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

Abstract

Provided is a technique capable of improving the cell growth rate in culture using microcarriers. 【Solution means】A cell line is established by introducing the following four genes into bovine precursor cells: CDK4 (R24C), TERT, CCND1, and p53 lacking a DNA binding domain.

Description

Technical Field

[0001] The present disclosure relates to the production of cultured meat.

Background Art

[0002] In recent years, techniques 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 suspending them using microcarriers.

Prior Art Documents

Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the technique described in Non-Patent Document 1, there is room for improvement in terms of the growth rate in culture using microcarriers. For this reason, there is a need for a technique capable of improving the growth rate of cells in culture using microcarriers.

Means for Solving the Problems

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

[0006] (1) According to one embodiment 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 the region encoding the DNA binding domain. According to this form of cell line, the cell growth rate can be improved in culture using microcarriers.

[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 cell growth rate can be improved in culture using microcarriers.

[0008] (3) According to another embodiment of the present disclosure, a method for producing bovine muscle cells is provided. This production method includes a step of introducing the following four genes into bovine muscle progenitor cells: CDK4(R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain. According to this form of production method, since the growth rate is excellent in culture using microcarriers, bovine muscle cells can be easily mass-cultured.

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

[0010] (5) According to another embodiment of the present disclosure, a method for producing bovine adipose cells is provided. This production method includes a step of introducing the following four genes into bovine adipose progenitor cells: CDK4(R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain. According to this form of production method, since the growth rate is excellent in culture using microcarriers, bovine adipose cells can be easily mass-cultured.

[0011] (6) According to another aspect of the present invention, a method for producing cultured meat is provided. This method for producing cultured meat includes a step of introducing, into bovine muscle progenitor cells, the following four genes: CDK4(R24C), TERT, CCND1, and p53 lacking a region encoding a DNA binding domain, using a transposon vector. According to this method for producing cultured meat, since the growth rate of cells can be improved in culture using microcarriers, bovine muscle cells can be easily cultured in large quantities, and as a result, the production efficiency of cultured meat can be improved.

[0012] Note that the present disclosure can be implemented in various forms. For example, it can be implemented in forms such as a method for establishing a cell line, a method for producing a cell line, cultured meat containing bovine muscle cells, cultured meat containing bovine fat cells, a method for producing cultured meat using microcarrier culture, and the use of a cell line for producing cultured meat.

Brief Description of the Drawings

[0013]

Figure 1

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Modes for Carrying Out the Invention

[0014] A. Definitions of Terms, etc. In the present disclosure, "bovine" means an animal belonging to the subfamily Bovinae, preferably Bos taurus of the domestic animal species. In the present disclosure, "bovine progenitor cells" means cells among bovine-derived cells that are expected to differentiate into specific types of bovine cells or form specific types of bovine tissues. The bovine progenitor cells in the present disclosure may preferably include mesenchymal stem cells having pluripotency to myocytes (muscle cells) or adipocytes (fat cells). The bovine progenitor cells are not particularly limited, and examples include progenitor cells derived from bovine tissues, such as muscle progenitor cells derived from muscle and fat progenitor cells derived from fat. The bovine muscle is not particularly limited, and examples include skeletal muscle, cardiac muscle, and smooth muscle, with skeletal muscle being preferred. The skeletal muscle is not particularly limited, and examples include muscle tissues of the cheek, temple, neck, back, chest, shoulder, waist, and thigh.

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

[0016] In the present disclosure, "CDK4" means a 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 cyclin. The sequence of bovine CDK4 (hereinafter also referred to as "bCDK4") (NM_001037594.2) is shown in SEQ ID NO: 1. In the present disclosure, "CDK4(R24C)" means a gene in which a mutation has been introduced such that the 24th R (arginine) in the encoded cyclin-dependent kinase 4 is replaced by C (cysteine). An example of the sequence of bovine CDK4(R24C) (hereinafter also referred to as "bCDK4(R24C)") is shown in SEQ ID NO: 2. Compared with SEQ ID NO: 1, the 70th C (cytosine) in SEQ ID NO: 2 is replaced by T (thymine). In addition to the replacement of the 70th C with T, the 72nd T in bCDK4(R24C) may be replaced by C. bCDK4(R24C) may have one or more bases deleted, substituted, or added to the sequence shown in SEQ ID NO: 2 as long as the desired function is not lost.

[0017] In the present disclosure, "TERT" means a gene encoding 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 SEQ ID NO: 3. bTERT may have one or more bases deleted, substituted, or added to the sequence shown in SEQ ID NO: 3 as long as the desired function is not lost.

[0018] In the present disclosure, "CCND1" means a 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, as long as the target function is not lost.

[0019] In the present disclosure, "p53" means a gene encoding the p53 tumor suppressor. The sequence of bovine p53 (hereinafter also referred to as "bp53") (NM_174201.2) is shown in SEQ ID NO: 5. In the present disclosure, "p53 lacking the region encoding the DNA binding domain" means p53 in which the region encoding the DNA binding domain is deleted with respect to wild-type p53. Since p53 lacking the region encoding the DNA binding domain is a dominant-negative type of p53, it is hereinafter also referred to as "DNp53". DNp53 includes a mode in which the DNA binding domain is not formed due to a partial deletion of the region encoding the DNA binding domain. In addition, DNp53 may have a partial deletion of a region other than the region encoding the DNA binding domain with respect to wild-type p53. An example of the sequence of bovine DNp53 (hereinafter also referred to as "bDNp53") is shown in SEQ ID NO: 6. bDNp53 may have one or more bases deleted, substituted, or added to the sequence shown in SEQ ID NO: 6, as long as the target function is not lost.

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

[0021] Bovine progenitor cells can be collected by treating excised bovine tissues with collagenase. More specifically, for example, bovine progenitor cells can be obtained by excising any tissue from the cheek or visceral fat of a slaughtered cow, cutting it finely with scissors, etc., and then digesting it using collagenase or the like. If necessary, specific cells may be separated using a cell sorter or the like. The obtained bovine progenitor cells are suspended in an arbitrary medium and may be cultured in a CO 2 incubator for about 1 to 7 days and then subjected to transfection. Note that the bovine progenitor cells may be subcultured and further cryopreserved before being subjected to transfection. The medium is not particularly limited, and for example, any medium that can be used for culturing animal cells, such as Eagle's MEM medium, Dulbecco's modified Eagle's MEM medium, Ham's medium F12, and mixed media thereof, may be used. Conditions such as the culture temperature, medium pH, and CO 2 concentration may be appropriately adopted as conditions generally used in animal cell culture.

[0022] As a method for introducing the gene, it is not particularly limited as long as it can introduce the above gene into bovine progenitor cells, but stable transfection is preferred rather than transient transfection. As a method for introducing the gene, for example, a method of introducing it using a vector can be mentioned. The vector is not particularly limited as long as it can introduce the above gene into bovine progenitor cells, but from the viewpoint of suppressing a decrease in safety, it is preferably a non-viral vector. The non-viral vector is not particularly limited, and for example, a transposon vector can be mentioned. The transposon vector is not particularly limited, and for example, PiggyBac vector, Sleeping Beauty vector, Tol2 vector, etc. can be mentioned, but from the viewpoint of gene introduction efficiency, it is preferable to use a PiggyBac vector.

[0023] The above four genes may be introduced separately or together. More specifically, when introducing using a transposon vector, a plurality of genes may be arranged in one vector and introduced, or each gene may be arranged in a separate vector and introduced. In addition, even when a plurality of genes are arranged in series in one vector and introduced, each protein is independently expressed.

[0024] The cells after the gene is introduced may be cultured in any medium that can be used for culturing animal cells. The medium is not particularly limited, and for example, Eagle's MEM medium, Dulbecco's modified Eagle's MEM medium, Ham's medium F12, and mixed media thereof can be mentioned. Conditions such as culture temperature, medium pH, and CO 2 Concentration and other conditions may be appropriately adopted under conditions generally used in animal cell culture.

[0025] According to the cell line of the present disclosure, it is excellent in suitability for microcarrier culture. More specifically, it is excellent in growth rate in culture using microcarriers.

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

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

[0028] Moreover, generally, the Cip / Kip family is composed of three proteins, p21 Cip1 , p27 Kip1 , and p57 Kip2 . These proteins inhibit the 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 the dominant-negative type of p53, it is considered that the action of the Cip / Kip system can be avoided by disturbing the function of the endogenous wild-type p53. As a result, it is presumed that it has the ability of infinite proliferation and can suppress the increase in doubling time.

[0029] Moreover, according to the cell line of the present disclosure, the differentiation efficiency is excellent. In particular, when bovine muscle progenitor cells are used, the muscle differentiation efficiency is excellent. The differentiation efficiency can be confirmed, for example, by observing the characteristic structure of the tissue using a microscope. In addition, the differentiation efficiency may be confirmed by examining the expression of various marker genes and the like by immunostaining or the like. Further, according to the cell line of the present disclosure, it is possible to suppress a decrease in the cell growth rate even in the absence of a coating agent.

[0030] Since the cell line of the present disclosure has an excellent growth rate in culture using microcarriers, large-scale culture becomes easy, and as a result, it is suitable for the production of cultured meat. The microcarriers used for culturing cells are not particularly limited, and examples thereof include microcarriers formed of resin materials such as polystyrene and polyvinyl alcohol, acrylamide, glass, collagen, cellulose, gelatin, polysaccharides, and the like. In addition, the cell line of the present disclosure is suitable for the production of cultured meat also in that it has an infinite proliferative ability and can suppress an increase in the doubling time. Furthermore, the cell line of the present disclosure is suitable for the production of cultured meat also in that it has excellent differentiation efficiency and can suppress a decrease in the cell growth rate in the absence of a coating agent. Note that the cell line of the present disclosure may be cultured by a culture method that does not use microcarriers. The culture method that does not use microcarriers is not particularly limited, and examples thereof include suspension culture and adherent culture that do not use microcarriers. From the viewpoint of large-scale culture, suspension culture is preferably used, and suspension culture using a bioreactor, an automatic culture device, or the like is more preferably used. In addition, the cell line of the present disclosure may be used not only for cultured meat but also for regenerative medicine, research materials, and the like.

[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 a step of introducing into bovine muscle progenitor cells the following four genes: CDK4 (R24C), TERT, CCND1, and p53 lacking a region encoding a DNA binding domain. Note that the bovine muscle progenitor cells may be cells that have undergone subculture, or cells that have undergone cryopreservation and subsequent thawing.

[0032] In the step of introducing the genes, the genes can be introduced, for example, using the method described above, but it is preferable to introduce the genes using a transposon vector, and more preferably to introduce the genes using a PiggyBac vector. The method for producing bovine muscle cells of the present disclosure may include a step of culturing the cells in a medium after the step of introducing the genes. The bovine muscle progenitor cells into which the four genes have been introduced are preferably selected using the expression of an antibiotic or the like as an indicator. That is, the method for producing bovine muscle cells of the present disclosure may include a step of selecting the cells into which the four genes have been introduced after the step of introducing the genes. Further, the method for producing bovine muscle cells of the present disclosure may include a step of subculturing the cells in a medium after the step of introducing the genes or after the step of selecting the cell line. Further, the method for producing bovine muscle cells of the present disclosure may include a step of culturing the cells using a microcarrier. The microcarrier to be used is not particularly limited, and examples thereof include microcarriers formed of resin materials such as polystyrene and polyvinyl alcohol, acrylamide, glass, collagen, cellulose, gelatin, polysaccharides, and the like. Note that in the method for producing bovine muscle cells of the present disclosure, the cells may be cultured by a culturing method without using a microcarrier, such as the one described above.

[0033] The method for producing bovine muscle cells of the present disclosure can improve the cell growth rate in culture using microcarriers, which facilitates large-scale culture and is thus suitable for the production of cultured meat. Also, it is suitable for the production of cultured meat because it has the ability to proliferate indefinitely and can suppress the increase in doubling time. Furthermore, it is suitable for the production of cultured meat due to its excellent differentiation efficiency and the ability to suppress the decrease in the cell growth rate in the absence of a coating agent. Note that the bovine muscle cells produced by the method for producing bovine muscle cells of the present disclosure may be used not only for the production of cultured meat but also for regenerative medicine, research materials, and the like.

[0034] D. Method for Producing Bovine Adipocytes According to another embodiment of the present disclosure, a method for producing bovine adipocytes is provided. The method for producing bovine adipocytes includes a step of introducing the following four genes: CDK4 (R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain into bovine adipose progenitor cells. The method for producing bovine adipocytes may be the same as the above-described method for producing bovine muscle cells except that the primary cells used are different. According to the method for producing bovine adipocytes of the present disclosure, the cell growth rate can be improved in culture using microcarriers, which facilitates large-scale culture and is thus suitable for the production of cultured meat. Also, it is suitable for the production of cultured meat because it has the ability to proliferate indefinitely and can suppress the increase in doubling time. Furthermore, it is suitable for the production of cultured meat due to its excellent differentiation efficiency and the ability to suppress the decrease in the cell growth rate in the absence of a coating agent. Note that the 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, and the like.

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

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

[0037] In addition, the method for producing cultured meat of the present disclosure preferably includes a step of culturing the cells into which the above four genes have been introduced and a step of forming a tissue with the proliferated cells. The culturing method in the step of culturing the cells is not particularly limited, and for example, the method as described above may be used. The method for forming a tissue is not particularly limited, and for example, the following method may be used. First, the proliferated myoblasts are induced to differentiate into myotubes. Thereby, the myoblasts are multinucleated by cell fusion with surrounding cells to form myotubes. The myotubes are further matured to form muscle fibers. In the method for producing cultured meat, for example, a three-dimensional tissue may be formed by laminating sheet-like muscle cells and adipose cells, or a three-dimensional tissue may be formed by embedding a cell mass containing muscle cells and adipose cells in a collagen gel or the like.

Examples

[0038] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples.

[0039] 1. Preparation of cells (1) Samples Bovine neck muscle was purchased from Tokyo Shibaura Zoki Corporation, and bovine muscle progenitor cells were isolated as follows. Muscle tissue was excised and minced using a mincer (manufactured by Bonny, 49 3683400500 2) and scissors. To the minced muscle tissue, a 0.2% collagenase solution (DMEM, high glucose, GlutaMAX Supplement, pyruvate (manufactured by Gibco, 10569-044) + 2 mg / mL collagenase (manufactured by Fujifilm Wako Pure Chemical Corporation, 032-22364) + 25 U / mL deoxyribonuclease I (manufactured by Sigma, D5025) + 10 mM HEPES (manufactured by Nacalai, 17557-94) + 1×Penicillin / Streptomycin / Amphotericin B (manufactured by Nacalai, 02892-54) + 2 μg / mL Gentamycin Sulfate Solution (manufactured by Nacalai, 16672-04)) was added, and the mixture was stirred at 37°C for 1 to 2 hours using a magnetic stirrer. The treated solution was filtered through a 59-μm nylon mesh (manufactured by AS ONE, PA-59μ), and the filtrate was centrifuged at 800 g for 5 minutes. The precipitate was suspended in HBSS (manufactured by Nacalai, 09735-75) containing 4% FBS and centrifuged again at 800 g for 5 minutes. The precipitate was suspended again in HBSS containing 4% FBS and filtered through a 100-μm cell strainer (manufactured by Corning, 352360) to obtain bovine muscle progenitor cells. Additionally, a cell sorter (FACS Aria II manufactured by Becton Dickinson) was used to separate specific cells as needed. The obtained bovine muscle progenitor cells were cultured for about 7 days under the culture conditions described below and then passaged once by treatment with trypsin (manufactured by Nacalai, 35553-74). The entire amount of these cells was seeded into another culture vessel and cultured for about 3 days, and the resulting cells were used for the transfection experiment. For the preparation of bovine adipose progenitor cells, adipose tissue excised from bovine visceral fat (pericardial fat) purchased from Tokyo Shibaura Zoki Corporation was used. The preparation of bovine adipose progenitor cells was performed in the same manner as the preparation of bovine muscle progenitor cells. The obtained bovine adipose progenitor cells were cultured for about 7 days under the culture conditions described below and then passaged once by treatment with trypsin (manufactured by Nacalai, 35553-74).The whole amount of this was seeded into another incubator, and the product cultured for about 3 days was used for the transfection experiment.

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

[0041] (3) Preparation of vectors The following five vectors were prepared. Four vectors other than 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. The 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 into which bCDK4 (R24C mutant), bTERT, and bCCND1 are inserted. CDK4 shown in SEQ 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 into which bCDK4 (WT), bTERT, and bCCND1 are inserted. The DNp53 vector (pPB[Exp]-Hygro-EF1A>cwTP53[NM_174201.2]*) shown in SEQ ID NO: 10 is a transposon plasmid into which bDNp53 is inserted. The control vector (pOF002_pPB-hEF1Ap-MCS (HygR)) shown in SEQ ID NO: 11 is a plasmid into which bDNp53 is not inserted relative to the DNp53 vector.

[0043] (4) Transfection Bovine muscle progenitor cells were collected by trypsin (manufactured by Nacalai, 35553-74) treatment and seeded at 2×10 4 cells / well in a 24-well plate (manufactured by IWAKI, 3820-024) and cultured for 1 day. Plasmid DNA (hyPBase vector + CDK4 R24C vector, or hyPBase vector + CDK4 WTThe (vector) was diluted in Opti-MEM (registered trademark) at 20 μg / mL. It was mixed 1:1 with 120 μL / mL ViaFect Transfection Reagent (manufactured by Promega, E4981) diluted in the same way and allowed to stand at room temperature for 15 minutes. In the negative control, pure water was added instead of the DNA solution. The culture medium in each well was replaced with 450 μL Opti-MEM (registered trademark), and 50 μL of the prepared liposome solution was added thereto (0.5 μg / well plasmid DNA). The cells were cultured in an incubator for 6 hours, and the culture medium was replaced with MM. The next day, the cells were passaged and seeded in a 6-well plate (manufactured by IWAKI, 3810-006N). The next day, the culture medium was replaced with MM supplemented with 3 μg / mL Puromycin (manufactured by Wako, 160-13151). The cells were cultured until the negative control cells died, and CDK4 R24C vector-introduced cells and CDK4 WT vector-introduced cells were obtained.

[0044] The CDK4 obtained above R24C For the vector-introduced cells obtained above, the DNp53 vector or the control vector was transfected by the same procedure (hyPBase vector + DNp53 vector, or hyPBase vector + control vector). For selection, 300 μg / mL Hygromycin B (manufactured by Wako, 089-06151) was used, and CDK4 R24C co-expressing cells of the vector and the DNp53 vector were prepared.

[0045] 2. Experimental methods and results <Experiment 1: Experiment on long-term subculture> Regarding bovine muscle progenitor cells, primary cells not introduced with the above vector, and cells introduced with the CDK4 WT vector (hereinafter also referred to as "CDK4 WT cells") and cells introduced with the CDK4 R24C vector (hereinafter also referred to as "CDK4 R24C cells") were used to conduct an experiment to confirm the number of divisions possible. The cells were adjusted to the same concentration (1×104 -3×10 4 cells / well) were seeded into 6-well plates, and then the cells were harvested by trypsin treatment at 3- or 4-day intervals. The harvested cells were mixed with an equal volume of trypan blue solution (manufactured by Invitrogen, T10282), and the viable cell count was measured using an automatic cell counter (Countess3, manufactured by Invitrogen). The harvested cells were diluted to an appropriate concentration and seeded into 6-well plates in the same manner. This was repeated until reaching approximately 100 cell divisions or until the division rate plateaued. In addition, experiments using primary cells and CDK4 WT cells and CDK4 R24C cells were conducted on a different day from those using primary cells.

[0046] Figure 1 is an explanatory diagram showing the results of long-term subculture. In Figure 1, the vertical axis represents the cumulative number of cell divisions (times), and the horizontal axis represents the number of culture days (days). The primary cells showed a significant decrease in the growth rate on the 21st day of culture, and the doubling time from the 18th to the 21st day of culture was approximately 144.5 hours. CDK4 WT cells showed a significant decrease in the growth rate on the 10th day of culture, and the doubling time from the 7th to the 10th day of culture was approximately 108.6 hours. In contrast, CDK4 R24C cells reached 100 cell divisions without a decrease in the growth rate, and the growth rate was maintained thereafter. CDK4 R24C cells reached a cumulative number of cell divisions of approximately 129.5 times on the 127th day of culture, and the doubling time from the 123rd to the 127th day of culture was approximately 22.5 hours. According to the above results, in primary cells and CDK4 WT cells co-expressing bCDK4, bTERT, and bCCND1, a significant decrease in the growth rate was observed during the culture period, indicating that the number of possible cell divisions is limited. In contrast, in CDK4 WT cells co-expressing bCDK4, bTERT, and bCCND1, cell divisions exceeding 100 times in cumulative were observed without a decrease in the growth rate, so it was expected to have infinite proliferative ability. R24C cells co-expressing bCDK4, bTERT, and bCCND1 R24C cells, cell divisions exceeding 100 times in cumulative were observed without a decrease in the growth rate, so it was expected to have infinite proliferative ability.

[0047] <Experiment 2: Comparative experiment on growth rate> CDK4 R24C Cells into which a control vector was further introduced (hereinafter also referred to as "control cells"), and CDK4 R24C Cells into which a DNp53 vector was further introduced (hereinafter also referred to as "CDK4 R24C / DNp53 cells") were used to compare the growth rates. Cultivation was performed in the same manner 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 showing a comparison of the growth results with or without DNp53. In Figure 2, the vertical axis indicates the cumulative number of divisions (times), and the horizontal axis indicates the number of days of culture (days). At all measurement days, the viable cell count of CDK4 R24C / DNp53 cells was higher than that of the control cells. On the 84th day of culture, the cumulative number of divisions of the control cells was about 108.4 times, while that of the CDK4 R24C / DNp53 cells was about 122.0 times. The average doubling time during the entire culture period was 19.4 hours for the control cells and 16.7 hours for the CDK4 R24C / DNp53 cells. According to the above results, it was found that the average doubling time of the CDK4 R24C / DNp53 cells was shortened by about 15% compared with that of the control cells. Therefore, it was shown that the growth rate of cells can be improved by further introducing a DNp53 vector into cells into which a CDK4 R24C vector has been introduced to express a dominant negative type of p53, thereby suppressing the action of endogenous p53.

[0049] <Experiment 3: Experiment on three-dimensional culture using microcarriers> CDK4 R24C cells and CDK4 R24CTo compare the suitability of 3D culture using microcarriers for / DNp53 cells, microcarrier culture at a 30 mL scale was performed. As the culture device, a disposable bioreactor (manufactured by ABLE, ABBWVS03A-6) was used, and as the microcarrier, Cytodex 1 (Cytodex is a registered trademark) (manufactured by Cytiva, 17044802) was used. Each cell was seeded so that the microcarrier density was 3 g / L and the cell density was 1.0×10 5 cells / mL. Then, intermittent stirring at 83 rpm (5 min) / 0 rpm (25 min) was repeated for a total of 4 hours, and then stirring was continued at 60 rpm. The culture was carried out in an incubator at 37°C and 5% CO 2 . During the culture period, sampling was performed daily, and the number of viable cells on the microcarriers was measured using a cell counter NC-200 (manufactured by ChemoMetec). For each cell, in the MC+ group, after each sampling, the microcarriers in the reactor were allowed to sediment naturally, 20 mL of the supernatant was removed, and the medium was exchanged with fresh medium. In the MC- group, the culture was continued without medium exchange. Also, 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 viable cell density (×10 5 cells / mL), and the horizontal axis represents the number of culture days (days). Figure 4 is an explanatory diagram showing a microscopic image of the cells in the MC+ group. For CDK4 R24C cells, although cell attachment to the microcarriers was confirmed by microscopic observation, the cells did not proliferate and the viable cell density did not improve. In contrast, for CDK4 R24C / DNp53 cells, cell attachment to the microcarriers was confirmed by microscopic observation, and the attached cells proliferated, resulting in an improvement in the viable cell density. The viable cell density on the 4th day of culture was 1.77×10 R24C cells / mL for CDK4 5 cells and 7.52×10 R24C cells / mL for CDK45 cells / mL, and in the MC group, CDK4 R24C cells were 1.46×10 5 cells / mL, and CDK4 R24C / DNp53 cells were 4.42×10 5 cells / mL.

[0051] CDK4 R24C cells, attachment to microcarriers was confirmed, but even on the 4th day of culture, the viable cell count only increased up to about 1.8-fold, indicating that the growth rate on microcarriers was extremely slow. In contrast, in CDK4 R24C / DNp53 cells, the viable cell count increased up to about 7.5-fold by the 4th day of culture, indicating an improved growth ability on microcarriers. Note that in CDK4 R24C / DNp53 cells, a decrease in the growth rate was observed on the 3rd - 4th day of culture. This was thought to be due to cell death and detachment caused by reaching 100% confluence on microcarriers. When comparing the doubling time on the 2nd - 3rd day of culture in the MC+ group, it was 120 hours in CDK4 R24C cells, while it was 27.7 hours in CDK4 R24C / DNp53 cells, indicating a clear improvement in the growth rate.

[0052] <Experiment 4: Confirmation experiment of myogenic differentiation efficiency> The above control cells and CDK4 R24CUsing CDK4 / DNp53 cells, the myogenic differentiation efficiency was compared. Cells were seeded at the same concentration in 6-well plates and then cultured in MM until they reached 100% confluence. Subsequently, the medium was changed to a 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 myogenic differentiation induction. The comparison of myogenic differentiation efficiency was performed by microscopically examining the area where myotubes were formed.

[0053] Figure 5 is an explanatory diagram showing microscopic images of cells cultured under myogenic differentiation induction. In Figure 5, myotubes are observed as elongated white regions. On the third day of myogenic differentiation induction, areas where myotubes were formed were seen in both cell types. When comparing the areas of the regions where myotubes were formed, CDK4 R24C / DNp53 cells formed myotubes in a significantly larger area than control cells. According to the above results, a certain myogenic differentiation region was observed in both cell types, but the area of that region was clearly larger in CDK4 R24C / DNp53 cells. Therefore, it was found that by further introducing a DNp53 vector into cells transfected with the CDK4 R24C vector to express dominant-negative p53 and thereby suppressing the action of endogenous p53, the myogenic differentiation efficiency can be improved.

[0054] <Experiment 5: Experiment on Cultivation in the Absence of a Coating Agent> Using the above control cells and CDK4 R24C / DNp53 cells, cell proliferation was compared under fibronectin-coated conditions (Fib+) and non-coated conditions (Fib-). Cells were seeded at 1.5×10 in fibronectin-coated or non-coated 6-well plates 4Cells were seeded at cells / well, and then harvested by trypsin treatment at 3- or 4-day intervals to measure the cell count. The harvested cells were diluted to an appropriate concentration and then seeded into 6-well plates in the same manner. This was repeated until approximately 100 divisions were reached.

[0055] Figure 6 is an explanatory diagram showing a comparison of the growth results with or without the 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 control cells, at all measurement days, the number of viable cells under the Fib+ condition was higher than that under the Fib- condition. CDK4 R24C / DNp53 cells showed a slightly faster growth rate under the Fib+ condition, but there were also measurement days when the number of viable cells under the Fib- condition was higher than that under the Fib+ condition. The average doubling time during the entire culture period was 17.8 hours under the Fib+ condition and 22.1 hours under the Fib- condition in control cells, and CDK4 R24C / DNp53 cells were 16.0 hours under the Fib+ condition and 17.1 hours under the Fib- condition. That is, in both cell types, there was a tendency for faster growth under the Fib+ condition compared to the Fib- condition, but the difference was more minor in CDK4 R24C / DNp53 cells. Therefore, CDK4 R24C By further introducing the DNp53 vector into cells transfected with the CDK4 vector to express dominant-negative p53 and thereby suppressing the function of endogenous p53, it was shown that the effect of the presence or absence of the coating agent on cell growth was reduced.

[0056] <Experiment 6: Comparative experiment on long-term subculture and growth rate in bovine adipose stem cells> Using preadipocytes isolated from bovine adipose tissue, in the same manner as the above method, cells transfected with the CDK4 R24C vector (hereinafter also referred to as "adipose CDK4 R24C cells") and cells transfected with the CDK4 R24C vector and the DNp53 vector (hereinafter referred to as "adipose CDK4 R24C(also referred to as "preadipocyte / DNp53 cells") were prepared. Primary cells without the above vector introduced (hereinafter also referred to as "adipose primary cells") and adipose CDK4 R24C cells and adipose CDK4 R24C / DNp53 cells were used to conduct an experiment for confirming the number of possible divisions and comparing the growth rates. The cells were seeded in a collagen-coated 6-well plate (manufactured by IWAKI, 4860-010) at the same concentration (3×10 4 cells / well) with each other, and then the cells were collected by trypsin treatment at intervals of 3 or 4 days. The collected cells were mixed with an equal volume of trypan blue solution, and the number of viable cells was measured using an automatic cell counter. The collected cells were diluted to an appropriate concentration and then seeded in a collagen-coated 6-well plate in the same manner, and subculture was repeated until the 39th day of culture.

[0057] FIG. 7 is an explanatory diagram showing the results of long-term subculture. In FIG. 7, the vertical axis represents the cumulative number of divisions (times), and the horizontal axis represents the number of culture days (days). Similar to the results of muscle-derived cells in Experiment 1, the growth rate of adipose primary cells significantly decreased on the 21st day of culture, and the doubling time from the 18th to the 21st day of culture was approximately 95.5 hours. In contrast, adipose CDK4 R24C cells and adipose CDK4 R24C / DNp53 cells maintained growth even on the 39th day of culture without a decrease in the growth rate. The cumulative number of divisions on the 39th day of culture was approximately 33.7 times for adipose CDK4 R24C cells and 38.4 times for adipose CDK4 R24C / DNp53 cells. In the comparison of the growth rates, the number of viable cells of adipose CDK4 R24C / DNp53 cells was more than that of adipose CDK4 R24C cells on all measurement days. The doubling time from the 35th to the 39th day of culture was 25.3 hours for CDK4 R24C cells and 20.7 hours for CDK4 R24C / DNp53 cells. According to the above results, it became clear that the same tendency as that of bovine muscle progenitor cells was also observed for adipose progenitor cells isolated from bovine adipose tissue.

[0058] The present invention is not limited to the above-described embodiments, and can be realized in various configurations without departing from the gist thereof. 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 appropriately replaced or combined in order to solve some or all of the above-described problems, or to achieve some or all of the above-described effects. Further, if the technical feature is not described as essential in this specification, it can be appropriately deleted.

Claims

1. A cell line comprising: It was established by introducing the following four genes into bovine progenitor cells: CDK4 (R24C), TERT, CCND1, and a p53 lacking the region encoding the DNA binding domain, The cell line, wherein the bovine progenitor cells are bovine muscle progenitor cells or bovine preadipocytes.

2. 2. The cell line of claim 1, The bovine progenitor cells are bovine muscle progenitor cells. cell line.

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

4. The method for producing bovine muscle cells according to claim 3, In the step, the gene is introduced using a transposon vector. A method for producing bovine muscle cells.

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

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