CN114134105B - Method for preparing cell culture meat by using high-activity muscle stem cells - Google Patents
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Abstract
The invention belongs to the field of stem cell application, and particularly relates to a method for preparing animal cell culture meat by utilizing high-activity muscle stem cells. The polypeptide fragment of Jagged1 is combined in the hydrogel cell scaffold in advance, so that Notch signal channels of stem cells are effectively activated, and the characteristics (dryness and growth differentiation potential) of the stem cells are maintained. The added Follistatin can inhibit the activity of myostatin, so that the volume, growth and differentiation of muscle cells are more potential, and the differentiated muscle fibers are thicker. These more mature and robust muscle fibers will enhance the texture and mouthfeel of the cell culture meat and produce more trophic factors specific to the mature muscle cells. Meanwhile, the hydrogel thin layers growing muscle cells are overlapped in a plurality of layers to form a three-dimensional muscle tissue, so that the differentiation of the cell myofibers under the low serum condition is continued, and the time and space advantages of the growth and differentiation of the muscle stem cells are more reasonably arranged.
Description
Technical Field
The invention belongs to the field of stem cell application, and particularly relates to a method for preparing cell culture meat by using high-activity muscle stem cells.
Background
Meat analogs, cell-based meat/CBM, have gained widespread attention in recent years from global public opinion and market. Global meat production and consumption continue to proliferate due to population growth, personal and socioeconomic demand growth. In 2012, the united states Food and Agricultural Organization (FAO) predicts that by 2050, global meat demand will reach 4.55 billion tons (76% increase over 2005). The growing meat demand worldwide causes increasing concerns and negative consequences due to the complexity of current large animal husbandry production methods and public health problems, as well as problems of greenhouse effect, environmental degradation, animal welfare, etc. associated with meat production. The large-scale development of animal husbandry is interrelated with the large-scale epidemic of food-borne diseases, antibiotic resistance and infectious diseases, which closely affect the physical health of humans. The livestock industry also exacerbates environmental problems including greenhouse gas emissions, as well as land and water resource utilization. There are also animal welfare and ethical issues, with billions of animals being destroyed by the operation of the human food system, either directly or indirectly, each year.
The negative problems associated with the production of these meats, and the rapid development of stem cell technology over the years, have prompted the intense discussion and study of new products such as meat analogs. Plant-based meat (PBM) and cell-based meat (CBM) methods can produce food from non-animal sources. Traditional PBM (e.g., tofu) has been in existence for centuries, but new PBM alternatives with enhanced organoleptic properties more meat-like have recently been commercialized. Recent years have seen the development of a new area of CBM cell agriculture, a strategy for producing meat analogue commodity products from cells rather than whole organisms or animals. CBM technology is based on advances in stem cell biology (e.g., induced pluripotent stem cells) and tissue engineering (e.g., in vitro skeletal muscle transplantation) originally used for medical applications. Once optimized, CBM production is expected to require less energy and resources and reduce waste compared to traditional meat production. CBM can replace some of the intensive animal industries to improve welfare of farm animals. Animal donors are only used to provide an initial source of cells, which can then be expanded in vitro. Donor animals are typically younger animals, with more healthy stem cells, and will be anesthetized by veterinarian and harvested for small amounts of tissue. The cells can be managed to proliferate indefinitely, eliminating the need for animal donors. Production of CBM generally involves four major components: (1) isolation and culture of muscle stem cells; (2) formulation of high-efficiency culture medium; (3) selection of a cell culture scaffold; (4) design of cell culture apparatus. The development of new technologies has spawned global companies that have focused on marketing CBM products. CBM has now changed from imagination in science fiction to a realistic tangible object. However, how to achieve efficient culture and differentiation lacks corresponding techniques, especially the screening of high-activity muscle stem cells at the beginning, and the time and space coordination problems of the two processes of rapid expansion and myogenic differentiation of cells in vitro.
Disclosure of Invention
Aiming at the problems of the cell culture meat at the present stage, the polypeptide fragment of Jagged1 is combined in the hydrogel cell scaffold in advance, so that the Notch signal channel of the stem cells is effectively activated, and the characteristics (the stem property and the growth differentiation potential) of the stem cells are maintained. The added Follistatin can inhibit the activity of myostatin, so that the volume, growth and differentiation of muscle cells are more potential, and the differentiated muscle fibers are thicker. These more mature and robust muscle fibers will enhance the texture and mouthfeel of the cell culture meat and produce more trophic factors specific to the mature muscle cells. Meanwhile, the hydrogel thin layers growing muscle cells are overlapped in a plurality of layers to form a three-dimensional muscle tissue, so that the differentiation of the cell myofibers under the low serum condition is continued, and the time and space advantages of the growth and differentiation of the muscle stem cells are more reasonably arranged.
Furthermore, the use of electrical stimulation at the cell differentiation stage can enhance the differentiation efficiency and maturity of muscle fibers.
The scheme of the invention is as follows:
a method for preparing cell culture meat by using high activity muscle stem cells, comprising the following steps:
(1) Screening high-activity muscle stem cells;
(2) The method adopts a hydrogel monolayer cell method to obtain the high-activity muscle stem cells: performing large-scale cell expansion by using a cell culture medium added with the Follistatin; a polypeptide fragment of the Jagged1 protein is combined in the hydrogel;
(3) Obtaining a three-dimensional multilayer structure of a muscle stem cell hydrogel thin layer;
(4) The three-dimensional structure tissue differentiated into mature muscle fiber cells, namely the cell culture meat, is obtained efficiently.
Further, the process of the step (1) is as follows: muscle stem cell MPCs from healthy young animal individuals are extracted and cultured, cells with higher ALDH1 enzymatic activity are screened using a flow cytometer, and conventional 2D surface culture is performed for initial expansion.
Further, the process of the step (2) is as follows: introducing ALDH1 high-expression muscle stem cells of 4 th to 6 th generation in logarithmic growth phase into a hydrogel thin-layer structure formed by collagen, and performing large-scale cell expansion by using a cell culture medium added with Follistatin; the polypeptide fragment of Jagged1 protein is combined in the hydrogel to stimulate the activation of a Notch1 signal channel of the muscle stem cells, so that the muscle stem cells are induced to maintain the stem cell characteristics and accelerate the growth, and the muscle stem cells are cultured for 5-7 days.
Further, the process of the step (3) is as follows: taking out the hydrogel monolayer of the muscle stem cells from the culture solution by using a cell shovel, paving the hydrogel monolayer in a new culture tray, stacking 4-6 layers of cell monolayers layer by layer, adding the cell culture solution containing the Follistatin, and continuing to perform stationary culture.
Further, the process of the step (4) is as follows: changing the three-dimensional multilayer structure of the muscle cells into a myogenic differentiation culture solution of the muscle cells for continuous culture for 6-10 days, and adding Follistatin into the culture solution; and meanwhile, intermittent low-frequency electrical stimulation is applied to the culture system to promote the differentiation of the muscle stem cells into mature muscle fiber cells.
Further, said step (1) further comprises initially culturing and expanding primary muscle stem cells from the animal to about 5x10 4 After the number of cells, the cells with the highest activity of about 15% of ALDH1, namely 1X10, are separated by separation and screening according to the activity of the ALDH1 4 And (3) cells.
Further, the thickness of the hydrogel thin layer structure in the step (2) is 1.5-2.5 mm; the concentration of the polypeptide fragment of the Jagged1 protein in the hydrogel is 0.2-1.0mg/mL; the concentration of the Follistatin in the 3D cell culture medium added with the Follistatin is 100-200ng/mL.
Further, the culture in the step (3) is carried out for 3 to 6 days.
Further, the Follistatin in the culture solution in the step (4) is the Follistatin 344 with the concentration of 100-200ng/mL; and (3) electric stimulation: frequency 20 Hz, time: 30-50 minutes twice a day; the culture solution is a DMEM cell culture medium, and the additive comprises 1-3% of the final volume fraction of fetal bovine serum and 0.5-2% of the final volume fraction of chicken embryo essence extract; the total volume of the cell culture medium was 10mL of culture solution/CM 3 And a cytoskeleton.
Compared with the prior art, the invention has the beneficial effects that:
the high-activity muscle stem cell is taken as a ALDH1 high-activity stem cell from young animal individuals, and has stronger oxidation resistance, apoptosis resistance and growth differentiation function than the conventional muscle stem cell. Thus, ALDH1+ cells can be more efficiently grown rapidly in vitro.
The polypeptide fragment of Jagged1 is combined in the hydrogel cell scaffold in advance, so that a Notch signal channel of the stem cells can be effectively activated, and the characteristics (the dryness and the growth differentiation potential) of the stem cells are better maintained.
The added Follistatin in the culture solution can inhibit the activity of myostatin, so that the volume, growth and differentiation of muscle cells are more potential, and the differentiated muscle fibers are thicker. These more mature and robust muscle fibers will enhance the texture and mouthfeel of the cell culture meat and produce more trophic factors specific to the mature muscle cells.
The multi-layer superposition of the hydrogel thin layers with the grown muscle cells is adopted to form the three-dimensional muscle tissue so as to continue the differentiation of the cell myogenic fibers under the low serum condition, thereby better reasonably arranging the time and space advantages of the growth and differentiation of the muscle stem cells. In addition, in the process of muscle cell differentiation, intermittent low-frequency electrical stimulation is applied to a culture system, so that the formation of muscle fibers can be more effectively promoted.
Drawings
Fig. 1: aldh+ muscle stem cells isolated from leg muscles of dogs of example 1 can rapidly grow and differentiate into muscle fibroblasts in the 2D plane. After 5 days of culture with the muscle stem cell growth medium, the culture medium is changed into a muscle cell differentiation medium for further 4 days. These cells can be demonstrated to have good ability to grow and differentiate into myotubes (muscle fibroblasts).
Fig. 2: aldh+ muscle stem cells isolated from leg muscles of dogs of example 1 grew rapidly in a 3D culture system. After the GFP-labeled muscle stem cells were transferred into the hydrogel sheet, they were cultured with the muscle stem cell growth medium for 3 days.
FIG. 3A multilayer superposition of thin layers of hydrogels grown by ALDH+ muscle stem cells of example 1 forms a three-dimensional multilayer structure. After 3 days of culture with the muscle stem cell growth medium, the culture was continued for 6 days after changing to the muscle cell differentiation medium (a, B). The general formation of mature myotubes (muscle fibroblasts) in the stereo structure is seen (C).
Detailed Description
The present invention will be described in detail with reference to specific examples, wherein the exemplary embodiments and descriptions of the present invention are provided for the purpose of illustration and are not intended to be limiting. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
a method for producing cell culture meat by using high-activity muscle stem cells of dogs, which comprises the following specific steps:
(1) Screening to obtain high-functional active muscle stem cells: primary muscle stem cells from hind limbs of experimental dogs (2 years old) were expanded to approximately 5x10 by preliminary culture 4 After the number of the cells, the cells with the highest ALDH1 activity (1 x 10) of about 15 percent are separated and screened according to the ALDH1 activity 4 A cell);
(2) Hydrogel monolayer cells for efficiently obtaining high-activity muscle stem cells: and when the ALDH1 high-expression muscle stem cell is cultured and amplified to the 4 th generation, introducing the cell into a hydrogel thin-layer structure (thickness of 2 mm) formed by collagen, and performing large-scale cell amplification by using the 3D cell culture medium. The hydrogel is combined with 0.5mg/mL Jagged1 polypeptide fragment to stimulate the activation of the Notch1 signal channel of the muscle stem cells, so that the muscle stem cells are induced to maintain the stem cell characteristics and accelerate the growth. Meanwhile, a certain amount of Follistatin (344, 150 ng/mL) is added into the culture solution to further promote the rapid growth of the muscle stem cells. The basic culture solution is a DMEM cell culture medium, and other additive materials comprise fetal bovine serum with a final volume fraction of 20% and chicken embryo essence extract (chicken embryo essential extracts/CEE) with a final volume fraction of 1%; culturing for 5 days;
(3) Obtaining a three-dimensional multilayer structure of muscle stem cell hydrogel thin layers: taking out the hydrogel monolayer of the muscle stem cells from the culture solution by using a cell shovel, spreading the hydrogel monolayer in a new culture tray, stacking 5 layers of cell monolayers layer by layer, adding the cell culture solution containing the Follistatin, and continuously standing and culturing for 3 days to form a stable three-dimensional multilayer structure of the muscle cells;
(4) Three-dimensional structure tissue (cell culture meat) differentiated into mature muscle fiber cells is efficiently obtained: the three-dimensional multi-layer structure of the muscle cells is changed into a myoblast differentiation culture solution of the muscle cells to be continuously cultured for 6 days, and the Follistatin (the Follistatin 344, 150 ng/mL) is continuously added during the period to promote the growth increase and differentiation of the muscle cells, and meanwhile, intermittent low-frequency electric stimulation (the frequency is 20 Hz; the time length is 40 minutes and twice a day) is applied in a culture system to promote the differentiation of the muscle stem cells into mature muscle fibroblasts. The basic culture solution is a DMEM cell culture medium, and other additive materials comprise fetal calf serum with a final volume fraction of 2% and chicken embryo essence extract (chicken embryo essential extracts/CEE) with a final volume fraction of 1%; and collecting the generated muscle cell tissues after the culture is finished, and detecting and identifying the morphology and the differentiation degree of the muscle cells.
A large number of mature muscle fibers were grown from the muscle cell tissue obtained in example 1, with an appearance resembling meat of direct animal origin. The observation and identification of the growth and differentiation process and the formation of the product of the muscle stem cells are carried out, and the related results are shown in the following figures 1-3; our invention can produce a natural meat-like product in vitro with relatively high efficiency by cell culture. The technology development of domestic artificial meat industrialization is promoted, and various problems of sanitation, health, environment, energy consumption, animal welfare and the like related to excessive development of animal husbandry are effectively buffered.
Example 2
A method for producing cell culture meat by using high-activity muscle stem cells of cattle comprises the following specific steps:
(1) Screening to obtain high-functional active muscle stem cells: primary muscle stem cells from the legs of farm cattle (Black Angus) were initially cultured and expanded to about 5X10 4 After the number of the cells, the cells with the highest ALDH1 activity (1.5x10) are separated and separated according to the activity of the ALDH1, wherein about 30 percent of the cells with the highest ALDH1 activity are separated 4 A cell);
(2) Hydrogel monolayer cells for efficiently obtaining high-activity muscle stem cells: and when the ALDH1 high-expression muscle stem cell is cultured and amplified to the 6 th generation, introducing the cell into a hydrogel thin-layer structure (thickness of 2 mm) formed by collagen, and performing large-scale cell amplification by using the 3D cell culture medium. The hydrogel has 0.5mg/mL of the polypeptide fragment of the Jagged1 protein bound thereto. Simultaneously, the quick growth of the muscle stem cells is further promoted by adding the Follistatin 344 (150 ng/mL) into the culture solution. The basic culture solution is a DMEM cell culture medium, and other additive materials comprise fetal bovine serum with a final volume fraction of 20% and chicken embryo essence extract (chicken embryo essential extracts/CEE) with a final volume fraction of 1%; culturing for 5 days;
(3) Obtaining a three-dimensional multilayer structure of muscle stem cell hydrogel thin layers: taking out the hydrogel monolayer of the muscle stem cells from the culture solution by using a cell shovel, spreading the hydrogel monolayer in a new culture tray, stacking 5 layers of cell monolayers layer by layer, adding the cell culture solution containing the Follistatin, and continuously standing and culturing for 3 days to form a stable three-dimensional multilayer structure of the muscle cells;
(4) Three-dimensional structure tissue (cell culture meat) differentiated into mature muscle fiber cells is efficiently obtained: the three-dimensional multi-layer structure of the muscle cells is changed into a myoblast differentiation culture solution of the muscle cells to be continuously cultured for 6 days, and the Follistatin 344 (150 ng/mL) is continuously added during the period to promote the growth increase and differentiation of the muscle cells, and meanwhile, intermittent low-frequency electric stimulation (frequency 20 Hz; time length: 40 minutes and twice daily) is applied in a culture system to promote the differentiation of the muscle stem cells into mature muscle fiber cells. The basic culture solution is a DMEM cell culture medium, and the final volume fraction of other additive substances including fetal calf serum is 2%; and collecting the generated muscle cell tissues after the culture is finished, and detecting and identifying the morphology and the differentiation degree of the muscle cells.
A large number of mature muscle fibers were grown in the bovine muscle cell tissue obtained in example 2, with a meat-like appearance. Thus, our invention can efficiently produce natural meat-like products in vitro by cell culture.
Claims (4)
1. A method for preparing cell culture meat by using high-activity muscle stem cells, which is characterized by comprising the following steps:
(1) Screening high-activity muscle stem cells;
(2) The method adopts a hydrogel monolayer cell method to obtain the high-activity muscle stem cells: performing large-scale cell expansion by using a cell culture medium added with the Follistatin; a polypeptide fragment of the Jagged1 protein is combined in the hydrogel;
(3) Obtaining a three-dimensional multilayer structure of a muscle stem cell hydrogel thin layer;
(4) The three-dimensional structure tissue differentiated into mature muscle fiber cells, namely cell culture meat, is obtained efficiently;
the process of the step (1) is as follows: extracting and culturing muscle stem cells MPC from healthy young animal individuals, screening cells with higher ALDH1 enzyme activity by using a flow cytometer, and performing conventional 2D surface culture for preliminary amplification;
the step (1) further comprises preliminary culture expansion of primary muscle stem cells from the animalUp to 5x10 4 After the number of the cells, separating and screening according to the activity of the ALDH1, and separating out 15% of cells with highest ALDH1 activity;
the process of the step (2) is as follows: introducing ALDH1 high-expression muscle stem cells of 4 th to 6 th generation in logarithmic growth phase into a hydrogel thin-layer structure formed by collagen, and performing large-scale cell expansion by using a cell culture medium added with Follistatin; the hydrogel is combined with a polypeptide fragment of Jagged1 protein to stimulate the activation of a Notch1 signal channel of the muscle stem cells, so that the muscle stem cells are induced to maintain stem cell characteristics and accelerate growth, and are cultured for 5-7 days;
the process of the step (3) is as follows: taking out the hydrogel monolayer of the muscle stem cells from the culture solution by using a cell shovel, paving the hydrogel monolayer in a new culture tray, stacking 4-6 layers of cell monolayers layer by layer, adding the cell culture solution containing the Follistatin, and continuing to perform stationary culture;
the process of the step (4) is as follows: changing the three-dimensional multilayer structure of the muscle cells into a myogenic differentiation culture solution of the muscle cells for continuous culture for 6-10 days, and adding Follistatin into the culture solution; and meanwhile, intermittent low-frequency electrical stimulation is applied to the culture system to promote the differentiation of the muscle stem cells into mature muscle fiber cells.
2. The method for preparing cell culture meat using high activity muscle stem cells as set forth in claim 1, wherein the hydrogel sheet structure in the step (2) has a thickness of 1.5-2.5 mm; the concentration of the polypeptide fragment of the Jagged1 protein in the hydrogel is 0.2-1.0mg/mL; the concentration of the Follistatin in the 3D cell culture medium added with the Follistatin is 100-200ng/mL.
3. The method for preparing cell culture meat using high activity muscle stem cells according to claim 1, wherein the culturing is performed for 3 to 6 days in the step (3).
4. The method for preparing cell culture meat using high activity muscle stem cells according to claim 1, which comprisesIs characterized in that the Follistatin in the culture solution in the step (4) is the Follistatin 344, and the concentration is 100-200ng/mL; and (3) electric stimulation: frequency 20 Hz, time: 30-50 minutes twice a day; the culture solution is a DMEM cell culture medium, and the additive comprises 1-3% of the final volume fraction of fetal bovine serum and 0.5-2% of the final volume fraction of chicken embryo essence extract; the total volume of the cell culture medium was 10mL of culture solution/CM 3 And a cytoskeleton.
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