Cell lines, methods for producing bovine muscle cells, methods for producing bovine adipocytes, methods for producing cultured meat

Abstract

This invention provides a technology that can improve the proliferation rate of cells in cultures using microcarriers. [Solution] The cell line is established by introducing the following four genes: CDK4(R24C), TERT, CCND1, and p53 lacking the DNA binding domain into bovine progenitor cells.

Description

Technical Field

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

Background Art

[0002] In recent years, a technique for artificially producing edible meat (cultured meat) by growing cells in vitro has 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 agitation suspension using microcarriers.

Prior Art Documents

Patent Documents

[0003] [[ID=2"2]]

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 from the viewpoint of the growth rate in culture using microcarriers. Therefore, a technique capable of improving the growth rate of cells in culture using microcarriers is required.

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: CDK4(R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain into bovine progenitor cells. According to this embodiment of the cell line, the proliferation rate of cells 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. This form of cell line can improve the cell proliferation rate in culture using microcarriers.

[0008] (3) Another form of the present disclosure provides a method for producing bovine muscle cells. This 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. This form of production 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) According to another form of the present disclosure, a method for producing bovine adipocytes is provided. This method includes introducing the following four genes: CDK4(R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain into bovine adipocyte precursor cells. According to this form of production, bovine adipocytes can be easily cultured in large quantities because they have excellent proliferation rates in culture using microcarriers.

[0011] (6) According to another embodiment of the present invention, a method for producing cultured meat is provided. 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. According to this embodiment of the method for producing cultured meat, the proliferation rate of cells can be improved in culture using microcarriers, so that bovine muscle cells can be easily cultured in large quantities, and as a result, the efficiency of cultured meat production can be improved.

[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. [Brief explanation of the drawing]

[0013] [Figure 1] This is an explanatory diagram showing the results of long-term subculturing. [Figure 2] This is an explanatory diagram comparing the proliferation results with and without DNp53. [Figure 3] This is an explanatory diagram showing the results of microcarrier culture. [Figure 4] This is an explanatory diagram showing microscopic images of cells from the MC+ group. [Figure 5] This is an explanatory diagram showing a microscopic image of cells cultured under muscle differentiation induction. [Figure 6] This is an explanatory diagram comparing the results of growth with and without a coating agent. [Figure 7] This is an explanatory diagram showing the results of long-term subculturing. [Modes for carrying out the invention]

[0014] A. Definitions of terms, etc. 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 derived from cattle 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. The sequence of bovine CDK4 (hereinafter also referred to as "bCDK4") (NM_001037594.2) is shown in Sequence ID No. 1. In this disclosure, "CDK4(R24C)" refers to the gene into which the encoding cyclin-dependent kinase 4 has been mutated so that the 24th R (arginine) is replaced with C (cysteine). An example of the sequence of bovine CDK4(R24C) (hereinafter also referred to as "bCDK4(R24C)") is shown in Sequence ID No. 2. In Sequence ID No. 2, the 70th C (cytosine) is replaced with T (thymine) compared to Sequence ID No. 1. In addition, bCDK4(R24C) may have a substitution of T at position 70, as well as a substitution of C at position 72. 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 the 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 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 Sequence ID No. 4. bCCND1 may have one or more bases deleted, substituted, or added to the sequence shown in Sequence ID No. 4, provided that the desired function is not lost.

[0019] In this disclosure, "p53" refers to 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" refers to 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 desired function is not lost.

[0020] B. Cell line 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 chopped with scissors, and then digested with collagenase to obtain bovine progenitor cells. If necessary, specific cells may be separated using a cell sorter or the like. The obtained bovine progenitor cells may be suspended in any medium and cultured in a CO2 incubator for 1 to 7 days before being subjected to transfection. Before being subjected to transfection, the bovine progenitor cells may be subcultured and further cryopreserved. The medium is not particularly limited, but any medium that can be used for culturing animal cells may be used, such as Eagle MEM medium, Dulbecco's modified Eagle MEM medium, Ham medium F12, and mixed media thereof. The conditions such as culture temperature, medium pH, and CO2 concentration 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 to introduce the gene 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, it is preferable to use a PiggyBac vector.

[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 into which genes have been introduced may be cultured in any medium suitable for animal cell culture. While not particularly limited, examples of suitable media include Eagle MEM medium, Dulbecco's modified Eagle MEM medium, Ham medium F12, and mixtures thereof. Conditions such as culture temperature, medium pH, and CO2 concentration 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] Furthermore, the cell line disclosed herein possesses unlimited proliferation ability and can suppress the increase in doubling time. Possessing unlimited proliferation ability means that immortalization has been achieved. The mechanism by which it possesses unlimited proliferation ability and suppresses the increase in doubling time is not yet clear, but the following hypothetical mechanisms are hypothesized.

[0027] 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 is a type of INK4. INK4 It inhibits the binding of cyclins to cyclin-dependent kinase 4 and the binding of cyclins to cyclin-dependent kinase 6. Mutant cyclin-dependent kinase 4 formed from the mutant CDK4(R24C) exhibits p16 compared to the wild type. INK4 The affinity for cyclins is reduced, and as a result, it is thought that the inhibition of cyclin binding can be suppressed. Therefore, it is presumed that the mutant CDK4(R24C) shows a suppression of the decrease in activity compared to wild-type CDK4.

[0028] Also, generally speaking, the Cip / Kip family is p21 Cip1 p27 Kip1 p57 Kip2 It is composed of three proteins. These proteins inhibit the activity of both cyclins and cyclin-dependent kinases by binding to them. The p53 tumor suppressor is p21 Cip1 Upstream of p21 Cip1 It controls the expression of p53. Dominant-negative p53 is thought to evade the effects of the Cip / Kip system by disrupting the function of wild-type endogenous p53. As a result, it is presumed to have unlimited proliferation capacity and suppress the increase in 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 Another embodiment of this disclosure provides a method for producing bovine muscle cells. 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 preferably 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 adipose tissue Other embodiments of this disclosure provide a method for producing bovine adipocytes. 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 this 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 cell proliferation rate in the absence of a coating agent. The bovine adipocytes produced by the method for producing bovine adipocytes of this 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: CDK4(R24C), TERT, CCND1, and p53 (DNp53) lacking the region encoding the DNA binding domain into bovine muscle progenitor cells using a PiggyBac vector.

[0036] The method for producing cultured meat according to this 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 of 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. [Examples]

[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 production (1) Sample We purchased beef cheek meat from Tokyo Shibaura Organ Co., Ltd. and isolated bovine muscle progenitor cells as follows: Muscle tissue was cut 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 1-2 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 treatment with trypsin (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 in the same manner as for 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 passed through once by treatment with trypsin (Nacalai, 35553-74).The entire amount was seeded in a separate incubator and cultured for about 3 days before being used in the transfection experiment.

[0040] (2)Cell culture Unless otherwise specified in the following description, the culture medium used was 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 the cells were cultured in a CO2 incubator (37°C, 5% CO2). 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 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 recovered 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 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, distilled water was used 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 6-well plates (IWAKI, 3810-006N). The following day, the culture medium was replaced with MM supplemented with 3 μg / mL Puromycin (Wako, 160-13151). The negative control cells were cultured until death, and CDK4 was used. 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> Regarding bovine muscle progenitor cells, primary cells that have not been introduced with the above vector 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 × 10 4 Cells 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 approximately 100 divisions were reached, or until the number of divisions plateaued. Note that experiments using primary cells and CDK4 were performed. 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 by 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 R24C Cells 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. The cells were cultured using the same method as in Experiment 1 above, and cell counts and subculturing were performed 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 measured 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 CDK4 cells. R24C The time for / DNp53 cells 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 rate of cell proliferation.

[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 device, 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 × 10⁶. 5 Each cell type was seeded to a cell / mL ratio, followed by intermittent stirring at 83 rpm (5 min) / 0 rpm (25 min) for a total of 4 hours, and then continued stirring at 60 rpm. Culture was carried out in an incubator at 37°C and 5% CO2. 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 type, 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 viable cell density (×10 5 The horizontal axis shows the number of days of culture (days), with the horizontal axis representing cells / mL. 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, resulting in an improvement in 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 are 7.52 × 10 5The cell / mL ratio is [value missing], and in the MC- group, CDK4 R24C The cells are 1.46 × 10 5 cells / mL, CDK4 R24C / DNp53 cells are 4.42 × 10 5 The value was cells / mL.

[0051] CDK4 R24C In the 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 very 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, The reason was thought to be that cell death occurred and cells detached because they reached 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: Experiment to confirm muscle differentiation efficiency> The above control cells and CDK4 R24CWe compared the muscle differentiation efficiency using / DNp53 cells. Cells were seeded in 6-well plates at the same concentration and then cultured in MM until 100% confluence was reached. Subsequently, the 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 under muscle differentiation induction for 3 days. 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 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 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 the control cells, the number of viable cells under Fib+ conditions was higher than the number of viable cells under Fib- conditions on all measurement days. CDK4 R24C In / DNp53 cells, the proliferation rate tended to be slightly faster under Fib+ conditions, but there were also days when the number of viable cells under Fib- conditions was higher than that under Fib+ conditions. The average doubling time over the entire culture period was 17.8 hours under Fib+ conditions and 22.1 hours under Fib- conditions 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 subculturing and proliferation rates of bovine adipose stem cells> Using adipose progenitor cells isolated from bovine adipose tissue, CDK4 was extracted using the same method as described above. 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 "adipophilic primary cells") and adipophilic CDK4 R24C Cells and fat CDK4 R24C Experiments were conducted to confirm the number of divisions possible and to compare the proliferation rates using / DNp53 cells. 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 adipocyte 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 / DNp53 cell viability count is adipose 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 a similar trend 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. In the cell line described in claim 1, The aforementioned bovine progenitor cells are bovine muscle progenitor cells. cell line.

3. A method for producing bovine muscle cells, The procedure 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. A method for producing bovine muscle cells.

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

5. A method for producing bovine adipose tissue, The procedure includes introducing the following four genes into bovine adipose progenitor cells: CDK4 (R24C), TERT, CCND1, and p53 lacking the region encoding the DNA binding domain. A method for producing bovine adipose tissue.

6. A method for producing cultured meat, The procedure 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 using a transposon vector. A method for producing cultured meat.

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