 and 10 ⁇ 8 M Vitamin K was added to determine the background osteocalcin present in the FCS used for each experiment.
  • To determine the background osteocalcin present in the FCS used for each experiment one ml of medium was incubated for 24 hrs in wells without cells and harvested at the same time as those with cells. After removal the medium was frozen in liquid nitrogen and stored at -70°C until required.
  • Osteocalcin was measured by using an osteocalcin radioimmunoassay kit (OSTK-PR, CIS Bio International, Cedex, France) . The result for each well was obtained by subtracting the value obtained from the media samples incubated in the absence of cells.
  • OSTK-PR osteocalcin radioimmunoassay kit
  • This assay was used to determine the amount of collagen and non-collagenous protein synthesised during a 24 hr labelling period.
  • the cell layer and medium were assayed separately and the total determined by addition of these two fractions.
  • the cells were labelled for 24 hrs with L-[5- 3 H] proline (23 Ci/mmol) 10 ⁇ Ci/ml of medium.
  • the medium was removed and added to 4.5 ml polypropylene tubes.
  • the cell layer washed twice with 0.5 ml of PBS' containing 1 mM proline and each of the washes were added to the appropriate tube.
  • the cell layer was removed by scraping with a rubber policeman into one ml of PBS'.
  • the cell suspension was then removed and added to separate polypropylene tubes.
  • the well was washed two more times with 0.5 ml of PBS' which was pooled with the cell suspension from that well. Both the media and cell layer samples were frozen in liquid nitrogen and stored at -70'C until required.
  • the samples were sonicated on ice for 5 sees using the microtip of a Branson sonifier at 20% maximum output power.
  • the protein was then precipitated by adding 2 mis of 20% TCA supplemented with 1 mM of proline to each tube which was then left at 4'C overnight.
  • the precipitate was then pelleted by centrifugation at 1600 x g at 4'C for 30 mins. The supernatant was discarded and the pellet resuspended in 1 ml of 5% TCA containing 1 mM proline and left for 60 mins at 4'C.
  • the TCA wash was repeated one more time and the pellet solubilised in 400 ⁇ l 0.2 M NaOH.
  • the pH was partially neutralised by addition of 300 ⁇ l 0.15 M HC1 and 200 ⁇ l 1M HEPES pH 7.2.
  • the samples were then divided in half by transfer of 450 ⁇ l to an additional tube to create two sets of tubes for each sample.
  • the tubes were then incubated for 2 hrs at 37"C following the addition of 50 ⁇ l of collagenase-buffer solution to each tube.
  • the collagenase-buffer solution contained 2.5 Iu/tube of highly purified clostridial collagenase and the second set contained buffer alone.
  • the buffer solutions with and without collagenase were prepared as shown in Table 1. Table 1 Preparation of Collagenase-buffer solutions
  • Tris-HCl (0.05 M pH 7.6) .+ CaCl 2 (5 mM) 625 ⁇ l 675 ⁇ l
  • the collagenase solution was prepared by dissolving 2,500 lu of the highly purified collagenase (EC in one ml of Tris-HCl (0.05 M pH 7.6)) containing 5 mM CaCl 2 and storing in 50 ⁇ l aliquots at -70°C until required.
  • the pellets were dissolved by incubation with 750 ⁇ l of 1 M NaOH at 60'C for 30 mins and then transferred to a 20 ml scintillation vial and the pH neutralised with 750 ⁇ l of 1 M HC1 and 15 mis of scintillin added.
  • the collagen component was contained within the supernatant and was calculated by subtracting the result obtained from the samples that were not treated with collagenase (i.e. background) from those that were (i.e. background + collagen) .
  • the non-collagenous protein was contained within the pellet in the collagenous treated samples.
  • the substrate specificity of the collagenous preparation used was assessed by determining its activity against tryptophan-labelled protein. This amino acid does not occur in collagen.
  • Cells were incubated for 24 hrs in one ml of medium containing 250 ⁇ mol of Na ascorbate and 10 ⁇ Ci of L-[G- 3 H]tryptophan (7.2 Ci/mmol) .
  • the cell layer and medium were processed as described for L-[5- 3 H] proline. After subtracting the background from the collagenase treated samples for six replicates, 0.4 ( ⁇ 0.02)% of the tryptophan was released from the cell layer and 0.1 ( ⁇ 0.01)% from the medium confirming that the preparation is largely free of non-specific proteolytic activity.
  • the cells in ASC 2P supplemented standard medium reached confluence sooner and there was a greater than 3 fold increase in cell number when the cells were passaged.
  • Alkaline Phosphatase Activity is associated with mineralisation and appears to be largely confined to bone cells that are actively synthesising matrix. It is therefore a useful marker of differentiation.
  • the cells cultured in ASC 2P in primary culture had reduced alkaline phosphatase activity when corrected for protein or DNA.
  • the alkaline phosphatase activity was increased when ASC 2P was withdrawn in secondary culture (see Figs. 5a, b, and c) . Osteocalcin Release
  • Osteocalcin is one of the non-collagenous proteins of bone and is probably the only one that is unique to calcified tissue.
  • groups not treated with 1 ,25(OH) VitD 3 and vitamin K there was a small base line level of osteocalcin which varied between 1-3 ng/ml. Following correction for either total protein or DNA these levels were constant between each of the groups. After 24 hrs incubation with D and K the total osteocalcin released was greatest in the AA treated group. This difference with the other culture conditions was also maintained when corrected for total protein and DNA.
  • the SA treated group was also significantly increased when corrected for DNA but not for protein.
  • the AS treated group was also elevated compared to SS except when corrected for protein (Figs. 6a, b and c) .
  • the extracellular matrix of bone constitutes 92-95% of the total volume of bone. It consists of both organic (22% by weight) and inorganic (69%) components. The organic matrix is largely collagen (90%) and the remainder is the heterogeneous group of non-collagenous proteins.
  • Vitamin D is essential for normal mineralisation.
  • Vitamin K is necessary in the formation of non-collagenous proteins of bone matrix.
  • ASC 2P in primary culture is necessary for the maintenance of stromal integrity within the explants. In addition it alters the cell population obtained by outgrowth from the explants. Cells which have been in the continual presence of ASC 2P proliferate faster and synthesise more collagen and non-collagenous proteins. They have a reduced amount of alkaline phosphatase but this can be readily induced with either 1 ,25(OH) 2 VitD 3 or dexamethasone. Osteocalcin release following stimulation with 1,25(OH) 2 VitD 3 is high. In further studies (results not shown) mineralisation and adipocytic differentiation were observed using light and electron microscopy.
  • SA standard medium in primary but 100 ⁇ M ASC 2P in secondary.
  • AS ASC 2P in primary culture and standard medium in secondary.
  • AA ASC 2P in both primary and secondary culture.
  • SA standard medium in primary but 100 ⁇ M ASC 2P in secondary.
  • AS ASC 2P in primary culture and standard medium in secondary.
  • AA ASC 2P in both primary and secondary culture.
  • SA standard medium in primary but 100 ⁇ M ASC 2P in secondary.
  • AS ASC 2P in primary culture and standard medium in secondary.
  • AA ASC 2P in both primary and secondary culture.
  • SA standard medium in primary but 100 ⁇ M ASC 2P in secondary.
  • AS ASC 2P in primary culture and standard medium in secondary.
  • AA ASC 2P in both primary and secondary culture.
  • FIG. 6a Effects of ASC 2P in primary culture on osteocalcin production.
  • SA standard medium in primary but 100 ⁇ M ASC 2P in secondary.
  • AS ASC 2P in primary culture and standard medium in secondary.
  • AA ASC 2P in both primary and secondary culture.

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Abstract

L'invention concerne un procédé pour maintenir des cellules osseuses ou des cellules ostéoformatrices en culture et consistant à produire des cellules, au stade de culture primaire, avec une source à action prolongée d'acide ascorbique L 2-phosphate. De préférence, au premier stade de passage, les cellules en culture sont introduites dans le corps comme une allogreffe, une autogreffe ou une hétérogreffe, avec le support d'une matrice biocompatible ou sous la forme d'une suspension.
PCT/GB1992/002185 1991-11-26 1992-11-26 Culture de cellules osseuses WO1993011225A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB919125052A GB9125052D0 (en) 1991-11-26 1991-11-26 Culture of bone cells
GB9125052.2 1991-11-26

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WO1993011225A1 true WO1993011225A1 (fr) 1993-06-10

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997014783A1 (fr) * 1995-10-20 1997-04-24 North Shore University Hospital Research Corporation Reparation osseuse par modification tissulaire faisant appel a des cellules periostiques cultivees
WO1999035242A1 (fr) * 1998-01-12 1999-07-15 Betagene, Inc. Milieu de culture de cellules neuroendocrines
US6152964A (en) * 1996-03-01 2000-11-28 Isotis B.V. Method for in vitro production of bone
EP2451963A4 (fr) * 2009-07-10 2013-01-23 Csl Ltd Procédé d'augmentation du rendement de l'expression de protéines dépendantes de la vitamine k
US20130267026A1 (en) * 2002-06-07 2013-10-10 P Tech, Llc Methods of building a body portion

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Publication number Priority date Publication date Assignee Title
EP0282746A1 (fr) * 1987-02-19 1988-09-21 Takeda Chemical Industries, Ltd. Procédé de préparation de tissu cultivé de manière artificielle
EP0339607A2 (fr) * 1988-04-29 1989-11-02 Samuel Dr. Itay Composition pour la réparation de cartilage et d'os et procédé pour sa préparation comme implant de tissu du squelette

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0282746A1 (fr) * 1987-02-19 1988-09-21 Takeda Chemical Industries, Ltd. Procédé de préparation de tissu cultivé de manière artificielle
EP0339607A2 (fr) * 1988-04-29 1989-11-02 Samuel Dr. Itay Composition pour la réparation de cartilage et d'os et procédé pour sa préparation comme implant de tissu du squelette

Non-Patent Citations (1)

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Title
BONE vol. 11, no. 5, 1990, NEW YORK, N.Y., US page 378 S.E. GRAVES ET AL. 'EFFECTS OF L-ASCORBIC ACID 2-PHOSPHATE ON HUMAN BONE DERIVED CELLS.' cited in the application *

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WO1997014783A1 (fr) * 1995-10-20 1997-04-24 North Shore University Hospital Research Corporation Reparation osseuse par modification tissulaire faisant appel a des cellules periostiques cultivees
US6152964A (en) * 1996-03-01 2000-11-28 Isotis B.V. Method for in vitro production of bone
WO1999035242A1 (fr) * 1998-01-12 1999-07-15 Betagene, Inc. Milieu de culture de cellules neuroendocrines
US20130267026A1 (en) * 2002-06-07 2013-10-10 P Tech, Llc Methods of building a body portion
US10294455B2 (en) * 2002-06-07 2019-05-21 P Tech, Llc Methods of building a body portion
US11613731B2 (en) 2002-06-07 2023-03-28 P Tech, Llc Scaffold and method for implanting cells
EP2451963A4 (fr) * 2009-07-10 2013-01-23 Csl Ltd Procédé d'augmentation du rendement de l'expression de protéines dépendantes de la vitamine k
US9212214B2 (en) 2009-07-10 2015-12-15 Csl Limited Methods of increasing the expression yield of vitamin K-dependent proteins

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AU2951892A (en) 1993-06-28

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