CN114958730A - Muscle stem cell proliferation medium, differentiation medium and application thereof - Google Patents
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Abstract
The invention provides a muscle stem cell proliferation culture medium, a muscle stem cell differentiation culture medium and application thereof. Comprises a muscle stem cell proliferation culture medium added with 3,2' -dihydroxyflavone and a muscle stem cell differentiation culture medium added with quercetin. When the muscle stem cell proliferation culture medium is adopted to culture the pig muscle stem cells in vitro in a short time, the proliferation speed is obviously improved, and the cell number obtained by culturing for 3 days is 1.35 times of that of a normal culture medium; the cell differentiation capacity is obviously improved and the expression level of muscle specific surface protein myosin heavy chain is obviously improved after the cell is induced and differentiated for 5 days in a muscle stem cell differentiation culture medium. The invention also provides application of the culture medium and a method for preparing the cultured meat based on the culture medium, which improve the amplification quantity and differentiation capacity of the short-term in-vitro culture of the muscle stem cells and are beneficial to the production of the cultured meat.
Description
Technical Field
The invention belongs to the technical field of stem cell culture, and particularly relates to a muscle stem cell proliferation culture medium, a muscle stem cell differentiation culture medium and application thereof.
Background
In recent years, as the global population has increased continuously and rapidly, animal husbandry as a traditional protein source has become non-sustainable and is no longer able to meet the needs of a large number of consumers. The cell culture meat serving as a subversive future food technology can not only solve the problems, but also relieve the problems of environmental damage, resource waste, animal welfare and the like caused by the traditional animal husbandry.
Production of cultured meat requires enrichment of large numbers of myogenic stem cells and further differentiation to produce muscle fibers. However, the muscle stem cells are faced with the problem of reduced proliferation and differentiation capacity during the in vitro culture process, which seriously hinders the production and development of cultured meat. Therefore, how to maintain the proliferation and differentiation capacity of the muscle stem cells in the in vitro culture process, rapidly proliferate the muscle stem cells in the proliferation stage to obtain the muscle stem cells with excellent differentiation potential, induce the muscle stem cells to efficiently differentiate into muscle fibers in the differentiation stage, and further generate the cultured meat products with excellent texture quality is a problem to be solved urgently in the research and industrial application of the cultured meat.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art and provide a culture medium for improving the proliferation capacity of muscle stem cells in an in vitro short-term culture process, a culture medium for improving the differentiation capacity of the muscle stem cells and application of the proliferation medium and/or the differentiation medium in a cell culture meat production process.
The first purpose of the invention is to provide an improved muscle stem cell proliferation culture medium, wherein the improved muscle stem cell proliferation culture medium comprises 3,2' -dihydroxyflavone.
Further, the concentration of the 3,2' -dihydroxyflavone in the culture medium for proliferating the modified muscle stem cells is 2.5-12.5 mu mol/L; preferably, the concentration of the 3,2 '-dihydroxyflavone in the modified muscle stem cell proliferation medium is 5-10. mu. mol/L, and more preferably, the concentration of the 3,2' -dihydroxyflavone in the modified muscle stem cell proliferation medium is 10. mu. mol/L.
Further, the improved muscle stem cell proliferation culture medium also comprises a conventional muscle stem cell proliferation culture medium;
preferably, the conventional muscle stem cell proliferation culture medium comprises 77-84 vol% of basal cell culture medium, 15-20 vol% of fetal bovine serum, 1-3 vol% of penicillin-streptomycin double-antibody solution and 1-10ng/ml of fibroblast growth factor 2; further preferably, the conventional muscle stem cell proliferation medium comprises 79 vol% of basal cell culture medium, 20 vol% of fetal bovine serum, 1 vol% of penicillin-streptomycin double antibody solution and 1-10ng/ml of fibroblast growth factor 2.
Further, the basal cell culture medium is selected from one of a DMEM medium, a MEM medium, a DMEM/F12 medium and a F10 medium.
Furthermore, in the penicillin-streptomycin double-resistant solution, the content of penicillin is 10000U/mL, and the content of streptomycin is 10 mg/mL.
The second purpose of the invention is to provide the application of the improved muscle stem cell proliferation medium in the in vitro proliferation culture of muscle stem cells.
Preferably, the in vitro proliferation culture of the muscle stem cells is in vitro short-term proliferation culture of the muscle stem cells.
Further, the improved muscle stem cell proliferation culture medium can increase the proliferation speed of the muscle stem cells in the in-vitro proliferation culture process and/or maintain the differentiation potential of the muscle stem cells in the in-vitro proliferation culture process.
The third purpose of the invention is to provide the application of the 3,2' -dihydroxyflavone in the in vitro proliferation culture of muscle stem cells.
Preferably, the in vitro proliferation culture of the muscle stem cells is an in vitro short-term proliferation culture of the muscle stem cells.
Further, carrying out in-vitro proliferation culture on the muscle stem cells by adopting a cell proliferation culture medium added with 3,2' -dihydroxyflavone; the 3,2' -dihydroxyflavone can improve the proliferation speed of the muscle stem cells in the in-vitro proliferation culture process and/or maintain the differentiation potential of the muscle stem cells in the in-vitro proliferation culture process.
Specifically, the application is to carry out in-vitro proliferation culture on the muscle stem cells by adopting an improved muscle stem cell proliferation culture medium added with 3,2 '-dihydroxyflavone, wherein the concentration of the 3,2' -dihydroxyflavone in the improved muscle stem cell proliferation culture medium is 2.5-12.5 mu mol/L; preferably, the concentration of the 3,2' -dihydroxyflavone in the modified muscle stem cell proliferation culture medium is 5-10 mu mol/L; more preferably, the concentration of the 3,2' -dihydroxyflavone in the medium for proliferating modified muscle stem cells is 10. mu. mol/L.
The fourth purpose of the invention is to provide an improved muscle stem cell differentiation medium, which comprises quercetin.
Further, the concentration of the quercetin in the improved muscle stem cell differentiation culture medium is 25-125 nmol/L;
preferably, the concentration of quercetin in the medium for differentiation of modified muscle stem cells is 50 nmol/L.
Further, the improved muscle stem cell differentiation medium also comprises a conventional muscle stem cell differentiation medium;
preferably, the conventional muscle stem cell differentiation medium comprises 92-97 vol% of basal cell culture medium, 2-5 vol% of horse serum and 1-3 vol% of penicillin streptomycin double antibody solution, and further preferably, the conventional muscle stem cell differentiation medium comprises 97 vol% of basal cell culture medium, 2 vol% of horse serum and 1 vol% of penicillin streptomycin double antibody solution.
Further, the basal cell culture medium is selected from one of a DMEM medium, a MEM medium, a DMEM/F12 medium and a F10 medium.
Furthermore, in the penicillin-streptomycin double-resistant solution, the content of penicillin is 10000U/ml, and the content of streptomycin is 10 mg/ml.
The fifth purpose of the invention is to provide the application of the improved muscle stem cell differentiation medium in the in vitro induction differentiation of muscle stem cells.
Preferably, the muscle stem cell is induced and differentiated in vitro into the muscle stem cell in vitro and induced and differentiated in short term.
Further, the improved muscle stem cell differentiation medium can improve the differentiation capacity of the muscle stem cells and/or improve the myotube forming capacity in inducing the muscle stem cell differentiation in vitro.
Furthermore, the muscle stem cells are obtained by adopting the improved muscle stem cell proliferation culture medium for in-vitro proliferation culture.
The sixth purpose of the invention is to provide the application of quercetin in vitro induction and differentiation of muscle stem cells.
Preferably, the muscle stem cell is induced and differentiated in vitro into the muscle stem cell in vitro and induced and differentiated in short term.
Further, adopting a cell differentiation medium added with quercetin to perform in-vitro induced differentiation on the muscle stem cells; the quercetin can improve the differentiation capability of the muscle stem cells and/or improve the myotube forming capability in the process of inducing the muscle stem cells to differentiate in vitro.
Furthermore, the muscle stem cells are obtained by adopting the improved muscle stem cell proliferation culture medium for in-vitro proliferation culture.
Specifically, the application is to adopt an improved muscle stem cell differentiation medium added with quercetin to perform in-vitro induced differentiation culture on muscle stem cells;
further, the concentration of the quercetin in the medium for differentiating the modified muscle stem cells is 25-125 nmol/L;
preferably, the concentration of quercetin in the medium for differentiation of modified muscle stem cells is 50 nmol/L.
It is a seventh object of the present invention to provide a method for preparing cultured meat, the method comprising the steps of:
s1: carrying out in-vitro proliferation culture on the muscle stem cells by adopting the improved muscle stem cell proliferation culture medium;
s2: mixing the muscle stem cells obtained in the step S1 with a gel system, and culturing the muscle stem cells to a pre-differentiation state by adopting the improved muscle stem cell proliferation culture medium; preferably, the muscle stem cells obtained in S1 are mixed with the gel system at a ratio of 4X 10 6 ～4.5×10 6 Individual cells/mL gel system;
s3: and (3) replacing the modified muscle stem cell proliferation culture medium with the modified muscle stem cell differentiation culture medium for induced differentiation to obtain the cultured meat, wherein the cultured meat comprises muscle cells, myotubes and muscle proteins.
Further, the gel system of S2 comprises bovine collagen, DMEM, NaOH and matrigel; preferably, the volume ratio of the bovine collagen, the DMEM, the NaOH and the matrigel in the gel system is 300:600:3: 80.
Further, S2 is that after muscle stem cells are mixed with a gel system, the muscle stem cells are cultured in a culture bracket to a pre-differentiation state;
preferably, the culture support is a culture meat production mold.
Further preferably, the culture scaffold is a porous netted culture meat production mold described in patent number zl201921875316.
An eighth object of the present invention is to provide a cultured meat produced by the aforementioned method for producing cultured meat.
The technical scheme of the invention has the following beneficial effects:
the invention discovers that the improved muscle stem cell proliferation culture medium added with 3,2' -dihydroxyflavone can accelerate the proliferation speed of muscle stem cells in the short-term culture process in vitro and maintain the differentiation potential of the cells. By adopting the improved muscle stem cell proliferation culture medium, the proliferation multiple of the muscle stem cells in the in-vitro short-term culture process can be enlarged by adding the 3,2' -dihydroxyflavone, and the cell proliferation multiple reaches 1.35 times of the number of cells cultured by a conventional culture medium; increasing the expression of paired box transcription factor PAX 7.
The improved muscle stem cell differentiation medium added with quercetin induces cells to differentiate, and obviously improves the expression quantity of muscle specific protein myosin heavy chain and the myotube forming capacity of the cells; by adopting the improved muscle stem cell differentiation medium, the differentiation capacity and myotube forming capacity of the muscle stem cells can be improved by adding quercetin.
The improved muscle stem cell proliferation culture medium and the improved muscle stem cell differentiation culture medium are combined to culture muscle stem cells and are used for production and production of cultured meat, the proliferation capacity of the muscle stem cells in the short-term culture process in vitro can be maintained, cell differentiation is promoted, and more muscle specific expression proteins are obtained. Meanwhile, the contractility of the cultured meat can be improved, the generation of muscle protein can be improved, and the contractility of the cultured meat, the expression of the muscle protein and the texture quality can be obviously improved, so that the muscle stem cells can be cultured in a short period under the two-dimensional (culturing cells in a culture dish) and three-dimensional (culturing cells in a gel system) culture conditions, the cell proliferation and differentiation capacity can be obviously improved, and a large amount of muscle cells and protein can be generated in the production of the cultured meat.
Drawings
FIG. 1 shows the expansion fold of muscle stem cells cultured in a conventional muscle stem cell proliferation medium and in a modified muscle stem cell proliferation medium supplemented with 3,2' -dihydroxyflavone at different concentrations for a short period of time.
FIG. 2 is the effect of conventional muscle stem cell proliferation medium, modified muscle stem cell proliferation medium supplemented with 10. mu. mol/L of 3,2' -dihydroxyflavone, on the sternness gene PAX7 characterizing the differentiation potential of muscle stem cells.
FIG. 3 shows the effect of conventional differentiation medium for myocyte and modified differentiation medium for myocyte supplemented with quercetin of different concentrations on the expression of the terminal differentiation marker gene MYHC for 5 days after inducing cell differentiation.
FIG. 4 shows the differentiation of cells cultured in a culture dish after immunofluorescence staining with MYHC.
FIG. 5 is a graph showing the effect on the relative contraction area of cultured meat when cultured meat is produced using a combination of a modified muscle stem cell proliferation medium and a conventional muscle stem cell differentiation medium (control group), and when cultured meat is produced using a combination of a modified muscle stem cell proliferation medium and a modified muscle stem cell differentiation medium (test group).
Fig. 6 is a graph showing the effect of producing cultured meat using a combination of modified muscle stem cell proliferation medium and conventional muscle stem cell differentiation medium (control group), and producing cultured meat using a combination of modified muscle stem cell proliferation medium and modified muscle stem cell differentiation medium (test group) on the water loss rate (fig. 6A) and myofibrillar fragmentation index (MFI value) (fig. 6B).
FIG. 7 is a graph showing the effect of MYHC expression in the production of cultured meat using a combination of modified muscle stem cell proliferation medium and conventional muscle stem cell differentiation medium (control group), and in the production of cultured meat using a combination of modified muscle stem cell proliferation medium and modified muscle stem cell differentiation medium (test group).
FIG. 8 shows the cultured meat after MYHC immunofluorescence staining.
Detailed Description
The term "vol%" as used herein means volume percent.
The cell culture methods used in the examples below are otherwise identical to conventional in vitro muscle stem cell culture methods, except for the culture medium.
The culture conditions used in the following examples are all CO 2 The concentration was 5% and the temperature was 37 ℃.
In the following examples:
(1) the formula of the conventional muscle stem cell proliferation culture medium is 79 vol% of F10 culture medium, 20 vol% of fetal calf serum, 1 vol% of penicillin-streptomycin double-antibody solution and 10ng/ml of fibroblast growth factor 2, wherein in the penicillin-streptomycin double-antibody solution, the content of penicillin is 10000U/ml, and the content of streptomycin is 10 mg/ml.
(2) The formula of the improved muscle stem cell proliferation culture medium comprises 79 vol% of F10 culture medium, 20 vol% of fetal calf serum, 1 vol% of penicillin-streptomycin double-antibody solution, 10ng/ml of fibroblast growth factor 2 and 3,2' -dihydroxyflavone with different concentrations, wherein in the penicillin-streptomycin double-antibody solution, the content of penicillin is 10000U/ml, and the content of streptomycin is 10 mg/ml.
(3) The formula of the conventional muscle stem cell differentiation medium is 97 vol% of DMEM cell culture medium, 2 vol% of horse serum and 1 vol% of penicillin streptomycin double antibody solution. In the penicillin-streptomycin double-resistant solution, the content of penicillin is 10000U/ml, and the content of streptomycin is 10 mg/ml.
(4) The formula of the improved muscle stem cell differentiation medium comprises 97 vol% of DMEM cell culture medium, 2 vol% of horse serum, 1 vol% of penicillin streptomycin double antibody solution and quercetin with different concentrations.
The differentiation culture dish (glass-bottomed dish) described in the following example was a 3.5cm dish precoated with 10-200. mu.g/ml matrigel (manufacturer: Corning, cat. No.: 356234).
The muscle stem cells used in the following examples were piglet muscle stem cells, and further, adherent piglet muscle stem cells.
The detection methods employed in the following examples are, unless otherwise indicated, those disclosed in the art.
Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified.
Example 1: detection of in vitro short-term culture proliferation capacity of pig muscle stem cells
In-vitro short-term culture of the porcine muscle stem cells:
this example is divided into 6 groups, respectively:
(1) a conventional muscle stem cell proliferation medium control group;
(2) an experimental group of a modified muscle stem cell proliferation culture medium with the concentration of 3,2' -dihydroxyflavone of 2.5 mu mol/L;
(3) an experimental group of the improved muscle stem cell proliferation culture medium with the concentration of 3,2' -dihydroxyflavone of 5 mu mol/L;
(4) an experimental group of modified muscle stem cell proliferation culture medium with the concentration of 3,2' -dihydroxyflavone of 7.5 mu mol/L;
(5) an experimental group of the improved muscle stem cell proliferation culture medium with the concentration of 3,2' -dihydroxyflavone of 10 mu mol/L;
(6) modified muscle stem cell proliferation medium test group with 3,2' -dihydroxyflavone concentration of 12.5 mu mol/L.
The above 6 groups of pig muscle stem cells were treated in a manner of 1.5X 10 5 The culture dish is inoculated into a 10cm culture dish which is respectively poured with a conventional muscle stem cell proliferation culture medium and a modified muscle stem cell proliferation culture medium containing 3,2' -dihydroxyflavone with corresponding concentration, liquid change is carried out for two days, digestion is carried out by 0.25% pancreatin on the 3 rd day, and counting is carried out by a blood cell counting plate.
The results show that: compared with the conventional culture medium, the muscle stem cell cultured in the improved muscle stem cell proliferation culture medium added with the 3,2' -dihydroxyflavone in a short term has the advantage that the proliferation speed is increased. When the concentration of the 3,2 '-dihydroxyflavone is 2.5, 5, 7.5, 10 and 12.5 mu mol/L, the amplification times of the porcine muscle stem cells are respectively 1.06, 1.28, 1.29, 1.35 and 1.10 times, wherein when the concentration is 5-10 mu mol/L, the significant difference exists, and the 3,2' -dihydroxyflavone can effectively improve the proliferation speed of the porcine muscle stem cells (figure 1).
Example 2: muscle stem cell differentiation potential assay
Taking the cells cultured by the conventional muscle stem cell proliferation culture medium in the example 1 as a control group, taking the cells cultured by the improved muscle stem cell proliferation culture medium with the concentration of 3,2' -dihydroxyflavone of 10 mu mol/L as an experimental group, extracting RNA in the cells, and respectively detecting the expression quantity of the sternness gene PAX7 for characterizing differentiation potential of the cells cultured by the control group and the experimental group by utilizing the RT-qPCR technology.
The results show that: the expression level of the porcine muscle stem cell PAX7 in the improved muscle stem cell proliferation medium with the concentration of the 3,2 '-dihydroxyflavone being 10 mu mol/L is 1.60 times that of the control group, and the difference is obvious, which indicates that the differentiation potential of the porcine muscle stem cell can be maintained when the concentration of the 3,2' -dihydroxyflavone is 10 mu mol/L (figure 2).
Example 3: detection of differentiation level of porcine muscle stem cells
This example is divided into 6 groups, respectively:
(1) a conventional muscle stem cell differentiation medium control group;
(2) an improved muscle stem cell differentiation culture medium experimental group with the quercetin concentration of 25 nmol/L;
(3) an improved muscle stem cell differentiation culture medium experimental group with the quercetin concentration of 50 nmol/L;
(4) an improved muscle stem cell differentiation culture medium experimental group with the quercetin concentration of 75 nmol/L;
(5) an improved muscle stem cell differentiation culture medium experimental group with the quercetin concentration of 100 nmol/L;
(6) modified muscle stem cell differentiation medium experimental group with quercetin concentration of 125 nmol/L.
The cells cultured in the modified proliferation medium supplemented with 10. mu. mol/L of 3,2' -dihydroxyflavone of example 1 were induced to differentiate.
The in vitro induced differentiation of the muscle stem cells comprises two stages:
the first stage is the proliferation stage of the pig muscle stem cell, the cell is cultured in vitro in the proliferation culture medium of the modified muscle stem cell added with 10 mu mol/L3, 2' -dihydroxyflavone, and the proliferation reaches the pre-differentiation stage for 5 days.
The second stage is the differentiation stage of the porcine muscle stem cells, the test group replaces the modified muscle stem cell proliferation medium of the previous stage with the modified muscle stem cell differentiation medium containing quercetin of corresponding concentration, and the control group is replaced with the conventional muscle stem cell differentiation medium. In the differentiation stage of the pig muscle stem cells, half liquid replacement is carried out every two days, namely, half of the original differentiation culture medium is sucked, half of the fresh differentiation culture medium is added, differentiation is finished after induced differentiation is carried out for 4-5 days, the terminal differentiation stage is reached, the gene representing the differentiation capacity in the stage is MYHC, and the expression quantity of the marker gene MYHC representing the differentiation level in the cells cultured in the control group and the test group is respectively detected by utilizing the RT-qPCR technology.
The results show that: inducing differentiation of cells cultured in the modified muscle stem cell proliferation medium added with 3,2' -dihydroxyflavone in a differentiation medium, wherein the modified muscle stem cell differentiation medium added with quercetin can remarkably up-regulate the expression of MYHC in cell terminal differentiation after inducing differentiation (figure 3), and the modified muscle stem cell differentiation medium containing 50nmol/L quercetin induces the highest expression level of MYHC in the differentiated muscle stem cells. Therefore, it can be concluded that the improved muscle stem cell differentiation medium added with quercetin can significantly improve the differentiation capacity of the pig muscle stem cells.
Example 4 measurement of myotube Forming ability of muscle Stem cell
This example was divided into 2 groups, control and test groups:
the cells cultured in the medium for proliferating 3,2' -dihydroxyflavone-modified muscle stem cells added at 10. mu. mol/L in example 1 were inoculated on a 3.5cm glass plate precoated with 10 to 200. mu.g/ml matrigel (manufacturer: Corning, cat. No. 356234), after the improved muscle stem cell proliferation culture medium is used for culturing for 5 days, the cells reach a pre-differentiation stage, the improved muscle stem cell proliferation culture medium of a control group is replaced by a conventional muscle stem cell differentiation culture medium, the improved muscle stem cell proliferation culture medium of a test group is replaced by the improved muscle stem cell differentiation culture medium containing 50nmol/L quercetin, and half liquid changing is carried out every two days, namely, half of the original differentiation medium is aspirated, half of the fresh differentiation medium is added, and after induced differentiation for 4 days, the terminal differentiation stage is reached, and the slender multinucleated myotubes are seen under a microscope. And (3) identifying the differentiated myotubes by using a MYHC antibody by using a cell immunofluorescence staining method.
The results show that: the muscle stem cells of the control group and the test group can be fused to form multinuclear myotubes to express muscle specific protein MYHC, and the multinuclear myotubes formed by the test group are more in number and stronger, which shows that the muscle stem cells have stronger myotube forming capability by the method provided by the invention (figure 4).
Example 5: culture meat texture characteristics and MYHC expression detection
This example was divided into 2 groups, control and test groups:
mixing the cells cultured in the culture medium for proliferating the modified muscle stem cells with the 3,2' -dihydroxyflavone added at 10 μmol/L in example 1 into the gel system, wherein the dosage of the gel system is 983 μ L, and the cells are in the gel systemHas a concentration of 4.5X 10 6 The gel system comprises 300 μ L of bovine collagen, 600 μ L of DMEM, 3 μ L of NaOH and 80 μ L of matrigel, and the mixture is uniformly mixed and then is put into a porous netted cultured meat production mold described in patent No. ZL201921875316.X, and the mixture is subjected to 5% CO production at 37 DEG C 2 Culturing in an incubator, culturing with the improved muscle stem cell proliferation culture medium for 24h to reach a pre-differentiation state, and replacing with a corresponding muscle stem cell differentiation culture medium.
Replacing the control group with a conventional muscle stem cell differentiation culture medium, replacing the test group with an improved muscle stem cell differentiation culture medium containing 50nmol/L quercetin, performing half liquid replacement every two days, namely, sucking half of the differentiation culture medium, adding half of a new differentiation culture medium, culturing for 7 days, reaching a terminal differentiation stage, and detecting the texture characteristics of the cultured meat and the expression of MYHC. The measurement method is improved on the basis of the industry standard 'objective evaluation method of meat eating quality' to detect the water loss rate and MFI value of the cultured meat and compare the water loss rate and MFI value with fresh meat. The expression of MYHC was detected by RT-qPCR technique.
The specific operation steps for measuring the water loss rate are as follows: weighing 0.1-0.3 g of meat sample by balance, and recording the weight as M1; placing a clean and smooth plastic plate on a flat desktop, placing three pieces of filter paper, and placing a meat sample on the filter paper; after 5min the weight of the meat sample was again recorded as M2. The water loss rate under pressure of the meat sample is calculated according to the water loss calculation formula (M1-M2)/M1 × 100% to characterize the water loss rate of the cultured meat.
The specific operation for measuring the MFI value is as follows: freezing and crushing 0.1-0.3 g of meat sample with 100ul precooled MFI buffer solution for 3 times, each time for 30s, and the interval for 60 s; homogenizing, centrifuging at 3000 Xg and 4 deg.C for 15min, and removing supernatant; adding 0.25ml buffer solution into the precipitate for homogenate, transferring into a new 1ml centrifuge tube, and washing the centrifuge tube with 100ul MFI buffer solution; obtaining homogenate as myofibrillar protein solution; and (3) measuring the concentration of the final protein filtrate by using a BCA method, adjusting the protein concentration of the myofibril extract to be 0.5mg/ml by using an MFI buffer solution, measuring an absorbance value at the wavelength of 540nm, and multiplying the absorbance value by 200 to obtain the MFI value.
The results show that: the cultured meat prepared from the pig muscle stem cells in the modified muscle stem cell differentiation medium supplemented with quercetin was more contractile than the control group (fig. 5). The water loss rate and the MFI value of the cultured meat of the test group are reduced by 11.14% and 35.60% respectively compared with those of the control group, and are closer to those of the fresh meat, which shows that the water loss rate and the MFI value are closer to those of the fresh meat (figure 6). Meanwhile, the expression level of MYHC in the cultured meat prepared from the pig muscle stem cells cultured in the modified muscle stem cell differentiation medium supplemented with quercetin was significantly increased, which was 3.74 times that of the control group (fig. 7).
Example 6 measurement of myotube-forming ability of cells in cultured meat
This example was divided into 2 groups, control and test groups:
mixing the cells cultured in the culture medium for proliferation of modified muscle stem cells supplemented with 10. mu. mol/L of 3,2' -dihydroxyflavone of example 1 into the gel system, wherein the dosage of the gel system is 983. mu.l, and the concentration of the cells in the gel system is 4.5X 10 6 The gel system comprises 300 mu L of bovine collagen, 600 mu L of DMEM, 3 mu L of NaOH and 80 mu L of matrigel, the components are uniformly mixed and then are evenly inoculated into a porous reticular culture meat production mold described in the patent number ZL201921875316.X, the culture is carried out for 24 hours by using an improved muscle stem cell proliferation culture medium to reach a pre-differentiation state, and the culture medium is replaced by a corresponding muscle stem cell differentiation culture medium.
Replacing a control group with a conventional muscle stem cell differentiation culture medium, replacing a test group with an improved muscle stem cell differentiation culture medium containing 50nmol/L quercetin, performing half-liquid replacement every two days, namely, sucking half of the differentiation culture medium, adding half of a new differentiation culture medium, culturing for 7 days, reaching a terminal differentiation stage, carefully picking out the cultured meat from a mold by using a clean forceps, cleaning for three times by using PBS, fixing the meat in a small dish by using PFA, and identifying myotubes after cell differentiation in the cultured meat by using an MYHC antibody by using an immunofluorescence staining method.
The results show that: both the control group and the test group can differentiate to form myotubes, and the cultured meat prepared by the pig muscle stem cells cultured by the cell modified differentiation medium added with quercetin has more and stronger myotubes than the control group (figure 8), which shows that the muscle stem cells cultured by the method provided by the invention can also keep higher differentiation capability in a three-dimensional gel system.
In conclusion, the invention discovers that the proliferation speed of the cell can be expanded by 1.35 times by using the improved muscle stem cell proliferation culture medium added with 3,2' -dihydroxyflavone in the in-vitro short-term culture process of the pig muscle stem cell; the improved muscle stem cell differentiation culture medium containing quercetin can be used for enabling cells to remarkably improve the expression quantity of muscle protein in the cell differentiation stage; in addition, the pork muscle stem cells cultured by the two culture media are used for producing the cultured meat, so that the contractility of the cultured meat and the expression quantity of muscle protein can be obviously improved, the water loss rate and the MFI value are reduced, and the quality of the meat is closer to that of fresh meat; in addition, the formation amount of myotubes in the cultured meat is more than that of the control group, which shows that the method provided by the invention is also suitable for the production of the cultured meat.
The examples disclosed above are intended to illustrate the disclosed embodiments of the present invention, but are not to be construed as limiting the invention, and many different antioxidants and differentiation inhibitors are listed herein and many different further combinations are possible without departing from the scope and spirit of the present invention, and thus the present invention is not limited to the disclosed embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (23)
1. An improved muscle stem cell proliferation culture medium, which is characterized by comprising 3,2' -dihydroxyflavone.
2. The modified muscle stem cell growth medium of claim 1, wherein the concentration of the 3,2' -dihydroxyflavone in the modified muscle stem cell growth medium is 2.5 to 12.5 μmol/L; preferably, the concentration of the 3,2 '-dihydroxyflavone in the modified muscle stem cell proliferation medium is 5-10. mu. mol/L, and more preferably, the concentration of the 3,2' -dihydroxyflavone in the modified muscle stem cell proliferation medium is 10. mu. mol/L.
3. The modified muscle stem cell proliferation medium of claim 1, wherein the modified muscle stem cell proliferation medium further comprises a conventional muscle stem cell proliferation medium; preferably, the conventional muscle stem cell proliferation culture medium comprises 77-84 vol% of basal cell culture medium, 15-20 vol% of fetal bovine serum, 1-3 vol% of penicillin-streptomycin double-antibody solution and 1-10ng/mL of fibroblast growth factor 2; further preferably, the conventional muscle stem cell proliferation medium comprises 79 vol% of basal cell culture medium, 20 vol% of fetal bovine serum, 1 vol% of penicillin-streptomycin double antibody solution and 1-10ng/mL of fibroblast growth factor 2.
4. The modified muscle stem cell growth medium according to claim 3, wherein the basal cell culture medium is one selected from the group consisting of DMEM medium, MEM medium, DMEM/F12 medium, and F10 medium.
5. The improved muscle stem cell proliferation medium of claim 3, wherein the penicillin-streptomycin double antibody solution contains 10000U/mL of penicillin and 10mg/mL of streptomycin.
6. The use of the modified muscle stem cell proliferation medium of claim 1 in muscle stem cell proliferation culture in vitro, preferably in muscle stem cell proliferation culture in vitro short term.
7. The use according to claim 6, wherein the modified muscle stem cell proliferation medium is capable of increasing the proliferation rate of muscle stem cells during in vitro proliferation culture and/or maintaining the differentiation potential of muscle stem cells during in vitro proliferation culture.
The application of the 3,2' -dihydroxyflavone in the in-vitro proliferation culture of the muscle stem cells is preferably used, and the in-vitro proliferation culture of the muscle stem cells is preferably used for the in-vitro short-term proliferation culture of the muscle stem cells.
9. The use of claim 8, wherein the muscle stem cells are cultured in vitro using a cell proliferation medium supplemented with 3,2' -dihydroxyflavone; the 3,2' -dihydroxyflavone can improve the proliferation speed of the muscle stem cells in the in-vitro proliferation culture process and/or maintain the differentiation potential of the muscle stem cells in the in-vitro proliferation culture process.
10. An improved muscle stem cell differentiation medium, which is characterized by comprising quercetin.
11. The modified muscle stem cell differentiation medium according to claim 10, wherein said quercetin is present in a concentration of 25-125nmol/L in the modified muscle stem cell differentiation medium; preferably, the concentration of quercetin in the medium for differentiation of modified muscle stem cells is 50 nmol/L.
12. The modified muscle stem cell differentiation medium according to claim 10, further comprising a conventional muscle stem cell differentiation medium; preferably, the conventional muscle stem cell differentiation medium comprises 92-97 vol% of basal cell culture medium, 2-5 vol% of horse serum and 1-3 vol% of penicillin streptomycin double antibody solution, and further preferably, the conventional muscle stem cell differentiation medium comprises 97 vol% of basal cell culture medium, 2 vol% of horse serum and 1 vol% of penicillin streptomycin double antibody solution.
13. The modified myostem cell differentiation medium according to claim 12, wherein said basal cell culture medium is selected from one of DMEM medium, MEM medium, DMEM/F12 medium, F10 medium.
14. The improved muscle stem cell differentiation medium according to claim 12, wherein said penicillin-streptomycin double antibody solution comprises penicillin in an amount of 10000U/mL and streptomycin in an amount of 10 mg/mL.
15. Use of the improved muscle stem cell differentiation medium according to claim 10 for the in vitro induced differentiation of muscle stem cells, preferably for the in vitro induced differentiation of muscle stem cells into in vitro short-term induced differentiation of muscle stem cells.
16. The use of claim 15, wherein the modified muscle stem cell differentiation medium is capable of increasing the differentiation capacity of muscle stem cells and/or increasing the myotube-forming capacity in inducing muscle stem cell differentiation in vitro.
17. The application of quercetin in vitro induced differentiation of muscle stem cells, preferably, the muscle stem cells are induced and differentiated in vitro into the muscle stem cells for short-term induced differentiation in vitro.
18. The use according to claim 17, wherein the induced differentiation of the muscle stem cells is performed in vitro using a cell differentiation medium supplemented with quercetin; the quercetin can improve the differentiation capability of the muscle stem cells and/or improve the myotube forming capability in the process of inducing the muscle stem cells to differentiate in vitro.
19. The use of claim 15 or 17, wherein the muscle stem cells are obtained by in vitro proliferation culture using the modified muscle stem cell proliferation culture medium of claim 1.
20. A method for preparing a cultured meat, comprising the steps of:
s1: performing in vitro proliferation culture on the muscle stem cells by using the modified muscle stem cell proliferation culture medium of claim 1;
s2: mixing the muscle stem cells obtained in S1 with gel system, and collectingCulturing muscle stem cells to a pre-differentiated state with the modified muscle stem cell proliferation medium of claim 1; preferably, the muscle stem cells obtained in S1 are mixed with the gel system at a ratio of 4X 10 6 ～4.5×10 6 Individual cells/mL gel system;
s3: changing the modified muscle stem cell proliferation medium to the modified muscle stem cell differentiation medium according to claim 10, and inducing differentiation to obtain the cultured meat.
21. The method of claim 20, wherein the components of the gel system of S2 include bovine collagen, DMEM, NaOH, matrigel; preferably, the volume ratio of the bovine collagen, the DMEM, the NaOH and the matrigel in the gel system is 300:600:3: 80.
22. The method according to claim 20, wherein S2 is the muscle stem cells mixed with the gel system and cultured in the culture scaffold to a pre-differentiation state; preferably, the culture support is a culture meat production mold.
23. The cultured meat produced by the production method according to claim 20.
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