WO2007052653A1 - Recipient de culture et appareil de culture - Google Patents

Recipient de culture et appareil de culture Download PDF

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Publication number
WO2007052653A1
WO2007052653A1 PCT/JP2006/321744 JP2006321744W WO2007052653A1 WO 2007052653 A1 WO2007052653 A1 WO 2007052653A1 JP 2006321744 W JP2006321744 W JP 2006321744W WO 2007052653 A1 WO2007052653 A1 WO 2007052653A1
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WIPO (PCT)
Prior art keywords
culture
fertilized egg
sealed space
cells
culture container
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PCT/JP2006/321744
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English (en)
Japanese (ja)
Inventor
Keiji Naruse
Norio Ishida
Hiroaki Inui
Jinji Mizuno
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Strex Incorporation
National University Corporation Okayama University
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Publication date
Application filed by Strex Incorporation, National University Corporation Okayama University filed Critical Strex Incorporation
Priority to JP2007542759A priority Critical patent/JPWO2007052653A1/ja
Publication of WO2007052653A1 publication Critical patent/WO2007052653A1/fr

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    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • 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
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/06—Bioreactors or fermenters specially adapted for specific uses for in vitro fertilization
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish

Definitions

  • the present invention relates to a culture vessel and a culture apparatus provided with the vessel. More particularly, the present invention relates to a microdevice (culture container) and a culture apparatus particularly suitable for culturing fertilized eggs important in reproductive medicine or regenerative medicine.
  • a technology has been developed in which fertilized eggs (zygotes) are produced by in vitro fertilization of sperm and eggs of animals, particularly mammals, and cultured to the stage of cleavage, morula, blastula, and even late blastocysts. ing.
  • a technology for obtaining offspring by transplanting fertilized eggs in the cleavage cyst embryo stage to the uterus has been developed, used for breeding in livestock, and also applied to human infertility treatment. .
  • infertility treatment generally requires ovum collection (in vitro maturation requires in vitro maturation), in vitro fertilization, and the process of returning a fertilized egg into the uterus.
  • the most important process is the process of generating fertilized eggs up to the developmental stage (4-8 cell stage, blastocyst stage) suitable for transplantation.
  • a fertilized egg is cultured on a culture plate such as a petri dish, or a stationary culture in which a well of a culture plate is filled with a culture solution of around 5001, and the fertilized egg is allowed to stand there.
  • a culture plate such as a petri dish, or a stationary culture in which a well of a culture plate is filled with a culture solution of around 5001, and the fertilized egg is allowed to stand there.
  • place the culture solution micro droplets 20 1
  • the uterine tissue is polar, has a powerful layered structure such as intimal cells and stromal cells, and there is a flow of internal fluid in the lumen of the uterus and fallopian tube. ing. Therefore, the well on the plate Compared to the inside, there is a power gap in its physical and biological environment. After culturing fertilized eggs for 2 to 5 days after fertilization under conditions far from the environment in the body, the quality of the development stage suitable for transplantation is high (low fragmentation). ) Fertilization It is not easy to obtain eggs at high frequency. That is, a fertilized egg is expressed in the living body and loses its specific function if it is simply cultured under conditions apart from the in vivo environment such as a petri dish.
  • Patent Document 1 discloses a carrier co-cultured with a fertilized egg for the purpose of inducing three-dimensional development of the fertilized egg.
  • This carrier is obtained by co-culturing animal cells, matrix constituents such as collagen gel and matrigel, and a fibrous mesh body having an opening enough to form a space for three-dimensional culture. It is called a built-in three-dimensional tissue reconstruction body! It is described that fertilized eggs are cultured by placing and co-culturing fertilized eggs in the space of this carrier (cell-integrated three-dimensional tissue reconstructed body).
  • Patent Document 1 JP 2001-340076 A
  • Patent Document 2 Japanese Patent Laid-Open No. 2002-143753
  • An object of the present invention is to provide a simple method and apparatus capable of culturing and maturing a cell, particularly a fertilized egg, in an oviduct which is a reproductive organ, in an environmental condition close to the inside of the uterus.
  • the present invention provides a rectangular box-shaped culture vessel formed of a deformable material, and the culture vessel includes an upper bottom, a lower bottom, and a side wall standing from the entire periphery of the lower bottom.
  • a rectangular box-shaped sealed space is formed from the upper and lower bottoms and the side wall,
  • the upper bottom and the lower bottom are permeable to oxygen and carbon dioxide, and a pair of opposing side walls has an engaging portion for engaging with an extension device that extends the culture vessel in a uniaxial horizontal direction. Each is formed.
  • the culture vessel is a tube that connects the rectangular box-shaped sealed space and the outside so that cells can be introduced into the rectangular box-shaped sealed space and the culture medium can be perfused. And means for blocking the communication.
  • the height of the rectangular box-shaped sealed space of the culture vessel is 250 ⁇ m to lcm.
  • the present invention further provides a culture device comprising any one of the culture vessels described above and an extension device provided with a holding portion that engages with the engagement portion of the culture vessel.
  • the present invention also provides a method for culturing a fertilized egg, the method comprising:
  • the culture vessel is periodically extended in a uniaxial horizontal direction.
  • the culture vessel includes a tube capable of communicating the sealed space and the outside, and the somatic cell culture process and the fertilized egg culture process are performed. Then, the culture solution is perfused into the sealed space through the tube.
  • the culture vessel includes an upper bottom, a lower bottom, and side walls standing from the entire periphery of the lower bottom, and a rectangular box-shaped sealed space is formed from the upper and lower bottoms and the side walls. Bottom bottom 1S Oxygen and carbon dioxide permeable, and a pair of opposing side walls It is preferable that an engagement portion for engaging with an extension device for extending the nutrient container in the uniaxial horizontal direction is formed.
  • the mammalian somatic cell is an eclampsia intima cell.
  • any one of the above culture vessels is used, and the culture vessel includes a holding portion that engages with the engagement portion of the culture vessel. It is held in
  • a culture container and a culture device having a simple structure capable of providing an environment close to the in vivo environment suitable for culturing cells, particularly fertilized eggs.
  • the fertilized egg culturing apparatus and method of the present invention do not require the use of a complex three-dimensional structure for culturing fertilized eggs.
  • fertilized eggs can be cultured and matured in environmental conditions close to the inside of the reproductive organs, the oviduct, which is a reproductive organ, by culturing fertilized eggs while applying stress in the horizontal direction under perfusion conditions. It is possible. Therefore, the success rate of infertility treatment can be increased.
  • FIG. 1 is a schematic cross-sectional view of one embodiment of the culture vessel of the present invention.
  • FIG. 2 is a perspective view of one embodiment of the culture container of the present invention.
  • FIG. 3 is a schematic cross-sectional view of another embodiment of the culture vessel of the present invention.
  • FIG. 4 is a perspective view of one embodiment of the culture apparatus of the present invention.
  • FIG. 5 is a graph showing the change over time in the incidence of mouse 2-cell stage ovum strength and scutellum when co-cultured using various culture vessels.
  • FIG. 6 is a graph showing the total number of cells and the number of internal cell masses (ICM) constituting mouse blastocysts measured by double fluorescence staining when co-cultured using various culture vessels.
  • FIG. 7 is a graph showing the pregnancy rate and offspring production rate when blastocysts obtained by co-culture using various culture vessels are transplanted into the embryonic female uterus.
  • FIG. 8 is a graph showing changes over time in the incidence of mouse 2-cell stage ovum and blastocysts when cultured in various culture vessels.
  • FIG. 9 Mau measured by double fluorescence staining when cultured in various culture vessels.
  • FIG. 3 is a graph showing the total number of cells and the number of internal cell masses (ICM) constituting a blastocyst.
  • ICM internal cell masses
  • the culture vessel 100 has a rectangular box shape made of a deformable material.
  • the culture vessel 100 is erected from the entire periphery of the upper base 110, the lower base 120, and the lower base 120.
  • a side wall 130 is provided, and a rectangular box-shaped sealed space 140 is formed from the upper base 110, the lower base 120 and the side wall 130.
  • a sensor is attached to the rectangular box-shaped sealed space 140 of the culture vessel 100, and various indicators (temperature, osmotic pressure, pH, useful substances, harmful substances, etc.) indicating the state of the culture solution are measured and realized. It can be configured to monitor (display and record) in time.
  • FIG. 2 is a perspective view of the culture vessel 100 shown in FIG.
  • the pair of side walls 130a and 130b are configured to be thicker than the other side walls 130c and 130d. Engagement forces 150a, 150b and 150c, 150d are formed on the side walls 130a and 130b. Up to this engagement rod 150 ⁇ , it is connected to an extension device 200 described later. Therefore, in order to engage and extend the engaging portion 150 to the extension device 200, the side walls 130a and 130b need to be thicker than the side walls 103c and 130d.
  • the thickness of the side walls 130c and 130d is preferably 0.1 to 30 mm, more preferably 1 to 2 mm.
  • the culture vessel 100 may be a microfluidic device made of a deformable material.
  • deformable means that the box-shaped space can be expanded and contracted without breaking the shape at least against horizontal pulling or pressing.
  • a silicone elastomer is typically used as the deformable material.
  • the silicone elastomer include elastomers such as polydimethylsiloxane (PDMS) and dimethylsiloxane.
  • PDMS polydimethylsiloxane
  • other materials include polyhydroxyethyl methacrylate, poly (N-burpyrosadone), polydimethylacrylamide, polyglycerol methacrylate, and polybutyl acrylate.
  • the upper base 110, the lower base 120, and the side wall 130 may be formed of the same material.
  • a combination of these materials may be used.
  • the method for producing such a container is not particularly limited.
  • a concave member having an upper base 110 and a half-height side wall 130 and a concave member having a lower base 120 and a half-height side wall 130 are vertically moved so as to form a sealed space inside. It can be manufactured by pasting together. When pasting together, a silicone adhesive that does not peel off even when the container is repeatedly extended may be used. Alternatively, apply a suitable organic solvent to the adhesive surface The surface of the above material may be dissolved by applying to and adhered to each other.
  • the upper base 110 and the lower base 120 forming the rectangular box-shaped sealed space 140 of the culture vessel 100 are preferably in the form of a thin film from the viewpoint of the permeability of oxygen and carbon dioxide. More preferably, the thickness is 50 to 500 m, more preferably 100 to 200 m.
  • Sarako, upper base 110 and lower base 120 are preferably transparent from the viewpoint of observation of the fertilized egg culture process (eg, observation with an optical microscope).
  • Such materials include oxygen and carbon dioxide permeable silicone elastomers.
  • examples of the silicone elastomer include polydimethylsiloxane (PDMS) elastomer.
  • the rectangular box-shaped enclosed space 140 has a height of 100 m or more (indicated by “h” in FIG. 1), preferably about 100 to 200 ⁇ m. is required. This height should be determined in consideration of the size of the fertilized egg.
  • the surface of the lower bottom 120 on the side of the rectangular box-shaped sealed space 140 is preferably coated so that cultured cells are difficult to peel off.
  • an extracellular matrix is preferably used.
  • the extracellular matrix include fibronectin, collagen, gelatin, laminin and the like.
  • the culture vessel 100 may be provided with a tube 160 that communicates the rectangular box-shaped sealed space 140 with the outside.
  • FIG. 3 is a schematic cross-sectional view of a culture vessel 100 ′ including a tube 160 that communicates the rectangular box-shaped sealed space 140 with the outside.
  • the culture medium can be perfused through the tube 160.
  • a peristaltic pump or the like may be used to adjust the flow rate with a personal computer (PC).
  • sensors can be attached to measure various indicators (temperature, osmotic pressure, pH, amount of useful substances, amount of harmful substances, etc.) that indicate the state of the culture, and can be monitored (displayed and recorded) in real time.
  • the cells to be cultured can be introduced into the rectangular box-shaped sealed space 140 via the tube 160. Therefore, the inner diameter of the tube 160 is preferably larger than at least the diameter of the cell to be introduced. Preferably it is 130-150 ⁇ ⁇ .
  • the material of the tube is not particularly limited, and examples thereof include polycarbonate.
  • the inner diameter surface of the tube is coated with Teflon (registered trademark).
  • a means for blocking the communication may be provided so that the hermetic seal in the culture vessel 100 'can be maintained. Examples of such means include a cap 170 and a clip sandwiching the tube 160 as shown in FIG.
  • the culture device of the present invention is configured by engaging the culture vessel 100 with the extension device 200.
  • FIG. 4 shows a culture apparatus 300 of the present invention.
  • the structure of the extension device 200 is not particularly limited as long as it can apply stress in the horizontal direction.
  • the extension device 200 includes a fixed plate 210, a movable plate 220, a step motor 230, a control device 240, and a coupler 250 that connects the step motor 230 and the movable plate 220.
  • the engaging plate 260a and 260b are erected on the fixed plate 210! /.
  • Engagement pins 270a and 270b are installed upright on the movable plate 220.
  • engaging pins 260 and 270 respectively provided on the fixed plate 210 and the movable plate 220 are provided with the engaging portions 110a to 110d of the culture vessel 100 as through holes or holes provided to a predetermined height. By inserting into this through hole or hole, the culture vessel 100 is suspended between the fixed plate 210 and the movable plate 220 and is fixed to the extension device 200.
  • the distance between the movable plate 220 and the fixed plate 210 changes.
  • the culture vessel 100 having deformable material power extends in a uniaxial horizontal direction, and cells (somatic cells 500 and fertilized eggs 400) placed on the surface of the bottom 120 of the rectangular box-shaped sealed space 140 Stress is applied to the.
  • the extension cycle is controlled by the controller 240.
  • the cycle depends on the cells being cultured. Preferably, it can be controlled to be the same as the heart rate of the animal from which the cultured cells are derived.
  • the strength of extension is preferably controlled so that the size of cells in culture is about 1.2 times that of non-extended cells.
  • the means for suspending the culture vessel 100 and the extension device 200 is not limited to the above pin structure or the like.
  • the culture apparatus 300 of the present invention can be installed in a CO incubator to control temperature, humidity, and the like. It is preferable in terms of culture control.
  • the culture apparatus 300 installed in such a CO incubator is referred to as a culture unit in this specification.
  • the panel of the culture unit has sensor force installed in a rectangular box-shaped enclosed space 140, and various indicators that indicate the state of the culture solution (eg, temperature, dissolved oxygen, amount of useful substances, ammonia concentration, etc.) Is displayed, and based on this, the amount of culture medium perfused into the culture vessel 100 is controlled by a computer.
  • various indicators that indicate the state of the culture solution (eg, temperature, dissolved oxygen, amount of useful substances, ammonia concentration, etc.) is displayed, and based on this, the amount of culture medium perfused into the culture vessel 100 is controlled by a computer.
  • a culture vessel composed of a thin film having oxygen and carbon dioxide permeability while controlling the temperature, humidity, and flow rate of the culture solution can be used. It is possible to culture while applying. As a result, culture can be performed under environmental conditions closer to the body. For example, in the culture of a fertilized egg, the flow in the eclampsia and fallopian tube is simulated, and the maturation / development operation can be performed under environmental conditions close to the body.
  • the culture container and culture apparatus of the present invention described above can be used for culturing various cells.
  • Such cells may be primary cultured cells, established cells, and the like. Specific examples include vascular endothelial cells, smooth muscle cells, cardiomyocytes, chondrocytes, bone cells, endometrial cells, fallopian tube epithelial cells, fibroblasts, egg cells (including fertilized eggs) and the like. In particular, it is preferably used in the fertilized egg culturing method of the present invention described in detail below.
  • the animal species from which these cells are derived is not particularly limited, but cells derived from mammals are preferred.
  • the method for culturing a fertilized egg uses a culture container having a sealed space formed of a deformable material and co-cultures a fertilized egg with a somatic cell while periodically extending the culture container in a uniaxial horizontal direction. Is done. If necessary, a culture vessel in which the culture solution in the sealed space can be perfused may be used.
  • the culture vessel used here is not particularly limited as long as it satisfies the above-mentioned conditions, but the culture vessel of the present invention is preferably used.
  • co-culture refers to culturing the same or different cells simultaneously.
  • the fertilized egg culturing method of the present invention means that mammalian somatic cells and mammalian eggs are cultured simultaneously.
  • Genital organ-derived cells, fibroblasts, and the like that are not particularly limited are used as somatic cells derived from mammals used in the method of the present invention.
  • somatic cells derived from reproductive organs for example, eclampsia endometrial cells are used.
  • the somatic cell is not necessarily a cell derived from the same species of animal as the fertilized egg, but is preferably derived from the same species of animal, preferably the same animal from which the fertilized egg was collected.
  • Co-culture of reproductive organ-derived somatic cells (particularly endometrial cells) obtained from the same animal with fertilized eggs is most preferred.
  • a fertilized egg derived from a mammal is preferably used.
  • mammals include humans, monkeys, mice, horses, hidges, goats, baboons, pigs, dogs, cats, rabbits, guinea pigs, rats, mice and the like.
  • the fertilized egg used for the culture may be in any stage of zygote, cleavage, morula, and blastula, but an egg in a life cycle other than the fertilized egg (eg, an egg in the follicle, ovulation) Eggs, etc.) may be cultured in a culture vessel and fertilized for use. Further, a fertilized egg after freezing and thawing may be used.
  • the culture solution is not particularly limited as long as it can culture desired cells, preferably cells derived from the above mammals, more preferably fertilized eggs and eclampsia endometrial cells.
  • desired cells preferably cells derived from the above mammals, more preferably fertilized eggs and eclampsia endometrial cells.
  • media such as GI BCO ⁇ -MEM, Medicalt IVC, and Irvine HTF are used.
  • the culture solution may further contain a useful substance for culture.
  • a useful substance for culture For example, j8 mercaptoethanol is used to prevent generation inhibition due to peroxidation due to oxygen in the gas phase.
  • toxic substances that can be generated during culture can be monitored as described below. Examples of such harmful substances include ammonia.
  • Culture conditions generally employed by those skilled in the art for culturing fertilized eggs are employed.
  • the temperature is about 37-38 ° C
  • the osmotic pressure is about 280 mOsmZkg.
  • the flow rate of the culture solution can be appropriately set in consideration of various conditions.
  • the sealed space 140 of the culture container 100 is filled with a culture solution using, for example, an injection needle, and the sealed space 140 is filled with the culture solution. It is preferable that no gas remains in the sealed space 140.
  • somatic cell 500 derived from a mammal is placed on the bottom 120 of the sealed space 140 of the culture vessel 100. For example, the cells are introduced and seeded using an injection needle or the like, and somatic cells 500 are cultured (proliferated). From this culture time, the extension device 200 is driven to periodically extend the culture vessel 100 in the uniaxial horizontal direction.
  • the fertilized egg 400 is placed on the cultured somatic cell 500 using, for example, an injection needle, and the fertilized egg 400 is cultured.
  • the extension device 200 is driven to periodically extend the culture vessel 100 in the uniaxial horizontal direction.
  • the stretching cycle can be controlled to be the same as the heart rate of the animal from which the cultured cells are derived, as described above, and the strength of the stretching is the size of the cells in culture (somatic cells 500 and fertilized eggs 400). It is preferable that the length is controlled to be about 1.2 times that of the non-stretched state.
  • gas replacement during the culture can be performed through the upper base 110 and the lower base 120.
  • the fertilized egg 400 cultured and matured to the blastocyst stage can be removed by breaking the upper base 110 and then transplanted into an ovary.
  • an environment similar to the matured environment of the fertilized egg can be provided.
  • the culture vessel 100 When using the culture vessel 100 'having the tube 160 communicating with the rectangular box-shaped sealed space 140 and the outside, it is preferable to perfuse the culture solution through the tube 160 during the culture.
  • a sensor for various measurements is attached, and the perfusion rate is determined in consideration of the measured temperature, osmotic pressure, pH, harmful substances, etc., to make it closer to the in vivo environment. Can do.
  • These controls can be performed via a computer. Therefore, it becomes possible to three-dimensionally mature a fertilized egg extremely easily and efficiently compared to the conventional method.
  • mouse ovarian endometrial cells MEC
  • mouse 2-cell stage eggs Fertilized eggs
  • This culture vessel is formed of a PDMS elastomer.
  • the culture apparatus 300 shown in Fig. 2 is installed in an incubator at 37 ° C, and an injection needle is punctured into the culture container 100 so that the sealed space 140 is filled with the culture solution so that no gas phase remains. Satisfied. Cultivation As a nutrient solution, ⁇ -MEM manufactured by GIBCO was used. In addition, 50 M of
  • Mouse endometrial cells were collected from 6-week-old ICR female mice purchased from CLEA Japan in accordance with methods commonly used by those skilled in the art and used for the test.
  • the culture vessel 100 was punctured using an injection needle, and 100 to 1000 pieces were seeded on the lower base 120.
  • the stretching apparatus 200 was driven and cultured for 96 hours while applying uniaxial periodic stretching (20%, 1 Hz).
  • the mouse 2 cell stage ovum was used to administer hormones to 6-week-old ICR female mice purchased from CLEA Japan in the usual way by those skilled in the art to promote superovulation, and the 6-week-old C57BL strain from CLEA Japan was also used. Two-cell embryos were mated with males and used for testing. It was placed on the cultured MEC using an injection needle.
  • the stretcher 200 was driven and cultured for 96 hours while applying uniaxial periodic stretching (20%, 1 Hz).
  • CCI Cell culture insert
  • MEC membrane structure at the bottom of the housing
  • ovum mouse 2-cell stage ovum (fertilized egg) was placed and left stationary. Time co-cultured. After culture, the blastocyst was transplanted into the uterus of a recipient female mouse. The incidence of blastocyst, total cell number and ICM number, pregnancy rate, and offspring production rate are shown in Figures 5-7, respectively.
  • FIG. 5 is a graph showing the change over time in the incidence of mouse 2-cell stage ovum blastocysts in Example 1 and Comparative Examples 1 and 2 above.
  • the culture using the apparatus of the present invention (Example 1) is more effective in the case of the conventional static co-culture (Comparative Example 1) and on-membrane static co-culture (Comparative Example 2).
  • FIG. 6 shows the total number of cells and the number of inner cell masses (ICM) constituting the mouse blastocyst measured by the double fluorescent staining method. From this result, it is clear that the total number of cells and the number of ICM are significantly higher in Example 1 using the culture apparatus of the present invention than in Comparative Examples 1 and 2. It was.
  • FIG. 7 shows the results of the obtained blastocyst transplantation test, that is, the pregnancy rate and the offspring production rate when transplanted into a recipient embryo. According to FIG. 7, it is clear that both the pregnancy rate and the litter production rate are significantly higher when cocultured with the method of Example 1 than with Comparative Examples 1 and 2. became.
  • Example 1 Using the same culture apparatus used in Example 1 above, only mouse 2-cell stage ova (fertilized eggs) that were not co-cultured with somatic cells were cultured. That is, the culture apparatus 300 shown in FIG. 2 was installed in an incubator at 37 ° C., and an injection needle was inserted into the culture container 100 to fill the sealed space 140 with the culture solution so that no gas phase remained.
  • Figure 8 shows the time course of the incidence of blastocysts. In addition, the total number of cells and the number of ICMs constituting mouse blastocysts were counted by double fluorescent staining.
  • Figure 9 shows the result.
  • FIG. 8-10 show the incidence of blastocysts, the total number of cells and the number of inner cell masses (ICM), and the pregnancy rate and offspring production rate, respectively.
  • a cell culture insert (manufactured by Corning Co., Ltd.) having a membrane structure at the bottom of the rod, put 2 ml of the same culture solution as in Example 1 above on the membrane at the bottom of the CCI.
  • the mouse 2 cell stage ovum (fertilized egg) was allowed to stand and cultured for 96 hours. After culture, the blastocyst was transplanted into the uterus of a recipient female mouse.
  • the blastocyst incidence, total cell number and ICM number, pregnancy rate, and litter production rate are shown in Figure 8 ⁇ : LO, respectively.
  • FIG. 8 is a graph showing the change over time in the incidence from mouse 2-cell stage ovum to blastocyst in Reference Example 1 and Reference Comparative Examples 1 and 2.
  • the culture using the apparatus of the present invention (Reference Example 1) is more effective in the case of conventional static culture (Reference Comparative Example 1) and on-membrane static culture (Reference Comparative Example 2).
  • Reference Comparative Example 1 the culture using the apparatus of the present invention
  • Reference Comparative Example 2 the culture using the apparatus of the present invention
  • FIG. 9 shows the total number of cells and the number of ICMs constituting the mouse blastocyst. From this result, it is clear that both the number of total cells and the number of ICM are significantly higher in Reference Example 1 using the culture apparatus of the present invention than in Reference Comparative Examples 1 and 2. became.
  • FIG. 10 shows the pregnancy rate and the offspring production rate when transplanted into embryonic female pupae.
  • Fig 10 According to the results, in both Reference Example 1, Reference Comparative Example 1 and Reference Comparative Example 2, both the pregnancy rate and offspring production rate resulted in the transfer of embryos that reached the blastocyst into the uterus of the recipient mouse. It was clear that both the production rate and the litter production rate were significantly higher when cultured by the method in Reference Example 1 than in Reference Comparative Examples 1 and 2.
  • Example 1 which is a co-culture system with somatic cells showed better pregnancy rate and offspring production rate. Therefore, it is considered that fertilized eggs can further increase the implantation rate and the birth rate when transplanted into the body by co-culture with somatic cells.
  • Reference Example 1 were inferior to the co-culture system of Example 1 above, an increase in the incidence of mouse eggs was confirmed. This result shows that even in a system without co-culture, the ovum generation ability can be enhanced by combining the perfusion of the present invention with the periodic extension of the culture vessel.
  • a culture vessel, a culture device, and a culture method suitable for culturing a fertilized egg are provided.
  • the culture container, culture apparatus, and culture method of the present invention can be used for recovery culture after freezing and thawing only by culturing fertilized eggs, establishment of embryonic stem cells, and analysis of the developmental mechanism of mammals after implantation (for artificial uterus). It can also be used as a new technique for development). In particular, it is extremely useful in fields such as reproductive medicine and regenerative medicine including infertility treatment. Moreover, it can be used for these new applications by changing the size Z scale of the culture vessel and the culture apparatus.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un appareil destiné à cultiver efficacement un œuf fécondé ainsi qu’un procédé de culture d’œuf fécondé. Le récipient de culture est notamment une boîte de forme rectangulaire, fabriquée en matériau déformable. Dans ledit récipient de culture, qui comprend une face supérieure, une face inférieure et des parois latérales s’étendant à la verticale à partir de chaque côté de la face inférieure, un espace clos en forme de boîte rectangulaire est formé par les faces inférieure et supérieure et par les parois latérales, les faces inférieure et supérieure sont perméables à l’oxygène et au dioxyde de carbone et deux parois latérales opposées sont munies de parties d’engagement permettant l’engagement du récipient de culture à une unité d’extension destinée à étendre le récipient de culture de manière uniaxiale, dans la direction horizontale. Il est possible de cultiver un œuf fécondé de manière très efficace en utilisant ledit récipient de culture, en plaçant les cellules végétatives provenant d’un mammifère et l’œuf fécondé sur la face inférieure de l’espace clos rempli de liquide de culture, puis en réalisant une co-culture tout en étendant périodiquement le récipient de culture de manière uniaxiale dans la direction horizontale. L’œuf fécondé obtenu grâce à ce procédé de culture présente un taux d’implantation extrêmement élevé.
PCT/JP2006/321744 2005-10-31 2006-10-31 Recipient de culture et appareil de culture WO2007052653A1 (fr)

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WO2008123508A1 (fr) * 2007-03-30 2008-10-16 Strex Incorporation Procédé d'incubation d'œuf fertilisé et appareil d'incubation d'œuf fertilisé
JP2009027973A (ja) * 2007-07-26 2009-02-12 Okayama Univ 卵細胞の培養装置及びその方法
JP2009055816A (ja) * 2007-08-30 2009-03-19 National Institute Of Advanced Industrial & Technology マイクロ流体チップ装置、マイクロ流体チップおよびこれに用いる流体送入用チューブ並びに試料導入導出方法
WO2012147878A1 (fr) * 2011-04-27 2012-11-01 株式会社メニコン Récipient de culture cellulaire
JP2013255494A (ja) * 2012-06-13 2013-12-26 sheng-nan Chang 生体力学的負荷測定用容器
JP2015216886A (ja) * 2014-05-19 2015-12-07 横河電機株式会社 細胞培養制御システム及び細胞培養制御方法
WO2016117281A1 (fr) * 2015-01-21 2016-07-28 東洋製罐グループホールディングス株式会社 Récipient de culture cellulaire, procédé de culture cellulaire et procédé d'utilisation du récipient de culture cellulaire
KR101932414B1 (ko) * 2017-03-24 2018-12-27 계명대학교 산학협력단 수정란의 메타볼리즘 측정장치 및 그 측정장치를 이용한 수정란의 메타볼리즘 측정방법
KR101961025B1 (ko) * 2017-09-15 2019-03-21 계명대학교 산학협력단 수정란 배양장치의 미세패턴 형성방법
WO2019054835A3 (fr) * 2017-09-15 2019-06-06 계명대학교 산학협력단 Appareil de culture d'œuf fertilisé qui imite la trompe de fallope et procédé de fabrication dudit appareil
KR102005807B1 (ko) * 2018-06-25 2019-07-31 단국대학교 산학협력단 난자 성장 장치 및 이를 이용한 난자 성장 방법
WO2022123220A1 (fr) * 2020-12-11 2022-06-16 Verso Biosense Group Limited Dispositif de commande de système de culture cellulaire
WO2023172887A1 (fr) 2022-03-07 2023-09-14 Deka Products Limited Partnership Systèmes de bioréacteurs de maturation de tissu à distribution de cellules uniforme
EP4174163A4 (fr) * 2020-07-28 2024-01-24 University of Yamanashi Appareil de culture d'oeufs congelés et procédé de culture d'oeufs congelés

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008271964A (ja) * 2007-03-30 2008-11-13 Strex Inc 受精卵の培養方法及び受精卵の培養装置
WO2008123508A1 (fr) * 2007-03-30 2008-10-16 Strex Incorporation Procédé d'incubation d'œuf fertilisé et appareil d'incubation d'œuf fertilisé
JP2009027973A (ja) * 2007-07-26 2009-02-12 Okayama Univ 卵細胞の培養装置及びその方法
JP2009055816A (ja) * 2007-08-30 2009-03-19 National Institute Of Advanced Industrial & Technology マイクロ流体チップ装置、マイクロ流体チップおよびこれに用いる流体送入用チューブ並びに試料導入導出方法
WO2012147878A1 (fr) * 2011-04-27 2012-11-01 株式会社メニコン Récipient de culture cellulaire
JPWO2012147878A1 (ja) * 2011-04-27 2014-07-28 株式会社メニコン 細胞培養器
JP6074860B2 (ja) * 2011-04-27 2017-02-08 国立大学法人 岡山大学 細胞培養器
JP2013255494A (ja) * 2012-06-13 2013-12-26 sheng-nan Chang 生体力学的負荷測定用容器
US9783774B2 (en) 2014-05-19 2017-10-10 Yokogawa Electric Corporation Cell culture control system, cell culture control method, and non-transitory computer readable storage medium
JP2015216886A (ja) * 2014-05-19 2015-12-07 横河電機株式会社 細胞培養制御システム及び細胞培養制御方法
WO2016117281A1 (fr) * 2015-01-21 2016-07-28 東洋製罐グループホールディングス株式会社 Récipient de culture cellulaire, procédé de culture cellulaire et procédé d'utilisation du récipient de culture cellulaire
KR101932414B1 (ko) * 2017-03-24 2018-12-27 계명대학교 산학협력단 수정란의 메타볼리즘 측정장치 및 그 측정장치를 이용한 수정란의 메타볼리즘 측정방법
KR101961025B1 (ko) * 2017-09-15 2019-03-21 계명대학교 산학협력단 수정란 배양장치의 미세패턴 형성방법
WO2019054835A3 (fr) * 2017-09-15 2019-06-06 계명대학교 산학협력단 Appareil de culture d'œuf fertilisé qui imite la trompe de fallope et procédé de fabrication dudit appareil
KR102005807B1 (ko) * 2018-06-25 2019-07-31 단국대학교 산학협력단 난자 성장 장치 및 이를 이용한 난자 성장 방법
EP4174163A4 (fr) * 2020-07-28 2024-01-24 University of Yamanashi Appareil de culture d'oeufs congelés et procédé de culture d'oeufs congelés
WO2022123220A1 (fr) * 2020-12-11 2022-06-16 Verso Biosense Group Limited Dispositif de commande de système de culture cellulaire
WO2023172887A1 (fr) 2022-03-07 2023-09-14 Deka Products Limited Partnership Systèmes de bioréacteurs de maturation de tissu à distribution de cellules uniforme

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