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WO2023225995A1 - Utilisation d'un échafaudage 3d à base d'alginate de sodium et de gélatine pour favoriser la différenciation des préadipocytes - Google Patents

Utilisation d'un échafaudage 3d à base d'alginate de sodium et de gélatine pour favoriser la différenciation des préadipocytes Download PDF

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WO2023225995A1
WO2023225995A1 PCT/CN2022/095563 CN2022095563W WO2023225995A1 WO 2023225995 A1 WO2023225995 A1 WO 2023225995A1 CN 2022095563 W CN2022095563 W CN 2022095563W WO 2023225995 A1 WO2023225995 A1 WO 2023225995A1
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differentiation
scaffold
gelatin
sodium alginate
medium
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Chinese (zh)
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郑勇
赖良学
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Shantou Debao Investment Co Ltd
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Shantou Debao Investment Co Ltd
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Priority to CN202280002824.5A priority Critical patent/CN115151634B/zh
Priority to PCT/CN2022/095563 priority patent/WO2023225995A1/fr
Publication of WO2023225995A1 publication Critical patent/WO2023225995A1/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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/256—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seaweeds, e.g. alginates, agar or carrageenan
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • A23L29/281—Proteins, e.g. gelatin or collagen
    • A23L29/284—Gelatin; Collagen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/73—Hydrolases (EC 3.)
    • 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
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate
    • 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
    • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment
    • C12N2537/10—Cross-linking

Definitions

  • This application belongs to the field of future food technology and specifically relates to the application of a sodium alginate-gelatin 3D scaffold in supporting the differentiation of adipose precursor cells.
  • animal meat occupies a large proportion of the world's dining tables.
  • the growing global demand for animal protein or animal meat poses a great challenge to traditional animal husbandry and even the global ecology.
  • relevant fields around the world have begun to devote themselves to research on cell-based meat, and gradually formed the main line of development of cell-cultured meat.
  • Cell cultured meat uses cells as the starting point and end point to "obtain high-quality primary cells", “edit and process cells to obtain seed cells that can maintain the ability to divide and differentiate for a long time”, “expand cells on a large scale and combine them with other tissues” Or the combination of substances to form meat products with a taste and flavor consistent with real animal meat” is carried out in three stages.
  • the technology includes harvesting stem cells with the ability to differentiate into muscle cells from different species, including induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs) and related unipotent stem cells, etc.; the resulting cells are modified with gene editing or small molecules Stable "seed cells” are formed through induction and other methods; using the seed cells as a starting point, the number of cells is expanded on a large scale through a cell fermentation tank; under a certain number of cells, cells with muscle texture and rich animal protein are obtained through differentiation induction. Meat.
  • hydrogels whose main components are gelatin or sodium alginate are widely used. Such hydrogel components have been widely used in the research and development of organoids, cell culture scaffolds, wound dressings, cultured meat, etc.
  • the working principle of hydrogel is to provide an environment for cultivating cells in vitro under the premise of simulating the physical and chemical environment and spatial structure of cells. In this environment, cells can still maintain their important physiological activities and functions, providing a good environment for cells. Be prepared to play a role in different directions later.
  • adipocytes as an indispensable ingredient in animal meat in terms of nutrition and flavor, has also attracted much attention in cell-based meat.
  • the growth and differentiation of adipocytes obtained from different species in vitro are quite stable. Therefore, the focus is on how to differentiate the adipocytes more efficiently into intracellular lipid droplets with a certain size and number. of mature adipocytes.
  • CN113768138A discloses a method for manufacturing an edible gellan gum/gelatin 3D scaffold for cell cultured meat. This method can be directly used for cell culture without freeze-drying. Without using toxic cross-linking agents, A 3D scaffold is formed through the electrostatic interaction between gellan gum and gelatin. The resulting gellan gum/gelatin 3D scaffold has good cell adhesion and compatibility and can be used for skeletal muscle cell culture. However, this scaffold cannot be used for Supports adipocyte precursor cell differentiation.
  • CN113208059A discloses a method for manufacturing an edible pectin/chitosan/collagen 3D scaffold for cell cultured meat.
  • the method is achieved by combining a certain concentration of pectin (high methoxy pectin and low methoxy pectin Pectin), chitosan and collagen were evenly mixed according to a certain proportion, fully reacted at room temperature for more than 2 hours, frozen overnight, and then freeze-dried to obtain a pectin/chitosan/collagen 3D scaffold.
  • This invention forms a 3D scaffold through the electrostatic interaction between pectin, chitosan and collagen.
  • the resulting pectin/chitosan/collagen 3D scaffold has good cell adhesion and compatibility.
  • the Scaffolds cannot be used to support adipocyte precursor cell differentiation.
  • This application provides the application of a sodium alginate-gelatin 3D scaffold in supporting the differentiation of adipose precursor cells.
  • the present application provides a hydrogel scaffold that supports the differentiation of adipocyte precursor cells, and also includes a method for performing differentiation of adipocyte precursor cells into intracellular lipid droplets of different sizes on the obtained scaffold.
  • this application provides the application of a sodium alginate-gelatin 3D scaffold in supporting the differentiation of adipocyte precursor cells.
  • This application provides a sodium alginate-gelatin 3D scaffold and finds that it can be used as a hydrogel scaffold to support the differentiation of adipocytes. It also includes performing differentiation of adipocyte cells into intracellular cells of different sizes on the obtained scaffold. Lipid droplet method. This application uses a lower-cost, higher-efficiency method to differentiate adipocyte precursor cells into mature adipocytes with a large number of lipid droplets. Moreover, sodium alginate, gelatin and cross-linking agents are all safe and non-toxic ingredients that are often used in large quantities in food. As part of food products, they can ensure food safety.
  • the scaffold with sodium alginate-gelatin content has a certain thickness and elasticity, so the scaffold can provide a certain degree of taste and support the taste of cell-based meat; in addition, the sodium alginate-gelatin scaffold and the differentiation completed on it Cells are conducive to subsequent mass production, storage, customized meat and other processes through the adipocyte-3D scaffold complex.
  • adipose precursor cells exist in a form that is adhered to the three-dimensional configuration of the scaffold.
  • the 3D configuration in the hydrogel has a large number of loose and porous structures.
  • This staggered arrangement of microfilaments has pores that are connected to each other.
  • the adipose precursor cells are supported by the microfilaments and located in the pores formed by them. And the acquisition of nutrients is completed through the connected pores, which provides the adhesion and nutritional environment required for the growth of fat precursor cells.
  • adipocyte precursor cells were differentiated more efficiently on 3D scaffolds.
  • a three-dimensional culture system can support more cells at the same time in the same base area.
  • the excellent connected pore structure allows the differentiation-inducing components in the culture medium to act more effectively and fully on adipocyte precursor cells, thereby achieving higher differentiation efficiency of differentiated adipocytes on the 3D scaffold, that is, simultaneously differentiating into adipocytes with Lipid droplets have a higher proportion of cells.
  • adhesion of adipocyte precursor cells in the 3D scaffold was not tight. This can provide more product forms for the later production of cell-based meat.
  • pure mature adipocytes can be isolated; with the myotube/myocyte-3D scaffold complex and adipocyte-3D scaffold studied in this laboratory The compound is extruded to obtain "fat and thin" cell meat.
  • the sodium alginate-gelatin 3D scaffold was prepared by the following method:
  • step (b) After removing the remaining cross-linking agent in the hydrogel scaffold obtained in step (a), soak it in ethanol, then irradiate it with an ultraviolet lamp, and finally rinse it with a buffer solution; and
  • step (c) Fragment the hydrogel scaffold treated in step (b) and place it in a buffer solution for shaking to obtain microfilaments of the sodium alginate-gelatin 3D scaffold.
  • the sodium alginate-gelatin 3D scaffold described in this application at least includes the main components of sodium alginate and gelatin, the cross-linking component of a cross-linking agent and ethanol, a three-dimensional structure formed by interlacing microfilaments, and a staggered arrangement of microfilaments. interconnected pores.
  • the mass ratio of sodium alginate, gelatin and water is (1-3):(1-4):(93-98), for example, it can be 1:1:98, 1:2:97 , 1:3:96, 1:4:95, 2:1:97, 2:2:96, 2:3:95, 2:4:94, 3:1:96, 3:2:95, 3 ⁇ 3:94, 3:4:93, etc.
  • the mixing temperature is 75-85°C, for example, it can be 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, etc.
  • the solidification temperature is -85 ⁇ -75°C, for example, it can be -85°C, -84°C, -83°C, -82°C, -81°C, -80°C, - 79°C, -78°C, -77°C, -76°C, -75°C, etc.; the solidification time is 8-16h, for example, it can be 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h wait.
  • the cross-linking agent solution is an aqueous solution of calcium chloride dihydrate
  • the concentration of the aqueous solution of calcium chloride dihydrate is 400-600 ⁇ M, for example, it can be 400 ⁇ M, 420 ⁇ M, 440 ⁇ M, or 460 ⁇ M. , 480 ⁇ M, 500 ⁇ M, 520 ⁇ M, 540 ⁇ M, 560 ⁇ M, 580 ⁇ M, 600 ⁇ M, etc.
  • the cross-linking temperature is -25 ⁇ -15°C, for example, it can be -25°C, -24°C, -23°C, -22°C, -21°C, -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, etc.; the cross-linking time is 8-16h, for example, it can be 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h , 16h, etc.
  • the energy of the ultraviolet lamp is 20-40mJ/cm 2 , for example, it can be 20mJ/cm 2 , 22mJ/cm 2 , 24mJ/cm 2 , 26mJ/cm 2 , 28mJ/cm 2 , 30mJ/cm 2 , 32mJ/cm 2 , 34mJ/cm 2 , 36mJ/cm 2 , 38mJ/cm 2 , 40mJ/cm 2 , etc.; the irradiation time is 1-3h, for example, it can be 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, 2.2h, 2.4h, 2.6h, 2.8h, 3h, etc.
  • the buffer solution is PBS.
  • the crushing is done by shearing, and the volume of the granular hydrogel fragments obtained by shearing is 1-9mm 2 , for example, it can be 1mm 2 , 2mm 2 , 3mm 2 , or 4mm 2 , 5mm 2 , 6mm 2 , 7mm 2 , 8mm 2 , 9mm 2 etc.
  • the buffer solution is PBS and/or DMEM.
  • step (c) the oscillation uses a Limbeer VORTEX-5 vortex mixer VORTEX-5.
  • the power of the oscillation is 40-60W, for example, it can be 40W, 42W, 44W, 46W, 48W, 50W, 52W, 54W, 56W, 58W, 60W, etc.
  • the oscillation time is 10 -15min, for example, it can be 10min, 11min, 12min, 13min, 14min, 15min, etc.
  • step (c) after the shaking, the following operations are required: oscillating and suspending the hydrogel fragments, which include tiny fragments obtained by oscillation and visible to the naked eye, and utilizing the different sizes of the hydrogel fragments to cause buffering
  • the sedimentation speed in the liquid is different.
  • the present application provides a method for differentiation of preadipocytes, which method includes the following steps: seeding preadipocytes on the sodium alginate-gelatin 3D scaffold for differentiation to obtain mature adipocytes.
  • the method for differentiation of adipocyte precursor cells specifically includes the following steps:
  • step (2) Add the mixture containing microfilaments and growth medium obtained in step (1) dropwise into the cell chamber, wait until the microfilaments have completely settled in the cell chamber, and remove the remaining liquid in the upper layer;
  • step (3) After resuspending the adipose precursor cells, inject the cells treated in step (2) into the cell chamber and hang it in a well plate, and then use growth medium to infiltrate the bottom of the cell chamber to inhibit contact; and
  • step (3) Replace the growth medium in step (3) with a differentiation medium, induce differentiation, and obtain mature adipocytes.
  • the growth medium consists of the following components in terms of mass percentage: DMEM 81-94%, FBS 5-15%, penicillin and streptomycin mixture 1 -4%.
  • the content of DMEM is 81-94%, for example, it can be 81%, 82%, 84%, 86%, 88%, 90%, 92%, 94% wait.
  • the content of the FBS is 5-15%, for example, it can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% , 13%, 14%, 15%, etc.
  • the content of the mixture of penicillin and streptomycin is 1-4%, for example, it can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% wait.
  • the mass ratio of microfilaments and growth medium of the sodium alginate-gelatin 3D scaffold is 1:(1 ⁇ 10 4 -6 ⁇ 10 4 ), for example, it can be 1:1 ⁇ 10 4 or 1:2 ⁇ 10 4 , 1:3 ⁇ 10 4 , 1:4 ⁇ 10 4 , 1:5 ⁇ 10 4 , 1:6 ⁇ 10 4 etc.
  • the specific step of dropping is: using a Pasteur pipette to extract the mixture containing microfilaments and growth medium obtained in step (1), and letting the pipette stand vertically for 3-5 seconds, for example It is 3s, 3.5s, 4s, 4.5s, 5s, etc.
  • the microfilament settles at the mouth of the Pasteur pipette, add it dropwise into the 24-well cell chamber.
  • the density of the adipose precursor cells is 10 6 -10 8 /mL, for example, it can be 10 6 /mL, 5 ⁇ 10 6 /mL, 10 7 /mL, 5 ⁇ 10 7 /mL, 10 8 /mL, etc.; the resuspension uses a growth medium solution, and the resuspension speed is 1000-1500rpm, for example, it can be 1000rpm, 1100rpm, 1200rpm, 1300rpm, 1400rpm, 1500rpm, etc.
  • the resuspension time is 5-10 minutes, for example, it can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, etc.
  • the number of adipose precursor cells injected into each chamber is 10 5 -10 7 , for example, it can be 10 5 /mL, 5 ⁇ 10 5 /mL, 10 6 / mL, 5 ⁇ 10 6 /mL, 10 7 /mL, etc.
  • the well plate is a 6-well plate.
  • the contact inhibition temperature is 36-38°C, for example, it can be 36°C, 36.5°C, 37°C, 37.5°C, 38°C, etc.
  • the contact inhibition time is 2-3 days, For example, it can be 2 days, 2.5 days, 3 days, etc.
  • the specific operation of inducing differentiation is: first replace the growth medium in step (3) with the first differentiation medium, and incubate at 36-38°C (for example, 36°C, 36.5 °C, 37 °C, 37.5 °C, 38 °C, etc.) to induce differentiation for 2-3 days (for example, it can be 2 days, 2.5 days, 3 days, etc.); then replace the first differentiation medium with the second differentiation medium, and Induce differentiation at 36-38°C (for example, it can be 36°C, 36.5°C, 37°C, 37.5°C, 38°C, etc.) for 8-10 days (for example, it can be 8 days, 9 days, 10 days, etc.), and 8-10
  • the second differentiation medium is replaced every 3-5 days (for example, it can be 3 days, 4 days, 5 days, etc.) within days (for example, it can be 8 days, 9 days, 10 days, etc.);
  • the first differentiation medium includes: IBMX, dexamethasone, insulin, FBS, DMEM-f12 differentiation medium containing double antibodies;
  • the second differentiation medium includes: insulin, FBS, DMEM-f12 containing double antibodies F12 differentiation medium.
  • the first differentiation medium includes the following components: 0.4-0.6mM IBMX, 0.2-0.3 ⁇ M dexamethasone, 0.8-1.2 ⁇ g/mL insulin, 8-12% FBS, 0.5-2% bis-containing DMEM-f12 differentiation medium.
  • the concentration of IBMX is 0.4-0.6mM, for example, it can be 0.4mM, 0.45mM, 0.5mM, 0.55mM, 0.6mM, etc.
  • the concentration of dexamethasone is 0.2-0.3 ⁇ M, for example, it can be 0.2 ⁇ M, 0.22 ⁇ M, 0.24 ⁇ M, 0.26 ⁇ M, 0.28 ⁇ M, 0.3 ⁇ M, etc.
  • the concentration of insulin is 0.8-1.2 ⁇ g/mL, for example, it can be 0.8 ⁇ g/mL, 0.9 ⁇ g/mL, 1.0 ⁇ g/mL, 1.1 ⁇ g/mL, 1.2 ⁇ g/mL, etc. .
  • the volume percentage of FBS is 8-12%, for example, it can be 8%, 9%, 10%, 11%, 12%, etc.
  • the percentage content of double antibodies in DMEM-f12 differentiation medium containing double antibodies is 0.5-2%, for example, it can be 0.5%, 0.8%, 1.0%, 1.2%, 1.5% , 1.8%, 2%, etc.
  • the second differentiation medium includes the following components: 0.8-1.2 ⁇ g/mL insulin, 8-12% FBS, and 0.5-2% DMEM-f12 differentiation medium containing double antibodies.
  • the concentration of insulin is 0.8-1.2 ⁇ g/mL, for example, it can be 0.8 ⁇ g/mL, 0.9 ⁇ g/mL, 1.0 ⁇ g/mL, 1.1 ⁇ g/mL, 1.2 ⁇ g/mL, etc. .
  • the volume percentage of FBS is 8-12%, for example, it can be 8%, 9%, 10%, 11%, 12%, etc.
  • the percentage content of double antibodies in the DMEM-f12 differentiation medium containing double antibodies is 0.5-2%, for example, it can be 0.5%, 0.8%, 1.0%, 1.2%, 1.5% , 1.8%, 2%, etc.
  • This application applies sodium alginate-gelatin 3D scaffold to support the differentiation of adipocyte precursor cells, and finds that a lower cost and higher efficiency method can be used to differentiate adipocyte precursor cells into cells with a large amount of intracellular lipids. Drops of mature adipocytes.
  • the sodium alginate, gelatin and cross-linking agent in the sodium alginate-gelatin 3D scaffold provided by this application are all safe and non-toxic ingredients. They are ingredients that are often used in large quantities in food. As part of food products, they can ensure consumption safety.
  • the sodium alginate-gelatin content scaffold provided by this application has a certain thickness and elasticity, so the scaffold can provide a certain degree of taste and support the taste of cell-based meat.
  • the sodium alginate-gelatin scaffold and the differentiated cells on the sodium alginate-gelatin scaffold of the present application are conducive to the subsequent mass production, storage, customized meat and other processes through the adipocyte-3D scaffold complex.
  • Figure 1 is a diagram showing the differentiation efficiency and lipid droplet size of adipocytes on the 9th day of differentiation on the scaffold provided in Example 1.
  • Figure 2 is a diagram showing the differentiation efficiency and lipid droplet size effect of adipocytes on the 9th day of differentiation on the scaffold provided in Example 2.
  • Figure 3 is a diagram showing the differentiation efficiency and lipid droplet size of adipocytes on the 9th day of differentiation on the scaffold provided in Example 3.
  • Figure 4 is a graph showing the differentiation efficiency and lipid droplet size of adipocytes on the 9th day of differentiation on the scaffold provided in Example 4.
  • Figure 5 is a diagram of the components of the scaffold (gelatin-sodium alginate microfilament) in simple Example 1.
  • This preparation example provides a sodium alginate-gelatin 3D scaffold, which is prepared by the following method:
  • step (c) Use high-temperature and high-pressure sterilized surgical scissors to cut the stent processed in step (b) into pieces with an average area of 2mm2 . Place the hydrogel fragments into a 50mL centrifuge tube, add 30mL of PBS in a oscillator Fully oscillate for 12 minutes at maximum power (30W);
  • (d) Oscillate and suspend the hydrogel fragments, which include tiny fragments obtained by oscillation and visible to the naked eye. Taking advantage of the different sizes of the hydrogel fragments resulting in different sedimentation speeds in the buffer, repeatedly shake the suspended fragments by hand and let them stand. 1s and the operation of removing tiny fragments from the upper layer to obtain the raw material microfilaments of the 3D hydrogel scaffold, and store them at 4°C.
  • This preparation example provides a sodium alginate-gelatin 3D scaffold, which is prepared by the following method:
  • step (c) Use high-temperature and high-pressure sterilized surgical scissors to cut the stent processed in step (b) into pieces with an average area of 1mm2 . Place the hydrogel fragments into a 50mL centrifuge tube, add 30mL of PBS, and shake Fully oscillate at the maximum power of the device (50W) for 12 minutes;
  • (d) Oscillate and suspend the hydrogel fragments, which include tiny fragments obtained by oscillation and visible to the naked eye. Taking advantage of the different sizes of the hydrogel fragments resulting in different sedimentation speeds in the buffer, repeatedly shake the suspended fragments by hand and let them stand. 2s and the operation of removing the tiny fragments of the upper layer to obtain the raw material microfilaments of the 3D hydrogel scaffold, and store them at 4°C.
  • This preparation example provides a sodium alginate-gelatin 3D scaffold, which is prepared by the following method:
  • step (c) Use high-temperature and high-pressure sterilized surgical scissors to cut the stent processed in step (b) into pieces with an average area of 2mm2 . Place the hydrogel fragments into a 50mL centrifuge tube, add 30mL of DMEM, and shake Fully oscillate at the maximum power of the device (30W) for 12 minutes;
  • (d) Oscillate and suspend the hydrogel fragments, which include tiny fragments obtained by oscillation and visible to the naked eye. Taking advantage of the different sizes of the hydrogel fragments resulting in different sedimentation speeds in the buffer, repeatedly shake the suspended fragments by hand and let them stand. After 3 seconds, remove the tiny fragments from the upper layer to obtain the raw material microfilaments of the 3D hydrogel scaffold, and store them at 4°C.
  • This preparation example provides a sodium alginate-gelatin 3D scaffold, which is prepared by the following method:
  • step (c) Use high-temperature and high-pressure sterilized surgical scissors to cut the stent processed in step (b) into pieces with an average area of 1mm2 . Place the hydrogel fragments into a 50mL centrifuge tube and add 50mL of FBS/DMEM (The mass ratio of FBS/DMEM is 1:10), fully oscillate for 12 minutes at the maximum power of the oscillator (20W);
  • This preparation example provides a sodium alginate-gelatin 3D scaffold, which is prepared by the following method:
  • step (c) Use high-temperature and high-pressure sterilized surgical scissors to cut the stent processed in step (b) into fragments with an average area of 0.5mm2 . Place the hydrogel fragments into a 50mL centrifuge tube, add 50mL of PBS, and place in the Fully oscillate at the maximum power of the oscillator (50W) for 12 minutes to obtain the raw materials of the 3D hydrogel scaffold, and store them at 4°C.
  • This preparation example provides a gellan gum/gelatin 3D scaffold, which is prepared by the following method:
  • step (b) Use high-temperature and high-pressure sterilized surgical scissors to cut the 3D scaffold obtained in step (a) into pieces with an average area of 1mm2 . Place the hydrogel fragments into a 50mL centrifuge tube, add 30mL of PBS, and shake Fully oscillate at the maximum power of the device (50W) for 12 minutes;
  • (c) Oscillate and suspend the above-mentioned scaffold, which contains tiny fragments obtained by oscillation and visible to the naked eye. Taking advantage of the different sedimentation speeds of fragments of different sizes, repeat the operations of oscillating and removing the upper hydrogel fragments to obtain the raw materials of the 3D hydrogel scaffold. microfilament and store it at 4°C.
  • This embodiment provides a method for differentiation of preadipocyte cells.
  • the method for differentiation of preadipocyte cells specifically includes the following steps:
  • step (1) Use a Pasteur pipette to extract the mixture of microfilament and growth medium in step (1). Place the Pasteur pipette vertically for 5 seconds. When the microfilament settles at the mouth of the Pasteur pipette, add it dropwise to the 24-well into the cell chamber; after the microfilaments have completely settled in the cell chamber, remove the remaining liquid in the upper layer;
  • DMEM-f12 differentiation medium containing 0.5mM IBMX, 0.25 ⁇ M dexamethasone, 1 ⁇ g/mL insulin, 10% FBS, and 1% double antibody, and allow the cells to differentiate here at 37°C. Induce differentiation in the culture medium for 2 days; then replace the differentiation medium with DMEM-f12 differentiation medium containing 1 ⁇ g/mL insulin, 10% FBS, and 1% double antibody, and continue differentiation at 37°C for 9 days, every 4 Change the fluid once a day.
  • This embodiment provides a method of differentiation of preadipocyte cells.
  • the method of differentiation of preadipocyte cells specifically includes the following steps:
  • step (1) Use a Pasteur pipette to extract the mixture of microfilament and growth medium in step (1), and let the Pasteur pipette stand vertically for 3 seconds.
  • the microfilament settles at the mouth of the Pasteur pipette, add it dropwise to the 24-well into the cell chamber; after the microfilaments have completely settled in the cell chamber, remove the remaining liquid in the upper layer;
  • adipocyte precursor cells 100 ⁇ L, that is, 1 ⁇ 10 6 adipocyte precursor cells, to each cell chamber; suspend the cell chamber in a 6-well plate, and add 12 mL of DMEM containing a mixture of 10% FBS, 1% penicillin, and streptomycin for growth. culture medium so that the bottom of the cell chamber is fully infiltrated by the culture medium and no air bubbles exist. The liquid level of the culture medium needs to cover the pores above the cell chamber, so that the adipocyte precursor cells are contact-inhibited at 37°C for 2 days;
  • DMEM-f12 differentiation medium containing 0.5mM IBMX, 0.25 ⁇ M dexamethasone, 1 ⁇ g/mL insulin, 10% FBS, and 1% double antibody, and allow the cells to differentiate here at 37°C. Induce differentiation in the culture medium for 2 days; then replace the differentiation medium with DMEM-f12 differentiation medium containing 1 ⁇ g/mL insulin, 10% FBS, and 1% double antibody, and continue differentiation at 37°C for 9 days, every 4 Change the fluid once a day.
  • This embodiment provides a method for differentiation of preadipocyte cells.
  • the method for differentiation of preadipocyte cells specifically includes the following steps:
  • step (1) Use a Pasteur pipette to extract the mixture of microfilament and growth medium in step (1). Place the Pasteur pipette vertically for 5 seconds. When the microfilament settles at the mouth of the Pasteur pipette, add it dropwise to the 24-well into the cell chamber; after the microfilaments have completely settled in the cell chamber, remove the remaining liquid in the upper layer;
  • adipocyte precursor cells 100 ⁇ L, that is, 1 ⁇ 10 6 adipocyte precursor cells, to each cell chamber; suspend the cell chamber in a 6-well plate, and add 12 mL of DMEM containing a mixture of 10% FBS, 1% penicillin, and streptomycin for growth. culture medium so that the bottom of the cell chamber is fully infiltrated by the culture medium and no air bubbles exist. The liquid level of the culture medium needs to cover the pores above the cell chamber, so that the adipocyte precursor cells are contact-inhibited at 37°C for 2 days;
  • DMEM-f12 differentiation medium containing 0.5mM IBMX, 0.25 ⁇ M dexamethasone, 1 ⁇ g/mL insulin, 10% FBS, and 1% double antibody, and allow the cells to differentiate here at 37°C. Induce differentiation in the culture medium for 2 days; then replace the differentiation medium with DMEM-f12 differentiation medium containing 1 ⁇ g/mL insulin, 10% FBS, and 1% double antibody, and continue differentiation at 37°C for 9 days, every 4 Change the fluid once a day.
  • This comparative example provides a method for differentiation of adipose precursor cells.
  • the only difference from Example 1 is that the scaffold microwires provided in Preparation Example 1 are replaced with the scaffold microwires provided in Comparative Preparation Example 1.
  • Test sample sodium alginate-gelatin 3D scaffold provided in Preparation Example 1-5;
  • Test method Use Young's modulus as a model to measure the elasticity of the stent
  • the sodium alginate-gelatin content stent provided by this application has a certain thickness, specifically 1-2mm, and has a certain elasticity, specifically 1.0-2.0kPa, so the stent can provide a certain degree of The taste provides support for the taste of cell-based meat.
  • Test samples the methods of differentiation of preadipocytes provided in Examples 1-3 and the method of differentiation of preadipocytes provided in Comparative Example 1;
  • Test method Nile red staining method
  • Test results A large number of lipid droplets appeared in the differentiated adipocyte precursor cells on the scaffold and were stained with Nile red.
  • Figure 1-4 shows Hoechest/NileRed staining, where Hoechest is blue, indicating the size, shape, and position of the cell nucleus; NileRed is red, indicating the size, position, and number of differentiated intracellular lipid droplets in the cytoplasm.
  • Example 1 100% 10.2
  • Example 2 100% 9.7
  • Example 3 100% 10.3 Comparative example 1 90% 5.6

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Abstract

La présente invention concerne l'utilisation d'un échafaudage 3D à base d'alginate de sodium et de gélatine pour favoriser la différenciation des préadipocytes. Les prépodocytes peuvent être différenciés en adipocytes matures avec un grand nombre de gouttelettes lipidiques selon un procédé moins coûteux et plus efficace. En outre, l'alginate de sodium, la gélatine et un agent de réticulation dans l'échafaudage 3D à base d'alginate de sodium et de gélatine de la présente invention sont tous des ingrédients sûrs et non toxiques fréquemment utilisés dans les produits alimentaires, et peuvent garantir la sécurité de consommation en tant que composant d'un produit alimentaire. L'échafaudage présente une certaine épaisseur et une certaine élasticité, étant ainsi capable de conférer un certain niveau de goût et de servir à renforcer le goût de la viande produite à partir de cellules.
PCT/CN2022/095563 2022-05-27 2022-05-27 Utilisation d'un échafaudage 3d à base d'alginate de sodium et de gélatine pour favoriser la différenciation des préadipocytes Ceased WO2023225995A1 (fr)

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PCT/CN2022/095563 WO2023225995A1 (fr) 2022-05-27 2022-05-27 Utilisation d'un échafaudage 3d à base d'alginate de sodium et de gélatine pour favoriser la différenciation des préadipocytes

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