WO2024007982A1 - 一种用于细胞培养肉生产的微流控仿生纤维及其制备方法和应用 - Google Patents

一种用于细胞培养肉生产的微流控仿生纤维及其制备方法和应用 Download PDF

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WO2024007982A1
WO2024007982A1 PCT/CN2023/104613 CN2023104613W WO2024007982A1 WO 2024007982 A1 WO2024007982 A1 WO 2024007982A1 CN 2023104613 W CN2023104613 W CN 2023104613W WO 2024007982 A1 WO2024007982 A1 WO 2024007982A1
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microfluidic
cells
fiber
bionic
protein
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French (fr)
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王洁
周光宏
丁希
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南京农业大学
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/22—Working-up of proteins for foodstuffs by texturising
    • A23J3/225—Texturised simulated foods with high protein content
    • A23J3/227—Meat-like textured foods
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/28—Formation of filaments, threads, or the like while mixing different spinning solutions or melts during the spinning operation; Spinnerette packs therefor
    • D01D5/30—Conjugate filaments; Spinnerette packs therefor
    • D01D5/34—Core-skin structure; Spinnerette packs therefor
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F1/00—General methods for the manufacture of artificial filaments or the like
    • D01F1/02—Addition of substances to the spinning solution or to the melt
    • D01F1/10—Other agents for modifying properties
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F8/00—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof
    • D01F8/02—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof from cellulose, cellulose derivatives, or proteins
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F8/00—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof
    • D01F8/18—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof from other substances
    • 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
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    • 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
    • C12N2533/72—Chitin, chitosan
    • 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
    • C12N2535/00—Supports or coatings for cell culture characterised by topography

Definitions

  • the invention belongs to the field of cell cultured meat, and specifically relates to microfluidic bionic fibers used for cell cultured meat production and their preparation methods and applications.
  • Meat is rich in essential nutrients such as protein, vitamins and minerals, and has become an important part of the diet of residents around the world. As the world's population and demand for meat increase year by year, the contradiction between the meat production method that relies on animal husbandry and ecological resource endowments, public health safety, and ethics and morals has become increasingly prominent. It is necessary to develop new types of meat that can replace traditional animal husbandry. Production technology is of great significance.
  • cell cultured meat stands out. It is a kind of meat that relies on tissue engineering and is based on the self-healing and regenerative ability of animal muscle tissue and the in vitro culture of relevant cells and tissues. It is expected to solve the future meat problem through in vitro culture. Class supply problems.
  • the way to produce cell-cultured meat is generally to give seed cells an appropriate carrier, and induce the seed cells to proliferate and differentiate on the carrier until they mature, and finally harvest the cell-cultured meat.
  • Common carrier materials used for cell-cultured meat production mainly include animal protein scaffolds, plant protein scaffolds, acellular plant scaffolds and bulk hydrogels.
  • scaffold materials still have deficiencies in seed cell adhesion, migration and fusion efficiency, and cannot accurately simulate the fibrous basic physiological structure of muscle fibers in natural skeletal muscle, causing seed cells to differentiate on the scaffold.
  • the ability to form mature muscle tissue is limited, making production inefficient.
  • Microfluidics is a classic technology in the field of tissue engineering. It can control trace amounts of liquid in micro-sized channels and is considered a powerful means to prepare fiber carriers.
  • the application of microfluidic technology in cell-cultured meat production has not yet been reported.
  • the present invention provides a microfluidic bionic fiber for the production of cell cultured meat.
  • the microfluidic bionic fiber prepared by the present invention effectively solves the long cycle of the existing cell cultured meat production method. , the process is cumbersome, and it cannot accurately simulate the in vivo growth environment of seed cells, there is less synthesis of related proteins, and the production efficiency of cell-cultured meat is low.
  • the invention also provides a preparation method and application of the microfluidic bionic fiber.
  • the microfluidic bionic fiber of the present invention has a "shell-core" structure; the shell of the microfluidic bionic fiber is formed by cross-linking a polymer with cell non-adhesion, The core wrapped by the outer shell is a hydrogel solution mixed with seed cells.
  • the polymer solution with cell non-adhesion includes but is not limited to any one or more of sodium alginate, chitosan, pectin, carrageenan, and gellan gum; the polymer solution with cell non-adhesion
  • concentration of the adhesive polymer solution is 10-50mg/mL.
  • the hydrogel solution contains a volume fraction of 30%-70% biological material, 0.01%-1% cross-linking agent, and the balance is calcium salt and 5 ⁇ 10 6 -5 ⁇ 10 8 seeds/mL. basal culture medium for cells.
  • the source of the seed cells includes but is not limited to any one or more of pigs, cattle, sheep, chickens, ducks, rabbits, fish, shrimps and crabs.
  • 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, muscle precursor cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, induced pluripotent stem cells, cardiomyocytes, adipose stem cells, Adipocyte precursor cells, bone marrow-derived adipocyte adult cells, fibroblasts, smooth muscle cells, vascular endothelial cells, epithelial cells, neural stem cells, glial cells, osteoblasts, chondrocytes, liver stem cells, hematopoietic stem cells, stromal cells, embryonic cells One or more types of stem cells and bone marrow stem cells.
  • the seed cells include but are not limited to one or more of muscle stem cells, muscle cells, muscle satellite cells, and muscle precursor cells.
  • the biological materials in the hydrogel solution are collagen, recombinant collagen, gelatin, Matrigel, hyaluronic acid, silk fibroin, elastin, spider silk protein, fibrin, fibrinogen, and silk fibroin.
  • Polypeptide polymerin, carboxymethylcellulose, polylactic acid, polyvinyl alcohol, lecithin, nanocellulose, soy protein, pea protein, gluten protein, rice protein, peanut protein, yeast protein, mycoprotein, wheat protein, potato One or more of egg white, zein, chickpea protein, mung bean protein, seaweed protein, almond protein, quinoa protein, and other materials that are biocompatible and
  • the biological material is one or more of collagen, recombinant collagen, gelatin, Matrigel, hyaluronic acid, and silk fibroin.
  • the basal culture medium is 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, one or more.
  • the basal culture medium is one or more of F-10, DMEM, MEM, F-12, DMEM/F-12.
  • the cross-linking agent described in step (1) includes but is not limited to NaOH, KOH, NaHCO 3 , HEPES balanced salt solution, EBSS balanced salt solution, HBSS balanced salt solution, PBS, DPBS, transglutaminase, tyrosine One or more of acidase, laccase, lysyl oxidase, polyphenol oxidase, catalase, thrombin, genipin and other chemical cross-linking agents.
  • 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 preparation method of microfluidic bionic fiber for cell cultured meat production according to the present invention includes the following steps:
  • the manufacturing materials of the microfluidic device in step (2) include but are not limited to crystalline silicon, polydimethoxysiloxane, glass, quartz, polyphthalamine, polymethylmethacrylate, polycarbonate , one or more of polystyrene, epoxy resin, acrylic, rubber and fluoroplastics.
  • the channel structure of the microfluidic device in step (2) can be a simple coaxial nesting of the internal phase channel and the external phase channel; or based on the coaxial nesting of the internal phase channel and the external phase channel,
  • the collection phase channel and the observation phase channel are constructed in the form of coaxial nesting.
  • the internal phase channel diameter of the microfluidic device described in step (2) ranges from 50 to 300 ⁇ m, and the external phase channel outlet diameter ranges from 200 to 800 ⁇ m.
  • step (2) the flow rate of the internal phase solution ranges from 0.5 to 10 mL/h, and the flow rate of the external phase solution ranges from 0.5 to 10 mL/h.
  • the two-phase fluid forms a stable laminar flow structure in the channel of the microfluidic device by adjusting the flow rate of the internal and external phases, and then squeezes it into the collection liquid, and then washes away the residual collection liquid to obtain bionic fibers;
  • the two-phase fluid can form a stable laminar flow structure in the channel of the microfluidic device by adjusting the flow rate of the internal and external phases.
  • the extruded bionic fibers are directly organized and integrated, and then soaked in the collection liquid to form bionic fibers. Three-dimensional organization.
  • the collection liquid in step (2) includes but is not limited to one or more of calcium salt, sodium salt, potassium salt, and magnesium salt solutions.
  • the internal and external phase fluids are filled into syringes respectively, and then polyethylene plastic tubes are used to connect the syringe outlet and the internal and external phase channel inlets of the microfluidic device, and then the syringe is fixed to the peristaltic pump; the peristaltic pump pushes the syringe piston.
  • the internal and external phase fluids flow into the microfluidic device through polyethylene plastic tubes, and are directly shaped into bionic fibers at the outlet of the device for subsequent organizational integration.
  • bionic fibers when preparing bionic fibers, choose to extend the outlet of the microfluidic device into the petri dish containing the collection liquid.
  • the two-phase fluid is directly squeezed into the collection liquid through the device outlet for cross-linking molding.
  • the shape and structure of the bionic fiber are in It is further stabilized in the collection liquid and can also provide a temporary storage container for the bionic fiber before being transferred to the cleaning liquid and culture medium to facilitate subsequent production operations.
  • the bionic fiber can be washed in a rinsing solution to fully remove the residual collection fluid.
  • the rinsing solution includes but is not limited to serum-containing culture medium, basal culture medium, phosphate buffer solution (PBS), physiological saline, and glucose. solution, sterile water.
  • microfluidic bionic fiber prepared by the preparation method of the present invention in the production of cell cultured meat.
  • the cell cultured meat production includes the following steps:
  • the generated bionic fibers directly clean the generated bionic fibers and transfer them to a petri dish containing a proliferation culture medium to ensure that the bionic fibers are completely immersed in the culture liquid. Place them in a 37°C, 5% CO 2 incubator for culture, and change them every two days. One culture medium.
  • the proliferation culture medium was replaced with a differentiation culture medium to continue differentiation culture, and 1/2 volume of the differentiation culture medium was replaced every two days; after 7 days of differentiation, the biomimetic fibers were harvested.
  • the channel size of the microfluidic device and the amount of cells used are optimized so that the cells in the generated fiber core are closely arranged;
  • the generated bionic fiber has cell non-adhesion properties, and cells are constrained to grow in the core; thirdly, under spatial constraints and close arrangement, cells tend to fuse with each other to form a whole during the culture process.
  • the components of the proliferation culture solution are 79-89% basic culture medium, 10-20% fetal bovine serum, and 1% penicillin-streptomycin, and then 1-10ng/mL alkaline ingredients are added to the above solution.
  • Fibrocyte growth factor (bFGF) Fibrocyte growth factor
  • the components of the differentiation culture medium are 94-97% basal culture medium, 2-5% horse serum and 1% penicillin-streptomycin.
  • the basal culture medium in the proliferation culture medium and differentiation culture medium 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 polymer part of the microfluidic bionic fiber can be removed to obtain pure cell fibers.
  • the specific method is to use alginate lyase, sodium citrate, ethylenediaminetetraacetic acid, chitosanase, and pectinase , one or more carrageenases.
  • the organizational integration methods include but are not limited to stacking, braiding, winding, bundling, folding, etc.
  • the food processing method 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, roasting, etc.
  • the polymer part of the mature biomimetic fiber can be removed to obtain pure cell fibers.
  • the removal method uses a lysis solution, the lysis solution includes alginate lyase, sodium citrate, ethylenediamine tetrahydrofuran. Acetic acid, chitosanase, pectinase or carrageenase.
  • the bionic fiber of the present invention can remove the shell after differentiation and maturity and use it for subsequent processing to obtain pure cell fiber with higher relative protein content and richer nutrition.
  • This invention utilizes the principle of bionic muscle fiber structure, the basic unit of natural skeletal muscle tissue, to give seed cells appropriate fiber carriers to ensure very good results in the efficient production of cell-cultured meat; and for the first time, microfluidics are used in the production of cell-cultured meat. technology, capable of manipulating minute amounts of liquids in micro-sized channels.
  • Muscle fiber is the most basic unit of skeletal muscle tissue. Countless muscle fibers are wrapped layer by layer by connective tissue membranes to form large skeletal muscle tissue.
  • the present invention uses muscle fiber bionics as the design principle and proposes a preparation method of bionic fiber carriers for cell cultured meat production based on microfluidic technology, and There have been no relevant reports in the field using this method to produce cell-cultured meat.
  • the present invention prepares bionic fibers based on 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. The directional growth and differentiation capabilities of seed cells growing in the fibers are improved. A huge improvement.
  • the present invention prepares bionic fibers with a "shell-core" structure based on microfluidic technology.
  • the seed cells are wrapped in a polymer shell with cell non-adhesion and exhibit highly directional and fused growth under the spatial constraints of the shell. Characteristics, 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 properties, so fibers prepared based on microfluidic technology have relatively high Good bionics.
  • the focus of the present invention is the preparation of microfluidic bionic fibers.
  • the present invention first designs and builds a coaxial nested microfluidic device; then prepares the inner and outer phase fluid materials, and adjusts the access of the inner and outer phase fluids. Sequence, flow rate and other parameters are used to stably generate bionic fibers and culture them; finally, the cultured bionic fibers are tissue-integrated and food-processed to obtain cell-cultured meat products.
  • seed cells are wrapped in a polymer shell with bionic fibers that do not have cell adhesion, and are placed in the hydrogel core under the spatial constraints of the shell. It exhibits highly directional and fusion growth characteristics, and its in vitro myogenic differentiation ability has also been significantly improved, improving production efficiency.
  • the present invention effectively improves the differentiation ability of livestock and poultry primary cells such as muscle stem cells in microfibers, increases the synthesis of muscle-related proteins, and further forms Mature muscle fibers, thereby improving the efficiency of cell-cultured meat production.
  • 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 present invention has the following advantages:
  • the present invention prepares continuous, large-scale, uniformly structured and controllable bionic fibers based on microfluidic technology.
  • the equipment involved has low cost, mild preparation conditions, simple operation, and rapid molding;
  • the present invention is inspired by the structure of muscle fibers in natural skeletal muscle tissue.
  • the bionic fibers prepared in this invention can provide good fiber carriers for seed cells, further simulate the three-dimensional growth environment of seed cells in the body, and have good bionic properties;
  • the bionic fiber shell prepared by the present invention does not have cell adhesion, so it can induce the directional arrangement, migration and fusion growth of seed cells in the core space of the fiber carrier, thereby significantly improving the differentiation ability of the seed cells.
  • Muscle-related proteins Increased synthesis improves cell-cultured meat production efficiency;
  • the bionic fiber prepared by the present invention has good organizational integration characteristics of fiber materials, and can be further used for deep processing operations such as braiding, winding, and stacking to realize the construction of large pieces of cultured meat.
  • Figure 1 is a schematic diagram of the preparation of microfluidic bionic fibers for cell cultured meat production according to the present invention
  • Figure 2 is a real-time image of the preparation process of the microfluidic bionic fiber used for cell cultured meat production according to the present invention and a structural diagram of the channel of the microfluidic device;
  • Figure 3 is a feasibility verification data chart for flexible adjustment of the size of microfluidic bionic fibers for cell-cultured meat production according to the present invention, in which (a) is a bright field image of bionic fibers with different core sizes, and the scale bar is 200 ⁇ m; (b) And (c) is the change diagram of the shell and core dimensions as the flow rate changes;
  • Figure 4 is a schematic diagram and photomicrograph of the channel structure of the microfluidic device used for tissue integration of the present invention, the scale bar is 200um;
  • Figure 5 is a microscope bright field view of the microfluidic bionic fiber culture process used for cell cultured meat production.
  • (a) is a microscope bright field view of the bionic fiber after incubation for 2 hours after preparation, and (b) is a proliferation culture for 2 hours.
  • the bright field view of Tianhou's bionic fiber microscope is the bright field view of the bionic fiber microscope after 3 days of differentiation and culture, (d) is the bright field view of the bionic fiber microscope after 7 days of differentiation and culture, the scale bar is 200 ⁇ m;
  • Figure 6 is a data diagram showing changes in cell differentiation-related genes and protein expression levels based on qPCR and Western Blot during the microfluidic bionic fiber differentiation and culture process used for cell cultured meat production, in which (a) is the MyoG gene, (b) MyHC -2a gene, (c) is MyHC-slow gene, (d) is related protein Western Blot band diagram, (e) gray value analysis of MyoG protein band, (f) is gray value analysis of Myosin protein band .
  • Figure 7 shows the immunofluorescence staining diagram and statistical diagram after the microfluidic bionic fiber culture is matured for cell cultured meat production.
  • (a) is the immunofluorescence staining diagram
  • i is the cell nucleus
  • ii is the cytoskeleton protein
  • iii is the muscle protein.
  • globulin, iv is the fusion image
  • the scale bar is 100 ⁇ m
  • (b) is the statistical analysis chart of cytoskeletal protein orientation
  • (c) is the statistical analysis chart of nuclear roundness and aspect ratio
  • (d) is the myosin-positive cells and muscle Pipe area statistical analysis chart;
  • Figure 8 is an electron microscope comparison of the microfluidic bionic fiber used for cell cultured meat production after maturation and commercially available pork.
  • (a) is the cultured and mature bionic fiber, and the scale bar is 40 ⁇ m;
  • (b) is commercially available pork. , scale bar is 100 ⁇ m;
  • Figure 9 is a comparison of the H&E staining of microfluidic bionic fibers used for cell-cultured meat production after maturation and commercial pork.
  • (a) is a longitudinal section of the cultured and mature bionic fibers, and (b) is commercially available pork. Longitudinal view,
  • (c) is a cross-section view of cultured mature bionic fiber,
  • (d) is a cross-section view of commercially available pork, the scale bar is 100 ⁇ m;
  • Figure 10 is a schematic diagram of the device used for microfluidic bionic fiber tissue integration for cell cultured meat production
  • Figure 11 shows the microfluidic bionic fiber tissue integration process (a) and finished product (b) for cell cultured meat production.
  • the scale bar is 1000 ⁇ m;
  • Figure 12 is a data chart comparing the contents of various amino acids after the microfluidic bionic fiber is matured for cell-cultured meat production with the control group and commercially available pork;
  • Figure 13 shows the polyacrylamide gel electrophoresis gel image of the protein composition of the produced cell-cultured meat and that of commercially available pork;
  • Figure 14 is a comparison data chart of the texture properties of the cell cultured meat after food processing and that of pork.
  • (a) is hardness
  • (b) is chewiness
  • (c) is elasticity
  • (d) is cohesiveness.
  • 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.
  • microfluidic bionic fiber The preparation process of microfluidic bionic fiber is shown in Figure 1.
  • a cylindrical glass capillary tube with an inner diameter of 580 ⁇ m and an outer diameter of 1000 ⁇ m is selected, and the outlet is drawn to an inner diameter of about 80 ⁇ m to serve as the internal phase channel; then a cylindrical glass capillary tube with an inner diameter of 1000 ⁇ m is selected.
  • a cylindrical glass capillary tube with an outer diameter of 580 ⁇ m and an outer diameter of 1000 ⁇ m is drawn into an inner diameter of about 200 ⁇ m as an external phase channel.
  • a circular glass capillary tube with an inner diameter of 0.8 mm and an outer diameter of 1 mm was selected as the collection phase channel; a square glass capillary tube with an inner edge length of 1.05 mm was selected as the observation phase channel.
  • Fix the observation square tube channel in the middle of the slide plane (the thickness of the slide is 1mm; the length of the slide is 30mm and the width is 25mm). You can observe the fiber movement in the channel by connecting a CCD camera. The situation is formed, and then insert the drawing end of the external phase channel and the collection phase channel through the two sections of the square tube channel respectively to ensure that the external phase channel is inserted into the collection phase channel and does not block each other.
  • one end is connected to the inlet of the internal phase channel of the microfluidic device. Then, fix the syringes containing the fluids of each phase on the peristaltic pump respectively, adjust the flow rate of the collected phase calcium chloride solution to 15mL/h, the flow rate of the internal phase hydrogel solution to 1.8mL/h, and the flow rate of the external phase sodium alginate solution. is 2mL/h, start the peristaltic pump.
  • the generation process of "shell-core" bionic fibers in microfluidic devices can be divided into two stages.
  • the internal phase fluid and the external phase fluid first converge between the internal phase channel outlet and the external phase channel outlet to form a coaxial laminar flow fluid, and then enter the collection phase channel and the collection phase solution converge again to form three layers of coaxial laminar flow fluid;
  • the sodium alginate solution in the outer phase begins to form a calcium alginate hydrogel in the presence of calcium ions in the collection phase and the inner phase solution and continues to diffuse into the inner layer.
  • the "shell-core" type bionic fiber It is continuously solidified and extruded into the collection liquid.
  • FIG. 2 microscopic observation shows obvious dividing lines between the collection phase fluid, the internal phase fluid and the external phase fluid, which is the phenomenon of laminar flow structure), and then through the collection phase
  • the channels are squeezed into the collection liquid to obtain bionic fibers.
  • the size of the prepared bionic fiber is controllable, continuous, and scalable, and can be flexibly regulated by changing the flow rate (Figure 3).
  • Figure 3 (a) is a bright field image of bionic fibers with different core sizes.
  • Figure 3 (b) as the flow rate of the internal phase solution increases, the internal phase diameter of the "shell-core" bionic fiber also increases.
  • bionic fibers can be rapidly and continuously generated from the outlet of the microfluidic device while ensuring sufficient internal and external phase fluids; by simply adjusting the outlet diameter of the microfluidic device, the flow rates of the internal and external phases can be controlled.
  • the size of the prepared bionic fiber; the glass capillary tubes, glass sheets, dispensing needles, etc. used to build the microfluidic device are common low-cost consumables.
  • the external phase solution needs to be introduced first and then the internal phase solution. If the order is reversed, fibers cannot be formed.
  • the microfluidic device built in this example is a simplified version of the microfluidic device built in Example 2. It does not contain a collection phase channel and an observation phase channel. It is a coaxial nested form of the internal phase channel and the external phase channel. It can be directly used for the organizational integration of bionic fibers; and the microfluidic device built in Example 2 is mainly used for real-time observation of the fluid state in the channel and the fiber generation process during the preparation of bionic fibers. In addition, there is no difference in structure, form, and function between the bionic fibers prepared based on the above two microfluidic devices.
  • 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 sucked away, and then the bionic fiber was washed 2-3 times with serum-free DMEM basic 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 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 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, respectively, 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.
  • samples fixed with 4% paraformaldehyde were dehydrated in gradients of 70%, 80%, 90% and absolute ethanol, and then xylene was used to gradiently replace the ethanol in the sample; the embedding agent paraffin was then used to replace the sample.
  • xylene in the sample embed the sample in fresh paraffin, slice it with a microtome and stain it with hematoxylin and eosin staining solution, observe and take pictures under an inverted microscope and compare it with commercially available pork ((a)-(d) in Figure 9 ). It can be seen from Figures 8 and 9 that exposed seed cells and myotube structures can be observed on the surface of the bionic fiber, and exhibit a tissue structure very similar to that of pork skeletal muscle fiber.
  • the coaxial nested microfluidic device constructed in Example 3 was integrated into the 3D printer nozzle moving system as a printing nozzle, and modified to obtain a microfluidic 3D printing device. Its structural schematic diagram is shown in Figure 10.
  • the printing device 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 moving optical axis is generally aluminum alloy, and then connect the x-axis moving optical axis 21 to z
  • the axis moves on the optical axis 22, that is, the printing movement system 2 is successfully assembled.
  • the printing nozzle 1 is clamped and fixed on the x-axis moving optical axis 21 in the 3D printing movement system 2, and is driven by the x-axis moving optical axis 21 to move in the x-axis direction; while the x-axis moving optical axis 21 and the z-axis moving optical axis 21
  • the shaft 22 is connected by 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.
  • the loading platform 3 is detachable to collect samples.
  • 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.
  • the injection pump 42 uses a Longer Pump LSP01-1A micro-injection pump
  • the sample loader 41 can use a syringe
  • the conduit 43 can use a polyethylene plastic tube.
  • 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.
  • the printing control display system 5 is mainly used to control printing leveling, the selection of printing programs, the issuance of printing instructions, and the position adjustment of the printing movement system 2;
  • the data transmission system 6 is used to transmit the printing instruction files into the 3D printer;
  • the data transmission system 6 Data transmission forms include USB transmission, memory card transmission or computer transmission.
  • the inner and outer phase printing materials are introduced into the microfluidic device through polyethylene plastic tubes. After the fiber is generated at the exit of the device (i.e., the exit of the external phase channel), select the printing program and start the 3D printing device. Then the 3D printer nozzle moving system drives the microfluidic device to move on the x and z axes, and the printed sample is on the y axis.
  • the stage is driven to move, and the moving speed of each optical axis is 5mm/s, so that the generated fibers are deposited on the stage 3 and stacked and formed according to the G-code printing instruction path.
  • a three-dimensional tissue is obtained, and 10 mg/mL chlorine is prepared.
  • Calcium chloride solution sterilized, is used as the collection liquid.
  • After printing the three-dimensional tissue take off the loading platform and slowly drop the above-mentioned calcium chloride solution onto the three-dimensional tissue until it is just submerged. After cross-linking treatment for 3 minutes, suck away the above-mentioned calcium chloride solution.
  • Calcium chloride solution the tissue integration process is shown in Figure 11(a), and the processed three-dimensional tissue is shown in Figure 11(b).
  • organizational integration can also adopt other methods such as stacking, braiding, wrapping, bundling or folding.
  • the three-dimensional tissue was transferred to proliferation culture medium (volume fraction 84% DMEM/F-12, 15% fetal calf serum, 1% penicillin-streptomycin and 5ng/mL fibroblast growth factor) for washing and infiltration for 10 minutes, and then The three-dimensional tissue transfer was placed in an incubator at 37°C and 5% CO2 for proliferation and culture for 2 days; under the bright field view of a microscope, when the porcine muscle stem cells in the three-dimensional tissue fully migrated and fused to form a fibrous structure, the proliferation culture was Aspirate off the liquid, and then wash the three-dimensional tissue 2-3 times with serum-free DMEM basic medium.
  • proliferation culture medium volume fraction 84% DMEM/F-12, 15% fetal calf serum, 1% penicillin-streptomycin and 5ng/mL fibroblast growth factor
  • differentiation culture medium volume fraction 97% DMEM, 2% horse serum, 1% penicillin-streptomycin
  • the cell-cultured meat, the amino acid analysis results ( Figure 12) show that the various amino acid contents of the cell-cultured meat are significantly higher than that of the control group (using the core hydrogel solution without seed cells, other preparation methods are consistent with Examples 1 and 2) , the content of Gly (glycine), Cys (cysteine) and Pro (proline) is very close to that of commercial pork.
  • meat-related cell-cultured meat is 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.
  • the shaped cell-cultured meat is then subjected to food pre-processing (cleaning, seasoning, color enhancement, modeling, sensory quality modification, etc.) and frying to obtain cell-cultured meat products.
  • food pre-processing cleaning, seasoning, color enhancement, modeling, sensory quality modification, etc.
  • frying to obtain cell-cultured meat products.
  • 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 Collect and assemble into large pieces of tissue to obtain a piece of cell-cultured meat composed of muscle fibers cultured in vitro.
  • Example 7 The preparation method of Example 7 is the same as that of Example 1, except that the polymer solution with cell non-adhesion is chitosan, and the concentration is 10 mg/mL.
  • the components of the hydrogel solution are gelatin with a volume fraction of 30%, genipin solution with a volume fraction of 1%, and F-10 culture medium containing calcium sulfate and 5 ⁇ 10 6 /mL bovine muscle stem cells with a volume fraction of 69%.
  • Example 8 The preparation method of Example 8 is the same as that of Example 1, except that the polymer solution with cell non-adhesiveness is pectin, and the concentration is 50 mg/mL.
  • the components of the hydrogel solution are 70% by volume hyaluronic acid, 1% by volume carbodiiminase solution, 29% by volume MEM medium containing calcium lactate, 5 ⁇ 10 8 cells/mL chicken muscle stem cells .
  • Example 9 The preparation method of Example 9 is the same as that of Example 1, except that the polymer solution with cell non-adhesion is carrageenan, and the concentration is 25 mg/mL.
  • the components of the hydrogel solution are fibrinogen with a volume fraction of 50%, thrombin solution with a volume fraction of 0.5%, DMEM/F-12 containing calcium chloride with a volume fraction of 49.5%, and 5 ⁇ 10 7 cells/mL sheep muscle stem cells. culture medium.
  • the above effects of the present invention can be achieved by using a certain amount of pig, cow, sheep, chicken, duck and other muscle stem cells for culture.

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Abstract

提供一种用于细胞培养肉生产的微流控仿生纤维及其制备方法和应用，该微流控仿生纤维具有"壳-核"结构，外壳由具有细胞非粘附性的高聚物交联后形成，外壳包裹的内核为混有种子细胞的水凝胶溶液。将内、外相流体分别通入微流控装置内、外相通道中，通过调节内、外相流速使两相流体在微流控装置通道形成稳定的层流结构，从装置出口处挤出获得仿生纤维。基于微流控技术构建仿生纤维，制备方法简单、成型快速、反应温和，制备的仿生纤维经过培养后可应用细胞培养肉生产，仿生纤维中的种子细胞在纤维载体中定向排列、迁移和融合生长，进而使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，提高细胞培养肉生产效率。

Description

一种用于细胞培养肉生产的微流控仿生纤维及其制备方法和应用 技术领域
本发明属于细胞培养肉领域，具体涉及用于细胞培养肉生产的微流控仿生纤维及其制备方法和应用。
背景技术
肉类含有丰富的蛋白质、维生素和矿物质等人体必需营养物质，已成为世界各国居民膳食宝塔的重要组成部分。随着世界人口数目与肉类需求的逐年上升，依赖畜牧养殖业的肉类生产方式同生态资源禀赋、公共卫生安全以及伦理道德等方面的矛盾日益突出，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。
在此背景下，细胞培养肉脱颖而出，它是一种依托组织工程，基于动物肌肉组织自愈再生能力，对相关细胞、组织进行体外培养而获取的肉类，有望通过体外培养的方式解决未来肉类供给难题。现阶段，生产细胞培养肉的方式一般是给予种子细胞适当的载体，并诱导种子细胞在载体上进行增殖、分化至成熟，最终收获即得到细胞培养肉。常见的用于细胞培养肉生产的载体材料主要有动物蛋白支架、植物蛋白支架、脱细胞植物支架和块状水凝胶等。尽管有不错的研究和应用进展，但支架材料在种子细胞贴壁、迁移和融合效率方面仍存在不足，且无法准确模拟天然骨骼肌中肌纤维的纤维状基本生理结构，致使种子细胞在支架上分化形成成熟肌肉组织的能力受到限制，从而生产效率低下。微流控是一项组织工程领域内的经典技术，其能够在微尺寸通道中操控微量液体，被认为是制备纤维载体的有力手段。然而，微流控技术在细胞培养肉生产方面的应用还未见报道。
发明内容
发明目的：针对现有技术存在的问题，本发明提供一种用于细胞培养肉生产的微流控仿生纤维，本发明制备的微流控仿生纤维有效解决了现有细胞培养肉生产方式周期长、过程繁琐，且无法准确模拟种子细胞的体内生长环境，相关蛋白合成少，细胞培养肉生产效率低的问题。
本发明还提供所述微流控仿生纤维的制备方法和应用。
技术方案：为了实现上述目的，本发明所述微流控仿生纤维具有“壳-核”结构；所述微流控仿生纤维的外壳由具有细胞非粘附性的高聚物交联后形成，所述外壳包裹的内核为混有种子细胞的水凝胶溶液。
其中，所述具有细胞非粘附性的高聚物溶液包括但不限于海藻酸钠、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高聚物溶液的浓度为10-50mg/mL。
其中，所述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。
其中，所述种子细胞来源包括但不限于猪、牛、羊、鸡、鸭、兔、鱼类、虾类和蟹类中的任意一种或者多种。
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。
其中，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、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，所述水凝胶溶液中的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粘附位点的材料均可。
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，所述基础培养基为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，步骤(1)中所述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、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、京尼平等化学交联剂中的一种或者多种。
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。
本发明所述的一种用于细胞培养肉生产的微流控仿生纤维的制备方法，包括如下步骤：
(1)配制微流控内、外相流体：配制具有细胞非粘附性的高聚物溶液作为外相流体，配制含有种子细胞的水凝胶溶液为内相流体；
(2)制备仿生纤维：将步骤(1)配制好的内、外相流体分别通入微流控装置的内、外相通道中，通过调节内、外相流速使两相流体在微流控装置的通道中形成稳定的层流结构，再由微流控装置挤出后经过收集液处理，获得仿生纤维。
其中，步骤(2)中所述微流控装置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。
其中，步骤(2)中所述微流控装置的通道结构可以是简易的内相通道与外相通道共轴嵌套的形式；或者在内相通道与外相通道共轴嵌套的基础上，再加上收集相通道和观察相通道构建的共轴嵌套的形式。
作为优选，步骤(2)中所述的微流控装置内相通道管径尺寸范围为50-300μm，外相通道出口管径尺寸范围为200-800μm。
进一步的，步骤(2)中内相溶液流速范围为0.5-10mL/h，外相溶液流速范围为0.5-10mL/h。
其中，步骤(2)中通过调节内、外相流速使两相流体在微流控装置通道中形成稳定的层流结构，再挤入到收集液中，洗去残余的收集液后获得仿生纤维；或者步骤(2)中通过调节内、外相流速使两相流体在微流控装置通道中形成稳定的层流结构，挤出的仿生纤维直接进行组织化集成，再浸泡在收集液中形成仿生纤维三维组织。
进一步地，步骤(2)中所述收集液包括但不限于钙盐、钠盐、钾盐、镁盐溶液中的一种或者多种。
具体制备时，将内、外相流体分别装填进注射器中，然后使用聚乙烯塑料管连接注射器出口和微流控装置内、外相通道入口，再将注射器固定到蠕动泵上；蠕动泵推动注射器活塞，内、外相流体经聚乙烯塑料管流入微流控装置，并在所述装置的出口处直接成型为仿生纤维，用于后续的组织化集成。
作为优选，制备仿生纤维时，选择将微流控装置的出口伸入装有收集液的培养皿中，两相流体经装置出口直接挤入到收集液中交联成型，仿生纤维形态、结构在收集液中进一步得到稳定，还可以在转移至清洗液和培养液之前为仿生纤维提供暂存的容器，便于后续生产操作。而直接进行仿生纤维的组织化集成时，可以在组织化集成过程中不使用收集液，选择在成型后使用收集液对三维组织进行整体交联和形状固定。
作为优选，可将仿生纤维置于漂洗液中清洗以充分去除残余的收集液，所述漂洗液包括但不限于含血清培养液、基础培养基、磷酸盐缓冲溶液(PBS)、生理盐水、葡萄糖溶液、无菌水。
本发明所述制备方法所制备的微流控仿生纤维在细胞培养肉生产中的应用。
其中，所述细胞培养肉生产包括如下步骤：
将仿生纤维转移至增殖培养液中进行增殖培养，待种子细胞在仿生纤维中自发融合形成纤维结构后，将增殖培养液替换为分化培养液；收集上述增殖、分化培养至成熟的仿生纤维，经组织化集成和食品化处理后用于细胞培养肉生产。
作为优选，直接将生成的仿生纤维清洗后，转移至装有增殖培液的培养皿中，保证仿生纤维被培养液完全浸没，放入37℃、5％CO2培养箱中培养，两天换一次培养液。
进一步地，仿生纤维增殖培养2天后，将增殖培养液换成分化培养液继续进行分化培养，每两天替换1/2体积的分化培养液；分化7天后，将仿生纤维收获。
本发明中为了保证待种子细胞在仿生纤维中融合形成纤维结构：第一，优选微流控装置通道尺寸和细胞使用量，使生成的纤维内核中细胞紧密排列；第二，所生成的仿生纤维外壳具有细胞非粘附特性，细胞被约束于内核中生长；第三，在空间约束和紧密排列的情况下，细胞在培养过程中有倾向于互相融合形成整体的生理特性。
其中，所述增殖培养液的成分为体积分数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(bFGF)。
其中，所述分化培养液的成分为体积分数94-97％基础培养基、2-5％马血清和1％青霉素-链霉素。
作为优选，所述增殖培养液和分化培养液中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。
进一步的，可去除微流控仿生纤维的高聚物部分来获取单纯的细胞纤维，具体方法为使用藻酸盐裂解酶、柠檬酸钠、乙二胺四乙酸、壳聚糖酶、果胶酶、卡拉胶酶中的一种或多种。
其中，所述组织化集成方式包括但不限于堆叠、编制、缠绕、捆绑、折叠等。
其中，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和烹饪包括煎、炸、煮、蒸、烤等。
进一步地，可去除所述成熟的仿生纤维的高聚物部分来获取单纯的细胞纤维，所述去除方法采用裂解液，所述裂解液包括藻酸盐裂解酶、柠檬酸钠、乙二胺四乙酸、壳聚糖酶、果胶酶或者卡拉胶酶。本发明仿生纤维可以在分化成熟后去除外壳，用于后续加工，获得纯细胞纤维，相对蛋白含量更高，营养更丰富。
本发明利用天然骨骼肌组织基本单元--肌纤维结构仿生的原理，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。
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。受天然骨骼肌组织中肌纤维这一基本单元纤维状结构的启发，本发明以肌纤维仿生为设计原理，提出一种基于微流控技术的用于细胞培养肉生产的仿生纤维载体的制备方法，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的高聚物外壳中，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。
具体而言，本发明中重点为微流控仿生纤维的制备，本发明首先设计并搭建如共轴嵌套式微流控装置；然后准备内、外相流体材料，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的高聚物外壳中，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。
本发明通过特定的“壳-核”结构、内外相组分调配以及整体方案设计，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。
有益效果：与现有技术相比，本发明具有如下优点：
(1)本发明基于微流控技术制备得到连续、规模化、结构均匀以及尺寸可控的仿生纤维，所涉及的设备装置成本低廉，制备条件温和、操作简单、成型迅速；
(2)本发明受天然骨骼肌组织中肌纤维结构的启发，制备的仿生纤维可为种子细胞提供良好的纤维载体，进一步模拟种子细胞在体内的三维生长环境，具有较好的仿生性；
(3)本发明制备的仿生纤维外壳不具有细胞贴附性，从而可诱导种子细胞在纤维载体内核空间中定向排列、迁移和融合生长，进而使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，提高了细胞培养肉生产效率；
(4)本发明制备的仿生纤维具有纤维材料良好的组织化集成特性，可进一步用于编制、缠绕、堆叠等精深加工操作，实现大块培养肉的构建。
附图说明
图1为本发明用于细胞培养肉生产的微流控仿生纤维的制备示意图；
图2为本发明用于细胞培养肉生产的微流控仿生纤维的制备过程实时图像以及微流控装置通道结构图；
图3为本发明用于细胞培养肉生产的微流控仿生纤维尺寸灵活调整的可行性验证数据图，其中，(a)为不同内核尺寸的仿生纤维明场图，比例尺为200μm；(b)和(c)为外壳、内核尺寸随流速改变的变化图；
图4为本发明用于组织化集成的微流控装置通道结构示意图和显微照片，比例尺为200um；
图5为用于细胞培养肉生产的微流控仿生纤维培养过程的显微镜明场视野图，其中，(a)为制备后孵育2小时仿生纤维显微镜明场视野图，(b)为增殖培养2天后仿生纤维显微镜明场视野图，(c)为分化培养3天后仿生纤维显微镜明场视野图，(d)为分化培养7天后仿生纤维显微镜明场视野图，比例尺为200μm；
图6为用于细胞培养肉生产的微流控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7为用于细胞培养肉生产的微流控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；
图8为用于细胞培养肉生产的微流控仿生纤维培养成熟后与市售猪肉的电镜对比图，其中，(a)为培养成熟的仿生纤维，比例尺为40μm；(b)为市售猪肉，比例尺为100μm；
图9为用于细胞培养肉生产的微流控仿生纤维培养成熟后与市售猪肉的H&E染色对比图，其中，(a)为培养成熟的仿生纤维纵切图，(b)为市售猪肉纵切图，(c)为培养成熟的仿生纤维横切图，(d)为市售猪肉横切图，比例尺为100μm；
图10为用于细胞培养肉生产的微流控仿生纤维组织化集成所用装置示意图；
图11为用于细胞培养肉生产的微流控仿生纤维组织化集成过程(a)及成品图(b)，比例尺为1000μm；
图12为用于细胞培养肉生产的微流控仿生纤维培养成熟后各类氨基酸含量与对照组和市售猪肉对比数据图；
图13为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14为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。
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
微流控内、外相流体的配制
(1)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。
(2)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。
实施例2
微流控仿生纤维的制备
微流控仿生纤维制备过程如图1所示，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。此外，选取一根圆形玻璃毛细管，毛细管内径为0.8mm，外径为1mm，作收集相通道；选取一根方形玻璃毛细管，内棱长为1.05mm，作观察相通道。将观察相方管通道固定在载玻片平面正中间的位置上(载玻片的厚度为1mm；载玻片的长为30mm，宽为25mm)，可以通过连接CCD摄像头，观察纤维在通道内的形成情况，然后将外相通道的拉制端和收集相通道分别经由方管通道两段插入，保证外相通道插入到收集相通道中并互不阻塞，在体式显微镜下调节外相通道和收集相通道至同一轴线(与玻璃毛细管长平行的横轴)，固定两管；接着将内相通道的拉制端从外相通道一边插入到外相通道中并固定，同样保证互不阻塞，调整四根玻璃毛细管的位置使其轴线(与玻璃毛细管长平行的横轴)重合。最后，在所有通道的接头处固定20G点胶针头，用AB胶粘连后即组装一种具有共轴嵌套形式的微流控装置，其结构如图2所示。
配制10mg/mL氯化钙溶液，灭菌，用作收集液。将上述收集液加入到注射器内，用一段聚乙烯塑料管一端连接注射器针头，一端连接微流控装置的收集相通道的入口；将实施例1中配制的海藻酸钠溶液加入到注射器内，用一段聚乙烯塑料管一端连接注射器针头，一端连接微流控装置的外相通道的入口；将实施例1配制的含有猪肌肉干细胞的水凝胶溶液加入到注射器内，聚乙烯塑料管一端连接注射器针头，一端连接微流控装置的内相通道的入口。然后，再将装有各相流体的注射器分别固定在蠕动泵上，调节收集相氯化钙溶液流速为15mL/h，内相水凝胶溶液流速为1.8mL/h，外相海藻酸钠溶液流速为2mL/h，启动蠕动泵。“壳-核”型仿生纤维在微流控装置内的生成过程可分为两个阶段。第一阶段，内相流体和外相流体首先在内相通道出口与外相通道出口之间汇聚形成同轴层流流体，然后进入收集相通道和收集相溶液再次汇聚形成三层同轴层流流体；第二阶段，三层流体形成后，外相海藻酸钠溶液在收集相和内相溶液钙离子的存在下开始形成海藻酸钙水凝胶并不断向内层扩散，“壳-核”型仿生纤维不断被固化挤出进入收集液。三相流体在装置中接触并形成稳定的层流结构(图2，显微观察可见收集相流体、内相流体和外相流体间明显的分界线，即为层流结构现象)，然后通过收集相通道挤入收集液中，即得到仿生纤维。所制备的仿生纤维尺寸可控，并且连续、可规模化，可通过改变流速来灵活调控(图3)。其中，图3中(a)为不同内核尺寸的仿生纤维明场图，如图3中(b)所示，随着内相溶液流速的增加，“壳-核”仿生纤维的内相直径也增加，而外相直径略微上升，受内相溶液流速的影响较小；如图3中(c)所示，随着外相溶液流速的增加，“壳-核”仿生纤维的内相直径随之减小，而外相直径同样略微上升，受外相溶液流速的影响较小。由此得出，“壳-核”仿生纤维内相的直径受各相流速影响，与内相溶液流速成正比，与外相溶液流速成反比；仿生纤维外相的直径几乎不受各相流速影响，受微流控装置出口口径的限制。
本实施例在保证内、外相流体充足的情况下，仿生纤维可从微流控装置出口处快速、持续生成；通过简单的调节微流控装置出口口径，内、外相流速的方式即可控制所制备的仿生纤维的尺寸；搭建微流控装置所使用的是玻璃毛细管、玻璃片、点胶针头等均为成本低廉的的常见耗材。此外，在制备时需要先通入外相溶液再通入内相溶液，如顺序相反无法形成纤维。
实施例3
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4所示，作为微流控装置用于实施例5三维组织的打印。
本实施例中搭建的微流控装置为实施例2中所搭建的微流控装置的简易版，不含有收集相通道和观察相通道，为内相通道与外相通道共轴嵌套的形式，可以直接用于仿生纤维的组织化集成；而实施例2中所搭建的微流控装置主要用于仿生纤维制备时通道内的流体状态和纤维生成过程的实时观察。此外，基于上述两种微流控装置制备的仿生纤维在结构、形态、功能上并无区别。
实施例4
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2制得的3根20cm左右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。
如图5所示，培养后2小时，种子细胞在“壳-核”仿生纤维的内核中依旧保持成球状并紧密排列(图5中(a))；增殖培养2天后，种子细胞在内核中完成迁移并相互融合形成纤维状结构(图5中(b))；分化培养3天后，细胞纤维相较于增殖培养2天时变细，并能看到细胞纤维中的肌管结构，说明种子细胞逐步开始分化(图5中(c))；分化培养7天后，细胞纤维相较于分化培养3天时进一步变细，细胞纤维中的肌管变长，种子细胞分化成熟(图5中(d))。如图5说明可观察到种子细胞融合形成纤维状结构，并被包裹于透明的高聚物外壳内。
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6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(Day7)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6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(day7)，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7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。
实施例5
微流控仿生纤维高聚物外壳的去除
称取4mg藻酸盐裂解酶干粉，加入1mL超纯水溶解后制得4mg/mL藻酸盐裂解酶溶液，使用0.22μm滤膜过滤除菌，并置于37℃水浴锅中备用。将实施例4中分化培养7天后得到成熟的仿生纤维的培养皿中的分化培养液吸去，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10mL不含血清的DMEM基础培养基，再向培养基中加入200μL藻酸盐裂解酶溶液，将培养皿置于37℃、5％CO2的培养箱中孵育20min。裂解完毕后，用弯头镊子将细胞纤维取出，PBS清洗后使用4％多聚甲醛和2.5％戊二醛进行组织固定。取2.5％戊二醛固定后的样品，置于50％，70％，80％，90％和无水乙醇中梯度脱水；将脱水后的样品浸入叔丁醇中置换，而后将样品冻干去除叔丁醇；使用离子溅射仪给样品表面喷金后用扫描电子显微镜进行观察拍照并与市售猪肉对比(图8中(a)和(b))。此外，取4％多聚甲醛固定后的样品，置于70％，80％，90％和无水乙醇中梯度脱水后，使用二甲苯梯度置换样品中的乙醇；再使用包埋剂石蜡置换样品中的二甲苯，用新鲜石蜡包埋样品，切片机切片后使用苏木精和伊红染色液进行染色，倒置显微镜观察拍照并与与市售猪肉对比(图9中(a)-(d))。从图8和9可以看出仿生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6
微流控仿生纤维的组织化集成
将实施例3构建的共轴嵌套形式的微流控装置集成到3D打印机喷头移动系统中作为打印喷头，改装得到微流控3D打印装置，其结构示意图如图10所示。
打印装置包括打印喷头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。
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，移动光轴一般是铝合金，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。打印喷头1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。
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，进样泵42采用注射泵为Longer Pump LSP01-1A微量注射泵，装样器41可采用注射器，导管43可采用聚乙烯塑料管。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。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。
用Auto CAD 2021软件建立打印模型，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。将实施例1中配制的海藻酸钠溶液加入到注射器内，用一段聚乙烯塑料管一端连接注射器针头，一端连接微流控装置的外相入口；将实施例1配制的含有猪肌肉干细胞的水凝胶溶液加入到注射器内，聚乙烯塑料管一端连接注射器针头，一端连接微流控装置的内相入口。然后，再将装有两相流体的注射器分别固定在两个Longer Pump LSP01-1A微量注射泵上，调节内相水凝胶溶液流速为1.8mL/h，外相海藻酸钠溶液流速为2mL/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装置。待纤维在装置出口(即外相通道出口)处生成后，选定打印程序并启动3D打印装置，随后3D打印机喷头移动系统带动微流控装置在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，使生成的纤维沉积于载物平台3并遵循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，用作收集液，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，组织化集成过程如图11中(a)，处理后的三维组织如图11中(b)。此外，组织化集成也可以采用堆叠、编制、缠绕、捆绑或者折叠等其他方式。
将三维组织转移至增殖培养液(体积分数84％DMEM/F-12、15％胎牛血清、1％青霉素-链霉素和5ng/mL成纤维细胞生长因子)中清洗、浸润10min，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；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15mL分化培养液(体积分数97％DMEM、2％马血清、1％青霉素-链霉素)，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，氨基酸分析结果(图12)显示细胞培养肉的各类氨基酸含量明显高于对照组(使用不含种子细胞的内核水凝胶溶液，其他制备方法和实施例1和2一致)，在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。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13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。
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。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。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14)。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。
本发明制备的微纤维在经过增殖、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、组装成大块组织即可得到体外培养的一块由肌纤维组成的细胞培养肉。
实施例7
实施例7与实施例1制备方法相同，不同之处在于：所述具有细胞非粘附性的高聚物溶液为壳聚糖，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％的含硫酸钙、含5×106个/mL牛肌肉干细胞F-10培养基。
实施例8
实施例8与实施例1制备方法相同，不同之处在于：所述具有细胞非粘附性的高聚物溶液为果胶，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％的碳二亚胺酶溶液，体积分数29％的含乳酸钙、5×108个/mL鸡肌肉干细胞MEM培养基。
实施例9
实施例9与实施例1制备方法相同，不同之处在于：所述具有细胞非粘附性的高聚物溶液为卡拉胶，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。
在本发明特定的微流控仿生纤维所具有的“壳-核”结构培养条件下，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上述效果。

Claims (19)

  1. 一种用于细胞培养肉生产的微流控仿生纤维，其特征在于，所述微流控仿生纤维具有“壳-核”结构；所述微流控仿生纤维的外壳由具有细胞非粘附性的高聚物交联后形成，所述外壳包裹的内核为混有种子细胞的水凝胶溶液。
  2. 根据权利要求1所述的用于细胞培养肉生产的微流控仿生纤维，其特征在于，所述具有细胞非粘附性的高聚物溶液为海藻酸钠、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高聚物溶液的浓度为10-50mg/mL。
  3. 根据权利要求1所述的用于细胞培养肉生产的微流控仿生纤维，其特征在于，所述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。
  4. 根据权利要求1所述的用于细胞培养肉生产的微流控仿生纤维，其特征在于，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。
  5. 根据权利要求1所述的用于细胞培养肉生产的微流控仿生纤维，其特征在于，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。
  6. 根据权利要求3所述的用于细胞培养肉生产的微流控仿生纤维，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。
  7. 根据权利要求3所述的用于细胞培养肉生产的微流控仿生纤维，其特征在于，所述基础培养基为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。
  8. 根据权利要求3所述的用于细胞培养肉生产的微流控仿生纤维，其特征在于，所述交联剂为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。
  9. 根据权利要求1所述的用于细胞培养肉生产的微流控仿生纤维，其特征在于，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。
  10. 一种权利要求1所述的用于细胞培养肉生产的微流控仿生纤维的制备方法，其特征在于，包括如下步骤：
    (1)配制微流控内、外相流体：配制具有细胞非粘附性的高聚物溶液作为外相流体，配制含有种子细胞的水凝胶溶液为内相流体；
    (2)制备仿生纤维：将步骤(1)配制好的内、外相流体分别通入微流控装置的内、外相通道中，通过调节内、外相流速使两相流体在微流控装置的通道中形成稳定的层流结构，再由微流控装置挤出后经过收集液处理，获得仿生纤维。
  11. 根据权利要求10所述的制备方法，其特征在于，步骤(2)中所述微流控装置的制作材料为晶体硅、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。
  12. 根据权利要求10所述的制备方法，其特征在于，步骤(2)中所述微流控装置的通道结构为内相通道与外相通道共轴嵌套的形式；或者在内相通道与外相通道共轴嵌套的基础上，再加上收集相通道和观察相通道构建的共轴嵌套的形式。
  13. 根据权利要求10所述的制备方法，其特征在于，步骤(2)中通过调节内、外相流速使两相流体在微流控装置通道中形成稳定的层流结构，再挤入到收集液中，洗去残余的收集液后获得仿生纤维；或者步骤(2)中通过调节内、外相流速使两相流体在微流控装置通道中形成稳定的层流结构，挤出的仿生纤维直接进行组织化集成，再浸泡在收集液中形成仿生纤维三维组织；所述收集液为钙盐、钠盐、钾盐、镁盐溶液中的一种或者多种。
  14. 一种权利要求1所述的用于细胞培养肉生产的微流控仿生纤维在细胞培养肉生产中的应用。
  15. 根据权利要求14所述的应用，其特征在于，所述细胞培养肉生产包括如下步骤：
    将仿生纤维转移至增殖培养液中进行增殖培养，待种子细胞在仿生纤维中融合形成纤维结构后，将增殖培养液替换为分化培养液；收集上述经过增殖、分化培养至成熟的仿生纤维，经组织化集成和食品化处理后用于细胞培养肉生产。
  16. 根据权利要求15所述的应用，其特征在于，所述增殖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；所述分化培养液的包括体积分数94-97％基础培养基、2-5％马血清和1％青霉素-链霉素。
  17. 根据权利要求15所述的应用，其特征在于，所述组织化集成采用堆叠、编制、缠绕、捆绑或者折叠方式。
  18. 根据权利要求15所述的应用，其特征在于，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。
  19. 根据权利要求15所述的应用，其特征在于，可去除所述成熟的仿生纤维的高聚物部分来获取单纯的细胞纤维，所述去除方法采用裂解液，所述裂解液包括藻酸盐裂解酶、柠檬酸钠、乙二胺四乙酸、壳聚糖酶、果胶酶或者卡拉胶酶。
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