WO2022045854A1 - Appareil de culture cellulaire pour la production de viande cultivée - Google Patents

Appareil de culture cellulaire pour la production de viande cultivée Download PDF

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WO2022045854A1
WO2022045854A1 PCT/KR2021/011613 KR2021011613W WO2022045854A1 WO 2022045854 A1 WO2022045854 A1 WO 2022045854A1 KR 2021011613 W KR2021011613 W KR 2021011613W WO 2022045854 A1 WO2022045854 A1 WO 2022045854A1
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canister
cell
cell culture
culture apparatus
differentiation
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PCT/KR2021/011613
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English (en)
Korean (ko)
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이희재
금준호
손예빈
조성천
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주식회사 씨위드
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Publication of WO2022045854A1 publication Critical patent/WO2022045854A1/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
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • C12M23/06—Tubular
    • 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
    • C12M23/26—Constructional details, e.g. recesses, hinges flexible
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • 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
    • C12M35/06—Magnetic means
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
    • 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
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts

Definitions

  • the present invention relates to a cell culture apparatus for producing cultured meat and a cultured meat production system including the apparatus.
  • Substitute meat (replacement meat, artificial meat) can be divided into plant meat made with plant materials such as soybeans, wheat, and rice to taste meat, and cultured meat (cultured meat) made by culturing animal cells.
  • Cultured meat refers to edible meat obtained by harvesting cells from living animals and then proliferating the cells using cell engineering technology, a field of cellular agriculture that obtains meat without raising livestock it can be said
  • Cultured meat production is a complex process that requires consideration of various engineering considerations, such as cells, scaffolds, media and growth factors, and bioreactors.
  • the dual bioreactor supplies oxygen and medium so that the cells attached to the scaffold can grow into cultured meat, the final product, and removes wastes.
  • a rotating bioreactor is often used, but various bioreactors may be used depending on the type of cultured meat to be produced, such as a tank stirring type or a stationary culture type.
  • the technology for producing cultured meat having the taste and texture of general meat is still lacking.
  • An object of the present invention is to provide an ingestible seaweed-derived cell culture support sheet, a cell culture apparatus using a plurality of cell culture support fibers, and a cultured meat production system using the cell culture apparatus.
  • an aspect of the present invention provides a cell culture device including a cell division canister, a cell differentiation canister, a canister fixed end, and a housing.
  • the cell division canister includes a multi-layered cell culture support sheet, an outer shell surrounding the support sheet to form a space separated from the outside, and a multi-channel tube connected to the outer shell.
  • the cell differentiation canister includes a plurality of cell culture support fibers aligned in one direction, an outer shell surrounding the support fibers to form a space separated from the outside, and a multi-channel tube connected to the outer shell.
  • the fixed end of the canister is connected to both ends of the cell differentiation canister to fix the cell differentiation canister, and can move up and down along the moving part of the fixed end of the canister.
  • the housing can be opened and closed, and a cell division canister and a cell division canister are positioned therein, and fixed end moving parts of the canister are installed on both walls.
  • the cell culture apparatus cultured cells selected from the group consisting of embryonic stem cells, muscle stem cells, mesenchymal stem cells, induced pluripotent stem cells, myoblasts and fibroblasts, and finally can produce muscle tissue.
  • the multi-channel tube may include a medium supply tube, a medium recovery tube, and a gas supply tube.
  • the cell differentiation canister may further include a coil for applying an electric field surrounding the canister, and the coil for applying the electric field may periodically apply an electric field of 30 to 90 mV simulating an action potential of a muscle.
  • the cell culture support sheet and the cell culture support fiber have a hydrogel structure containing alginate and cellulose left after removing the cell components from the medulla of seaweed, and the seaweed may include at least one of seaweed, kelp, and shiitake. there is.
  • the cell division canister and the cell differentiation canister may further include sensors for measuring the glucose concentration and pH of the medium.
  • the cell culture apparatus may further include a controller that adjusts the temperature and carbon dioxide concentration in the housing and/or an actuator that moves the fixed end of the canister.
  • the cell differentiation canister contracts and relaxes by the movement of the fixed end of the canister, and the outer shells of the two canisters are made of a polymer material that can be contracted and relaxed so as not to damage the outer shell of the canister in the process, and the tensile strength is 700 % or more, and the elongation may be 20 kN/m or more.
  • Another aspect of the present invention provides a cultured meat production system comprising the following configuration:
  • At least one cell medium supply unit connected to the multi-channel tube of the cell culture device;
  • a cell medium recovery unit connected to the multi-channel tube of the cell culture device
  • a gas mixing unit connected to the multi-channel tube of the cell culture apparatus and including a gas tank and a mixer;
  • one or more peristaltic pumps connected to the cell medium supply unit and the cell medium recovery unit;
  • a microcontroller that controls the operation of the cell culture device, peristaltic pump, and gas mixing section.
  • the cell medium supply unit may include a division medium supply unit and a differentiation medium supply unit.
  • the cell culture apparatus of the present invention uses a cell culture support derived from seaweed that can be ingested, and has the advantage of being able to apply physical and electrical stimulation necessary for differentiation into muscle tissue to cells to be cultured.
  • FIG. 1 schematically shows the structure of a cell culture apparatus including a cell division canister, a cell differentiation canister, a canister fixed end, and a housing.
  • FIG. 2 shows a state in which a cell culture support sheet is formed in multiple layers in a cell division canister (A) and the overall structure of the canister (B).
  • FIG 3 shows a state in which a plurality of cell culture support fibers are aligned in one direction in a cell differentiation canister (A) and the overall structure of the canister (B).
  • FIG. 4 shows a view of a cell differentiation canister connected to a fixed end of the canister.
  • FIG. 5 shows a process of seeding cells in a cell differentiation canister and then differentiating them into muscle tissue through physical and electrical stimulation.
  • FIG. 6 shows a cultured meat production system according to an example of the present invention.
  • the embodiments of the present invention specifically represent ideal embodiments of the present invention. As a result, various modifications of the drawings are expected. Accordingly, the embodiment is not limited to a specific shape of the illustrated area, and includes, for example, a shape modification by manufacturing.
  • the cell culture apparatus 100 of the present invention includes a cell division canister 110 , a cell differentiation canister 120 , a canister fixing end 130 , and a housing 140 .
  • the cell culture apparatus 100 may be used for culturing cells selected from the group consisting of embryonic stem cells, muscle stem cells, mesenchymal stem cells, induced pluripotent stem cells, myoblasts and fibroblasts, and finally the cells After culturing, it can be differentiated to form muscle tissue (cultured meat).
  • the cell division canister 110 will be described with reference to FIGS. 2 and 6 .
  • the cell division canister 110 includes a multi-layered cell culture support sheet 111 , a shell 112 surrounding the support sheet to form a space separated from the outside, and a multi-channel tube 113 connected to the shell.
  • a plurality of cell division canisters 110 may be included in the housing.
  • the cell division canister 110 performs a function of proliferating the seeded cells.
  • Cell division medium is supplied using the medium supply tube 113-1, which is one of the multi-channel tubes 113, and the cells are seeded on the multi-layered cell culture support sheet 111, followed by general cell culture conditions (37° C., Cells can be cultured in 5% CO 2 ).
  • general cell culture conditions 37° C., Cells can be cultured in 5% CO 2 .
  • FIG. 2 it can be seen that the cell culture support sheet of the cell division canister is multi-layered (A) and the overall structure (B).
  • the multi-layered cell culture support sheet 111 may be used as at least one layer or more, and may include clip-type fixing devices 111-1 at both ends of the sheet so that the multi-layer structure can be maintained.
  • the cell division canister may further include a sensor 114 for measuring the glucose concentration and pH of the medium, and when the sensor detects a decrease in the glucose concentration and pH of the medium, one of the multi-channel tubes 113
  • the old medium may be removed through the phosphorus medium recovery tube 113-2, and the fresh cell division medium may be supplied through the medium supply tube 113-1.
  • the cell division canister 110 may use one of the multi-channel tubes 113 as the gas supply tube 113-3 to receive nitrogen, carbon dioxide, and oxygen necessary for cell culture.
  • the multi-channel tube 113 of the cell division canister 110 includes a medium supply tube 113-1, a medium recovery tube 113-2, and a gas supply tube 113-3.
  • Each of the medium supply tube 113 - 1 , the medium recovery tube 113 - 2 , and the gas supply tube 113 - 3 is connected to the division medium supply unit, the medium recovery unit, and the gas mixing unit outside the housing.
  • the cells proliferated in the cell division canister 110 are recovered and re-seeded in the cell division canister 120 .
  • the cell differentiation canister 120 will be described with reference to FIGS. 3 and 6 .
  • the cell differentiation canister 120 includes a plurality of cell culture support fibers 121 aligned in one direction, an outer shell 122 surrounding the support fibers to form a space separated from the outside, and a multi-channel tube 123 connected to the outer shell. includes A plurality of cell differentiation canisters 120 may be included in the housing.
  • FIG. 3 it can be seen that a plurality of cell culture support fibers are aligned in one direction in the cell differentiation canister (A) and the overall structure of the canister (B).
  • the cell differentiation canister 120 performs a function of differentiating the seeded cells into muscle tissue. After supplying the cell differentiation medium to the medium supply tube 123-1, which is one of the multi-channel tubes 123, and seeding the cells on a plurality of cell culture support fibers 121, general cell culture conditions (37° C., 5% The cells can be cultured in CO 2 ). At this time, differentiation promoting factors such as insulin and dexamethasone may be added to the cell differentiation medium for efficient cell differentiation.
  • the cell differentiation canister 120 further includes a fixing device 125, for example, a fixing device such as a cap or a clip, that collects a plurality of cell culture support fibers 121 existing inside the outer shell so as not to separate them. can do.
  • a fixing device for example, a fixing device such as a cap or a clip
  • the cylindrical cell differentiation canister 120 can be connected to the multi-channel tube while fixing the cell culture support fiber 121 through the fixing device connected to the outer skin.
  • the fixing device may be in the form of a cap, for example, a structure that is detachable, such as a bottle cap.
  • the cell division canister 120 may further include a sensor 124 for measuring the glucose concentration and pH of the medium, and when the sensor detects a decrease in the glucose concentration and pH of the medium, the multi-channel tube 123 ), the old medium may be removed through the medium recovery tube 123-2, and a fresh cell differentiation medium may be supplied through the medium supply tube 123-1.
  • the cell division canister may use one of the multi-channel tubes 123 as the gas supply tube 123-3 to receive nitrogen, carbon dioxide, and oxygen necessary for cell culture.
  • the multi-channel tube 123 of the cell differentiation canister 120 includes a medium supply tube 123-1, a medium recovery tube 123-2, and a gas supply tube 123-3.
  • the medium supply tube 123-1 and the gas supply tube 123-3 may be connected to the fixing device 125 at the top of the cell differentiation canister 120, and the medium recovery tube 123- 2) may be connected to the fixing device 125 at the bottom of the cell differentiation canister 120 .
  • the cell differentiation canister 120 may further include a coil 124 for applying an electric field surrounding the canister, and the coil 124 for applying the electric field generates an electric field of 30 to 90 mV simulating an action potential of a muscle. It can be applied periodically to the cell differentiation canister. Periodically applying the electric field means applying the electric field of the above intensity for a predetermined time at regular time intervals.
  • the electric field application coil 124 is controlled by a microcontroller (MCU) of the cultured meat production system, and receives power.
  • MCU microcontroller
  • the cell differentiation canister 120 may contract and relax to promote cell differentiation, and for this, the cell culture apparatus includes a canister fixing end 130 .
  • An actuator 150 may be additionally included in order to move the fixed end of the canister.
  • the canister fixed end 130 is connected to both ends of the cell differentiation canister or the fixing device 125 to fix the canister, and can move up and down along the canister fixed end moving part 141 .
  • FIG. 5 shows the process of seeding cells in the cell differentiation canister and then differentiating them into muscle tissue through physical and electrical stimulation. indicates.
  • the cell culture support has a hydrogel structure including alginate and cellulose left after removing the cellular components from the medullary part of the seaweed, and the seaweed is seaweed, kelp, and seaweed.
  • the seaweed is seaweed, kelp, and seaweed.
  • the method for preparing the cell culture support includes a seaweed pretreatment step, an SDS treatment step, a cortex separation step, a PBS washing step, a gelation step, a freeze drying step, a sterilization step, and a manufacturing step.
  • the seaweed pretreatment step includes an ozone treatment step and a cutting step as a pretreatment step for inducing a decellularization process of seaweed individuals to be treated.
  • the pretreatment solution is a solution to properly swell the tissue of the seaweed, and it is produced by dissolving 1-3% (w/v) sodium chloride (NaCl) in distilled water, and preferably the concentration of sodium chloride is 1-2% (w/v). ) to make and use a pretreatment solution.
  • the ozone treatment step is a process of immersing a seaweed object in a pretreatment solution and performing ozone treatment to sterilize microorganisms present on the surface of the seaweed object. At this time, ozone is irradiated for about 1 minute to 1 minute and 30 seconds.
  • the cutting step is a process of cutting the seaweed individual into a desired size, and the shape and size of the cutting may vary depending on the purpose of the finally manufactured cell culture support.
  • the step of treating SDS is a process of weakening the cortex tissue of seaweed by immersing the cut seaweed in an aqueous SDS solution.
  • concentration of the SDS aqueous solution used is usually maintained in the range of 1 to 5% (w/v), and when immersing seaweed individuals, an SDS aqueous solution having a concentration of preferably 1 to 2% (w/v) is used. .
  • the cortex separation step is a process for separating the cortex layer of seaweed after treatment with an anionic detergent, and is carried out while slowly shaking the SDS solution in which the seaweed is immersed for 15 to 30 minutes. Through this process, the densely arranged cortical part and the medullar layer of the loose cellulose structure of the seaweed individual are separated from each other.
  • the phosphate buffered saline (PBS) washing step is a process of removing the cortex in the cortical part separation step and washing the remaining medulla with PBS
  • the gelation step is a process of gelling the remaining medulla after washing with PBS.
  • the immersion step is a process of immersing the water quality part in an aqueous solution of calcium chloride (CaCl2) in a concentration range of 0.5 to 10% (w/v) for about 1 to 10 minutes.
  • the treatment concentration and treatment time depend on the type of cell culture support and the type of cells to be cultured. can be changed. Through this process, Ca 2+ ions in aqueous solution are used for cross-linking of alginate, and the stiffness of the gel varies according to the degree of cross-linking.
  • the washing step is a process of taking out the water quality part after immersion and washing with PBS.
  • the water quality part becomes a transparent gel state. Since this gel has a unique network structure of seaweed, it is possible to make a sponge-type cell culture support sheet by freeze-drying the gel.
  • Cell culture support fibers can be prepared by cutting a freeze-dried support sheet to a predetermined length.
  • each shell is made of a polymer, and is a material that can contract and relax.
  • a polymer having a tensile strength of 700% or more and an elongation of 20 kN/m or more may be used. there is.
  • a medical polymer having excellent contractility and elongation ability may be used, and examples of the medical polymer are described in Table 1 below.
  • the housing 140 has a cell division canister and a cell differentiation canister positioned therein, and canister fixed end moving parts 141 are installed on both walls.
  • the housing 140 since the housing 140 has to maintain the inside of the housing in a constant environment for cell culture and differentiation, it is possible to create a closed environment with the outside through an openable and openable door.
  • the cell culture apparatus 100 may further include a controller 160 for controlling the temperature and carbon dioxide concentration inside the housing.
  • the cultured meat production system of the present invention includes a cell culture apparatus 100; At least one cell medium supply unit 200 connected to the cell division canister and the multi-channel tube of the cell differentiation canister in the cell culture apparatus; a cell medium recovery unit 300 connected to the cell division canister and the multi-channel tube of the cell differentiation canister in the cell culture apparatus; a gas mixing unit 400 connected to the cell division canister and the multi-channel tube of the cell differentiation canister in the cell culture apparatus, and including a gas tank and a mixer; One or more peristaltic pumps 500 connected to the cell medium supply unit and the cell medium recovery unit; and a microcontroller 600 for controlling the operation of the cell culture device, the peristaltic pump, and the gas mixing unit.
  • the cell medium supply unit 200 is connected to the cell division canister and the multi-channel tubes 113 and 123 of the cell differentiation canister in the cell culture apparatus, and specifically is connected to the medium supply tubes 113-1 and 123-1. Meanwhile, the cell medium supply unit may include a division medium supply unit 210 and a differentiation medium supply unit 220 .
  • the cell medium recovery unit 300 is connected to the cell division canister and the multi-channel tubes 113 and 123 of the cell differentiation canister in the cell culture apparatus, and specifically is connected to the medium recovery tubes 113-2 and 123-2. .
  • the gas mixing unit 400 is connected to the cell division canister and the multi-channel tubes 113 and 123 of the cell differentiation canister in the cell culture apparatus, and specifically is connected to the gas supply tubes 113-3 and 123-3. do.
  • the gas mixing unit 400 includes a gas tank and a mixer.
  • a medium is supplied to the canister for cell division, and animal stem cells (eg, muscle stem cells) derived from mammals are seeded on the cell culture support. After the seeded cells are proliferated for a certain period of time to the target number of cells, the support itself after cell culture is completed is taken out of the canister.
  • animal stem cells eg, muscle stem cells
  • the cells proliferating on the cell culture support are manually separated.
  • the alginate component of the cell culture support is dissolved, and the cells are separated by treatment with trypsin.
  • the separated cells are seeded again in a canister for cell differentiation, and differentiation is induced for a certain period of time by applying physical, electrical, and chemical stimuli.
  • the differentiated cultured meat (cell culture support + cultured muscle tissue) deposited on the cell culture support by deposition from the cell differentiation canister is taken out, processed/cooked, and commercialized into cultured meat.

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Abstract

La présente invention concerne un appareil de culture cellulaire pour la production de viande cultivée et un système de production de viande cultivée comprenant l'appareil, l'appareil de culture cellulaire utilisant un support de culture cellulaire comestible dérivé d'algues, et ayant l'avantage de pouvoir appliquer des stimulations physiques et électriques nécessaires pour différencier des cellules cultivées en tissu musculaire.
PCT/KR2021/011613 2020-08-28 2021-08-30 Appareil de culture cellulaire pour la production de viande cultivée WO2022045854A1 (fr)

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KR10-2020-0109231 2020-08-28

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050006147A (ko) * 2002-04-08 2005-01-15 밀레늄 바이올로직스 인코포레이티드 자동화된 조직 엔지니어링 시스템
KR100824357B1 (ko) * 2006-12-26 2008-04-23 주식회사 바이오트론 소형 생물반응기
US20170260489A1 (en) * 2014-09-17 2017-09-14 Toyo Seikan Group Holdings, Ltd. Cell culture apparatus
CN108860966A (zh) * 2018-05-09 2018-11-23 汤玉 一种干细胞运输容器

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Publication number Priority date Publication date Assignee Title
US7662615B2 (en) * 2004-07-27 2010-02-16 Chung Yuan Christian University System and method for cultivating cells
BR112013025086A2 (pt) * 2011-03-29 2016-08-23 Yongxin Zhang sistema e métodos de biorreator multifuncional para classificação e cultura de células

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
KR20050006147A (ko) * 2002-04-08 2005-01-15 밀레늄 바이올로직스 인코포레이티드 자동화된 조직 엔지니어링 시스템
KR100824357B1 (ko) * 2006-12-26 2008-04-23 주식회사 바이오트론 소형 생물반응기
US20170260489A1 (en) * 2014-09-17 2017-09-14 Toyo Seikan Group Holdings, Ltd. Cell culture apparatus
CN108860966A (zh) * 2018-05-09 2018-11-23 汤玉 一种干细胞运输容器

Non-Patent Citations (1)

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Title
TOM BEN-ARYE, LEVENBERG SHULAMIT: "Tissue Engineering for Clean Meat Production", FRONTIERS IN SUSTAINABLE FOOD SYSTEMS, vol. 3, pages 46, XP055762193, DOI: 10.3389/fsufs.2019.00046 *

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KR20220029490A (ko) 2022-03-08

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