Cell line, method for producing bovine muscle cell, method for producing bovine adipocyte, and method for producing cultured meat

Abstract

Provided is a technique with which it is possible to improve the proliferation rate of cells in culture in which microcarriers are used. A cell line is established by introducing, into bovine progenitor cells, the following four genes: CDK4 (R24C), TERT, CCND1, and p53 that lacks a DNA binding domain.

Description

Cell line, method for producing bovine muscle cells, method for producing bovine adipose cells, method for producing cultured meat

[0001] The present disclosure relates to the production of cultured meat. This application is based on Japanese Patent Application No. 2024-172844 filed on October 1, 2024, the contents of which are incorporated herein by reference.

[0002] In recent years, technologies for artificially producing edible meat (cultured meat) by growing cells in vitro have attracted attention. In order to produce cultured meat at an industrial level, it is required to culture cells in large quantities. For example, Non-Patent Document 1 discloses culturing cells by stirring and suspension using microcarriers.

[0003] Sakaguchi Katsuhisa, Tanaka Ry一郎, "Stirring and suspension culture of bovine myoblasts for cultured meat production", Journal of the Society of Biotechnology, 2022, Vol. 100, No. 4, p. 169-172

[0004] In the technology described in Non-Patent Document 1, there is room for improvement from the perspective of the growth rate in culture using microcarriers. Therefore, there is a need for a technology that can improve the growth rate of cells in culture using microcarriers.

[0005] The present disclosure can be realized in the following forms.

[0006] (1) According to one aspect of the present invention, a cell line is provided. This cell line is established by introducing the following four genes into bovine progenitor cells: CDK4 (R24C), TERT, CCND1, and p53 lacking a region encoding a DNA binding domain. According to this form of cell line, the growth rate of cells in culture using microcarriers can be improved.

[0007] (2) In the cell line described in (1) above, the bovine progenitor cells may be bovine muscle progenitor cells. According to this form of cell line, the growth rate of cells in culture using microcarriers can be improved.

[0008] (3) Another embodiment of the present disclosure provides a method for producing bovine muscle cells. This method includes introducing the following four genes into bovine muscle progenitor cells: CDK4 (R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain. This embodiment of the production method allows for easy mass culture of bovine muscle cells because of the excellent proliferation rate in culture using microcarriers.

[0009] (4) In the method for producing bovine muscle cells described in (3) above, the gene may be introduced using a transposon vector in the step. With this method of production, the gene is introduced using a transposon vector, so the decrease in safety can be suppressed.

[0010] (5) Another embodiment of the present disclosure provides a method for producing bovine adipocytes. This method includes introducing the following four genes into bovine adipocyte progenitor cells: CDK4 (R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain. This embodiment of the production method allows for easy mass culture of bovine adipocytes because of the excellent proliferation rate in culture using microcarriers.

[0011] (6) Another embodiment of the present invention provides a method for producing cultured meat. This method for producing cultured meat includes the step of introducing the following four genes: CDK4 (R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain into bovine muscle progenitor cells using a transposon vector. This embodiment of the method for producing cultured meat can improve the cell proliferation rate in culture using microcarriers, thereby enabling easy mass culture of bovine muscle cells and improving the efficiency of cultured meat production.

[0012] Furthermore, this disclosure can be implemented in various forms, for example, in the form of a method for establishing a cell line, a method for manufacturing a cell line, cultured meat containing bovine muscle cells, cultured meat containing bovine adipocytes, a method for manufacturing cultured meat using microcarrier culture, and the use of cell lines for manufacturing cultured meat.

[0013] This is an explanatory diagram showing the results of long-term subculturing. This is an explanatory diagram comparing the proliferation results with and without DNp53. This is an explanatory diagram showing the results of microcarrier culture. This is an explanatory diagram showing microscopic images of cells in the MC+ group. This is an explanatory diagram showing microscopic images of cells cultured under muscle differentiation induction. This is an explanatory diagram comparing the proliferation results with and without a coating agent. This is an explanatory diagram showing the results of long-term subculturing.

[0014] A. Definitions of Terms In this disclosure, "cattle" means animals belonging to the subfamily Bovinae, preferably the domesticated breed of cattle (Bos taurus). In this disclosure, "bovine progenitor cells" means cells of cattle origin that are expected to differentiate into a specific type of bovine cell or form a specific type of bovine tissue. Bovine progenitor cells in this disclosure may also preferably include mesenchymal stem cells that have the ability to differentiate into myocytes (muscle cells) or adipocytes (fat cells). Examples of bovine progenitor cells are not particularly limited, but include progenitor cells derived from bovine tissue, such as muscle progenitor cells derived from muscle and fat progenitor cells derived from fat. Examples of bovine muscle are not particularly limited, but include skeletal muscle, cardiac muscle, smooth muscle, etc., but skeletal muscle is preferred. Skeletal muscles are not particularly limited, but examples include the muscle tissue of the cheeks, temples, neck, back, chest, shoulders, lower back, and thighs.

[0015] In this disclosure, the term "muscle progenitor cell" may be replaced with the terms "muscle tissue-derived progenitor cell," "muscle stem cell," or "myogenic progenitor cell." In this disclosure, muscle progenitor cells may include somatic stem cells present in muscle tissue. Muscle progenitor cells may also be myosatellite cells or myoblasts. Muscle satellite cells are precursors of skeletal muscle cells. Satellite cells can differentiate into myoblasts. In this disclosure, the term "adipose progenitor cell" may be replaced with the terms "adipose stem cell," "stromal vascular fraction (SVF)," or "adipose tissue-derived stem cells (ADSC)." In this disclosure, adipose progenitor cells may include somatic stem cells present in adipose tissue.

[0016] In this disclosure, "CDK4" refers to the gene encoding cyclin-dependent kinase 4. Cyclin-dependent kinase 4 regulates the transition from the G1 phase to the S phase of the cell cycle. Cyclin-dependent kinase 4 is activated by binding to cyclins. Sequence ID 1 shows the sequence of bovine CDK4 (hereinafter also referred to as "bCDK4"). In this disclosure, "CDK4 (R24C)" refers to the gene into which a mutation has been introduced in which the 24th R (arginine) in the encoded cyclin-dependent kinase 4 is replaced with C (cysteine). Sequence ID 2 shows an example of the sequence of bovine CDK4 (R24C) (hereinafter also referred to as "bCDK4 (R24C)"). Sequence ID 2 shows that, compared to Sequence ID 1, the 70th C (cytosine) in Sequence ID 2 is replaced with T (thymine). In addition to the substitution of the 70th C in bCDK4(R24C) with a T, the 72nd T may also be substituted with a C. bCDK4(R24C) may have one or more bases deleted, substituted, or added to the sequence shown in Sequence ID No. 2, provided that the desired function is not lost.

[0017] In this disclosure, "TERT" means a gene that codes for telomerase reverse transcriptase. Telomerase reverse transcriptase adds telomere sequences to DNA. The coding sequence (CDS_NM_001046242.1) of bovine TERT (hereinafter also referred to as "bTERT") is shown in Sequence ID No. 3. bTERT may have one or more bases deleted, substituted, or added to the sequence shown in Sequence ID No. 3, provided that the desired function is not lost.

[0018] In this disclosure, "CCND1" means the gene encoding cyclin D1. Cyclin D1 is involved in the transition from the G1 phase to the S phase of the cell cycle by forming a complex with CDK4 or CDK6. The sequence of bovine CCND1 (hereinafter also referred to as "bCCND1") (NM_001046273.2) is shown in SEQ ID NO: 4. bCCND1 may have one or more bases deleted, substituted, or added to the sequence shown in SEQ ID NO: 4, provided that the desired function is not lost.

[0019] In this disclosure, "p53" means the gene encoding the p53 tumor suppressor. The sequence (NM_174201.2) of bovine p53 (hereinafter also referred to as "bp53") is shown in Sequence ID No. 5. In this disclosure, "p53 lacking the region encoding the DNA binding domain" means p53 in which the region encoding the DNA binding domain is deleted compared to wild-type p53. Since p53 lacking the region encoding the DNA binding domain is a dominant-negative type of p53, it will also be referred to as "DNp53" in the following description. DNp53 also includes a form in which the DNA binding domain is not formed due to the deletion of a portion of the region encoding the DNA binding domain. Furthermore, DNp53 may also have deletions in a portion of the region other than the region encoding the DNA binding domain compared to wild-type p53. An example of the sequence of bovine DNp53 (hereinafter also referred to as "bDNp53") is shown in Sequence ID No. 6. bDNp53 may have one or more bases deleted, substituted, or added to the sequence shown in Sequence ID No. 6, provided that the intended function is not lost.

[0020] B. Cell Lines According to one embodiment of the present disclosure, a cell line is provided which is established by introducing the following four genes into bovine progenitor cells: CDK4 (R24C), TERT, CCND1, and p53 (DNp53) lacking the region encoding the DNA binding domain. Of the above four genes, TERT and CCND1 are endogenous genes that are naturally expressed in bovine progenitor cells. Therefore, the introduction of TERT and CCND1 increases the expression levels of telomerase reverse transcriptase and cyclin D1 compared to the wild type. Of the above four genes, CDK4 (R24C) and DNp53 are exogenous genes that are not naturally expressed in bovine progenitor cells.

[0021] Bovine progenitor cells can be obtained by treating excised bovine tissue with collagenase. More specifically, for example, any tissue can be excised from the cheek or visceral fat of a slaughtered cow, finely cut with scissors, and then digested with collagenase to obtain bovine progenitor cells. If necessary, specific cells may be separated using a cell sorter. The obtained bovine progenitor cells are suspended in any culture medium and CO2 is added. ２ The cells may be cultured in an incubator for 1 to 7 days before being subjected to transfection. The bovine progenitor cells may be subcultured and cryopreserved before being subjected to transfection. The culture medium is not particularly limited, but any medium suitable for culturing animal cells may be used, such as Eagle MEM medium, Dulbecco's modified Eagle MEM medium, Ham medium F12, and mixtures thereof. Culture temperature, medium pH, CO2 ２ The concentration and other conditions may be those commonly used in animal cell culture.

[0022] The method of gene introduction is not particularly limited as long as it is capable of introducing the above gene into bovine progenitor cells, but stable transfection rather than transient transfection is preferred. One example of a gene introduction method is using a vector. The vector is not particularly limited as long as it is capable of introducing the above gene into bovine progenitor cells, but from the viewpoint of suppressing a decrease in safety, a non-viral vector is preferred. Non-viral vectors are not particularly limited, but examples include transposon vectors. Transposon vectors are not particularly limited, but examples include PiggyBac vectors, Sleeping Beauty vectors, Tol2 vectors, etc., but from the viewpoint of gene introduction efficiency, PiggyBac vectors are preferred.

[0023] The four genes mentioned above may be introduced separately or together. More specifically, when introducing them using a transposon vector, multiple genes may be placed in a single vector, or each gene may be placed in a separate vector. Even when multiple genes are introduced in series in a single vector, each protein will be expressed independently.

[0024] Cells after gene introduction may be cultured in any medium suitable for animal cell culture. The medium is not particularly limited, but examples include Eagle MEM medium, Dulbecco's modified Eagle MEM medium, Ham medium F12, and mixtures thereof. Culture temperature, medium pH, CO2 ２ The concentration and other conditions may be those commonly used in animal cell culture.

[0025] The cell lines disclosed herein exhibit excellent suitability for microcarrier culture. More specifically, they exhibit superior growth rates in cultures using microcarriers.

[0026] In addition, according to the cell line of the present disclosure, it has an infinite proliferation ability and can suppress an increase in the doubling time. Having an infinite proliferation ability means that immortalization has been acquired. Although the mechanism by which it has an infinite proliferation ability and can suppress an increase in the doubling time is not clear, for example, the following putative mechanisms are presumed.

[0027] Here, generally, the family of cyclin-dependent kinase inhibitors (CKIs) includes INK4 (inhibitors of CDK4) and Cip / Kip (CDK interacting protein / Kinase inhibitory protein). Generally, p16, which is a type of INK4 ＩＮＫ４ inhibits the binding of cyclin to cyclin-dependent kinase 4 and the binding of cyclin to cyclin-dependent kinase 6. Mutant cyclin-dependent kinase 4 formed from mutant CDK4 (R24C) has a reduced affinity for p16 ＩＮＫ４ compared to the wild type, and as a result, it is considered that the inhibition of binding to cyclin can be suppressed. Therefore, it is presumed that according to mutant CDK4 (R24C), a decrease in activity is suppressed compared to wild-type CDK4.

[0028] In addition, generally, the Cip / Kip family is composed of three proteins, p21 Ｃｉｐ１ , p27 Ｋｉｐ１ , and p57 Ｋｉｐ２ . These proteins inhibit activity by binding to both cyclin and cyclin-dependent kinase. The p53 tumor suppressor controls the expression of p21 Ｃｉｐ１ upstream of p21 Ｃｉｐ１ . According to dominant-negative p53, it is considered that the action of the Cip / Kip system can be avoided by disturbing the function of endogenous wild-type p53. As a result, it is presumed that it has an infinite proliferation ability and can suppress an increase in the doubling time.

[0029] Furthermore, the cell lines disclosed herein exhibit superior differentiation efficiency. In particular, when bovine muscle progenitor cells are used, muscle differentiation efficiency is excellent. Differentiation efficiency can be confirmed, for example, by observing the characteristic structure of the tissue using a microscope. Alternatively, differentiation efficiency may be confirmed by examining the expression of various marker genes, etc., by immunostaining. In addition, the cell lines disclosed herein can suppress the decrease in cell proliferation rate even in the absence of a coating agent.

[0030] The cell lines disclosed herein exhibit excellent growth rates in culture using microcarriers, making large-scale culture easy and thus suitable for the production of cultured meat. While not particularly limited, the microcarriers used for cell culture include, for example, resin materials such as polystyrene and polyvinyl alcohol, acrylamide, glass, collagen, cellulose, gelatin, and polysaccharides. Furthermore, the cell lines disclosed herein are suitable for the production of cultured meat because they possess unlimited growth potential and can suppress the increase in doubling time. In addition, the cell lines disclosed herein are suitable for the production of cultured meat because they exhibit excellent differentiation efficiency and can suppress the decrease in cell growth rate in the absence of a coating agent. The cell lines disclosed herein may also be cultured using methods that do not use microcarriers. While not particularly limited, methods that do not use microcarriers include suspension culture and adherent culture without microcarriers; however, from the viewpoint of large-scale culture, suspension culture is preferred, and suspension culture using a bioreactor or automated culture device is more preferred. Furthermore, the cell lines disclosed herein may be used not only for cultured meat but also for regenerative medicine, research materials, and other applications.

[0031] C. Method for Producing Bovine Muscle Cells According to other embodiments of the present disclosure, a method for producing bovine muscle cells is provided. The method for producing bovine muscle cells includes the step of introducing the following four genes: CDK4 (R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain into bovine muscle progenitor cells. The bovine muscle progenitor cells may be cells that have undergone subculturing, or cells that have undergone cryopreservation and subsequent thawing.

[0032] In the gene introduction step, for example, the gene can be introduced using the method described above, but it is preferable to introduce the gene using a transposon vector, and more preferable to introduce the gene using a PiggyBac vector. The method for producing bovine muscle cells of this disclosure may include a step of culturing the cells in a culture medium after the gene introduction step. Bovine muscle progenitor cells into which the four genes have been introduced are preferably selected based on the expression of antibiotics or the like. That is, the method for producing bovine muscle cells of this disclosure may include a step of selecting cells into which the four genes have been introduced after the gene introduction step. Furthermore, the method for producing bovine muscle cells of this disclosure may include a step of subculturing the cells in a culture medium after the gene introduction step or after the cell line selection step. Furthermore, the method for producing bovine muscle cells of this disclosure may include a step of culturing the cells using a microcarrier. The microcarrier used is not particularly limited, but examples include microcarriers formed from resin materials such as polystyrene and polyvinyl alcohol, acrylamide, glass, collagen, cellulose, gelatin, polysaccharides, etc. In addition, in the method for producing bovine muscle cells according to this disclosure, the cells may be cultured by a culture method that does not use microcarriers, such as the one described above.

[0033] The bovine muscle cell production method disclosed herein is suitable for the production of cultured meat because it can improve the cell proliferation rate in culture using microcarriers, thereby facilitating large-scale culture. Furthermore, it is suitable for the production of cultured meat because it possesses unlimited proliferation ability and can suppress the increase in doubling time. It is also suitable for the production of cultured meat because it exhibits excellent differentiation efficiency and can suppress the decrease in cell proliferation rate in the absence of a coating agent. The bovine muscle cells produced by the bovine muscle cell production method disclosed herein may be used not only for the production of cultured meat, but also for regenerative medicine, research materials, and other applications.

[0034] D. Method for Producing Bovine Adipocytes According to other embodiments of the present disclosure, a method for producing bovine adipocytes is provided. The method for producing bovine adipocytes includes the step of introducing the following four genes: CDK4 (R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain into bovine adipocyte precursor cells. The method for producing bovine adipocytes may be the same as the method for producing bovine muscle cells described above, except that the primary cells used are different. The method for producing bovine adipocytes of the present disclosure can improve the cell proliferation rate in culture using microcarriers, making it easy to culture on a large scale and thus suitable for the production of cultured meat. It is also suitable for the production of cultured meat because it has unlimited proliferation ability and can suppress the increase in doubling time. Furthermore, it is also suitable for the production of cultured meat because it has excellent differentiation efficiency and can suppress the decrease in the cell proliferation rate in the absence of a coating agent. Bovine adipocytes produced by the method for producing bovine adipocytes of the present disclosure may be used not only for the production of cultured meat, but also for regenerative medicine, research materials, etc.

[0035] E. Method for producing cultured meat According to other embodiments of the present disclosure, a method for producing cultured meat is provided. The method for producing cultured meat includes introducing the following four genes into bovine muscle progenitor cells using a PiggyBac vector: CDK4 (R24C), TERT, CCND1, and p53 (DNp53) lacking a region encoding a DNA binding domain.

[0036] The method for producing cultured meat according to the present disclosure preferably further includes the step of introducing the following four genes: CDK4 (R24C), TERT, CCND1, and p53 (DNp53) lacking the region encoding the DNA binding domain into bovine adipose progenitor cells using a PiggyBac vector.

[0037] Furthermore, the method for producing cultured meat according to this disclosure preferably includes the steps of culturing cells into which the four genes described above have been introduced, and forming tissue using the proliferated cells. The method of culturing cells in the cell culture step is not particularly limited, but for example, the method described above may be used. The method for forming tissue is not particularly limited, but for example, the following method may be used. First, the proliferated myoblasts are differentiated into myotubes. This causes the myoblasts to become multinucleated by cell fusion with surrounding cells, forming myotubes. Muscle fibers are formed by further maturing the myotubes. In addition, in the method for producing cultured meat, for example, three-dimensional tissue may be formed by stacking sheet-like muscle cells and adipocytes, or three-dimensional tissue may be formed by embedding a cell mass containing muscle cells and adipocytes in a collagen gel or the like.

[0038] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0039] 1. Cell Preparation (1) Samples Cow cheek meat was purchased from Tokyo Shibaura Organ Co., Ltd., and bovine muscle progenitor cells were isolated as follows: Muscle tissue was cut out and shredded using a mincer (Bonny, 49 3683400500 2) and scissors. To the shredded muscle tissue, a 0.2% collagenase solution (DMEM, high glucose, GlutaMAX Supplement, pyruvate (Gibco, 10569-044) + 2 mg / mL collagenase (Fujifilm Wako Pure Chemical Industries, 032-22364) + 25 U / mL deoxyribonuclease I (Sigma, D5025) + 10 mM HEPES (Nacalai, 17557-94) + 1 × Penicillin / Streptomycin / Amphotericin B (Nacalai, 02892-54) + 2 μg / mL Gentamycin Sulfate Solution (Nacalai, 16672-04)) was added, and the mixture was stirred at 37°C for 1 to 2 hours using a magnetic stirrer. The treatment solution was filtered through a 59 μm nylon mesh (AS ONE, PA-59μ), and the filtrate was centrifuged at 800 g for 5 minutes. The precipitate was suspended in HBSS (Nacalai, 09735-75) with 4% FBS added, and centrifuged again at 800 g for 5 minutes. The precipitate was again suspended in HBSS with 4% FBS added, and filtered through a 100 μm cell strainer (Corning, 352360) to obtain bovine muscle progenitor cells. If necessary, a cell sorter (Becton Dickinson FACS Aria II) was used to isolate specific cells. The obtained bovine muscle progenitor cells were cultured for about 7 days under the culture conditions described below, and then passed through once by trypsin treatment (Nacalai, 35553-74). The entire amount was seeded in a separate incubator and cultured for about 3 days before being used for transfection experiments. For the preparation of bovine adipose progenitor cells, adipose tissue excised from bovine visceral fat (pericardial fat) purchased from Tokyo Shibaura Organ Co., Ltd. was used. The preparation of bovine adipose progenitor cells was carried out using the same method as for the preparation of bovine muscle progenitor cells.The obtained bovine adipose progenitor cells were cultured for approximately 7 days under the culture conditions described below, and then passed through once by treatment with trypsin (Nacalai, 35553-74). The entire amount was seeded in a separate incubator and cultured for approximately 3 days before being used for transfection experiments.

[0040] (2) Cell Culture Unless otherwise specified in the following description, the culture medium shall be MM (Maintenance Medium; DMEM, high glucose, GlutaMAX Supplement, pyruvate (Gibco, 10569-044) + 20% Fetal Bovine Serum (South Africa Origin) (Biosera, 556-33865 (FB-1003 / 500)) + 10 mM HEPES (Nacalai, 17557-94) + 1 × Penicillin / Streptomycin / Amphotericin B (Nacalai, 02892-54) + 2 μg / mL Gentamycin Sulfate Solution (Nacalai, 16672-04) + 5 ng / mL bFGF (ReproCell, RCHEOT003)) and CO ２ Cultured in an incubator (37°C, 5% CO2) ２ The incubators used were coated with 0.1 mg / mL Fibronectin bovine plasma (Merck, F4759).

[0041] (3) Vector Preparation The following five vectors were prepared. Four of the vectors, excluding the control vector, were synthesized by VectorBuilder Inc. The control vector was prepared by removing the inserted gene from the vector synthesized by VectorBuilder Inc.

[0042] The hyPBase vector (pRP[Exp]-mCherry-CAG>hyPBase) shown in SEQ ID NO: 7 is a transposase expression helper plasmid. CDK4 shown in SEQ ID NO: 8 Ｒ２４ＣThe vector (pPB[Exp]-Puro-EF1A>cwTERT[NM_001046242.1](ns):T2A:cwCDK4[NM_001037594.2](ns)*:T2A:cwCCND1[NM_001046273.2]) is a transposon plasmid in which bCDK4 (R24C variant), bTERT, and bCCND1 are inserted. CDK4 is shown in Sequence ID No. 9. ＷＴ The vector (pPB[Exp]-Puro-EF1A>cwTERT[NM_001046242.1](ns):T2A:cwCDK4[NM_001037594.2](ns):T2A:cwCCND1[NM_001046273.2]) is a transposon plasmid in which bCDK4 (WT), bTERT, and bCCND1 are inserted. The DNp53 vector shown in Sequence ID No. 10 (pPB[Exp]-Hygro-EF1A>cwTP53[NM_174201.2]*) is a transposon plasmid in which bDNp53 is inserted. The control vector shown in Sequence ID No. 11 (pOF002_pPB-hEF1Ap-MCS (HygR)) is a plasmid in which bDNp53 is not inserted compared to the DNp53 vector.

[0043] (4) Transfected bovine muscle progenitor cells were harvested by treatment with trypsin (Nacalai, 35553-74) and placed in a 24-well plate (IWAKI, 3820-024) in 2 × 10⁶ units. ４ Cells were seeded in a cell / well and cultured for one day. Plasmid DNA (hyPBase vector + CDK4) Ｒ２４Ｃ Vector, or hyPBase vector + CDK4 ＷＴThe vector was diluted to 20 μg / mL in Opti-MEM®. It was then mixed 1:1 with similarly diluted 120 μL / mL ViaFect Transfection Reagent (Promega, E4981) and allowed to stand at room temperature for 15 minutes. For the negative control, pure water was added instead of the DNA solution. The culture medium in each well was replaced with 450 μL of Opti-MEM®, and 50 μL of the prepared liposome solution was added (0.5 μg / well plasmid DNA). The cells were incubated for 6 hours, and then the culture medium was replaced with MM. The following day, the cells were subcultured and seeded into a 6-well plate (IWAKI, 3810-006N). The following day, the culture medium was replaced with MM supplemented with 3 μg / mL Puromycin (Wako, 160-13151). The cells were cultured until the negative control cells died, and then CDK4 4 was added. Ｒ２４Ｃ Vector-transformed cells and CDK4 ＷＴ Vector-introduced cells were obtained.

[0044] CDK4 obtained above Ｒ２４Ｃ Vector-transfected cells were transfected with either the DNp53 vector or a control vector using the same procedure (hyPBase vector + DNp53 vector, or hyPBase vector + control vector). For selection, 300 μg / mL Hygromycin B (Wako, 089-06151) was used, and CDK4 was selected. Ｒ２４Ｃ Co-expression cells of the vector and the DNp53 vector were generated.

[0045] 2. Experimental Methods and Results <Experiment 1: Experiment on Long-Term Subculturing> Bovine muscle progenitor cells were subjected to the introduction of the above vector into primary cells and CDK4 ＷＴ Cells into which the vector has been introduced (hereinafter referred to as "CDK4") ＷＴ (Also called "cells") and CDK4 Ｒ２４Ｃ Cells into which the vector has been introduced (hereinafter referred to as "CDK4") Ｒ２４Ｃ An experiment was conducted to determine the number of times cells (also called "cells") could divide. The cells were subjected to the same concentration (1 × 10⁻¹⁶). ４ -3 x 10 ４Cells were seeded in a 6-well plate at a concentration of cells / well, and then harvested by trypsin treatment at 3 or 4-day intervals. The harvested cells were mixed with an equal volume of trypan blue solution (Invitrogen, T10282), and the number of viable cells was counted using an automated cell counter (Countess3, Invitrogen). The harvested cells were diluted to an appropriate concentration and seeded in a 6-well plate again. This was repeated until the cells reached approximately 100 divisions or until the number of divisions plateaued. Note that the experiment using primary cells and CDK4 ＷＴ Cells and CDK4 Ｒ２４Ｃ The experiment using cells was conducted on a different day.

[0046] Figure 1 is an explanatory diagram showing the results of long-term subculturing. In Figure 1, the vertical axis represents the cumulative number of divisions (times), and the horizontal axis represents the number of culture days (days). Primary cells showed a significant decrease in proliferation rate on day 21 of culture, and the doubling time from day 18 to 21 of culture was approximately 144.5 hours. CDK4 ＷＴ The cell proliferation rate decreased significantly on day 10 of culture, and the doubling time from day 7 to day 10 of culture was approximately 108.6 hours. In contrast, CDK4 Ｒ２４Ｃ The cells underwent 100 divisions without any decrease in proliferation rate, and the proliferation rate was maintained thereafter. (CDK4) Ｒ２４Ｃ The cells reached a cumulative number of divisions of approximately 129.5 on day 127 of culture, and the doubling time from day 123 to day 127 of culture was approximately 22.5 hours. Based on these results, primary cells and bCDK4 ＷＴ CDK4 co-expressing bTERT and bCCND1 ＷＴ In cells, a significant decrease in proliferation rate was observed during the culture period, indicating a limited number of possible divisions. In contrast, bCDK4 Ｒ２４Ｃ CDK4 co-expressing bTERT and bCCND1 Ｒ２４Ｃ In the cells, more than 100 cumulative cell divisions were observed without any decrease in the proliferation rate, suggesting that they possess unlimited proliferative capacity.

[0047] <Experiment 2: Comparison of Growth Rates> CDK4 Ｒ２４ＣCells into which a control vector has been further introduced (hereinafter also referred to as "control cells") and CDK4 Ｒ２４Ｃ Cells into which the DNp53 vector has been further introduced (hereinafter referred to as "CDK4") Ｒ２４Ｃ We used cells (also known as "DNp53 cells") to compare their proliferation rates. Culturing was performed using the same method as in Experiment 1 above, and cell counts and subculturing were carried out at 3 or 4-day intervals.

[0048] Figure 2 is an explanatory diagram comparing the growth results with and without DNp53. In Figure 2, the vertical axis shows the cumulative number of divisions (times), and the horizontal axis shows the number of culture days (days). CDK4 was used on all measurement days. Ｒ２４Ｃ The number of viable DNp53 cells was higher than that of control cells. On day 84 of culture, the cumulative number of divisions in control cells was approximately 108.4, compared to CDK4 Ｒ２４Ｃ The cumulative number of divisions of DNp53 cells was approximately 122.0. The average doubling time over the entire culture period was 19.4 hours for control cells and 19.4 hours for CDK4 cells. Ｒ２４Ｃ The DNp53 cell lifespan was 16.7 hours. Based on these results, CDK4 Ｒ２４Ｃ The average doubling time of DNp53 cells was found to be approximately 15% shorter compared to control cells. Therefore, CDK4 Ｒ２４Ｃ It was shown that by further introducing the DNp53 vector into cells that had been introduced with the vector, dominant-negative p53 could be expressed, thereby suppressing the action of endogenous p53 and improving the cell proliferation rate.

[0049] <Experiment 3: Experiment on 3D culture using microcarriers> CDK4 Ｒ２４Ｃ Cells and CDK4 Ｒ２４ＣTo compare the suitability of DNp53 cells for 3D culture using microcarriers, a 30 mL scale microcarrier culture was performed. A disposable bioreactor (ABLE, ABBWVS03A-6) was used as the culture apparatus, and Cytodex 1 (Cytodex is a registered trademark) (Cytiva, 17044802) was used as the microcarrier. The microcarrier density was 3 g / L, and the cell density was 1.0 × 10⁻⁶. ５ Each cell was seeded to a concentration of cells / mL, followed by intermittent stirring at 83 rpm (5 min) / 0 rpm (25 min) for a total of 4 hours, after which stirring was continued at 60 rpm. The culture was carried out at 37°C and 5% CO2. ２ The culture was performed in an incubator. During the culture period, samples were taken daily, and the number of viable cells on the microcarriers was measured using a cell counter NC-200 (ChemoMetec). For each cell group, in the MC+ group, after each sampling, the microcarriers in the reactor were allowed to settle naturally, and 20 mL of the supernatant was removed and replaced with fresh medium to perform a medium change. In the MC- group, culture was continued without medium changes. Furthermore, cell attachment to the microcarriers was confirmed by microscopic observation.

[0050] Figure 3 is an explanatory diagram showing the results of microcarrier culture. In Figure 3, the vertical axis represents the live cell density (×10). ５ The horizontal axis shows the number of cells / mL, and the horizontal axis shows the number of culture days. Figure 4 is an explanatory diagram showing microscopic images of cells in the MC+ group. CDK4 Ｒ２４Ｃ In cells, while microscopic observation confirmed the attachment of cells to microcarriers, proliferation did not occur, and the density of viable cells did not improve. In contrast, CDK4 Ｒ２４Ｃ In DNp53 cells, microscopic observation confirmed the colonization of cells to microcarriers, and the colonized cells proliferated, improving the viable cell density. On day 4 of culture, the viable cell density in the MC+ group was CDK4 Ｒ２４Ｃ The cells are 1.77 × 10 ５ cells / mL, CDK4 Ｒ２４Ｃ / DNp53 cells were 7.52 × 10 ５cells / mL, and in the MC- group, CDK4 Ｒ２４Ｃ The cells are 1.46 × 10 ５ cells / mL, CDK4 Ｒ２４Ｃ / DNp53 cells were 4.42 × 10 ５ The value was cells / mL.

[0051] CDK4 Ｒ２４Ｃ In cells, colonization to the microcarrier was confirmed, but even on day 4 of culture, the number of viable cells had only increased by about 1.8 times, revealing that the proliferation rate on the microcarrier was extremely slow. In contrast, CDK4 co-expressing DNp53 Ｒ２４Ｃ In DNp53 cells, the number of viable cells increased by approximately 7.5 times by day 4 of culture, indicating improved proliferation ability on microcarriers. Furthermore, CDK4 was observed on days 3-4 of culture. Ｒ２４Ｃ A decrease in proliferation rate was observed in DNp53 cells, which was thought to be due to cell death and detachment after reaching 100% confluence on the microcarrier. Comparing the doubling times on days 2-3 of culture in the MC+ group, CDK4 Ｒ２４Ｃ In cells, it is 120 hours, whereas in CDK4 Ｒ２４Ｃ In DNp53 cells, the time was 27.7 hours, indicating a clear improvement in proliferation rate.

[0052] <Experiment 4: Confirmation experiment of muscle differentiation efficiency> Control cells and CDK4 Ｒ２４ＣWe compared the muscle differentiation efficiency using DNp53 cells. Cells were seeded at the same concentration in 6-well plates and then cultured in MM until 100% confluence was reached. Subsequently, the culture medium was changed to 2% FBS differentiation induction medium (DMEM, high glucose, GlutaMAX Supplement, pyruvate + 2% Fetal Bovine Serum (South Africa Origin) + 10 mM HEPES + 1× Penicillin / Streptomycin / Amphotericin B + 2 μg / mL Gentamycin Sulfate Solution), and the cells were cultured for 3 days under muscle differentiation induction. The muscle differentiation efficiency was compared by confirming the areas where myotubes were formed under a microscope.

[0053] Figure 5 is an explanatory diagram showing a microscopic image of cells cultured under myotubation induction. In Figure 5, myotubes are observed as elongated white regions. On day 3 of myotubation induction, regions where myotubes were formed were observed in both cell types. Comparing the area where myotubes were formed, CDK4 Ｒ２４Ｃ In DNp53 cells, myotubes were formed over a significantly larger area than in control cells. Based on these results, a certain degree of muscle differentiation was observed in both cell types, but the area of ​​that region was significantly larger than that of CDK4 cells. Ｒ２４Ｃ / DNp53 cells were found to be significantly broader. Therefore, CDK4 Ｒ２４Ｃ It was shown that by further introducing the DNp53 vector into cells that had been introduced with the vector, dominant-negative p53 could be expressed, thereby suppressing the action of endogenous p53 and improving muscle differentiation efficiency.

[0054] <Experiment 5: Experiment on cell culture in the absence of coating agent> The above control cells and CDK4 Ｒ２４Ｃ We compared cell proliferation under fibronectin-coated (Fib+) and uncoated (Fib-) conditions using DNp53 cells. Cells were placed in 1.5 × 10⁶ well plates in either fibronectin-coated or uncoated conditions. ４Cells were seeded at a rate of cells / well, and then harvested by trypsin treatment at 3 or 4-day intervals to count the number of cells. The harvested cells were diluted to an appropriate concentration and then seeded again in a 6-well plate. This process was repeated until the cells reached approximately 100 divisions.

[0055] Figure 6 is an explanatory diagram comparing the proliferation results with and without a coating agent. In Figure 6, the vertical axis represents the cumulative number of divisions (times), and the horizontal axis represents the number of culture days (days). In the control cells, the number of viable cells under the Fib+ condition was higher than the number of viable cells under the Fib- condition on all measurement days. CDK4 Ｒ２４Ｃ In DNp53 cells, the proliferation rate tended to be slightly faster under the Fib+ condition, but there were also measurement days where the number of viable cells under the Fib- condition was higher than that under the Fib+ condition. The average doubling time over the entire culture period was 17.8 hours under the Fib+ condition and 22.1 hours under the Fib- condition for control cells, and CDK4 Ｒ２４Ｃ In DNp53 cells, the growth time was 16.0 hours under Fib+ conditions and 17.1 hours under Fib- conditions. In other words, growth tended to be faster under Fib+ conditions compared to Fib- conditions in all cell types, but the difference was due to CDK4 Ｒ２４Ｃ The DNp53 cells showed less severe symptoms. Therefore, CDK4 Ｒ２４Ｃ It was shown that by further introducing the DNp53 vector into vector-introduced cells, dominant-negative p53 was expressed, thereby suppressing the action of endogenous p53, and thus reducing the effect of the presence or absence of a coating agent on cell proliferation.

[0056] <Experiment 6: Comparison of Long-Term Subculture and Proliferation Rates of Bovine Adipose Stem Cells> Using adipose progenitor cells isolated from bovine adipose tissue, the same method as described above was used to compare CDK4 Ｒ２４Ｃ Cells into which the vector has been introduced (hereinafter referred to as "fatty acid CDK4") Ｒ２４Ｃ (Also called "cells") and CDK4 Ｒ２４Ｃ Cells into which the vector and DNp53 vector have been introduced (hereinafter referred to as "fatty acid CDK4") Ｒ２４ＣWe created primary cells (also called "DNp53 cells") into which the above vector had not been introduced (hereinafter also called "adipocyte primary cells") and adipocyte CDK4 Ｒ２４Ｃ Cells and fat CDK4 Ｒ２４Ｃ Using DNp53 cells, experiments were conducted to confirm the number of divisions possible and to compare the proliferation rates. The cells were subjected to the same concentration (3 × 10⁻¹⁶). ４ Cells were seeded in a collagen-coated 6-well plate (IWAKI, 4860-010) at a rate of cells / well, and then harvested by trypsin treatment at 3 or 4-day intervals. The harvested cells were mixed with an equal volume of trypan blue solution, and the number of viable cells was counted using an automated cell counter. The harvested cells were diluted to an appropriate concentration, and then seeded again in a collagen-coated 6-well plate, and subculturing was repeated until day 39 of culture.

[0057] Figure 7 is an explanatory diagram showing the results of long-term subculturing. In Figure 7, the vertical axis shows the cumulative number of divisions (times), and the horizontal axis shows the number of culture days (days). Similar to the results for muscle-derived cells in Experiment 1, the proliferation rate of adipose primary cells decreased significantly on day 21 of culture, and the doubling time from day 18 to 21 of culture was approximately 95.5 hours. In contrast, adipose CDK4 Ｒ２４Ｃ Cellular and adipose-derived CDK4 Ｒ２４Ｃ / DNp53 cells maintained their proliferation even on day 39 of culture without any decrease in proliferation rate. The cumulative number of divisions on day 39 of culture was as follows: Ｒ２４Ｃ Cells react approximately 33.7 times, fat CDK4 Ｒ２４Ｃ The number of DNp53 cells was 38.4. In the comparison of proliferation rates, on all measurement days, adipocyte CDK4 Ｒ２４Ｃ / The number of viable cells in DNp53 cells is related to adipocyte CDK4 Ｒ２４Ｃ The number of viable cells was greater than the number of viable cells. The doubling time for culture days 35-39 was CDK4 Ｒ２４Ｃ The cells were 25.3 hours old, and CDK4 Ｒ２４Ｃ The DNp53 cell lifespan was 20.7 hours. Based on these results, it became clear that adipose progenitor cells isolated from bovine adipose tissue exhibited similar trends to bovine muscle progenitor cells.

[0058] The present invention is not limited to the embodiments described above, and can be realized in various configurations without departing from its spirit. For example, the technical features in the embodiments and examples corresponding to the technical features in each form described in the summary of the invention can be replaced or combined as appropriate in order to solve some or all of the above-described problems, or to achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate.

Claims

1. A cell line established by introducing the following four genes: CDK4 (R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain into bovine progenitor cells.

2. A cell line according to claim 1, wherein the bovine progenitor cells are bovine muscle progenitor cells.

3. A method for producing bovine muscle cells, comprising the step of introducing the following four genes: CDK4 (R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain into bovine muscle progenitor cells.

4. A method for producing bovine muscle cells according to claim 3, wherein the step involves introducing the gene using a transposon vector.

5. A method for producing bovine adipocytes, comprising the step of introducing the following four genes: CDK4 (R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain into bovine adipocytes.

6. A method for producing cultured meat, comprising the step of introducing p53, which lacks the following four genes: CDK4 (R24C), TERT, CCND1, and the region encoding the DNA binding domain, into bovine muscle progenitor cells using a transposon vector.

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