WO2017082025A1 - Cell support composite, and method for producing cell support composite - Google Patents

Cell support composite, and method for producing cell support composite Download PDF

Info

Publication number
WO2017082025A1
WO2017082025A1 PCT/JP2016/081390 JP2016081390W WO2017082025A1 WO 2017082025 A1 WO2017082025 A1 WO 2017082025A1 JP 2016081390 W JP2016081390 W JP 2016081390W WO 2017082025 A1 WO2017082025 A1 WO 2017082025A1
Authority
WO
WIPO (PCT)
Prior art keywords
collagen
cell support
cell
cells
substrate
Prior art date
Application number
PCT/JP2016/081390
Other languages
French (fr)
Japanese (ja)
Inventor
文彦 北川
Original Assignee
日機装株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 日機装株式会社 filed Critical 日機装株式会社
Publication of WO2017082025A1 publication Critical patent/WO2017082025A1/en

Links

Images

Classifications

    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/06—Plates; Walls; Drawers; Multilayer plates
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • 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

Definitions

  • the present invention relates to a cell support complex, particularly a cell support complex using tubular epithelial cells.
  • tubular epithelial cells isolated from the kidney by enzyme treatment cannot maintain their original cylindrical cell structure due to the disappearance of the in vivo environment or changes in cell characteristics due to culture on a petri dish. Specifically, it is known that when tubule epithelial cells are seeded on a petri dish or an artificial membrane, the monolayer epithelial structure is lost and a gap is formed between the cells, or the cells are stratified. When such a phenomenon occurs, the reabsorption function of useful components in the plasma of the bioartificial kidney deteriorates.
  • Patent Document 1 As a technique for avoiding stratification and contact inhibition of tubular epithelial cells, a bioartificial tubule disclosed in Patent Document 1 is known. This is due to the application of MEK inhibitors to suppress contact inhibition and stratification of tubule epithelial cells, and to form a confluent monolayer on the inner surface of the artificial membrane. (Patent Document 1).
  • the MEK inhibitor is administered at a non-optimal timing while it is unclear when the tubular epithelial cells reach confluence on the artificial membrane. For this reason, MEK inhibitors may be allowed to act in a state where gaps are formed between tubular epithelial cells or in a layered state. As a result, there was a problem that a high-performance bioartificial kidney could not be obtained.
  • the present invention has been made in view of these problems, and an object of the present invention is to provide a cell support complex having improved stability of a single-layer epithelial structure.
  • a cell support complex includes a base material formed of an artificial material, a collagen molecule or a fragment thereof that adheres to at least a part of the base material, and a collagen molecule or a part thereof. And cultured cells attached to the substrate through the fragments. The cultured cells form a confluent monolayer without substantial stratification.
  • a confluent monolayer epithelial structure of cultured cells can be formed on the base material without observing under a microscope.
  • a cell support complex with improved stability of the monolayer epithelial structure.
  • the cultured cells may form a confluent monolayer without substantial stratification.
  • substantially means that the single-layer structure is maintained to such an extent that a decrease in mass transfer efficiency due to layering does not become a problem, and does not necessarily mean that no layering has occurred at all. .
  • the collagen molecule may be selected from collagen I, collagen IV, or two or more of these.
  • Collagen molecules are all adhesion amount to the substrate is 50 [mu] g / cm 2 or more 138 ⁇ g / cm 2 or less collagen I, adhesion amount to the substrate is 19.2 ⁇ g / cm 2 ultra 121 ⁇ g / cm 2 or less collagen IV, of the Or one of them may be selected.
  • Another aspect of the present invention is a method for producing a cell support complex.
  • the method for producing the cell support complex includes a step of coating a collagen molecule or a fragment thereof on at least a part of a substrate formed of an artificial material, and a step of seeding cultured cells on the collagen molecule or a fragment thereof. Culturing the cultured cells to form a monolayer structure of the cultured cells.
  • a confluent monolayer epithelial structure of cultured cells can be formed on the base material without observing under a microscope.
  • a cell support complex with improved stability of the monolayer epithelial structure.
  • the collagen molecule may be selected from collagen I, collagen IV, or two or more thereof.
  • FIG. 1A is a view showing a conventional cell support complex using a general coating agent.
  • FIG. 1 (B) is a view showing a conventional cell support complex without using a coating agent. It is a figure which shows typically the structure of the cell support complex which concerns on embodiment.
  • FIG. 2A is a diagram showing a configuration of a cell support complex according to an embodiment in which a base material has water permeability.
  • FIG. 2 (B) is a diagram showing a case where the substrate is cultured for a short time in the cell support complex according to the embodiment in which the substrate does not have water permeability.
  • FIG. 1A is a view showing a conventional cell support complex using a general coating agent.
  • FIG. 1 (B) is a view showing a conventional cell support complex without using a coating agent. It is a figure which shows typically the structure of the cell support complex which concerns on embodiment.
  • FIG. 2A is a diagram showing a configuration of a cell support complex according to an embodiment in which a base material has water
  • FIG. 2C is a diagram showing a case where the substrate is cultured for a long time in the cell support complex according to the embodiment having no water permeability.
  • FIG. 3A to FIG. 3C are diagrams showing a part of the structure of the device using the cell support complex according to the embodiment.
  • FIG. 3A is a diagram showing a part of the structure of an apparatus using Transwell as a base material.
  • FIG. 3B is a view showing a part of the structure of a dialysis apparatus using a hollow fiber membrane as a base material.
  • FIG. 3C is a diagram showing a part of the structure of an apparatus using a petri dish, a well plate, a fine channel, a microcarrier, or a hollow fiber membrane as a base material.
  • FIG. 4 (A) to 4 (D) are diagrams showing the state of cell proliferation over time when the concentration of adhesion molecules coated on the petri dish is changed.
  • FIG. 4A is a diagram illustrating a result of using Synthemax which is a comparative example.
  • FIG. 4B is a diagram showing the results using Poly-L-lysine, which is a comparative example.
  • FIG. 4C is a diagram showing the results using collagen I.
  • FIG. FIG. 4D is a diagram showing the results using collagen IV.
  • FIG. 1 is a diagram schematically showing the structure of a cell support complex according to the prior art.
  • FIG. 1A is a view showing a conventional cell support complex using a general coating agent.
  • FIG. 1 (B) is a view showing a conventional cell support complex without using a coating agent.
  • tubular epithelial cells may be layered, or conversely, gaps may be left between the cells. there were.
  • the useful substance was not efficiently transferred from the cell apical membrane side to the cell basement membrane side via the transporter (arrow P).
  • concentration-dependent mass transfer occurs through the artificial membrane from the gap between the cells (arrow Q).
  • the conventional cell support complex not using the coating agent shown in FIG.
  • FIG. 2 is a diagram schematically showing the configuration of the cell support complex 10 according to the embodiment.
  • FIG. 2A is a diagram showing a configuration of a cell support complex according to an embodiment in which a base material has water permeability.
  • FIG. 2 (B) is a diagram showing a case where the substrate is cultured for a short time in the cell support complex according to the embodiment in which the substrate does not have water permeability.
  • FIG. 2C is a diagram showing a case where the substrate is cultured for a long time in the cell support complex according to the embodiment having no water permeability.
  • the cell support complex 10 includes a base material 20 formed of an artificial material, a collagen molecule 30 that adheres to at least a part of the base material, and a collagen molecule 30 or its molecule. And cultured cells 40 attached to the base material 20 via the fragments.
  • “at least a part of the substrate 20” means, for example, “at least one surface of the substrate 20 having a flat surface or a curved surface”.
  • the base material 20 has a flat plate structure, it means at least one surface thereof.
  • the base material 20 has a cylindrical structure, it refers to at least one of the inner surface and the outer surface.
  • the base material 20 is a module used for culturing tubular epithelial cells.
  • FIG. 2A shows a case where the substrate 20 has water and various ion permeability. In this case, it is preferable that the base material 20 also has the permeability
  • the average pore diameter of the substrate 20 is 5 ⁇ m or less.
  • Transwell Corning Co., Ltd .: average pore diameter of 0.4 or 3.0 ⁇ m
  • An average pore size of 5 ⁇ m or more is not preferable because cells may pass through the substrate 20.
  • the shape of the substrate 20 is not particularly limited, but is preferably an artificial membrane such as a hollow fiber membrane, transwell, or flat membrane, a fine channel chip, solid particles, or hollow particles. An example of these will be described later with reference to FIG.
  • the substrate when examining the type and concentration of adhesion molecules, or when evaluating the amount of drug taken up by cultured cells 40, the substrate does not necessarily have water permeability. You don't have to.
  • the collagen molecule 30 or a fragment thereof may be coated on a petri dish or well plate that does not have water permeability as the substrate 20, and the cultured cells 40 may be cultured thereon.
  • the cultured cells 40 take in the useful substance 50 (FIG. 2B).
  • the cell layer is lifted to form a dome by releasing the incorporated useful substance 50 from the transporter 44 on the cell basement membrane side (FIG. 2 (C)). ).
  • the base material 20 is not particularly limited as long as it is made of an artificial material.
  • the collagen molecule 30 is an adhesion molecule that is coated on at least a part of the substrate.
  • the collagen molecule 30 includes collagen I and collagen IV. These can be obtained from Nitta Gelatin Co., Ltd., for example.
  • the concentration of the collagen molecule 30 is adjusted as appropriate so that the cell support complex 10 can maintain its performance during a practical period. Further, such a period is preferably 2 weeks or more from the start of culture, more preferably 3 weeks or more, and most preferably 4 weeks or more. The concentration of the collagen molecule 30 will be described later in the manufacturing method.
  • the 750 [mu] g / ml Ultra-3000 ⁇ g / ml following were coated with concentrations of about 50 [mu] g / cm 2 ultra 138 ⁇ g / cm 2 adhesive of the following concentrations.
  • the monolayer structure of the cultured cells can be maintained for 15 days or longer. More preferably, coating is performed at a concentration of 1000 ⁇ g / ml or more and 3000 ⁇ g / ml or less to obtain an adhesion amount of 65.4 ⁇ g / cm 2 or more and 138 ⁇ g / cm 2 or less.
  • the monolayer structure of a cultured cell can be maintained for 28 days or more.
  • collagen IV it is preferable to coat at a concentration of more than 500 ⁇ g / ml and not more than 3000 ⁇ g / ml to obtain an adhesion amount of more than 19.2 ⁇ g / cm 2 and not more than 121 ⁇ g / cm 2 .
  • the monolayer structure of the cultured cells can be maintained for 15 days or longer.
  • the bonding amount of about 25 [mu] g / cm 2 or more 121 ⁇ g / cm 2 following concentrations were coated at a concentration 750 [mu] g / ml or more 3000 ⁇ g / ml.
  • the monolayer structure of a cultured cell can be maintained for 28 days or more.
  • the collagen molecule 30 may be used as a fragment thereof, or may be used by sequentially coating the base material 20 with or without mixing collagen I and collagen IV.
  • the amount of collagen molecule 30 may be an amount corresponding to the molecular weight of full-length collagen molecule 30 described above.
  • the cultured cell 40 adheres to the base material 20 through the collagen molecule 30 or a fragment thereof.
  • the transporter 42 on the cell apical membrane side and the cell basement membrane It is necessary to express the transporter 44 on the side (FIG. 2).
  • the type of the cultured cell 40 is not particularly limited, but is preferably, for example, a human tubular epithelial cell, or a tubular epithelial cell derived from a human iPS cell or ES cell.
  • tubular epithelial cells that can be collected and separated from the kidney, and tubular epithelial-like cells that have been induced to differentiate and introduced genes from iPS cells or ES cells are envisaged.
  • Proximal tubule cells are mainly assumed as tubule epithelial cells, but they may be not only proximal tubule cells but also distal tubule epithelial cells and collecting duct epithelial cells. .
  • tubular epithelial cells instead of tubular epithelial cells, immortalization of tubular cells and expression of cell lines (MDCK cells, LLC-PK1 cells, JTC-12 cells, HK-2 cells) or specific transporters In order to achieve this, cells transfected with these cells may be used.
  • tubular cells derived from other animal species may be used in place of human tubular epithelial cells.
  • REGM (Medium etc.) REGM (Lonza) can be suitably used as a medium for culturing the cultured cells 40.
  • tubular cell culture media such as EpiCM (ScienCell) or Keratinocyte SFM (Life Technologies) can be used.
  • EpiCM ScrenCell
  • Keratinocyte SFM Life Technologies
  • the materials used in the conventional cell support complex can be preferably used.
  • the method for producing the cell support complex 10 includes the step 1 of coating the collagen molecule 30 or a fragment thereof on at least a part of the substrate 20 made of an artificial material, and the collagen molecule 30 or a fragment thereof adhered to the substrate 20. Step 2 of seeding the cultured cells 40 with respect to the above and Step 3 of forming the monolayer structure of the cultured cells 40 by culturing the cultured cells 40 are included.
  • the “at least part of the base material 20” here is as described above.
  • the collagen molecule 30 to be coated in step 1 is preferably selected from collagen I, collagen IV, fragments thereof, or two or more of these.
  • the preferred concentration of these collagen molecules 30 is as described above.
  • step 2 cells are seeded by a normal method.
  • the cell density to be seeded is preferably about 1.0 ⁇ 10 5 to about 1.0 ⁇ 10 8 cells / ml.
  • the cultured cells 40 grow to confluence and then maintain a confluent state. Therefore, in the production of the cell support complex 10, the number of cells to be seeded is not a great restriction.
  • the culture conditions in Step 3 are 37 ° C. and 5% CO 2 using REGM (Lonza) as a medium, for example.
  • REGM Longza
  • the cultured cells 40 are preferably cultured for about 5 days or longer before the cultured cells 40 become confluent.
  • FIG. 3A to FIG. 3C are diagrams showing a part of the structure of the device using the cell support complex according to the embodiment.
  • FIG. 3A is a diagram showing a part of the structure of an apparatus using Transwell as a base material.
  • FIG. 3B is a view showing a part of the structure of a dialysis apparatus using a hollow fiber membrane as a base material.
  • FIG. 3C is a diagram showing a part of the structure of an apparatus using a petri dish, a well plate, a fine channel, a microcarrier, or a hollow fiber membrane as a base material.
  • the useful substance 50 in this liquid is removed from the artificial membrane by using the mechanism shown in FIG. 2 by flowing the liquid to the side where the cultured cells 40 are arranged. Move to the other side.
  • This dialysis apparatus can be used as a drug evaluation module for examining the function of cells and the uptake and discharge of drugs with a small amount of liquid.
  • This dialysis apparatus can be used as a bioartificial dialysis module that collects useful substances from plasma components filtered by a hemofilter.
  • the device includes, in addition to one or more of these structures, a cartridge that houses them.
  • the material of the cartridge may be made of the same material as that of a cartridge such as a conventional plasma filter.
  • the shape of the cartridge preferably takes a suitable form according to the application. When used for the purpose of dialysis, the shape of the cartridge is preferably the shape of a dialyzer, and when used as a drug evaluation module, it is preferably a Transwell shape or a fine channel shape.
  • this embodiment it is possible to form a confluent monolayer epithelial structure of cultured cells on a substrate without observing with a microscope. As a result, it is possible to provide a cell support complex with improved stability of the monolayer epithelial structure. Moreover, it is possible to prevent pores from being formed in the monolayer epithelial structure of the cell support complex.
  • a 24-well plate (made of polystyrene: CELLSTAR, GreinerBio-one) was coated with Type I Collagen (Cellmatrix Type I-A, Nitta Gelatin Co., Ltd.) or Type IV Collagen (Cellmatrix Type IV, Nitta Gelatin Co., Ltd.).
  • each collagen solution diluted with 1N HCl to 100000, 5000, 3000, 2000, 1000, 500, 100, 75, 50, 25, 10, 5 ⁇ g / ml was added to the wells and allowed to stand overnight at 4 ° C. Overnight.
  • FIG. 4 shows the results after seeding 1, 5, 7, 9, 12, 15, 18, 24, and 28 days.
  • the adhesion molecule is coated by a method in which the coating solution containing the adhesion molecule is left standing overnight at 4 ° C. (overnight: a part of the adhesion molecule is coated), and the coating solution containing the adhesion molecule Was carried out by leaving the solution at 4 ° C. until the solution was completely dried (air-drying: the entire amount of adhesion molecules were coated).
  • a 24-well plate made of polystyrene: CELLSTAR, GreinerBio-one
  • Synthemax Coregen
  • Each Synthemax solution diluted with REGM to 100000, 5000, 3000, 2000, 1000, 500, 100, 75, 50, 25, 10, 5 ⁇ g / ml was added to the well and allowed to stand at room temperature. After 2 hours, the Synthemax solution was sucked and dried at room temperature for 2 hours.
  • 1.0 ⁇ 10 5 human proximal tubule cells (Lonza) were seeded, and cultured under conditions of 37 ° C. and 5% CO 2 using REGM (Lonza). The medium was changed every 2 days.
  • FIG. 4 shows the results after seeding 1, 5, 7, 9, 12, 15, 18, 24, and 28 days.
  • a 24-well plate made of polystyrene: CELLSTAR, GreinerBio-one
  • Poly-L-lysine ScienCell
  • the coating was performed by adding each Poly-L-lysine solution diluted with PBS ( ⁇ ) to 100,000, 5000, 3000, 2000, 1000, 500, 100, 75, 50, 25, 10, 5 ⁇ g / ml to the wells. It carried out by leaving still. After 2 hours, the Poly-L-lysine solution was sucked and dried at room temperature for 2 hours.
  • 1.0 ⁇ 10 5 human proximal tubule cells (Lonza) were seeded and cultured in REGM (Lonza) at 37 ° C. and 5% CO 2 . The medium was changed every 2 days.
  • FIG. 4 shows the results after seeding 1, 5, 7, 9, 12, 15, 18, 24, and 28 days.
  • M-PER Mammalian Protein Extraction Reagent (Thermo SCIENTIFIC), which is a protein solubilizer, was added thereto, followed by pipetting to peel off and collect the adhesion molecules from the petri dish surface. This was quantified by labeling the adhesion molecule with FITC using 2-D Quant Kit (GE Healthcare) or FluoReporter FITC protein Labeling Kit (invitrogen). Wallac 1420ARVO MX / LIGHT (Perkin Elmer) was used for quantification of adhesion molecules.
  • FIGS. 4 (A) to 4 (D) are diagrams showing the state of cell proliferation over time when the concentration of adhesion molecules coated on the petri dish is changed.
  • FIG. 4A is a diagram illustrating a result of using Synthemax which is a comparative example.
  • FIG. 4B is a diagram showing the results using Poly-L-lysine, which is a comparative example.
  • FIG. 4C is a diagram showing the results using collagen I.
  • FIG. FIG. 4 (D) is a diagram showing the results using collagen IV with reduced growth factors.
  • maximum adhesion amount (air-dried) means that after the coating solution is completely evaporated, the coating solution is allowed to stand at 4 ° C. until the collagen molecules or fragments thereof are coated on the substrate.
  • the adhesion molecular weight of the measured collagen molecule is shown. In this case, since the collagen molecules or fragments thereof contained in the coating solution are all coated on the substrate, the molecular weight of adhesion is equal to the total amount of collagen molecules or fragments contained in the coating solution before drying.
  • overweight indicates the amount of adhesion molecule after coating by allowing the coating solution containing the adhesion molecule to stand at 4 ° C. overnight.
  • the adhesion molecular weight corresponding to some of the actually coated collagen molecules or fragments thereof among the adhesion molecules is measured.
  • ⁇ indicates that the proliferation of the cells closed the pores of the monolayer epithelial structure of the cell support complex and maintained the monolayer epithelial structure of the cells.
  • A indicates “having a tendency to agglomerate”, but no pores are formed, but the cells are partially laminated.
  • the coating amount is 500 ⁇ g / ml or more and 3000 ⁇ g / ml or less to obtain an adhesion amount of more than 19.2 ⁇ g / cm 2 and 121 ⁇ g / cm 2 or less, about 15 days
  • the monolayer structure of the cultured cells was maintained. If 750 [mu] g / ml or more 3000 ⁇ g / ml to about 25 [mu] g / cm 2 or more was coated at a concentration 121 ⁇ g / cm 2 adhesive of the following concentrations, a single-layer structure of more than 28 days in culture the cells could be maintained.
  • the function of collagen IV may not be exhibited. Further, at a high concentration exceeding 3000 ⁇ g / ml, the viscosity of collagen IV is high, so that uniform coating may be difficult.
  • 10 cell support complex, 20 ... substrate, 30 ... collagen molecule, 40 ... cultured cell.
  • the present invention relates to a cell support complex, particularly a cell support complex using tubular epithelial cells.

Landscapes

  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)
  • External Artificial Organs (AREA)

Abstract

This cell support composite 10 is provided with: a substrate 20 formed from an artificial material; collagen molecules 30 or fragments thereof which are bonded to at least a portion of the substrate 20; and cultured cells 40 which are adhered to the substrate 20 via the collagen molecules 30 or the fragments thereof. The cultured cells 40 form a confluent monolayer with substantially no stratification. The collagen molecules 30 improve stability of the monolayer epithelial structure of the cultured cells 40 in the cell support composite 10. Furthermore, the formation of holes in the monolayer epithelial structure is inhibited.

Description

細胞支持複合体および細胞支持複合体の製造方法Cell support complex and method for producing cell support complex
　本発明は、細胞支持複合体、特に、尿細管上皮細胞を用いた細胞支持複合体に関する。 The present invention relates to a cell support complex, particularly a cell support complex using tubular epithelial cells.
　近年、急性及び、慢性腎不全患者の腎機能を代替するバイオ人工腎臓として、中空糸膜等のポリマー膜と尿細管上皮細胞をハイブリッド化したモジュールの開発が進められている。特に、バイオ人工腎臓の製造や供給、使用を考慮すると、数週間以上にわたって腎機能を維持できるバイオ人工腎臓が必要になる。 Recently, as a bioartificial kidney that replaces the renal function of patients with acute and chronic renal failure, a module in which a polymer membrane such as a hollow fiber membrane and a tubular epithelial cell are hybridized has been developed. In particular, considering the manufacture, supply, and use of bioartificial kidneys, bioartificial kidneys that can maintain kidney function for several weeks or more are required.
　しかし、腎臓から酵素処理により分離された尿細管上皮細胞は、生体内環境の消失やシャーレ上における培養による細胞の特徴の変化などから、本来の円柱状の細胞構造を維持できない。具体的には、尿細管上皮細胞をシャーレや人工膜上に播種すると、単層上皮構造を消失して細胞間に隙間が生じたり、細胞が重層化することが知られている。このような現象が生じることで、バイオ人工腎臓の血漿中の有用成分の再吸収機能が劣化する。 However, tubular epithelial cells isolated from the kidney by enzyme treatment cannot maintain their original cylindrical cell structure due to the disappearance of the in vivo environment or changes in cell characteristics due to culture on a petri dish. Specifically, it is known that when tubule epithelial cells are seeded on a petri dish or an artificial membrane, the monolayer epithelial structure is lost and a gap is formed between the cells, or the cells are stratified. When such a phenomenon occurs, the reabsorption function of useful components in the plasma of the bioartificial kidney deteriorates.
　尿細管上皮細胞の重層化や接触阻害を回避する手法として、特許文献１に開示されたバイオ人工尿細管が知られている。これは、ＭＥＫ阻害剤の適用により、尿細管上皮細胞の接触阻害や重層化を抑制し、コンフルエント（細胞が容器いっぱいに隙間なく増殖した状態）な単層を、人工膜内面に持続的に形成させる技術である（特許文献１）。 As a technique for avoiding stratification and contact inhibition of tubular epithelial cells, a bioartificial tubule disclosed in Patent Document 1 is known. This is due to the application of MEK inhibitors to suppress contact inhibition and stratification of tubule epithelial cells, and to form a confluent monolayer on the inner surface of the artificial membrane. (Patent Document 1).
特開２００８－５３９８０４号公報JP 2008-539804 A
　しかし、特許文献１の手法では、尿細管上皮細胞を人工膜上に播種後、コンフルエントに達するまで顕微鏡観察して確認した後に、ＭＥＫ阻害剤を作用させる必要がある。ところが特許文献１で使用される中空糸膜等の人工膜は、光を透過しない材質が多い。また、たとえ顕微鏡観察可能な中空糸膜を用いた場合でも、バイオ人工尿細管は中空糸膜を束ねてモジュール化した構造を有するため、顕微鏡観察で中空糸膜内腔に細胞がコンフルエントな状態にて接着していることを確認することは難しい。よって、特許文献１の手法では、人工膜上で尿細管上皮細胞がコンフルエントに達する時期が不明確のまま、最適ではないタイミングにてＭＥＫ阻害剤を投与することになる。そのため、尿細管上皮細胞間に隙間が生じている状態や、重層化している状態でＭＥＫ阻害剤を作用させる場合があった。その結果、性能のよいバイオ人工腎臓を得られないという問題点があった。 However, in the technique of Patent Document 1, it is necessary to act on the MEK inhibitor after confirming by observing the tubular epithelial cells by microscopic observation until they reach confluence after seeding on the artificial membrane. However, many artificial membranes such as hollow fiber membranes used in Patent Document 1 do not transmit light. Even when a hollow fiber membrane that can be observed with a microscope is used, the bioartificial tubule has a modular structure in which the hollow fiber membranes are bundled, so that the cells are confluent in the hollow fiber membrane lumen by microscopic observation. It is difficult to confirm that they are adhered. Therefore, in the technique of Patent Document 1, the MEK inhibitor is administered at a non-optimal timing while it is unclear when the tubular epithelial cells reach confluence on the artificial membrane. For this reason, MEK inhibitors may be allowed to act in a state where gaps are formed between tubular epithelial cells or in a layered state. As a result, there was a problem that a high-performance bioartificial kidney could not be obtained.
　本発明はこうした課題に鑑みてなされたものであり、単層上皮構造の安定性が向上した細胞支持複合体を提供することを目的とする。 The present invention has been made in view of these problems, and an object of the present invention is to provide a cell support complex having improved stability of a single-layer epithelial structure.
　上記課題を解決するために、本発明のある態様の細胞支持複合体は、人工材料で形成された基材と、基材の少なくとも一部に接着するコラーゲン分子もしくはその断片と、コラーゲン分子もしくはその断片を介して基材に付着した培養細胞と、を有する。培養細胞は、実質的に重層化することなくコンフルエントな単層を形成している。 In order to solve the above problems, a cell support complex according to an aspect of the present invention includes a base material formed of an artificial material, a collagen molecule or a fragment thereof that adheres to at least a part of the base material, and a collagen molecule or a part thereof. And cultured cells attached to the substrate through the fragments. The cultured cells form a confluent monolayer without substantial stratification.
　この態様によると、顕微鏡観察をすることなく、基材上に培養細胞のコンフルエントな単層上皮構造を形成させることができる。その結果、単層上皮構造の安定性が向上した細胞支持複合体を提供することができる。また、細胞支持複合体の単層上皮構造に孔が形成されることを防止することができる。 According to this embodiment, a confluent monolayer epithelial structure of cultured cells can be formed on the base material without observing under a microscope. As a result, it is possible to provide a cell support complex with improved stability of the monolayer epithelial structure. Moreover, it is possible to prevent pores from being formed in the monolayer epithelial structure of the cell support complex.
　培養細胞は、実質的に重層化することなくコンフルエントな単層を形成していてもよい。なお、「実質的に」とは、重層化による物質の移動効率の低下が問題とならない程度に単層構造が維持されていることを意味し、必ずしも重層化が全く生じていないことを意味しない。 The cultured cells may form a confluent monolayer without substantial stratification. Note that “substantially” means that the single-layer structure is maintained to such an extent that a decrease in mass transfer efficiency due to layering does not become a problem, and does not necessarily mean that no layering has occurred at all. .
　コラーゲン分子は、コラーゲンＩ、コラーゲンＩＶ、またはこれらのうち２つ以上から選択されてもよい。 The collagen molecule may be selected from collagen I, collagen IV, or two or more of these.
　コラーゲン分子は、基材に対する接着量が５０μｇ／ｃｍ２以上１３８μｇ／ｃｍ２以下のコラーゲンＩ、基材に対する接着量が１９．２μｇ／ｃｍ２超１２１μｇ／ｃｍ２以下のコラーゲンＩＶ、のうちのいずれか１つから選択されてもよい。 Collagen molecules are all adhesion amount to the substrate is 50 [mu] g / cm 2 or more 138μg / cm 2 or less collagen I, adhesion amount to the substrate is 19.2μg / cm 2 ultra 121μg / cm 2 or less collagen IV, of the Or one of them may be selected.
　本発明の別の態様は、細胞支持複合体の製造方法である。この細胞支持複合体の製造方法は、人工材料で形成された基材の少なくとも一部に、コラーゲン分子もしくはその断片をコーティングするステップと、コラーゲン分子もしくはその断片に対して培養細胞を播種するステップと、培養細胞を培養することにより、培養細胞の単層構造を形成するステップと、を含む。 Another aspect of the present invention is a method for producing a cell support complex. The method for producing the cell support complex includes a step of coating a collagen molecule or a fragment thereof on at least a part of a substrate formed of an artificial material, and a step of seeding cultured cells on the collagen molecule or a fragment thereof. Culturing the cultured cells to form a monolayer structure of the cultured cells.
　この態様によると、顕微鏡観察をすることなく、基材上に培養細胞のコンフルエントな単層上皮構造を形成させることができる。その結果、単層上皮構造の安定性が向上した細胞支持複合体を提供することができる。また、細胞支持複合体の単層上皮構造に孔が形成されることを防止することができる。 According to this embodiment, a confluent monolayer epithelial structure of cultured cells can be formed on the base material without observing under a microscope. As a result, it is possible to provide a cell support complex with improved stability of the monolayer epithelial structure. Moreover, it is possible to prevent pores from being formed in the monolayer epithelial structure of the cell support complex.
　この細胞支持複合体の製造方法において、コラーゲン分子は、コラーゲンＩ、コラーゲンＩＶ、またはこれらのうち２つ以上から選択されてもよい。 In this method for producing a cell support complex, the collagen molecule may be selected from collagen I, collagen IV, or two or more thereof.
　コラーゲン分子もしくはその断片をコーティングするステップは、コラーゲンＩを７５０μｇ／ｍｌ超３０００μｇ／ｍｌ以下の濃度でコーティングして５０μｇ／ｃｍ２超１３８μｇ／ｃｍ２以下の濃度の接着量とすること、コラーゲンＩＶを５００μｇ／ｍｌ以上３０００μｇ／ｍｌ以下の濃度でコーティングして１９．２μｇ／ｃｍ２超１２１μｇ／ｃｍ２以下の濃度の接着量とすることのいずれか１つを含んでもよい。 The step of coating the collagen molecule or fragment thereof, to an adhesive amount of collagen I coated at a concentration 750 [mu] g / ml Ultra-3000μg / ml 50μg / cm 2 ultra 138μg / cm 2 or less of the concentration, the collagen IV Any one of coating at a concentration of 500 μg / ml or more and 3000 μg / ml or less to have an adhesion amount of more than 19.2 μg / cm 2 and 121 μg / cm 2 or less may be included.
　本発明によれば、単層上皮構造の安定性が向上した細胞支持複合体を提供することができる。 According to the present invention, it is possible to provide a cell support complex with improved stability of a single-layer epithelial structure.
従来技術の細胞支持複合体の構成を模式的に示す図である。図１（Ａ）は、一般的なコーティング剤を用いた従来の細胞支持複合体を示す図である。図１（Ｂ）は、コーティング剤を用いない従来の細胞支持複合体を示す図である。It is a figure which shows typically the structure of the cell support composite_body | complex of a prior art. FIG. 1A is a view showing a conventional cell support complex using a general coating agent. FIG. 1 (B) is a view showing a conventional cell support complex without using a coating agent. 実施の形態に係る細胞支持複合体の構成を模式的に示す図である。図２（Ａ）は、基材が水透過性を有する実施の形態に係る細胞支持複合体の構成を示す図である。図２（Ｂ）は、基材が水透過性を有さない実施の形態に係る細胞支持複合体において、短時間培養した場合を示す図である。図２（Ｃ）は、基材が水透過性を有さない実施の形態に係る細胞支持複合体において、長時間培養した場合を示す図である。It is a figure which shows typically the structure of the cell support complex which concerns on embodiment. FIG. 2A is a diagram showing a configuration of a cell support complex according to an embodiment in which a base material has water permeability. FIG. 2 (B) is a diagram showing a case where the substrate is cultured for a short time in the cell support complex according to the embodiment in which the substrate does not have water permeability. FIG. 2C is a diagram showing a case where the substrate is cultured for a long time in the cell support complex according to the embodiment having no water permeability. 図３（Ａ）～図３（Ｃ）は、実施の形態に係る細胞支持複合体を用いた装置の構造の一部を示す図である。図３（Ａ）は、基材としてＴｒａｎｓｗｅｌｌを用いた装置の構造の一部を示す図である。図３（Ｂ）は、基材として中空糸膜を用いた透析装置の構造の一部を示す図である。図３（Ｃ）は、基材としてシャーレ、ウェルプレート、微細流路、マイクロキャリア、または中空糸膜を用いた装置の構造の一部を示す図である。FIG. 3A to FIG. 3C are diagrams showing a part of the structure of the device using the cell support complex according to the embodiment. FIG. 3A is a diagram showing a part of the structure of an apparatus using Transwell as a base material. FIG. 3B is a view showing a part of the structure of a dialysis apparatus using a hollow fiber membrane as a base material. FIG. 3C is a diagram showing a part of the structure of an apparatus using a petri dish, a well plate, a fine channel, a microcarrier, or a hollow fiber membrane as a base material. 図４（Ａ）～図４（Ｄ）は、シャーレに対してコーティングする接着分子の濃度を変えた場合における細胞の増殖状態を経時的に示す図である。図４（Ａ）は、比較例であるＳｙｎｔｈｅｍａｘを用いた結果を示す図である。図４（Ｂ）は、比較例であるＰｏｌｙ－Ｌ－ｌｙｓｉｎｅを用いた結果を示す図である。図４（Ｃ）は、コラーゲンＩを用いた結果を示す図である。図４（Ｄ）は、コラーゲンＩＶを用いた結果を示す図である。FIGS. 4 (A) to 4 (D) are diagrams showing the state of cell proliferation over time when the concentration of adhesion molecules coated on the petri dish is changed. FIG. 4A is a diagram illustrating a result of using Synthemax which is a comparative example. FIG. 4B is a diagram showing the results using Poly-L-lysine, which is a comparative example. FIG. 4C is a diagram showing the results using collagen I. FIG. FIG. 4D is a diagram showing the results using collagen IV.
　以下、図面を参照しながら、本発明を実施するための形態について詳細に説明する。なお、説明において同一の要素には同一の符号を付し、重複する説明を適宜省略する。 Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings. In the description, the same elements are denoted by the same reference numerals, and repeated descriptions are omitted as appropriate.
　図１は、従来技術の細胞支持複合体の構成を模式的に示す図である。図１（Ａ）は、一般的なコーティング剤を用いた従来の細胞支持複合体を示す図である。図１（Ｂ）は、コーティング剤を用いない従来の細胞支持複合体を示す図である。たとえば図１に示すように、尿細管上皮細胞および一般的なコーティング剤を用いた従来の細胞支持複合体では、尿細管上皮細胞が重層化したり、逆に細胞間に隙間が空いてしまうことがあった。その結果、重層化した領域では、細胞頂端膜側から細胞基底膜側へのトランスポーターを介した有用物質の移動が効率よく行われないという問題があった（矢印Ｐ）。一方、細胞間に空いた隙間からは、人工膜を介して、濃度依存的な物質移動が生じるという問題があった（矢印Ｑ）。この点、図１（Ｂ）に示すコーティング剤を用いない従来の細胞支持複合体でも同様の問題があった。 FIG. 1 is a diagram schematically showing the structure of a cell support complex according to the prior art. FIG. 1A is a view showing a conventional cell support complex using a general coating agent. FIG. 1 (B) is a view showing a conventional cell support complex without using a coating agent. For example, as shown in FIG. 1, in a conventional cell support complex using tubular epithelial cells and a general coating agent, tubular epithelial cells may be layered, or conversely, gaps may be left between the cells. there were. As a result, in the layered region, there was a problem that the useful substance was not efficiently transferred from the cell apical membrane side to the cell basement membrane side via the transporter (arrow P). On the other hand, there is a problem that concentration-dependent mass transfer occurs through the artificial membrane from the gap between the cells (arrow Q). In this regard, the conventional cell support complex not using the coating agent shown in FIG.
［実施の形態］
　図２は、実施の形態に係る細胞支持複合体１０の構成を模式的に示す図である。図２（Ａ）は、基材が水透過性を有する実施の形態に係る細胞支持複合体の構成を示す図である。図２（Ｂ）は、基材が水透過性を有さない実施の形態に係る細胞支持複合体において、短時間培養した場合を示す図である。図２（Ｃ）は、基材が水透過性を有さない実施の形態に係る細胞支持複合体において、長時間培養した場合を示す図である。 [Embodiment]
FIG. 2 is a diagram schematically showing the configuration of the cell support complex 10 according to the embodiment. FIG. 2A is a diagram showing a configuration of a cell support complex according to an embodiment in which a base material has water permeability. FIG. 2 (B) is a diagram showing a case where the substrate is cultured for a short time in the cell support complex according to the embodiment in which the substrate does not have water permeability. FIG. 2C is a diagram showing a case where the substrate is cultured for a long time in the cell support complex according to the embodiment having no water permeability.
　図２（Ａ）に示すように、細胞支持複合体１０は、人工材料で形成された基材２０と、基材の少なくとも一部に接着するコラーゲン（Ｃｏｌｌａｇｅｎ）分子３０と、コラーゲン分子３０もしくはその断片を介して基材２０に付着した培養細胞４０と、を有する。ここでいう「基材２０の少なくとも一部」とは、たとえば「平面または曲面を有する基材２０の少なくとも１つの面」をいう。基材２０が平板状構造の場合には、その少なくとも一方の面をいう。基材２０が円筒構造を有する場合には、その内側面または外側面の少なくとも一方をいう。 As shown in FIG. 2A, the cell support complex 10 includes a base material 20 formed of an artificial material, a collagen molecule 30 that adheres to at least a part of the base material, and a collagen molecule 30 or its molecule. And cultured cells 40 attached to the base material 20 via the fragments. Here, “at least a part of the substrate 20” means, for example, “at least one surface of the substrate 20 having a flat surface or a curved surface”. When the base material 20 has a flat plate structure, it means at least one surface thereof. When the base material 20 has a cylindrical structure, it refers to at least one of the inner surface and the outer surface.
（基材２０）
　基材２０は、尿細管上皮細胞の培養に用いられるモジュールである。図２（Ａ）には、基材２０が水や各種イオンの透過性を有する場合を示す。この場合、基材２０は、糖や低分子タンパク質の透過性も有することが好ましい。そのため、基材２０には、孔が形成されている。基材２０の平均孔径は、５μｍ以下である。このような基材２０として、たとえば、Ｔｒａｎｓｗｅｌｌ（Ｃｏｒｎｉｎｇ社：平均孔径０．４または３．０μｍ）を用いることができる。平均孔径が５μｍ以上の場合には、基材２０を細胞が通過してしまう場合があるため好ましくない。 (Substrate 20)
The base material 20 is a module used for culturing tubular epithelial cells. FIG. 2A shows a case where the substrate 20 has water and various ion permeability. In this case, it is preferable that the base material 20 also has the permeability | transmittance of sugar or a low molecular protein. Therefore, holes are formed in the base material 20. The average pore diameter of the substrate 20 is 5 μm or less. As such a substrate 20, for example, Transwell (Corning Co., Ltd .: average pore diameter of 0.4 or 3.0 μm) can be used. An average pore size of 5 μm or more is not preferable because cells may pass through the substrate 20.
　基材２０の形状は特に限定されないが、たとえば中空糸膜、Ｔｒａｎｓｗｅｌｌ、平膜等の人工膜や、微細流路チップ、中実粒子、中空粒子であることが好ましい。これらの一例を、図３を用いて後述する。図２（Ｂ）および（Ｃ）に示すように、接着分子の種類や濃度を検討したり、培養細胞４０による薬物の取込量を評価する際には、基材は必ずしも水透過性を有していなくともよい。この場合には、基材２０として水透過性を有さないシャーレやウェルプレートなどの上にコラーゲン分子３０もしくはその断片をコーティングし、その上で培養細胞４０を培養してもよい。短時間培養した場合には、培養細胞４０は有用物質５０を内部に取り込む（図２（Ｂ））。一方、長時間培養した場合には、取り込んだ有用物質５０を細胞基底膜側のトランスポーター４４から放出することなどによって、細胞層が浮き上がり、ドームを形成する現象が見られる（図２（Ｃ））。 The shape of the substrate 20 is not particularly limited, but is preferably an artificial membrane such as a hollow fiber membrane, transwell, or flat membrane, a fine channel chip, solid particles, or hollow particles. An example of these will be described later with reference to FIG. As shown in FIGS. 2 (B) and 2 (C), when examining the type and concentration of adhesion molecules, or when evaluating the amount of drug taken up by cultured cells 40, the substrate does not necessarily have water permeability. You don't have to. In this case, the collagen molecule 30 or a fragment thereof may be coated on a petri dish or well plate that does not have water permeability as the substrate 20, and the cultured cells 40 may be cultured thereon. When cultured for a short time, the cultured cells 40 take in the useful substance 50 (FIG. 2B). On the other hand, in the case of culturing for a long time, the cell layer is lifted to form a dome by releasing the incorporated useful substance 50 from the transporter 44 on the cell basement membrane side (FIG. 2 (C)). ).
　基材２０は、人工材料で形成されていれば素材は特に限定されないが、たとえばポリスチレン、ポリカーボネート（ＰＣ）、ポリエステル（ＰＥＴ）、ポリエステル系ポリマーアロイ（ＰＥＰＡ）、エチレン－ビニルアルコール共重合体（ＥＶＡＬ）、ポリエチレン、ポリスルホン（ＰＳｆ）、ポリエーテルスルホン（ＰＥＳ）であることが好ましい。 The base material 20 is not particularly limited as long as it is made of an artificial material. For example, polystyrene, polycarbonate (PC), polyester (PET), polyester polymer alloy (PEPA), ethylene-vinyl alcohol copolymer (EVAL) ), Polyethylene, polysulfone (PSf), and polyethersulfone (PES).
（コラーゲン分子３０）
　コラーゲン分子３０は、基材の少なくとも一部にコーティングされる接着分子である。本実施の形態では、コラーゲン分子３０として、コラーゲンＩ、コラーゲンＩＶが含まれる。これらはたとえば新田ゼラチン株式会社から入手することができる。 (Collagen molecule 30)
The collagen molecule 30 is an adhesion molecule that is coated on at least a part of the substrate. In the present embodiment, the collagen molecule 30 includes collagen I and collagen IV. These can be obtained from Nitta Gelatin Co., Ltd., for example.
　コラーゲン分子３０の濃度は、細胞支持複合体１０が実用に耐える期間、性能を維持できるように適宜調整する。また、このような期間は、培養開始から２週間以上であることが好ましく、３週間以上であることがさらに好ましく、４週間以上であることが最も好ましい。コラーゲン分子３０の濃度は、製造方法において後述する。 The concentration of the collagen molecule 30 is adjusted as appropriate so that the cell support complex 10 can maintain its performance during a practical period. Further, such a period is preferably 2 weeks or more from the start of culture, more preferably 3 weeks or more, and most preferably 4 weeks or more. The concentration of the collagen molecule 30 will be described later in the manufacturing method.
　コラーゲンＩの場合、７５０μｇ／ｍｌ超３０００μｇ／ｍｌ以下の濃度でコーティングして約５０μｇ／ｃｍ２超１３８μｇ／ｃｍ２以下の濃度の接着量とすることが好ましい。これにより、１５日以上培養細胞の単層構造を維持することができる。１０００μｇ／ｍｌ以上３０００μｇ／ｍｌ以下の濃度でコーティングして６５．４μｇ／ｃｍ２以上１３８μｇ／ｃｍ２以下の濃度の接着量とすることがさらに好ましい。これにより、２８日以上培養細胞の単層構造を維持することができる。７５０μｇ／ｍｌ未満では、コラーゲンＩの機能が発揮できない可能性があるため、および３０００μｇ／ｍｌを超える高濃度ではコラーゲンＩの粘性が高いために均一なコーティングが難しい場合があるためである。 For collagen I, it is preferable that the 750 [mu] g / ml Ultra-3000μg / ml following were coated with concentrations of about 50 [mu] g / cm 2 ultra 138μg / cm 2 adhesive of the following concentrations. Thereby, the monolayer structure of the cultured cells can be maintained for 15 days or longer. More preferably, coating is performed at a concentration of 1000 μg / ml or more and 3000 μg / ml or less to obtain an adhesion amount of 65.4 μg / cm 2 or more and 138 μg / cm 2 or less. Thereby, the monolayer structure of a cultured cell can be maintained for 28 days or more. This is because if it is less than 750 μg / ml, the function of collagen I may not be exerted, and if the concentration is higher than 3000 μg / ml, the viscosity of collagen I is high and uniform coating may be difficult.
　コラーゲンＩＶの場合、５００μｇ／ｍｌ超３０００μｇ／ｍｌ以下の濃度でコーティングして１９．２μｇ／ｃｍ２超１２１μｇ／ｃｍ２以下の濃度の接着量とすることが好ましい。これにより、１５日以上培養細胞の単層構造を維持することができる。７５０μｇ／ｍｌ以上３０００μｇ／ｍｌ以下の濃度でコーティングして約２５μｇ／ｃｍ２以上１２１μｇ／ｃｍ２以下の濃度の接着量とすることがさらに好ましい。これにより、２８日以上培養細胞の単層構造を維持することができる。５００μｇ／ｍｌ未満では、コラーゲンＩＶの機能が発揮できない可能性があるため、および３０００μｇ／ｍｌを超える高濃度ではコラーゲンＩＶの粘性が高いために均一なコーティングが難しい場合があるためである。 In the case of collagen IV, it is preferable to coat at a concentration of more than 500 μg / ml and not more than 3000 μg / ml to obtain an adhesion amount of more than 19.2 μg / cm 2 and not more than 121 μg / cm 2 . Thereby, the monolayer structure of the cultured cells can be maintained for 15 days or longer. It is further preferable that the bonding amount of about 25 [mu] g / cm 2 or more 121μg / cm 2 following concentrations were coated at a concentration 750 [mu] g / ml or more 3000μg / ml. Thereby, the monolayer structure of a cultured cell can be maintained for 28 days or more. This is because if the concentration is less than 500 μg / ml, the function of collagen IV may not be exhibited, and if the concentration is higher than 3000 μg / ml, the viscosity of collagen IV is high, so that uniform coating may be difficult.
　なお、コラーゲン分子３０は、その断片が使用されてもよいし、コラーゲンＩとコラーゲンＩＶが混合されて、または混合されずに基材２０に対して順にコーティングされることによって使用されてもよい。コラーゲン分子３０の断片が使用される場合には、コラーゲン分子３０の量は、上述した完全長のコラーゲン分子３０の分子量に相当する量であってもよい。 The collagen molecule 30 may be used as a fragment thereof, or may be used by sequentially coating the base material 20 with or without mixing collagen I and collagen IV. When a fragment of collagen molecule 30 is used, the amount of collagen molecule 30 may be an amount corresponding to the molecular weight of full-length collagen molecule 30 described above.
（培養細胞４０）
　培養細胞４０は、コラーゲン分子３０もしくはその断片を介して基材２０に付着する。細胞支持複合体１０に使用されるためには、培養細胞４０は、コラーゲン分子３０もしくはその断片を介して基材２０に固定された場合に、細胞頂端膜側のトランスポーター４２と、細胞基底膜側のトランスポーター４４とを発現する必要がある（図２）。 (Cultivated cells 40)
The cultured cell 40 adheres to the base material 20 through the collagen molecule 30 or a fragment thereof. In order to be used for the cell support complex 10, when the cultured cell 40 is fixed to the substrate 20 via the collagen molecule 30 or a fragment thereof, the transporter 42 on the cell apical membrane side and the cell basement membrane It is necessary to express the transporter 44 on the side (FIG. 2).
　培養細胞４０の種類は特に限定されないが、たとえばヒト尿細管上皮細胞、またはヒトｉＰＳ細胞もしくはＥＳ細胞由来の尿細管上皮様細胞であることが好ましい。具体的には、腎臓から採取・分離可能な尿細管上皮細胞、およびｉＰＳ細胞もしくはＥＳ細胞より分化誘導および遺伝子導入した尿細管上皮様細胞が想定される。尿細管上皮様細胞として、近位尿細管の細胞が主に想定されるが、近位尿細管の細胞だけではなく、遠位尿細管の上皮細胞や、集合管の上皮細胞であってもよい。または、尿細管上皮細胞の代わりに、尿細管細胞の不死化、および株化細胞（ＭＤＣＫ細胞、ＬＬＣ－ＰＫ１細胞、ＪＴＣ－１２細胞、ＨＫ－２細胞）または特定のトランスポーター等のタンパク質を発現させるために、これらの細胞に遺伝子導入した細胞を用いてもよい。または、ヒト尿細管上皮細胞の代わりに、他動物種由来尿細管細胞を用いてもよい。 The type of the cultured cell 40 is not particularly limited, but is preferably, for example, a human tubular epithelial cell, or a tubular epithelial cell derived from a human iPS cell or ES cell. Specifically, tubular epithelial cells that can be collected and separated from the kidney, and tubular epithelial-like cells that have been induced to differentiate and introduced genes from iPS cells or ES cells are envisaged. Proximal tubule cells are mainly assumed as tubule epithelial cells, but they may be not only proximal tubule cells but also distal tubule epithelial cells and collecting duct epithelial cells. . Or, instead of tubular epithelial cells, immortalization of tubular cells and expression of cell lines (MDCK cells, LLC-PK1 cells, JTC-12 cells, HK-2 cells) or specific transporters In order to achieve this, cells transfected with these cells may be used. Alternatively, tubular cells derived from other animal species may be used in place of human tubular epithelial cells.
（培地など）
　培養細胞４０の培養用の培地として、ＲＥＧＭ（Ｌｏｎｚａ社）を好適に使用することができる。これに代えて、ＥｐｉＣＭ（ＳｃｉｅｎＣｅｌｌ社）やＫｅｒａｔｉｎｏｃｙｔｅＳＦＭ（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）等、尿細管細胞培養培地を用いこともできる。その他の材料についても、従来の細胞支持複合体に用いられていた材料を好適に使用することができる。 (Medium etc.)
REGM (Lonza) can be suitably used as a medium for culturing the cultured cells 40. Alternatively, tubular cell culture media such as EpiCM (ScienCell) or Keratinocyte SFM (Life Technologies) can be used. As for other materials, the materials used in the conventional cell support complex can be preferably used.
（製造方法）
　次に、細胞支持複合体１０の製造方法について説明する。細胞支持複合体１０の製造方法は、人工材料で形成された基材２０の少なくとも一部に、コラーゲン分子３０もしくはその断片をコーティングするステップ１と、基材２０に接着したコラーゲン分子３０もしくはその断片に対して培養細胞４０を播種するステップ２と、培養細胞４０を培養することにより、前記培養細胞４０の単層構造を形成するステップ３と、を含む。ここでいう「基材２０の少なくとも一部」は、上述のとおりである。 (Production method)
Next, a method for producing the cell support complex 10 will be described. The method for producing the cell support complex 10 includes the step 1 of coating the collagen molecule 30 or a fragment thereof on at least a part of the substrate 20 made of an artificial material, and the collagen molecule 30 or a fragment thereof adhered to the substrate 20. Step 2 of seeding the cultured cells 40 with respect to the above and Step 3 of forming the monolayer structure of the cultured cells 40 by culturing the cultured cells 40 are included. The “at least part of the base material 20” here is as described above.
　ステップ１においてコーティングされるコラーゲン分子３０は、コラーゲンＩ、コラーゲンＩＶ、これらの断片、またはこれらのうち２つ以上から選択されることが好ましい。これらのコラーゲン分子３０の好ましい濃度は、上述のとおりである。 The collagen molecule 30 to be coated in step 1 is preferably selected from collagen I, collagen IV, fragments thereof, or two or more of these. The preferred concentration of these collagen molecules 30 is as described above.
　ステップ２では、通常の方法にて細胞を播種する。播種する細胞密度は、約１．０×１０５～約１．０×１０８個／ｍｌであることが好ましい。コラーゲン分子３０を適切に選択すると、培養細胞４０はコンフルエントになるまで増殖したあとコンフルエントな状態を維持する。そのため、細胞支持複合体１０の製造において、播種する細胞数は大きな制約とはならない。 In step 2, cells are seeded by a normal method. The cell density to be seeded is preferably about 1.0 × 10 5 to about 1.0 × 10 8 cells / ml. When the collagen molecule 30 is appropriately selected, the cultured cells 40 grow to confluence and then maintain a confluent state. Therefore, in the production of the cell support complex 10, the number of cells to be seeded is not a great restriction.
　ステップ３における培養条件は、たとえば培地としてＲＥＧＭ（Ｌｏｎｚａ社）を用いて３７℃、５％ＣＯ２である。培養細胞４０がコンフルエントになるまでに、培養細胞４０を約５日以上培養することが好ましい。なお、培地は定期的に交換することが好ましい。たとえば２日毎に交換する。 The culture conditions in Step 3 are 37 ° C. and 5% CO 2 using REGM (Lonza) as a medium, for example. The cultured cells 40 are preferably cultured for about 5 days or longer before the cultured cells 40 become confluent. In addition, it is preferable to change a culture medium regularly. For example, change every two days.
（作用）
　尿細管細胞として機能する培養細胞４０が単層上皮構造をとると、細胞頂端膜側のトランスポーター４２と細胞基底膜側のトランスポーター４４とを介した有用物質５０の移動がおこり、基材２０を介して有用物質５０の再吸収が促進される（図２）。 (Function)
When the cultured cell 40 functioning as a tubular cell has a single-layer epithelial structure, the useful substance 50 moves through the transporter 42 on the cell apical membrane side and the transporter 44 on the cell basement membrane side, and the base material 20 Thus, reabsorption of the useful substance 50 is promoted (FIG. 2).
（装置）
　図３（Ａ）～図３（Ｃ）は、実施の形態に係る細胞支持複合体を用いた装置の構造の一部を示す図である。図３（Ａ）は、基材としてＴｒａｎｓｗｅｌｌを用いた装置の構造の一部を示す図である。図３（Ｂ）は、基材として中空糸膜を用いた透析装置の構造の一部を示す図である。図３（Ｃ）は、基材としてシャーレ、ウェルプレート、微細流路、マイクロキャリア、または中空糸膜を用いた装置の構造の一部を示す図である。 (apparatus)
FIG. 3A to FIG. 3C are diagrams showing a part of the structure of the device using the cell support complex according to the embodiment. FIG. 3A is a diagram showing a part of the structure of an apparatus using Transwell as a base material. FIG. 3B is a view showing a part of the structure of a dialysis apparatus using a hollow fiber membrane as a base material. FIG. 3C is a diagram showing a part of the structure of an apparatus using a petri dish, a well plate, a fine channel, a microcarrier, or a hollow fiber membrane as a base material.
　図３（Ａ）のＴｒａｎｓｗｅｌｌを用いた透析装置では、培養細胞４０が配置された側に液体を流すことによって、図２に示したメカニズムを用いて、この液体中にある有用物質５０を人工膜の反対側に移動させる。この透析装置は、細胞の機能や、薬物の取込・排出を微量液量で調べる薬物評価モジュールとして使用可能である。 In the dialysis apparatus using Transwell of FIG. 3 (A), the useful substance 50 in this liquid is removed from the artificial membrane by using the mechanism shown in FIG. 2 by flowing the liquid to the side where the cultured cells 40 are arranged. Move to the other side. This dialysis apparatus can be used as a drug evaluation module for examining the function of cells and the uptake and discharge of drugs with a small amount of liquid.
　図３（Ｂ）の中空糸膜を用いた透析装置では、中空糸膜の管腔内に液体を流すことによって、図２に示したメカニズムを用いて、この液体中にある有用物質５０を管腔外へと移動させる。この透析装置は、血液濾過器で濾過した血漿成分中から、有用物質を回収するバイオ人工透析モジュールとして使用可能である。 In the dialysis apparatus using the hollow fiber membrane of FIG. 3 (B), the liquid 50 is caused to flow through the lumen of the hollow fiber membrane, thereby using the mechanism shown in FIG. Move out of the cavity. This dialysis apparatus can be used as a bioartificial dialysis module that collects useful substances from plasma components filtered by a hemofilter.
　図３（Ｃ）のシャーレ、ウェルプレート、微細流路、マイクロキャリア、中空粒子を用いた装置では、培養細胞４０が配置された側に微量の液体を流すことによって、細胞の有用物質５０の取り込みを確認できる。この装置は、細胞の機能や、薬物の取込・排出を微量液量で調べる薬物動態・薬効評価モジュールとして利用可能である。 In the apparatus using the petri dish, well plate, fine flow path, microcarrier, and hollow particles in FIG. 3C, a small amount of liquid is flowed to the side where the cultured cells 40 are arranged, thereby taking up the useful substance 50 of the cells. Can be confirmed. This device can be used as a pharmacokinetic / drug efficacy evaluation module that examines cell functions and drug uptake / discharge in a small amount of liquid.
　装置は、これらの構造の１つ以上に加えて、これらを内部に収めるカートリッジを含む。カートリッジの材質は、従来の血漿濾過器等のカートリッジと同じ材料で作製されてよい。カートリッジの形状は、用途に応じて好適な形態をとることが好ましい。透析を目的に使用する場合は、カートリッジの形状はダイアライザの形状であることが好ましく、薬物評価モジュールとして使用する場合は、Ｔｒａｎｓｗｅｌｌ形状や微細流路形状であることが好ましい。 The device includes, in addition to one or more of these structures, a cartridge that houses them. The material of the cartridge may be made of the same material as that of a cartridge such as a conventional plasma filter. The shape of the cartridge preferably takes a suitable form according to the application. When used for the purpose of dialysis, the shape of the cartridge is preferably the shape of a dialyzer, and when used as a drug evaluation module, it is preferably a Transwell shape or a fine channel shape.
　本実施の形態によれば、顕微鏡観察をすることなく、基材上に培養細胞のコンフルエントな単層上皮構造を形成させることができる。その結果、単層上皮構造の安定性が向上した細胞支持複合体を提供することができる。また、細胞支持複合体の単層上皮構造に孔が形成されることを防止することができる。 According to this embodiment, it is possible to form a confluent monolayer epithelial structure of cultured cells on a substrate without observing with a microscope. As a result, it is possible to provide a cell support complex with improved stability of the monolayer epithelial structure. Moreover, it is possible to prevent pores from being formed in the monolayer epithelial structure of the cell support complex.
［実施例］
　２４ウェルプレート（ポリスチレン製：ＣＥＬＬＳＴＡＲ、ＧｒｅｉｎｅｒＢｉｏ－ｏｎｅ社）をＴｙｐｅ　Ｉ　Ｃｏｌｌａｇｅｎ（Ｃｅｌｌｍａｔｒｉｘ　Ｔｙｐｅ　Ｉ－Ａ、新田ゼラチン株式会社）またはＴｙｐｅ　ＩＶ　Ｃｏｌｌａｇｅｎ（Ｃｅｌｌｍａｔｒｉｘ　Ｔｙｐｅ　ＩＶ、新田ゼラチン株式会社）にてコーティングした。コーティングは、１０００００、５０００、３０００、２０００、１０００、５００、１００、７５、５０、２５、１０、５μｇ／ｍｌに１Ｎ　ＨＣｌで希釈した各コラーゲン溶液をウェルに添加し、４℃にて一晩静置（ｏｖｅｒｎｉｇｈｔ）した。細胞播種前に、コラーゲン溶液を吸引し、ＰＢＳ（－）で２回洗浄後、ヒト近位尿細管細胞（Ｌｏｎｚａ社）を１．０×１０５個播種して、ＲＥＧＭ（Ｌｏｎｚａ社）を用いて３７℃、５％ＣＯ２の条件下で培養した。培地は２日毎に交換した。播種１、５、７、９、１２、１５、１８、２４、２８日経過後の結果を図４に示す。 [Example]
A 24-well plate (made of polystyrene: CELLSTAR, GreinerBio-one) was coated with Type I Collagen (Cellmatrix Type I-A, Nitta Gelatin Co., Ltd.) or Type IV Collagen (Cellmatrix Type IV, Nitta Gelatin Co., Ltd.). . For coating, each collagen solution diluted with 1N HCl to 100000, 5000, 3000, 2000, 1000, 500, 100, 75, 50, 25, 10, 5 μg / ml was added to the wells and allowed to stand overnight at 4 ° C. Overnight. Prior to cell seeding, the collagen solution is aspirated and washed twice with PBS (−), then 1.0 × 10 5 human proximal tubule cells (Lonza) are seeded, and REGM (Lonza) is used. The cells were cultured at 37 ° C. and 5% CO 2 . The medium was changed every 2 days. FIG. 4 shows the results after seeding 1, 5, 7, 9, 12, 15, 18, 24, and 28 days.
　接着分子のコーティングは、接着分子を含むコーティング溶液を４℃で一晩静置してコーティングする方法（一晩静置（ｏｖｅｒｎｉｇｈｔ）：接着分子の一部がコーティング）と、接着分子を含むコーティング溶液を４℃で溶液が完全に乾燥するまで静置する方法（風乾：接着分子全量がコーティング）で実施した。 The adhesion molecule is coated by a method in which the coating solution containing the adhesion molecule is left standing overnight at 4 ° C. (overnight: a part of the adhesion molecule is coated), and the coating solution containing the adhesion molecule Was carried out by leaving the solution at 4 ° C. until the solution was completely dried (air-drying: the entire amount of adhesion molecules were coated).
　比較例として、２４ウェルプレート（ポリスチレン製：ＣＥＬＬＳＴＡＲ、ＧｒｅｉｎｅｒＢｉｏ－ｏｎｅ社）をＳｙｎｔｈｅｍａｘ（Ｃｏｒｎｉｎｇ社）にてコーティングした。１０００００、５０００、３０００、２０００、１０００、５００、１００、７５、５０、２５、１０、５μｇ／ｍｌにＲＥＧＭで希釈した各Ｓｙｎｔｈｅｍａｘ溶液をウェルに添加し、室温で静置した。２時間経過後、Ｓｙｎｔｈｅｍａｘ溶液を吸引し、室温で２時間乾燥させた。ここに、ヒト近位尿細管細胞（Ｌｏｎｚａ社）を１．０×１０５個播種して、ＲＥＧＭ（Ｌｏｎｚａ社）を用いて３７℃、５％ＣＯ２の条件下で培養した。培地は２日毎に交換した。播種１、５、７、９、１２、１５、１８、２４、２８日経過後の結果を図４に示す。 As a comparative example, a 24-well plate (made of polystyrene: CELLSTAR, GreinerBio-one) was coated with Synthemax (Corning). Each Synthemax solution diluted with REGM to 100000, 5000, 3000, 2000, 1000, 500, 100, 75, 50, 25, 10, 5 μg / ml was added to the well and allowed to stand at room temperature. After 2 hours, the Synthemax solution was sucked and dried at room temperature for 2 hours. Here, 1.0 × 10 5 human proximal tubule cells (Lonza) were seeded, and cultured under conditions of 37 ° C. and 5% CO 2 using REGM (Lonza). The medium was changed every 2 days. FIG. 4 shows the results after seeding 1, 5, 7, 9, 12, 15, 18, 24, and 28 days.
　比較例として、２４ウェルプレート（ポリスチレン製：ＣＥＬＬＳＴＡＲ、ＧｒｅｉｎｅｒＢｉｏ－ｏｎｅ社）をＰｏｌｙ－Ｌ－ｌｙｓｉｎｅ（ＳｃｉｅｎＣｅｌｌ社）にてコーティングした。コーティングは、１０００００、５０００、３０００、２０００、１０００、５００、１００、７５、５０、２５、１０、５μｇ／ｍｌにＰＢＳ（－）で希釈した各Ｐｏｌｙ－Ｌ－ｌｙｓｉｎｅ溶液をウェルに添加し、室温で静置して実施した。２時間経過後、Ｐｏｌｙ－Ｌ－ｌｙｓｉｎｅ溶液を吸引し、室温で２時間乾燥させた。ここに、ヒト近位尿細管細胞（Ｌｏｎｚａ社）を１．０×１０５個播種して、ＲＥＧＭ（Ｌｏｎｚａ社）にて３７℃、５％ＣＯ２の条件下で培養した。培地は２日毎に交換した。播種１、５、７、９、１２、１５、１８、２４、２８日経過後の結果を図４に示す。 As a comparative example, a 24-well plate (made of polystyrene: CELLSTAR, GreinerBio-one) was coated with Poly-L-lysine (ScienCell). The coating was performed by adding each Poly-L-lysine solution diluted with PBS (−) to 100,000, 5000, 3000, 2000, 1000, 500, 100, 75, 50, 25, 10, 5 μg / ml to the wells. It carried out by leaving still. After 2 hours, the Poly-L-lysine solution was sucked and dried at room temperature for 2 hours. Here, 1.0 × 10 5 human proximal tubule cells (Lonza) were seeded and cultured in REGM (Lonza) at 37 ° C. and 5% CO 2 . The medium was changed every 2 days. FIG. 4 shows the results after seeding 1, 5, 7, 9, 12, 15, 18, 24, and 28 days.
（接着分子の質量測定）
　上述した手法でシャーレに接着分子をコーティング後、ＰＢＳ（－）で１回洗浄した。ここにタンパク質可溶化剤であるＭ－ＰＥＲ　Ｍａｍｍａｌｉａｎ　Ｐｒｏｔｅｉｎ　Ｅｘｔｒａｃｔｉｏｎ　Ｒｅａｇｅｎｔ（Ｔｈｅｒｍｏ　ＳＣＩＥＮＴＩＦＩＣ）を添加後、ピペッティングして接着分子をシャーレ表面より剥離、回収した。これを２－Ｄ　Ｑｕａｎｔ　Ｋｉｔ（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）または、ＦｌｕｏＲｅｐｏｒｔｅｒ　ＦＩＴＣ　ｐｒｏｔｅｉｎ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ（ｉｎｖｉｔｏｒｏｇｅｎ）にて接着分子をＦＩＴＣ標識して定量した。接着分子の定量には、ｗａｌｌａｃ　１４２０ＡＲＶＯ　ＭＸ／ＬＩＧＨＴ（Ｐｅｒｋｉｎ　Ｅｌｍｅｒ社）を用いた。 (Measurement of adhesion molecule mass)
The petri dish was coated with adhesion molecules by the method described above, and then washed once with PBS (−). M-PER Mammalian Protein Extraction Reagent (Thermo SCIENTIFIC), which is a protein solubilizer, was added thereto, followed by pipetting to peel off and collect the adhesion molecules from the petri dish surface. This was quantified by labeling the adhesion molecule with FITC using 2-D Quant Kit (GE Healthcare) or FluoReporter FITC protein Labeling Kit (invitrogen). Wallac 1420ARVO MX / LIGHT (Perkin Elmer) was used for quantification of adhesion molecules.
　図４（Ａ）～図４（Ｄ）は、シャーレに対してコーティングする接着分子の濃度を変えた場合における細胞の増殖状態を経時的に示す図である。図４（Ａ）は、比較例であるＳｙｎｔｈｅｍａｘを用いた結果を示す図である。図４（Ｂ）は、比較例であるＰｏｌｙ－Ｌ－ｌｙｓｉｎｅを用いた結果を示す図である。図４（Ｃ）は、コラーゲンＩを用いた結果を示す図である。図４（Ｄ）は、成長因子が低減されたコラーゲンＩＶを用いた結果を示す図である。 FIGS. 4 (A) to 4 (D) are diagrams showing the state of cell proliferation over time when the concentration of adhesion molecules coated on the petri dish is changed. FIG. 4A is a diagram illustrating a result of using Synthemax which is a comparative example. FIG. 4B is a diagram showing the results using Poly-L-lysine, which is a comparative example. FIG. 4C is a diagram showing the results using collagen I. FIG. FIG. 4 (D) is a diagram showing the results using collagen IV with reduced growth factors.
　なお、表中で「最大接着量（風乾）」とは、コーティング溶液が完全に蒸発することによってコラーゲン分子もしくはその断片が基材にコーティングされるまで、コーティング溶液を４℃に静置した後に、測定したコラーゲン分子の接着分子量を示す。この場合、コーティング溶液に含まれるコラーゲン分子もしくはその断片がすべて基材にコーティングされるため、接着分子量は乾燥前にコーティング溶液に含まれたコラーゲン分子もしくはその断片の全量に等しい。一方、「接着量（ｏｖｅｒｎｉｇｈｔ）」は、接着分子を含むコーティング溶液を４℃にて一晩静置することによってコーティングした後の接着分子量を示す。この場合、接着分子のうち実際にコーティングされた一部のコラーゲン分子もしくはその断片に相当する接着分子量が測定される。また、「○」は、細胞の増殖によって細胞支持複合体の単層上皮構造の孔が塞がり、かつ細胞の単層上皮構造が維持されたことを示す。「△」の「凝集化傾向あり」とは、孔は生じていないが、一部分で細胞が積層してきた場合を示す。 In the table, “maximum adhesion amount (air-dried)” means that after the coating solution is completely evaporated, the coating solution is allowed to stand at 4 ° C. until the collagen molecules or fragments thereof are coated on the substrate. The adhesion molecular weight of the measured collagen molecule is shown. In this case, since the collagen molecules or fragments thereof contained in the coating solution are all coated on the substrate, the molecular weight of adhesion is equal to the total amount of collagen molecules or fragments contained in the coating solution before drying. On the other hand, “overweight” indicates the amount of adhesion molecule after coating by allowing the coating solution containing the adhesion molecule to stand at 4 ° C. overnight. In this case, the adhesion molecular weight corresponding to some of the actually coated collagen molecules or fragments thereof among the adhesion molecules is measured. In addition, “◯” indicates that the proliferation of the cells closed the pores of the monolayer epithelial structure of the cell support complex and maintained the monolayer epithelial structure of the cells. “A” indicates “having a tendency to agglomerate”, but no pores are formed, but the cells are partially laminated.
　図４（Ａ）より、Ｓｙｎｔｈｅｍａｘでは濃度をどのように調節しても、７日を超えて培養細胞の単層構造が維持できなかった。同様に、図４（Ｂ）より、Ｐｏｌｙ－Ｌ－ｌｙｓｉｎｅでは濃度をどのように調節しても、７日を超えて培養細胞の単層構造が維持できなかった。 From FIG. 4 (A), it was not possible to maintain the monolayer structure of the cultured cells over 7 days no matter how the concentration was adjusted with Synthemax. Similarly, from FIG. 4 (B), it was not possible to maintain the monolayer structure of the cultured cells over 7 days no matter how the concentration was adjusted with Poly-L-lysine.
　図４（Ｃ）より、コラーゲンＩの場合、７５０μｇ／ｍｌ超３０００μｇ／ｍｌ以下の濃度でコーティングして約５０μｇ／ｃｍ２超１３８μｇ／ｃｍ２以下の濃度の接着量とすれば、約１５日以上培養細胞の単層構造が維持できた。１０００μｇ／ｍｌ以上３０００μｇ／ｍｌ以下の濃度でコーティングして６５．４μｇ／ｃｍ２以上１３８μｇ／ｃｍ２以下の濃度の接着量とすれば、２８日以上培養細胞の単層構造が維持できた。７５０μｇ／ｍｌ未満では、コラーゲンＩの機能が発揮できない可能性がある。また、３０００μｇ／ｍｌを超える高濃度ではコラーゲンＩの粘性が高いために均一なコーティングが難しい場合がある。 4 from (C), when collagen I, if 750 [mu] g / ml Ultra-3000μg / ml following were coated with concentrations of about 50 [mu] g / cm 2 ultra 138μg / cm 2 adhesive of the following concentrations, about 15 days or more The monolayer structure of cultured cells could be maintained. When coating was performed at a concentration of 1000 μg / ml or more and 3000 μg / ml or less to give an adhesion amount of 65.4 μg / cm 2 or more and 138 μg / cm 2 or less, the monolayer structure of cultured cells could be maintained for 28 days or more. If it is less than 750 μg / ml, the function of collagen I may not be exhibited. Moreover, since the viscosity of collagen I is high at a high concentration exceeding 3000 μg / ml, uniform coating may be difficult.
　図４（Ｄ）より、コラーゲンＩＶの場合、５００μｇ／ｍｌ以上３０００μｇ／ｍｌ以下の濃度でコーティングして１９．２μｇ／ｃｍ２超１２１μｇ／ｃｍ２以下の濃度の接着量とすれば、約１５日以上培養細胞の単層構造が維持できた。７５０μｇ／ｍｌ以上３０００μｇ／ｍｌ以下の濃度でコーティングして約２５μｇ／ｃｍ２以上１２１μｇ／ｃｍ２以下の濃度の接着量とすれば、２８日以上培養細胞の単層構造が維持できた。５００μｇ／ｍｌ未満では、コラーゲンＩＶの機能が発揮できない可能性がある。また、３０００μｇ／ｍｌを超える高濃度ではコラーゲンＩＶの粘性が高いために均一なコーティングが難しい場合がある。 As shown in FIG. 4 (D), in the case of collagen IV, if the coating amount is 500 μg / ml or more and 3000 μg / ml or less to obtain an adhesion amount of more than 19.2 μg / cm 2 and 121 μg / cm 2 or less, about 15 days As described above, the monolayer structure of the cultured cells was maintained. If 750 [mu] g / ml or more 3000μg / ml to about 25 [mu] g / cm 2 or more was coated at a concentration 121μg / cm 2 adhesive of the following concentrations, a single-layer structure of more than 28 days in culture the cells could be maintained. If it is less than 500 μg / ml, the function of collagen IV may not be exhibited. Further, at a high concentration exceeding 3000 μg / ml, the viscosity of collagen IV is high, so that uniform coating may be difficult.
　以上、本発明を実施例にもとづいて説明した。本発明は上記実施の形態に限定されず、種々の設計変更が可能であり、様々な変形例が可能であること、またそうした変形例も本発明の範囲にあることは、当業者に理解されるところである。 The present invention has been described above based on the embodiments. It is understood by those skilled in the art that the present invention is not limited to the above-described embodiment, and various design changes are possible, and various modifications are possible, and such modifications are within the scope of the present invention. It is a place.
　１０…細胞支持複合体、２０…基材、３０…コラーゲン分子、４０…培養細胞。 10 ... cell support complex, 20 ... substrate, 30 ... collagen molecule, 40 ... cultured cell.
　本発明は、細胞支持複合体、特に、尿細管上皮細胞を用いた細胞支持複合体に関する。 The present invention relates to a cell support complex, particularly a cell support complex using tubular epithelial cells.

Claims (7)

  1. 　人工材料で形成された基材と、
    　前記基材の少なくとも一部に接着するコラーゲン分子もしくはその断片と、
    　前記コラーゲン分子もしくはその断片を介して前記基材に付着した培養細胞と、を有することを特徴とする細胞支持複合体。     A substrate made of artificial material;
    Collagen molecules or fragments thereof that adhere to at least a portion of the substrate;
    And a cultured cell attached to the substrate via the collagen molecule or a fragment thereof.
  2. 　前記培養細胞は、実質的に重層化することなくコンフルエントな単層を形成していることを特徴とする請求項１に記載の細胞支持複合体。 The cell-supporting complex according to claim 1, wherein the cultured cells form a confluent monolayer without substantial stratification.
  3. 　前記コラーゲン分子は、コラーゲンＩ、コラーゲンＩＶ、またはこれらのうち２つ以上から選択されることを特徴とする請求項１または２に記載の細胞支持複合体。 The cell support complex according to claim 1 or 2, wherein the collagen molecule is selected from collagen I, collagen IV, or two or more thereof.
  4. 　前記コラーゲン分子は、前記基材に対する接着量が５０μｇ／ｃｍ２以上１３８μｇ／ｃｍ２以下のコラーゲンＩ、前記基材に対する接着量が１９．２μｇ／ｃｍ２超１２１μｇ／ｃｍ２以下のコラーゲンＩＶのうちのいずれか１つから選択されることを特徴とする請求項３に記載の細胞支持複合体。 Wherein the collagen molecule is adhered amount of 50 [mu] g / cm 2 or more 138μg / cm 2 or less of collagen I with respect to the substrate, the adhesion amount with respect to the substrate of 19.2μg / cm 2 ultra 121μg / cm 2 or less collagen IV The cell support complex according to claim 3, wherein the cell support complex is selected from any one of the following.
  5. 　人工材料で形成された基材の少なくとも一部に、コラーゲン分子もしくはその断片をコーティングするステップと、
    　前記基材に接着した前記コラーゲン分子もしくはその断片に対して培養細胞を播種するステップと、
    　前記培養細胞を培養することにより、前記培養細胞の単層構造を形成するステップと、を含むことを特徴とする細胞支持複合体の製造方法。     Coating at least a portion of a substrate formed of an artificial material with collagen molecules or fragments thereof;
    Seeding cultured cells against the collagen molecules or fragments thereof adhered to the substrate;
    Forming a monolayer structure of the cultured cells by culturing the cultured cells, and a method for producing a cell support complex.
  6. 　前記コラーゲン分子は、コラーゲンＩ、コラーゲンＩＶ、またはこれらのうち２つ以上から選択されることを特徴とする請求項５に記載の細胞支持複合体の製造方法。 6. The method for producing a cell-supporting complex according to claim 5, wherein the collagen molecule is selected from collagen I, collagen IV, or two or more thereof.
  7. 　前記コラーゲン分子もしくはその断片をコーティングするステップは、７５０μｇ／ｍｌ超３０００μｇ／ｍｌ以下の濃度でコーティングして５０μｇ／ｃｍ２超１３８μｇ／ｃｍ２以下の濃度の接着量とすること、コラーゲンＩＶを５００μｇ／ｍｌ超３０００μｇ／ｍｌ以下の濃度でコーティングして１９．２μｇ／ｃｍ２超１２１μｇ／ｃｍ２以下の濃度の接着量とすることのいずれか１つを含むことを特徴とする請求項６に記載の細胞支持複合体の製造方法。 The step of coating the collagen molecule or fragment thereof, to a 750 [mu] g / ml Ultra-3000μg / ml and coated at a concentration 50 [mu] g / cm 2 ultra 138μg / cm 2 adhesive of the following concentrations, collagen IV 500 [mu] g / 7. The method according to claim 6, comprising any one of coating at a concentration of more than 3000 μg / ml and an amount of adhesion of more than 19.2 μg / cm 2 and not more than 121 μg / cm 2 . A method for producing a cell support complex.
PCT/JP2016/081390 2015-11-10 2016-10-24 Cell support composite, and method for producing cell support composite WO2017082025A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2015220405A JP6755085B2 (en) 2015-11-10 2015-11-10 Cell-supported complex and method for producing cell-supported complex
JP2015-220405 2015-11-10

Publications (1)

Publication Number Publication Date
WO2017082025A1 true WO2017082025A1 (en) 2017-05-18

Family

ID=58695085

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2016/081390 WO2017082025A1 (en) 2015-11-10 2016-10-24 Cell support composite, and method for producing cell support composite

Country Status (2)

Country Link
JP (1) JP6755085B2 (en)
WO (1) WO2017082025A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019181920A1 (en) * 2018-03-22 2019-09-26 日機装株式会社 Cell culturing method, method for producing cell support composite, cultured cell, and cell support composite
US12024720B2 (en) 2017-04-06 2024-07-02 Nikkiso Co., Ltd. Cell cultivation method, cell support composite production method, cultivated cells, and cell support composite

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6932051B2 (en) * 2017-09-05 2021-09-08 日機装株式会社 Method of culturing cells and method of producing cell-supporting complex

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07135961A (en) * 1993-11-19 1995-05-30 Sumitomo Bakelite Co Ltd Substrate for culture of vascular endothelial cell and its production
WO2008047760A1 (en) * 2006-10-17 2008-04-24 Tokai University Educational System Bioartificial renal tubule
JP2009515558A (en) * 2005-11-16 2009-04-16 ユニバーシティ オブ ノース カロライナ アット チャペル ヒル Extracellular matrix components for proliferation or differentiation of hepatic progenitor cells
JP2014515270A (en) * 2011-05-27 2014-06-30 アドバンスト・テクノロジーズ・アンド・リジェネレイティブ・メディスン・エルエルシー Bioartificial proximal tubule system and method of use thereof
JP2015047076A (en) * 2013-08-29 2015-03-16 独立行政法人産業技術総合研究所 Cell culture substrate, method for differentiation-inducing of osteoblasts using the same and method for producing osteoblasts
JP2015180202A (en) * 2004-04-28 2015-10-15 サノフィ パスツール ヴァックスデザイン コーポレーション Artificial immune system: methods for making and use

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07135961A (en) * 1993-11-19 1995-05-30 Sumitomo Bakelite Co Ltd Substrate for culture of vascular endothelial cell and its production
JP2015180202A (en) * 2004-04-28 2015-10-15 サノフィ パスツール ヴァックスデザイン コーポレーション Artificial immune system: methods for making and use
JP2009515558A (en) * 2005-11-16 2009-04-16 ユニバーシティ オブ ノース カロライナ アット チャペル ヒル Extracellular matrix components for proliferation or differentiation of hepatic progenitor cells
WO2008047760A1 (en) * 2006-10-17 2008-04-24 Tokai University Educational System Bioartificial renal tubule
JP2014515270A (en) * 2011-05-27 2014-06-30 アドバンスト・テクノロジーズ・アンド・リジェネレイティブ・メディスン・エルエルシー Bioartificial proximal tubule system and method of use thereof
JP2015047076A (en) * 2013-08-29 2015-03-16 独立行政法人産業技術総合研究所 Cell culture substrate, method for differentiation-inducing of osteoblasts using the same and method for producing osteoblasts

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12024720B2 (en) 2017-04-06 2024-07-02 Nikkiso Co., Ltd. Cell cultivation method, cell support composite production method, cultivated cells, and cell support composite
WO2019181920A1 (en) * 2018-03-22 2019-09-26 日機装株式会社 Cell culturing method, method for producing cell support composite, cultured cell, and cell support composite
JPWO2019181920A1 (en) * 2018-03-22 2021-02-04 日機装株式会社 Cell culture method, cell support complex production method, cultured cells and cell support complex
JP2021191305A (en) * 2018-03-22 2021-12-16 日機装株式会社 Culture method of cells
JP7147031B2 (en) 2018-03-22 2022-10-04 日機装株式会社 Cell culture method

Also Published As

Publication number Publication date
JP2017085972A (en) 2017-05-25
JP6755085B2 (en) 2020-09-16

Similar Documents

Publication Publication Date Title
WO2017082024A1 (en) Cell support composite, and method for producing cell support composite
JP6995112B2 (en) Method for culturing cells and method for producing a cell-supporting complex
Wung et al. Hollow fibre membrane bioreactors for tissue engineering applications
JP7147031B2 (en) Cell culture method
Bettahalli et al. Integration of hollow fiber membranes improves nutrient supply in three-dimensional tissue constructs
JP6605618B2 (en) Cell support complex and method for producing cell support complex
EP2075019B1 (en) Bioartificial renal tubule
WO2017082025A1 (en) Cell support composite, and method for producing cell support composite
JPWO2008143149A1 (en) Cell sheet having dimensions, manufacturing method thereof, and cell culture carrier therefor
Ray et al. Surface engineering of a bioartificial membrane for its application in bioengineering devices
Palakkan et al. Evaluation of polypropylene hollow-fiber prototype bioreactor for bioartificial liver
JP2009082005A (en) Method for producing cell sheet with suppressed shrinkage
Rizki-Safitri et al. Bioengineered kidney models: Methods and functional assessments
JP6932051B2 (en) Method of culturing cells and method of producing cell-supporting complex
JP7062052B2 (en) Method of culturing cells and method of producing cell-supporting complex
WO2019221172A1 (en) Cell scaffold material
Han et al. Transmission and regulation of biochemical stimulus via a nanoshell directly adsorbed on the cell membrane to enhance chondrogenic differentiation of mesenchymal stem cell
WO2023248934A1 (en) Method for producing cell support composite, method for producing drug evaluation device, drug evaluation method and drug evaluation device
EP3313985A1 (en) A multilayer sheet of cells
JP2004344002A (en) Method for culturing cell with porous membrane
Matsusaki et al. Cell surface engineering using a layer-by-layer nanofilm for biomedical applications
WO2004050824A1 (en) Tissue culture apparatus

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16863989

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16863989

Country of ref document: EP

Kind code of ref document: A1