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WO2005095577A1 - Conteneur de culture, méthode de culture des chondrocytes et méthode d'évaluation des chondrocytes - Google Patents

Conteneur de culture, méthode de culture des chondrocytes et méthode d'évaluation des chondrocytes Download PDF

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Publication number
WO2005095577A1
WO2005095577A1 PCT/JP2005/006707 JP2005006707W WO2005095577A1 WO 2005095577 A1 WO2005095577 A1 WO 2005095577A1 JP 2005006707 W JP2005006707 W JP 2005006707W WO 2005095577 A1 WO2005095577 A1 WO 2005095577A1
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WIPO (PCT)
Prior art keywords
collagen
culture
chondrocytes
gel layer
layer
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PCT/JP2005/006707
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English (en)
Japanese (ja)
Inventor
Masahito Taya
Masahiro Kino-Oka
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Japan Tissue Engineering Co., Ltd.
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Publication date
Application filed by Japan Tissue Engineering Co., Ltd. filed Critical Japan Tissue Engineering Co., Ltd.
Priority to JP2006511874A priority Critical patent/JP4825666B2/ja
Publication of WO2005095577A1 publication Critical patent/WO2005095577A1/fr

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Classifications

    • 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
    • A61L27/3817—Cartilage-forming cells, e.g. pre-chondrocytes
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • C12N5/0655—Chondrocytes; Cartilage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • 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 a culture vessel, a method for culturing chondrocytes, a method for evaluating chondrocytes, a method for producing a three-dimensional culture of chondrocytes, and a human implant.
  • chondrocytes grown in two-dimensional culture represented by planar culture (monolayer culture) into a joint in the form of a cell suspension.
  • planar culture monolayer culture
  • chondrocytes are dedifferentiated into fibroblast-like cells when continuous culture is continued, techniques for culturing chondrocytes while maintaining the characteristics of the chondrocytes have been studied.
  • chondrocytes are culturing chondrocytes on UV-crosslinked type II collagen-coated surfaces. Shakibaei et al, "Cell Biology International", 1997, 21, 2, p.115-125). Also disclosed is a method of regenerating chondrocytes by embedding in collagen gel and performing three-dimensional culture even on dedifferentiated chondrocytes (Japanese Translation of PCT International Publication No. 2003-534792). It is also known that chondrocytes can be supported on a scaffold composed of artificial materials or extracellular matrix components, and that the characteristics of chondrocytes can be maintained by three-dimensional culture.
  • wrap chondrocytes in collagen gel There is a technique for treating joint diseases by implanting cultured cartilage cultured in an implanted state into the joint (Mitsuo Ochi et al., "Nippon Medical Shinbun", 1998, 3875, p.33-36 ).
  • differentiation is promoted by culturing epithelial cells in a culture vessel having a culture surface covered with a cross-linked collagen film (Japanese Patent Application Laid-Open No. 08-038165). Disclosure of the invention
  • Still another object is to provide a method for efficiently producing a three-dimensional culture of chondrocytes.
  • the inventors of the present invention have repeatedly studied the above problems, and have found that culturing chondrocytes on the surface of a collagen gel layer can suppress dedifferentiation and proliferate while maintaining the characteristics well. Completed the invention. According to the present invention, the following means are provided.
  • a culture vessel comprising a collagen layer containing a collagen in an uncrosslinked or low crosslinked state.
  • the collagen layer preferably contains at least 0.5 mg of collagen per 1 cm 2 of the surface.
  • the collagen layer contains type I collagen.
  • Any of these culture vessels is preferably a culture vessel for culturing chondrocytes, and is preferably in a form for two-dimensional culture typified by planar culture (monolayer culture).
  • the collagen layer may be dried.
  • the collagen layer may be a layer that forms a network structure of a collagen fibrous body when a condition for gelling the collagen is given.
  • the collagen layer may be a collagen layer obtained by drying collagen in the collagen solution supplied to the culture surface of the culture vessel without completely crosslinking the collagen.
  • a culture container including a collagen gel layer containing collagen in an uncrosslinked or low crosslinked state.
  • the collagen in the collagen layer is preferably fibrillated, and the collagen gel layer preferably has a network structure of a collagen fibrous body.
  • the collagen layer containing uncrosslinked or low crosslinked collagen mainly contains an inert gas. It may be obtained by being gelled after being stored in an inert gas atmosphere.
  • a method for producing a culture vessel comprising a step of drying a collagen solution without completely crosslinking the collagen solution to form a collagen layer in the culture vessel.
  • a step of storing the collagen layer in an inert gas atmosphere mainly containing an inert gas may be provided.
  • the method may further include a step of gelling the collagen layer.
  • the collagen gel layer preferably contains 0.5 mg or more of collagen per cm 2 of the surface.
  • the collagen gel layer preferably contains type I collagen.
  • the collagen gel layer may have a network structure of a collagen fibrous body.
  • the collagen gel layer is a layer formed by supplying a liquid to a collagen layer obtained by drying without completely cross-linking the collagen in the collagen solution supplied to the culture surface of the culture vessel. Can be done.
  • a liquid may be supplied to the collagen layer of any of the culture vessels to form a collagen gel layer.
  • chondrocytes are seeded on the surface of a collagen gel layer containing uncrosslinked or low-crosslinked collagen on the surface. Sowing process,
  • a method for evaluating chondrocytes comprising the steps of:
  • the chondrocytes to be seeded in the seeding step can be cells cultured on a culture surface having no collagen gel layer.
  • the collagen gel layer contains at least 0.5 mg of collagen per cm 2 of the surface.
  • the evaluation step is a step of evaluating based on the circularity of the cells.
  • the circularity can be a value obtained by dividing the projected image perimeter of each cell by the circumference corresponding to the projected area circle.
  • cells having the circularity equal to or greater than a predetermined threshold value are expressed as chondrocyte traits. It is preferable to determine that it is maintained.
  • the circularity is preferably 0.8 or more.
  • the collagen gel layer may have a network structure of a collagen fibrous body.
  • the evaluation step may be a step of evaluating a ratio (%) of the number of cells having a circularity of 0.8 or more to the total number of measured cells.
  • a method for producing a three-dimensional culture of chondrocytes wherein the chondrocytes are formed on a collagen gel layer having uncrosslinked or lowly crosslinked collagen on the surface.
  • a production method comprising: In this production method, the two-dimensional culture step is preferably a subculture step.
  • these production methods include an evaluation step of evaluating the state of differentiation based on the individual cell shape on the surface of the collagen gel layer in the culturing step, and the three-dimensional culturing step is performed in the evaluation step.
  • the step may be a step of performing three-dimensional culture using cells determined to maintain chondrocyte traits.
  • an artificial implant including a collagen layer containing collagen in an uncrosslinked or low crosslinked state.
  • an artificial implant comprising: a collagen gel layer containing collagen in an uncrosslinked or low crosslinked state; and chondrocytes on the surface of the collagen gel layer.
  • a method for producing a surface-modified artificial transplant comprising: forming a collagen gel layer containing uncrosslinked or low-crosslinked collagen on the surface of the artificial implant; Culturing chondrocytes with
  • FIG. 1 is a view showing a production example of the culture vessel of the present invention.
  • FIG. 2 is a diagram showing an example of a cell observation device used in the method for evaluating chondrocytes of the present invention.
  • FIG. 3 is a diagram showing an SEM image of chondrocytes during two-dimensional culture in Example 1.
  • FIG. 4 is a diagram showing an SEM image of chondrocytes during two-dimensional culture in Comparative Example 1.
  • FIG. 5 is a diagram showing a type II collagen immunostaining image of Example 1.
  • FIG. 6 is a diagram showing a type II collagen immunostaining image of Comparative Example 1.
  • FIG. 7 is a graph showing changes in cell numbers after 168 hours and 504 hours in the three-dimensional culture of Example 1 and Comparative Example 1.
  • FIG. 8 is a diagram showing changes in the number of cells after 168 hours and 504 hours in the three-dimensional culture of Example 2 and Comparative Example 2.
  • FIG. 9 is a diagram showing changes in cell numbers after 168 hours and 504 hours in the three-dimensional culture of Example 3 and Comparative Example 3.
  • FIG. 10 is a diagram ((a) to (c)) showing the state of culturing chondrocytes on collagen gels having different collagen concentrations.
  • FIG. 11 is a graph showing the amount of GAG production with respect to collagen concentration.
  • FIG. 12 is a graph showing the ratio of the number of chondrocytes having a predetermined circularity to the collagen concentration.
  • FIG. 13 is a diagram ((a), (b)) showing a histogram indicating the distribution of circularity.
  • FIG. 14 is a view showing a step of preserving a collagen gel layer in Example 6.
  • FIG. 15 is a graph showing the percentage of circular cells on the surface of the collagen gel stored under each storage condition.
  • FIG. 16 is an electron micrograph showing the state of collagen in a collagen gel under each storage condition.
  • 10 indicates a cell observation device
  • 11 indicates a support
  • 13 indicates an LED lamp
  • 15 indicates a CCD camera
  • 16 indicates a three-dimensional stage
  • 17 indicates an incubator
  • 19 indicates a control device
  • 20 indicates a culture vessel.
  • the culture vessel of the present invention includes a collagen layer containing uncrosslinked or lowly crosslinked collagen.
  • the collagen gel layer obtained by gelling the collagen layer can proliferate cells while maintaining the characteristics of the chondrocytes even when the chondrocytes are cultured in a plane. .
  • a collagen gel layer having a network structure of fibrous collagen can be formed.
  • this collagen layer in an inert gas atmosphere mainly composed of inert gas, it is possible to form a network structure of fibrous collagen by subsequent gelation.
  • an inert gas atmosphere mainly composed of inert gas
  • the culture container of the present invention will be described, and a method for producing the culture container, a method for culturing chondrocytes using the culture container of the present invention, a method for evaluating the same, and the like will be described.
  • the culture vessel of the present invention various conventionally known culture vessels can be used without particular limitation.
  • a plane or curved surface suitable for forming a collagen layer is provided as a portion corresponding to a culture surface.
  • examples of such a culture vessel include various kinds of vessels for flat culture (for monolayer culture), and specific examples include a T-shaped flask, a square flask, a ⁇ D-shaped flask, and a petri dish. it can.
  • the material used for the culture surface can be used without limitation.
  • the main culture vessel is preferably used for the growth of chondrocytes, it is preferably a culture vessel used for culturing chondrocytes.
  • the site of the culture vessel where the collagen layer is formed can be the surface inside the culture vessel that comes into contact with the culture solution and the cultured cells, but is preferably the site that forms the bottom surface of the culture cavity.
  • the main culture vessel has a collagen layer having uncrosslinked or low crosslinked collagen on the surface.
  • Collagen in an uncrosslinked state or a low crosslinked state means a state in which collagen constituent molecules (such as ⁇ chains) are not crosslinked with each other or are contained at a low degree of crosslinking. Therefore, collagen in an uncrosslinked or low crosslinked state includes collagen constituent molecules that are present without being crosslinked.
  • the cross-linked state of collagen is formed by cross-linking its constituent molecules with each other by an electron beam such as ultraviolet rays, heat or various cross-linking agents.
  • Such a collagen layer containing collagen in an uncrosslinked or low crosslinked state can be obtained by performing a collagen layer forming step without a crosslinking reaction or a collagen layer forming step with a crosslinking reaction with a limited degree of crosslinking.
  • the step of forming a collagen layer that does not involve a cross-linking reaction means not only performing no cross-linking reaction, either physical cross-linking or chemical cross-linking, but also using an ultraviolet ray Avoiding exposure to etc. can also be included.
  • the step of forming a collagen layer involving a crosslinking reaction with a limited degree of crosslinking involves performing a crosslinking reaction by adjusting the amount of electron beam irradiation and the amount of a crosslinking agent added so that uncrosslinked collagen constituent molecules can remain. Can be implemented. In the case of crosslinking, the crosslinking may be performed before, during, or after drying the collagen solution.
  • the collagen in the collagen layer is in an uncrosslinked or low crosslinked state that is effective in the present invention, that is, an effective amount of uncrosslinked collagen constituent molecules that allow culturing while maintaining the characteristics of the cultured cells remains.
  • a good culture of chondrocytes and fibroblast-like cells derived from chondrocytes can be formed.
  • chondrocyte traits in collagen gel-embedded cultures of chondrocyte-derived fibroblast-like cells observed to be dedifferentiated in a normal culture vessel with a polystyrene surface When cells capable of being cultured are cultured on the surface of a collagen gel layer obtained by gelling the collagen layer, preferably, 50% or more of the cell number can be observed as a spherical form.
  • the cells having recovered the characteristics of the chondrocytes and the surface of the collagen gel layer can be obtained.
  • the ratio of the number of cells having a spherical shape is more preferably 80% or more, and further preferably about 100%. Note that whether or not the cell has a spherical shape can be determined based on the circularity of the cell as described later.
  • collagen is not cross-linked or low-cross-linked, it is possible to maintain the differentiation state of chondrocytes and to cultivate or to realize the potential of differentiation. It is considered that as a result, it is possible to determine whether or not they have potential differentiation potential. It has been found that when the collagen layer of the culture vessel of the present invention is gelled, fibrosis proceeds even though collagen is not cross-linked or low-cross-linked, and a network structure of fibrous collagen is formed ( See Example 6.) Although not limiting the present invention, it is presumed that such a network structure of uncross-linked or low-cross-linked collagen can make the differentiation ability of cultured cells visible.
  • the collagen layer may be formed to such an extent that a uniform collagen gel layer can be formed on the culture surface by the moisture of the culture solution or the like during the culture.
  • the collagen layer uniformly covers the entire culture surface.
  • the thickness of the collagen layer is not particularly limited, but is preferably a thickness that does not hinder observation from below the culture vessel.
  • Collagen that forms the collagen layer is available without particular limitation Alternatively, a collagen that can be prepared can be used.
  • enzyme-soluble collagen such as acid-soluble collagen, neutral salt-soluble collagen, and atherocollagen, and alkali-solubilized collagen, and these collagens were chemically modified by succinylation, acylation, and methylation.
  • Chemically modified collagen can be used.
  • a synthetic peptide having a collagen motif artificial collagen
  • an enzyme-soluble collagen such as acid-soluble collagen or atelocollagen is used. Acid-soluble collagen is preferable from the viewpoint of safety, and enzyme-solubilized collagen is preferable from the viewpoint of easiness of operation for preparing a collagen layer.
  • the type of collagen constituting the collagen layer is not particularly limited, and it is possible to use type I, type II, type III, type IV and type V alone or in combination of two or more types.
  • type I collagen is collagen secreted by dedifferentiated chondrocytes
  • type II collagen is collagen secreted by chondrocytes that have maintained differentiation.
  • type I collagen is used. This is because type I collagen can not only maintain differentiation but also obtain sufficient cell proliferation ability. It is also preferable to use atelocollagen treated with an enzyme such as protease or pepsin.
  • acidic collagen it is preferable to use acidic collagen.
  • the collagen layer preferably has a collagen of 0.5 mg or more per lcm 2 of surface. If the collagen concentration is less than 0.0 Smg / cm 2 , chondrocyte traits (particularly morphological traits such as cell morphology, type II collagen production ability, glycosaminodalican production, etc.) in two-dimensional This is because it becomes difficult to maintain the ability to live. On the other hand, when it is not less than 0. SmgZ cm 2 , even a chondrocyte which has not exhibited the external trait of the chondrocyte by ordinary planar culture can be cultured with the external trait displayed. Collagen concentration is preferably, l mg Z cm 2 or more.
  • the collagen concentration it is preferable to increase the collagen concentration.
  • the collagen concentration is too high, it may be difficult to prepare the collagen layer or the cell growth rate may be low.Therefore, set the collagen concentration after conducting preliminary experiments in advance. Is preferred.
  • the collagen layer having collagen in an uncrosslinked or low crosslinked state may be formed, for example, without supplying a collagen solution to a culture surface, without gelling or after gelling, without crosslinking, or without crosslinking. It can be obtained by drying after removing water by suppressing ⁇ . Preferably, the collagen solution is dried without gelling in order to maintain uncrosslinked or low crosslinked.
  • FIG. 1 illustrates a process for producing a culture vessel having a collagen layer on the culture surface of the culture vessel.
  • a collagen solution adjusted to a predetermined concentration is supplied to and laid down on the bottom of the culture vessel so that the surface has a collagen concentration of 0.5 mgZcm 2 or more.
  • the required amount of the collagen solution was V (ml) for the required amount of collagen solution, A (cm 2 ) for the collagen-coated surface, C CL (mgZcm 2 ) for the desired collagen application concentration, and the collagen solution to be laid.
  • the drying method is not particularly limited.
  • the collagen layer (dry body) can be obtained by air-drying at room temperature for several days and finally drying by completely removing moisture by a vacuum dryer. By doing so, it is possible to obtain a culture vessel having a collagen layer at a concentration set on the culture surface.
  • air drying is not always necessary, the air drying period in the case of air drying depends on the collagen concentration. It is preferable that the higher the concentration, the longer the air drying period. Also, if the drying process is performed rapidly, Because of sponge formation, sponge formation is prevented by slow drying, that is, air drying over time. In addition, in these series of steps, in order to avoid crosslinking of collagen, light and heat are blocked.
  • the collagen layer may be composed of collagen alone, but may contain other components as long as the object of the present invention is not hindered.
  • biocompatible polymers bioabsorbable polymers, biodegradable polymers, and the like can be included.
  • saccharides such as hyaluronic acid, chitin, chitosan mucopolysaccharide, synthetic polymers such as polylactic acid and polyglycolic acid, high water-containing polymers such as hydroxyethyl ethyl methacrylate, and fibrin and albumin.
  • the proteins can be mixed.
  • a collagen gel layer suitable for culturing cultured cells such as chondrocytes can be formed by supplying a liquid to the collagen layer.
  • the collagen gel layer has a network structure of fibrous collagen while being uncrosslinked or lowly crosslinked.
  • a culture vessel provided with a collagen gel layer formed by gelling the collagen layer is also included in one embodiment of the present invention.
  • a neutral buffer such as PBS is supplied to the collagen layer to make the collagen layer neutral in the presence of water. This allows the collagen to gel.
  • the collagen layer can be stored until gelled. When preserving, it is preferable to preserve under an inert gas atmosphere mainly containing an inert gas.
  • the inert gas includes, for example, nitrogen, argon, helium, and a mixed gas thereof, and is preferably nitrogen.
  • the inert gas mainly contains an inert gas by 50% or more. It is more preferably at least 70%, further preferably at least 80%, and still more preferably at least 90%.
  • the storage period and temperature for the preferred collagen layer storage step are not particularly limited. It can be from a few days to a week, or about a few weeks.
  • the temperature can generally be about 20-30 ° C.
  • the gas atmosphere to be preserved is preferably dried, for example, at a humidity of 10% RH or less. More preferably, it is at most 5% RH. This is because moisture in the storage step may promote unintended collagen crosslinking and suppress fibrosis.
  • the storage step it is preferable to shield light as in the drying step. This is because light may promote crosslinking.
  • an artificial implant provided with a collagen layer containing collagen in an uncrosslinked or undercrosslinked state.
  • the collagen layer is synonymous with the collagen layer in the culture vessel of the present invention described above, and the collagen constituting the collagen layer is synonymous with the collagen constituting the collagen layer of the culture vessel.
  • the collagen layer included in the artificial implant gels to form a collagen gel when a liquid is supplied and given gelling conditions.
  • an artificial implant including a collagen gel layer in which the collagen layer is gelled is also included in one embodiment of the present invention.
  • the collagen layer or the collagen gel layer is preferably provided corresponding to the culture surface.
  • the surface of the collagen layer or collagen gel layer may form a uniform flat surface, Various shapes such as a shape, a column shape, a spherical shape, a cone shape, and a rectangular shape can be adopted.
  • the three-dimensional form of the artificial implant can include an external form of a body part to be replaced (for example, an artificial bone, an artificial joint, an artificial meniscus, etc.). It can be spherical, conical, or rectangular parallelepiped.
  • the artificial implant can be composed of, for example, a metal material, a ceramic material, a synthetic resin material, a biological material, and the like. Preferably, a material having excellent biocompatibility or biodegradability is used.
  • an artificial implant having a surface modified with a chondrocyte layer or a method for producing a surface-modified artificial implant (a method for modifying the surface of an artificial implant) is also provided. Is done.
  • the chondrocyte culture method of the present invention comprises a seeding step of seeding chondrocytes on the surface of a collagen gel layer containing uncrosslinked or low-crosslinked collagen, and a culture step of growing chondrocytes on the surface of the gel.
  • a collagen gel layer the collagen that can be used for the above-mentioned collagen layer can be used similarly, and the collagen that is preferably used for the above-mentioned collagen layer can also be used preferably.
  • the collagen gel layer is formed by adding a liquid (typically a culture solution, water, buffer solution, etc.) to collagen, and adding any of temperature, concentration, pH, salt concentration, etc., depending on the type of collagen, or a combination thereof. It can be obtained by gelling under gelling conditions consisting of: In addition, the collagen gel layer It can also be obtained by gelling by supplying a liquid to the lage layer and giving necessary gelling conditions. Therefore, prior to the seeding step, a collagen solution is supplied to the surface of the culture vessel or the artificial implant, dried without crosslinking to form a collagen layer, and then a liquid is supplied to the collagen layer. A collagen gel layer can be made. In addition, a collagen gel layer can be produced by supplying a liquid to a culture vessel or an artificial implant having the above-mentioned collagen layer on the culture surface in advance and gelling the collagen layer.
  • a liquid typically a culture solution, water, buffer solution, etc.
  • chondrocytes are cultured on the surface of the collagen gel layer.
  • it depends on the surface morphology of the collagen gel layer, it is basically a two-dimensional culture form on a flat or curved surface. Therefore, unlike three-dimensional culture such as so-called collagen gel embedding culture, it is possible to easily observe the morphology of the cultured cells and to obtain a favorable cell growth rate. From the viewpoint of ease of cell observation and the like, it is preferable to use planar culture.
  • chondrocytes maintain their characteristics by culturing chondrocytes on the surface of a collagen gel layer having uncrosslinked or low-crosslinked collagen on the surface.
  • GAG glycosaminoglycan
  • type I collagen it can be grown while presenting a spherical cell morphology (circular cell morphology in the projected image) as an indicator on the outer shape. Therefore, it is preferable for a method of culturing chondrocytes, and particularly preferable for a method of culturing by subculture.
  • chondrocytes (at least at least) that have lost the characteristic spherical cell morphology in appearance while maintaining the characteristics of chondrocytes by the conventional two-dimensional culture. Externally, it can be said to be a potential chondrocyte.) Can be proliferated while displaying its external trait again. (Chondrocyte evaluation method)
  • the method for evaluating chondrocytes of the present invention comprises a step of sowing chondrocytes on a surface of a collagen gel layer containing collagen in a low cross-linked state on the surface, a step of culturing chondrocytes on the surface of the gel, and a step of culturing the surface of the gel.
  • An evaluation step for evaluating the state of differentiation based on the shape of each cell is provided.
  • the present evaluation method includes a seeding step and a culture step in the above-described culture method, and these are also provided with an evaluation step.
  • the evaluation process that is characteristic of the present evaluation method will be described.
  • the evaluation step evaluates chondrocytes based on the circularity of the cells.
  • the circularity of a cell can be calculated from the aspect ratio of each cell or the like, but is preferably a value obtained by dividing the circumference of the projected image of each cell by the circumference corresponding to the projected area circle.
  • the projected image circumference means the circumference of the projected image of the cell
  • the projected area circle equivalent circumference means the circumference of a perfect circle corresponding to the area of the projected image of the cell. ing.
  • the projected image circumference of the cell and the circumference equivalent to the projected area circle are obtained by imaging the projected image of chondrocytes cultured on the surface of the collagen gel with an imaging device such as a CCD camera, and further from this captured image or from the captured image. It can be easily obtained by adding image processing.
  • JP-A-08-287261 describes a system for recognizing the shape of an individual cell and the concept of the circularity of a cell.
  • the chondrocyte trait is maintained when the circularity is equal to or greater than a predetermined threshold.
  • the predetermined threshold here refers to the degree of circularity with which it can be determined that chondrocytes maintain the trait, and can be set based on the resolution of an imaging device such as a CCD camera or the flow of image processing. , It is preferable to set based on a preliminary experiment performed in advance. For example, it is preferable to determine that the chondrocyte trait is maintained when the circularity is 0.8 or more.
  • the threshold value for determining the circularity is less than 0.8, cells that do not produce type II collagen, which is a characteristic product of chondrocytes, are included, which is not preferable.
  • the value is 0.8 or more, especially when the above-described projected image circumference and the projected area circle are made circular, the chondrocyte differentiation state (whether or not the chondrocyte-like trait is maintained) is highly accurate. Can be determined. More preferably, it is 0.9 or more.
  • the cell circularity can be, for example, 0.8 or more, and preferably 0.9 or more.
  • the cell observation device 10 includes a support 11 for supporting a culture vessel 20, an LED lamp 13 for illuminating the culture vessel 20, a CCD camera 15 for photographing the bottom surface of the culture vessel 20, and a predetermined temperature inside the enclosure. It has an incubator 17 for maintaining and a control device 19 for analyzing images taken by the CCD camera 15.
  • the support 11 supports the culture vessel 20 to which the chondrocytes adhere to the bottom surface, with the bottom surface visually exposed downward.
  • the support stand 11 supports only the bottom edge of the culture vessel 20 or makes a portion corresponding to the bottom face of the culture vessel 20 a transparent member so that photographing by the CCD camera 15 is not hindered. It is configured.
  • the LED lamp 13 projects individual cells in the culture container 20 onto the bottom surface of the culture container 20 by illuminating the culture container 20 supported by the support base 11 from above.
  • the CCD camera 15 is located below the culture vessel 20 so that the bottom of the culture vessel 20 can be photographed. It is installed in one. When the CCD camera 15 receives the command signal from the control device 19, the CCD camera 15 photographs the bottom of the culture vessel 20 illuminated from above by the LED lamp 13, and the individual cells in the culture vessel 20 are cultivated.
  • a projection image projected on the bottom surface is obtained, and data of the projection image is transmitted to the control device 19.
  • the CCD camera 15 is attached to the three-dimensional stage 16 and can move up, down, left, right, back and forth.
  • the three-dimensional stage 16 is operated by a command signal from a control device 19 to position the CCD camera 15 at a predetermined position.
  • Incubator 17 is a support 11 and LED lamp 13 and the temperature-adjustable case containing therein a CCD camera 15, the line for supplying the 5% C0 2 containing culture container 20 which is supported air to support 11 And a line for discharging gas from the culture vessel 20.
  • the control device 19 includes a well-known CPU, ROM, RAM and the like.
  • the control device 19 outputs a command signal to the three-dimensional stage 16 to position the CCD camera 15 at a predetermined position, and then outputs a command signal to the CCD camera 15 to shoot a projection image, and project the projection image.
  • the image is input from the CCD camera 15 and subjected to image analysis processing, and the circularity, cell concentration, and the like are calculated based on the projected image after the image analysis processing.
  • the image processing can be performed, for example, as follows.
  • the control device 19 of the cell observation device 10 can clarify the individual cell image by performing a closing process and a filling hole process after binarizing the projected image of the bottom surface of the culture vessel 20.
  • binarization means that pixels with a gray level value below a certain threshold are set to a value of 1 and the other pixels are set to a value of 0 to partition the image into two regions, white and black. This is the process to be performed.
  • a black body of a predetermined size or more is regarded as a cell, and the black body is filled with small holes by closing processing to smooth the boundary, and further, by filling isolated holes by filling hole processing.
  • cell Image is regarded as a cell, and the black body is filled with small holes by closing processing to smooth the boundary, and further, by filling isolated holes by filling hole processing.
  • the method is suitable for the evaluation of chondrocytes, and a good growth state can be obtained.
  • evaluation based on continuous observation can be easily performed. It is known that when a chondrocyte maintains a trait, it maintains a substantially spherical shape.However, in this evaluation method, it is easy to observe the cell shape of the chondrocyte, Whether or not the trait is maintained can be easily evaluated.
  • chondrocytes by culturing chondrocytes on the surface of the collagen gel layer having collagen in a low cross-linked state on the surface, the appearance of chondrocytes maintaining their traits is The cells can be grown while presenting a spherical cell morphology (a circular cell morphology in the projected image) as an index. Therefore, the differentiation state of chondrocytes can be evaluated with high accuracy. Further, according to the present evaluation method, by culturing latent chondrocytes in two-dimensional culture such as ordinary planar culture, it is possible to present a spherical cell morphology unique to chondrocytes.
  • the cells can potentially retain the characteristics of chondrocytes. Can be determined. Therefore, according to this evaluation method, it is determined whether the cells exhibiting fibroblast-like morphology are potential chondrocytes or cells that have completely de-differentiated from chondrocytes by culturing the cells before seeding on the surface of the collagen gel layer. Regardless of the history, it can be easily determined. Conventionally, such a determination cannot be made without three-dimensional culture.
  • the potential chondrocytes in this specification are chondrocytes having a fibroblast-like morphology on a normal culture surface such as polystyrene, and have a high probability of becoming spheres after transition to three-dimensional culture. It refers to chondrocytes that can be expected or differentiated into chondrocytes of a specific shape.
  • the production method comprises a step of sowing chondrocytes on the surface of a collagen gel layer having uncrosslinked or low crosslinked collagen on the surface, and a two-dimensional culture step of growing chondrocytes on the surface of the collagen gel layer. And a three-dimensional culture step of performing three-dimensional culture using the chondrocytes grown in the culture step.
  • this production method in the two-dimensional culturing step, dedifferentiation of the cultured chondrocytes is suppressed and the chondrocytes are cultured at a sufficient growth rate, so that a three-dimensional culture can be obtained efficiently. .
  • the two-dimensional culture step it can be easily determined from the cell morphology whether or not the chondrocytes maintain their differentiation state, so that the chondrocytes can be surely proliferated.
  • the evaluation step is performed, and the three-dimensional culturing step is performed using the chondrocytes determined to maintain the trait of chondrocytes.
  • the three-dimensional culture in the three-dimensional culture step includes, for example, culture on a scaffold material having an arbitrary shape such as a collagen sponge, and embedding culture on a collagen gel-agarose gel having no arbitrary shape. . From the above, according to the present invention, three-dimensional images obtained by these production methods can be obtained. A culture is also provided.
  • the two-dimensional culture space of the present invention constitutes a pseudo three-dimensional culture space.
  • a 25 cm 2 T-shaped flask was used as a culture vessel to prepare a culture vessel having a collagen layer.
  • a culture vessel equipped with a collagen layer having an uncrosslinked collagen of 05 mgZcm 2 was prepared.
  • a cell suspension having a cell density of 2.5 ⁇ 10 6 cellsZml prepared using the cultured cells obtained in Examples 1 and 2 and a 3 wt% atelocollagen solution were prepared. : 4 volume ratio (collagen concentration after mixing: 0.4 wt%), and this mixed solution was seeded into a 48-well plate at 220 ⁇ in a dome shape (5 ⁇ 10 5 cells / ml). The thickness at this time was about 2 mm.
  • Example 1 After chondrocytes seeded, allowed to gel by 1 hour standing at 37 ° C, followed by addition of liquid medium, subjected to three-dimensional culture at 37 ° C, 5% C0 2 , of the actual Example 1 and 2 A three-dimensional culture was obtained. Furthermore, in Example 1, seeding was performed at a seeding density of 2 X IC cells / ml, and three-dimensional culture was performed in the same manner to obtain a three-dimensional culture of Example 3.
  • a three-dimensional culture by embedding collagen gel was performed using egosyn chondrocytes that were passaged three and five times by two-dimensional culture in the same manner as in Example except that the culture was performed on polystyrene. 1-3 three-dimensional cultures were obtained. Also, as a control, three-dimensional culture using collagen gel embedding was performed using primary rabbit heron chondrocytes. The results of the two-dimensional culture are shown in FIGS. 3 to 6, and the results of the three-dimensional culture are shown in FIGS.
  • Figures 3 and 4 show SEM images of Example 1 (chondrocytes cultured two-dimensionally on the collagen gel layer surface) and Comparative Example 1 (chondrocytes cultured two-dimensionally on polystyrene). Is shown.
  • Figures 5 and 6 show the immunostained images of type 1 collagen of Example 1 (chondrocytes cultured two-dimensionally on the collagen gel layer surface) and Comparative Example 1 (cultured on a polystyrene surface, respectively). 1 shows a type II collagen immunostaining image of chondrocytes). As is evident from Figs.
  • chondrocytes cultured two-dimensionally on the surface of the collagen gel layer maintain a spherical cell morphology, which is an indicator of the trait maintenance of soft cells, and produce type II collagen.
  • chondrocytes cultured on polystyrene showed a flat fibroblast-like morphology, and hardly produced type II collagen, an extracellular matrix produced by chondrocytes. From the above, it was found that by culturing chondrocytes on the surface of the collagen gel layer in an uncrosslinked state, dedifferentiation can be suppressed, the differentiated state can be well maintained, proliferated, and subcultured.
  • FIGS. 7 to 9 show changes in the cell numbers of Examples 1 to 3 and Comparative Examples 1 to 3 and the control example after 168 hours and 504 hours in the three-dimensional culture.
  • Table 1 shows the doubling time at this time. The doubling time was calculated from the cell number after 168 hours and 504 hours.
  • Example 1 + Comparative Example 1 Three passages during two-dimensional culture
  • Example 3 In Example 3 and Comparative Example 3 (the inoculation density during three-dimensional culture was 2 ⁇ lC ⁇ cells / ml), the doubling time of cells subcultured on collagen was 5.01 [day]. In contrast, the doubling time of cells passaged on polystyrene was 19.92 [day]. That is, when three-dimensional culture was performed by inoculating cells at a high concentration, in Example 3, no remarkable increase in the cell doubling time was observed, and the cells grew with sufficient proliferation ability. In No. 3, the cell doubling time was remarkably increased, and a decrease in the cell proliferation ability was evident.
  • a 25 cm 2 T-shaped flask was used as a culture vessel to prepare a culture vessel having a collagen layer.
  • 3 X 10 _3 mgZcm 2 (b) 1.
  • collagen concentration is 5. 3X 10- 4 mg / cm 2 , 5. 3 X 10 "3 mg / cm 2 5. 3 X 10" 2 mg / cm 1 ⁇ 05mgZcm 2
  • a culture vessel provided with a collagen layer having uncrosslinked collagen at four different concentrations was prepared, and perforated chondrocytes were prepared at a concentration of 1.OX 10 4 cells / cm 2 in the same manner as in Example 4. seeded, and cultured at 37 ° C, 5% Rei_0 2.
  • GAG glycosaminodalican
  • FIG. 11 shows the amount of GAG production with respect to the collagen gel concentration (C CL ). Specifically, the average GAG generation rate (V / v 0 ) on days 20-31 of culture is shown. GAG production rate can be used as an indicator of chondrocyte trait retention.
  • V is the amount of GAG produced when chondrocytes are cultured on the polystyrene surface. And the production volume V at each concentration is dimensionless.
  • collagen concentration of about Olmg / cm 2 in v / v. There be about 1, but is substantially the same GAG production amount and the culture conditions in polystyrene surface, the collagen concentration 0. 5mg / cm 2 ⁇ l.
  • FIG. 12 shows the ratio of the number of chondrocytes having a predetermined circularity to the collagen gel concentration (C CL ).
  • N r is the number of cells with a circularity of 0.9 or more. und , and the total cell number was N all .
  • the method of calculating the circularity will be described later.
  • FIG. 12 when the collagen concentration is low, the ratio of the number of cells having a circularity of 0.9 or more is extremely low, which supports the observation results of the cell morphology shown in FIG. 10 (a). In contrast, from the vicinity of about 0. Smg / cm 2 becomes remarkable increase of the cell number ratio, at about LmgZcm 2 has become the number of cells ratio is approximately 1, cell morphology observation shown in FIG. 10 (c) The results well supported the results.
  • Fig. 13 is a histogram showing the distribution of circularity with respect to collagen concentration.
  • Fig. 13 (a) shows the results obtained when cultivating egret chondrocytes in the same manner on the polystyrene surface
  • Fig. 13 (b) shows the results.
  • the results are for a culture surface with a collagen concentration of 1.05 gZcm 2 . Since the cells are of the same generation, the histogram should show the same tendency.
  • cells incubated on a high-concentration collagen gel have higher circularity (chondrocyte cells). Cells that maintained the phenotype). These cells were considered to be cells that proliferate while maintaining the characteristics of chondrocytes even after transition to collagen gel-embedded three-dimensional culture.
  • This example demonstrates the relationship between the collagen layer preservation step and chondrocyte culture. Is confirmed.
  • a preservation step was performed, and then the collagen layer was gelled to form a collagen gel layer, and chondrocytes were seeded.
  • the collagen layer For the collagen layer, apply 0.5 wt% of a 0.5 wt% acidic collagen solution (manufactured by Koken Co., Ltd.) to the bottom of a T-flask (25 ml) with a female pipette so that the collagen concentration is 1.05 mgZcm 2, and then shade It was dried at about 25 ° C for about 3 days under lOPa force and lOOPa in the reduced pressure drying apparatus thus formed to form a collagen layer. Then, immediately after adding PBS, gelation was carried out. In addition, a storage step was carried out under air atmosphere and nitrogen atmosphere for 4 days and 7 days, respectively, and then gelation was carried out similarly.
  • a 0.5 wt% acidic collagen solution manufactured by Koken Co., Ltd.
  • All storage steps were performed at 25 ° C and 10% RH or less in a light-shielded state.
  • After preparing each collagen gel layer immediately wash the collagen layer twice with PBS, and then subculture three times in a polystyrene culture vessel using DMEM medium containing fetal bovine serum. were seeded so that 10 4 concentration of cel lsZcm 2, and cultured at 37 ° C, 5% C0 2 .
  • the number of cells having a circularity of 0.9 or more was measured, and the ratio to the total number of cells was determined.
  • the measurement of the number of cells and the measurement of circularity were performed in the same manner as in the previous example.
  • a polystyrene T-flask without a collagen layer and a collagen gel layer was subjected to the same operation without a storage step. The results are shown in FIG.
  • the collagen layer derived from the collagen layer stored under a nitrogen atmosphere showed a better circular cell rate than the collagen gel layer derived from the collagen layer stored under an air atmosphere.
  • the fibrous body formed by twisting a plurality of filaments formed a network structure
  • the collagen gel layer of (b) In the collagen gel layer, although a filament-like one was observed, a flat, void-free surface was formed instead of a network structure.
  • the collagen gel layer obtained by gelation can no longer form a network structure with appropriate pores. It was thought that it was difficult to maintain the differentiation ability of chondrocytes and to maintain the potential differentiation ability of chondrocytes.
  • the collagen gel layer obtained by storing for 7 days was also observed, and showed the same structure as that of each stored for 4 days.

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Abstract

L'intention est de fournir un conteneur de culture dans lequel les cellules peuvent proliférer tout en conservant leurs caractères de chondrocytes. Notamment, un conteneur de culture ayant une couche de collagène qui contient un collagène dans un état de liaison non croisée ou faiblement croisée à sa surface. En cultivant les cellules dans ce conteneur de culture, les caractères des chondrocytes peuvent être maintenus ou les caractères des cellules ayant potentiellement des chondrocytes peuvent s'exprimer.
PCT/JP2005/006707 2004-03-31 2005-03-30 Conteneur de culture, méthode de culture des chondrocytes et méthode d'évaluation des chondrocytes WO2005095577A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015213515A (ja) * 2006-07-28 2015-12-03 ド クレシー, ウッドDE CRECY, Eudes より適切な細胞変異体を選択可能であり、継続的に培養液を製造する移動型容器を備える連続培養装置
WO2018021362A1 (fr) * 2016-07-25 2018-02-01 宇部興産株式会社 Procédé de suppression de la dédifférenciation de cellules qui se dédifférencient facilement, procédé de préparation desdites cellules, et procédé de production de substance

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06292565A (ja) * 1993-03-15 1994-10-21 Bio Material Kenkyusho:Kk 細胞培養用基質材料
JPH08163979A (ja) * 1994-10-12 1996-06-25 Sumitomo Bakelite Co Ltd 細胞培養器の包装方法
JPH09500298A (ja) * 1993-07-07 1997-01-14 スミス アンド ネフュー ピーエルシー 埋込型プロステーシス、キットおよびそれを製造するための装置
JP2002142752A (ja) * 2000-11-13 2002-05-21 Asahi Techno Glass Corp コラーゲンコート細胞培養容器及びその製造方法
JP2002233567A (ja) * 2000-12-06 2002-08-20 Mitsuo Ochi 移植用組織等価物及びその製造方法
JP2003135056A (ja) * 2001-10-30 2003-05-13 Mitsuo Ochi 移植用組織等価物の製造方法及びその製造用器具

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06292565A (ja) * 1993-03-15 1994-10-21 Bio Material Kenkyusho:Kk 細胞培養用基質材料
JPH09500298A (ja) * 1993-07-07 1997-01-14 スミス アンド ネフュー ピーエルシー 埋込型プロステーシス、キットおよびそれを製造するための装置
JPH08163979A (ja) * 1994-10-12 1996-06-25 Sumitomo Bakelite Co Ltd 細胞培養器の包装方法
JP2002142752A (ja) * 2000-11-13 2002-05-21 Asahi Techno Glass Corp コラーゲンコート細胞培養容器及びその製造方法
JP2002233567A (ja) * 2000-12-06 2002-08-20 Mitsuo Ochi 移植用組織等価物及びその製造方法
JP2003135056A (ja) * 2001-10-30 2003-05-13 Mitsuo Ochi 移植用組織等価物の製造方法及びその製造用器具

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ALBERTS B. ET AL.: "Saibo no Bunshi Seibutsugaku.", 3RD EDITION, KABUSHIKI KAISHA NYUTON PURESU, 20 July 1995 (1995-07-20), pages 1180 - 1181, XP002997059 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015213515A (ja) * 2006-07-28 2015-12-03 ド クレシー, ウッドDE CRECY, Eudes より適切な細胞変異体を選択可能であり、継続的に培養液を製造する移動型容器を備える連続培養装置
WO2018021362A1 (fr) * 2016-07-25 2018-02-01 宇部興産株式会社 Procédé de suppression de la dédifférenciation de cellules qui se dédifférencient facilement, procédé de préparation desdites cellules, et procédé de production de substance
CN109477069A (zh) * 2016-07-25 2019-03-15 宇部兴产株式会社 抑制易脱分化的细胞的脱分化的方法、所述细胞的调制方法、及物质的产生方法
JPWO2018021362A1 (ja) * 2016-07-25 2019-05-23 宇部興産株式会社 脱分化しやすい細胞の脱分化を抑制する方法、当該細胞の調製方法、及び物質の産生方法
CN109477069B (zh) * 2016-07-25 2022-04-15 宇部兴产株式会社 抑制易脱分化的细胞的脱分化的方法、所述细胞的调制方法、及物质的产生方法

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