CN117343899B - Cell culture meat based on collagen scaffold and preparation method thereof - Google Patents
Cell culture meat based on collagen scaffold and preparation method thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
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Abstract
The application relates to the field of cell culture meat, in particular to cell culture meat based on a collagen scaffold and a preparation method thereof. A method for preparing cell culture meat based on a collagen scaffold, comprising the following steps: mixing collagen with the mixed solution to obtain a soaking solution; soaking plant wiredrawing proteins in a soaking solution, taking out and culturing to obtain a collagen scaffold; mixing a collagen scaffold with a buffer solution; regulating the number of muscle stem cells by using a growth medium to obtain a muscle stem cell solution; and mixing the muscle stem cell solution with the collagen scaffold, and culturing and differentiating to obtain the cell culture meat. The preparation method can enable the cells to be rapidly and uniformly attached to the collagen scaffold, and proliferate and differentiate along the collagen scaffold in a large quantity to form the required cell culture meat, so that the cost can be greatly reduced, and the quality and the yield can be improved.
Description
Technical Field
The application relates to the field of cell culture meat, in particular to cell culture meat based on a collagen scaffold and a preparation method thereof.
Background
The cell culture meat technology is a new food synthesis biotechnology which has been developed in recent years, and is characterized in that animal cells are cultured on a large scale to obtain tissues such as muscle, fat and the like, and then the tissues are processed and produced into meat products through food. Compared with the traditional cultivation mode, the cell culture meat technology has great advantages in the aspects of environmental resource protection, animal welfare, public health and the like.
The three-dimensional shaping is used as the most important link of the production of the meat culture product, and is a process of inoculating cells obtained by large-scale expansion on a cell bracket for three-dimensional culture, and differentiating the cells to form the meat culture product. Various three-dimensional cell culture and tissue shaping techniques are currently available for the preparation of cultured meat products, including solid scaffolds or molds, 3D bioprinting, electrospinning, and sheet stacking techniques, among others.
The plant wiredrawing protein is a fibrous plant protein which is formed by the production and processing of plant protein through a special process and has the texture similar to muscle fiber, and the advantages of the plant wiredrawing protein are as follows: giving the meat texture to the product; replace meat products, reduce cost and improve use cost performance; the taste improvement effect is good; therefore, it has potential as an edible cell scaffold, but is not good as a plant protein itself, and it is difficult for cells to adhere thereto and proliferate and differentiate. Therefore, there is still a need for improvement.
Disclosure of Invention
In order to solve the problems, the application provides cell culture meat based on a collagen scaffold and a preparation method thereof.
In a first aspect, the present application provides a method for preparing cell culture meat based on a collagen scaffold, which adopts the following technical scheme:
a method for preparing cell culture meat based on a collagen scaffold, comprising the following steps:
cutting plant wiredrawing protein, sterilizing, drying and freezing for later use;
mixing the collagen solution with the mixed solution to obtain a soaking solution; the pH value of the soaking solution is 7.0-7.5, and the concentration of the collagen is 1-3mg/mL;
soaking plant wiredrawing protein in the soaking solution, taking out, and culturing at 37+ -3deg.C for 15-45min to obtain collagen scaffold;
mixing the collagen scaffold with a buffer solution for later use;
muscle stem cells were adjusted to (2.5-3.5). Times.10 using growth medium 6 Obtaining a muscle stem cell solution by using the total volume of the cell;
mixing the muscle stem cell solution with the collagen scaffold, and culturing and differentiating at 37+/-3 ℃ to obtain the cell culture meat.
In the related art, there is disclosed a technical scheme of premixing muscle stem cells with collagen and then grafting a protein scaffold. In the technical scheme, when the muscle stem cells are premixed with the collagen, the muscle stem cells wrap the collagen mostly or completely, so that substances obtained by premixing still cannot be uniformly and firmly adhered on the surface of the plant wiredrawing protein for proliferation and differentiation, and the obtained cell culture meat has low quality and high cost.
By adopting the technical scheme, the collagen and the plant wiredrawing protein are mixed, which is equivalent to coating a layer of adhesive on the surface of the plant wiredrawing protein, the subsequent muscle stem cells are adhered on the layer of adhesive and uniformly distributed on the surface of the collagen bracket, and then the muscle stem cells proliferate and differentiate along the collagen bracket, so that uniform cell protein meat is formed.
The cell culture meat provided by the application can be suitable for smooth muscle cells and fibroblasts besides muscle stem cells.
Preferably, the mixed solution at least comprises one or more of a non-essential amino acid solution, a GlutaMAX additive and bFGF.
Preferably, the mixed solution is a mixture of a non-essential amino acid solution, a GlutaMAX additive, bFGF and DMEM medium;
based on the volume of the collagen solution, the consumption of the non-essential amino acid solution is 0.5-1.5%, the consumption of the Glutamax additive is 0.5-1.5%, the use concentration of bFGF is 5-20ng/mL, and the concentration of collagen in the soaking solution is regulated to the target concentration by supplementing the DMEM culture medium.
By adopting the technical scheme, the mixed solution is composed of specific raw materials and specific dosage, and after being mixed with collagen, the mixed solution is favorable for promoting the collagen to be covered on the surface of the plant wiredrawing protein quickly in the follow-up process, and is uniformly distributed in the lines on the surface of the plant wiredrawing protein, so that the mixed solution is combined with the plant wiredrawing protein more comprehensively and firmly. The collagen scaffold prepared by the mixed solution can firmly adhere muscle stem cells for a long time and is not easy to fall off; the muscle stem cells are uniformly dispersed on all parts of the surface of the collagen scaffold, so that a large number of proliferation and differentiation are effectively realized, and the cell culture meat with a directional structure is formed.
Preferably, the concentration of collagen in the soaking solution is 1.5-2.5mg/mL.
By adopting the technical scheme, the concentration of the collagen is further limited, and the concentration of the collagen corresponds to the unit number of the muscle stem cells, and is not too viscous or too thin. Collagen can be uniformly covered on the surface of plant wiredrawing protein, and the formed collagen scaffold has enough adhesion sites for adhering muscle stem cells, and also has enough adhesive force, so that the muscle stem cells cannot fall off easily.
Preferably, when the soaked plant wiredrawing protein is taken out from the soaking liquid, the soaked plant wiredrawing protein is suspended and shaken, so that the superfluous collagen in the pores flows out.
If the redundant collagen moves along with the collagen scaffold, the redundant collagen is easy to be too thick and excessively filled into holes of the plant wiredrawing protein, and the plant wiredrawing protein has no position for attaching muscle stem cells, so that the effect of formed cell meats is affected.
Preferably, when the plant wiredrawing protein is soaked in the soaking liquid, the plant wiredrawing protein is shaken by adopting a crisscross method.
By adopting the technical scheme, the plant wiredrawing protein is shaken by adopting the crisscross method, so that the collagen can be adhered to the surface of the plant wiredrawing protein more quickly and fully.
Preferably, the muscle stem cell solution is poured from the top down when the muscle stem cell solution is mixed with the collagen scaffold.
By adopting the technical scheme, the muscle stem cell solution is poured downwards from the top, so that the muscle stem cells can be more fully contacted with the collagen scaffold, and the subsequent proliferation and differentiation of the muscle stem cells along the collagen scaffold are facilitated.
Preferably, the growth medium comprises FBS, DMEM/F12.
Preferably, FBS accounts for 15-25% of the total mass of the growth medium in terms of mass percent, and DMEM/F12 is supplemented to 100%.
Preferably, after the muscle stem cell solution is mixed with the collagen scaffold, the method comprises the following culture steps:
placing the strain in 3-8% CO2 at 37+ -3deg.C, and culturing for 18-28 hr to obtain initial inoculated protein scaffold;
taking out the initial collagen scaffold, replacing with new growth medium, and placing in 3-8% CO at 37+ -3deg.C 2 Continuously culturing for 4-8d; the growth medium was changed every 2 days.
By adopting the technical scheme, the culture is carried out under proper conditions, which is favorable for the rapid and uniform proliferation of the muscle stem cells at all parts of the collagen scaffold.
Preferably, after the muscle stem cell solution is mixed with the collagen scaffold, the method comprises the following differentiation steps:
sucking out the growth medium for culture, and continuously differentiating and culturing for 8-15d by using differentiating and culturing conditions based on 37+ -3deg.C and 3-8% CO 2; the differentiation medium was changed every 2 days.
Preferably, the differentiation medium comprises FBS, DMEM/F12, and green streptomycin.
Preferably, the differentiation medium comprises 1-3% FBS, 0.5-2% penicillin and DMEM/F12 supplemented to 100% by mass.
In a second aspect, the present application provides a cell culture meat prepared by a method for preparing a collagen scaffold-based cell culture meat.
In summary, the present application has the following beneficial effects:
1. the collagen and the plant wiredrawing protein are mixed, namely, a layer of adhesive is coated on the surface of the plant wiredrawing protein, the subsequent muscle stem cells are adhered on the layer of adhesive and uniformly distributed on the surface of the collagen bracket, and then the muscle stem cells proliferate and differentiate along the collagen bracket, so that uniform cell protein meat is formed.
2. The mixed solution composed of specific raw materials and specific dosage is mixed with collagen to promote the collagen to rapidly cover the surface of the plant wiredrawing protein, and the collagen is uniformly distributed in the grains on the surface of the plant wiredrawing protein, so that the collagen is more comprehensively and firmly combined with the plant wiredrawing protein. Moreover, the muscle stem cells can be firmly adhered for a long time, and are not easy to fall off; the muscle stem cells are uniformly dispersed on all parts of the surface of the collagen scaffold, so that a large number of proliferation and differentiation are effectively realized, and the cell culture meat with a directional structure is formed.
Drawings
FIG. 1 is a diagram showing the plant fiber drawing protein in step 1 a) of example 1 of the present application after cleavage.
FIG. 2 shows a cell culture meat obtained in example 1 of the present application.
FIG. 3 is a view showing the proliferation of step 2 c) in example 1 of the present application for 3 days.
FIG. 4 is a view showing the differentiation for 3 days in step 2 d) of example 1 of the present application.
FIG. 5 is a view of comparative example 1, step 7) from the third day.
Fig. 6 is a view of the control group 1.
Fig. 7 is a view of control group 2.
Detailed Description
The present application is described in further detail below with reference to the drawings and examples.
The raw materials used in the following examples and comparative examples are all commercially available products.
Examples
Example 1
A method for preparing cell culture meat based on a collagen scaffold, comprising the following steps:
step 1): manufacturing a collagen scaffold:
step 1 a): plant wire drawing proteins were cut into pieces (about 1 cm. Times.1 cm. Times.1.5 cm in size) with scissors, see in particular FIG. 1.
Then sterilizing the cut plant wiredrawing protein at 121 ℃ for 30min, drying in a 50 ℃ oven, and then freezing at-20 ℃ to obtain the protein scaffold for later use.
Step 1 b): mixing collagen solution (collagen type I, derived from pig tendon, concentration of 3 mg/mL) with the mixed solution, adjusting pH to 7.4 and collagen concentration of 2mg/mL to obtain soaking solution, wherein the soaking solution is 4mL; and (5) standby application.
The mixed solution is a mixture of non-essential amino acid solution, glutamax additive, bFGF and DMEM culture medium. Based on the volume of the collagen solution, the dosage of the non-essential amino acid solution is 1%, the dosage of the Glutamax additive is 1%, the use concentration of bFGF is 10ng/mL, and the dosage of the DMEM culture medium is added according to the adjustment of the collagen concentration of the soaking solution to 2 mg/mL.
In this example, the non-essential amino acid solution was NEAA (100X), the product number BL1115A, the GlutaMAX additive was GlutaMAX (100X), the product number 35050079, and bFGF was purchased from gibco, and the product number PHG0368.
Step 1 c): placing the protein scaffold in a 6-hole plate, adding the soaking solution, completely immersing the protein scaffold, and standing for 1min.
Then, the edge of the protein scaffold is clamped by forceps, and the protein scaffold is gently shaken for 5 times by using a crisscross method, so that the protein scaffold is fully contacted with the soaking liquid.
This step is operated under aseptic, low temperature (kept low temperature using dry ice or crushed ice) conditions throughout.
Step 1 d): taking out the protein scaffold attached with the type one collagen, suspending and slightly shaking left and right to enable the excessive part of the type one collagen in the pores of the protein scaffold to flow out.
Then placing in a sterile culture dish, transferring into a cell culture box, and placing for 30min at 37 ℃.
Step 1 e): after 30min of culture, the collagen type I is formed and adhered on a protein scaffold, and the collagen scaffold is obtained.
PBS buffer is added into the culture dish, and the addition amount is based on that the PBS buffer completely submerges the collagen scaffold. Then placing in a refrigerator at 4 ℃ for standby.
Step 2): muscle stem cell inoculation:
step 2 a): use of growth medium to modulate muscle stem cells to 3×10 6 Each ml, a muscle stem cell solution was obtained.
The growth medium consisted of 20% FBS+80% DMEM/F12.
Step 2 b): the collagen scaffold was placed in a 6-well plate, 4mL of the muscle stem cell solution was slowly poured from the top, as many muscle stem cells as possible were seeded onto the collagen scaffold, and the collagen scaffold was fully immersed in the muscle stem cell solution. Placing back into cell incubator at 37deg.C with 5% CO 2 Is cultured for 24 hours.
Step 2 c): after 24h of culture, the muscle stem cells inoculated on the collagen scaffold are stably attached to obtain the initial inoculated protein scaffold.
The initial inoculum protein scaffold was removed and placed in a new 6-well plate,5mL of growth medium was added and then returned to the cell incubator at 37℃with 5% CO 2 The culture was continued for another 5 days, and the growth medium was changed every 2 days.
Step 2 d): after 5 days of continuous culture, the growth medium was aspirated, 5mL of differentiation medium was added, and the mixture was incubated at 37℃with 5% CO 2 The differentiation medium was changed every 2 days, and the culture was continued for 10 days to obtain cell culture meat. With particular reference to fig. 2.
The differentiation medium consisted of 2% FBS+97% DMEM/F12+1% Streptomyces lividans.
The observations of 3 days of proliferation in step 2 c) are shown in FIG. 3, and the observations of 3 days of differentiation in step 2 d) are shown in FIG. 4.
Example 2
A method for preparing cell culture meat based on a collagen scaffold, comprising the following steps:
step 1): manufacturing a collagen scaffold:
step 1 a): plant wire drawing proteins were cut into pieces (about 1 cm. Times.1 cm. Times.1.5 cm in size) with scissors.
Then sterilizing the cut plant wiredrawing protein at 121 ℃ for 30min, drying in a 50 ℃ oven, and then freezing at-20 ℃ to obtain the protein scaffold for later use.
Step 1 b): mixing collagen solution (collagen type I, derived from pig tendon, concentration of 3 mg/mL) with the mixed solution, and adjusting pH to 7.0 and collagen concentration of 1.5mg/mL to obtain soaking solution, wherein the soaking solution is 4mL; and (5) standby application.
The mixed solution is a mixture of non-essential amino acid solution, glutamax additive, bFGF and DMEM culture medium. Based on the volume of the collagen solution, the dosage of the non-essential amino acid solution is 1.5%, the dosage of the GlutaMAX additive is 0.5%, the use concentration of bFGF is 5ng/mL, and the dosage of the DMEM culture medium is added according to the adjustment of the collagen concentration of the soaking solution to 1.5 mg/mL.
In this example, the non-essential amino acid solution was NEAA (100X), the product number BL1115A, the GlutaMAX additive was GlutaMAX (100X), the product number 35050079, and bFGF was purchased from gibco, and the product number PHG0368.
Step 1 c): placing the protein scaffold in a 6-hole plate, adding the soaking solution, completely immersing the protein scaffold, and standing for 1min.
Then, the edge of the protein scaffold is clamped by forceps, and the protein scaffold is gently shaken for 5 times by using a crisscross method, so that the protein scaffold is fully contacted with the soaking liquid.
This step is operated under aseptic, low temperature (kept low temperature using dry ice or crushed ice) conditions throughout.
Step 1 d): taking out the protein scaffold attached with the type one collagen, suspending and slightly shaking left and right to enable the excessive part of the type one collagen in the pores of the protein scaffold to flow out.
Then placing in a sterile culture dish, transferring into a cell culture box, placing at 40 ℃ and culturing for 15min.
Step 1 e): after 15min of culture, the collagen type I is formed and adhered on a protein scaffold, and the collagen scaffold is obtained.
PBS buffer is added into the culture dish, and the addition amount is based on that the PBS buffer completely submerges the collagen scaffold. Then placing in a refrigerator at 4 ℃ for standby.
Step 2): muscle stem cell inoculation:
step 2 a): muscle stem cells were adjusted to 2.5X10 using growth medium 6 Each ml, a muscle stem cell solution was obtained.
The growth medium consisted of 20% FBS+80% DMEM/F12.
Step 2 b): the collagen scaffold was placed in a 6-well plate, 4mL of the muscle stem cell solution was slowly poured from the top, as many muscle stem cells as possible were seeded onto the collagen scaffold, and the collagen scaffold was fully immersed in the muscle stem cell solution. Placing back into cell incubator at 40deg.C with 3% CO 2 Is cultured for 18 hours.
Step 2 c): after 18h of culture, the muscle stem cells inoculated on the collagen scaffold are stably attached to obtain the initial inoculated protein scaffold.
The initial inoculum protein scaffold was removed and placed in a new 6-well plate, 5mL of growth medium was added, and then returned to the cell incubator at 40℃at 3%CO 2 The culture was continued for 8 days again, and the growth medium was changed every 2 days.
Step 2 d): after 8 days of continuous culture, the growth medium was aspirated, 5mL of differentiation medium was added, and the mixture was incubated at 40℃with 3% CO 2 The differentiation medium was changed every 2 days, and the culture was continued for 8 days to obtain cell culture meat.
The differentiation medium consisted of 2% FBS+97% DMEM/F12+1% Streptomyces lividans.
Example 3
A method for preparing cell culture meat based on a collagen scaffold, comprising the following steps:
step 1): manufacturing a collagen scaffold:
step 1 a): plant wire drawing proteins were cut into pieces (about 1 cm. Times.1 cm. Times.1.5 cm in size) with scissors.
Then sterilizing the cut plant wiredrawing protein at 121 ℃ for 30min, drying in a 50 ℃ oven, and then freezing at-20 ℃ to obtain the protein scaffold for later use.
Step 1 b): mixing collagen solution (collagen type I, derived from pig tendon, concentration of 3 mg/mL) with the mixed solution, and adjusting pH to 7.5 and collagen concentration of 2.5mg/mL to obtain soaking solution, wherein the soaking solution is 4mL; and (5) standby application.
The mixed solution is a mixture of non-essential amino acid solution, glutamax additive, bFGF and DMEM culture medium. Based on the volume of the collagen solution, the amount of the non-essential amino acid solution was 0.5%, the amount of the GlutaMAX additive was 1.5%, the bFGF concentration was 20ng/mL, and the amount of the DMEM medium was added in such a manner that the collagen concentration in the soaking solution was adjusted to 2.5mg/mL.
In this example, the non-essential amino acid solution was NEAA (100X), the product number BL1115A, the GlutaMAX additive was GlutaMAX (100X), the product number 35050079, and bFGF was purchased from gibco, and the product number PHG0368.
Step 1 c): placing the protein scaffold in a 6-hole plate, adding the soaking solution, completely immersing the protein scaffold, and standing for 1min.
Then, the edge of the protein scaffold is clamped by forceps, and the protein scaffold is gently shaken for 5 times by using a crisscross method, so that the protein scaffold is fully contacted with the soaking liquid.
This step is operated under aseptic, low temperature (kept low temperature using dry ice or crushed ice) conditions throughout.
Step 1 d): taking out the protein scaffold attached with the type one collagen, suspending and slightly shaking left and right to enable the excessive part of the type one collagen in the pores of the protein scaffold to flow out.
Then placing in a sterile culture dish, transferring into a cell culture box, placing at 34 ℃ and culturing for 45min.
Step 1 e): after 45min of culture, the collagen type I is formed and adhered to a protein scaffold, and the collagen scaffold is obtained.
PBS buffer is added into the culture dish, and the addition amount is based on that the PBS buffer completely submerges the collagen scaffold. Then placing in a refrigerator at 4 ℃ for standby.
Step 2): muscle stem cell inoculation:
step 2 a): muscle stem cells were adjusted to 3.5X10 using growth medium 6 Each ml, a muscle stem cell solution was obtained.
The growth medium consisted of 20% FBS+80% DMEM/F12.
Step 2 b): the collagen scaffold was placed in a 6-well plate, 4mL of the muscle stem cell solution was slowly poured from the top, as many muscle stem cells as possible were seeded onto the collagen scaffold, and the collagen scaffold was fully immersed in the muscle stem cell solution. Put back into the cell incubator at 34 ℃ and 8% CO 2 Is cultured for 28 hours.
Step 2 c): after 28h of culture, the muscle stem cells inoculated on the collagen scaffold are stably attached to obtain the initial inoculated protein scaffold.
The initial inoculum protein scaffold was removed and placed in a new 6-well plate, 5mL of growth medium was added, and then returned to the cell incubator at 34℃with 8% CO 2 The culture was continued for another 4 days, and the growth medium was changed every 2 days.
Step 2 d): after 4 days of continuous culture, the growth medium was aspirated, 5mL of differentiation medium was added, and the medium was incubated at 34℃with 8% CO 2 The differentiation medium was changed every 2 days, and the culture was continued for 15 days to obtain cell culture meat.
The differentiation medium consisted of 2% FBS+97% DMEM/F12+1% Streptomyces lividans.
Example 4
A method for preparing cell culture meat based on a collagen scaffold, which is different from example 1 in that: in the mixed solution, the amount of the non-essential amino acid solution was 0.1%, the amount of the Glutamax additive was 1.9%, and the bFGF was used at a concentration of 25ng/mL.
Example 5
A method for preparing cell culture meat based on a collagen scaffold, which is different from example 1 in that: the non-essential amino acid solution in the mixed solution is replaced by a Glutamax additive, namely the use amount of the non-essential amino acid solution is 0, and the use amount of the Glutamax additive is 2%.
Example 6
A method for preparing cell culture meat based on a collagen scaffold, which is different from example 1 in that: the GlutaMAX additive in the mixed solution was replaced with L-glutamine, i.e., the amount of L-glutamine used was 1% and the amount of GlutaMAX additive used was 0.
Example 7
A method for preparing cell culture meat based on a collagen scaffold, which is different from example 1 in that: the bFGF in the mixed solution was replaced with the kgf recombinant protein, i.e., the bFGF was used in an amount of 0 and the bFGF recombinant protein was used in an amount of 10ng/mL.
Example 8
A method for preparing cell culture meat based on a collagen scaffold, which is different from example 1 in that: the concentration of the collagen solution is 10mg/mL, and the concentration of the collagen in the soaking solution is 6mg/mL.
Example 9
A method for preparing cell culture meat based on a collagen scaffold, comprising the following steps:
step 1): manufacturing a collagen scaffold:
step 1 a): plant wire drawing proteins were cut into pieces (about 1 cm. Times.1 cm. Times.1.5 cm in size) with scissors.
Then sterilizing the cut plant wiredrawing protein at 121 ℃ for 30min, drying in a 50 ℃ oven, and then freezing at-20 ℃ to obtain the protein scaffold for later use.
Step 1 b): mixing collagen solution (collagen type I, derived from pig tendon, concentration of 3 mg/mL) with the mixed solution, and adjusting pH to 7.4 and collagen concentration of 2mg/mL to obtain soaking solution; and (5) standby application.
The mixed solution is a mixture of non-essential amino acid solution, glutamax additive, bFGF and DMEM culture medium. Based on the volume of the collagen solution, the dosage of the non-essential amino acid solution is 1%, the dosage of the Glutamax additive is 1%, the use concentration of bFGF is 10ng/mL, and the dosage of the DMEM culture medium is added according to the adjustment of the collagen concentration of the soaking solution to 2 mg/mL.
In this example, the non-essential amino acid solution was NEAA (100X), the product number BL1115A, the GlutaMAX additive was GlutaMAX (100X), the product number 35050079, and bFGF was purchased from gibco, and the product number PHG0368.
Step 1 c): placing the protein scaffold in a 6-hole plate, adding the soaking solution, completely immersing the protein scaffold, and standing for 1min.
This step is operated under aseptic, low temperature (kept low temperature using dry ice or crushed ice) conditions throughout.
Step 1 d): taking out the soaked protein scaffold, placing the protein scaffold in a sterile culture dish, transferring the protein scaffold into a cell incubator, and placing the protein scaffold at 37 ℃ for 30min.
Step 1 e): after 30min of culture, the collagen type I is formed and adhered on a protein scaffold, and the collagen scaffold is obtained.
PBS buffer is added into the culture dish, and the addition amount is based on that the PBS buffer completely submerges the collagen scaffold. Then placing in a refrigerator at 4 ℃ for standby.
Step 2): muscle stem cell inoculation:
step 2 a): use of growth medium to modulate muscle stem cells to 3×10 6 Obtaining muscle stem cell solution per ml。
The growth medium consisted of 20% FBS+80% DMEM/F12.
Step 2 b): the collagen scaffold was placed in a 6-well plate, 4mL of the muscle stem cell solution was slowly poured onto the collagen scaffold from the top, so that as many muscle stem cells as possible were seeded onto the collagen scaffold, and the collagen scaffold was fully immersed in the muscle stem cell solution. Placing back into cell incubator at 37deg.C with 5% CO 2 Is cultured for 24 hours.
Step 2 c): after 24h of culture, the muscle stem cells inoculated on the collagen scaffold are stably attached to obtain the initial inoculated protein scaffold.
The initial inoculum protein scaffold was removed and placed in a fresh 6-well plate, 5mL of growth medium was added, and then returned to the cell incubator at 37℃with 5% CO 2 The culture was continued for another 5 days, and the growth medium was changed every 2 days.
Step 2 d): after 5 days of continuous culture, the growth medium was aspirated, 5mL of differentiation medium was added, and the mixture was incubated at 37℃with 5% CO 2 The differentiation medium was changed every 2 days, and the culture was continued for 10 days to obtain cell culture meat.
The differentiation medium consisted of 2% FBS+97% DMEM/F12+1% Streptomyces lividans.
Comparative example
Comparative example 1
A method for preparing cell culture meat based on a collagen scaffold, comprising the following steps:
step 1): use of growth medium to modulate muscle stem cells to 3×10 6 Each ml, a muscle stem cell solution was obtained.
The growth medium consisted of 20% FBS+80% DMEM/F12.
Step 2): mixing collagen solution (collagen type I, derived from pig tendon, concentration of 3 mg/mL) with the mixed solution, adjusting pH to 7.4 and collagen concentration of 2mg/mL to obtain soaking solution, wherein the soaking solution is 4mL; and (5) standby application.
The mixed solution is a mixture of non-essential amino acid solution, glutamax additive, bFGF and DMEM culture medium. Based on the volume of the collagen solution, the dosage of the non-essential amino acid solution is 1%, the dosage of the Glutamax additive is 1%, the use concentration of bFGF is 10ng/mL, and the dosage of the DMEM culture medium is added according to the adjustment of the collagen concentration of the soaking solution to 2 mg/mL.
In this example, the non-essential amino acid solution was NEAA (100X), the product number BL1115A, the GlutaMAX additive was GlutaMAX (100X), the product number 35050079, and bFGF was purchased from gibco, and the product number PHG0368.
Step 3): 4mL of the muscle stem cell solution was mixed with the soak solution, and cultured at 37℃for 30min to obtain a premix.
Step 4): plant wire drawing proteins were cut into pieces (about 1 cm. Times.1 cm. Times.1.5 cm in size) with scissors.
Then sterilizing the cut plant wiredrawing protein at 121 ℃ for 30min, drying in a 50 ℃ oven, and then freezing at-20 ℃ to obtain the protein scaffold for later use.
Step 5): pouring the premix liquid onto the protein scaffold slowly from the top, inoculating as many muscle stem cells as possible onto the protein scaffold, fully immersing the protein scaffold into the muscle stem cell solution, and standing for 1min.
Then, the edge of the protein scaffold is clamped by forceps, and the protein scaffold is gently shaken for 5 times by using a crisscross method, so that the protein scaffold is fully contacted with the soaking liquid.
This step is operated under aseptic, low temperature (kept low temperature using dry ice or crushed ice) conditions throughout.
Step 6): taking out the protein scaffold attached with the type one collagen, suspending and slightly shaking left and right to enable the excessive part of the type one collagen in the pores of the protein scaffold to flow out.
Then placing in a sterile culture dish, transferring into a cell incubator, and transferring into 5% CO at 37deg.C 2 Is cultured for 24 hours.
Step 7): after 24h of culture, an initial inoculum protein scaffold was obtained.
The initial inoculum protein scaffold was removed and placed in a fresh 6-well plate, 5mL of growth medium was added, and then returned to the cell incubator at 37℃with 5% CO 2 Is continuous under the condition of (2)The culture was performed for 5 days, with the growth medium being changed every 2 days.
The observation on the third day is shown in fig. 5.
Step 8): after 5 days of continuous culture, the growth medium was aspirated, 5mL of differentiation medium was added, and the mixture was incubated at 37℃with 5% CO 2 The differentiation medium was changed every 2 days, and the culture was continued for 10 days to obtain cell culture meat.
The differentiation medium consisted of 2% FBS+97% DMEM/F12+1% Streptomyces lividans.
Performance test
qPCR detects the expression level of Myog gene:
1) Detecting an object:
control group 1: muscle stem cells were conditioned to 3×10 using growth medium (20% fbs+80% dmem/F12) 6 And (3) culturing the cells in a normal adherence mode at a concentration of each ml, and differentiating the cells for 0 d.
The observations of control group 1 are shown in FIG. 6.
Control group 2: muscle stem cells were conditioned to 3×10 using growth medium (20% fbs+80% dmem/F12) 6 Cells were obtained by normal adherent culture, differentiation for 3d, and 0.25% pancreatin digestion at each ml.
The observations of control group 2 are shown in fig. 7.
Experimental groups 1-10: examples 1-9, comparative example 1, differentiation was performed for 3d, and on the third day, digestion was performed using 0.1% collagenase to obtain cells seeded on a collagen scaffold.
2) The detection method comprises the following steps:
1. RNA was extracted using the Omega total RNA extraction kit, reverse transcribed into cDNA, and qPCR was performed using gapdh as an internal reference gene.
2. The qPCR procedure was: pre-denaturation at 95℃for 10min, denaturation at 95℃for 15s, annealing at 60℃for 1min, and circulation 40 times.
3. The relative expression amounts of the control group 2 and the experimental groups 1-10 are calculated by taking the control group 1 as a control, the relative expression amounts of the target genes in different cDNAs are calculated by using 2-delta Ct, and the difference significance analysis is performed by using single-factor variance analysis of SPSS software.
The relative expression levels of the Myog genes in each group are recorded in table 1.
TABLE 1
As can be seen from comparison of the detection data of example 1 (experimental group 1) with that of control group 2 in Table 1, the relative expression amount of example 1 was higher. As can be seen from comparison of the proliferation and differentiation pictures of the control group 1 and the control group 2 with the proliferation and differentiation picture of the example 1, the proliferation and differentiation effect of the example 1 is better than that of the common adherence proliferation and differentiation effect. The preparation method of the application can indeed promote the muscle stem cells to have faster, more uniform and better proliferation and differentiation effects on the collagen scaffold, thereby preparing the cell culture meat with better quality.
Comparative example 1 cell culture meat was prepared by premixing collagen with muscle stem cells and then mixing with a protein scaffold. As can be seen from comparison of the detection data of example 1 with that of comparative example 1 (experimental group 10), the relative expression level of example 1 was much higher than that of comparative example 1. It can also be seen from a comparison of the cell proliferation observations of example 1 and comparative example 1 that example 1 has a better proliferation effect.
Examples 4 to 7 were obtained by changing the composition and amount of the mixed solution and then applying the mixed solution to the cell culture meat, and comparing the measured data with those of example 1, it was found that the relative expression amounts of examples 4 to 7 were decreased to a different extent from those of example 1. It is described that the composition and the compounding amount of the mixed solution are required to be further limited, so that a good compounding effect can be exerted between the mixed solution and the collagen or the plant wiredrawing protein.
Example 8 is based on example 1, wherein the concentration of collagen in the soaking solution was increased. In the actual research and development process, the collagen concentration is found to be too large, so that the viscosity is too large and too viscous, and the subsequent adhesion to the surface of the plant wiredrawing protein is not facilitated, thereby affecting the adhesion of muscle stem cells. This conclusion is also demonstrated by comparison of the test data from example 8 with example 1.
As can be seen from comparison of the test data of example 9 with that of example 1 in Table 1, the relative expression level of example 1 was higher than that of example 9. The method further limits the contact mode of the protein scaffold and the soaking liquid, shakes out the redundant collagen from the protein scaffold and the like, and is beneficial to improving the distribution and adhesion of the collagen on the protein scaffold, so that the effect of attaching muscle stem cells to the collagen scaffold is improved.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (7)
1. A method for preparing cell culture meat based on a collagen scaffold, which is characterized by comprising the following steps:
mixing the collagen solution with the mixed solution to obtain a soaking solution; the pH value of the soaking solution is 7.0-7.5, and the concentration of collagen in the soaking solution is 1.5-2.5mg/ml;
soaking plant wiredrawing protein in the soaking solution, taking out, and culturing at 37+ -3deg.C for 15-45min to obtain collagen scaffold;
mixing the collagen scaffold with a buffer solution for later use;
muscle stem cells were adjusted to (2.5-3.5). Times.10 using growth medium 6 Obtaining a muscle stem cell solution per ml;
mixing the muscle stem cell solution with the collagen scaffold, and culturing and differentiating the mixture at 37+/-3 ℃ to obtain cell culture meat;
the mixed solution is prepared by mixing a non-essential amino acid solution, a Glutamax additive, bFGF and a DMEM culture medium;
based on the volume of the collagen solution, the consumption of the non-essential amino acid solution is 0.5-1.5%, the consumption of the Glutamax additive is 0.5-1.5%, the use concentration of bFGF is 5-20ng/mL, and the concentration of collagen in the soaking solution is regulated to the target concentration by supplementing the DMEM culture medium.
2. The method for preparing a collagen scaffold-based cell culture meat according to claim 1, wherein: and (3) when the soaked plant wiredrawing protein is taken out from the soaking liquid, suspending and shaking the plant wiredrawing protein so as to enable the redundant collagen in the pores to flow out.
3. The method for preparing a collagen scaffold-based cell culture meat according to claim 1, wherein: when the plant wiredrawing protein is soaked in the soaking liquid, the plant wiredrawing protein is shaken by adopting a crisscross method.
4. The method for preparing a collagen scaffold-based cell culture meat according to claim 1, wherein: when the muscle stem cell solution is mixed with the collagen scaffold, the muscle stem cell solution is poured from the top down.
5. The method for preparing a collagen scaffold-based cell culture meat according to claim 1, wherein: after the muscle stem cell solution is mixed with the egg collagen white scaffold, the method comprises the following culture steps:
placing at 37+ -3deg.C and 3-8% CO 2 Culturing for 18-28h to obtain an initial inoculated protein scaffold;
taking out the initial collagen scaffold, replacing with new growth medium, and placing in 3-8% CO at 37+ -3deg.C 2 Is cultured continuously for 4-8d.
6. The method for preparing a collagen scaffold-based cell culture meat according to claim 1, wherein: after the muscle stem cell solution is mixed with the collagen scaffold, the method comprises the following differentiation steps:
the growth medium used for the culture was aspirated and differential culture was used based on 37.+ -. 3 ℃ and 3-8% CO 2 Is subjected to continuous differentiation culture for 8-15d.
7. A cell culture meat prepared based on the method for preparing a cell culture meat based on a collagen scaffold according to any one of claims 1 to 6.
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