CN115747144A - Three-dimensional culture method of pseudosciaena crocea muscle satellite cells - Google Patents
Three-dimensional culture method of pseudosciaena crocea muscle satellite cells Download PDFInfo
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Abstract
The invention discloses a three-dimensional culture method of pseudosciaena crocea muscle satellite cells, and relates to the technical field of cell culture. The method comprises the following steps: (1) Preparing separated pseudosciaena crocea muscle satellite cells into cell suspension; (2) Adding the cell suspension into a DMEM high-sugar medium containing fibrinogen, adding antibiotics into the DMEM high-sugar medium, adding thrombin, uniformly mixing to obtain a mixed suspension, adding the mixed suspension into a cell culture plate, and solidifying to form cell-fibrin hydrogel; (3) And adding a culture medium into the hydrogel system for cell culture. The invention provides a scaffold material for growth and differentiation of pseudosciaena crocea muscle satellite cells, which utilizes natural protein hydrogel-fibrin hydrogel to carry out three-dimensional culture on the pseudosciaena crocea muscle satellite cells, realizes the three-dimensional culture of the pseudosciaena crocea muscle satellite cells for the first time and promotes the development of cell culture fish.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a three-dimensional culture method of pseudosciaena crocea muscle satellite cells.
Background
With the increasing demand for proteins and the increased environmental hazards of traditional farming, it is critical to develop sustainable alternative proteins to meet the ever-increasing demand for meat consumption. In recent years, cell culture meat has become a source of alternative proteins that can meet future demands for meat proteins while also addressing the environmental, ethical and health challenges associated with traditional meat.
Cell-cultured meat is meat cultured from animal stem cells, and simulates the process of proliferation and differentiation of cells in vivo, thereby producing a product having the same nutritional and organoleptic properties as conventional meat products. In 2013, the Mark Post doctor team in the netherlands produced the first piece of cell culture meat using stem cell technology. Subsequently, more and more research has focused on the production of cell culture meat.
The production process of cell culture meat can be roughly divided into four steps. First, it is necessary to isolate a cell line from a target species that can differentiate into muscle fibers, adipocytes and other important cell types that make up meat, such as myosatellite cells, which are flat, protruding cells in skeletal muscle except for skeletal muscle fibers (muscle cells), which are on the surface of muscle fibers and can proliferate and differentiate when the muscle fibers are damaged, and participate in the repair of muscle fibers, thus having stem cell properties. Secondly, there is a need to develop a medium formulation with low cost and high proliferation rate. Thirdly, for some biological processes and product types, it is necessary to develop a food safety scaffold simulating the function of Extracellular matrix (ECM), the biological scaffold is used to make cells form the tissue structure of meat, and the common carrier materials for cell culture meat production mainly include animal protein scaffold, plant protein scaffold, acellular plant scaffold, block hydrogel and the like, such as CN114645012A and CN113768138A. Finally, the bioreactor and the biological process are developed and expanded, and the edible quality such as texture, color and the like similar to the natural meat paste is formed through food processing.
Currently, cell culture beef and cell culture pork are the most studied, and less so for some non-mammals such as fish. The fish meat has high nutritive value, is rich in high-quality protein and unsaturated fatty acid, and has low cholesterol content and rich mineral content. Therefore, the development of cell culture fish is not slow. Aiming at the traditional marine economic fish-large yellow croaker in China, no report related to cell culture development is found.
Disclosure of Invention
The invention aims to provide a three-dimensional culture method suitable for pseudosciaena crocea muscle satellite cells aiming at the vacancy of the existing pseudosciaena crocea muscle satellite cell three-dimensional culture research, so as to be applied to the production of cell culture fish.
In order to achieve the purpose, the invention adopts the following technical scheme:
a three-dimensional culture method of pseudosciaena crocea muscle satellite cells comprises the following steps:
(1) Preparing separated pseudosciaena crocea muscle satellite cells into cell suspension;
(2) Adding the cell suspension into a DMEM high-sugar medium containing fibrinogen, adding antibiotics into the DMEM high-sugar medium, adding thrombin, uniformly mixing to obtain a mixed suspension, adding the mixed suspension into a cell culture plate, and solidifying to form cell-fibrin hydrogel;
(3) And adding a culture medium into the hydrogel system for cell culture.
The invention provides a three-dimensional space bracket for migration and growth of pseudosciaena crocea muscle satellite cells by utilizing fibrin hydrogel, and realizes proliferation and differentiation of the pseudosciaena crocea muscle satellite cells in a three-dimensional environment. Fibrin is formed by the conversion of fibrinogen under the action of thrombin and has a certain three-dimensional network structure. Fibrin hydrogel has excellent biocompatibility, and contains natural arginine-glycine-aspartic acid (RGD) sites to facilitate cell adhesion.
Furthermore, the pseudosciaena crocea muscle satellite cells are separated from the supraaxial muscles of the pseudosciaena crocea.
In the step (1), pseudosciaena crocea muscle satellite cells with good growth state are selected, when the cell confluence reaches 80%, trypsin is added for digestion and centrifugation, and cells are collected to prepare cell suspension.
In the step (2), the cell suspension is mixed with the fibrinogen solution, then the thrombin solution is added, the thrombin converts the fibrinogen into the fibrin to form fibrin hydrogel, and the pseudosciaena crocea muscle satellite cells are uniformly dispersed in the fibrin hydrogel for migration and growth. When the fibrinogen solution and the thrombin solution are prepared, a DMEM high-sugar culture medium is used as a solvent, and provides proper survival conditions for pseudosciaena crocea muscle satellite cells.
Preferably, the concentration of the fibrinogen in the mixed suspension is 5-15mg/mL, and the concentration of the thrombin is 2.5-7.5U/mL. The research of the invention shows that the fibrin hydrogel prepared under the conditions is suitable for the growth of pseudosciaena crocea muscle satellite cells, and the aspects of cell growth speed, cell density, cell activity and the like all reach the experimental requirements.
More preferably, the concentration of fibrinogen in the mixed suspension is 10mg/mL and the concentration of thrombin is 5U/mL.
Preferably, the concentration of cells in the mixed suspension is 1.0X 10 6 Per mL-5.0X 10 6 Per mL, too low a cell density may affect the differentiation of cells in a three-dimensional environment. More preferably, the concentration of cells in the mixed suspension is 3.0X 10 6 Per mL-4.0X 10 6 One per mL.
According to the invention, antibiotics are added in the culture system to avoid microbial contamination. Specifically, 100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate are added into the DMEM high-glucose medium.
In the step (2), the mixed suspension is added into a cell culture plate to be solidified into a water drop shape. The hydrogel culture system is placed on the culture plate in a drop-shaped form, specifically, the volume of each culture system is 10-40. Mu.L.
Preferably, the conditions for hydrogel coagulation are 25-27 ℃ and 4-6% CO 2 Hydrogel placed in CO 2 And (3) solidifying in the cell culture box to ensure normal growth of cells in the hydrogel solidification process. The solidification time is 20-60min.
In step (3), after adding the culture medium to the hydrogel system, the cell culture plate is placed in CO 2 Culturing the cells in a cell culture chamber under 25-27 deg.C and 4-6% 2 Multiplication cultureAfter 3-5 days, differentiation culture is carried out, and the culture medium is replaced every two days.
Preferably, the proliferation medium used in the proliferation culture is a DMEM high-sugar medium containing 10% fetal bovine serum, 10ng/mL basic fibroblast growth factor/FGF 2 blocking polypeptide, 1 × antibiotics (100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate), and the differentiation medium used in the differentiation culture is a DMEM/F12 medium containing 8% horse serum, 10ng/mL human recombinant insulin-like growth factor-1, 50nM necrosul fonamide, 200 μ M ascorbic acid, 1 × antibiotics (100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate).
The invention has the following beneficial effects:
the invention provides a scaffold material for growth and differentiation of pseudosciaena crocea muscle satellite cells, which is used for carrying out three-dimensional culture on the pseudosciaena crocea muscle satellite cells by utilizing natural protein hydrogel-fibrin hydrogel, so that the proliferation and differentiation of the pseudosciaena crocea muscle satellite cells in a three-dimensional environment are realized for the first time, and the development of cell culture fish is promoted.
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FIG. 1 is a photomicrograph of the cells of example 1 taken at days 1, 3, 5, 7 and 9 of the culture, wherein the first row is a photomicrograph of the cells at days 1, 3 and 5 from left to right, and the second row is a photomicrograph of the cells at days 7 and 9 from left to right and a magnified view of a portion of the photomicrograph of the cells at day 7 of the culture.
FIG. 2 is a photograph of immunofluorescent staining after 7 days of differentiation in example 1, which is a photograph of desmin immunofluorescent staining, a photograph of nuclear immunofluorescent staining and a combined desmin/nucleus photograph in the order from left to right.
FIG. 3 is a photomicrograph of the cells from days 1, 3, 5, 7 and 9 of example 2, in which the first row is a photomicrograph of the cells from day 1, 3 and 5 of the cell culture, and the second row is a photomicrograph of the cells from day 7 and 9 of the cell culture and a magnified view of a portion of the photomicrograph of the cell culture at day 7 of the cell culture.
FIG. 4 is a microphotograph (first row) of the cell culture at day 3 in example 3 and a staining pattern (second row) of live and dead cells after 7 days of differentiation.
Detailed Description
The present invention is further illustrated by the following examples. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and nature of the invention.
The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
DMEM high glucose medium (BL 304A, biosharp); DMEM/F12 medium (PM 150312, procell); fetal bovine serum (WISENT); horse serum (BL 209A, biosharp); basic fibroblast growth factor/FGF 2 blocking polypeptide (bs-0217P, bioss); human recombinant insulin-like growth factor-1 (P00048, solibao); necrosulfonamide (N872612-5 mg, meclin); ascorbic acid (a 103534-100g, alatin); 100 × antibiotic (P1400, solibao); fibrinogen (BS 943-1g, biosharp); thrombin (BS 903-1000u, biosharp); type IV collagenase (17104019, gibco);
the primary pseudosciaena crocea muscle satellite cell is prepared by a conventional method in a laboratory, muscle tissue is separated from the upper shaft part of adult pseudosciaena crocea muscle, then the muscle tissue is digested by 0.1 percent of type IV collagenase solution and 0.1 percent of trypsin solution respectively, the digested tissue is filtered by 70 mu m and 40 mu m cell sieves respectively, the tissue is centrifuged for 5 minutes at 300g, the cell precipitate is resuspended by complete culture medium, and then the cell precipitate is prepared according to the proportion of 1 multiplied by 10 6 Inoculating into 6-well plate at a concentration of individual cells/mL, and culturing at 27 deg.C and 5% 2 Cultured in an incubator.
Example 1
Firstly, preparing fibrinogen mother liquor and thrombin mother liquor by utilizing DMEM high-glucose solution containing 1 × antibiotics under aseptic conditions, wherein the concentrations are 50mg/mL and 200U/mL respectively, and storing in a refrigerator at 4 ℃ for later use.
Selecting pseudosciaena crocea muscle satellite cells with good growth state, and adding trypsin when the cell confluence reaches 80%Digestion, centrifugation, cell collection and counting. Taking 40 mu L of fibrinogen mother liquor, adding 75 mu L of DMEM high-glucose solution containing 1 × antibiotics, adding 80 mu L of cell suspension, finally adding 5 mu L of thrombin mother liquor, and blowing and beating up and down by using a pipette after the addition is finished, and mixing uniformly. The whole system was 200. Mu.L, the final fibrinogen concentration was 10mg/mL, the thrombin concentration was 5U/mL, and the cell concentration was 4.0X 10 6 one/mL.
Adding 10. Mu.L of the cell-fibrinogen suspension obtained above to a 48-well plate in the form of droplets, and determining CO content at 27 ℃ 5% 2 Standing in cell culture box for 20min to coagulate fibrinogen. Then, 500. Mu.L of the proliferation medium was added and then placed in an incubator for culture. Proliferation culture was carried out for 4 days, and the differentiation medium was changed on day 4, followed by differentiation culture for 7 days, with the medium being changed every two days. And the morphology of the cultured cells was observed with an inverted microscope on days 1, 3, 5, 7 and 9. On the seventh day of differentiation culture, the fibrin hydrogel containing the cells was immunofluorescent-stained.
Wherein the proliferation culture medium is DMEM high-sugar medium containing 10% fetal calf serum, 10ng/mL basic fibroblast growth factor/FGF 2 blocking polypeptide and 1 × antibiotics (100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate); the differentiation medium was DMEM/F12 medium containing 8% horse serum, 10ng/mL human recombinant insulin-like growth factor-1, 50nM necrosulinamide, 200. Mu.M ascorbic acid, 1 Xantibiotic (100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate).
The microphotographs of the cultured cells of this example on days 1, 3, 5, 7 and 9 are shown in FIG. 1, and it can be seen that the cells are uniformly distributed in the gel and are in a three-dimensional growth state under the microscope. With the increase of the culture time, the cells gradually grow and differentiate, and finally, the cells are also in a three-dimensional network structure. The immunofluorescence staining pattern of the cell-fibrin hydrogel at the seventh day of differentiation is shown in figure 2, knot protein/cell nucleus staining can show that the pseudosciaena crocea muscle satellite cells are differentiated in the fibrin hydrogel to form myotubes, which shows that the fibrin hydrogel is suitable for the growth and differentiation of the pseudosciaena crocea muscle satellite cells.
Example 2
Firstly, preparing fibrinogen mother liquor and thrombin mother liquor by utilizing DMEM high-glucose solution containing 1 × antibiotics under aseptic conditions, wherein the concentrations are 50mg/mL and 200U/mL respectively, and storing in a refrigerator at 4 ℃ for later use.
Selecting pseudosciaena crocea muscle satellite cells in good growth state, adding trypsin for digestion and centrifugation when the cell confluence reaches 80%, collecting cells and counting. Taking 40 mu L of fibrinogen mother liquor, adding 75 mu L of DMEM high-glucose solution containing 1 × antibiotics, adding 80 mu L of cell suspension, finally adding 5 mu L of thrombin mother liquor, and blowing and beating up and down by using a pipette after the addition is finished, and mixing uniformly. The whole system was 200. Mu.L, the final fibrinogen concentration was 10mg/mL, the thrombin concentration was 5U/mL, and the cell concentration was 4.0X 10 6 one/mL.
Adding 40 μ L of the cell-fibrinogen suspension obtained above into 48-well plate in dropwise manner, at 27 deg.C, 5% 2 Standing in cell incubator for 20min to coagulate fibrinogen. Then 500. Mu.L of the enrichment medium was added thereto and then placed in an incubator for culture. Proliferation culture was carried out for 4 days, and the differentiation medium was changed on day 4, followed by differentiation culture for 7 days, with the medium being changed every two days. And the morphology of the cultured cells was observed with an inverted microscope on days 1, 3, 5, 7 and 9.
The microphotographs of the cultured cells of this example at days 1, 3, 5, 7 and 9 are shown in FIG. 3, and it can be seen under the microscope that the cells are uniformly distributed in the gel and in a three-dimensional growth state. As the culture time increased, the cells gradually expanded and proliferated in the fibrin hydrogel, and after changing the differentiation medium, the cells fused to form myotubes. Relatively large gel systems are also suitable for cell growth.
Example 3
Firstly, preparing fibrinogen mother liquor and thrombin mother liquor by utilizing DMEM high-sugar solution containing 1 × antibiotics under aseptic conditions, wherein the concentrations are 50mg/mL and 200U/mL respectively, and storing the fibrinogen mother liquor and the thrombin mother liquor in a refrigerator at 4 ℃ for later use.
Selecting pseudosciaena crocea muscle satellite cells in good growth state, adding trypsin for digestion and centrifugation when the cell confluence reaches 80%, collecting cells and counting. Taking fibrinogen mother liquor 2Adding 97.5 mu L of DMEM high-sugar solution containing 1 Xantibiotics into 0 mu L of the mixed solution, adding 80 mu L of the cell suspension, finally adding 2.5 mu L of thrombin mother solution, and blowing and beating the mixture up and down by using a liquid transfer gun after the addition is finished. The whole system is 200 μ L, the final fibrinogen concentration is 5mg/mL, the thrombin concentration is 2.5U/mL, and the cell concentration is 4.0X 10 6 one/mL.
Adding 10. Mu.L of the cell-fibrinogen suspension obtained above to a 48-well plate in the form of droplets, and determining CO content at 27 ℃ 5% 2 Standing in cell incubator for 20min to coagulate fibrinogen. Then 500. Mu.L of the enrichment medium was added thereto and then placed in an incubator for culture. Proliferation culture was carried out for 4 days, and the differentiation medium was changed on day 4, followed by differentiation culture for 7 days, with the medium being changed every two days. And the cultured cell morphology was observed with an inverted microscope on day 3. After the culture was completed, the fibrin hydrogel containing the cells was stained for live and dead cells.
The microphotograph and the live-dead cell staining photograph of the 3 rd day of the cells cultured in this example are shown in FIG. 4, and it can be seen under the microscope that the cells are uniformly distributed in the gel and in a three-dimensional growth state. The live and dead cell staining pictures show that the cell growth state is good, indicating that the fibrin hydrogel with the concentration is suitable for the growth of the cells.
The foregoing is a further detailed description of the invention and is not to be taken in a limiting sense as the invention is defined by the appended claims. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.
Claims (9)
1. A three-dimensional culture method of pseudosciaena crocea muscle satellite cells is characterized by comprising the following steps:
(1) Preparing separated pseudosciaena crocea muscle satellite cells into cell suspension;
(2) Adding the cell suspension into a DMEM high-sugar medium containing fibrinogen, adding antibiotics into the DMEM high-sugar medium, adding thrombin, uniformly mixing to obtain a mixed suspension, adding the mixed suspension into a cell culture plate, and solidifying to form cell-fibrin hydrogel;
(3) And adding a culture medium into the hydrogel system for cell culture.
2. The three-dimensional culture method of pseudosciaena crocea muscle satellite cells according to claim 1, wherein in the step (2), the concentration of fibrinogen in the mixed suspension is 5-15mg/mL, and the concentration of thrombin is 2.5-7.5U/mL.
3. The three-dimensional culture method of pseudosciaena crocea muscle satellite cells according to claim 2, wherein the concentration of fibrinogen and the concentration of thrombin in the mixed suspension are 10mg/mL and 5U/mL, respectively.
4. The three-dimensional culture method of pseudosciaena crocea muscle satellite cells according to any one of claims 1 to 3, wherein in the step (2), the cell concentration in the mixed suspension is 1.0 x 10 6 Per mL-5.0X 10 6 one/mL.
5. The three-dimensional culture method of pseudosciaena crocea muscle satellite cells according to claim 1, wherein in the step (2), 100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate are added into the DMEM high-sugar medium.
6. The three-dimensional culture method of pseudosciaena crocea muscle satellite cells according to claim 1, wherein in the step (2), the mixed suspension is added into a cell culture plate to be solidified into a drop shape, and the volume is 10-40 μ L.
7. The method for three-dimensional culture of pseudosciaena crocea muscle satellite cells according to claim 1 or 6, wherein the hydrogel is coagulated at 25-27 ℃ and 4-6% CO 2 The setting time is 20-60min.
8. The method for three-dimensional culture of pseudosciaena crocea muscle satellite cells according to claim 1, wherein in step (3), the cell culture conditions are 25-27 ℃ and 4-6% by weight of CO 2 Proliferation ofAfter culturing for 3-5 days, differential culture is carried out.
9. The three-dimensional culture method of pseudosciaena crocea muscle satellite cells according to claim 8, wherein the proliferation culture medium adopted by the proliferation culture is a DMEM high-sugar medium containing 10% fetal bovine serum, 10ng/mL basic fibroblast growth factor/FGF 2 blocking polypeptide, 100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate, and the differentiation culture medium adopted by the differentiation culture is a DMEM/F12 medium containing 8% horse serum, 10ng/mL human recombinant insulin-like growth factor-1, 50nM neocrosulfonamide, 200 μ M ascorbic acid, 100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate.
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