KR102563045B1 - Method for proliferating or cultivating muscle satellite cells for production of cultured meat - Google Patents

Abstract

The present invention relates to a method for mass-proliferating or culturing muscle stem cells for the production of cultured meat, and more particularly, to a method for mass-proliferating or culturing muscle stem cells through regulation of intracellular signaling pathways rather than genetic manipulation. it's about

Description

METHOD FOR PROLIFERATING OR CULTIVATING MUSCLE SATELLITE CELLS FOR PRODUCTION OF CULTURED MEAT}

The present invention relates to a method for proliferating or culturing muscle stem cells to produce cultured meat, and more particularly, to a method for mass-proliferating or long-term culturing muscle stem cells through regulation of intracellular signaling pathways rather than genetic manipulation. it's about

Cultured meat means making meat by differentiating muscle cells into muscles in a laboratory. The most important but unresolved part in the cultured meat research that has been conducted so far is the problem of mass proliferation and long-term culture of muscle stem cells. There is no standardized culture method for mass growth.

On the other hand, pigs are an important food material for humans and an excellent animal model for disease research, and studying the properties of pig muscle stem cells is important not only for biotechnology research but also for pork production.

Muscle stem cells (Satellite cells) are a heterogeneous population of adult stem cells located in skeletal muscle (Almada et al., 2016), and have been reported to be essential for muscle growth and regeneration of damaged muscle fibers. Although many studies have been conducted for muscle stem cell culture, studies on the self-renewal and indefinite growth of stem cells in vitro are still unclear (Wilschut et al., 2010). In addition, since research on embryonic stem cells is used for embryonic development or cell therapy using animal-derived cells, a lot of investment has already been carried out, and numerous studies on muscle stem cells have been conducted in humans and mice. . However, studies on muscle stem cells of pigs that are actually consumed as food by humans are lacking. The culture conditions currently used for culturing muscle stem cells were established based on the results obtained through research on mouse or human muscle stem cells, but the culture conditions established from heterogeneous cell lines are actually used for culturing pig muscle stem cells. There are limits to its application as it is.

As meat consumption increases with the increase in world population and the food shortage problem becomes serious, research on cultured meat, which produces meat by culturing animal cells, is attracting attention as one of the future alternative foods, but small amounts of meat are produced. Due to the disadvantage that it takes a long time to do, it is not commercially available.

Post, M. J. (2012). Cultured meat from stem cells: Challenges and prospects. Meat science, 92(3), 297-301.

In order to solve the above problems, the present invention provides a method for proliferating or culturing muscle stem cells by regulating the Wnt signaling pathway. In addition, the present invention provides a medium composition for proliferation or culture of muscle stem cells containing an activator of the Wnt signaling pathway.

In addition, the present invention provides a cultured meat production method comprising the step of proliferating or culturing muscle stem cells using an activator of the Wnt signaling pathway.

The present invention provides a method for proliferating or culturing muscle stem cells by regulating the Wnt signaling pathway.

The method can be used for cultured meat production.

The regulation of the Wnt signaling pathway may be performed by adding an activator of the Wnt signaling pathway to the muscle stem cell culture medium.

The activator may be CHIR99021.

The CHIR99021 may be a compound represented by Formula 1 below.

[Formula 1]

In addition, the present invention provides a medium composition for proliferation or culture of muscle stem cells containing an activator of the Wnt signaling pathway.

In addition, the present invention provides a cultured meat production method comprising the step of proliferating or culturing muscle stem cells using an activator of the Wnt signaling pathway.

The present invention can provide a culture method capable of maintaining muscle stem cells in a satellite cell state for a long period of time and proliferating them in large quantities without differentiating them. In addition, since the present invention increases the proliferation rate of cells through regulation of intracellular signaling pathways rather than genetic manipulation, conventional problems in developing cultured meat can be improved. In addition, since the beginning of research on cultured meat is to extract and culture satellite cells from biological tissues to obtain a large amount of muscle fibers, the present invention can therefore be usefully applied to research on cultured meat production.

Figure 1 shows the results of comparing the morphological appearance of muscle stem cells according to the culture conditions.
Figure 2 shows the results of comparing cell proliferation rates according to culture conditions.
Figure 3 shows the result of comparing the viability of cells according to the culture conditions.
Figure 4 shows the result of comparing the expression of key transcription factors in muscle stem cells according to culture conditions.
5 shows the protein expression results of key transcription factors according to culture conditions.
Figure 6 shows the cell cycle comparison results of muscle stem cells according to culture conditions.
7 shows a comparison of apoptosis stages of muscle stem cells according to culture conditions.

Hereinafter, the present invention will be described in detail.

Cultured meat means making meat by differentiating muscle cells into muscles in a laboratory. The most important but unresolved part in the cultured meat research that has been conducted so far is the problem of mass proliferation and long-term culture of muscle stem cells. There is no standardized culture method for mass growth.

Accordingly, the present inventors intend to provide a method for mass-proliferating or long-term culturing muscle stem cells (satellite cells) for the production of cultured meat.

The present invention provides a method for mass-proliferating or long-term culturing muscle stem cells (satellite cells) through regulation of the intracellular signal transduction (Wnt signal transduction) pathway rather than genetic manipulation. Satellite cells (SCs), called muscle stem cells, play an important role in muscle growth and development as a source of nucleus during new muscle generation. ) exists between them.

Since the present invention can increase the proliferation rate of cells through regulation of the intracellular signal transduction (Wnt signal transduction) pathway rather than genetic manipulation, problems in the prior art related to the development of cultured meat can be improved. In addition, cultured meat research begins with obtaining a large amount of muscle fibers by extracting and culturing satellite cells from biological tissues, and therefore, the present invention can be usefully applied to cultured meat production research.

In the present invention, the regulation of the Wnt signaling pathway can be performed by adding an activator of the Wnt signaling pathway to the muscle stem cell culture medium.

Research on Wnt signaling is mainly conducted in cancer-related fields (cancer cells, diseases, cancer-related genes, etc.), and there are few cases of application to muscle stem cells.

The activator may be CHIR99021. CHIR99021 is a compound acting as an inhibitor of the enzyme GSK-3 and may be represented by the following formula (1).

[Formula 1]

The method can be applied not only to pig muscle stem cells but also to muscle stem cells of other livestock such as chickens and cattle.

In addition, the present invention provides a medium composition for proliferation or culture of muscle stem cells containing an activator of the Wnt signaling pathway.

In addition, the present invention provides a cultured meat production method comprising the step of proliferating or culturing muscle stem cells using an activator of the Wnt signaling pathway. Since the description of the activator of the Wnt signaling pathway is the same as that described above, description thereof will be omitted.

Hereinafter, the present invention will be described in more detail through examples. Objects, features, and advantages of the present invention will be easily understood through the following examples. The present invention is not limited to the embodiments described herein and may be embodied in other forms. The embodiments introduced here are provided to sufficiently convey the spirit of the present invention to those skilled in the art to which the present invention belongs. Therefore, the present invention should not be limited by the following examples.

<Example>

Materials and Methods

Porcine muscle satellite cell (PMSC) derivation and culture

Muscle tissue was extracted from the femurs of 1-day-old piglets and immediately washed three times with DPBS (Dulbecco's phosphate buffer saline, Gibco, #14190-144) containing 10% PS (Penicillin streptomycin, Gibco, #15140-122). . The muscle tissue was chopped into small pieces and separated into digestive juices; Collagenase D (Roche, #11088858001, 2 mg/ml), dispase II (Roche, #4942078001, 1 U/ml), and 0.25% trypsin in DMEM/F12 (Gibco, #11320-033) supplemented with 10% PS. (trypsin)-EDTA (Gibco, # 25200-072) at 37°C for 1 hour. The homogenate was filtered through a 70 μm cell strainer and neutralized with F12 medium containing 15% fetal bovine serum (FBS, Gibco, #16000-044). After centrifugation at 1100 rpm for 5 minutes, ACK lysing buffer (Gibco, # A10492-01) was added to the supernatant and placed on ice for 5 minutes. After one more centrifugation, the supernatant was discarded, and the cell pellet was incubated with 15% FBS, 1% PSG (Penicillin streptomycin glutamine, Gibco, #10378-016), and bFGF (basic fibroblast growth factor, Gibco, #13256-029). , 10 ng/ml) was resuspended in F12 medium supplemented. Cells were uniformly seeded on a culture plate and cultured for 1 hour in a 37° C. incubator. In order to isolate satellite cells, the medium (suspended cells) was collected and transferred to another culture plate coated with 0.1% gelatin.

Treatment of inhibitor (XAV939) and activator (CHIR99021)

XAV939 (Tankyrase inhibitor, Sigma-Aldrich, #3004) and CHIR99021 (GSK-3 inhibitor, Stemgent, #04-0004-02) to regulate the Wnt/β-catenin signaling pathway in PMSC were treated with inhibitors and activators, respectively. Control cells (SC group) were cultured in a medium (DMEM/F12 containing 15% FBS, 1% PSG and 10 ng/ml bFGF). XAV939 was added to the medium at a final concentration of 1 μM (XAV group) and CHIR99021 was added to the medium at a final concentration of 3 μM (CHIR group).

Cell viability assay

Cell viability was analyzed using CCK-8 (Cell Counting Kit-8, Dojindo, #CK04-11). Briefly, cells were cultured in a 96-well plate at a density of 5 x 10 ⁴ cells per well and cultured until the cells reached about 80-90% confluency. Then, the cells were treated with CCK-8 solution according to the manufacturer's instructions and incubated at 37°C for 4 hours. The OD value of each well was measured using a microplate reader at a wavelength of 450 nm.

cell proliferation assay

PMSCs were cultured in a 10 cm ² dish at a density of 1 x 10 ⁶ cells/dish and cultured for 3 days. Cells were detached with 0.25% trypsin-EDTA and counted under an inverted microscope using a hemocytometer. Experiments were performed three times.

Protein extraction and western blotting

Cells were cultured in 10 cm ² dishes at a density of 1×10 ⁶ cells/dish and collected for western blot analysis. Total protein was extracted from satellite cells with RIPA buffer (Biosesang, Sungnam, Korea) supplemented with protease inhibitors. Protein concentration was measured using the DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Proteins extracted from the cells were separated by SDS-PAGE using a 12% acrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk with TBST buffer at room temperature, then incubated overnight at 4°C with primary antibody and incubated with secondary antibody for 1 hour and 30 minutes at room temperature (incubating). The primary antibodies used in this study were GAPDH (1:5000, monoclonal, MA5-15738, Invitrogen, Carlsbad, CA, USA), Pax7 (1:1000, monoclonal, DSHB, Iowa, IA, USA), MyoD (1 :1000, polyclonal, 18943-1-AP, Proteintech, Rosemont, IL, USA), and secondary antibodies were goat anti-mouse IgG (HRP-conjugate, #31430, Thermo Fisher, San Jose, CA, USA) and goat Anti-rabbit IgG (HRP-conjugate, #31460, Thermo Fisher, San Jose, CA, USA) was used. Protein expression was visualized using the ECL chromogenic kit (SuperSignal WestPico Plus, Thermo Fisher, San Jose, CA, USA) and protein density was measured using the iBright CL100 Imaging System (Thermo Fisher, San Jose, CA, USA). USA).

Immunofluorescence staining of PMSCs

PMSCs were cultured on a confocal microscope dish and fixed with 4% paraformaldehyde for 20 minutes at room temperature. After washing the cells with PBS, they were permeabilized and blocked with 0.3% Triton X-100 and 3% BSA in PBS for 1 hour at room temperature. Then, they were incubated overnight at 4°C with anti-MyoD (Polyclonal, 1:200, Proteintech, Rosemont, IL, USA) and anti-Pax7 (Monoclonal, 1:50, DSHB, Iowa, IA, USA) primary antibodies. . After washing, the primary antibody was visualized with a fluorescently labeled secondary antibody (Alexa Fluor 488 or 568; Molecular Probes, Eugene, OR, USA). Cells were then stained with DAPI (4'-6-diamidino-2-phenylindole) for 5 minutes at room temperature. Fluorescent images were collected using a super-resolution confocal laser scanning microscope (SR-CLSM) installed at the University-Wide Research Facility (CURF), Chonbuk National University.

flow cytometry

Cell cycle and apoptosis assays were performed using a FACS caliber flow cytometry (Becton, Dickinson Company, CA, USA) and BD Cell Quest Pro Software. For cell cycle analysis, samples were fixed in cold 70% ethanol for 5 min at 4°C. Cells were then stained with a diluted propidium iodide (PI) solution containing 100 μg/ml RNase A (Bio Legend, # 421301, San Diego, CA, USA) and immediately analyzed by flow cytometry. Apoptosis was assayed using the Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit and PI (Bio Legend, # 640914, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, cells were detached with 0.25% trypsin-EDTA and washed with cold PBS containing 1% BSA. Cells were then double stained with FITC Annexin V and PI solutions for 15 min at room temperature in the dark. Stained cells were mixed with Annexin V Binding Buffer and analyzed by flow cytometry.

statistical analysis

Data were analyzed using SAS software version 9.4 (SAS Institute Inc., USA). Statistical differences were determined by Student's t-test or analysis of variance (ANOVA) followed by Duncan's multiple range test for post hoc comparisons. All values are expressed as mean ± standard error (SE).

Example 1. Analysis of the effect of XAV939 and CHIR99021 on proliferation and viability of PMSC

To evaluate the effects of XAV939 and CHIR99021 on PMSC (porcine muscle satellite cells), XAV939 (XAV), an inhibitor of the Wnt signaling pathway, and CHIR99021 (CHIR), an activator, were used. Cell proliferation rate and survival rate were compared after culturing by adding to muscle stem cell (SC) culture medium. Depending on each culture condition, there was a difference in the growth rate of the cells that could be confirmed with the naked eye (FIG. 1). In addition, the cell proliferation rate was significantly increased in the CHIR group (treated with CHIR) (p < 0.01) and decreased in the XAV group (p < 0.01) compared to the SC group (control) (Figs. 1 and 2). The cell viability was significantly higher in the CHIR group than in the SC group (p < 0.01) and the lowest in the XAV group (p < 0.01) (FIG. 3).

Example 2. Analysis of the effect of XAV939 and CHIR99021 on Pax7 and MyoD protein expression in PMSC

To investigate the expression of key transcription factors induced by Wnt signaling pathway regulators, the expression of Pax7 and MyoD proteins was analyzed using immunofluorescence staining and Western blotting. Through cell immunostaining, the expression of Pax7, a representative marker of satellite cells, and MyoD, a representative marker of myogenesis, were observed using a confocal microscope. As a result of the observation, depending on the culture conditions It was confirmed that there was a difference in expression. Specifically, as a result of immunostaining, in the SC group without addition of XAV939 or CHIR99021, Pax7 and MyoD were highly expressed in most cells compared to other groups (FIG. 4). In the XAV group, the expression of Pax7 and MyoD decreased compared to the SC group, but the number of cells expressing each transcription factor was similar (FIG. 4). On the other hand, the expression of Pax7 in the CHIR group was slightly decreased compared to the SC group, but the expression of MyoD was completely decreased (FIG. 4). The expression levels of Pax7 and MyoD proteins in PMSCs by Western blot analysis were consistent with the immunostaining results (FIG. 5). XAV939 significantly reduced the expression of Pax7 and MyoD proteins compared to the SC group (p < 0.01), and CHIR99021 significantly reduced the expression of MyoD while maintaining Pax7 expression similar to that in the SC group (p < 0.01). .

Example 3. Effects of XAV939 and CHIR99021 on cell cycle and apoptosis

The effects of Wnt signaling pathway inhibitors and activators on the cell cycle and apoptosis of PMSCs were determined using flow cytometry. The PMSC ratio in the G0/G1 phase was significantly higher in the XAV group than in the SC and CHIR groups (p < 0.01) (FIG. 6). On the other hand, the PMSC ratios of the SC and CHIR groups were significantly higher than those of the XAV group in S phase (p < 0.01) and G2/M phase (p < 0.05) (FIG. 6). Addition of XAV939 and CHIR99021 also affected PMSC apoptosis. The proportion of viable cells was the highest in the CHIR group (p < 0.01), and the proportions of late apoptosis and dead cells were significantly higher in the SC and XAV groups than in the CHIR group (p < 0.01). The rate of necrosis was significantly higher in the SC group than in the other groups (p < 0.05), and there was no significant difference in early apoptosis (FIG. 7).

Through the above experimental results, it can be confirmed that the addition of CHIR to the satellite cell culture medium increases the survival rate and proliferation rate of cells, which is advantageous for mass proliferation and long-term culture of muscle stem cells.

Claims (7)

A cultured meat production method comprising the step of proliferating or culturing muscle stem cells using an activator of the Wnt signaling pathway,
The regulation of the Wnt signaling pathway is to add an activator of the Wnt signaling pathway to the culture medium of muscle stem cells,
The activator is a method for producing cultured meat that is CHIR99021 represented by the compound of Formula 1 below.
[Formula 1]

delete delete delete delete A medium composition for proliferation or culture of muscle stem cells containing an activator of the Wnt signaling pathway,
The activator is CHIR99021 represented by the compound of Formula 1 below.
[Formula 1]
delete