CN116555033A - A mold and a method for arraying and growing muscle stem cells and their application - Google Patents

Abstract

The invention provides a mould, a method for arranging and growing muscle stem cells and application thereof, belonging to the technical field of cell culture; the die comprises a groove and needle-shaped assemblies vertically inserted into two ends of the groove. In the invention, the groove is used for containing gel; the needle-shaped component is used for fixing the gel, and after the two ends of the gel are fixed, the cells are mechanically acted, and grow towards the fixed direction, so that the directionality of the cell growth is controlled. The invention can realize the arrangement growth of cells in three-dimensional culture by utilizing the mould formed by combining the grooves and the needle-shaped components, wherein the needle-shaped components have a certain anchoring effect, can control the directionality of cell growth, and enable the growth and differentiation of muscle stem cells into orderly arranged myotube tissues. The invention realizes the arrangement, growth and differentiation of the fish muscle satellite cells in a three-dimensional environment for the first time, and promotes the development of cell culture fish meat.

Description

A mold and a method for arraying and growing muscle stem cells and their application

technical field

The invention belongs to the technical field of cell culture, and in particular relates to a mold, a method for arraying and growing muscle stem cells and an application thereof.

Background technique

Artificial meat is an alternative protein source, and it is an emerging product to meet the future demand for meat protein and alleviate the environmental pressure brought by animal husbandry. Strictly speaking, there is no very clear definition of artificial meat at present, and most countries are classified as "meat substitutes". The "artificial meat" is mainly divided into two categories, one is plant-based meat (Plant-based Meat) with high content of plant protein as the main component, and the other is cell cultured meat (Cultured meat) cultivated by animal stem cells. Since the cells are cultured in vitro to mimic the process of cell growth and differentiation in vivo, it is possible to produce products with the same nutritional and organoleptic properties as traditional meat products.

As early as the 1930s, some researchers proposed the concept of cell cultured meat. The first demonstration of cell-cultured meat took place in 2002. In 2013, Dr. MarkPost's team used bovine myoblasts to grow the first artificial steak and make it into a hamburger, but the cost was high (85 grams, about $330,000). In 2016, Mem-phis Meats, an American food technology company, developed artificial beef balls and artificial chicken for the first time in the world. In 2019, Zhou Guanghong's team at Nanjing Agricultural University and others cultivated pig muscle stem cells to obtain China's first piece of artificial meat. Today, a growing number of companies (at least 70 by mid-2021) are working to commercialize and scale up cell-based meat, but many challenges lie ahead. Among them, it is a big challenge to make cell-cultured meat closer to real meat products in terms of nutrition, taste and flavor, and it is also the main research and development direction of artificial meat in the future.

In order to develop cell-cultured meat that resembles real meat, it is necessary to consider the structure of naturally occurring muscle tissue. In most edible vertebrates (pigs, cattle, chickens, fish, etc.), there are three kinds of muscle tissues, namely skeletal muscle, smooth muscle and heart muscle, among which skeletal muscle is the largest tissue in the animal body and is an important source of meat . The main functional unit of skeletal muscle is the muscle fiber (also called myocyte); the muscle fiber is organized into bundles called fascicles; the muscle bundles are organized into muscles. The well-aligned muscle fibers, fat, structured connective tissue, and other structural elements of muscle combine to produce its mechanical properties, which would be considered texture if muscle were eaten as meat. Therefore, well-arranged muscle fibers are a major feature of muscle tissue. Therefore, it is necessary to develop a device and/or method to realize the arrangement and growth of muscle stem cells under three-dimensional culture conditions.

Contents of the invention

The object of the present invention is to provide a mold and a method for arranging and growing muscle stem cells and its application. Using the mold, the muscle stem cells can be arranged and grown in a three-dimensional environment.

The present invention provides a mold, which includes a groove and needle-shaped components vertically inserted into two ends of the groove.

Preferably, the material of the groove includes silica gel; the shape of the groove includes U-shape.

Preferably, the needle-shaped components include pins; the distance between the needle-shaped components is 3-9mm.

The present invention also provides a method for arraying and growing muscle stem cells, comprising the following steps:

1) mixing the cell suspension of the muscle stem cells, the pregel matrix and thrombin to obtain a mixed suspension;

2) adding the mixed suspension into the aseptic mold described in the above scheme, solidifying to form cell fibrin hydrogel;

3) adding a proliferation medium to the cell fibrin hydrogel for cell culture;

The pregel matrix is based on DMEM high glucose medium and also contains fibrinogen and antibiotics.

Preferably, the concentration of fibrinogen in the mixed suspension is 8-10 mg/mL.

Preferably, the concentration of muscle stem cells in the mixed suspension is 4×10 ⁶ to 9×10 ⁶ cells/mL; the concentration of thrombin in the mixed suspension is 4 to 5 U/mL.

Preferably, the antibiotics include penicillin G sodium salt and streptomycin sulfate; the concentration of penicillin G sodium salt in the pregel matrix is 100U/mL; the concentration of streptomycin sulfate in the pregel matrix is 0.1 mg/mL.

Preferably, the coagulation conditions include: the temperature is 25-27° C., the volume concentration of CO ₂ is 4%-6%, and the time is 20-60 minutes.

Preferably, the temperature of the cell culture is 25-27°C; the volume concentration of CO ₂ in the cell culture is 4%-6%; the cell culture includes sequential proliferation culture and differentiation culture; the proliferation culture The time for the differentiation culture is 3-5 days; the time for the differentiation culture is 6-8 days.

The present invention also provides the application of the mold described in the above scheme or the method described in the preparation of cell culture meat.

The present invention provides a mold, which includes a groove and needle-shaped components vertically inserted into two ends of the groove. In the present invention, the groove is used to hold the gel; the needle-shaped assembly is used to fix the gel. After the two ends of the gel are fixed, the cells will grow towards the fixed direction under mechanical action, thereby controlling directionality of cell growth. In the present invention, the mold composed of grooves and needle-shaped components can realize the arrangement and growth of cells in three-dimensional culture, wherein the needle-shaped components have a certain anchoring effect, can control the directionality of cell growth, and make the growth and differentiation of muscle stem cells into an orderly arrangement. of myotube tissue. The invention realizes the arrangement, growth and differentiation of fish muscle satellite cells in a three-dimensional environment for the first time, and promotes the development of cell cultured fish meat.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without paying creative labor.

Figure 1 is a comparison of three-dimensional cell culture models in Examples and Comparative Examples, wherein, A: silicone tube model culture, B: round drop culture;

Figure 2 is the immunofluorescence staining diagram after 7 days of differentiation in Example 1, from left to right are desmin immunofluorescence staining diagram, cell nucleus immunofluorescence staining diagram and desmin/nucleus combined diagram;

Figure 3 is the immunofluorescence staining diagram after 7 days of differentiation in Example 2, from left to right are desmin immunofluorescence staining diagram, cell nucleus immunofluorescence staining diagram and desmin/nucleus combined diagram;

Figure 4 is the immunofluorescence staining diagram after 7 days of differentiation in Example 3, from left to right are desmin immunofluorescence staining diagram, cell nucleus immunofluorescence staining diagram and desmin/nucleus combined diagram;

Figure 5 is the immunofluorescence staining diagram after 7 days of differentiation in Comparative Example 1, from left to right are desmin immunofluorescence staining diagram, cell nucleus immunofluorescence staining diagram and desmin/nuclei combination diagram;

Figure 6 is the immunofluorescence staining diagram after 7 days of differentiation in Comparative Example 2, from left to right are desmin immunofluorescence staining diagram, cell nucleus immunofluorescence staining diagram and desmin/nucleus combination diagram;

Fig. 7 is the immunofluorescence staining image after 7 days of differentiation in Comparative Example 3, from left to right is desmin immunofluorescence staining image, cell nucleus immunofluorescence staining image and desmin/nucleus combined image.

Detailed ways

The present invention provides a mold, which includes a groove and needle-shaped components vertically inserted into two ends of the groove.

In the present invention, the material of the groove includes silica gel, and other biosafety grades, materials that can be sterilized, can be installed with fixed brackets, and can contain hydrogel can also replace silica gel; the shape of the groove includes U-shaped ; The shape of the cross section of the groove is preferably a semicircle; The groove is used to hold the gel.

In the present invention, the pitch of the needle-shaped components is preferably 3-9mm; the material of the needle-shaped components is preferably stainless steel or non-metallic material; the non-metallic material preferably includes plastic; the length of the needle-shaped components Preferably 10mm; the diameter of the needle assembly is preferably 0.7mm; the vertical insertion is preferably vertical insertion; the needle assembly is preferably a pin; the needle assembly is vertically inserted into both ends of the groove to form a fixed point , which acts as an anchor and is used to fix the gel. After the two ends of the gel are fixed, the cells will grow in a fixed direction under mechanical action, thereby controlling the directionality of cell growth.

In the present invention, the preparation method of the mold preferably comprises the following steps:

Cut the silicone tube in half lengthwise to get grooves;

Insert pins vertically at both ends of the groove to obtain a mold.

In the present invention, the silicone tube is preferably a food-grade silicone tube; the inner diameter of the silicone tube is preferably 3.2 mm; the outer diameter of the silicone tube is preferably 6.4 mm; the silicone tube is from conventional commercially available sources. During the specific implementation of the present invention, the silicone tube was purchased online at the Runze flagship store.

Before the silicone tube is longitudinally cut into two halves, the present invention preferably also includes cutting the silicone tube transversely into silicone tube sections; the length of the groove is preferably 12-18mm, more preferably 15mm, and this length is for The model is placed in a 24-well plate for culture length. If it is cultured in other well plates, the length will be changed according to the diameter of the well plate.

After obtaining the mould, the present invention preferably also includes sterilizing the mould; the sterilizing method preferably includes placing the mould in an aqueous ethanol solution with a volume concentration of 75% for sterilization; After the sterilization, the present invention preferably also includes cleaning the sterilized mold; the reagent used in the cleaning is preferably sterile PBS; after the cleaning, the present invention preferably also includes cleaning the mold Drying; the drying method is preferably air drying in a sterile environment. In the specific implementation process of the present invention, the sterile environment is preferably a ultra-clean workbench with ultraviolet light turned on.

The present invention also provides a method for arraying and growing muscle stem cells, comprising the following steps:

1) mixing the cell suspension of the muscle stem cells, the pregel matrix and thrombin to obtain a mixed suspension;

2) adding the mixed suspension into the aseptic mold described in the above scheme, solidifying to form cell fibrin hydrogel;

3) adding a proliferation medium to the cell fibrin hydrogel for cell culture;

The pregel matrix is based on DMEM high glucose medium and also contains fibrinogen and antibiotics.

In the invention, firstly, the cell suspension of muscle stem cells, the pregel matrix and thrombin are mixed to obtain a mixed suspension.

In the present invention, the muscle stem cells are preferably muscle stem cells derived from fish; the muscle stem cells derived from fish are preferably fish muscle satellite cells; the fish muscle satellite cells are preferably large yellow croaker muscle satellite cells; The yellow croaker muscle satellite cells are preferably isolated from large yellow croaker muscle tissue; the large yellow croaker muscle tissue is preferably juvenile; the large yellow croaker muscle tissue is preferably muscle tissue isolated from the upper axis of the large yellow croaker muscle. The present invention has no special limitation on the source and isolation method of the large yellow croaker muscle satellite cells.

In the present invention, the mixing of the cell suspension of muscle stem cells, the pre-gel matrix and thrombin preferably includes mixing the cell suspension of muscle stem cells and the pre-gel matrix to obtain a mixture; Thrombin mix.

In the present invention, the concentration of muscle stem cells in the mixed suspension is preferably 4×10 ⁶ -9×10 ⁶ cells/mL. In the present invention, the concentration of fibrinogen in the mixed suspension is preferably 8-10 mg/mL; the concentration of thrombin in the mixed suspension is preferably 4-5 U/mL. In the present invention, the antibiotic preferably includes penicillin G sodium salt and streptomycin sulfate; the concentration of penicillin G sodium salt in the pregel matrix is 100U/mL; The concentration is 0.1mg/mL.

After the mixed suspension is obtained, the present invention adds the mixed suspension into the aseptic mold described in the above scheme, and solidifies to form the cell fibrin hydrogel.

In the present invention, based on the groove length being 15 mm and depth being 1.5 mm, the volume of adding the mixed suspension into the aseptic mold described in the above scheme is preferably 50-80 μL, more preferably 60-70 μL.

In the present invention, the coagulation conditions preferably include: the temperature is 25-27° C., the volume concentration of CO ₂ is 4%-6%, and the time is 20-60 minutes.

The material used in the present invention is fibrin hydrogel, which has excellent biocompatibility and can be used for cell proliferation and differentiation, while collagen hydrogel has the risk of breaking.

After the cell fibrin hydrogel is formed, the present invention adds a proliferation medium to the cell fibrin hydrogel for cell culture.

In the present invention, the temperature of the cell culture is preferably 25-27°C, more preferably 26°C; the volume concentration of _CO2 for the cell culture is preferably 4%-6%, preferably 5%; the cell The culture preferably includes sequential proliferation culture and differentiation culture; the time of the proliferation culture is preferably 3-5 days, more preferably 4 days; the time of the differentiation culture is preferably 6-8 days, more preferably 7 days.

In the present invention, the proliferation medium used in the proliferation culture is based on DMEM high-glucose medium, and preferably also includes components at the following concentrations: fetal bovine serum with a volume concentration of 10%, 10ng/mL human recombinant Basic fibroblast growth factor, 100 U/mL penicillin G sodium salt and 0.1 mg/mL streptomycin sulfate. In the present invention, the differentiation medium used in the differentiation culture is based on DMEM/F12 medium, and preferably also includes the following components at concentrations: horse serum with a volume concentration of 8%, 10ng/mL human recombinant insulin Like growth factor-1, 50nMnecrosulfonamide, 200μM ascorbic acid, 100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate.

The present invention also provides the application of the mold described in the above scheme or the method described in the preparation of cell culture meat. In the present invention, the cell-cultured meat preferably includes cell-cultured fish.

In order to further illustrate the present invention, a mold provided by the present invention and a method for arraying and growing muscle stem cells and their applications are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

The present invention will be further described below in conjunction with specific embodiments. The following examples are only used to illustrate the present invention, and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention belong to the scope of the present invention.

The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

DMEM high glucose medium (BL304A, Biosharp), DMEM/F12 medium (PM150312, Procell); fetal bovine serum (WISENT); horse serum (BL209A, Biosharp); human recombinant basic fibroblast growth factor (P5453-10μg , Biyuntian); human recombinant insulin-like growth factor-1 (P00048, Suo Lai Bao); necrosulfonamide (N872612-5mg, McLean); ascorbic acid (A103534-100g, Aladdin); 100 × antibiotics (P1400, Su Lai Bao );

Other reagents: fibrinogen (BS943-1g, Biosharp); thrombin (BS903-1000u, Biosharp); type IV collagenase (17104019, Gibco);

The fish muscle satellites used in the following examples are large yellow croaker muscle satellite cells. The primary cells of large yellow croaker muscle satellite cells are prepared by conventional methods in the laboratory. The muscle tissue is isolated from the upper axis of the juvenile large yellow croaker muscle, and then digested with 0.1% type IV collagenase solution and 0.1% trypsin solution respectively. The obtained tissues were filtered with 70 μm and 40 μm cell sieves respectively, centrifuged at 300 g for 5 min, and the cell pellet was resuspended with complete medium, and then inoculated in a 6-well plate at a concentration of 1×10 ⁶ cells/mL, and placed in a culture condition of 27 Cultivate in an incubator with 5% _CO2 .

Example 1

First, take an imported food-grade silicone tube with a specification of 3.2*6.4mm (inner diameter*outer diameter), cut it into small sections about 15mm long with scissors, and then cut it in half (longitudinal) with a blade to obtain a grooved tube. The chamber system; stainless steel pins with a length of 10 mm and a diameter of 0.7 mm were inserted into the tube as fixed points, and the distance between the pins was 8 mm. After the preparation is completed, put it into 75% ethanol for sterilization, wash it with sterile PBS after the sterilization, and finally put it into a 24-well plate to dry for use.

Then, under aseptic conditions, the fibrinogen mother solution and the thrombin mother solution were prepared with DMEM high-sugar solution containing 1×antibiotics, the concentrations were 50mg/mL and 200U/mL, respectively, and stored in a refrigerator at 4°C until use.

Fish muscle satellite cells in good growth state were selected, and when the cell confluence reached 80%, trypsin was added for digestion, centrifuged, and the cells were collected and counted. Take 160 μL of fibrinogen mother solution and add 300 μL of DMEM high-glucose medium containing 1× antibiotics (100 U/mL penicillin G sodium salt and 0.1 mg/mL streptomycin sulfate), then add 320 μL of cell suspension, and finally add 20 μL of thrombin mother solution After the addition, pipette up and down with a pipette to mix evenly. The whole system was 800 μL, the final concentration of fibrinogen was 10 mg/mL, the concentration of thrombin was 5 U/mL, and the concentration of cells was 9.0×10 ⁶ cells/mL.

Take 60 μL of the cell-fibrinogen suspension obtained above and add it to the silica gel tube template in a 24-well plate, and place it in a 5% _CO2 cell culture incubator at 27°C for 40 minutes to solidify the fibrinogen. Then add 1000 μL of proliferation medium and put it into the incubator for cultivation. Proliferation culture was performed for 3 days, and the differentiation medium was replaced on the third day, followed by differentiation culture for 7 days, and the medium was replaced every two days. On the seventh day of differentiation culture, immunofluorescent staining was performed on the fibrin hydrogels containing the cells.

The proliferation medium is DMEM high glucose containing 10% fetal bovine serum, 10 ng/mL human recombinant basic fibroblast growth factor, 1× antibiotics (100 U/mL penicillin G sodium salt and 0.1 mg/mL streptomycin sulfate) Medium; Differentiation medium is containing 8% horse serum, 10ng/mL human recombinant insulin-like growth factor-1, 50nMnecrosulfonamide, 200μM ascorbic acid, 1× antibiotics (100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate ) of DMEM/F12 medium.

In this example, the cells cultured using the silicone tube template are shown in A in Figure 1 . The immunofluorescence staining image of cells-fibrin hydrogel on the seventh day of differentiation is shown in Figure 2. Desmin/nucleus staining shows that fish muscle satellite cells have differentiated into myotubes in fibrin hydrogel, and muscle The tubes are arranged in an orderly manner and have a certain directionality.

Example 2

First, take an imported food-grade silicone tube with a specification of 3.2*6.4mm (inner diameter*outer diameter), cut it into small sections about 15mm long with scissors, and then cut it in half (longitudinal) with a blade to obtain a grooved tube. A chamber system; a stainless steel pin with a length of 10 mm and a diameter of 0.7 mm was inserted into the tube as a fixed point, and the distance between the pins was 6 mm. After the preparation is completed, put it into 75% ethanol for sterilization, wash it with sterile PBS after the sterilization, and finally put it into a 24-well plate to dry for use.

Then, under aseptic conditions, the fibrinogen mother solution and the thrombin mother solution were prepared with DMEM/high glucose solution containing 1×antibiotics, the concentrations were 50 mg/mL and 200 U/mL respectively, and they were stored in a refrigerator at 4°C until use.

Fish muscle satellite cells in good growth state were selected, and when the cell confluence reached 80%, trypsin was added for digestion, centrifuged, and the cells were collected and counted. Take 128 μL of fibrinogen mother solution and add 336 μL of DMEM high-glucose medium containing 1×antibiotics, then add 320 μL of cell suspension, and finally add 16 μL of thrombin mother solution. After adding, pipette up and down to mix evenly. The whole system was 800 μL, the final fibrinogen concentration was 8 mg/mL, the thrombin concentration was 4 U/mL, and the cell concentration was 9.0×10 ⁶ cells/mL.

Take 50 μL of the cell-fibrinogen suspension obtained above and add it to the silica gel tube template in a 24-well plate, and place it in a 27° C., 5% CO ₂ cell culture incubator for 40 minutes to solidify the fibrinogen. Then add 1000 μL of proliferation medium and put it into the incubator for cultivation. Proliferation culture was performed for 3 days, and the differentiation medium was replaced on the third day, followed by differentiation culture for 7 days, and the medium was replaced every two days. On the seventh day of differentiation culture, immunofluorescent staining was performed on the fibrin hydrogels containing the cells.

The proliferation medium is DMEM high glucose containing 10% fetal bovine serum, 10 ng/mL human recombinant basic fibroblast growth factor, 1× antibiotics (100 U/mL penicillin G sodium salt and 0.1 mg/mL streptomycin sulfate) Medium; Differentiation medium is containing 8% horse serum, 10ng/mL human recombinant insulin-like growth factor-1, 50nMnecrosulfonamide, 200μM ascorbic acid, 1× antibiotics (100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate ) of DMEM/F12 medium.

In this example, the immunofluorescent staining of the cell-fibrin hydrogel on the seventh day of differentiation is shown in Figure 3. The desmin/nucleus staining shows that the fish muscle satellite cells are differentiated into fibrin hydrogel and arranged neatly. of myotubes.

Example 3

First, take an imported food-grade silicone tube with a specification of 3.2*6.4mm (inner diameter*outer diameter), cut it into small sections about 15mm long with scissors, and then cut it in half (longitudinal) with a blade to obtain a grooved tube. A chamber system; stainless steel pins with a length of 10 mm and a diameter of 0.7 mm were inserted into the tube as fixed points, and the distance between the pins was 4 mm. After the preparation is completed, put it into 75% ethanol for sterilization, wash it with sterile PBS after the sterilization, and finally put it into a 24-well plate to dry for use.

Then, under aseptic conditions, the fibrinogen mother solution and the thrombin mother solution were prepared with DMEM/high glucose solution containing 1×antibiotics, the concentrations were 50 mg/mL and 200 U/mL respectively, and they were stored in a refrigerator at 4°C until use.

Fish muscle satellite cells in good growth state were selected, and when the cell confluence reached 80%, trypsin was added for digestion, centrifuged, and the cells were collected and counted. Take 160 μL of fibrinogen mother solution and add 470 μL of DMEM high-glucose medium containing 1× antibiotics, then add 150 μL of cell suspension, and finally add 20 μL of thrombin mother solution. After the addition, pipette up and down to mix evenly. The whole system was 800 μL, the final concentration of fibrinogen was 10 mg/mL, the concentration of thrombin was 5 U/mL, and the concentration of cells was 4.0×10 ⁶ cells/mL.

Take 50 μL of the cell-fibrinogen suspension obtained above and add it to the silica gel tube template in a 24-well plate, and place it in a 27° C., 5% CO ₂ cell culture incubator for 40 minutes to solidify the fibrinogen. Then add 1000 μL of proliferation medium and put it into the incubator for cultivation. Proliferation culture was performed for 3 days, and the differentiation medium was replaced on the third day, followed by differentiation culture for 7 days, and the medium was replaced every two days. On the seventh day of differentiation culture, immunofluorescent staining was performed on the fibrin hydrogels containing the cells.

The proliferation medium is DMEM high glucose containing 10% fetal bovine serum, 10 ng/mL human recombinant basic fibroblast growth factor, 1× antibiotics (100 U/mL penicillin G sodium salt and 0.1 mg/mL streptomycin sulfate) Medium; Differentiation medium is containing 8% horse serum, 10ng/mL human recombinant insulin-like growth factor-1, 50nMnecrosulfonamide, 200μM ascorbic acid, 1× antibiotics (100U/mL penicillin G sodium salt and 0.1mg/mL streptomycin sulfate ) of DMEM/F12 medium.

In this example, the immunofluorescent staining of the cell-fibrin hydrogel on the seventh day of differentiation is shown in Figure 4. The desmin/nucleus staining shows that the fish muscle satellite cells are differentiated into a neat arrangement in the fibrin hydrogel of myotubes.

Comparative example 1

This comparative example is similar to that in Example 1, the difference being that instead of using a silica gel model for culture, 30 μL of the cell-fibrinogen suspension in Example 1 was added dropwise into a 48-well plate for subsequent culture.

The drop-shaped hydrogel-embedded cells cultured in this comparative example are shown in B in Figure 1 . The immunofluorescence staining image of cells-fibrin hydrogel on the seventh day of differentiation is shown in Figure 5. Desmin/nucleus staining shows that fish muscle satellite cells have differentiated into myotubes in fibrin hydrogel, but muscle The tubes are anisotropic and distributed randomly. This is quite different from the myotubes formed by cells cultured using silica models. Therefore, the method adopted in the present invention can well form myotubes arranged in the same direction, which is closer to the myotube tissue of real meat.

Comparative example 2

This comparative example is similar to that in Example 1, with two differences. The first is that the silica gel model is not used for culture, but 30 μL of cell-fibrinogen suspension is added to a 48-well plate in a drop form for subsequent culture. ; The second is that the cell concentration in the cell-fibrinogen suspension is 7.0×10 ⁶ cells/mL.

The immunofluorescence staining image of the cell-fibrin hydrogel on the seventh day of differentiation in this comparative example is shown in FIG. 6 . Although the cell concentration was changed, the myotubes formed by the cells were still arranged in disorder.

Comparative example 3

This comparative example is similar to that in Example 2, except that the cell concentration in the cell-fibrinogen suspension is 3.0×10 ⁶ cells/mL.

The immunofluorescence staining image of the cell-fibrin hydrogel on the seventh day of differentiation in this comparative example is shown in FIG. 7 . When the cell concentration is reduced within the preferred range, although myotubes can also be formed, compared with Example 2, the number of multinucleated myotubes formed is small, the length is short, and the arrangement effect is not good.

The above is a further detailed description of the present invention, and should not be regarded as a limitation to the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims (10)

1. A mold, characterized in that it comprises a groove and a needle assembly vertically inserted into two ends of the groove. 2. The mold according to claim 1, wherein the material of the groove comprises silica gel; the shape of the groove comprises a U shape. 3 . The mold according to claim 1 , wherein the needle-shaped components comprise pins; and the distance between the needle-shaped components is 3-9 mm. 4 . 4. A method for the growth of muscle stem cells, comprising the following steps: 1) mixing the cell suspension of the muscle stem cells, the pregel matrix and thrombin to obtain a mixed suspension; 2) adding the mixed suspension into the aseptic mold according to any one of claims 1 to 3, solidifying to form cell fibrin hydrogel; 3) adding a proliferation medium to the cell fibrin hydrogel for cell culture; The pregel matrix is based on DMEM high glucose medium and also contains fibrinogen and antibiotics. 5. The method according to claim 4, characterized in that the concentration of fibrinogen in the mixed suspension is 8-10 mg/mL. 6. The method according to claim 4 or 5, characterized in that the concentration of muscle stem cells in the mixed suspension is 4×10 ⁶ to 9×10 ⁶ cells/mL; the thrombin in the mixed suspension The concentration is 4~5U/mL. 7. method according to claim 4, is characterized in that, described antibiotic comprises penicillin G sodium salt and streptomycin sulfate; The concentration of penicillin G sodium salt is 100U/mL in the described pregel matrix; The concentration of streptomycin sulfate in the gel matrix was 0.1 mg/mL. 8 . The method according to claim 4 , wherein the coagulation conditions include: the temperature is 25-27° C., the volume concentration of CO ₂ is 4%-6%, and the time is 20-60 minutes. 9. method according to claim 4 is characterized in that, the temperature of described cell culture is 25～27 ℃; The volume concentration of the _CO of described cell culture is 4%～6%; Described cell culture comprises successively Proliferation culture and differentiation culture are carried out; the time of the proliferation culture is 3-5 days; the time of the differentiation culture is 6-8 days. 10. Application of the mold according to any one of claims 1 to 3 or the method according to any one of claims 4 to 9 in the preparation of cell culture meat.