Method for establishing pig skeletal muscle satellite cell and pig precursor fat cell co-culture system

Abstract

The invention discloses a method for establishing a co-culture system of porcine skeletal muscle satellite cells (PSCs) and porcine precursor fat cells (ADSCs). The method comprises the following steps: selecting a PSCs and ADSCs co-culture proliferation culture medium and a co-culture differentiation culture medium, and establishing an optimal cell co-culture ratio. According to the optimal co-culture proliferation and differentiation culture medium for the PSCs and the ADSCs and the established co-culture proportion, mass proliferation and efficient differentiation of cells can be promoted, the influence of addition of fat cells with different concentrations on muscle cell differentiation can be researched, fat deposition in co-culture is regulated by regulating the proportion of the fat cells, and the differentiation of the muscle cells is promoted. And an earlier-stage foundation is provided for explaining a signal mechanism theoretical basis between cells under growth and development conditions of muscle and adipose tissues and exploring cell culture meat in a later stage.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to a method for establishing a co-culture system of porcine skeletal muscle satellite cells (PSCs) and porcine preadipocytes (ADSCs). Background Art

[0002] As the global population increases and the demand for animal protein increases, traditional animal husbandry faces many sustainability and ethical issues (Santos et al. 2023). Artificial meat technology uses cellular agriculture technology to produce meat, which requires fewer resources such as land, water, and feed, while producing lower levels of greenhouse gas emissions and water pollution (Tilman et al. 2017, Jara et al. 2023), without the need to raise and slaughter animals, providing a sustainable and ethically viable alternative (Porter et al. 2019).

[0003] Texture is a critical consideration in the research and development of artificial meat. Intramuscular fat (IMF) content is a key factor in determining muscle quality characteristics. In order to produce artificial marbled meat with ideal texture and taste, it is crucial to develop methods that can effectively integrate engineered adipose tissue into muscle tissue (Louis et al. 2023). Previous studies have shown that co-culture of myoblasts and adipocytes can more accurately simulate the in vivo environment and enhance the study of cell interactions. Myoblasts and adipocytes have different nutritional requirements for optimal growth, which highlights the need to tailor specific culture conditions for each. Cell differentiation is affected by multiple factors, and cells from different sources exhibit different differentiation patterns. The unique induction differentiation culture media of myoblasts and adipocytes pose challenges to studying the differentiation mechanisms of these cells (Kuppusamy et al. 2020, Cheng et al. 2023), (Song et al. 2023). Myoblasts show higher differentiation efficiency in serum-free or low-serum medium, while adipocytes generally require high-serum medium and may benefit from the addition of fatty acids for optimal differentiation (Stout et al. 2022, Cheng et al. 2023). Currently, there is no recognized culture medium that can induce synchronous differentiation of these two cell types. Therefore, it is urgent to establish a co-culture protocol that can accurately control the progression and key stages of cell differentiation.

[0004] Additionally, the protocol should focus on maintaining a balanced ratio of myoblasts to adipocytes during co-culture to achieve successful co-differentiation of these cell types. Some studies have attempted to supplement muscle tissue with adipose tissue and increase the production of proteins such as myoglobin, myosin, and actin, which contribute to the textural properties and flavor of traditional meat. However, achieving cultured meat with a texture and mouthfeel comparable to pork remains a long-term goal. (Panduranganand Kim2015)

[0005] Therefore, establishing a co-culture protocol to maintain the proliferation and differentiation of myoblasts and adipocytes and detecting the balanced ratio of myoblasts and adipocytes during co-culture to achieve successful co-differentiation of these cell types are crucial to understand the mutual influence during muscle and adipose tissue generation, ultimately promoting the advancement of meat quality and sensory attributes in cultured meat production. Summary of the invention

[0006] The purpose of the present invention is to select a co-culture proliferation medium and a co-culture differentiation medium for PSCs and ADSCs and to establish an optimal cell co-culture ratio, and to provide a method for establishing a co-culture system of porcine skeletal muscle satellite cells (PSCs) and porcine preadipocytes (ADSCs).

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0008] A method for establishing a co-culture system of porcine skeletal muscle satellite cells (PSCs) and porcine preadipocytes (ADSCs) comprises the following steps:

[0009] 1) Isolation and purification of PSCs seed cells: PSCs cells were isolated from healthy piglets aged 3-7 days. The isolated PSCs cells were cultured in DMEM medium containing 20% ​​fetal bovine serum (FBS) for 3-6 days; at this time, the growth density of PSCs reached 65%-80%, and flow sorting technology was used to purify PSCs using CD31, CD45, CD56, and CD29 antibodies to obtain F-PSCs cells; they were cultured in DMEM medium containing 20% ​​FBS for 3-6 days, and when the growth density of F-PSCs reached 70%-80%, they were digested with trypsin and transferred to a T25 cell culture flask for use.

[0010] 2) Isolation and culture of ADSCs seed cells: ADSCs cells were isolated from healthy piglets aged 3-7 days, and the obtained ADSCs cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 3-6 days. When the growth density of ADSCs was expected to reach 75%-85%, they were digested with trypsin and transferred to a T25 cell culture flask for use.

[0011] 3) Preparation of co-culture differentiation medium: The co-culture differentiation medium is divided into solution A and solution B, which are used successively; wherein, solution A is added with 10mM / mL dexamethasone (DEX) + 10mM / mL rosiglitazone + 1mg / mL insulin

[0012] Solution B is DMEM medium containing FBS or HS supplemented with 10 mM / mL 3-isobutyl-1-methylxanthine (IBMX); Solution B is DMEM medium containing FBS or HS supplemented with 1 mg / mL insulin.

[0013] 4) Inoculation and culture: F-PSCs and ADSCs are inoculated at a cell ratio of 9:1 to 1:9, and cultured in the co-culture differentiation medium A solution prepared in step 3) for 3 to 5 days, and then the solution is discarded; solution B is added to continue culturing for 1 to 2 days, and 3 to 5 cycles are repeated to complete the induction of myogenic differentiation of F-PSCs and adipogenic differentiation of ADSCs. Preferably, in the method for establishing a co-culture system of PSCs and ADSCs, the optimal co-culture differentiation medium components include: co-culture differentiation medium A solution: 5% HS DMEM medium + 10mM / mL dexamethasone (DEX) + 10mM / mL rosiglitazone + 1mg / mL insulin + 10mM / mL 3-isobutyl-1-methylxanthine (IBMX); co-culture differentiation medium B solution: 5% HS + 1mg / mL insulin DMEM medium (DM2).

[0014] Preferably, in the method for establishing a co-culture system of PSCs and ADSCs, the optimal ratio of the number of PSCs and ADSCs in co-culture is 5:1 to 2:1.

[0015] Preferably, in the method for establishing a co-culture system of PSCs and ADSCs, the optimal ratio of the number of PSCs and ADSCs in co-culture is 7:3.

[0016] The beneficial effects of the present invention are:

[0017] The selected PSCs and ADSCs co-culture proliferation medium and co-culture differentiation medium and the optimal cell co-culture cell ratio established by the present invention enable PSCs and ADSCs to proliferate and differentiate simultaneously in in vitro culture, especially when the cell number ratio of F-PSCs to ADSCs in the cell population is 7:3 under 2D cell culture conditions, the co-culture model has a large amount of muscle generation and fat deposition. It is very important to understand the mutual influence in the process of muscle and adipose tissue generation, clarify the signal mechanism between cells under the conditions of muscle and adipose tissue growth and development, and promote the progress of meat quality and sensory attributes in cultured meat production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1is a line graph showing the ratio of Pax7+ cells after flow cytometry sorting of PSCs and F-PSCs at different passage times in Example 1, and the statistical time is when the passage times of PSCs and F-PSCs are the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, and 9th generations;

[0019] Figure 2 After the 3rd, 6th and 9th generation F-PSCs were sorted by flow cytometry in Example 1, Pax7 + The cell immunofluorescence images of F-PSCs were collected at the 3rd, 6th, and 9th passages of F-PSCs;

[0020] Figure 3 From left to right are MYHC in Example 1 + The cell immunofluorescence images, the bar graphs of cell differentiation index and myotube fusion index were collected at the 3rd, 6th and 9th passages of F-PSCs;

[0021] Figure 4 From left to right are the bright field images and Oil Red O staining images of ADSCs-induced adipogenic differentiation in Example 1, and the images were collected on the 4th, 8th and 12th days of cell differentiation;

[0022] Figure 5 The middle left picture is the DiO and DIL staining pictures of ADSCs and F-PSCs co-cultured in 4 ratios under 5 kinds of proliferation culture media in Example 2, and the upper and lower right pictures are the bar chart comparison of the counting analysis of F-PSCs and ADSCs, respectively, collected at the 0th day and the 8th day after inoculation;

[0023] Figure 6 From left to right, the cell proliferation rate analysis of F-PSCs in 4 co-culture proliferation medium conditions and 1 empty DMEM medium with cell number ratios of F-PSCs and ADSCs (10:1, 5:1, 2:1, 1:2);

[0024] Figure 7 The left picture shows the DiO and DIL staining of F-PSCs and ADSCs under 4 co-culture proliferation medium and 1 empty DMEM medium according to 6 cell number ratios of ADSCs and F-PSCs; the upper and lower right sides show the cell proliferation rate analysis of the corresponding co-cultured F-PSCs and ADSCs;

[0025] Figure 8 It is a bright field image of the ratio of the number of F-PSCs and ADSCs in four co-culture differentiation media, and the acquisition time is 12 days of cell co-culture differentiation;

[0026] Fig. 9 The upper and lower rows are the cell immunofluorescence images and Oil Red O staining detection images of MyHC protein expression of F-PSCs and ADSCs at four co-culture cell number ratios in the optimal co-culture differentiation medium, respectively. The collection time is 12 days of cell co-culture differentiation;

[0027] Fig.10 The immunofluorescence images of MYHC protein expression cells and the Oil Red O staining detection images of lipid droplets at 11 different ratios of F-PSCs and ADSCs co-cultured cells. The acquisition time was 4 days (left) and 8 days (right) of cell co-culture differentiation.

[0028] Fig.11 This is a bar chart of lipid droplet size and lipid area percentage of ADSCs under 11 ratios of F-PSCs and ADSCs co-cultured cells, and the statistical time is the 8th day;

[0029] Fig.12 This is a bar chart of the MYHC+ nuclear ratio and myotube diameter of F-PSCs under 11 F-PSCs and ADSCs co-culture cell number ratios, and the statistical time is the 4th day;

[0030] Fig.13 It is a bar chart of the MYHC+ nuclear ratio and myotube diameter of F-PSCs under 11 different cell number ratios of F-PSCs and ADSCs co-culture, and the statistical time is the 4th day. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further explained below in conjunction with examples.

[0032] Trypsin-EDTA (0.25%) (Gibco, #25200114)

[0033] Collagenase type I (Gibco, #17018029)

[0034] DMEM medium (Gibco, #12491015)

[0035] Horse serum (HS, Gibco, #16050122)

[0036] Fetal bovine serum (FBS, Gibco, #A5670701)

[0037] PBS (Gibco, #10010023)

[0038] Double antibody (Gibco, #15070063)

[0039] Dexamethasone (DEX, MCE, #HY-14648G)

[0040] Rosiglitazone (MCE, #HY-17386)

[0041] Insulin (Insulin, MCE, #HY-P0035)

[0042] Phosphodiesterase inhibitor (IBMX, MCE, #HY-12318)

[0043] Oil Red O powder (sigma, #O0625)

[0044] 4% paraformaldehyde solution (Servicebio, #G1101-500ML)

[0045] Myosin heavy chain primary antibody (MyHC, Santa Cruz, #sc-376157)

[0046] Pax7 primary antibody (abcam, #ab187339)

[0047] Cell Counting Kit-8 (CCK-8 kit, Beyotime, #C0037)

[0048] Dil (cell membrane red fluorescent probe, Beyotime, #C1036)

[0049] Dio (cell membrane green fluorescent probe, Beyotime, #C1038)

[0050] Cy3 labeled goat anti-mouse IgG (Servicebio, #GB21401)

[0051] Example 1: Acquisition of F-PSCs and ADSCs Seed Cells

[0052] To obtain PSCs, the piglets were bled and the skin surface was cleaned and disinfected. The two longissimus muscles and leg muscles were extracted from the piglets, and the excess fascia, blood vessels and other tissues were removed. The muscles were washed with 1xPBS containing 2% penicillin-streptomycin (PS). The muscle tissue was cut into pieces with surgical scissors and 1x PBS solution was added to dilute the muscle. Centrifuged at 2000rpm for 5min and the supernatant was discarded. 2mg / mL type I collagenase was added to the precipitate and incubated at 37℃ for about 2 hours. After digestion, the cells were transferred to DMEM medium supplemented with 10% FBS and filtered through 100μm, 70μm and 40μm cell filters in turn. After differential centrifugation of the filtered precipitate for 2h, the cells were resuspended in DMEM medium containing 20% ​​FBS and transferred to a culture plate covered with BD matrix gel (Biozellen).

[0053] The cells were cultured with a new medium every 2 to 3 days, and were passaged to the third generation when the confluence reached 60%, and cell sorting was performed by flow cytometry. During sorting, the cells were suspended in FACS buffer, CD29, CD56, CD31, and CD45 antibodies were added, and incubated on ice for 30-45 minutes. After antibody incubation, the cells were washed twice with cold PBS 1x and flow sorted to obtain F-PSCs cells.

[0054] During the passaging process, the proportion of PSCs expressing the Pax7 marker gene in the original generation decreased significantly, from 78% to 1.2%. Therefore, flow cytometry sorting used surface marker antibodies CD31-CD45-CD56+CD29+ to separate a high-purity PSCs population and obtain F-PSCs. After sorting, the Pax7+ cell population was enriched to 97%, and the proportion of Pax7+ cells in subsequent passaging remained above 70%. In order to evaluate the differentiation potential of F-PSCs, we induced differentiation of cells at passages 3, 6, and 9. The expression of myosin heavy chain (MyHC) was used as a marker of myofiber formation ability. The results showed that the stemness of F-PSCs was maintained after flow cytometry sorting and serial passaging. Therefore, in subsequent experiments, F-PSCs purified by flow cytometry were used as seed cells for co-culture.

[0055] In order to obtain ADSCs, healthy piglets of 7 days old were killed, the skin surface was cleaned and disinfected, the back fat and abdominal fat of the pigs were surgically cut off, and the excess fascia and other excess tissues were picked out. The fat was cut off, washed with lx PBS solution (2000g) for 5min, and the upper floating fat was removed with 2mg / mL type I collagenase at 37°C for 2h. After digestion, DMEM medium supplemented with 10% FBS and 2% PS was added to terminate the digestion and centrifuged at 2000g for 5min. The cell precipitate was filtered through 100μm, 70μm and 40μm cell filters successively. After differential centrifugation for 2h after filtration, the precipitate was inoculated in a cell culture dish. When its growth density was 75% to 85%, the "cocktail" adipogenic induction method was used to induce adipogenic differentiation of ADSCs. Preparation of the "cocktail" induction method The components of the adipogenic differentiation induction medium include: A solution: 10% FBS DMEM medium + 10mM / mL dexamethasone (DEX) + 10mM / mL rosiglitazone + 1mg / mL insulin + 10mM / mL 3-isobutyl-1-methylxanthine (IBMX); B solution: 10% FBS + 1mg / mL insulin DMEM medium, according to the rule of culturing A solution for 3 days and B solution for 1 day, repeat three times. The induced differentiated cells were stained with Oil Red O, and the Oil Red O powder was dissolved in isopropanol to prepare Oil Red O concentrated solution, and the concentrated solution: H2O = 3:2 was prepared into Oil Red O dilution solution, and after filtering with filter paper, it was prepared into Oil Red O standard solution. After ADSCs were fixed with 4% paraformaldehyde solution for 30 minutes, Oil Red O standard solution was added, stained for 15 minutes, and finally washed with 60% isopropanol for 15 seconds, and observed with a fluorescent inverted microscope.

[0056] The ADSCs obtained produced a large amount of lipid droplets on the 12th day of induced differentiation. Therefore, in subsequent experiments, the ADSCs cultured to a growth density of 75% to 85% after the above separation were used as seed cells for co-culture.

[0057] Figure 1 It is a line chart showing the proportion of Pax7+ cells after F-PSCs of different passage times were sorted by flow cytometry in Example 1. During the passage process, the proportion of F-PSCs expressing the Pax7 marker gene in the original generation decreased significantly from 78% to 1.2%. The statistical time is the passage times of F-PSCs: 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, and 9th generations; Figure 2 After the 3rd, 6th and 9th generation F-PSCs were sorted by flow cytometry in Example 1, Pax7 +Immunofluorescence images of F-PSCs. After sorting, the Pax7+ cell population was enriched to 97%, and the proportion of Pax7+ cells in subsequent passages remained above 70%. The images were collected when F-PSCs were passaged 3, 6, and 9; Figure 3 MYHC in Example 1 + The cell immunofluorescence images of the cells and the bar graphs comparing the cell differentiation index and the myotube fusion index showed that after flow cytometry sorting and serial passaging, the differentiation ability of F-PSCs was maintained and they were able to successfully differentiate and fuse into myotubes. The collection time was the 3rd, 6th, and 9th passages of F-PSCs. Figure 4 These are the bright field images and Oil Red O staining images of ADSCs-induced adipogenic differentiation in Example 1. A large number of lipid droplets are produced on the 12th day of differentiation, and the images were collected on the 4th, 8th and 12th days of cell differentiation.

[0058] Example 2: Selection of proliferation medium for co-culture of F-PSCs and ADSCs

[0059] To explore the optimal co-culture proliferation conditions for porcine skeletal muscle satellite cells and porcine adipose pre-stem cells. The study design included a control group using DMEM and experimental groups using different growth medium (GM) combinations: GM1 with 5% FBS, GM2 with 10% FBS, GM3 with 15% FBS, and GM4 with 20% FBS. Six different mixed cell number ratios of F-PSCs and ADSCs (10:0, 10:1, 5:1, 2:1, 1:2, 0:10) were initially established to determine the optimal co-culture cell number ratio. The goal of this optimal ratio is to simulate the in vivo environment and promote effective interactions between the two cell types. In order to distinguish the two cell types, F-PSCs were stained with green fluorescent DiO and ADSCs were stained with red fluorescent DiI. The results showed that the proliferation activity of F-PSCs was significantly enhanced with the increase of all GM serum concentrations. At GM4, the proliferation activity of F-PSCs was the strongest, and at GM3, the proliferation activity of ADSCs was the strongest, that is, adipocytes require less serum. In the co-culture system, regardless of the cell ratio and GM, the proliferation rate of F-PSCs was significantly inhibited compared with single culture. The proliferation rate of ADSCs in co-culture was gradually enhanced compared with single culture. This phenomenon was particularly evident at high serum concentrations (GM4). When the cell number ratio of F-PSCs to ADSCs reached 2:1 or 1:2, the proliferation rate of ADSCs increased significantly. At this time, ADSCs became the dominant cells in the co-culture process, which should be avoided because it may lead to excessive proliferation of one cell type. Therefore, GM3 was selected as the most suitable co-culture medium because it can promote the synchronous growth of both cell types.

[0060] Figure 5 The DiO and DIL staining images of ADSCs and F-PSCs co-cultured in 5 proliferation culture media at 4 cell number ratios (10:1, 5:1, 2:1, 1:2) in Example 2, as well as the counting analysis of F-PSCs and ADSCs, the cell number of F-PSCs is the largest at GM4, and the cell number of ADSCs is the largest at GM3. The collection time is day 0 and day 8 after inoculation; Figure 6 is the cell proliferation rate analysis of F-PSCs and ADSCs under 4 co-culture proliferation medium conditions and 1 empty DMEM medium at 4 cell number ratios (10:1, 5:1, 2:1, 1:2). In the co-culture system, regardless of the cell ratio and GM, the proliferation rate of F-PSCs was significantly inhibited compared with single culture; Figure 7 The cell proliferation rate of ADSCs co-cultured with F-PSCs and ADSCs in 4 co-culture proliferation culture media and 1 empty DMEM culture medium at 6 cell number ratios (10:0, 10:1, 5:1, 2:1, 1:2, 0:10) was analyzed. When the cell number ratio of F-PSCs to ADSCs reached 2:1 or 1:2, the proliferation rate of ADSCs was significantly improved.

[0061] Example 3: Selection of differentiation medium for co-culture of F-PSCs and ADSCs

[0062] In order to explore the optimal co-culture differentiation conditions of porcine skeletal muscle satellite cells and porcine pre-adipocyte stem cells. Four co-culture differentiation media (DM1-DM4) were established in this study. The four co-culture differentiation media were composed of A solution and B solution used successively. The four A solutions were: DMEM medium containing 2% HS (A1), DMEM medium containing 5% HS (A2), DMEM medium containing 2% FBS (A3) and DMEM medium containing 5% FBS (A4) added with 10mM / mL dexamethasone (DEX) + 10mM / mL rosiglitazone + 1mg / mL insulin + 10mM / mL 3-isobutyl-1-methylxanthine (IBMX); the four B solutions were: DMEM medium containing 2% HS (B1), DMEM medium containing 5% HS (B2), DMEM medium containing 2% FBS (B3) and DMEM medium containing 5% FBS (B4) added with 1mg / mL insulin. In order to explore the ability of 4 co-culture differentiation media (DM1-DM4) to induce co-differentiation. This example preliminarily established four co-culture ratios of F-PSCs and ADSCs, and the ratio of cultured cells was F-PSCs:ADSCs = 10:1, 5:1, 2:1, 1:2. The culture method is: after 3 days of culture in liquid A, the liquid is discarded, and liquid B is added to continue culture for 1 day, and repeated 3 times; the fusion of F-PSCs and the number of lipid droplets produced by ADSCs are observed every 4 days.

[0063] In DM1 (2% HS), a large number of myotubes were observed with very low lipid droplets. In contrast, a large number of myotubes and lipid droplets appeared in DM2 (5% HS). As the serum concentration increased, the lipid droplet levels increased in DM3 (2% FBS) and DM4 (5% FBS), resulting in a decrease in the number and complexity of myotubes. Therefore, DM2 was selected as the most suitable co-culture differentiation medium because it can promote the differentiation of both cell types.

[0064] At the final stage of differentiation, bright field microscopy showed mature adipocytes surrounded by myotubes. Immunofluorescence staining and Oil Red staining showed that the cells were co-cultured and differentiated at cell number ratios of 10:1, 5:1, 2:1, and 1:2. Notably, myotubes were significantly formed in the 10:1 cell number ratio system, despite the absence of lipid droplets. In contrast, myotube formation was clearly observed in the co-culture systems with cell number ratios of 5:1 and 2:1, while it was less obvious in the co-culture system with a cell number ratio of 1:2. In addition, the co-culture systems with cell number ratios of 5:1 and 2:1 showed better cell viability and differentiation ability.

[0065] Figure 8It is the bright field image of F-PSCs and ADSCs in 6 co-culture ratios under 4 co-culture differentiation media, and the acquisition time is 12 days of cell co-culture differentiation; Fig. 9 These are the cell immunofluorescence images and Oil Red O staining detection images of MyHC protein expression in four co-culture ratios of F-PSCs and ADSCs in the optimal co-culture differentiation medium. The collection time was 12 days of cell co-culture differentiation.

[0066] Example 4: Establishment of the co-culture ratio of F-PSCs and ADSCs

[0067] In order to further explore the co-culture ratio of F-PSCs and ADSCs. The co-culture ratio of F-PSCs and ADSCs was refined, and F-PSCs and ADSCs were cultured for 4 days and 8 days respectively with different ratios, and the culture ratio was 10:0 to 0:10 (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10). After using the DM2 co-culture differentiation medium and the induced co-culture differentiation method in Example 3 (one cycle) for 4 days and (two cycles) for 8 days, the expression of MYHC protein and the distribution ratio of lipid droplets were detected using cell immunofluorescence technology and Oil Red O technology. During the co-culture process, the overall trend is that as the proportion of PSCs cells gradually decreases, myoblast fusion and myotube maturation are significantly reduced. When the ratio of PSCs to ADSCs co-cultured cells was 7:3, the proportion of MYHC+ positive cells was not significantly different from that of 100%, 90%, 80%, and 60% F-PSCs at 4 and 8 days of co-culture differentiation, and the proportion was significantly higher than that of other cell ratios; at 4 and 8 days of co-culture differentiation, the degree of fusion of myotubes was significantly different from that of 100% F-PSCs, but not significantly different from that of 80% and 90% F-PSCs, and significantly higher than that of F-PSCs added at other cell ratios. On the 8th day of co-culture differentiation, the distribution area of ​​lipid droplets and the relative size of lipid droplets were significantly lower than those of 70%, 80%, 90%, and 100% ADSCs, and significantly higher than those of 0% ADSCs compared with other cell ratios, and not significantly different from those of other cell ratios. Therefore, the co-culture ratio of F-PSCs to ADSCs = 7:3 was selected as the most suitable co-culture medium because it is the best choice to promote the differentiation of both cell types.

[0068] Fig.10 The immunofluorescence images of MYHC protein expression cells and the Oil Red O staining detection images of lipid droplets at 11 co-culture ratios of F-PSCs and ADSCs were collected at 4 and 8 days of cell co-culture differentiation. Fig.11This is a bar chart of lipid droplet size and lipid area percentage of ADSCs under 11 ratios of F-PSCs and ADSCs co-cultured cells, and the statistical time is the 8th day; Fig.12 This is a bar chart of the MYHC+ nuclear ratio and myotube diameter of F-PSCs under 11 F-PSCs and ADSCs co-culture cell number ratios, and the statistical time is the 4th day; Fig.13 It is a bar chart of the MYHC+ nuclear ratio and myotube diameter of F-PSCs under 11 different cell number ratios of F-PSCs and ADSCs co-culture, and the statistical time is the 4th day.

[0069] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians 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

1. A method for establishing a co-culture system of porcine skeletal muscle satellite cells and porcine preadipocytes, characterized in that: The following steps are involved: 1) Isolation and purification of porcine skeletal muscle satellite PSCs seed cells: PSCs cells were isolated from healthy piglets aged 3-7 days; the isolated PSCs cells were cultured in DMEM medium containing 10% FBS for 3-6 days; at this time, the growth density of PSCs reached 65%-80%, and flow sorting technology was used to purify PSCs using CD31, CD45, CD56, and CD29 antibodies to obtain F-PSCs cells; Culture the cells in DMEM medium containing 20% ​​FBS for 3 to 6 days. When the growth density of F-PSCs reaches 70% to 80%, digest them with trypsin and transfer them to a T25 cell culture flask for later use. 2) Isolation and culture of porcine preadipocyte ADSCs seed cells: ADSCs cells were isolated from healthy piglets aged 3-7 days, and the obtained ADSCs cells were cultured in DMEM medium containing 10% FBS for 3-6 days. When the growth density of ADSCs was expected to reach 75%-85%, they were digested with trypsin and transferred to T25 cell culture flasks for later use; 3) Preparation of co-culture differentiation medium: The co-culture differentiation medium is divided into solution A and solution B, which are used successively; wherein solution A is a DMEM medium containing 2% to 5% FBS or HS supplemented with 10 mM / mL dexamethasone + 10 mM / mL rosiglitazone + 1 mg / mL insulin + 10 mM / mL 3-isobutyl-1-methylxanthine; solution B is a DMEM medium containing 2% to 5% FBS or HS supplemented with 1 mg / mL insulin; 4) Inoculation and culture: F-PSCs and ADSCs are inoculated at a cell ratio of 9:1 to 1:9, respectively, and cultured in the co-culture differentiation medium A solution prepared in step 3) for 3 to 5 days, then the solution is discarded; solution B is added and cultured for 1 to 2 days, and 3 to 5 cycles are repeated to complete the induction of myogenic differentiation of F-PSCs and adipogenic differentiation of ADSCs.

2. The method for establishing a co-culture system of porcine skeletal muscle satellite cells and porcine preadipocytes according to claim 1, characterized in that: Step 3) The co-culture differentiation medium components include: co-culture differentiation medium A solution is: DMEM medium containing 5% HS + 10mM / mL dexamethasone + 10mM / mL rosiglitazone + 1mg / mL insulin + 10mM / mL 3-isobutyl-1-methylxanthine; co-culture differentiation medium B solution is: DMEM medium containing 5% HS + 1mg / mL insulin.

3. The method for establishing a co-culture system of porcine skeletal muscle satellite cells and porcine preadipocytes according to claim 1, characterized in that: In step 4), the ratio of the number of F-PSCs and ADSCs co-cultured cells is 10:1 to 1:

2.

4. The method for establishing a co-culture system of porcine skeletal muscle satellite cells and porcine preadipocytes according to claim 1, characterized in that: Step 4) The ratio of the number of F-PSCs and ADSCs co-cultured cells is 5:1 to 2:

1.

5. The method for establishing a co-culture system of porcine skeletal muscle satellite cells and porcine preadipocytes according to claim 1, characterized in that: Step 4) The ratio of the number of F-PSCs and ADSCs co-cultured cells is 7:3.

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