CN114874978A - Personalized customization of cell culture meat based on porous scaffold material and production method thereof - Google Patents
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Abstract
The invention discloses a personalized customization of cell culture meat based on a porous scaffold material and a production method thereof. The porous scaffold material constructed by the invention has good biocompatibility and mechanical property, and higher porosity, connectivity and a proper pore size structure, is suitable for information transfer between cells and sufficient exchange of nutrient substances in the cell culture process, and is beneficial to the fate processes of cell growth, proliferation, differentiation, maturity and the like. In addition, edible qualities such as texture, taste and nutritional value of the cell culture meat can be improved by inoculating different types of cells and changing the cell inoculation ratio, so that personalized customization of the cell culture meat is realized. The porous scaffold material provided by the invention has important guiding significance for the personalized development of the cell culture meat production industry.
Description
Technical Field
The invention belongs to the field of cell culture meat, relates to a production method of personalized cell culture meat, and particularly relates to personalized customization of cell culture meat based on a porous support material and a production method thereof.
Background
The cell culture meat is based on animal cell large-scale culture technology and tissue engineering technology, and is formed by extracting stem cells or adult cells from animals and carrying out in-vitro proliferation, differentiation and culture construction. As a novel cell agricultural product, the cell culture medium is not dependent on livestock breeding, and the real meat is obtained by direct cell culture, so that the utilization rate of environmental resources is greatly reduced, and the problem of environmental pollution caused by livestock breeding is solved. In the face of continuously rising meat demand, animal epidemic diseases such as swine fever and avian influenza also bring a serious challenge to meat safety, so efficient and green cell culture meat needs to be researched and developed urgently.
The formation of mature muscle tissue through in vitro large-scale induced differentiation is one of the most critical links in the cell culture meat technology, and the method mainly adopted at present is to inoculate the livestock seed cells obtained by expanding culture onto a three-dimensional material, attach the cells onto the material, proliferate and differentiate to maturity. In the three-dimensional material for producing cell culture meat, the porous scaffold material has wide application potential in the field of cell culture meat due to low preparation cost, simple and environment-friendly method and convenient and efficient process. As a three-dimensional carrier for in vitro cell culture, the structural characteristics of the porous scaffold material are crucial to the cell fate process. Among these, pore structure is the primary reference characteristic of porous scaffold materials. The porous scaffold utilizes the pore structure to provide a place for three-dimensional cultured cells to obtain nutrients, exchange gases, excrete waste and grow metabolism. In addition, based on the anisotropic muscle tissue structure, the porous scaffold material provides an important support for three-dimensional culture of muscle stem cells, and the porous structure can be used for guiding and stimulating the functional expression of cells, including cytoskeleton arrangement, striated myotube formation, myogenic protein expression and the like. Therefore, the porous scaffold material with proper pore size and higher porosity and connectivity can provide a comfortable growth environment and a moderate contact and inhibition space for cells through the pore structure, and is further favorable for the fate process of cell growth, proliferation, differentiation, maturation and the like. However, most of the existing porous scaffold materials for constructing cell culture meat often neglect the requirement of pore structure, which is not favorable for sufficient exchange of nutrients, information transmission among cells and three-dimensional culture fate of cells in the cell culture process. Therefore, it is desirable to construct porous scaffold materials with appropriate pore structures for cell culture meat production.
The mouthfeel, texture and appearance of meat are important factors in measuring the quality of meat, wherein the mouthfeel texture is not only due to the chewiness provided by muscle fibers, but also the toughness of connective tissue junctions. Muscle tissue, connective tissue, capillary tissue, and the like can be constructed by culturing stem cells such as muscle stem cells, smooth muscle cells, fibroblasts, and endothelial cells, or adult cells. Therefore, selection of different cell cultures can be used to improve the eating quality of the cell culture meat. In addition, the different types of cell seeding ratios have a significant impact on the quality of meat through cell-cell interactions. However, most of the cell culture meat still stays in the construction mode of muscle stem cell single culture, and the effect of the cell type and the inoculation ratio on the taste, texture and appearance of the cell culture meat is ignored, so that the overall eating quality and sensory evaluation of the cell culture meat are influenced.
Object of the Invention
The invention content is as follows: aiming at the problems in the prior art, the invention provides a personalized customization of cell culture meat based on a porous scaffold material and a production method thereof, which effectively solve the problems that the pore structure of the existing porous scaffold material is not suitable for life activities such as proliferation and differentiation of muscle stem cells and the like and the eating quality of the cell culture meat is too single. The invention adopts different pore-forming modes and inoculates different cell types and cell proportions to realize the personalized customization of the cell culture meat.
The technical scheme is as follows: in order to achieve the aim, the invention provides a personalized customization of cell culture meat based on porous scaffold material and a production method thereof, which comprises the following steps:
(1) preparing a porous scaffold material: preparing a uniformly dispersed solution from a safe and edible material suitable for cell growth, prefabricating the uniformly dispersed solution into hydrogel in a physical, chemical or enzymatic crosslinking mode, adding a pore-forming agent and/or repeatedly freezing and thawing at a low temperature, and freezing and drying to construct a porous scaffold material; specifically, a pore-forming agent is added before hydrogel is formed, and then the porous scaffold material is constructed by freeze drying, or the porous scaffold material is constructed by directly freezing and thawing at low temperature repeatedly after the hydrogel is formed, or the pore-forming agent is added before the hydrogel is formed, and then the porous scaffold material is constructed by freezing and thawing at low temperature repeatedly after the hydrogel is formed;
(2) inoculating and culturing cells: after the porous scaffold material is sterilized, cleaned and soaked, inoculating cell suspension, and slowly supplementing and adding proliferation culture solution to carry out in-vitro three-dimensional proliferation culture on cells after the cells are attached to the scaffold; inducing cell differentiation culture after cell proliferation culture to form culture meat tissue; the in vitro three-dimensional proliferation culture of the cells is to select the stem cells or adult cells of the livestock or aquatic animals of a single cell type or different cell types according to different cell inoculation ratios to carry out three-dimensional culture so as to meet the personalized customization of later cell culture meat;
(3) personalized customization: collecting the cell culture meat after the differentiation process is finished, performing cell culture meat food treatment, and cooking according to the need to obtain the culture meat product. The safe edible material suitable for the adherent growth of the muscle stem cells comprises safe edible protein, polysaccharide or protein-polysaccharide compound and other safe edible materials.
Preferably, the safe and edible protein comprises natural proteins such as collagen, soybean protein, proteolytic peptide and the like, or in-vitro recombinant proteins expressed and secreted by escherichia coli, yeast, mammalian cell lines and the like; the polysaccharide comprises sodium alginate, agar, chitosan, etc.
Further, dispersing a safe and edible material suitable for cell growth in water to prepare a suspension in the step (1), and filtering to remove insoluble components after dissolving; filtering, adding a pore-forming agent, adjusting the pH to be an alkaline environment by adding NaOH or adding transglutaminase into the filtrate, and forming hydrogel at room temperature; then vacuum freeze-drying to obtain a porous scaffold material;
or, dispersing a safe and edible material suitable for cell growth in water to prepare a suspension, dissolving, and filtering to remove insoluble components; filtering, adding NaOH to adjust the pH value to be an alkaline environment or adding transglutaminase into the filtrate, and forming hydrogel at room temperature; constructing a porous structure on the hydrogel in a low-temperature repeated freeze thawing mode, and then performing vacuum freeze drying to obtain a porous scaffold material;
or, dispersing a safe and edible material suitable for cell growth in water to prepare a suspension, dissolving, and filtering to remove insoluble components; filtering, adding a pore-forming agent, adjusting the pH of the solution to be an alkaline environment or adding transglutaminase into the solution, and forming hydrogel at room temperature; and constructing a porous structure on the hydrogel in a low-temperature repeated freeze-thaw mode, and then performing vacuum freeze-drying to obtain the porous scaffold material.
Wherein the pore-forming agent comprises one or more of glycerol, polyethylene glycol (PEG), polyethylene oxide (PEO), polyvinyl alcohol (PVA), sucrose, sodium chloride and calcium carbonate particles. Other non-toxic porogens and the like may also be used.
Wherein the low-temperature repeated freeze-thawing is a repeated process of forming, thawing and re-forming ice crystals.
Preferably, the low-temperature repeated freezing and thawing is to freeze to form ice crystals within the range of-200 to-20 ℃, melt at 20 to 40 ℃, and freeze within the range of-200 to-20 ℃ to finish the repeated freezing and thawing process.
The porous scaffold material meeting the requirements of the pore structure can be obtained by pore-forming modes such as repeated freeze thawing, pore-forming agent addition and the like, wherein the pore structure is obtained through repeated processes of ice crystal formation, thawing and reformation, and the pore size is adjusted by adjusting the addition proportion of the pore-forming agent.
Wherein, the cells in the step (2) comprise muscle stem cells, progenitor cells thereof, nerve cells, smooth muscle cells, endothelial cells, fibroblasts, mesenchymal stem cells, or stem cells or adult cells of other animals.
Furthermore, the inoculated cells of the porous scaffold material can be derived from livestock and poultry animals such as pigs, cows, chickens, ducks and rabbits or aquatic animals such as fishes and shrimps.
Wherein, the individual customization of the cell culture meat can be realized according to the inoculation of different cell types and different cell inoculation ratios.
Wherein the culture medium for cell proliferation and differentiation culture in the step (2) is selected according to the type of inoculated cells, and comprises F10, DMEM/F12, DMEM or MEM basal culture solution which are prepared according to the requirement.
Further, the sterilization method of the porous scaffold material comprises sterilization modes such as 75% ethanol, ultraviolet radiation and ozone permeation.
Further, after the cell culture meat tissue is processed by food processing, such as seasoning pickling, flour pasting, monascus red coloring and other food additives, the cell culture meat is cooked by different methods, including cooking methods such as steaming, frying and the like.
Specifically, the personalized customization and production method of the cell culture meat based on the porous scaffold material comprises the following steps:
(1) preparing a porous scaffold material: a safe edible material suitable for the adherent growth of muscle stem cells is adopted, a uniformly dispersed solution is prepared according to the dissolution characteristics and is prefabricated to form hydrogel, and a porous scaffold material with high porosity, connectivity, a proper pore structure and good mechanical properties is constructed on the basis of a freeze-drying technology.
(2) Inoculating and culturing cells: after the porous scaffold material is treated by the steps of sterilization, cleaning, soaking and the like, selecting cell types according to needs, inoculating cell suspension with proper volume and density, slowly supplementing and adding proliferation culture solution to carry out in-vitro three-dimensional proliferation culture on cells after the cells are attached to the scaffold, wherein the cell number is increased in a proliferation stage, and then inducing the cells to differentiate to form cultured meat tissues; selecting the livestock stem cells or adult cells of single type or different cell types and different cell inoculation ratios to carry out three-dimensional culture so as to meet the individual customization of cell culture meat;
(3) personalized customization: collecting the cell culture meat after the differentiation process is finished, performing cell culture meat food treatment in different modes, and cooking according to the need to obtain the culture meat product.
The invention provides a cell culture meat personalized customization based on a porous scaffold material and a production method thereof, the porous scaffold material has a proper pore structure and is suitable for three-dimensional cell culture in vitro, muscle stem cells can be promoted to proliferate, differentiate and mature, and cell culture meat production with different textures, tastes and nutritional values can be met by selectively inoculating different types of cells and changing the cell inoculation proportion, so that the personalized customization of the cell culture meat is realized.
The material adopted in the invention selects a proper physical, chemical or enzymatic crosslinking mode according to the requirement to construct a hydrogel system with a three-dimensional network structure and a stable gel state, and the constructed porous scaffold material has good biocompatibility and mechanical property, and higher porosity, connectivity and a proper pore size structure thereof, is suitable for information transfer between cells and sufficient exchange of nutrient substances in the cell culture process, and is further favorable for the fate processes of cell growth, proliferation, differentiation, maturation and the like. In addition, edible qualities such as texture, taste and nutritional value of the cell culture meat can be improved by inoculating different types of cells and changing the cell inoculation ratio, so that personalized customization of the cell culture meat is realized. The porous scaffold material provided by the invention has important guiding significance for the personalized development of the cell culture meat production industry. The invention can adopt different pore-forming modes according to different taste textures and nutritional requirements, selectively inoculate different types of cells and change the cell inoculation proportion to carry out in-vitro three-dimensional culture according to requirements, can realize personalized customization of cell culture meat, and effectively solves the problem that the cell culture meat products are too single in quality.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the material for constructing the porous scaffold has low cost, is safe and edible, the preparation method is simple and environment-friendly, the preparation process is convenient and efficient, the reaction condition is mild, expensive instruments and experimental devices are not needed, and the time and the capital consumption of research and development production of cell culture meat are reduced to a great extent;
(2) the invention can adjust the porosity and the pore size by selecting different pore-forming modes (such as adding pore-forming agent, repeated freezing and thawing or combined use) to construct the porous scaffold material with a proper pore structure, thereby being beneficial to the fate processes of information transmission between cells, full exchange of nutrient substances, cell growth and proliferation, differentiation and maturation and the like in the cell culture process;
(3) according to the porous scaffold material constructed by the invention, edible qualities such as texture, taste and nutritional value of the cell culture meat can be improved by inoculating different types of cells and changing the cell inoculation ratio, so that individual customization of the cell culture meat is realized, and diversified and nutritional development requirements of the cell culture meat are met.
Drawings
FIG. 1 is a flow chart of the process for preparing the porous scaffold material and producing cell culture meat according to the present invention;
FIG. 2 is a scanning electron microscope image of a porous scaffold material prepared by adding different porogens according to the present invention, wherein (a) is a pore-forming agent without being added, (b) is a pore-forming agent with PEO being added, (c) is a pore-forming agent with PEG being added, and (d) is a pore-forming agent with PVA being added, and the scale bar is 100 μm;
FIG. 3 is a scanning electron microscope image of the porous support material of the present invention, wherein (a) is a cross-sectional scanning electron microscope image, and (b) is a longitudinal-sectional scanning electron microscope image with a scale bar of 100 μm;
FIG. 4 is an immunofluorescence chart of the porous scaffold material of the present invention inoculated with muscle stem cells cultured to the terminal stage of differentiation, with the scale bar being 100 μm;
FIG. 5 is a photograph showing a cooked product of cell-cultured meat constructed according to the present invention.
Detailed Description
The invention is further illustrated by the following figures and examples.
The raw materials and reagents used in the examples are commercially available.
The collagen, the transglutaminase, the muscle stem cells, the smooth muscle cells and the fibroblasts are all commercially available. Wherein, the food-grade collagen is purchased from Ten Kate Protein Technologies; transglutaminase available from Solarbio; the muscle stem cells are porcine skeletal muscle stem cells, and both smooth muscle cells and fibroblasts are porcine cells, and other poultry and aquatic animals can also be adopted.
Example 1
Preparation of food-grade collagen porous scaffold material
The preparation method of the porous scaffold material for cell culture meat production is shown in figure 1, and comprises the following steps:
firstly, dispersing food-grade collagen in ultrapure water to prepare suspension with the mass fraction of 4%, fully dissolving the suspension in a warm water bath at 50 ℃ for 0.5h, and filtering to remove insoluble components; uniformly mixing a pore-foaming agent water solution (PEO (MW 300000), PEG (MW 400) or PVA (MW 85000)) with the filtered collagen solution according to the volume ratio of 1: 2; uniformly dispersing a 10% by mass glutamine transferase (100u/g) water solution and the filtered collagen solution in a protein-pore-forming agent mixed solution according to the volume ratio of 1:10, and forming hydrogel at room temperature for 40 min. And (3) freezing the sample with the stable gel state by adopting liquid nitrogen at the temperature of-196 ℃ for 5min, re-melting at the temperature of 25 ℃ for 8min, freezing at the temperature of-80 ℃ for 5h, and repeatedly freezing and thawing at a low temperature to construct a porous structure, and then freeze-drying in vacuum for 24h to obtain the porous scaffold material.
The porous scaffold material prepared by the implementation is spongy and fluffy and porous. Microscopic observation is carried out on the pore structure of the porous support material through a scanning electron microscope, as shown in figure 2, the communicated porous structure of the support material can be observed, no pore-foaming agent group is added, the pore size distribution is 10-60 mu m, and a relatively wider pore size distribution range (10-100 mu m) can be obtained by adding PEG; the PVA is added to obtain a pore structure with uniform size; in addition, the pore structure is changed according to the different types of the added pore-foaming agents, PEO is used as the pore-foaming agent, and the constructed porous support material has a highly-communicated network structure.
Example 2
Preparation of food-grade collagen porous scaffold material
Firstly, dispersing food-grade collagen in water to prepare suspension with the mass concentration of 4%, fully dissolving the suspension in a warm water bath at 50 ℃ for 0.5h, and filtering to remove insoluble components; dispersing transglutaminase (100u/g) in water to prepare a solution with the mass fraction of 10%, uniformly mixing the solution with the filtered collagen solution according to the volume ratio of 10:1, and forming hydrogel with stable gel state at room temperature for 30 min; freezing with liquid nitrogen at-196 deg.C for 5min to form ice crystals, re-melting at 25 deg.C for 8min, freezing at-80 deg.C for 5h to construct porous structure, and vacuum lyophilizing the hydrogel in frozen state for 24h to obtain porous scaffold material. The pore structure of the porous support material is microscopically observed by a scanning electron microscope, as shown in figure 3, the transverse and longitudinal sections all show pore communication, the pore size distribution is uniform, and the pore diameter of a channel is about 10-60 mu m at most.
Example 3
In vitro cell culture of porous scaffold material
A method for producing cell culture meat based on a porous scaffold material, comprising the steps of:
sterilizing the porous scaffold material prepared in example 2 with 75% ethanol, then performing PBS immersion cleaning for 3 times, and finally irradiating for 40min under a closed condition by an ultraviolet lamp; after radiation sterilization, 100ul of 1X 10 are taken 8 The porcine skeletal muscle and muscle stem cells per ml are inoculated on a porous bracket material (the size is about 10mm multiplied by 1mm) cut by a surgical knife blade; inoculating muscle stem cells with the porous scaffold material for about 1h, slowly adding 2ml of F10 proliferation culture solution containing 20% fetal calf serum, 1% penicillin streptomycin double antibody solution and 5ng/ml fibroblast growth factor into the pore plate, placing the culture plate in saturated humidity, 37 ℃ and 5% CO 2 Culturing in an incubator for 3 days, and replacing the proliferation culture solution every two days; when the proliferation of the muscle stem cells reaches the pre-differentiation density, replacing a differentiation culture solution, wherein the differentiation culture solution comprises 2% horse serum, 97% DMEM cell culture medium and 1% penicillin streptomycin double antibody solution, replacing 1/2 differentiation culture solution every two days, allowing the cells to enter a differentiation process, and continuing to differentiate for 14 days.
And (3) taking the cell culture meat at the final stage of differentiation (differentiation for 14 days) for immunofluorescence detection, and observing and collecting images under a confocal microscope. FIG. 4 shows the F-actin immunofluorescent staining result secreted by the muscle stem cells loaded by the porous scaffold material, and as shown in FIG. 4, the food-grade collagen porous scaffold material has good biocompatibility, supports the proliferation and differentiation of the muscle stem cells, and expresses a large amount of F-actin at the end of differentiation, which indicates that the scaffold material is suitable for the growth, proliferation and differentiation of the muscle stem cells and has the expression of marker proteins.
In addition, in order to realize the diversified construction of the cell culture meat, the muscle stem cells, the smooth muscle cells and the fibroblasts of the pigs are also co-cultured in vitro by utilizing the porous scaffold material. Cell type and number ratio muscle stem cells: smooth muscle cells: fibroblast cells were 2:1: 1. The porous support material is used for multi-cell co-culture, so that the expression of the muscle stem cell marker protein and the generation of related extracellular matrix can be promoted, and the sensory characteristics, texture and mouthfeel of cell culture meat are improved, thereby being closer to the edible experience of natural muscle tissues.
Example 4
Processing cell culture meat into food:
the method for processing the cell culture meat into food comprises the following steps:
cutting the porous scaffold material prepared in example 2 into a cylinder with a diameter of about 2.2cm and a height of 1.5mm by using a surgical blade, sterilizing the porous scaffold material by using 75% ethanol, performing PBS (phosphate buffer solution) immersion cleaning for 3 times, and finally irradiating for 40min by using an ultraviolet lamp under a closed condition; after radiation sterilization, 100ul of 1X 10 are taken 9 Inoculating each/ml of the porcine skeletal muscle and muscle stem cells on the treated porous scaffold material; inoculating muscle stem cells with the porous scaffold material for about 1h, slowly adding 2ml of F10 proliferation culture solution containing 20% fetal calf serum, 1% penicillin streptomycin double antibody solution and 5ng/ml fibroblast growth factor into the pore plate, placing the culture plate in saturated humidity, 37 ℃ and 5% CO 2 Culturing in an incubator for 3 days, and replacing the proliferation culture solution every two days; when the proliferation of the muscle stem cells reaches the pre-differentiation density, replacing a differentiation culture solution, wherein the differentiation culture solution comprises 2% of horse serum, 97% of DMEM cell culture medium and 1% of penicillin streptomycin double antibody solution, replacing 1/2 differentiation culture solution every two days, enabling the cells to enter a differentiation process, and differentiating for 14 days.
After the 14-day differentiation process, the porous scaffold material structure is kept intact, and the tissue texture of the cell culture meat is endowed with elasticity. Harvesting the cultured meat tissue and slowly washing for a plurality of times to remove the components of the culture solution; selecting red yeast rice red to color, cooking the cultured meat in a frying mode, placing the cultured meat in a frying pan, frying the cultured meat in the front and the back, cooking the cultured meat in the dark yellow on two sides, and taking out the cooked cultured meat. The graph of a cooked cell culture meat constructed by the porous scaffold material is shown in fig. 5, after frying, the cell culture meat is glossy, the appearance performance is not different from that of a normal muscle tissue, the taste is similar to that of a fried ham, and the cell culture meat has a small specification, a light meat taste and slightly low fragrance.
According to the method, the cells are replaced by the porcine skeletal muscle stem cells, the porcine smooth muscle cells and the porcine fibroblasts in a ratio of 2:1: 1. After the 14-day differentiation process is finished, the structure of the porous scaffold material is kept complete, the cultured meat tissue is harvested and slowly cleaned for multiple times to remove the components of the culture solution; selecting monascus red for coloring treatment, cooking the cultured meat in a frying mode, placing the cultured meat in a frying pan, frying the cultured meat in the front and the back, cooking the cultured meat in the dark yellow on two sides, and taking out the cooked cultured meat; the cell culture meat obtained based on the multi-cell co-culture not only has mechanical properties similar to those of natural muscle tissues, but also the secreted extracellular matrix gives it an apparent luster, exhibits sensory properties similar to those of natural muscle tissues, and the cooked cell culture meat has a typical meat-like taste and a pleasant meat flavor.
Example 5
Dispersing food-grade collagen or gelatin in water to prepare suspension with mass concentration of 6%, dissolving in warm water bath at 50 deg.C for 0.5 hr, and filtering to remove insoluble components; taking a polysaccharide (sodium alginate, agar or chitosan) water solution with the mass fraction of 2%, protein: the polysaccharide is mixed evenly according to the volume ratio of 2: 1. Adopting 10% of transglutaminase by mass to induce protein molecules to be crosslinked, wherein the volume ratio of the transglutaminase to the mixed liquid is 1:10, standing for 2h at room temperature, and forming hydrogel with stable gel state through electrostatic interaction between protein and polysaccharide; freezing at-80 deg.C for 5h, re-melting at 25 deg.C for 8min, freezing at-80 deg.C for 5h to construct porous structure, and vacuum lyophilizing the hydrogel in frozen state for 24h to obtain porous scaffold material.
Example 6
Dispersing food-grade collagen or gelatin in ultrapure water to prepare suspension with mass fraction of 6%, dissolving in 50 deg.C warm water bath for 0.5 hr, and filtering to remove insoluble components; uniformly mixing a pore-foaming agent (glycerol or calcium carbonate) with the filtered collagen solution according to the mass ratio of 1: 3; uniformly dispersing a 10 mass percent transglutaminase aqueous solution and the filtered collagen solution in a protein-pore-forming agent mixed solution according to a volume ratio of 1:10, and forming hydrogel at room temperature for 40 min. The samples with stable gel state are frozen overnight at-80 ℃ and then freeze-dried in vacuum for 24h to obtain the porous scaffold material.
Claims (10)
1. A personalized customization and production method of cell culture meat based on porous scaffold materials are characterized by comprising the following steps:
(1) preparing a porous scaffold material: preparing a uniformly dispersed solution from a safe and edible material suitable for cell growth, prefabricating the uniformly dispersed solution into hydrogel in a physical, chemical or enzymatic crosslinking mode, adding a pore-forming agent and/or repeatedly freezing and thawing at a low temperature, and freezing and drying to construct a porous scaffold material;
(2) inoculating and culturing cells: after the porous scaffold material is sterilized, cleaned and soaked, inoculating cell suspension, and slowly supplementing and adding proliferation culture solution to carry out in-vitro three-dimensional proliferation culture on cells after the cells are attached to the scaffold; inducing cell differentiation culture after cell proliferation culture to form culture meat tissue; the in vitro three-dimensional proliferation culture of the cells is to select the stem cells or adult cells of livestock and poultry, aquatic animals of a single cell type or different cell types according to different cell inoculation ratios to carry out three-dimensional culture so as to meet the personalized customization of later cell culture meat;
(3) personalized customization: collecting the cell culture meat after the differentiation process is finished, performing cell culture meat food treatment, and cooking according to the need to obtain the culture meat product.
2. The personalized customization of cell culture meat based on porous scaffold material and the production method thereof according to claim 1, characterized in that the material suitable for cell growth and safe to eat in step (1) comprises safe to eat protein, polysaccharide or protein-polysaccharide complex.
3. The personalized customization of cell culture meat based on porous scaffold material and the production method thereof according to claim 1, characterized in that, in step (1), the material which is suitable for cell growth and safe and edible is dispersed in water to prepare suspension, and after dissolution, insoluble components are removed by filtration; filtering, adding a pore-forming agent, adjusting the pH of the solution to be an alkaline environment or adding transglutaminase into the solution, and forming hydrogel at room temperature; then vacuum freeze-drying to obtain a porous scaffold material;
or, dispersing a safe and edible material suitable for cell growth in water to prepare a suspension, dissolving, and filtering to remove insoluble components; adjusting the pH of the solution to be in an alkaline environment or adding transglutaminase into the solution to form hydrogel at room temperature; constructing a porous structure on the hydrogel in a low-temperature repeated freeze thawing mode, and then performing vacuum freeze drying to obtain a porous scaffold material;
or, dispersing a safe and edible material suitable for cell growth in water to prepare a suspension, dissolving, and filtering to remove insoluble components; filtering, adding a pore-forming agent, adjusting the pH of the solution to be an alkaline environment or adding transglutaminase into the solution, and forming hydrogel at room temperature; and constructing a porous structure on the hydrogel in a low-temperature repeated freeze-thaw mode, and then performing vacuum freeze-drying to obtain the porous scaffold material.
4. The personalized customization of cell culture meat based on porous scaffold material and the production method thereof according to claim 3, characterized in that the pore-forming agent preferably comprises one or more of glycerol, polyethylene glycol (PEG), polyethylene oxide (PEO), polyvinyl alcohol (PVA), sucrose, sodium chloride, calcium carbonate particles.
5. The personalized customization of cell culture meat based on porous scaffold material and its production method according to claim 3, characterized in that the low temperature repeated freeze-thaw is an repeated process by ice crystal formation, thawing, re-formation.
6. The personalized customization of cell culture meat based on porous scaffold material and the production method thereof as claimed in claim 3, characterized in that the low temperature repeated freezing and thawing is freezing to form ice crystals within the set temperature range of-200 to-20 ℃, and the repeated freezing and thawing process is completed after thawing at 20 to 40 ℃.
7. The method for personalized customization of cell culture meat based on porous scaffold material and its production according to claim 1, characterized in that the cells of step (2) comprise one or more of muscle stem cells, progenitor cells, nerve cells, smooth muscle cells, endothelial cells, fibroblasts, mesenchymal stem cells; the inoculated cells are derived from livestock and poultry animals or aquatic animals.
8. The personalized customization of cell culture meat based on porous scaffold material and its production method as claimed in claim 1, characterized in that step (2) can realize personalized customization according to different cell types and different cell seeding ratio, wherein the cell types are one or more of the cell types as claimed in claim 7.
9. The personalized customization of cell culture meat based on porous scaffold material and its production method of claim 1, characterized in that, the culture medium of step (2) of cell proliferation and differentiation culture is selected according to the type of inoculated cells, and is prepared as required by using F10, DMEM/F12, DMEM or MEM basal medium.
10. The personalized customization of cell culture meat based on porous scaffold material and the production method thereof according to claim 1, characterized in that the food processing of cell culture meat in step (3) comprises seasoning curing, flour pasting or monascus red coloring; and then, cooking the cell culture meat in different ways, including steaming, frying or frying.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115612159A (en) * | 2022-10-25 | 2023-01-17 | 中国海洋大学 | Edible medium for 3D culture of cells and crosslinking method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150250923A1 (en) * | 2012-09-18 | 2015-09-10 | Corticalis As | Hydrogel coated scaffold |
| CN113215090A (en) * | 2020-11-05 | 2021-08-06 | 西北农林科技大学 | Manufacturing method of edible chitosan/sodium alginate/gelatin 3D scaffold for cell culture meat |
| CN114438013A (en) * | 2022-01-26 | 2022-05-06 | 上海食未生物科技有限公司 | Method for preparing cell culture meat biological scaffold through physical crosslinking |
-
2022
- 2022-05-07 CN CN202210496027.9A patent/CN114874978A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150250923A1 (en) * | 2012-09-18 | 2015-09-10 | Corticalis As | Hydrogel coated scaffold |
| CN113215090A (en) * | 2020-11-05 | 2021-08-06 | 西北农林科技大学 | Manufacturing method of edible chitosan/sodium alginate/gelatin 3D scaffold for cell culture meat |
| CN114438013A (en) * | 2022-01-26 | 2022-05-06 | 上海食未生物科技有限公司 | Method for preparing cell culture meat biological scaffold through physical crosslinking |
Non-Patent Citations (3)
| Title |
|---|
| HODA KHALESI 等: ""New insights into food hydrogels with reinforced mechanical properties: A review on innovative strategies"", 《ADVANCES IN COLLOID AND INTERFACE SCIENCE》, pages 1 - 18 * |
| 中国食品药品检定研究院: "《药用辅料和药品包装材料检验技术》", 中国医药科技出版社, pages: 170 * |
| 吴霞 等: ""胶原-PVA 复合水凝胶的制备"", 《生物医学工程与临床》, vol. 16, no. 4, pages 309 - 313 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115612159A (en) * | 2022-10-25 | 2023-01-17 | 中国海洋大学 | Edible medium for 3D culture of cells and crosslinking method thereof |
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