WO2024007984A1 - 一种基于微流控3d打印技术的细胞培养肉生产设备及其应用 - Google Patents

一种基于微流控3d打印技术的细胞培养肉生产设备及其应用 Download PDF

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Publication number
WO2024007984A1
WO2024007984A1 PCT/CN2023/104677 CN2023104677W WO2024007984A1 WO 2024007984 A1 WO2024007984 A1 WO 2024007984A1 CN 2023104677 W CN2023104677 W CN 2023104677W WO 2024007984 A1 WO2024007984 A1 WO 2024007984A1
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Prior art keywords
printing
microfluidic
cells
cell
protein
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PCT/CN2023/104677
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English (en)
French (fr)
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周光宏
王洁
丁希
丁世杰
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南京周子未来食品科技有限公司
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Publication of WO2024007984A1 publication Critical patent/WO2024007984A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/20—Apparatus for additive manufacturing; Details thereof or accessories therefor
    • B29C64/227—Driving means
    • B29C64/232—Driving means for motion along the axis orthogonal to the plane of a layer
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P30/00—Shaping or working of foodstuffs characterised by the process or apparatus
    • A23P30/20—Extruding
    • A23P30/25—Co-extrusion of different foodstuffs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/10—Processes of additive manufacturing
    • B29C64/106—Processes of additive manufacturing using only liquids or viscous materials, e.g. depositing a continuous bead of viscous material
    • B29C64/112—Processes of additive manufacturing using only liquids or viscous materials, e.g. depositing a continuous bead of viscous material using individual droplets, e.g. from jetting heads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/20—Apparatus for additive manufacturing; Details thereof or accessories therefor
    • B29C64/227—Driving means
    • B29C64/236—Driving means for motion in a direction within the plane of a layer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/20—Apparatus for additive manufacturing; Details thereof or accessories therefor
    • B29C64/245—Platforms or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/30—Auxiliary operations or equipment
    • B29C64/307—Handling of material to be used in additive manufacturing
    • B29C64/321—Feeding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/30—Auxiliary operations or equipment
    • B29C64/386—Data acquisition or data processing for additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y30/00—Apparatus for additive manufacturing; Details thereof or accessories therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y40/00—Auxiliary operations or equipment, e.g. for material handling
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y50/00—Data acquisition or data processing for additive manufacturing
    • 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
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2533/50—Proteins
    • C12N2533/56—Fibrin; Thrombin
    • 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
    • C12N2533/80—Hyaluronan
    • 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

  • the invention belongs to the field of cell cultured meat, and specifically relates to cell cultured meat production equipment based on microfluidic 3D printing technology and its application.
  • Meat is the main source of protein in the diet of residents around the world. With the growth of the world's population and the improvement of living standards of residents in developing countries, people's demand for meat is also increasing, which will force the traditional animal husbandry industry to expand production scale to meet the growing demand for meat.
  • traditional livestock breeding will cause serious occupation of water and land resources and emit a large amount of greenhouse gases.
  • the food-borne diseases and animal welfare issues caused by intensive breeding should not be underestimated. Therefore, it is of great significance to develop new meat production technologies that can replace traditional livestock farming.
  • Cell-cultured meat is an emerging meat production technology born at the intersection of cell engineering, tissue engineering and food engineering. It is obtained through large-scale expansion of muscle stem cells in vitro, induction of differentiation, product collection and food processing. meat. Compared with traditional animal husbandry, cell-cultured meat can significantly reduce energy consumption, water resource abuse and greenhouse gas emissions, and eliminate the occupation of approximately 99% of land resources. Since Professor Mark Post of the Netherlands announced the birth of the world's first cell-cultured meat in 2013, there has been an upsurge in the research and industrialization of cell-cultured meat worldwide. Despite good research progress, existing known production methods are still very limited in terms of muscle fiber formation capabilities, meat structure simulation, and scale.
  • tissue engineering technologies have been used in the production of cell-cultured meat, such as animal and plant protein scaffolds, hydrogels, cell sheet engineering, and 3D bioprinting.
  • bio-3D printing is an advanced technology that is driven by three-dimensional model instructions and based on the principles of additive manufacturing to position and assemble biomaterials or cell units to construct tissue engineering scaffolds and tissues and organs. It has great potential in the construction of cell-cultured meat tissues. .
  • biological 3D printer equipment on the market is generally expensive and has high maintenance costs.
  • the structure and type of printing nozzles are single, the flexibility of nozzle replacement is poor, and there are major restrictions on batch printing; in addition, there are currently no solutions for printing on the market.
  • Special 3D printing equipment for cell cultured meat production Therefore, it is worth looking forward to developing special 3D printing equipment suitable for the production of cell-cultured meat to achieve low-cost, customized, large-scale, convenient and efficient production of cell-cultured meat.
  • the present invention provides a cell cultured meat production equipment based on microfluidic 3D printing technology, which effectively solves the high cost of existing biological 3D printer equipment, single printing nozzle function, poor flexibility and The problem of limited scale has further filled the gap in 3D printing equipment for cell cultured meat production.
  • the present invention also provides the application of the cell cultured meat production equipment based on microfluidic 3D printing technology in preparing cell cultured meat.
  • the present invention is a cell cultured meat production equipment based on microfluidic 3D printing technology.
  • the equipment includes a printing nozzle, a printing movement system, a loading platform, a sampling system, and a base;
  • the printing mobile system is placed on a base and consists of multiple movable optical axes, the printing nozzle is fixed on one of the movable optical axes, and the loading platform is connected to the other movable optical axis;
  • the sample introduction system It is connected to the printing nozzle, which is a microfluidic chip.
  • the microfluidic chip is a device that can manipulate, process and control trace amounts of liquid or samples in the channel.
  • the printing movement system includes an x-axis moving optical axis, a z-axis moving optical axis, and a y-axis moving optical axis.
  • the z-axis moving optical axis and the y-axis moving optical axis are fixed on the base, and the x-axis moving optical axis Shift with z axis
  • the printing nozzle is fixed on the x-axis moving optical axis, and the loading platform is connected to the y-axis moving optical axis).
  • the printing moving system is a moving axis that can drive the printing nozzle to move in two directions on the x-axis moving optical axis and the z-axis moving optical axis, and drive the loading platform to move on the y-axis moving optical axis, and its configured movement
  • the coordinate system includes any one of Cartesian coordinate system, trigonometric coordinate system, polar coordinate system and plane joint coordinate system.
  • the printing nozzle is integrated on the x-axis moving optical axis in the printing movement system.
  • the x-axis moving optical axis is connected to the z-axis moving optical axis.
  • the printing nozzle is driven by the x-axis and z-axis moving optical axes at xz. Move within the plane.
  • the types of microfluidic chips on the printing mobile system can be flexibly replaced, and integrated printing can be performed according to different production needs.
  • the printing mobile system can integrate one or more microfluidic chips as printing nozzles, further develop a multi-nozzle parallel printing device, and realize the construction of a multi-nozzle microfluidic 3D printing device.
  • microfluidic chips when multiple microfluidic chips are integrated on the printing mobile system, microfluidic chips with the same channel structure or different channel structures can be used.
  • microfluidic chip is integrated into the printing movement system through clamping, buckling, plugging, magnetic attraction, tenoning, riveting, threaded connection or bayonet connection.
  • the loading platform is a detachable structure, assembled in the printing movement system, and connected with the y-axis moving optical axis for assembly line printing;
  • the material of the loading platform is copper, aluminum, iron, steel, alloy, glass , ceramic or carbon fiber plates.
  • the carrier platform is combined with the y-axis moving optical axis in the printing movement system and is a detachable structure.
  • the y-axis moving optical axis drives the carrier platform and the printed matter formed on the carrier platform to move in the y-axis direction.
  • the loading platform and the y-axis moving optical axis are connected through a snap-type connection and can be flexibly disassembled.
  • the sampling system consists of a sample loader, a sample injection pump and a conduit.
  • the sample loader is fixed on the sample pump, and the conduit connects the outlet of the sample loader and the sample inlet of the printing nozzle.
  • the sampling system includes a sample loader, a sample pump and a conduit.
  • the sample pump is placed on a horizontal table, the sample loader is fixed on the sample pump, one end of the conduit is connected to the outlet of the sample loader, and the other end is connected to the printer
  • the sampling port of the nozzle, the feeding method of the sampling system includes piston extrusion, pneumatic extrusion or screw extrusion.
  • the sample loader is fixed on the sampling pump through buckles and can be flexibly disassembled.
  • One end of the conduit is connected to the outlet of the sample loader, and the other end is connected to the inlet of the printing nozzle.
  • a print control display system and a data transmission system are embedded in the base; the data transmission system is connected to the print control display system through wireless or data lines, and the print control display system is connected to the printing mobile system through wireless or data lines.
  • the printing control display system includes a control display screen, which is mainly used to control printing leveling (zeroing, resetting the print nozzle, returning to zero, adjusting it to the level of the printing platform, etc.), selecting the printing program, and issuing printing instructions. , Printing mobile system position adjustment. By operating on the display screen, functions such as selecting the printing instruction file, adjusting the printing speed, the position of the print nozzle, and the position of the loading platform can be realized.
  • the data transmission system is used to transmit the printing instruction file into the 3D printing device;
  • the data transmission form of the data transmission system includes USB transmission, memory card transmission or computer transmission.
  • the data transmission system includes insertion ports for storage devices such as USB and memory cards, which are mainly used to import print command files to the print control display system in the printer.
  • the print control display system and the data transmission system are integrated with the base.
  • the front of the base is opened and the print control display system is embedded.
  • the top of the base is drilled and the data transmission system interface is embedded. After the printing control display system and data transmission system are embedded in the base, they are connected to power together with the base.
  • the rectangular parallelepiped base and embed a fixed print control display system and various data transmission connections as a data transmission system; then, place the base on a horizontal table and use bolts to move the y-axis to the optical axis and the z-axis moving optical axis are fixed on the base, and then the x-axis moving optical axis is connected to the z-axis moving optical axis, that is, the printing moving system is successfully assembled; then, the printing nozzle is fixed on the x-axis moving optical axis, and the object is loaded
  • the platform is connected to the y-axis moving optical axis.
  • the sampling system consists of a sample loader, a sample pump and a conduit.
  • the sample loader is fixed on the sample pump, and the conduit connects the sample loader outlet and the printing nozzle inlet.
  • the printing nozzle is driven by the x-axis moving optical axis to move on the x-axis.
  • the x-axis moving optical axis can slide on the z-axis moving optical axis.
  • the printed tissue on the loading platform is driven by the y-axis moving optical axis.
  • the moving optical axes in three directions cooperate with each other to stack the fibers generated by the printing nozzle in the x, y, and z-axis directions.
  • the microfluidic chip is a device that can manipulate, process and control trace amounts of liquid or sample (generally 10 -6 -10 -15 L) in a channel on a microscopic scale.
  • the printing material is loaded into the sampler of the sampling system, and the sampler is fixed on the injection pump, and then the sampler is connected to the printing nozzle with a conduit, and the sample is loaded with the sampler using the injection pump.
  • the material in the container is pumped into the print head.
  • the printing instruction file is imported into the printing control display system through the data transmission system.
  • the entire 3D printing production equipment is started; the generated fibers are deposited on the loading platform and are printed on the object according to the printing instruction file.
  • the three directions of x, y and z are stacked and formed.
  • the loading platform is disassembled and the printed products are collected for subsequent processing operations.
  • the manufacturing materials of the microfluidic chip include but are not limited to crystalline silicon, polydimethoxysiloxane, glass, quartz, polyphthalamine, polymethylmethacrylate, polycarbonate, polystyrene, One or more of epoxy resin, acrylic, rubber and fluoroplastic; the manufacturing method of the microfluidic chip includes glass capillary assembly method, mechanical processing method, etching method or mold method.
  • the microfluidic chip type is a single-channel type, a coaxial nested type or a multi-channel parallel type.
  • the microfluidic chip can be based on microfluidic chips with different structures, and can produce solid, "shell-core", hollow, multi-component, spiral, and bead-type fibers for microfluidic applications. 3D printing.
  • the present invention can generate fibers with different structures by building microfluidic chips with different structures and adjusting and designing the type, flow rate, etc. of the incoming fluid.
  • the microfluidic chip is a coaxial nested microfluidic chip.
  • the inner diameter of the outlet of the microfluidic chip ranges from 200 to 2000 ⁇ m.
  • the cell-cultured meat production equipment based on microfluidic 3D printing technology of the present invention is used in the preparation of cell-cultured meat.
  • the process of preparing cell-cultured meat is:
  • step (2) Import the printing instruction file into the printing device, and fill the bio-ink prepared in step (1) into the sampling system; connect the sampling system to the entrance of the printing nozzle, that is, the channel entrance of the microfluidic chip, and The bio-ink is squeezed into the printing nozzle, which is the microfluidic chip, through the conduit; after the fibers are generated at the outlet of the microfluidic chip, select the instruction file to be printed and start the entire device; the fibers generated at the printing nozzle outlet are processed in each part of the printing mobile system. It is deposited on the loading platform driven by the optical axis, and is stacked and formed following the printing instruction path;
  • step (3) Disassemble the loading platform, cross-link and solidify the three-dimensional tissue printed in step (2), and then transfer it to the corresponding culture medium for proliferation and differentiation culture;
  • step (3) Harvest the mature three-dimensional tissue cultured in step (3), wash and remove the culture medium, and then process it into food to obtain cell-cultured meat.
  • step (2) the print instruction file is imported from the data transmission system to the printer, the bio-ink prepared in step (1) is filled into the sampler and connected to the conduit, and then the sampler is fixed to the injection pump Go up; connect the other end of the catheter to the inlet of the printing nozzle, that is, the channel entrance of the microfluidic chip, and use the injection pump to squeeze the bioink in the sample container into the microfluidic chip through the catheter; wait until the fiber is in the microfluidic chip After the chip exit is generated, select the instruction file to be printed in the print control display system and start the 3D printing device.
  • the print control display system will display "Print” or " Cancel” two commands, click "Print” again to start the entire device.
  • the fibers generated by the exit of the printing nozzle are deposited on the loading platform driven by the x-axis moving optical axis, z-axis moving optical axis and y-axis moving optical axis of the printing movement system, and are stacked following the printing instruction path. forming;
  • the bioink in step (1) includes a material solution with cell non-adhesion and a hydrogel solution containing seed cells; the hydrogel solution containing seed cells contains a volume fraction of 30%-70% Biological materials, 0.01%-1% cross-linking agent, and the balance is basic culture medium containing calcium salt and 5 ⁇ 10 6 -5 ⁇ 10 8 /mL seed cells.
  • the material solution with cell non-adhesiveness in the bioink is any one or more of sodium alginate, chitosan, pectin, carrageenan, and gellan gum; the material solution with cell non-adhesion is The concentration of the adhesive material solution is 10-50 mg/mL.
  • the seed cells in the bioink are derived from any one or more of pigs, cattle, sheep, chickens, ducks, rabbits, fish, shrimps and crabs; the seed cells are muscle stem cells, myoblasts , muscle satellite cells, muscle precursor cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, induced pluripotent stem cells, cardiomyocytes, adipose stem cells, adipose precursor cells, bone marrow-derived adipose adult cells, fibroblasts, One or more of smooth muscle cells, vascular endothelial cells, epithelial cells, neural stem cells, glial cells, osteoblasts, chondrocytes, liver stem cells, hematopoietic stem cells, stromal cells, embryonic stem cells, and bone marrow stem cells.
  • the seed cells are derived from any one or more of pigs, cattle, sheep, chickens, and ducks; the seed cells include but are not limited to muscle stem cells, muscle cells, muscle satellite cells, and muscle precursor cells. One or more.
  • the biological material in step (1) is collagen, recombinant collagen, gelatin, Matrigel, hyaluronic acid, silk fibroin, elastin, spider silk protein, fibrin, fibrinogen, silk fibroin, layer Adhesin, fibronectin, integrin, cadherin, nestin, acellular extracellular matrix, chondroitin sulfate, heparin, keratan sulfate, dermatan sulfate, heparan sulfate, keratin, keratin sulfate, cellulose, Polymer, carboxymethyl cellulose, polylactic acid, polyvinyl alcohol, lecithin, nanocellulose, soy protein, pea protein, gluten protein, rice protein, peanut protein, yeast protein, fungal protein, wheat protein, potato protein, One or more of zein, chickpea protein, mung bean protein, seaweed protein, almond protein, quinoa protein and other materials that are biocompatible and can provide adhesion sites for
  • the biological material is one or more of collagen, recombinant collagen, gelatin, Matrigel, hyaluronic acid, and silk fibroin.
  • the basic culture medium used in the bioink component includes but is not limited to F-10, DMEM, MEM, F-12, DMEM/F-12, DMEM/F-12 One or more of GlutamMAX TM , F-12K, RPMI 1640, IMDM, L-15, 199, MCDB 131, LHC, and McCoy's 5A.
  • the basal culture medium is one or more of F-10, DMEM, MEM, F-12, DMEM/F-12.
  • the cross-linking agents used in the bioink components include but are not limited to NaOH, KOH, NaHCO 3 , HEPES balanced salt solution, EBSS balanced salt solution, HBSS balanced salt solution, PBS, DPBS and other pH regulators, transglutamine Any one or more of amidase, tyrosinase, laccase, lysyl oxidase, polyphenol oxidase, catalase, thrombin, and genipin.
  • the cross-linking agent includes one or more of NaOH, KOH, and NaHCO3.
  • the hydrogel solution includes biological materials, cross-linking agents, basic culture media containing calcium salts, and seed cells; each 1 mL of hydrogel solution contains 290-699 ⁇ L of 4-8 mg/mL biological materials, 1-10 ⁇ L 1-2mol/L cross-linking agent, 300-700 ⁇ L basic culture medium containing 15-25mg/mL calcium salt, 1 ⁇ 10 7 -1 ⁇ 10 8 seed cells;
  • the biological materials are collagen, recombinant collagen One or more of protein, gelatin, Matrigel, hyaluronic acid, and silk fibroin;
  • the cross-linking agent is one or more of NaOH, KOH, and NaHCO 3 ;
  • the calcium salt is calcium chloride , one or more of calcium carbonate, calcium sulfate, and calcium nitrate;
  • the basal culture medium is one or more of F-10, DMEM, MEM, F-12, DMEM/F-12;
  • the seed cells are one or more of muscle stem cells, myoblasts, muscle satellite
  • the hydrogel solution includes biological materials, cross-linking agents, basic culture media containing calcium salts, and seed cells; the biological material is 150-650 ⁇ L 6 mg/mL per 1 mL of hydrogel solution.
  • Collagen and 40-149 ⁇ L Matrigel the cross-linking agent is 1-10 ⁇ L 1mol/L alkali solution
  • the basic culture medium containing calcium salt and seed cells is 300-700 ⁇ L DMEM solution containing 15-25mg/mL calcium salt, resuspend 1.5 ⁇ 10 6 -1.5 ⁇ 10 8 muscle stem cells.
  • the seed cells are porcine muscle stem cells;
  • the hydrogel solution includes biological materials, cross-linking agents, calcium salts, and basal culture medium of seed cells;
  • the biological materials in 1 mL of hydrogel solution are 600 ⁇ L 6mg/mL collagen and 97 ⁇ L Matrigel, the cross-linking agent is 3 ⁇ L 1mol/L NaOH solution, the basal medium containing calcium salt and seed cells is 300 ⁇ L DMEM solution containing 20mg/mL calcium chloride, resuspend 1.5 ⁇ 10 7 pigs Muscle stem cells.
  • the cross-linking agent includes one or more of NaOH, KOH, and NaHCO3.
  • the hydrogel solution containing seed cells includes biological materials, cross-linking agents, calcium salts, and basic culture medium containing seed cells; each 1 mL of hydrogel solution contains 290-699 ⁇ L of 4-8 mg/mL.
  • the biological material 1-10 ⁇ L 1-2 mol/L cross-linking agent, 300-700 ⁇ L basic culture medium containing 15-25 mg/mL calcium salt, 1 ⁇ 10 7 -1 ⁇ 10 8 seed cells;
  • the biological material is collagen One or more of protein, recombinant collagen, gelatin, Matrigel, hyaluronic acid, and silk fibroin;
  • the cross-linking agent is one or more of NaOH, KOH, and NaHCO 3 ;
  • the calcium salt It is one or more of calcium chloride, calcium carbonate, calcium sulfate, and calcium nitrate;
  • the basal culture medium is one or more of F-10, DMEM, MEM, F-12, and DMEM/F-12. species;
  • the seed cells are one or more
  • the hydrogel solution containing seed cells includes biological materials, cross-linking agents, calcium salts, and basic culture medium containing seed cells;
  • the biological materials in 1 mL of hydrogel solution are 150- 650 ⁇ L 6mg/mL collagen and 40-149 ⁇ L Matrigel, the cross-linking agent is 1-10 ⁇ L 1mol/L alkali solution, the basic culture medium containing calcium salts and seed cells is 300-700 ⁇ L DMEM containing 15-25mg/mL calcium salts Resuspend 1.5 ⁇ 10 6 -1.5 ⁇ 10 8 muscle stem cells in the solution.
  • the seed cells are porcine muscle stem cells;
  • the hydrogel containing seed cells is The solution includes biological materials, cross-linking agents, basic culture media containing calcium salts, and seed cells;
  • the biological materials in 1 mL hydrogel solution are 600 ⁇ L 6 mg/mL collagen and 97 ⁇ L Matrigel, and the cross-linking agent is 3 ⁇ L 1 mol/L.
  • NaOH solution, calcium salt-containing, and seed cell basal medium were 300 ⁇ L of DMEM solution containing 20 mg/mL calcium chloride to resuspend 1.5 ⁇ 10 7 porcine muscle stem cells.
  • step (2) the moving speed range of each moving optical axis in the printing moving system is 0.5-50 mm/s.
  • the method of cross-linking and curing treatment in step (3) includes but is not limited to one or more of temperature-induced cross-linking, electrostatic interaction cross-linking, ionic cross-linking, and enzymatic cross-linking.
  • the basal culture medium used when preparing the culture medium in step (3) includes but is not limited to F-10, DMEM, MEM, F-12, DMEM/F-12, DMEM/F-12GlutamMAX TM , F-12K , RPMI 1640, IMDM, L-15, 199, MCDB 131, LHC, McCoy's 5A.
  • the proliferation culture solution described in step (3) includes a volume fraction of 79-89% basal culture medium, 10-20% fetal bovine serum, and 1% penicillin-streptomycin, and then 1-10ng/ mL basic fibroblast growth factor; the components of the differentiation culture medium are 94-97% basal medium, 2-5% horse serum and 1% penicillin-streptomycin.
  • the method of food processing in step (4) includes pre-processing and cooking.
  • the pre-processing includes cleaning, seasoning, color enhancement, modeling or sensory quality modification, etc.
  • the cooking includes frying, frying, boiling, steaming, Roast etc.
  • the invention is based on the 3D stacking molding method of fiber materials as the design principle.
  • the main idea is to modify the conventional extrusion 3D printer and use a microfluidic chip to replace the printing nozzle of the extrusion 3D printer, thereby using the original extrusion 3D printer.
  • the stacked plastic fiber materials become cell-loaded bionic fibers to further build three-dimensional tissues.
  • the invention effectively solves the problems of high cost of existing biological 3D printer equipment, single printing nozzle function, poor flexibility and limited scale, and fills the gap of 3D printing equipment in the field of cell cultured meat production.
  • the present invention uses microfluidic chips to replace the printing nozzles of extrusion 3D printers and develop microfluidic 3D printing devices; different from the integrated printing nozzles on various 3D printers on the market, the microfluidic nozzles used as printing nozzles
  • the type and channel structure of the fluidic chip can be flexibly designed, and the number of microfluidic chips used for printing can be flexibly increased or decreased.
  • the materials for building microfluidic chips are low-cost, convenient and easy to obtain.
  • the present invention is based on the fact that muscle fiber is the most basic unit of skeletal muscle tissue, and numerous muscle fibers are wrapped layer by layer by connective tissue membranes to form large skeletal muscle tissue; the cell cultured meat proposed by the present invention based on microfluidic 3D printing technology is based on bionic fiber
  • the three-dimensional tissue is formed after being stacked layer by layer.
  • the bionic fiber is to the three-dimensional tissue just like the muscle fiber is to the muscle, so it has good bionic properties.
  • the growth of seed cells in three-dimensional tissues further simulates their growth in natural tissues, and the stacking direction of bionic fibers can be adjusted through the printing path, which helps to reproduce the tissue orientation of skeletal muscle tissue in vitro. opposite sex.
  • the present invention improves the industrial 3D printer nozzle, replaces it with a microfluidic chip with low cost and customizable structure, and designs the connection method between the microfluidic chip and the 3D printer, so that the printing device can be used as a
  • the number and form of microfluidic chips in the nozzle can be flexibly changed.
  • This invention relies on 3D printing technology.
  • the printing accuracy (the minimum size of the extruded fiber) depends on the size of the microfluidic chip outlet.
  • the inner diameter of the microfluidic chip outlet ranges from 200 to 2000 ⁇ m.
  • the minimum size has reached the 3D printing slicing software standard. Accuracy of 0.2mm.
  • cell-cultured meat products printed from different batches have almost no changes in size and can accurately reproduce the printing path of the 3D model after slicing in the slicing software.
  • the present invention utilizes the principle of muscle fiber structure, the basic unit of natural skeletal muscle tissue, to provide seed cells with appropriate fiber carriers to ensure very good results in the efficient production of cell-cultured meat;
  • microfluidic technology is used in cell cultured meat production, which can control trace amounts of liquid in micro-sized channels.
  • This invention uses muscle fiber bionics as the design principle and proposes a bionic fiber carrier based on 3D printing combined with microfluidic technology for the production of cell cultured meat. There are no relevant reports in the field using this method to produce cell cultured meat.
  • the present invention prepares bionic fibers based on 3D printing microfluidic technology and is used for cell cultured meat production. The fiber preparation process is continuous and rapid.
  • the prepared fibers have good bionic properties, and the seed cells growing in the fibers have directional growth ability and differentiation.
  • Ability has been greatly improved.
  • the present invention prepares bionic fibers with a "shell-core" structure based on microfluidic technology.
  • the seed cells are wrapped in a shell of cell-non-adhesive material, and exhibit highly directional and fusion growth characteristics under the spatial constraints of the shell.
  • In vitro Myogenic differentiation ability has also been significantly improved, improving production efficiency, and the prepared fibers are very similar to natural skeletal muscle fibers in terms of shape and physiological characteristics. Therefore, fibers prepared based on microfluidic technology have better bionic properties. sex.
  • the present invention first designs and builds, for example, a coaxial nested microfluidic chip; then prepares materials, and adjusts the input sequence, flow rate and other parameters of the internal and external fluids to stably generate bionic fibers and Culture is carried out; finally, the cultured bionic fibers are tissue-integrated and food-processed to obtain cell-cultured meat products.
  • seed cells are wrapped in a shell of bionic fibers that do not have cell adhesion, and are highly visible in the hydrogel core under the spatial constraints of the shell.
  • the directional and integrated growth characteristics and in vitro myogenic differentiation ability have also been significantly improved, improving production efficiency.
  • cell culture is carried out in bionic fibers with a specific "shell-core" structure formed by 3D printing, which effectively improves the differentiation ability of primary livestock and poultry cells in the microfibers, such as muscle stem cells, and increases the synthesis of muscle-related proteins. And further form mature muscle fibers, thereby improving the production efficiency of cell-cultured meat.
  • the microfibers prepared by the present invention undergo spontaneous contraction and beating after 2 days of proliferation culture and 14 days of differentiation culture. This is due to the migration and fusion of primary myoblasts in a specific core wrapped in a calcium alginate shell to form a multinucleated myotube.
  • the tubes further differentiate and highly express myosin, thereby forming mature muscle fibers and exhibiting certain physiological functions.
  • the comparison results of the scanning electron microscope and H&E tissue staining of the microfibers and pork showed that the microfibers had a tight structure and obvious myotubes could be observed on the surface. Structure, tissue sections also show staining characteristics that are close to those of pork fiber, and are very close to pork fiber on the whole.
  • spontaneous contraction and beating of microfibers also appeared under bright field observation. This was under in vitro culture conditions. Achieving the leap from cells to mature tissue shows that the microfibers composed of seed cells have fully matured to form muscle fibers and have the contractile function of natural muscle fibers.
  • the present invention has the ability to culture a muscle fiber in vitro. These muscle fibers cultured in vitro are By collecting and assembling large pieces of tissue, you can theoretically obtain a piece of cell-cultured meat composed of muscle fibers cultured in vitro.
  • the present invention has the following advantages:
  • the present invention proposes a cell-cultured meat production equipment based on microfluidic 3D printing technology. This equipment is used for the preparation of cell-cultured meat. It is easy to operate, rapid in molding, has a high degree of mechanization, and requires low equipment and device costs. And the printed products have high precision and stable quality;
  • the three-dimensional tissue prepared by the present invention based on microfluidic 3D printing has good tissue anisotropy, further simulates the growth of seed cells in natural tissue, and has good bionics;
  • the seed cells are directionally arranged in the fibrous basic structure of the three-dimensional tissue and further migrate and fuse, which contributes to the differentiation and maturation of the seed cells and the synthesis of related proteins, further improving the cell cultured meat. production efficiency;
  • the cell cultured meat constructed based on microfluidic 3D printing technology developed by the present invention is highly programmable, and its size, shape and other characteristics can be adjusted at will in the modeling software, further developing different types of cell cultured meat products. , helping to improve market consumer acceptance of cell-cultured meat;
  • the microfluidic chip used as a printing nozzle can be flexibly replaced, and the chip channel structure can also be customized according to production needs, thereby producing cells with different structures and components.
  • Cultured meat has great potential for the diversification and personalized customization of cell-cultured meat products;
  • the microfluidic 3D printing device developed by the present invention can integrate multiple microfluidic chips on the 3D printing mobile system as needed, and the carrier platform can also be combined with the transmission system to realize multi-nozzle assembly line printing, which is helpful To promote the scale and industrialization of cell-cultured meat.
  • Figure 1 is a schematic diagram of a device for cell cultured meat produced based on microfluidic 3D printing technology according to the present invention
  • Figure 2 is a schematic diagram and photo of the microfluidic chip channel structure of the cell-cultured meat production equipment based on microfluidic 3D printing technology of the present invention.
  • the scale bar is 200 ⁇ m;
  • Figure 3 is a physical diagram of the cell-cultured meat produced based on microfluidic 3D printing technology according to the present invention.
  • (a) is a physical diagram of the printing process;
  • (b) is a physical diagram of the finished product, and the scale bar is 1000 ⁇ m;
  • Figure 4 shows printed products of different shapes constructed based on microfluidic 3D printing technology according to the present invention, in which (a) is a triangle, (b) is a hexagon, (c) is a circle, and (d) is a heart shape.
  • Scale bar is 1000 ⁇ m;
  • Figure 5 is a physical diagram of the cell cultured meat produced based on the multi-nozzle microfluidic 3D printing technology of the present invention, in which (a) is a physical diagram of the printing process, (b) is a physical diagram of the finished product, and the scale bar is 1000 ⁇ m;
  • Figure 6 is a bright field view of a microscope during the cell culture meat culture process produced based on microfluidic 3D printing technology.
  • (a) is a bright field view of a three-dimensional tissue microscope 4 hours after printing is completed, and
  • (b) is a view of the culture for 2 hours.
  • Bright field view of Tianhou's three-dimensional tissue microscope, scale bar is 400 ⁇ m;
  • Figure 7 is an immunofluorescent staining diagram of tissue after maturation of cell-cultured meat produced based on microfluidic 3D printing technology of the present invention, in which (a) is the cell nucleus, (b) is the cytoskeletal protein, (c) is myosin, (d) is the fused image, the scale bar is 100 ⁇ m;
  • Figure 8 is a polyacrylamide gel electrophoresis gel imaging diagram of the protein composition of cell-cultured meat produced based on microfluidic 3D printing technology of the present invention and that of commercially available pork;
  • Figure 9 is a comparative view of the appearance of the finished product of cell cultured meat produced based on microfluidic 3D printing technology and commercially available pork, in which pork (left) and cell cultured meat (right), the scale bar is 2000 ⁇ m;
  • Figure 10 is a comparison data chart of the texture properties of the finished product of cell cultured meat produced based on microfluidic 3D printing technology and that of pork.
  • (a) is the hardness
  • (b) is the chewiness
  • (c) is the elasticity.
  • (d) is cohesion;
  • Figure 11 is a data chart showing changes in cell differentiation-related genes and protein expression levels based on qPCR and Western Blot during the bionic fiber differentiation and culture process, in which (a) is the MyoG gene, (b) MyHC-2a gene, (c) is MyHC- slow gene, (d) is the Western Blot band diagram of the related protein, (e) is the gray value analysis of the MyoG protein band, (f) is the gray value analysis of the Myosin protein band.
  • Figure 12 shows the immunofluorescence staining diagram and statistical diagram after the bionic fiber culture has matured.
  • (a) is the immunofluorescence staining diagram, i is the cell nucleus, ii is the cytoskeletal protein, iii is myosin, iv is the fusion image, and the scale bar is 100 ⁇ m;
  • (b) is a statistical analysis chart of cytoskeleton protein orientation,
  • (c) is a statistical analysis chart of nuclear roundness and aspect ratio,
  • (d) is a statistical analysis chart of myosin-positive cells and myotube area.
  • the raw materials and reagents used in the examples are all commercially available.
  • the seed cells are all obtained using existing conventional separation and purification methods or directly commercially available.
  • the equipment includes a printing nozzle 1, a printing movement system 2, a loading platform 3, a sampling system 4, a printing control and display system 5, a data transmission system 6, and a base 7.
  • the printing nozzle 1 is a microfluidic chip, which is clamped and fixed on the x-axis moving optical axis 21 in the 3D printing movement system 2.
  • the x-axis moving optical axis 21 drives it to move in the x-axis direction; and the x-axis moving optical axis 21 is connected to the z-axis moving optical axis 22 through bolts, and the z-axis moving optical axis 22 drives it to move in the z-axis direction.
  • the carrier platform 3 is assembled on the y-axis moving optical axis 23 in the printing movement system 2 through buckles.
  • the y-axis moving optical axis 23 drives the carrier platform 3 and the printed products formed on the carrier platform 3 in the y-axis direction.
  • Mobile, the loading platform 3 is detachable to collect samples.
  • the moving optical axis, loading platform and base are generally made of aluminum alloy.
  • the sampling system 4 includes a sample loader 41, a sample pump 42 and a conduit 43.
  • the sample loader 41 is fixed on the sample pump 42 and can be flexibly disassembled for loading printing materials.
  • One end of the conduit 43 is connected to the outlet of the sample loader 41, and the other end of the conduit 43 is connected to the outlet of the sample loader 41. Connect to the inlet of print head 1.
  • the present invention has no special restrictions on the type of the sampling pump 42. It is enough to use a syringe pump that is well known to those skilled in the art and can be used with syringes.
  • the sampling pump 42 is Longer Pump LSP01-1A Micro Injection. Pump.
  • the sample loader 41 uses a syringe that is well known to those skilled in the art, and there are no special restrictions on the brand, type, and size of the syringe; the conduit 43 uses a polyethylene plastic tube that is well known to those skilled in the art. There are no special restrictions on the brand, type, and size of the pipe.
  • the outer diameter of the polyethylene plastic pipe is 1.3 mm and the inner diameter is 0.9 mm.
  • the print control and display system 5 and the data transmission system 6 are integrated with the base 7.
  • the front of the base 7 is opened and the print control display system 5 is embedded.
  • the top of the base is drilled and embedded with the data transmission system 6 interface.
  • the data transmission system 6 is connected to the base 7 through a data cable.
  • the printing control display system 5 is connected to the printing mobile system 2 through a data line connection. Specifically, the front and top edges of the cuboid base 7 are open and connected to the print control display system 5 and the data transmission system 6.
  • the print control display system 5 is mainly used to control print leveling, select print programs, and issue print instructions.
  • the position of the printing movement system 2 is adjusted; the data transmission system 6 is used to transmit the printing instruction file to the 3D printer; the data transmission form of the data transmission system 6 includes USB transmission, memory card transmission or computer transmission.
  • Microfluidic chips can be single-channel devices used to print solid fibers. Draw the outlet of a glass capillary tube to an outer diameter of 200 ⁇ m and an inner diameter of 100 ⁇ m. Use AB glue to stick the drawn glass capillary tube to the glass sheet to build a single-channel microfluidic chip.
  • the microfluidic chip can also be a co-nested device, used to print hollow type, "shell-core" fiber, spiral fiber, bead type, etc.
  • the present invention has no special limitation on the number of channels of the catheter in the microfluidic chip. The number of channels of the catheter can be double channels, three channels or four channels.
  • a coaxial nested microfluidic chip is specifically used.
  • the coaxial nested microfluidic chip includes an internal phase glass capillary for introducing the internal phase solution, and an external phase glass capillary for introducing the external phase solution.
  • the coaxial nested microfluidic chip consists of a glass capillary tube, a dispensing needle and a glass piece.
  • the glass capillary tube is cylindrical and the dispensing needle is a 20G dispensing needle. There are no special restrictions on the type and size of the glass piece.
  • the glass slide is a commercially available slide, and the thickness of the slide is 1 mm; the length of the slide is 30 mm, and the width is 25 mm.
  • the specific method is: select a cylindrical glass capillary tube with an inner diameter of 580 ⁇ m and an outer diameter of 1000 ⁇ m, and draw the outlet to an inner diameter of about 80 ⁇ m as an internal phase channel; then select a cylindrical glass capillary tube with an inner diameter of 580 ⁇ m and an outer diameter of 1000 ⁇ m. Glass capillary tube, the outlet is drawn to an inner diameter of about 200 ⁇ m, used as an external phase channel. Fix the external phase channel in the middle position on the glass slide, and then insert the pulled end of the internal phase channel from one end of the external phase channel to ensure that the two phase channels do not block each other. Adjust the external phase channel and the internal phase channel to the same position under the stereomicroscope.
  • the printing nozzle 1 in this embodiment is configured to be composed of multiple microfluidic chips to perform multi-nozzle microfluidic 3D printing.
  • the materials for making microfluidic chips can be replaced by crystalline silicon, polydimethoxysiloxane, quartz, polyamide, polymethylmethacrylate, polycarbonate, polystyrene, epoxy resin, acrylic , rubber and fluoroplastics.
  • Preparation of microfluidic external phase fluid Take an appropriate amount of sodium alginate powder and place it in a ultra-clean workbench for ultraviolet irradiation sterilization overnight. Use a pipette to measure 20mL of sterile water into a centrifuge tube. Weigh 0.6g of sodium alginate powder using an electronic balance on a clean workbench and pour it into the centrifuge tube. Use a vortexer to mix evenly, and then place the centrifuge tube in the centrifuge tube.
  • Preparation of microfluidic internal phase fluid Weigh 0.1g calcium chloride into a centrifuge tube, add 5mL of DMEM basic medium containing phenol red (C11995500CP, Gibco) to dissolve, and prepare a DMEM solution containing 20mg/mL calcium chloride. , use a 0.22 ⁇ m filter membrane to filter and sterilize, and store on ice for later use; weigh 0.2g NaOH in a centrifuge tube, add 5mL of ultrapure water to dissolve, and prepare a 1mol/L NaOH solution, use a 0.22 ⁇ m filter membrane to filter and sterilize, Store on ice until later use.
  • the same preparation method as above can be used, except that: the cell non-adhesive material solution is chitosan with a concentration of 10 mg/mL; the component of the hydrogel solution is gelatin with a volume fraction of 30% , genipin solution with a volume fraction of 1%, F-10 culture medium containing calcium chloride and 5 ⁇ 10 6 cells/mL bovine muscle stem cells with a volume fraction of 69%.
  • the cell non-adhesive material solution is pectin with a concentration of 50 mg/mL
  • the components of the hydrogel solution are hyaluronic acid with a volume fraction of 70% and carbodiazepine with a volume fraction of 1%.
  • the cell non-adhesive material solution is carrageenan with a concentration of 25 mg/mL; the components of the hydrogel solution are fibrinogen with a volume fraction of 50% and coagulation with a volume fraction of 0.5%.
  • the effects of the present invention can be achieved by using a certain amount of muscle stem cells from pigs, cattle, sheep, chickens, ducks, etc. for culture.
  • Example 1 Using the coaxial nested microfluidic chip constructed in Example 1, add the sodium alginate solution prepared in Example 2 into a 5mL syringe, and use a section of polyethylene plastic tube to connect one end of the syringe needle and the other end to the microfluidic chip.
  • the external phase inlet add the hydrogel solution containing porcine muscle stem cells prepared in Example 2 into a 2 mL syringe, connect one end of the polyethylene plastic tube to the syringe needle, and connect the other end to the internal phase inlet of the microfluidic chip.
  • the syringes containing the two-phase fluid were fixed on the two pumps respectively, and the flow rate of the inner phase hydrogel solution was adjusted to 1.7 mL/h, and the concentration of the outer phase sodium alginate solution was 1.8 mL/h.
  • the internal and external phase printing materials are introduced into the microfluidic chip through the polyethylene plastic tube, so that the two-phase fluid forms a stable laminar flow structure in the device from the outlet of the microfluidic chip (i.e., the outlet of the external phase channel).
  • Biomimetic fiber with "shell-core" structure Biomimetic fiber with "shell-core" structure.
  • the printing program is selected through the printing control display system 5 and the entire microfluidic 3D printing device is started. Then the printing movement system 2 of the microfluidic 3D printing device drives the microfluidic process.
  • the control chip moves on the x and z axes, and the printed sample is driven by the stage on the y axis.
  • the moving speed of each optical axis is 5mm/s, so that the generated fibers are deposited on the stage to follow the G-code printing instruction path.
  • Stack the molding After printing, the three-dimensional tissue is obtained. Prepare a 10 mg/mL calcium chloride solution, sterilize it, and use it as an ionic cross-linking agent.
  • Auto CAD 2021 software can be used to build three-dimensional models of different shapes. After slicing and printing, three-dimensional structures of different shapes such as triangles, hexagons, circles and heart shapes can be obtained ( Figure 4).
  • magnetic integration means can be further used to install multiple microfluidic chips on the x-axis moving optical axis of the printing movement system 2, and multi-nozzle microfluidic 3D printing can be performed according to the above single-nozzle printing operation.
  • Figure 5 shows real-time photos and finished photos of cell-cultured meat constructed by a multi-nozzle microfluidic 3D printing device integrating four microfluidic chips.
  • the three-dimensional tissue after the final printing process in Example 3 was transferred to a 10 cm diameter petri dish containing proliferation culture medium (proliferation culture medium: volume fraction 84% DMEM/F-12 (Gibco, 11550043), 15% fetal bovine serum (Gibco, 10270-106), 1% penicillin-streptomycin (Gibco, 15140122), containing fibroblast growth factor bFGF (R&D, 233-FB-500/CF) at a final concentration of 5ng/mL, washed and infiltrated for 10 minutes , the amount of proliferation culture medium in the culture dish is enough to submerge the three-dimensional tissue, and then transfer the three-dimensional tissue to an incubator at 37°C and 5% CO 2 for proliferation and culture for 2 days;
  • proliferation culture medium proliferation culture medium: volume fraction 84% DMEM/F-12 (Gibco, 11550043), 15% fetal bovine serum (Gibco, 10270-106), 1% penicillin-streptomycin (
  • the proliferation culture medium was aspirated, and then used with DMEM without serum. Wash the three-dimensional tissue 2-3 times with basal medium. After cleaning, add differentiation culture medium (volume fraction 97% DMEM (C11995500CP, Gibco), 2% horse serum (Hyclone, SH30074.02), 1% penicillin-streptomycin (Gibco, 15140122)) to the culture dish. The amount of differentiation culture medium in the culture dish is just enough to submerge the three-dimensional tissue. Place it at 37°C and 5% CO2 to continue differentiation culture.
  • differentiation culture medium volume fraction 97% DMEM (C11995500CP, Gibco)
  • horse serum Hyclone, SH30074.02
  • penicillin-streptomycin Gabco, 15140122
  • the Myosin protein of seed cells in the fiber was significantly higher than that of the ordinary two-dimensional culture group (the ordinary two-dimensional culture group directly used the conventional porcine muscle stem cell differentiation and culture method to proliferate and differentiate the porcine muscle stem cells, and its cells
  • the usage amount, proliferation, differentiation and culture time are completely consistent with the above-mentioned tissue culture), further indicating that the differentiation ability of seed cells has been significantly improved, and the synthesis of muscle-related proteins has increased, which helps to improve the production efficiency of cell-cultured meat, which can be observed by observing the fiber surface.
  • exposed seed cells and myotube structures and showed a tissue structure very similar to that of pork skeletal muscle fibers.
  • the meat-related aspects of cell cultured meat are The types and band positions of proteins (myosin heavy chain, actin and myosin light chain proteins, etc.) are similar to those of commercially available pork; and amino acid analysis shows that the various amino acid contents of cell-cultured meat are different from those of commercially available pork. Not much, especially the contents of Gly (glycine), Cys (cysteine) and Pro (proline) are very close to commercially available pork.
  • the bionic fiber was transferred to a medium containing proliferation culture medium (volume fraction 84% F-10 (Gibco, 11550043), 15% fetal bovine serum (Gibco, 10270-106), 1% penicillin-streptomycin (Gibco , 15140122), in a 10cm sterile cell culture dish containing fibroblast growth factor bFGF (R&D, 233-FB-500/CF) at a final concentration of 5ng/mL, and then place the culture dish at 37°C and 5% CO 2 Proliferation culture was carried out in an incubator for 2 days. Observe under the bright field of the microscope.
  • the proliferation culture medium was aspirated, and then the DMEM basic medium without serum was used. Clean the bionic fiber 2-3 times. After cleaning, add differentiation culture medium (volume fraction 97% DMEM (C11995500CP, Gibco), 2% horse serum (Hyclone, SH30074.02), 1% penicillin-streptomycin (Gibco, 15140122)) to the culture dish. The differentiation culture was continued under the conditions of 37°C and 5% CO2 , and then 1/2 of the differentiation culture medium in the culture dish was replaced every two days. Mature bionic fibers were obtained after 7 days of differentiation and culture.
  • RT-qPCR and Western Blot were used to evaluate the changes in differentiation-related gene and protein expression of seed cells grown on biomimetic fibers and two-dimensional plates from the molecular biology level.
  • Two of the The seed cells in the dimensional dish are conventionally directly used to differentiate and culture porcine muscle stem cells.
  • the porcine muscle stem cells are inoculated onto a sterile 3.5cm diameter culture dish covered with Matrigel for proliferation and differentiation culture. The cell usage, proliferation, Differentiation and culture time are completely consistent with bionic fibers.
  • Trizol was used to lyse the cells in the biomimetic fibers and two-dimensional dishes, and the cultured cell total RNA extraction kit of Tiangen Biochemical Co., Ltd. was used to extract RNA from the lysed cells; assay After determining the concentration of RNA in the sample, use a reverse transcription kit to perform reverse transcription on the RNA to obtain cDNA.
  • the reverse transcription program is set to 37°C for 15 minutes and 85°C for 5 seconds; then, use an RT-qPCR kit to reversely transcribe the cDNA. Carry out qPCR reaction.
  • the target genes are MyoG, MyHC-2a and MyHC-slow.
  • the reaction program is 95°C for 30s, 95°C for 5s, and 60°C for 30s.
  • Myogenin, MyoG myogenin gene
  • the expression of myogenin gene (Myogenin, MyoG) in seed cells cultured on bionic fibers at the beginning of differentiation (Day 0) is more than 300 times higher than that in the two-dimensional plate culture control.
  • the expression of muscle maturation markers-myosin synthesis-related genes MyHC-2a and MyHC-slow in seed cells cultured in bionic fibers was significantly higher than that in the two-dimensional plate culture control group.
  • RIPA lysis solution to lyse the cells in the bionic fiber and two-dimensional dish on ice to obtain cellular protein samples.
  • the collected protein samples were centrifuged at 12,000 rpm for 5 minutes at 4°C and the supernatant was collected.
  • the BCA kit was used to determine the protein concentration of the samples. Dilute the sample protein concentration to 1.25 mg/mL, then add a quarter volume of 5 ⁇ Loading buffer to the sample, mix and heat at 95°C for 5 minutes to denature the protein. Take 20 ⁇ L of denatured protein from each sample for SDS-PAGE gel electrophoresis.
  • the electrophoresis conditions are 80V for 30min and 120V for 70min.
  • PVDF membrane of appropriate size, use rapid wet transfer to transfer the membrane, cut out the band corresponding to the protein molecular weight (MyHC: 220kDa; MYOG: 34kDa; GAPDH: 36kDa), use 5% skim milk powder to block the membrane, and use the primary antibody Incubate overnight at 4°C, and incubate with the secondary antibody at room temperature for 2 hours; mix developer solution A and solution B at a ratio of 1:1, drop it onto the strip, and incubate in the dark for 5 minutes, then aspirate the developer solution, use an imager to develop and Take pictures and use imageJ software to analyze the gray value of protein bands.
  • MyHC 220kDa
  • MYOG 34kDa
  • GAPDH 36kDa
  • the MyoG protein of seed cells in the bionic fiber was 2.2 times and 2.4 times higher than that of the two-dimensional culture group respectively; at the early stage (day 0), middle stage (day 3) and end stage of differentiation ( Day 7), the Myosin protein of seed cells in the bionic fiber was 2.66, 1.78 and 2 times higher than that of the two-dimensional culture group, indicating that the differentiation ability of the seed cells has been significantly improved, and the synthesis of muscle-related proteins has increased, which helps to improve the production efficiency of cell cultured meat. .
  • the cytoskeletal proteins in the bionic fibers are oriented along the direction of the fiber (it can be observed that the direction of F-actin is consistent with the direction of the fiber, and the seed cells are Highly directional growth), the myogenic marker protein myosin has a high expression, indicating that the seed cells are directionally arranged, migrated and fused in the bionic fibers, and their differentiation ability has been significantly improved, and the synthesis of muscle-related proteins has increased.

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Abstract

本发明公开了一种基于微流控3D打印技术的细胞培养肉生产设备及其应用，该设备包括打印喷头、打印移动系统、载物平台、进样系统、底座，打印移动系统置于底座上并由多个可移动光轴组成，打印喷头设置在移动光轴上，进样系统与打印喷头相连接，打印喷头为微流控芯片。本发明基于微流控3D打印，生物墨水经微流控芯片挤出形成打印纤维，并在打印移动系统的带动下堆叠成型，实现一步式构建具有组织各向异性的整块三维组织用于细胞培养肉生产，其操作简单、成型迅速，微流控芯片的结构、数量等可按需设计和灵活替换、增减，且种子细胞在打印三维组织的纤维状基本结构中定向排列并进一步迁移、融合，提高了种子细胞的分化成熟及相关蛋白的合成效率。

Description

一种基于微流控3D打印技术的细胞培养肉生产设备及其应用 技术领域
本发明属于细胞培养肉领域，具体涉及基于微流控3D打印技术的细胞培养肉生产设备及其应用。
背景技术
肉类是世界各国居民膳食中主要的蛋白质来源。随着世界人口的增长和发展中国家居民生活水平的提高，人们对肉类的需求也愈来愈大，这将迫使传统畜牧养殖业扩大生产规模来满足日益增长的肉类需求。然而，作为一种资源密集型产业，传统畜牧养殖会带来严重的水、土地资源占用，排放大量温室气体，同时集约化养殖带来的食源性疾病和动物福利问题也不容小觑。因此，开发能够替代传统畜牧养殖的新型肉类生产技术具有重要意义。
细胞培养肉是一项诞生于细胞工程、组织工程和食品工程等学科交叉下的新兴肉类生产技术，其通过对肌肉干细胞进行体外大规模扩增、诱导分化、产物收集和食品化处理而获取肉类。相较于传统畜牧养殖业，细胞培养肉可大幅降低能源消耗、水资源滥用和温室气体排放，并解除约99％的土地资源占用。自2013年荷兰Mark Post教授宣布世界第一块细胞培养肉诞生起，世界范围内便掀起对细胞培养肉研究和产业化的热潮。尽管有不错的研究进展，但是现有已知生产手段在肌纤维形成能力、肉类结构仿真和规模化上仍十分有限。
迄今，越来越多组织工程技术被运用于细胞培养肉的生产，比如动、植物蛋白支架、水凝胶、细胞片工程、生物3D打印等。其中，生物3D打印是一种在三维模型指令的驱动下，依据增材制造原理定位装配生物材料或细胞单元，构建组织工程支架和组织器官的先进技术，在细胞培养肉组织构建方面具有重大潜力。现阶段，市面上生物3D打印机设备普遍造价高昂、维护成本高，打印喷头结构、种类单一，喷头更换灵活性差，且在批量打印上存在较大的限制；此外，目前市场上还未见到针对细胞培养肉生产的专用3D打印设备。因此，开发适用于细胞培养肉生产的专用3D打印设备为实现低成本、定制化、规模化、方便高效生产细胞培养肉值得期待。
发明目的
发明内容：针对现有技术存在的问题，本发明提供一种基于微流控3D打印技术的细胞培养肉生产设备，有效解决了现有生物3D打印机设备成本高、打印喷头功能单一、灵活性差以及规模化有限的问题，进一步填补了针对细胞培养肉生产领域3D打印设备的空白。
本发明还提供所述基于微流控3D打印技术的细胞培养肉生产设备在制备细胞培养肉中的应用。
技术方案：为了实现上述目的，本发明所述一种基于微流控3D打印技术的细胞培养肉生产设备，所述设备包括打印喷头、打印移动系统、载物平台、进样系统、底座；所述打印移动系统置于底座上并由多个可移动光轴组成，所述打印喷头固定在其中一个可移动光轴上，载物平台与另一个可移动光轴相连接；所述进样系统与打印喷头相连接，所述打印喷头为微流控芯片，所述微流控芯片为可在通道中对微量液体或样品在进行操纵、处理与控制的装置。
作为优选，所述打印移动系统包括x轴移动光轴、z轴移动光轴和y轴移动光轴，所述z轴移动光轴和y轴移动光轴固定在底座上，x轴移动光轴与z轴移 动光轴连接，所述打印喷头固定在x轴移动光轴上，载物平台置于与y轴移动光轴)上相连接。
其中，所述打印移动系统为可带动打印喷头在x轴移动光轴、z轴移动光轴上两个方向移动，带动载物平台在y轴移动光轴上移动的移动轴，其配置的移动坐标系包括笛卡尔坐标系、三角坐标系、极坐标系和平面关节坐标系中的任意一种。
作为优选，所述打印喷头集成于打印移动系统中的x轴移动光轴上，x轴移动光轴又与z轴移动光轴相连接，打印喷头由x、z轴移动光轴带动着在xz平面内移动。
其中，所述打印移动系统上微流控芯片类型可以灵活替换，根据不同生产需求进行集成打印。
进一步地，所述打印移动系统上可集成一个或者多个微流控芯片作打印喷头，进一步开发多喷头并行打印装置，实现多喷头微流控3D打印装置的搭建。
进一步地，所述打印移动系统上集成多个微流控芯片时，可使用通道结构相同或者通道结构各异的微流控芯片。
进一步地，所述微流控芯片通过夹持、扣接、插接、磁吸、榫接、铆合、螺纹连接或者卡口连接集成到打印移动系统上。
其中，所述载物平台为可拆卸结构，装配于打印移动系统中，与y轴移动光轴连接，进行流水线打印；所述载物平台的材质为铜、铝、铁、钢、合金、玻璃、陶瓷或者碳纤维板材。
作为优选，载物平台与打印移动系统中的y轴移动光轴相结合，为可拆卸结构，y轴移动光轴带动载物平台及成型在载物平台上的打印品在y轴方向移动。
进一步地，所述所述载物平台与y轴移动光轴通过卡扣式连接，可灵活拆卸。
作为优选，所述进样系统由装样器、进样泵和导管组成，装样器固定在进样泵上，导管将装样器的出口和打印喷头的进样口连接。
其中，所述进样系统包括装样器、进样泵和导管，所述进样泵置于水平桌面，装样器固定在进样泵上，导管一端连接装样器的出口，一端连接打印喷头的进样口，所述进样系统的进料方式包括活塞式挤入、气动式挤入或者螺杆式挤入。
作为优选，所述装样器通过卡扣固定在进样泵上，可灵活拆卸，导管一端与装样器的出口连接，一端与打印喷头的进口连接。
作为优选，所述底座中嵌入安装打印控制显示系统和数据传输系统；数据传输系统通过无线或者数据线连接至打印控制显示系统，打印控制显示系统通过无线或者数据线连接与打印移动系统连接。其中，所述打印控制显示系统包括控制显示屏，主要用于控制打印调平(调零，让打印喷头复位、归零、调至与打印平台水平等)、打印程序的选择、打印指令的下达、打印移动系统位置调整。可通过在显示屏上操作，实现选择打印指令文件、调整打印速度、打印喷头位置、载物平台的位置等功能。
其中，所述数据传输系统用于将打印指令文件传输进3D打印设备；所述数据传输系统的数据传输形式包括USB传输、内存卡传输或者电脑传输。所述 数据传输系统包括USB、储存卡等存储设备的插入口，主要用于向打印机中打印控制显示系统导入打印指令文件。
进一步地，打印控制显示系统和数据传输系统与底座为一个整体，底座前方开口后嵌入打印控制显示系统，底座上方打孔后嵌入数据传输系统接口。打印控制显示系统和数据传输系统嵌入底座后，跟随底座一起接电。
作为优选，将长方体底座前部和顶部边缘处开口，嵌入固定打印控制显示系统和多种数据传输连接口作数据传输系统；然后，将底座置于水平桌面上，使用螺栓将y轴移动光轴和z轴移动光轴固定在底座上，再将x轴移动光轴连接至z轴移动光轴上，即组装成功打印移动系统；然后，将打印喷头固定在x轴移动光轴上，载物平台则连接至y轴移动光轴。进样系统由装样器、进样泵和导管组成，装样器固定在进样泵上，导管将装样器出口和打印喷头进样口连接。打印过程中，打印喷头被x轴移动光轴带动在x轴上移动，x轴移动光轴又可在z轴移动光轴上滑动，载物平台上的打印组织被y轴移动光轴带动在y轴上，3个方向的移动光轴互相配合，将打印喷头生成的纤维在x，y，z轴方向堆叠成型。其中，微流控芯片是一种可在通道中对微量液体或样品(体积一般为10-6-10-15L)在微观尺度上进行操纵、处理与控制的装置。在打印过程中，将打印材料装入进样系统的装样器中，并将装样器固定在进样泵上，再用导管将装样器与打印喷头连接，利用进样泵将装样器中的材料泵入打印喷头。而打印指令文件通过数据传输系统导入打印控制显示系统中，在打印控制显示系统中选择目标打印指令文件后，启动整个3D打印生产设备；生成的纤维在载物平台上沉积并依据打印指令文件在x、y、z三个方向堆叠成型，完成后拆卸载物平台，收取打印成品用于后续加工操作。
其中，所述微流控芯片的制作材料包括但不限于晶体硅、聚二甲氧基硅氧烷、玻璃、石英、聚酞胺、聚甲基丙烯酸甲酯、聚碳酸酯、聚苯乙烯、环氧树脂、丙烯酸、橡胶和氟塑料中一种或者多种；所述微流控芯片的制作方法包括玻璃毛细管组装法、机械加工法、刻蚀法或者模具法。
其中，所述微流控芯片类型为所述微流控芯片类型为单通道型、共轴嵌套型或者多通道并行型。
作为优选，所述微流控芯片可基于不同结构的微流控芯片，可生成实心型、“壳-核”型、空心型、多组分型、螺旋形、串珠型纤维用于微流控3D打印。本发明通过搭建不同结构的微流控芯片，对通入的流体种类、流速等进行调整与设计，即可生成不同结构的纤维。
作为优选，所述微流控芯片为共轴嵌套型微流控芯片。
进一步地，所述微流控芯片出口内径尺寸范围为200-2000μm。
本发明所述的基于微流控3D打印技术的细胞培养肉生产设备在制备细胞培养肉中应用，所述制备细胞培养肉的过程为：
(1)配制微流控3D打印所需的生物墨水，备用；
(2)将打印指令文件导入打印设备，将步骤(1)配制的生物墨水装填入进样系统；将进样系统连接至打印喷头的进口处，即微流控芯片的通道入口处，将生物墨水经导管挤入打印喷头即微流控芯片；待纤维在微流控芯片出口处生成后，选定需要打印的指令文件，启动整个设备；打印喷头出口生成的纤维在打印移动系统的各个光轴的带动下沉积于载物平台，并遵循打印指令路径堆叠成型；
(3)将载物平台拆卸，并对步骤(2)打印得到的三维组织进行交联固化处理，然后转移至相应的培养液中进行增殖、分化培养；
(4)将步骤(3)中培养成熟的三维组织收获，清洗去除培养液后，进行食品化处理即可得到细胞培养肉。
作为优选，步骤(2)中将打印指令文件从数据传输系统处导入打印机，将步骤(1)配制的生物墨水装填入装样器中并连接导管，再将装样器固定在进样泵上；将导管另一端连接至打印喷头的进口处，即微流控芯片的通道入口处，使用进样泵将装样器中的生物墨水经导管挤入微流控芯片；待纤维在微流控芯片出口处生成后，在打印控制显示系统中选定需要打印的指令文件，启动3D打印设备，具体可以在打印控制显示系统上点击打印指令文件后，打印控制显示系统会出现“打印”或者“取消”两个指令，再次点击“打印”即启动整个设备。设备启动后，由打印喷头的出口处生成的纤维在打印移动系统的x轴移动光轴、z轴移动光轴和y轴移动光轴的带动下沉积于载物平台，并遵循打印指令路径堆叠成型；
其中，步骤(1)中所述生物墨水包括具有细胞非粘附性的材料溶液和含有种子细胞的水凝胶溶液；所述含有种子细胞的水凝胶溶液中含体积分数30％-70％生物材料、0.01％-1％交联剂，余量为含钙盐、含5×106-5×108个/mL种子细胞的基础培养基。
作为优选，所述生物墨水中所述具有细胞非粘附性材料溶液为海藻酸钠、壳聚糖、果胶、卡拉胶、结冷胶中的任意一种或多种；所述具有细胞非粘附性的材料溶液的浓度为10-50mg/mL。
其中，所述生物墨水中所述种子细胞来源于猪、牛、羊、鸡、鸭、兔、鱼、虾和蟹中的任意一种或者多种；所述种子细胞为肌肉干细胞、成肌细胞、肌卫星细胞、肌肉前体细胞、骨髓源间充质干细胞、脂肪源间充质干细胞、诱导多能干细胞、心肌细胞、脂肪干细胞、脂肪前体细胞、骨髓源脂肪成体细胞、成纤维细胞、平滑肌细胞、血管内皮细胞、上皮细胞、神经干细胞、神经胶质细胞、成骨细胞、软骨细胞、肝脏干细胞、造血干细胞、基质细胞、胚胎干细胞、骨髓干细胞中的一种或者多种。
作为优选，所述种子细胞来源猪、牛、羊、鸡、鸭中的任意一种或者多种；所述种子细胞包括但不限于肌肉干细胞、肌细胞、肌卫星细胞、肌肉前体细胞中的一种或者多种。
其中，步骤(1)所述生物材料为胶原蛋白、重组胶原蛋白、明胶、基质胶、透明质酸、丝素蛋白、弹性蛋白、蛛丝蛋白、纤维蛋白、纤维蛋白原、丝纤蛋白、层粘连蛋白、纤粘连蛋白、整合蛋白、钙粘蛋白、巢蛋白、脱细胞外基质、硫酸软骨素、肝素、硫酸角质素、硫酸皮肤素、硫酸乙酰肝素、角蛋白、硫酸角蛋白、纤维素、聚合素、羧甲基纤维素、聚乳酸、聚乙烯醇、卵磷脂、纳米纤维素、大豆蛋白、豌豆蛋白、面筋蛋白、大米蛋白、花生蛋白、酵母蛋白、真菌蛋白、小麦蛋白、土豆蛋白、玉米蛋白、鹰嘴豆蛋白、绿豆蛋白、海藻蛋白、杏仁蛋白、藜麦蛋白中的一种或者多种以及其他具有生物相容性和能够为种子细胞提供黏附位点的材料。
作为优选，所述生物材料为胶原蛋白、重组胶原蛋白、明胶、基质胶、透明质酸、丝素蛋白中的一种或者多种。其中，所述生物墨水成分中使用的基础培养基包括但不限于F-10、DMEM、MEM、F-12、DMEM/F-12、DMEM/F-12  GlutamMAXTM、F-12K、RPMI 1640、IMDM、L-15、199、MCDB 131、LHC、McCoy's 5A中的一种或者多种。
作为优选，所述基础培养基为F-10、DMEM、MEM、F-12、DMEM/F-12中的一种或者多种。其中，所述生物墨水成分中使用的交联剂包括但不限于NaOH、KOH、NaHCO3、HEPES平衡盐溶液、EBSS平衡盐溶液、HBSS平衡盐溶液、PBS、DPBS等pH调节剂，转谷氨酰胺酶、酪氨酸酶、漆酶、赖氨酰氧化酶、多酚氧化酶、过氧化氢酶、凝血酶、京尼平中的任意一种或者多种。
作为优选，所述交联剂包括NaOH、KOH、NaHCO3中的一种或者多种。
作为优选，所述水凝胶溶液中包括生物材料、交联剂，含钙盐、含种子细胞的基础培养基；每1mL水凝胶溶液中含290-699μL 4-8mg/mL的生物材料，1-10μL 1-2mol/L交联剂，300-700μL含15-25mg/mL钙盐、1×107-1×108种子细胞的基础培养基；所述生物材料为胶原蛋白、重组胶原蛋白、明胶、基质胶、透明质酸、丝素蛋白中的一种或者多种；所述交联剂为NaOH、KOH、NaHCO3中的一种或者多种；所述钙盐为氯化钙、碳酸钙、硫酸钙、硝酸钙中的一种或者多种；所述基础培养基为F-10、DMEM、MEM、F-12、DMEM/F-12中的一种或者多种；所述种子细胞为猪、牛、羊、鸡、鸭的肌肉干细胞、成肌细胞、肌卫星细胞、肌肉前体细胞中的一种或者多种。
更为优选地，所述水凝胶溶液中包括生物材料、交联剂，含钙盐、含种子细胞的基础培养基；每1mL水凝胶溶液中所述生物材料为150-650μL 6mg/mL胶原蛋白和40-149μL基质胶，交联剂为1-10μL 1mol/L碱溶液，含钙盐、种子细胞的基础培养基为300-700μL含15-25mg/mL钙盐的DMEM溶液重悬1.5×106-1.5×108个肌肉干细胞。
进一步地，所述种子细胞为猪肌肉干细胞；所述水凝胶溶液中包括生物材料、交联剂，含钙盐、种子细胞的基础培养基；1mL水凝胶溶液中所述生物材料为600μL 6mg/mL胶原和97μL基质胶，交联剂为3μL 1mol/L NaOH溶液，含钙盐、种子细胞的基础培养基为300μL含20mg/mL氯化钙的DMEM溶液重悬1.5×107个猪肌肉干细胞。
作为优选，所述交联剂包括NaOH、KOH、NaHCO3中的一种或者多种。
作为优选，所述含有种子细胞的水凝胶溶液中包括生物材料、交联剂，含钙盐、含种子细胞的基础培养基；每1mL水凝胶溶液中含290-699μL 4-8mg/mL的生物材料，1-10μL 1-2mol/L交联剂，300-700μL含15-25mg/mL钙盐、1×107-1×108种子细胞的基础培养基；所述生物材料为胶原蛋白、重组胶原蛋白、明胶、基质胶、透明质酸、丝素蛋白中的一种或者多种；所述交联剂为NaOH、KOH、NaHCO3中的一种或者多种；所述钙盐为氯化钙、碳酸钙、硫酸钙、硝酸钙中的一种或者多种；所述基础培养基为F-10、DMEM、MEM、F-12、DMEM/F-12中的一种或者多种；所述种子细胞为猪、牛、羊、鸡、鸭的肌肉干细胞、成肌细胞、肌卫星细胞、肌肉前体细胞中的一种或者多种。
更为优选地，所述含有种子细胞的水凝胶溶液中包括生物材料、交联剂，含钙盐、含种子细胞的基础培养基；每1mL水凝胶溶液中所述生物材料为150-650μL 6mg/mL胶原蛋白和40-149μL基质胶，交联剂为1-10μL 1mol/L碱溶液，含钙盐、种子细胞的基础培养基为300-700μL含15-25mg/mL钙盐的DMEM溶液重悬1.5×106-1.5×108个肌肉干细胞。
进一步地，所述种子细胞为猪肌肉干细胞；所述含有种子细胞的水凝胶溶 液中包括生物材料、交联剂，含钙盐、种子细胞的基础培养基；1mL水凝胶溶液中所述生物材料为600μL 6mg/mL胶原和97μL基质胶，交联剂为3μL 1mol/L NaOH溶液，含钙盐、种子细胞的基础培养基为300μL含20mg/mL氯化钙的DMEM溶液重悬1.5×107个猪肌肉干细胞。
其中，进一步的，步骤(2)中打印移动系统中的各个移动光轴的移动速度范围为0.5-50mm/s。
其中，步骤(3)中所述交联固化处理的方式包括但不限于温度诱导交联、静电相互作用交联、离子交联、酶交联中的一种或者几种。
其中，步骤(3)中所述培养液配制时使用的基础培养基包括但不限于F-10、DMEM、MEM、F-12、DMEM/F-12、DMEM/F-12GlutamMAXTM、F-12K、RPMI 1640、IMDM、L-15、199、MCDB 131、LHC、McCoy's 5A。
其中，步骤(3)中所述增殖培养液包括体积分数为79-89％基础培养基、10-20％胎牛血清、1％青霉素-链霉素，再向上述溶液中加入1-10ng/mL碱性成纤维细胞生长因子；所述分化培养液的成分为94-97％基础培养基、2-5％马血清和1％青霉素-链霉素。
其中，步骤(4)中所述食品化处理的方法包括前处理和烹饪，所述前处理包括清洗、调味、增色、造型或者感官品质修饰等，所述烹饪包括煎、炸、煮、蒸、烤等。
本发明以纤维材料的3D堆叠成型方式为设计原理，主要思路是对常规的挤出型3D打印机进行改装，使用微流控芯片对挤出型3D打印机的打印喷头进行替换，从而将原本用于堆叠成型的塑料纤维材料变为负载细胞的仿生纤维，进一步构建三维组织。本发明有效解决了现有生物3D打印机设备成本高、打印喷头功能单一、灵活性差以及规模化有限的问题，且填补了针对细胞培养肉生产领域3D打印设备的空白。
本发明使用微流控芯片对挤出型3D打印机的打印喷头进行替换，开发微流控3D打印装置；不同于市场上各类3D打印机上的一体化打印喷头，所述用作打印喷头的微流控芯片的种类、通道结构可灵活设计，用于打印的微流控芯片数量可灵活增减，而且搭建微流控芯片的材料成本低，便捷易得。
本发明基于肌纤维是骨骼肌组织的最基本组成单元，无数条肌纤维经结缔组织膜层层包裹形成大块骨骼肌组织；本发明提出的基于微流控3D打印技术构建的细胞培养肉以仿生纤维为基本单元，层层堆叠后形成三维组织，仿生纤维之于三维组织就像肌纤维之于肌肉，故具有较好的仿生性。相较于仿生纤维，种子细胞在三维组织中生长进一步模拟了其在天然组织中的生长情况，且仿生纤维堆叠的方向可通过打印路径调整，有助于体外重现骨骼肌组织的组织各向异性。
本发明通过对工业3D打印机喷头进行改进，使用成本低、可定制结构的微流控芯片进行替换，并通过对微流控芯片和3D打印机之间的连接方式进行设计，使打印装置上用作喷头的微流控芯片的数量和形式可灵活改变。本发明依托3D打印技术，打印精度(挤出纤维的最小尺寸)取决于微流控芯片出口大小，所述微流控芯片出口内径尺寸范围为200-2000μm，最小尺寸已达到3D打印切片软件标准精度的0.2mm。此外，不同批次打印的细胞培养肉产品在尺寸上几乎无变化，并能准确再现三维模型在切片软件中切片后的打印路径。
本发明3D打印时利用天然骨骼肌组织基本单元--肌纤维结构的原理，给予种子细胞适当的纤维载体保证在细胞培养肉高效生产方面具有非常好的效果； 并首次在细胞培养肉生产中采用微流控技术，能够在微尺寸通道中操控微量液体。本发明以肌纤维仿生为设计原理，提出一种基于3D打印结合微流控的技术用于细胞培养肉生产的仿生纤维载体，且领域内还未见到相关报道使用该手段生产细胞培养肉。本发明基于3D打印微流控技术制备仿生纤维，并用于细胞培养肉生产，纤维的制备过程连续且快速，所制备的纤维具有良好的仿生性，在纤维内生长的种子细胞定向生长能力和分化能力得到极大的提升。本发明基于微流控技术制备具有“壳-核”结构的仿生纤维，种子细胞被包裹于具有细胞非粘附性材料外壳中，并在外壳的空间约束下呈现高度定向、融合生长特性，体外成肌分化能力也得到显著提升，提高了生产效率，并且不论是形状上，还是生理特性上，制备的纤维与天然骨骼肌纤维都十分相似，因此基于微流控技术制备的纤维具有较好的仿生性。
在本发明的3D打印中，本发明首先设计并搭建例如共轴嵌套型微流控芯片；然后准备材料，通过调整内、外相流体的通入顺序、流速等参数，从而稳定生成仿生纤维并进行培养；最后将培养得到的仿生纤维进行组织化集成和食品化处理得到细胞培养肉产品。相较于常见的使用支架、块状水凝胶载体等生产方式，种子细胞被包裹于仿生纤维不具有细胞粘附性材料外壳中，并在外壳的空间约束下在水凝胶内核中呈现高度定向、融合生长特性，体外成肌分化能力也得到显著提升，提高了生产效率。
在本发明中由3D打印形成的具有特定“壳-核”结构的仿生纤维中进行细胞培养，有效提升了微纤维中畜禽原代细胞如肌肉干细胞的分化能力，使肌肉相关蛋白合成增加，并进一步形成成熟的肌纤维，从而提高细胞培养肉生产效率。本发明制备的微纤维经增殖培养2天、分化培养14天后出现自发性收缩跳动现象，这是原代成肌细胞在海藻酸钙外壳包裹的特定内核中迁移、融合形成多核肌管，多核肌管进一步分化并高度表达肌球蛋白(Myosin)，从而形成成熟肌纤维且展现出一定的生理功能的体现。
本发明形成的仿生纤维在经过增殖、分化培养后，在分化7天时，微纤维和猪肉的扫描电镜和H&E组织染色的对比结果显示，微纤维结构紧实，且表面能观察到明显的肌管结构，组织切片也展现出与猪肉纤维接近的染色特性，整体上与猪肉纤维十分接近；在分化14天时，微纤维下明场观察下还出现自发性收缩跳动现象，这是在体外培养条件下实现从细胞到成熟组织的跨越，说明由种子细胞组成的微纤维已经充分成熟形成肌纤维并具有天然肌纤维的收缩功能，本发明已经具有体外培养出一根肌纤维的能力，将这些体外培养的肌纤维进行收集、组装成大块组织理论上即可得到体外培养的一块由肌纤维组成的细胞培养肉。
有益效果：与现有技术相比，本发明具有如下优点：
(1)本发明提出了一种基于微流控3D打印技术的细胞培养肉生产设备，该设备用于细胞培养肉的制备，操作便捷、成型迅速、机械化程度高、所需设备装置成本低，且打印成品精度高、质量稳定；
(2)本发明基于微流控3D打印制备的三维组织具有良好的组织各向异性，进一步模拟了种子细胞在天然组织中的生长情况，具有较好的仿生性；
(3)本发明制备的细胞培养肉中，种子细胞在三维组织的纤维状基本结构中定向排列并进一步迁移、融合，有助于种子细胞的分化成熟及相关蛋白的合成，进一步提高细胞培养肉的生产效率；
(4)本发明开发的基于微流控3D打印技术构建的细胞培养肉具有高度可编程性，其尺寸、形状等特征可在建模软件中随意调整，进一步开发品类各异的细胞培养肉产品，有助于提高细胞培养肉的市场消费者接受度；
(5)本发明开发的微流控3D打印装置，用作打印喷头的微流控芯片可灵活替换，在芯片通道结构上还可按生产需求进行定制，从而生产结构、组分各异的细胞培养肉，对细胞培养肉产品的多元化、个性化定制方面具有重大潜力；
(6)本发明开发的微流控3D打印装置，其3D打印移动系统上可以按需集成多个微流控芯片，载物平台还可以与传动系统相结合，实现多喷头流水线打印，有助于推动细胞培养肉的规模化、产业化进程。
附图说明
图1为本发明基于微流控3D打印技术生产的细胞培养肉的装置示意图；
图2本发明基于微流控3D打印技术的细胞培养肉生产设备的微流控芯片通道结构示意图和照片，比例标尺为200μm；
图3为本发明基于微流控3D打印技术生产的细胞培养肉实物图，其中，(a)为打印过程实物图；(b)为成品实物图，比例尺为1000μm；
图4为本发明基于微流控3D打印技术构建的不同形状的打印成品，其中，(a)为三角形，(b)为六边形，(c)为圆形，(d)为心形，比例尺为1000μm；
图5为本发明基于多喷头微流控3D打印技术生产的细胞培养肉实物图，其中，(a)为打印过程实物图，(b)为成品实物图，比例尺为1000μm；
图6为本发明基于微流控3D打印技术生产的细胞培养肉培养过程显微镜明场视野图，其中，(a)为打印完成后4小时三维组织显微镜明场视野图，(b)为培养2天后三维组织显微镜明场视野图，比例尺为400μm；
图7为本发明基于微流控3D打印技术生产的细胞培养肉培养成熟后组织免疫荧光染色图，其中，(a)为细胞核，(b)为细胞骨架蛋白，(c)为肌球蛋白，(d)为融合图像，比例尺为100μm；
图8为本发明基于微流控3D打印技术生产的细胞培养肉蛋白质组成与市售猪肉的聚丙烯酰胺凝胶电泳凝胶成像图；
图9为本发明基于微流控3D打印技术生产的细胞培养肉食品化处理后成品与市售猪肉的外观对比图，其中，猪肉(左)，细胞培养肉(右)，比例尺为2000μm；
图10为本发明基于微流控3D打印技术生产的细胞培养肉食品化处理后成品与猪肉的质构特性对比数据图，(a)为硬度，(b)为咀嚼性，(c)为弹性，(d)为凝聚性；
图11为仿生纤维分化培养过程中基于qPCR和Western Blot的细胞分化相关基因和蛋白表达水平变化数据图，其中，(a)为MyoG基因，(b)MyHC-2a基因，(c)为MyHC-slow基因，(d)为相关蛋白Western Blot条带图，(e)MyoG蛋白条带灰度值分析，(f)为Myosin蛋白蛋白条带灰度值分析。
图12为仿生纤维培养成熟后免疫荧光染色图及统计图，其中，(a)为免疫荧光染色图，i是细胞核，ii是细胞骨架蛋白，iii是肌球蛋白，iv是融合图像，比例尺为100μm；(b)为细胞骨架蛋白定向性统计分析图，(c)为细胞核圆度、纵横比统计分析图，(d)为肌球蛋白阳性细胞和肌管区域统计分析图。
具体实施方式
以下结合附图和实施例对本发明做进一步说明。
实施例中所使用的原料和试剂等都是市售可得。其中种子细胞均采用现有常规分离纯化方法获得或者直接市售获得。
实施例1
基于微流控3D打印技术的细胞培养肉生产设备示意图如图1所示：
该设备包括打印喷头1、打印移动系统2、载物平台3、进样系统4、打印控制显示系统5、数据传输系统6、底座7。
打印喷头1为微流控芯片，夹持固定于3D打印移动系统2中的x轴移动光轴21上，由x轴移动光轴21带动其在x轴方向上移动；而x轴移动光轴21又与z轴移动光轴22通过螺栓相连接，由z轴移动光轴22带动其在z轴方向上移动。载物平台3通过卡扣装配在打印移动系统2中的y轴移动光轴23上，由y轴移动光轴23带动载物平台3及成型在载物平台3上的打印品在y轴方向移动，载物平台3可拆卸，以便收取样品，移动光轴、载物平台和底座一般是铝合金。
进样系统4包括装样器41、进样泵42和导管43，装样器41固定在进样泵42上，可灵活拆卸以便装填打印材料，导管43一端与装样器41出口连接，一端与打印喷头1的进口连接。本发明对所述进样泵42的种类无特殊的限制，采用本领域技术人员熟知的能够适用于注射器的注射泵即可，在本实施例中进样泵42为Longer Pump LSP01-1A微量注射泵。装样器41采用本领域技术人员熟知的注射器，并对所述注射器的品牌、种类、尺寸并无特殊的限制；导管43采用本领域技术人员熟知的聚乙烯塑料管，对所述聚乙烯塑料管的品牌、种类、尺寸并无特殊的限制，在本实施例中聚乙烯塑料管外径为1.3mm，内径为0.9mm。
打印控制显示系统5和数据传输系统6与底座7为一个整体，底座7前方开口后嵌入打印控制显示系统5，底座上方打孔后嵌入数据传输系统6接口，数据传输系统6通过数据线连接至打印控制显示系统5，打印控制显示系统5通过数据线连接与打印移动系统2连接。具体而言，长方体底座7前部和顶部边缘处开口，连接打印控制显示系统5和数据传输系统6，打印控制显示系统5主要用于控制打印调平、打印程序的选择、打印指令的下达、打印移动系统2位置调整；数据传输系统6用于将打印指令文件传输进3D打印机；数据传输系统6的数据传输形式包括USB传输、内存卡传输或者电脑传输。将底座7置于水平桌面上，使用螺栓将y轴移动光轴23和z轴移动光轴22固定在底座7上，再将x轴移动光轴21连接至z轴移动光轴22上，即组装成功打印移动系统2。
微流控芯片可以为单通道装置，用于打印实心纤维。将1根玻璃毛细管出口处拉制成外径为200μm，内径为100μm尺寸，用AB胶将拉制后的玻璃毛细管粘至玻璃片上即搭建成型单通道微流控芯片。微流控芯片也可以为共嵌套装置，用于打印空心型、“壳-核”型纤维、螺旋型纤维、串珠型等。本发明对微流控芯片中导液管的通道个数并无特殊的限制，导液管的通道个数可以为双通道、三通道或四通道。
在本实施例中，具体采用共轴嵌套型微流控芯片，共轴嵌套型微流控芯片包括内相玻璃毛细管用于通入内相溶液，外相玻璃毛细管用于通入外相溶液。共轴嵌套型微流控芯片由玻璃毛细管、点胶针头和玻璃片组成，玻璃毛细管为圆柱形，点胶针头为20G点胶针头；对玻璃片的种类和尺寸并无特殊的限制， 玻璃片为市售载玻片，载玻片的厚度为1mm；载玻片的长为30mm，宽为25mm。具体方法为：选取一根内径为580μm，外径为1000μm的圆柱形玻璃毛细管，将出口拉制成内径约80μm，作内相通道；再选取一根内径为580μm，外径为1000μm的圆柱形玻璃毛细管，将出口拉制成内径约200μm，作外相通道。将所述外相通道固定在载玻片上的正中间位置，然后将内相通道拉制端从外相通道一端插入，保证两相通道互不阻塞，在体式显微镜下调节外相通道和内相通道至同一轴线，固定两管；然后，两相通道的接头处固定20G点胶针头，用AB胶粘连后即组装完成，其结构示意图和显微照片如图2所示，作为微流控芯片用于实施例3三维组织的打印。
将本实施例中的打印喷头1设置成由多个微流控芯片组合而成，进行多喷头微流控3D打印。此外，微流控芯片的制作材料可以替换为晶体硅、聚二甲氧基硅氧烷、石英、聚酞胺、聚甲基丙烯酸甲酯、聚碳酸酯、聚苯乙烯、环氧树脂、丙烯酸、橡胶和氟塑料。
实施例2
微流控3D打印材料的配制
微流控外相流体的配制：取适量海藻酸钠粉末，置于超净工作台中紫外照射灭菌，过夜。用移液管量取20mL无菌水于离心管中，在超净工作台内用电子天平称取0.6g海藻酸钠粉末并倒入离心管，使用涡旋仪混匀，再将离心管放入37℃恒温水浴锅中，孵育15min后取出再次涡旋，重复上述操作3-5次至海藻酸钠粉末完全溶解后配得30mg/mL海藻酸钠溶液，3000×g离心5min去除海藻酸钠溶液中的气泡，备用(用于实施例3三维组织的打印)。
微流控内相流体的配制：称取0.1g氯化钙于离心管中，加入5mL含酚红的DMEM基础培养基(C11995500CP，Gibco)溶解，配得含20mg/mL氯化钙的DMEM溶液，使用0.22μm滤膜过滤除菌，冰上保存备用；称取0.2g NaOH于离心管中,加入5mL超纯水溶解，配得1mol/L NaOH溶液，使用0.22μm滤膜过滤除菌后，冰上保存备用。
以1mL内相流体体系为例，取含1.5×107个猪肌肉干细胞细胞悬液于离心管中，300×g离心5min，去除上清，将细胞沉淀置于冰上保存备用。用300μL含20mg/mL氯化钙的DMEM溶液重悬1.5×107个猪肌肉干细胞，向细胞悬液中加入600μL 6mg/mL胶原(来源于牛皮的胶原，Sigma，型号C2124)后，整体转移至装含有3μL 1mol/L NaOH溶液的2mL离心管中，再加入97μL基质胶(标准型Matrigel，Corning试剂公司)，用1mL枪头轻轻吹打混匀，最后将得到的水凝胶溶液置于冰上保存备用(用于实施例3三维组织的打印)。
此外，可以采用上述相同的制备方法相同，不同之处在于：所述具有细胞非粘附性材料溶液为壳聚糖，浓度为10mg/mL；水凝胶溶液的成分为体积分数30％的明胶，体积分数1％的京尼平溶液，体积分数69％的含氯化钙、含5×106个/mL牛肌肉干细胞F-10培养基。
或者不同之处在于：所述具有细胞非粘附性材料溶液为果胶，浓度为50mg/mL；水凝胶溶液的成分为体积分数70％的透明质酸，体积分数1％的碳二亚胺溶液，体积分数29％的含氯化钙、5×108个/mL鸡肌肉干细胞MEM培养基。
或者不同之处在于：所述具有细胞非粘附性材料溶液为卡拉胶，浓度为25mg/mL；水凝胶溶液的成分为体积分数50％的纤维蛋白原，体积分数0.5％的凝 血酶溶液，体积分数49.5％的含氯化钙、5×107个/mL羊肌肉干细胞DMEM/F-12培养基。
在本发明特定3D打印技术下，采用一定量的猪、牛、羊、鸡、鸭等肌肉干细胞进行培养均可以实现本发明效果。
实施例3
三维组织的打印
用Auto CAD 2021软件建立打印模型，模型的尺寸为15mm×20mm×2mm，将模型导出为.stl模型文件格式；再将.stl模型文件导入Cura切片软件，设置打印间距为0.7mm，打印速度为5mm/s，运行切片程序得到G-code打印指令文件；将G-code打印指令文件保存至移动盘，经数据传输系统6导入3D打印设备的打印控制显示系统5，备用。
采用实施例1构建的共轴嵌套型微流控芯片，将实施例2中制备的海藻酸钠溶液加入到5mL注射器内，用一段聚乙烯塑料管一端连接注射器针头，一端连接微流控芯片的外相入口；将实施例2制备的含有猪肌肉干细胞的水凝胶溶液加入到2mL注射器内，聚乙烯塑料管一端连接注射器针头，一端连接微流控芯片的内相入口。然后，再将装有两相流体的注射器分别固定在两个泵上，调节内相水凝胶溶液流速为1.7mL/h，外相海藻酸钠溶液浓度为1.8mL/h。在泵的推动下，内、外相打印材料经聚乙烯塑料管被引入微流控芯片，使两相流体在装置内形成稳定的层流结构从微流控芯片出口处(即外相通道出口)形成“壳-核”结构的仿生纤维。待纤维在打印喷头出口处(外相通道出口)生成后，通过打印控制显示系统5选定打印程序并启动整个微流控3D打印设备，随后微流控3D打印设备的打印移动系统2带动微流控芯片在x、z轴上移动，打印样品在y轴上由载物台带动移动，各个光轴移动速度为5mm/s，使生成的纤维在载物平台上沉积遵循G-code打印指令路径堆叠成型，打印完成后得到三维组织，配制10mg/mL氯化钙溶液，灭菌，用作离子交联剂，将打印完成后的三维组织，取下载物平台，向三维组织上缓慢滴加上述氯化钙溶液直至刚好浸没，交联处理3min后，吸去上述氯化钙溶液，处理后的三维组织如图3所示。
此外，利用Auto CAD 2021软件可建立不同形状的三维模型，经切片、打印后得到三角形、六边形、圆形和心形等不同形状的三维组织(图4)。
在本实施例中，还可以进一步采用磁吸式集成手段在打印移动系统2的x轴移动光轴上安装多个微流控芯片，并按上述单喷头打印操作进行多喷头微流控3D打印，图5展示了集成4个微流控芯片的多喷头微流控3D打印装置构建细胞培养肉的实时照片和成品照片。
实施例4
三维组织的培养
将实施例3最后打印处理后的三维组织转移至装有增殖培养液的直径10cm的培养皿中(增殖培养液：体积分数84％DMEM/F-12(Gibco，11550043)、15％胎牛血清(Gibco,10270-106)、1％青霉素-链霉素(Gibco,15140122)，含终浓度5ng/mL成纤维细胞生长因子bFGF(R&D，233-FB-500/CF))中清洗、浸润10min，培养皿中增殖培养液的量刚好浸没三维组织即可，再将三维组织转移置于37℃、5％CO2的培养箱中进行增殖培养2天；
如图6所示，在显微镜明场视野下观察，当三维组织中猪肌肉干细胞充分迁移、融合形成纤维状结构后，将增殖培养液吸去，然后用不含血清的DMEM 基础培养基清洗三维组织2-3遍。清洗完毕后，向培养皿中加入分化培养液(体积分数97％DMEM(C11995500CP，Gibco)、2％马血清(Hyclone,SH30074.02)、1％青霉素-链霉素(Gibco,15140122))，其中培养皿中分化培养液的量刚好浸没三维组织即可，置于37℃、5％CO2条件下继续进行分化培养，之后每隔两天将培养皿中的分化培养液替换1/2，分化7天后进行免疫荧光染色观察，如图7所示，三维组织中猪肌肉干细胞骨架蛋白沿打印纤维方向定向排列(可观察到F-actin方向与纤维走向一致，种子细胞呈高度定向生长)，成肌标志蛋白myosin具有较高的表达，说明种子细胞在三维组织中定向排列、迁移和融合生长，且保持着较强的成肌分化能力，有助于种子细胞的分化成熟及相关蛋白的合成。同时经过测试纤维中种子细胞Myosin蛋白明显高出普通二维培养组数倍(其中普通二维培养组为常规的直接采用猪肌肉干细胞分化培养手段，将猪肌肉干细胞进行增殖、分化培养，其细胞使用量、增殖、分化培养时间等与上述组织培养完全一致)，进一步说明种子细胞的分化能力得到显著提升，肌肉相关蛋白合成增加，有助于提高细胞培养肉生产效率，通过观测纤维表面可观察到裸露的种子细胞和肌管结构，并展现出与猪肉骨骼肌纤维十分相似的组织结构。
实施例5
三维组织的食品化处理
收获分化成熟的三维组织，用超纯水清洗去除残余的分化培养液得到初步的细胞培养肉，如图8的SDS-PAGE蛋白凝胶电泳凝胶成像图所示，细胞培养肉中肉类相关蛋白(肌球蛋白重链、肌动蛋白和肌球蛋白轻链蛋白等)种类、条带位置与市售猪肉相似；并且通过氨基酸分析，显示细胞培养肉的各类氨基酸含量与市售猪肉差异不大，尤其是Gly(甘氨酸)、Cys(半胱氨酸)和Pro(脯氨酸)含量上十分接近市售猪肉。
配制30mg/mL的海藻酸钠溶液，50mg/mL的明胶溶液和100mg/mL的转谷氨酰胺酶溶液，10mg/mL氯化钙溶液备用；将上述明胶溶液和转谷氨酰胺酶溶液按体积比9：1混合，滴加到初步的细胞培养肉上，使其在初步的细胞培养肉表面充分包被，置于37℃孵育2h后，再将其浸入海藻酸钠溶液中3s后捞出，再置于氯化钙溶液中交联3min，清洗去残余的氯化钙溶液即得到塑形成功的细胞培养肉(图9)。塑形处理后进行相应的肉类品质分析，细胞培养肉在硬度、弹性、咀嚼性和内聚性4个质构指标上与市售猪肉无显著性差异(图10)。塑形处理的细胞培养肉再进行食品前处理(清洗、调味、增色、造型、感官品质修饰等)和煎制处理得到细胞培养肉产品。
实施例6
微流控仿生纤维的培养
向直径10cm无菌细胞培养皿中加入20mL F-10基础培养基作漂洗液，将实施例3制得的3根20cm左右的形成“壳-核”结构的仿生纤维用弯头镊子夹住一端，置于漂洗液中清洗2-3遍以充分去除残余的收集液。清洗完毕后，将仿生纤维转移至盛有增殖培养液(体积分数84％F-10(Gibco，11550043)、15％胎牛血清(Gibco,10270-106)、1％青霉素-链霉素(Gibco,15140122)，含终浓度5ng/mL成纤维细胞生长因子bFGF(R&D，233-FB-500/CF))的10cm无菌细胞培养皿中，再将培养皿置于37℃、5％CO2的培养箱中进行增殖培养2天。在显微镜明场视野下观察，当仿生纤维中猪肌肉干细胞充分迁移、融合形成纤维状结构后，将增殖培养液吸去，然后用不含血清的DMEM基础培养基 清洗仿生纤维2-3遍。清洗完毕后，向培养皿中加入分化培养液(体积分数97％DMEM(C11995500CP，Gibco)、2％马血清(Hyclone,SH30074.02)、1％青霉素-链霉素(Gibco,15140122))置于37℃、5％CO2条件下继续进行分化培养，之后每隔两天将培养皿中的分化培养液替换1/2，分化培养7天后得到成熟的仿生纤维。
在分化第0天、第3天和第7天使用RT-qPCR和Western Blot从分子生物学水平分别评估生长在仿生纤维和二维平皿的种子细胞分化相关基因和蛋白表达的变化情况，其中二维平皿的种子细胞为常规的直接采用猪肌肉干细胞分化培养手段，将猪肌肉干细胞接种至铺有基质胶的无菌直径3.5cm的培养皿上进行增殖、分化培养，其细胞使用量、增殖、分化培养时间等与仿生纤维完全一致。在分化第0天、第3天和第7天，使用Trizol裂解仿生纤维和二维平皿中的细胞，并使用天根生化有限公司的培养细胞总RNA提取试剂盒提取裂解细胞中的RNA；测定样品中RNA浓度后，使用反转录试剂盒对RNA进行逆转录，得到cDNA，反转录程序设为37℃15min，85℃5s；然后，使用RT-qPCR试剂盒对反转录得到的cDNA进行qPCR反应，目的基因为MyoG、MyHC-2a和MyHC-slow，反应程序为95℃30s、95℃5s、60℃30s。如图11中(a)-(c)所示，培养于仿生纤维的种子细胞在分化开始(Day 0)时肌生成素基因(Myogenin，MyoG)比二维平皿培养对照表达高出300余倍；分化末期(Day 7)时，种子细胞培养于仿生纤维中肌肉成熟标志-myosin合成相关基因MyHC-2a和MyHC-slow表达均显著高于二维平皿培养对照组。进一步的，使用RIPA裂解液冰上裂解仿生纤维和二维平皿中的细胞获取细胞蛋白样品，收集的蛋白样品在4℃12000rpm转速离心5min后收集上清液，使用BCA试剂盒测定样品蛋白浓度，并将样品蛋白浓度稀释到1.25mg/mL，然后加入样品四分之一体积的5×Loading buffer，混匀后在95℃下加热5min使蛋白变性。每个样品取20μL变性蛋白进行SDS-PAGE凝胶电泳，电泳条件为80V 30min，120V 70min。然后，切取适宜大小的PVDF膜，使用快速湿转移进行转膜，切取对应蛋白分子量的条带(MyHC：220kDa；MYOG：34kDa；GAPDH：36kDa)，使用5％脱脂奶粉对膜进行封闭，一抗4℃孵育过夜，二抗室温孵育2h；将显影液A液和B液按1：1混合，将其滴加在条带上，避光孵育5min，然后吸去显影液，使用成像仪显影并拍照，使用imageJ软件进行蛋白条带的灰度值分析。如图11中(d)-(f)所示，种子细胞分化相关蛋白表达与基因表达呈现相同的趋势。综上，种子细胞在仿生纤维中生长时，分化相关基因和蛋白表达(MyoG和Myosin蛋白表达)显著高于二维培养组。在分化初期(day 0)和末期(day 7)，仿生纤维中种子细胞MyoG蛋白分别高出二维培养组2.2倍核2.4倍；在分化初期(day 0)、中期(day 3)和末期(day 7)，仿生纤维中种子细胞Myosin蛋白分别高出二维培养组2.66、1.78和2倍，说明种子细胞的分化能力得到显著提升，肌肉相关蛋白合成增加，有助于提高细胞培养肉生产效率。
此外，在分化7天后对仿生纤维进行免疫荧光染色观察及分析。使用4％多聚甲醛对分化7天仿生纤维进行固定，固定后的样品用0.5％Triton X-100通透30min，通透后用5％BSA溶液封闭30min；一抗4℃孵育过夜，二抗室温孵育2h，并进一步孵育鬼笔环肽对F-actin进行染色30min；最后向样品上滴加含有DAPI细胞核染料的封片剂进行封片，使用激光共聚焦显微镜观察并拍照。如图12中(a)-(d)所示，相较于二维培养组，仿生纤维中的细胞骨架蛋白沿纤维方向定向排列(可观察到F-actin方向与纤维走向一致，种子细胞呈 高度定向生长)，成肌标志蛋白myosin具有较高的表达，说明种子细胞在仿生纤维中定向排列、迁移和融合生长，且分化能力得到显著提升，肌肉相关蛋白合成增加。

Claims (25)

  1. 一种基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述设备包括打印喷头(1)、打印移动系统(2)、载物平台(3)、进样系统(4)、底座(7)；所述打印移动系统(2)置于底座(7)上并由多个可移动光轴组成，所述打印喷头(1)固定在其中一个可移动光轴上，载物平台(3)与另一个可移动光轴相连接；所述进样系统(4)与打印喷头(1)相连接，所述打印喷头(1)为微流控芯片，所述微流控芯片为可在通道中对微量液体或样品在进行操纵、处理与控制的装置。
  2. 根据权利要求1所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述打印移动系统(2)包括x轴移动光轴(21)、z轴移动光轴(22)和y轴移动光轴(23)，所述z轴移动光轴(22)和y轴移动光轴(23)固定在底座(7)上，x轴移动光轴(21)与z轴移动光轴(22)连接，所述打印喷头(1)固定在x轴移动光轴(21)上，载物平台(3)置于与y轴移动光轴(23)上相连接。
  3. 根据权利要求2所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述打印移动系统(2)由可带动打印喷头(1)在x轴移动光轴(21)、z轴移动光轴(22)上两个方向移动的移动轴和带动载物平台(3)在y轴移动光轴(23)上移动的移动轴组成，其配置的移动坐标系包括笛卡尔坐标系、三角坐标系、极坐标系和平面关节坐标系中的任意一种。
  4. 根据权利要求2所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述载物平台(3)为可拆卸结构，装配于打印移动系统(2)中，与y轴移动光轴(23)连接，进行流水线打印；所述载物平台(3)的材质为铜、铝、铁、钢、合金、玻璃、陶瓷或者碳纤维板材。
  5. 根据权利要求1所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述进样系统(4)包括装样器(41)、进样泵(42)和导管(43)，所述进样泵(42)置于水平桌面，装样器(41)固定在进样泵(42)上，导管(43)一端连接装样器(41)的出口，一端连接打印喷头(1)的进样口，所述进样系统(4)的进料方式包括活塞式挤入、气动式挤入或者螺杆式挤入。
  6. 根据权利要求1所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述底座(7)中嵌入安装打印控制显示系统(5)和数据传输系统(6)；数据传输系统(6)通过无线或者有线连接至打印控制显示系统(5)，打印控制显示系统(5)通过无线或者有线连接与打印移动系统(2)连接。
  7. 根据权利要求6所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述打印控制显示系统(5)用于控制打印调平、打印程序的选择、打印指令的下达、打印移动系统(2)位置调整；所述数据传输系统(6)用于将打印指令文件传输进3D打印设备；所述数据传输系统(6)的数据传输形式包括USB传输、内存卡传输或者电脑传输。
  8. 根据权利要求6所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，打印控制显示系统(5)和数据传输系统(6)嵌入底座(7)后，跟随底座(7)一起接电。
  9. 根据权利要求1所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述打印移动系统(2)上微流控芯片类型可以灵活替换，根据不同生产需求进行集成打印。
  10. 根据权利要求9所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述打印移动系统(2)上可集成一个或者多个微流控芯片作打印喷头(1)，集成多个微流控芯片时，可使用通道结构相同或者通道结构各异的微流控芯片。
  11. 根据权利要求1所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述微流控芯片通过夹持、扣接、插接、磁吸、榫接、铆合、螺纹连接或者卡口连接集成到打印移动系统(2)上。
  12. 根据权利要求1所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述微流控芯片的制作材料包括晶体硅、聚二甲氧基硅氧烷、玻璃、石英、聚酞胺、聚甲基丙烯酸甲酯、聚碳酸酯、聚苯乙烯、环氧树脂、丙烯酸、橡胶和氟塑料中一种或者多种；所述微流控芯片的制作方法包括玻璃毛细管组装法、机械加工法、刻蚀法或者模具法。
  13. 根据权利要求1所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述微流控芯片类型为所述微流控芯片类型为单通道型、共轴嵌套型或者多通道并行型。
  14. 根据权利要求1所述的基于微流控3D打印技术的细胞培养肉生产设备，其特征在于，所述微流控芯片基于不同通道结构，可生成实心型、“壳-核”型、空心型、多组分型、螺旋形、串珠型纤维用于微流控3D打印。
  15. 一种权利要求1所述的基于微流控3D打印技术的细胞培养肉生产设备在制备细胞培养肉中应用，所述制备细胞培养肉的过程为：
    (1)配制微流控3D打印所需的生物墨水，备用；
    (2)将打印指令文件导入打印设备，将步骤(1)配制的生物墨水装填入进样系统(4)；将进样系统(4)连接至打印喷头(1)的进口处，即微流控芯片的通道入口处，将生物墨水经导管(43)挤入打印喷头(1)即微流控芯片；待纤维在微流控芯片出口处生成后，选定需要打印的指令文件，启动整个设备；打印喷头(1)出口生成的纤维在打印移动系统(2)的各个光轴的带动下沉积于载物平台(3)，并遵循打印指令路径堆叠成型；
    (3)将载物平台(3)拆卸，并对步骤(2)打印得到的三维组织进行交联固化处理，然后转移至相应的培养液中进行增殖、分化培养；
    (4)将步骤(3)中培养成熟的三维组织收获，清洗去除培养液后，进行食品化处理即可得到细胞培养肉。
  16. 根据权利要求15所述的应用，其特征在于，步骤(1)中所述生物墨水为含有种子细胞的水凝胶溶液、或者为具有细胞非粘附性材料和含有种子细胞的水凝胶溶液组合；所述具有细胞非粘附性材料可以与含有种子细胞的水凝胶溶液直接混合或者所述具有细胞非粘附性材料包裹含有种子细胞的水凝胶溶液；所述含有种子细胞的水凝胶溶液中含体积分数30％-70％生物材料、0.01％-1％交联剂，余量为含钙盐、含5×106-5×108个/mL种子细胞的基础培养基。
  17. 根据权利要求16所述的应用，其特征在于，所述生物墨水中所述具有细胞非粘附性材料为海藻酸钠、壳聚糖、果胶、卡拉胶、结冷胶中的任意一种或多种；所述具有细胞非粘附性的材料溶液的浓度为10-50mg/mL。
  18. 根据权利要求16所述的应用，其特征在于，所述生物墨水中所述种子细胞来源于猪、牛、羊、鸡、鸭、兔、鱼、虾和蟹中的任意一种或者多种；所述种子细胞为肌肉干细胞、成肌细胞、肌卫星细胞、肌肉前体细胞、骨髓源间充质干细胞、脂肪源间充质干细胞、诱导多能干细胞、心肌细胞、脂肪干细 胞、脂肪前体细胞、骨髓源脂肪成体细胞、成纤维细胞、平滑肌细胞、血管内皮细胞、上皮细胞、神经干细胞、神经胶质细胞、成骨细胞、软骨细胞、肝脏干细胞、造血干细胞、基质细胞、胚胎干细胞、骨髓干细胞中的一种或者多种。
  19. 根据权利要求16所述的应用，其特征在于，所述生物材料为胶原蛋白、重组胶原蛋白、明胶、基质胶、透明质酸、丝素蛋白、弹性蛋白、蛛丝蛋白、纤维蛋白、纤维蛋白原、丝纤蛋白、层粘连蛋白、纤粘连蛋白、整合蛋白、钙粘蛋白、巢蛋白、脱细胞外基质、硫酸软骨素、肝素、硫酸角质素、硫酸皮肤素、硫酸乙酰肝素、角蛋白、硫酸角蛋白、纤维素、聚合素、羧甲基纤维素、聚乳酸、聚乙烯醇、卵磷脂、纳米纤维素、大豆蛋白、豌豆蛋白、面筋蛋白、大米蛋白、花生蛋白、酵母蛋白、真菌蛋白、小麦蛋白、土豆蛋白、玉米蛋白、鹰嘴豆蛋白、绿豆蛋白、海藻蛋白、杏仁蛋白、藜麦蛋白中的一种或者多种。
  20. 根据权利要求16所述的应用，其特征在于，所述生物墨水成分中使用的基础培养基为F-10、DMEM、MEM、F-12、DMEM/F-12、DMEM/F-12 GlutamMAXTM、F-12K、RPMI 1640、IMDM、L-15、199、MCDB 131、LHC、McCoy's 5A中的一种或者多种。
  21. 根据权利要求16所述的应用，其特征在于，所述生物墨水成分中使用的交联剂包括NaOH、KOH、NaHCO3、HEPES平衡盐溶液、EBSS平衡盐溶液、HBSS平衡盐溶液、PBS、DPBS、转谷氨酰胺酶、酪氨酸酶、漆酶、赖氨酰氧化酶、多酚氧化酶、过氧化氢酶、凝血酶、京尼平中的任意一种或者多种。
  22. 根据权利要求16所述的应用，其特征在于，所述含有种子细胞的水凝胶溶液中包括生物材料、交联剂，含钙盐、含种子细胞的基础培养基；每1mL水凝胶溶液中含290-699μL 4-8mg/mL的生物材料，1-10μL 1-2mol/L交联剂，300-700μL含15-25mg/mL钙盐、1×107-1×108种子细胞的基础培养基；所述生物材料为胶原蛋白、重组胶原蛋白、明胶、基质胶、透明质酸、丝素蛋白中的一种或者多种；所述交联剂为NaOH、KOH、NaHCO3中的一种或者多种；所述钙盐为氯化钙、碳酸钙、硫酸钙、硝酸钙中的一种或者多种；所述基础培养基为F-10、DMEM、MEM、F-12、DMEM/F-12中的一种或者多种；所述种子细胞为猪、牛、羊、鸡、鸭的肌肉干细胞、成肌细胞、肌卫星细胞、肌肉前体细胞中的一种或者多种。
  23. 根据权利要求15所述的应用，其特征在于，步骤(3)中所述交联固化处理的方式优选包括温度诱导交联、静电相互作用交联、离子交联、酶交联中的一种或者几种。
  24. 根据权利要求15所述的应用，其特征在于，步骤(3)中所述增殖培养的培养液包括体积分数79-89％基础培养基、10-20％胎牛血清、1％青霉素-链霉素，其中含有1-10ng/mL碱性成纤维细胞生长因子；所述分化培养的培养液包括体积分数94-97％基础培养基、2-5％马血清和1％青霉素-链霉素。
  25. 根据权利要求15所述的应用，其特征在于，步骤(4)中所述食品化处理的方法包括前处理和烹饪，所述前处理包括清洗、调味、增色、造型或者感官品质修饰中的一种或者多种，所述烹饪包括煎、炸、煮、蒸或者烤。
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