WO2007094511A1 - Procédé de culture de cellules animales utilisant un transporteur d'oxygène à base d'hémoglobine - Google Patents

Procédé de culture de cellules animales utilisant un transporteur d'oxygène à base d'hémoglobine Download PDF

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WO2007094511A1
WO2007094511A1 PCT/JP2007/053240 JP2007053240W WO2007094511A1 WO 2007094511 A1 WO2007094511 A1 WO 2007094511A1 JP 2007053240 W JP2007053240 W JP 2007053240W WO 2007094511 A1 WO2007094511 A1 WO 2007094511A1
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hemoglobin
culture
cells
oxygen carrier
animal cell
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PCT/JP2007/053240
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English (en)
Japanese (ja)
Inventor
Yasuyuki Sakai
Hirosuke Naruto
Katsuji Ohta
Atsushi Mizuno
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Oxygenix Co., Ltd.
The University Of Tokyo
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Publication of WO2007094511A1 publication Critical patent/WO2007094511A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions

Definitions

  • the present invention relates to a method for culturing animal cells with improved oxygen supply capacity. Specifically, the present invention relates to an animal cell culture method for constructing a biological tissue that can be used in the field of artificial organs and regenerative medicine. Background art
  • a living body is a three-dimensional collection of cellular tissues, and the entire collection functions as a system.
  • the control of the generation, regeneration, and maintenance of these tissues has become a major issue in the field of human organs and regenerative medicine.
  • the problem to be solved by the present invention is that in an animal cell culture method for artificially reconstructing a high-density and Z- or large-sized tissue apparatus suitable for the field of artificial organs or regenerative medicine, particularly oxygen
  • the inventor of the present invention which is to provide a culture method excellent in supply ability, has intensively studied to solve the above problems.
  • erythrocytes which has a high oxygen dissolution amount per unit volume and exhibits a sigmoid-type release ability under physiological conditions, is optimal (Fig. 1).
  • erythrocytes are often hemolyzed in the nra'iro cell culture system due to the unstable membrane structure, and the released free hemoglobin is toxic to the cultured cells.
  • the “artificial erythrocyte of a type in which hemoglobin (Hb) is encapsulated in a lipid bilayer (ribosome)” was developed for blood transfusion.
  • Hb hemoglobin
  • ribosome lipid bilayer
  • the inventors have found that the above-described problems can be solved by adding the artificial red blood cells to the medium in the conventional cell culture method, and have completed the present invention. That is, the present invention is as follows. '
  • a method for culturing animal cells comprising culturing animal cells in the presence of a medium containing a hemoglobin-containing oxygen carrier.
  • the oxygen carrier include those in which hemoglobin is encapsulated in ribosomes.
  • examples of the hemoglobin include human hemoglobin, and examples of the ribosome include those modified with polyethylene glycol.
  • examples of the animal cells include hepatocytes, and examples of the hepatocytes include those derived from humans, rats, or pigs.
  • examples of the culture method of the present invention include a method of culturing by irrigating the medium, and specifically, a method using a flat plate perfusion culture apparatus.
  • the medium contains, for example, a polymer containing a polymer that inhibits or suppresses the cytotoxicity of the carrier to the animal cells, specifically, the transport into the animal cells. Anything that inhibits or prevents the body from being taken in can be used.
  • examples of the polymer include ushi serum albumin.
  • the culture method of the present invention can be performed, for example, to maintain or promote the functional activity of the animal cells and / or increase the number of animal cells.
  • the culture method of the present invention can be performed, for example, to construct a biological tissue used for an artificial organ or a regenerative medical organ.
  • An animal cell culture kit comprising a medium containing a hemoglobin-containing oxygen carrier.
  • the kit of the present invention can be used, for example, to construct a biological tissue used for an artificial organ or a regenerative medical organ.
  • Figure 1 is a graph showing the oxygen saturation curve.
  • FIG. 2 is a schematic cross-sectional view showing the structure of polyethylene glycol-modified ribosome-encapsulating human hemoglobin (Oxygen Carrier).
  • FIG. 3 is a schematic diagram showing a cell culture system using a flat plate perfusion culture apparatus.
  • FIG. 4 is a graph showing the influence of Oxygen Carrier (albumin secretion amount) on cells subjected to static culture.
  • A is a graph showing the effect on rat normal hepatocytes.
  • B is a graph showing the effect on Hep G2 cells, showing [alb ( ⁇ )] before adding lucalbumin (BSA) to the medium and [alb (+)] after adding.
  • FIG. 5 is a graph showing the effect of Oxygen Carrier (total albumin secretion) on rat normal hepatocytes cultured using a flat plate perfusion culture device.
  • Figure 6 shows the effects of Oxygen Carrier on the normal rat hepatocytes cultured using a flat plate perfusion culture device (the presence or absence of Oxygen Carrier and the effect of Oxygen Carrier on the distance from the inlet of the culture tank). is there.
  • A is a graph showing the predicted oxygen concentration gradient from the inlet to the outlet of the culture tank.
  • B is a photograph showing the cell morphology before the start of culture.
  • C is a photograph showing cell morphology near the entrance and exit of the culture tank after culturing.
  • FIG. 7 is a diagram showing the influence of Oxygen Carrier (the presence or absence of Oxygen Carrier and the effect of Oxygen Carrier on the distance from the culture tank inlet) on Hep G2 cells cultured using a flat plate perfusion culture apparatus. Specifically, it is a photograph showing the cell morphology near the entrance and exit of the culture tank after culturing.
  • Figure 8 shows the time course of dulcose consumption of rat hepatocytes cultured in monolayers for 6 days in medium with or without ribosomal hemoglobin (LEH) (LEH (+)) or without (LEH (-)). It is a graph shown in.
  • Figure 9 shows a graph showing the amount of albumin produced (secreted) over time in rat hepatocytes cultured in monolayers for 6 hours in a medium with or without LEH (LEH (+)) or without (LEH (-)). It is. .
  • Figure 10 shows the amount of DNA in rat hepatocytes cultured in monolayers in LEH-added (LEH (+)) or non-supplemented (LEH (-)) medium at the start of culture (after 0 days) and 6 days. It is a graph comparing after cultivation (after 6 days).
  • liver tissue of the same density as the living body (2xl0 8 cells cm 3 -tissue) is about 15 cm 3 Is the limit.
  • an oxygen carrier containing hemoglobin as in a living body (a hemoglobin-based oxygen carrier (also referred to as a hemoglobin-containing oxygen carrier)).
  • a hemoglobin-based oxygen carrier also referred to as a hemoglobin-containing oxygen carrier
  • an artificial red blood cell polyethylene glycol (PEG) modified ribosome-encapsulated human hemoglobin; Oxygen Carrier
  • PEG polyethylene glycol
  • Oxygen Carrier oxygen carrier
  • the animal cell culture method of the present invention is a method characterized by culturing animal cells in the presence of a medium containing a hemoglobin-containing oxygen carrier as described above.
  • a medium containing a hemoglobin-containing oxygen carrier as described above.
  • the hemoglobin-containing oxygen carrier used in the method of the present invention is not limited as long as hemoglobin having oxygen binding ability is supported on an arbitrary carrier (carrier).
  • the mode of loading depends on the form of the carrier and is not particularly limited, and examples thereof include all known modes of loading such as bonding, association, and inclusion.
  • the type of carrier is not particularly limited, and examples include all known carriers such as lipid vesicles (liposomes), micelles, emulsions, and organic polymers. Among them, ribosomes are preferred.
  • the oxygen carrier used in the method of the present invention is preferably one in which hemoglobin is included in the ribosome (ribosomal endoglobin type hemoglobin). Can be mentioned.
  • the oxygen carrier used in the method of the present invention will be described in detail by taking liposomal endoplasmic hemoglobin as an example.
  • the type of biological origin and molecular structure of the hemoglobin that can be used for liposomal endocystic hemoglobin is not limited as long as it has an oxygen binding ability.
  • Examples of biological origin include human origin, rat origin, mouse origin, ushi origin, and pig origin, among which human origin is preferable.
  • normal hemoglobin As the type of molecular structure, for example, in the case of human hemoglobin, it is usually preferable to use normal hemoglobin. However, there is no limitation, and if necessary, all or part of the hemoglobin used may become abnormal. Moglobin can also be used. Normal hemoglobin includes HbF composed of two ⁇ chains and two y chains, and HbA composed of two chains and two; 3 chains. These include HbS (cause of sickle cell disease) and HbH (cause of thalassemia). Furthermore, as the above-mentioned hemoglobin, it is usually preferable to use oxyhemoglobin in a state in which it is bound to oxygen molecules. However, in some cases, deoxyhenioglobin in a state that is not bound to oxygen molecules is used. It may be included. Deoxyhemoglobin can be used as oxyhemoglobin by binding oxygen molecules by a known treatment during or after culture.
  • Lipid endoplasmic reticulum (ribosome) Lipid endoplasmic reticulum (hereinafter sometimes simply referred to as “endoplasmic reticulum”) is a membrane-constituting lipid selected from various known amphipathic molecules capable of forming a bilayer structure in an aqueous medium.
  • the above-mentioned lipids can be used, preferably natural or synthetic saturated phospholipids and unsaturated phospholipids, and combinations thereof, and synthetic saturated phospholipids are more preferable.
  • Saturated phospholipids are not limited, but include natural extracts such as extracts from extracellular membranes such as hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, liver plasma membrane, erythrocyte membrane, Escherichia coli membrane, and intracellular membranes.
  • natural extracts such as extracts from extracellular membranes such as hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, liver plasma membrane, erythrocyte membrane, Escherichia coli membrane, and intracellular membranes.
  • acyl phosphatidylethanolamine dimyristoyl phosphatidyl choline, dipalmitoyl Scan choline, distearoyl phosphatidylcholine is more preferable.
  • unsaturated phospholipids include, but are not limited to, natural unsaturated phospholipids such as egg yolk lecithin and soybean lecithin, 1-palmitoyl, 2-oleoyl phosphatidyl choline, geoleoyl phosphatidinorecholine Preferred examples include synthetic unsaturated phospholipids such as 1,2-di (octadeca-trans-2, trans-4-dienoyl) phosphatidylcholine, and polymerizable phospholipids such as 1,2-bisleostearoylphosphatidylcholine.
  • natural unsaturated phospholipids such as egg yolk lecithin and soybean lecithin
  • 1-palmitoyl 2-oleoyl phosphatidyl choline
  • geoleoyl phosphatidinorecholine Preferred examples include synthetic unsaturated phospholipids such as 1,2-di (octadeca-trans-2, trans-4-dienoyl) phosphatidy
  • the polymerizable phospholipid may have a non-polymerizable long chain, and the non-polymerizable long chain is not limited, but, for example, a straight chain or branched chain having 2 to 24 carbon atoms. And an alkyl group, an acyl group, a non-polymerizable alkenyl group, a non-polymerizable alkenoyl group, and the like.
  • the membrane constituent lipid of the endoplasmic reticulum preferably includes a polyethylene glycol (PEG) type lipid, that is, a lipid having a PEG chain bound thereto.
  • PEG polyethylene glycol
  • the formed lipid vesicle is modified with PEG, specifically, has a PEG chain on the surface of the vesicle.
  • the content ratio is not limited, but is preferably 0.01 to 10 mol%, more preferably 0.1 to I mol%, with respect to the total membrane constituent lipid. preferably from 0.1 to 0.3 mol 0/0.
  • the molecular weight of the PEG chain in the PEG type lipid is not limited, but is preferably 1,000 to 12,000, and more preferably 2,000 to 5,000, for example. If the content ratio of the PEG type lipid and the molecular weight of the PEG chain are within the above ranges, the above-described effects can be easily obtained.
  • the membrane-constituting lipid of the endoplasmic reticulum may contain a negatively charged lipid (anionic lipid).
  • the negatively charged lipid include, but are not limited to, diacylphosphatidylglycerolone, diacylphosphatidic acid ', diacylphosphatidylinositol, diacylphosphatidylserine, and fatty acid.
  • the fatty acid is not limited, but for example, a saturated or unsaturated fatty acid having 12 to 20 carbon atoms, specifically, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid. Examples include acid or octadeca-2,4-genic acid.
  • the membrane-constituting lipid of the endoplasmic reticulum may contain a lipid component as a stabilizer.
  • stabilizers include, but are not limited to, sterols. Specific examples include ergosterol and cholesterol, among which cholesterol is preferred.
  • the content is preferably 5 to 50 mol% with respect to the total lipid constituting the membrane, and is preferable. Is 15-40 mol%.
  • DPPG dipalmitoylphosphatidylglycerol
  • PA palmitic acid
  • lipid (anionic lipid) serving as a stabilizer a carboxylic acid type lipid having no phosphate group can also be used.
  • carboxylic acid type lipids include lipids represented by the following general formula (1).
  • RK 2 and R 3 has the following general formula ()
  • M is a hydrogen atom or a monovalent cation
  • m is an integer of 1 to 5 indicating a methylene chain length.
  • AA 2 and A 3 are selected from C (0) O, CONH, or NHCO independently of each other All may be the same or any of the four or two forces may be different from the others,
  • n is an integer of 1 to 3 representing the methylene chain length.
  • chain hydrocarbon group examples include a linear or branched acyclic saturated hydrocarbon group or acyclic unsaturated hydrocarbon group (including acyclic terpenes) having 2 to 20 carbon atoms.
  • chain hydrocarbon group examples include various hydrocarbon groups classified into alkyl groups, alkenyl groups, alkynyl groups, alkadienyl groups, alkylidene groups, alkylidyne groups, and the like.
  • the binding sites of Ri, R 2 and R 3 are lysine, asparagine, gnoletamine, aspartic acid, glutamic acid, serine, threonine,
  • a trifunctional amino acid such as mouth shin is preferable.
  • a trifunctional amino acid having one reactive functional group and two equal reactive functional groups i.e. aspartic acid or glutamic acid having one terminal amino group and two terminal carboxyl groups, or one terminal Lysine, asparagine, glutamine, etc. having a carboxyl group and two terminal amino groups are preferred.
  • Aspartic acid and glutamic acid are more preferred, and homocystine and dartathione may also be used.
  • carboxylic acid type lipid represented by the general formula (1)
  • DHSG (1,5-0-dihexadecyl-N-succinyl-L-glutamate) represented by the following formula.
  • the ribosomal endoplasmic hemoglobin may contain other components as appropriate in addition to the above-mentioned lipid endoplasmic reticulum and hemoglobin encapsulated therein, and is not limited.
  • Other components include, for example, Alosteretta eta-ter, amino acids, saccharides, reducing agents, vitamins, bases, etc., contained in the inner aqueous phase of lipid vesicles.
  • a method for preparing the ribosomal endovesicular type hemoglobin mouth that is, the method of encapsulating hemoglobin in the endoplasmic reticulum formed by a lipid bilayer membrane
  • a known general method used for preparing a lipid endoplasmic reticulum can be used.
  • the microfluidizer method, etc. can be selected or combined as appropriate.
  • the means and conditions to be used can be appropriately selected and set based on the common general technical knowledge of those skilled in the art.
  • a desired film-forming lipid (generally in powder form) is added to a hemoglobin-containing liquid that is an inclusion substance, hydrated and swollen, and then allowed to stand, and then vortex mixer
  • lipids By dispersing lipids with a forced stirrer, ultrasonic irradiator (homogenizer, etc.), microfluidizer, high-pressure extruder (Etastruder), or freeze-thawing, etc.
  • a dispersion of vesicles can be obtained.
  • freeze-thawing or a combination of freeze-thaw and a high-pressure extruder is preferable because the number of coating layers can be effectively reduced, and the permeability of the filter and thus the processing time are significantly improved.
  • the particle size of the finally obtained liposomal endocyst-type hemog-mouth bottle is often reduced more than that obtained at this stage. It is also preferable in terms of productivity such as yield.
  • the particle size (number average particle size) of lipoglobin endovesicle type finally obtained is not limited, but it is possible to obtain effects such as encapsulation efficiency, sterilization treatment, blood dynamics, etc.
  • the thickness is preferably 30 to 450 nm, more preferably 80 to 350 nm, still more preferably 150 to 300 nm, and particularly preferably 200 to 300 nm.
  • lipid vesicles can be formed in the presence of a suitable pH buffer and / or a surfactant. This makes it possible to stably maintain the functional activity of the encapsulated hemoglobin, even if it is outside the stable pH region of the medium (culture medium) containing liposomal endocystic hemoglobin and hemoglobin. It is preferable because the culture efficiency can be improved.
  • pH buffer examples include glycine buffer, phthalate-potassium buffer, succinate buffer, monopotassium citrate buffer, tris (human roxymethyl) aminomethane buffer, folate buffer, acetate buffer, etc. Is mentioned.
  • the stable pH range of hemoglobin is preferably 6-8.
  • the amount of pH buffer used is not particularly limited It can be set as appropriate.
  • the surfactant examples include polyethylene sorbitan monooleate such as polysorbate 80, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan trioleate. Ethylene sorbitan is preferred. By using an appropriate amount of such a surfactant, an effect that leads to the stability of the encapsulated hemoglobin can be obtained without adversely affecting the formation of lipid vesicles.
  • the above-mentioned surfactant generally includes all compounds belonging to emulsifiers and solubilizers.
  • lipid vesicles in addition to the above-described pH buffer solution and surfactant, other various additives may be mixed in advance and used without any limitation.
  • liposomal endoplasmic hemoglobin In order to prevent denaturation (decrease in activity) of encapsulated hemoglobin, it is usually preferable to prepare liposomal endoplasmic hemoglobin at a temperature of 20 ° C or lower in the process of handling hemoglobin. No limitation.
  • hemoglobin-containing oxygen carriers that can be used in the present invention
  • particularly preferred specific examples include polyethylene glycol-modified ribosome-encapsulated human hemoglobin having a diameter of 200 to 300 ⁇ .
  • This oxygen carrier is available from Oxygenitas Co., Ltd. (Product name: Oxygen Carrier) (H. Sakai et al, J. Biochem., 131, 611-617 (2002) JG Riess, Chem. Rev., 101, 2797-2919 (2001)).
  • the culture method of the present invention can be applied to culture of all known animal cells.
  • human cells, rat cells, mouse cells, pig cells, monkey cell cells are preferable, and human cells, rats Origin, mouse-derived animal cells are more preferred.
  • cell types include cells derived from various organs or tissues, such as cells derived from liver, cornea, heart, skin, stem cells, kidneys, blood vessels, bones, and cartilage.
  • hepatocytes in culture method of the invention the cardiac cells are preferably les, 0
  • the animal cell cultured by the method of the present invention may be any cultured cell. . That is, the cultured cells may be, for example, normal cells or abnormal cells (for example, cancer cells), and these may be primary cultured cells or cultured cells ⁇ . Also good.
  • the animal cell cultured by the method of the present invention is preferably a cell that can be used to construct a biological tissue used for an artificial organ or a regenerative medical organ.
  • the medium (culture solution) that can be used in the method of the present invention is a suitable medium known in the technical field, depending on the type of cultured cells to be used, except that it contains the above-described hemoglobin-containing oxygen carrier. Can do.
  • medium containing various hormones insulin, dexamethasone, EGF
  • DME medium supplemented with 10% FBS
  • DMEM supplemented with 10% rabbit fetal serum (FBS) 20 mM HEPES, 1% NEAA, 10 7 M insulin, 10 ⁇ 6 M dexamethasone, 10 ng / ml mouse epithelial cell growth factor (EGF) and any antibiotics
  • 0.5 mM ascorubic acid diphosphate essential trace metals (e.g., 10 '8 M selenium, 10 ⁇ 6 Micromax zinc, 10 ⁇ 7 Micromax copper and the like) and the like.
  • the content of the hemoglobin-containing oxygen carrier in the medium is not limited, for example, 5 to 50 ° /. ( ⁇ / ⁇ ) is preferred, and more preferably 10 to 20% ( ⁇ / ⁇ ). Further, the hemoglobin content in the medium is preferably S SOg dl / 1 , more preferably ⁇ SOg dL- 1 .
  • the method of the culture method of the present invention is not limited, and may be any of well-known culture methods such as stationary culture, shaking culture, and perfusion culture, depending on the type of target cultured cell and the like.
  • perfusion culture is preferable for static culture.
  • perfusion culture is preferable because it is possible to reduce the culture efficiency and cost and to easily adjust the cell culture conditions.
  • Perfusion culture is a culture technique that involves aseptic operation of loading and unloading the medium into and from the culture vessel, and is a technique that is originally suitable for tissue culture. 1 To construct biological tissue for use in artificial organs or regenerative medical organs High density important to It can also be suitably used for cell culture. Examples of perfusion culture include reflux culture, fed-batch culture, chemostat, and perfusion.
  • the culture method of the present invention can be suitably used for maintaining or promoting the functional activity of animal cells that are cultured cells and / or increasing the number of animal cells. This is because the method of the present invention is extremely excellent in oxygen supply ability, and is particularly important for cell culture for constructing a living tissue.
  • the culture method of the present invention may include, for example, a hemoglobin-containing oxygen carrier (hemoglobin-based oxygen carrier): Even when added to the culture medium continuously for 6 days, the oxygen carrier showed an effective oxygen supply capacity. This result indicates that the oxygen carrier is useful for constructing a biological tissue used for an artificial organ or a regenerative medical organ.
  • the culture method of the present invention can be suitably used for constructing a biological tissue used for an artificial organ or a regenerative medical organ.
  • preferred examples of the artificial organ or the regenerative medical organ include cornea, artificial heart, artificial liver, human skin, stem cell, artificial kidney, artificial blood vessel, artificial bone, and cartilage.
  • the animal cell culture kit of the present invention is a culture kit comprising a medium containing a hemoglobin-containing oxygen carrier as described above.
  • the kit of the present invention may appropriately contain other optional components considered necessary for animal cell culture, and is not limited.
  • the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
  • rat hepatocytes were isolated from adult rats (5-6 weeks old, male) by collagenase perfusion.
  • the culture medium was a serum-free DME medium (low-glucose) with various hormones (insulin, dexamethasone, EGF) added.
  • Hep G2 cells were used as human liver cancer-derived cells.
  • 10% FBS-added caro DME medium was used as the culture solution.
  • a small-scale flat plate perfusion culture device (Fig. 3) capable of forming an oxygen-deficient region was constructed (AW TUles et al, Biotechnol. Bioeng., 73, 379-389 (200 l) KL Murad et al., Blood, 93, 2121-2127 (1999)) 0
  • This culture device has a structure that can be fitted with slide glass carrying cells, and the culture fluid flows through a 0.5 mm high layer from the surface of this slide glass. It is a mechanism.
  • Rat normal hepatocytes (lxlO 5 cells cm 2 ) or Hep G2 cells (5xl0 4 cells cm- 2 ) were preliminarily placed on a slide glass in advance (24 hr) to form a monolayer. After that, a cell-supporting slide glass was placed in the culture apparatus, and an Oxygen Carrier-added culture solution (
  • Perfusion culture was continued for 24 hr. Thereafter, for normal rat hepatocytes, the cells on the slide glass removed from the device were observed with a phase contrast microscope. For Hep G2 cells, the cells on the slide glass removed from the apparatus were fixed with 4% paraformaldehyde (PFA), immunostained for albumin, and then observed with a microscope.
  • PFA paraformaldehyde
  • Rat normal hepatocytes were cultured for 24 hr using a flat plate perfusion culture device, and total albumin secretion was measured as an indicator of cell functional activity (ELISA method).
  • OxyC (-) system compared to OxyC (+) system. About 3 times the amount of secretion was confirmed in the system (Fig. 5).
  • Fig. 6 shows the oxygen concentration gradient predicted by model equation (1) (Fig. 6 (A)) and the observed cell morphology of normal rat hepatocytes (before culture: Fig. 6 (B), after culture: Figure 6 (C)) is also shown.
  • a hemoglobin-based oxygen carrier such as Oxygen Carrier
  • Oxygen Carrier such acidic cell damage does not occur and tissue can be reconstructed under physiological conditions.
  • an ia w'to? Culture system with a hemoglobin concentration of about 13% was used.
  • Oxygen Carrier The effectiveness of a hemoglobin-based oxygen carrier (Oxygen Carrier) was evaluated using a two-dimensional hepatocyte culture system. As a result, it was shown that the improvement of oxygen supply capacity, which is one of the three issues for the reconstruction of large tissues, can be sufficiently solved by perfusion culture using a culture solution containing Oxygen Carrier.
  • rat hepatocytes were generated by normal collagenase perfusion 5-6 weeks old Wistar males. Isolated from rat liver.
  • the basic culture medium is DMEM supplemented with 10% ushi fetal serum (FBS), 20 mM HEPES, 1% NEAA, 10 ' 7 M insulin, 10 ⁇ 6 M dexamethasone, 10 ng / ml mouse epidermal growth factor (EGF) And antibiotics.
  • Rat hepatocytes (1.2X10 5 cells / cm 2 ) were cultured in collagen-coated 12-well culture plates (3.8 cm 2 ; IWAKI, Japan) for 24 hours in the basal medium, and then the medium was treated with 20% (v / v) Changed daily to LEH (+/-) medium.
  • the glucose concentration in the culture medium was measured with a glucose analyzer (Glucose Analyzer 2; Beckman Instruments, Inoreland).
  • the concentration of rat serum albumin was measured by sandwich enzyme immunoassay (ELISA).
  • the DNA concentration was measured after 0 days (before LEH addition) and after 6 days by 4 ′, 6-diamidino-2-phenylindole (DAPI) fluorometric analysis.
  • PEGylated LEH was obtained from Oxygenitas Corporation (Japan). LEH was packed in N 2 filled bottles and stored at 4 ° C. The present inventors sucked LEH with a sterile needle (Terumo, Japan) and filtered it through a 0.45 / im cellulose acetate filter (Advantech, Japan). LEH was suspended in 0.9% NaCl solution.
  • the concentrations of Hb and lipid were 10 g / dl and 7 g / dl, respectively.
  • the average diameter of LEH was 269 ⁇ .
  • the metHb level was 8.5%.
  • Usi Serum albumin (BSA) was predicted to inhibit the toxicity of LEH.
  • FIGS. 8 and 9 the two functions (glucose consumption and albumin production) were slightly enhanced by the addition of LEH.
  • Figure 10 shows the effect of LEH (+/-) on the amount of DNA in rat hepatocytes. The amount of DNA decreases during 6 days of culture. The amount of DNA in cultures with LEH added was 1.2 times higher than in the control group.
  • an animal cell culture method for artificially reconstructing a high-density and / or large-sized tissue (organ) suitable for the field of artificial organs or regenerative medicine is particularly excellent in oxygen supply ability.
  • the animal cell culture method of the present invention is extremely useful in that a culture method can be provided.

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Abstract

L'invention concerne un procédé de culture de cellules animales en vue de reconstruire artificiellement un tissu (organe) ayant une haute densité et/ou une grande taille, lequel procédé est adapté au domaine des organes artificiels ou de la médecine régénérative, en particulier un procédé de culture qui permet avantageusement un apport d'oxygène. Ce procédé de culture de cellules animales est caractérisé par la mise en culture d'une cellule animale en présence d'un milieu de culture additionné d'un transporteur d'oxygène contenant de l'hémoglobine. L'invention concerne également un kit de culture de cellules animales qui comprend un milieu de culture additionné d'un transporteur d'oxygène contenant de l'hémoglobine.
PCT/JP2007/053240 2006-02-15 2007-02-15 Procédé de culture de cellules animales utilisant un transporteur d'oxygène à base d'hémoglobine WO2007094511A1 (fr)

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CN111411070A (zh) * 2020-05-15 2020-07-14 河南医学高等专科学校 一种培养基的基层物质、其制备方法、培养基及其应用
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CN109844097A (zh) * 2016-07-11 2019-06-04 塞莱斯有限公司 培养类器官的方法
JP2019521707A (ja) * 2016-07-11 2019-08-08 セレス リミテッド オルガノイドを培養するための方法
US11401501B2 (en) 2016-07-11 2022-08-02 Cellesce Limited Methods for culturing organoids
JP7123412B2 (ja) 2016-07-11 2022-08-23 セレス リミテッド オルガノイドを培養するための方法
CN109844097B (zh) * 2016-07-11 2023-10-31 塞莱斯有限公司 培养类器官的方法
JPWO2019131806A1 (ja) * 2017-12-26 2020-12-17 株式会社マイオリッジ 心筋細胞の薬剤応答性試験方法
US11726083B2 (en) 2017-12-26 2023-08-15 Myoridge Co. Ltd. Method for testing drug response of cardiomyocytes
JP7341478B2 (ja) 2017-12-26 2023-09-11 株式会社マイオリッジ 心筋細胞の薬剤応答性試験方法
WO2021230261A1 (fr) * 2020-05-11 2021-11-18 国立大学法人大阪大学 Système de libération d'oxygène en réponse à une stimulation
CN111411070A (zh) * 2020-05-15 2020-07-14 河南医学高等专科学校 一种培养基的基层物质、其制备方法、培养基及其应用

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