CN211394496U - Porous netted cultivation meat production mould - Google Patents
Porous netted cultivation meat production mould Download PDFInfo
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Abstract
The utility model relates to a porous netted cultivation meat production mould. The production mold comprises an outer wall, a first groove, a second groove and a micro-column array formed by regularly arranging a plurality of micro-columns. The first groove is arranged on the inner side of the outer wall, and the second groove is arranged in the first groove; the bottom of the second groove is provided with a plurality of micro-columns which are regularly arranged to form a micro-column array. The design of the micro-column array considers the mechanical change in the later mixed gel compaction process, enhances the diffusion of nutrient substances to cells, guides the local three-dimensional cell arrangement by controlling the spatial pattern of mechanical tension and promotes the differentiation process. The second recess is a culture space for culturing meat muscle tissue. The design of the first groove is beneficial to the demolding process of the male mold, and the using amount of the culture solution can be enlarged, so that the exchange of nutrient substances is promoted. By using the mould, larger reticular culture meat muscle tissues can be produced and obtained.
Description
Technical Field
The invention belongs to the technical field of stem cell and animal cell cultured meat, and particularly relates to a porous netted cultured meat production mold.
Background
China is the world's first country of meat production and consumption. The demand for meat products is further increasing. However, the traditional animal husbandry production has the problems of low protein conversion rate of food raw materials, environmental pollution, threat to the health of animals and human beings due to the epidemic of some viruses, and the like. When the meat is cultured and produced, the stem cells of the animals are extracted to produce the meat in vitro, so that the potential of relieving a plurality of problems of the traditional animal husbandry is realized.
One of the most important techniques in the in vitro production of cultured meat is the in vitro production of muscle tissue. Traditional muscle tissue engineering utilizes the principle of bionics to pull muscle tissue in vitro through two anchor points. Such muscle tissue is generally small and is intended for applications in muscle tissue engineering or screening of muscle pharmaceuticals.
Culturing meat requires the production of a large muscle tissue in vitro for consumption, and it is quite difficult to arrange many muscle cell layers in a large area. The present mould for producing cultured meat has a cylindrical single-column mould and a double-column mould drawn by two anchor points. By using the single-column mould, a muscle bundle tightly attached to the cylinder can be formed around the cylinder, and a muscle bundle can be formed after the muscle bundle is unfolded. The double-column type mold also forms a muscle bundle between the two column-type anchor points. However, since the transportation distance limit of oxygen and other nutrients and wastes is 100-.
Disclosure of Invention
In order to solve the technical problem, the utility model provides a porous netted cultivation meat production mould of microcolumn array is arranged to great size, law, places myogenic cell and aquogel muscle tissue glue in wherein and cultivates, can obtain a porous netted muscle tissue of cultivateing the meat and using. The mould can also enlarge and adjust the length and the width of the outer wall, the first groove and the second groove while keeping the size and the arrangement mode of the microcolumns and the heights of the first groove 2 and the second groove 3 in the range limited by the application, thereby realizing the production of a larger cultured meat product which accords with the size of future industrial production in vitro.
The utility model discloses technical scheme as follows:
a porous netted cultivation meat production mould, the upper end of said mould is open, including outer wall 1, the first groove 2, the second groove 3, microcolumn 4;
the longitudinal section of the outer wall 1 is of a downward two-stage ladder shape, the first stage is the bottom of the first groove 2, and the second stage is the bottom of the second groove 3.
The second groove 3 is arranged at the bottom of the first groove 2; the second recess is a culture area for adding mainly mixed gel required for culturing meat.
A plurality of microcolumns 4 are vertically arranged at the bottom of the second groove 3; the micro-columns 4 are regularly staggered and arranged at intervals to form a micro-column array, mechanical change in the later mixed gel culture process is considered in the design of the micro-column array, diffusion of nutrients to cells is enhanced, local three-dimensional cell arrangement is guided by controlling the spatial pattern of mechanical tension, and the differentiation process is promoted.
Furthermore, the length of the microcolumn 4 is 1-5 mm, the width is 0.5-1.5 mm, and the height is 1-5 mm.
Furthermore, the regular arrangement mode of the microcolumns 4 is staggered and arranged at intervals.
Further, the staggered interval arrangement of the microcolumns 4 is specifically as follows: the micro-pillars 4 of adjacent rows are spaced by 1-3mm, the micro-pillars 4 of adjacent rows are offset in parallel by 1-4mm, and the micro-pillars 4 in each row are spaced by 0.5-3 mm.
Based on the size, arrangement mode and spacing distance of the micro-columns 4, even if the size of the mould is enlarged, the gel can be contracted between different columns to form muscle bundles by combining the gel formation of the mixture containing myogenic cells and the mechanical change in the gel compaction process, and the proper transportation distance of oxygen, other nutrient components and metabolic waste is given, so that the gel can be contracted between different columns to form muscle bundles, the contraction space formed by the muscle bundles around the micro-columns can enhance the diffusion of nutrient substances to cells, and the local three-dimensional cell arrangement is guided by controlling the spatial pattern of mechanical tension to promote the differentiation process, thereby obtaining larger muscle tissues.
The number of rows of the microcolumns 4 is more than or equal to 2; it is preferable that the microcolumns 4 are disposed as much as possible according to the area of the bottom of the second groove 3 within the above range of the size and arrangement of the microcolumns 4.
Further, the cross section of the micro-column 4 is rectangular or elliptical.
Further, the mold is a cuboid or a cylinder with an opening at the upper end.
Further, the first groove 2 is centrally arranged inside the outer wall 1.
Further, the height of the first groove 2 is 3-7 mm. Within this height range, the first recess may provide a suitable medium storage space and facilitate demolding when preparing the present mold.
Further, the width of the bottom of the first groove 2 is 2-4 mm. This width facilitates demolding when preparing the present mold.
Further, the second groove 3 is centrally arranged at the bottom of the first groove 2.
Further, the height of the second groove 3 is 2-6mm, that is, the height of the second groove 3 is the same as or slightly higher than the height of the microcolumn 4.
Further, in some embodiments, the outer wall 1 has a length of 20 to 100 mm; the width is 20-100 mm; the height is 10-18mm, and a cuboid with an opening at the upper end is formed.
Further, in some embodiments, the length of the first groove 2 is 16 to 92 mm; the width is 16-92 mm; the height is 3-7 mm; the width of the bottom is 2-4 mm.
Further, in some embodiments, the length of the second groove 3 is 10-84 mm; the width is 10-84 mm; the height is 2-6 mm.
The mould can also adjust the length and the width of the outer wall 1 of the mould, the first groove 2 and the second groove 3 while keeping the size and the arrangement mode of the microcolumns 4 and the heights of the first groove 2 and the second groove 3 within the range limited by the application, thereby realizing the production of a larger muscle tissue for eating in vitro and producing a cultured meat product which meets the size of future industrial production.
The utility model discloses the beneficial effect that technical scheme realized does:
the porous netted culture meat production die with the micro-column arrays regularly arranged is designed, and fully considers that in the in-vitro culture process of culture meat, a mixed solution containing myogenic cells forms hydrogel muscle tissues and mechanical changes in the hydrogel muscle tissue compaction process, so that the hydrogel muscle tissues can be contracted between different columns to form muscle bundles, the contraction space formed by the muscle bundles around the micro-columns can enhance the diffusion of nutrient substances to cells, and the local three-dimensional cell arrangement is guided by controlling the space pattern of mechanical tension, so that the differentiation process is promoted. The mould can also adjust the length and width of the outer wall of the mould, the first groove and the second groove while keeping the size and arrangement mode of the microcolumns and the heights of the first groove and the second groove within the range limited by the application, and can be properly enlarged, so that a larger cultured meat product which is in line with the size of future industrial production can be produced in vitro.
Drawings
Fig. 1 is a schematic structural view of the mold for producing cultured meat according to the present invention.
FIG. 2 growth of reticular muscle tissue in a cultured meat production mold.
In the figure: 1 outer wall, 2 first grooves, 3 second grooves and 4 microcolumns.
Detailed Description
The present invention will be further clarified by the following description with reference to the attached drawings and specific examples, which should be understood as being merely illustrative of the present invention and not limiting the scope of the present invention, and modifications of various equivalent forms of the present invention by those skilled in the art after reading the present invention, all fall within the scope defined by the appended claims of the present application.
In the description of the present invention, "a plurality" means two or more unless specifically limited otherwise.
Example 1
A porous netted cultivation meat production mould, the said mould is the cuboid with open-ended upper end, including outer wall 1, the first groove 2, the second groove 3, microcolumn 4;
the longitudinal section of the outer wall 1 is of a downward two-stage ladder shape, the first stage from top to bottom is the bottom of the first groove 2, and the second stage is the bottom of the second groove 3.
The length of the outer wall 1 is 25 mm; the width is 25 mm; the height is 12mm, and a cuboid with an opening at the upper end is formed.
The first groove 2 is arranged in the middle of the inner side of the outer wall 1, and the length of the first groove 2 is 20 mm; the width is 20 mm; the height is 5 mm. The width of the bottom edge of the first groove 2 is 2 mm.
The second groove 3 is arranged at the bottom of the first groove 2 in the middle; the length of the second groove 3 is 16 mm; the width is 16 mm; the height is 3mm and the thickness of the bottom of the second groove 3 is 4 mm.
25 microcolumns 4 are vertically arranged at the bottom of the second groove 3; the micro-column 4 is 2mm in length, 1mm in width and 3mm in height; the cross section of the micro-column is rectangular, and 25 micro-columns 4 are regularly arranged to form a micro-column array;
the arrangement mode of the micro-pillars 4 is staggered and spaced, the micro-pillar array is 7 rows, each row of micro-pillars is sequentially arranged by 4-3-4 micro-pillars, the space between the micro-pillars 4 in the adjacent rows is 1mm, the micro-pillars 4 in the adjacent rows are parallelly offset by 1mm, and the space between the micro-pillars 4 in each row is 1.5mm (figure 1).
Example 2
A porous netted cultivation meat production mould, the said mould is the cuboid with open-ended upper end, including outer wall 1, the first groove 2, the second groove 3, microcolumn 4;
the length of the outer wall 1 is 30 mm; the width is 30 mm; the height is 15mm, forms the cuboid of upper end opening.
The longitudinal section of the outer wall 1 is of a downward two-stage ladder shape, the first stage from top to bottom is the bottom of the first groove 2, and the second stage is the bottom of the second groove 3.
The first groove 2 is arranged in the middle of the inner side of the outer wall 1, and the length of the first groove 2 is 26 mm; the width is 26 mm; the height is 6 mm. The width of the bottom edge of the first groove 2 is 4 mm.
The second groove 3 is arranged at the bottom of the first groove 2 in the middle; the length of the second groove 3 is 18 mm; the width is 18 mm; the height is 4mm and the thickness of the bottom of the second groove 3 is 5 mm.
18 microcolumns 4 are vertically arranged at the bottom of the second groove 3; the microcolumn 4 has a length of 3mm, a width of 1.5mm and a height of 4 mm; the cross section of the microcolumn is rectangular; the 18 micro-columns 4 are regularly arranged to form a micro-column array;
the arrangement mode of the micro-columns 4 is staggered and spaced, the micro-column array is 5 rows, each row of micro-columns is respectively arranged in 4-3-4 rows in sequence, the space between the micro-columns 4 in the adjacent rows is 1.5mm, the micro-columns 4 in the adjacent rows are offset in parallel by 2mm, and the space between the micro-columns 4 in each row is 1 mm.
Example 3
A porous netted cultivation meat production mould, the said mould is the cuboid with open-ended upper end, including outer wall 1, the first groove 2, the second groove 3, microcolumn 4;
the length of the outer wall 1 is 20 mm; the width is 20 mm; the height is 10mm, forms the cuboid of upper end opening.
The longitudinal section of the outer wall 1 is of a downward two-stage ladder shape, the first stage from top to bottom is the bottom of the first groove 2, and the second stage is the bottom of the second groove 3.
The first groove 2 is arranged in the middle of the inner side of the outer wall 1, and the length of the first groove 2 is 16 mm; the width is 16 mm; the height is 3 mm. The width of the bottom edge of the first groove 2 is 3 mm.
The second groove 3 is arranged at the bottom of the first groove 2 in the middle; the length of the second groove 3 is 10 mm; the width is 10 mm; the height is 3mm and the thickness of the bottom of the second groove 3 is 4 mm.
18 microcolumns 4 are vertically arranged at the bottom of the second groove 3; the micro-column 4 is 1.5mm in length, 0.5mm in width and 2mm in height; the cross section of the microcolumn is rectangular; the 18 micro-columns 4 are regularly arranged to form a micro-column array;
the arrangement mode of the micro-pillars 4 is staggered and spaced, the micro-pillar array is 5 rows, each row of micro-pillars is respectively arranged in 4-3-4 rows in sequence, the space between the micro-pillars 4 in the adjacent rows is 1mm, the micro-pillars 4 in the adjacent rows are parallelly offset by 1mm, and the space between the micro-pillars 4 in each row is 0.5 mm.
Example 4
The mold of this example is the same as that of example 1 except that the microcolumn 4 has an elliptical cross-section.
Example 5
A porous netted cultivation meat production mould, the said mould is the cuboid with open-ended upper end, including outer wall 1, the first groove 2, the second groove 3, microcolumn 4;
the length of the outer wall 1 is 100 mm; the width is 100 mm; the height is 18mm, and a cuboid with an opening at the upper end is formed.
The longitudinal section of the outer wall 1 is of a downward two-stage ladder shape, the first stage from top to bottom is the bottom of the first groove 2, and the second stage is the bottom of the second groove 3.
The first groove 2 is arranged in the middle of the inner side of the outer wall 1, and the length of the first groove 2 is 92 mm; the width is 92 mm; the height is 7 mm. The width of the bottom edge of the first groove 2 is 4 mm.
The second groove 3 is arranged at the bottom of the first groove 2 in the middle; the length of the second groove 3 is 84 mm; the width is 84 mm; the height is 6mm and the thickness of the bottom of the second groove 3 is 5 mm.
250 microcolumns 4 are vertically arranged at the bottom of the second groove 3; the microcolumn 4 has a length of 4mm, a width of 1.5mm and a height of 5 mm; the cross section of the microcolumn is rectangular; 250 micro-columns 4 are regularly arranged to form a micro-column array;
the arrangement mode of the micro-columns 4 is staggered and spaced, the micro-column array is 20 rows, each row of micro-columns is respectively arranged in 13-12-13 rows in sequence, the micro-columns 4 in adjacent rows are spaced by 2.5mm, the micro-columns 4 in adjacent rows are offset in parallel by 3mm, and the micro-columns 4 in each row are spaced by 2 mm.
Example 6 production of cultured meat Using Male mold
1) Constructing a three-dimensional CAD model of a male mold with regularly arranged columnar grooves by using three-dimensional drawing software, and performing data processing on the model based on a refined STL model method; a male die for manufacturing the mould for producing the cultured meat is designed, the longitudinal section of the male die is of a three-step structure, the first step from bottom to top is used for forming the outer wall 1 of the mould, the second step is used for forming the first groove 2 of the mould, and the third step is used for forming the second groove 3 and the microcolumn 4. The height of the second layer of steps of the male die is 3-7mm, the height of the third layer of steps is 2-6mm, the third layer of steps is provided with a plurality of columnar grooves (used for producing the microcolumn 4), the arrangement mode of the columnar grooves is staggered and spaced, the columnar grooves of adjacent rows are offset in parallel by 1-4mm, the length and width of the columnar grooves are (1-5) × (0.5-1.5) × (1-5) mm, the interval between the columnar grooves of the adjacent rows is 1-3mm, the interval between each columnar groove in each row is 0.5-3mm, and the length and width of the rest of the columnar grooves can be correspondingly adjusted according to the required porous reticular cultured meat production die.
2) Carrying out layered slicing processing on the obtained STL model file by using slicing software to obtain a motion control Gcode code file of the printer;
3) and (3) importing the Gcode code file into an FDM printer, executing a processing instruction on the printer, extruding thermoplastic plastic wires such as PLA (polylactic acid) or ABS (acrylonitrile butadiene styrene), and performing point-by-point accumulation molding to form male mold structures of various sizes for manufacturing cultured meat production molds.
4) Weighing the solution A and the solution B of the Dow Corning Sylgard 184 silicon rubber PDMS, and mixing according to the mass ratio of 10: 1. The well-mixed PDMS (polydimethylsiloxane) was carefully poured into and filled into the male mold. Placing PDMS and the male mold in a vacuum degasser, and degassing for 1-16 h. The PDMS and the master were simultaneously set at 25 ℃ for 24 h. Then, a contact part of PDMS and the male mold is cut by an operating blade, the PDMS and the male mold are pried along the periphery of the mold by the other end of the weighing scoop, the culture meat production mold and the micro-columns regularly arranged on the culture meat production mold can be completely taken out by extruding the periphery of the PDMS and the male mold, the ratio of the number of the fractures when the columns are taken out is 0-20%, and the rest number of the micro-columns is more than 90% so as to be used for the subsequent production of culture meat. The mold taken out is the mold for producing the culture meat.
And (3) cleaning the mould, sterilizing at 121 ℃ for 15min, taking out and drying. The sterilized PDMS mold was soaked in 0.2% (mass to volume) Pluronic F-127 solution for 1 hour to prevent the hydrogel from adhering to the PDMS mold. And then taking out the PDMS mold, washing the PDMS mold for 3 times by using PBS, and airing the PDMS mold for later use.
Example 7 growth of reticular muscle tissue in a cultured meat production mould.
(1) Mixing type I collagen (concentration of 3.35-3.73mg/ml), DMEM culture medium containing phenol red, 1M NaOH and matrigel, and making into mixed solution. The volume ratio of the collagen to the DMEM medium containing phenol red to the NaOH solution to the matrigel is 50:40:1.5:8, and the pH of the mixed solution is 7.3-7.5. In this example, the specific addition amounts of collagen, DMEM medium containing phenol red, NaOH solution, and matrigel were 500ul, 400ul, 15ul, and 8ul, respectively.
(2) Mixing myoblast cell line C2C12 (source: ATCC, American culture Collection) with the mixed solution to obtain mixed solution containing cells, wherein the density of myoblast cell line C2C12 in the mixed solution containing cells is 1x105Per ml-1x107One per ml.
(3) The cell-containing mixed solution was slowly added to the cultured meat production mold prepared in example 1 in an amount not higher than the height of the microcolumn 4 of the mold, and gently shaken to remove air bubbles.
Placing the culture meat production mold filled with the mixed solution at 37 deg.C and 5% CO2Cultured in an incubator for 2h to form hydrogel muscle tissue. Then 2.5ml growth medium was added to fill the whole culture meat production mold. The growth medium is a culture medium comprising 79 vol% F-10, 20 vol% fetal calf serum and 1 vol% penicillin-streptomycin double antibody, and the growth medium comprises 1-10ng/ml fibroblast growth factor 2. Hydrogel muscle tissue after 1-3 days of culture, the growth medium was changed to 2.5ml of differentiation medium to fill the entire cultured meat production mold. The differentiation medium comprises 97 vol% of DMEM medium, 2 vol% of horse serum and 1 vol% of penicillin-streptomycin double antibody. Reticular muscle tissue was obtained after 5-7 days of growth (figure 2).
The experimental results showed that the reticular muscle obtained by this example has a size of about 12mm in length, 12mm in width, 0.1-0.3mm in thickness, and a single reticular muscle of about 0.12-0.15g in weight, and that the reticular muscle tissue was also found to have a long myotube-like structure by staining with Phalloidin (Phalloidin), and the obtained reticular muscle tissue was used as a raw material source for cultured meat.
Example 8
The experimental method was the same as in example 7, except that the production mold was replaced with the cultured meat production mold prepared in example 5, and the amounts of the mixed solution, cells and medium were adjusted in equal proportion.
The results show that: the mesh muscles obtained by this embodiment are approximately 80mm long, 80mm wide and 0.2-0.5mm thick. The weight of each reticular muscle is about 3.2g, and the reticular muscle tissue is also shown to have a longer myotube-like structure by using the Phalloidin (Phalloidin) staining, and the obtained reticular muscle tissue can be used as a raw material source of the cultured meat.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A porous netted cultivation meat production mould is characterized in that the upper end of the mould is open and comprises an outer wall (1), a first groove (2), a second groove (3) and a microcolumn (4);
the first groove (2) is arranged on the inner side of the outer wall (1),
the second groove (3) is arranged at the bottom of the first groove (2);
a plurality of micro-columns (4) are vertically arranged at the bottom of the second groove (3), and the micro-columns (4) are arranged at intervals in a staggered mode to form a micro-column array.
2. The porous netted cultured meat production mold according to claim 1, wherein the microcolumn (4) has a length of 1 to 5mm, a width of 0.5 to 1.5mm and a height of 1 to 5 mm.
3. The porous netted cultured meat production jig according to claim 1, wherein the plurality of microcolumns (4) are arranged in staggered intervals: the micro columns (4) in adjacent rows are spaced by 1-3mm, the micro columns (4) in adjacent rows are offset in parallel by 1-4mm, and the micro columns (4) in each row are spaced by 0.5-3 mm.
4. The porous mesh-shaped cultured meat production mold according to claim 1, wherein the cross section of the microcolumns (4) is rectangular or elliptical.
5. The porous netted cultured meat production mold according to claim 1, wherein the mold is a rectangular parallelepiped or a cylinder having an upper end opened.
6. The porous netted cultured meat production mold according to claim 1, wherein the first groove (2) is provided centrally inside the outer wall (1).
7. The porous mesh-shaped cultured meat production mold according to claim 1, wherein the height of the first groove (2) is 3-7 mm.
8. The porous mesh-shaped cultured meat production mold according to claim 1, wherein the width of the bottom of the first groove (2) is 2-4 mm.
9. The porous netted cultured meat production mold according to claim 1, wherein the second groove (3) is provided centrally at the bottom of the first groove (2).
10. The porous netted cultured meat production mold according to claim 1, wherein the height of the second groove (3) is 2-6 mm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553148A (en) * | 2020-12-24 | 2021-03-26 | 南京农业大学 | Improved culture medium for in vitro amplification culture of meat seed cells and application of improved culture medium |
| CN112708591A (en) * | 2020-12-24 | 2021-04-27 | 南京农业大学 | Culture medium with definite chemical components for in-vitro differentiation of muscle stem cells |
| CN113604359A (en) * | 2021-08-09 | 2021-11-05 | 中国肉类食品综合研究中心 | Biological meat cultivating device |
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2019
- 2019-11-03 CN CN201921875316.XU patent/CN211394496U/en active Active
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553148A (en) * | 2020-12-24 | 2021-03-26 | 南京农业大学 | Improved culture medium for in vitro amplification culture of meat seed cells and application of improved culture medium |
| CN112708591A (en) * | 2020-12-24 | 2021-04-27 | 南京农业大学 | Culture medium with definite chemical components for in-vitro differentiation of muscle stem cells |
| CN113604359A (en) * | 2021-08-09 | 2021-11-05 | 中国肉类食品综合研究中心 | Biological meat cultivating device |
| CN113604359B (en) * | 2021-08-09 | 2023-07-14 | 中国肉类食品综合研究中心 | Biological meat cultivating device |
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Assignee: Nanjing Zhouzi future food technology Co.,Ltd. Assignor: NANJING AGRICULTURAL University Contract record no.: X2021980004101 Denomination of utility model: A kind of porous mesh mold for meat production Granted publication date: 20200901 License type: Exclusive License Record date: 20210528 |