Method for producing a cell population containing immortalized mesenchymal cells and a cell population containing immortalized mesenchymal cells

Abstract

To provide methods for stably producing cell populations containing immortalized mesenchymal cells capable of subculture, and to provide cell populations containing immortalized mesenchymal cells.SOLUTION: The present invention provides a method for producing a cell population containing immortalized mesenchymal cells, which comprises: a) preparing a cell population containing mesenchymal cells; b) culturing the cell population of a) with a medium containing a platelet lysate; and c) introducing a plasmid with a gene encoding adenoviral E1A and E1B regions respectively, a gene encoding the SV40 large T antigen, and a gene encoding the Bcl-xL protein to a cell constituting the cell population containing the mesenchymal cells of b).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a cell population containing immortalized mesenchymal cells that can be utilized in the fields of medicine, biochemistry, and the like and that can be stably subcultured, and to immortalized mesenchymal cells. [Background technology]

[0002] Mesenchymal stem cells, which are included in interstitial cells, are known as somatic stem cells that exist in bone marrow, adipose tissue, dental pulp, and fetal appendages such as the placenta, umbilical cord, and fetal membrane. Somatic stem cells have been particularly applied to regenerative medicine, and in recent years, new therapies that promote the regeneration and functional improvement of tissues and organs by administering cells such as mesenchymal cells to patients have been rapidly put into practical use. For example, bone marrow mesenchymal cell preparations for patients with acute graft-versus-host disease (GVHD) and spinal cord injury, and skeletal myoblast cell sheets for patients with severe heart failure are being sold in Japan as regenerative medicine products.

[0003] Mesenchymal cells usually have the property of adhering to a culture substrate, and in the production of the cell preparation, in addition to the step of adhering the cells to the culture substrate and culturing them, a step of detaching the cells adhered to the culture substrate is required. The above-mentioned bone marrow mesenchymal cells also have the property of adhering to a culture substrate, and after the cells are grown and cultured in a state of being adhered to the culture substrate, a step of detaching the cells from the culture substrate is carried out to recover the cell suspension. Examples of the method of detaching the cells from the culture substrate include chemical means using proteolytic enzymes such as trypsin or chemicals, and physical means using a tool such as a cell scraper that physically detaches the cells. For example, Patent Document 1 discloses a method of adding trypsin to a culture substrate and detaching mesenchymal cells. Patent Document 2 discloses a method of detaching cells from a culture substrate by temperature change using a culture substrate whose surface is coated with a temperature-responsive polymer whose cell adhesiveness changes depending on the temperature.

[0004] Mesenchymal cells usually have a limited number of cell divisions. In order to permanently use highly useful mesenchymal cells, particularly mesenchymal stem cells, there are known techniques for immortalizing mesenchymal cells such as bone marrow mesenchymal cells and amniotic mesenchymal cells. Non-Patent Document 1 shows that the life span of bone marrow stem cells is extended by introducing genes having base sequences encoding bmi-1, hTERT, and E6 and E7 of human papillomavirus (HPV) into bone marrow stem cells. In addition, Patent Document 3 discloses a method for generating a permanent amniotic cell line, which includes introducing at least one nucleic acid that causes expression of gene products of the E1A and E2B regions into amniotic cells. Patent Document 4 discloses a method for generating a permanent human amniotic cell line, which includes a step of introducing a nucleic acid molecule containing a base sequence encoding adenovirus gene functions E1A and E1B into primary human amniotic cells, followed by a step of introducing a nucleic acid molecule containing a base sequence encoding SV40 large T antigen or Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1).

[0005] Many of the recombinant polypeptides or proteins used in pharmaceutical compositions and the like are produced using animal cells, particularly mammalian cells, and particularly cells derived from humans. Research is also being conducted on the production of recombinant peptides or recombinant proteins using mammalian, particularly human-derived mesenchymal cells as hosts. Patent Document 5 discloses an adenovirus-producing cell line in which an expression construct containing (a) a promoter operable in animal cells, and (b) an adenovirus E1 coding modified gene sequence is introduced into animal cells such as A549, HER, HeLa, and CHO. It is also disclosed that human amniotic cells can be used as the above-mentioned animal cells. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 5394932 [Patent Document 2] International Publication No. 2001 / 068799 [Patent Document 3] Special table 2003-514526 [Patent Document 4] Special table 2012-516690 [Patent Document 5] Special table 2015-500007 [Non-patent literature]

[0007] [Non-Patent Document 1] Mori, T. et al. Combination of hTERT and bmi-1, E6, or E7 induces prolongation of the life span of bone marrow stromal cells from an elderly donor without affecting their neurogenic potential, Mol. Cell Biol., 25: 5183-5195, (2005). Summary of the Invention [Problem to be solved by the invention]

[0008] Methods for obtaining immortalized mesenchymal cells that can be subcultured permanently are known, such as those described in Patent Documents 3 and 4 and Non-Patent Document 1. However, the present inventors have found that mesenchymal cells into which an immortalization gene has been introduced by known methods do not necessarily become stably immortalized cells. Therefore, a method for obtaining more stably immortalized mesenchymal cells is desired.

[0009] An objective of the present invention is to provide a method for stably and efficiently producing a cell population containing immortalized mesenchymal cells, and a cell population containing immortalized mesenchymal cells. [Means for solving the problem]

[0010] As a result of extensive investigations, the present inventors have found that a cell population containing mesenchymal cells is cultured in a medium containing a platelet lysate, and then the cells constituting the cell population are transfected with genes encoding the E1A and E1B regions of adenovirus, a gene encoding the SV40 large T antigen, and Bcl-x L The present inventors have found that a cell population containing stably immortalized mesenchymal cells can be obtained by introducing a plasmid carrying a gene encoding the above-mentioned gene, thereby completing the present invention. That is, according to the present specification, the following invention is provided.

[0011] (1)a) preparing a cell population containing mesenchymal cells; b) culturing the cell population of a) with a medium containing a platelet lysate; and c) A cell that constitutes the cell population containing mesenchymal cells of b) is treated with a gene encoding the E1A region and the E1B region of adenovirus, a gene encoding the SV40 large T antigen, and Bcl-x L introducing a plasmid carrying a gene encoding the protein; A method for producing a cell population comprising immortalized mesenchymal cells. (2) The method according to (1), wherein in the cell population prepared in step a), the proportion of CD324-positive mesenchymal cells is 70% or more, the proportion of CD90-positive mesenchymal cells is 90% or more, and the cell population contains cells expressing the TERT gene. (3) The method according to (1) or (2), wherein the proportion of CD326-positive mesenchymal cells in the cell population prepared in step a) is 10% or less. (4) Any of the methods (1) to (3), wherein in the cell population prepared in step a), the proportion of CD73-positive mesenchymal cells is 80% or more, the proportion of CD166-positive mesenchymal cells is 80% or more, the proportion of CD45-positive mesenchymal cells is 10% or less, and the proportion of CD105-positive mesenchymal cells is 70% or more. (5) Any of the methods (1) to (4), wherein the mesenchymal cells are derived from a fetal appendage. (6) Any of the methods (1) to (5), further comprising a step of culturing the cell population in suspension in a serum-free medium. (7) A method for producing a cell population containing cells that produce the adeno-associated virus, the method comprising the step of introducing a gene for producing the adeno-associated virus into cells that constitute the cell population produced by any one of the methods (1) to (6). (8) A cell population containing immortalized mesenchymal cells, produced by any of the methods (1) to (6). (9) A cell population comprising immortalized mesenchymal cells, the cell population comprising an adenovirus E1A region, an E1B region, an SV40 large T antigen, and Bcl-x L A cell population comprising cells that contain at least one protein-encoding gene and that express the TERT gene. Effect of the Invention

[0012] According to the present invention, it is possible to provide a method for stably and efficiently producing a cell population containing immortalized mesenchymal cells that can be permanently subcultured, and a cell population containing immortalized mesenchymal cells. [Brief description of the drawings]

[0013] [Figure 1] 1 is a micrograph (magnification: 40x) of a cell population containing non-transformed amnion-derived mesenchymal cells. The non-transformed mesenchymal cells had a spindle-shaped cell morphology. [Diagram 2] Figure 2 is a micrograph (magnification 40x) of a cell population containing amnion-derived mesenchymal cells transformed with an immortalization plasmid. The transformed immortalized amnion mesenchymal cells had a cobblestone-like cellular morphology. [Diagram 3] 3 is a micrograph (magnification 40x) of a cell population containing non-transformed bone marrow-derived mesenchymal cells. The non-transformed bone marrow mesenchymal cells had a spindle-shaped cell morphology. [Figure 4]Figure 4 is a micrograph (magnification 40x) of a cell population containing bone marrow-derived mesenchymal cells transformed with an immortalization plasmid. The cellular morphology of the transformed immortalized bone marrow mesenchymal cells became similar to that of cobblestones. [Diagram 5] 5 is a graph comparing the AAV2 genome concentration produced in immortalized amnion mesenchymal cells (amniotic cells), immortalized bone marrow mesenchymal cells (bone marrow cells), and HEK293T cells transfected with an adeno-associated virus (AAV)-producing gene. It was confirmed that AAV2 was produced in all of the cells. [Figure 6] Figure 6 is a micrograph (magnification 40x) of a cell population containing amnion-derived immortalized mesenchymal cells cultured in suspension in 293 SFM II. The cell population containing amnion-derived immortalized mesenchymal cells was cultured in suspension in a dispersed single cell state. [Figure 7] Figure 7 is a micrograph (magnification 40x) of HEK293T cells cultured in suspension in FreeStyleF17 Expression Medium. Multiple cells were cultured in a clumped state. [Figure 8] Figure 8 is a micrograph (magnification 40x) of HEK293T cells cultured in suspension in FreeStyle293 Expression Medium. Multiple cells were cultured in a clumped state. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] [1] Explanation of terms As used herein, the term "mesenchymal cells" includes "mesenchymal stem cells (MSCs)" and refers to cells that meet the following definition.

[0015] As "mesenchymal cells", somatic cells (tissue cells) that can be collected from various tissues and organs and that satisfy the following definition can be used. Examples of the somatic cells include bone marrow cells, bone marrow stem cells, adipocytes, adipocyte stem cells, dental pulp cells, dental pulp stem cells, nerve cells, nerve stem cells, cardiac muscle cells, cardiac muscle stem cells, hepatic cells, hepatic stem cells, epithelial cells, epithelial stem cells, skeletal muscle cells, skeletal muscle stem cells, hematopoietic cells, hematopoietic stem cells, mesenchymal cells, mesenchymal stem cells, mesenchymal stem cells derived from amniotic epithelium, mesenchymal cells derived from amniotic epithelium, digestive tract epithelial cells, digestive tract epithelial stem cells, osteoblasts, chondrocytes, synovial cells, synovial stem cells, etc.

[0016] Definition of Mesenchymal Cells i) They exhibit adhesion to plastic under culture conditions in standard medium, which is a basal medium (e.g., αMEM medium) supplemented with serum, serum replacement reagents, or growth factors (e.g., human platelet lysate, which is a serum replacement reagent). ii) The surface antigens CD73 and CD90 are positive, and CD45 and CD326 are negative.

[0017] The "mesenchymal cells" may satisfy the above definitions i) and ii), and there is no particular limitation as to whether or not they have the ability to differentiate into bone, cartilage, fat, etc. In this specification, the "mesenchymal cells" also include cells that have the ability to differentiate into bone, cartilage, and fat, such as mesenchymal stem cells. The "mesenchymal cells" also include cells that meet the above definition but do not have the ability to differentiate into bone, cartilage, or fat. The "mesenchymal cells" also include cells that meet the above definition but differentiate into only one or two of bone, cartilage, and fat.

[0018] As used herein, "immortalized" mesenchymal cells refer to cells that can stably divide and grow without dying under conditions that allow the cells to grow. As used herein, immortalized mesenchymal cells are capable of cell division at least 60 times, 70 times, 80 times, 90 times, 100 times, 110 times, 120 times, 130 times, 140 times, 150 times, 160 times, 170 times, 180 times, 190 times, or 200 times.

[0019] In this specification, "fetal appendages" refers to the fetal membrane, placenta, umbilical cord, and amniotic fluid. Furthermore, the "fetal membrane" is a gestational sac containing fetal amniotic fluid, and is composed of the amnion, chorion, and decidua from the inside out. Of these, the amnion and chorion originate from the fetus. The "amnion" is a transparent thin film with few blood vessels that is the innermost layer of the fetal membrane, and its inner wall is covered with a single layer of epithelial cells with secretory function and secretes amniotic fluid. The inner layer of the amnion (also called the epithelial cell layer) is covered with a single layer of epithelial cells with secretory function and secretes amniotic fluid, and the outer layer of the amnion (also called the extracellular matrix layer, equivalent to the interstitium) contains mesenchymal cells.

[0020] The form of the "cell population containing mesenchymal cells" as used herein is not particularly limited, and examples include cell pellets, cell aggregates, cell sheets, cell suspensions, cell suspensions, frozen products thereof, and the like.

[0021] As used herein, the "proportion of mesenchymal cells that are positive for a given surface antigen" refers to the proportion of cells that are positive for a given surface antigen analyzed by flow cytometry, as described in the Examples below. As used herein, the proportion of cells that are positive for a given surface antigen may be referred to as the "positive rate," and the proportion of cells that are negative for a given surface antigen may be referred to as the "negative rate."

[0022] As used herein, "mesenchymal cells," "cell populations comprising mesenchymal cells," and "fetal appendages" are preferably derived from humans.

[0023] In the present specification, adenovirus "E1A" is a protein that is expressed first in the replication of adenovirus, and "E1B" is a protein that is expressed next. Adenovirus E1A and E1B are known to bind to and inactivate tumor suppressor genes Rb and p53 in animal cells, thereby transforming the cells into cancerous cells and enabling them to proliferate. The E1A protein has an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3, or has an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the sequence represented by any one of SEQ ID NOs: 1 to 3. In addition, the gene encoding the E1A protein has a base sequence that encodes a protein having an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3, or has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the sequence represented by any one of SEQ ID NOs: 1 to 3. The base sequence of the gene encoding the E1A protein is not limited as long as it encodes these proteins, but may be, for example, a sequence represented by SEQ ID NO: 4 or a sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence represented by SEQ ID NO: 4. The E1B protein has an amino acid sequence represented by any of SEQ ID NOs: 5 to 8, or has an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence represented by any of SEQ ID NOs: 5 to 8. The gene encoding the E1B protein has a base sequence encoding a protein having an amino acid sequence represented by any of SEQ ID NOs: 5 to 8, or has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence represented by any of SEQ ID NOs: 5 to 8. The base sequence of the gene encoding the E1B protein is not limited as long as it encodes these proteins, but can be, for example, a sequence represented by SEQ ID NO:9 or a sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence of SEQ ID NO:9.

[0024] In this specification, "SV40 (Simian Virus 40)" refers to a DNA virus belonging to polyomaviruses, which is known to infect cells of mice and the like and cause some of the cells to become cancerous. "SV40 large T antigen" (hereinafter also referred to as "LTAg") is known to bind to and inactivate tumor suppressor genes Rb and p53, similar to adenovirus E1A and E1B proteins. LTAg has an amino acid sequence represented by SEQ ID NO: 10, or has an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the sequence represented by SEQ ID NO: 10. In addition, a gene encoding LTAg has a base sequence encoding a protein having an amino acid sequence represented by SEQ ID NO: 10, or an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with the sequence represented by SEQ ID NO: 10. The base sequence of the gene encoding LTAg is not limited as long as it encodes these proteins, but can be, for example, a sequence represented by SEQ ID NO:11 or a sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence of SEQ ID NO:11.

[0025] As used herein, "Bcl-x L " is a protein belonging to the Bcl2 family that suppresses apoptosis, and Bcl-x L The genes that code for Bcl-x are known as oncogenes. L The gene encoding Bcl-x may be derived from any animal, but for example, a gene derived from chicken is preferably used. L The protein has an amino acid sequence represented by SEQ ID NO: 12 or has an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence represented by SEQ ID NO: 12. LThe gene encoding Bcl-x has a nucleotide sequence encoding a protein having an amino acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence represented by SEQ ID NO: 12. L The base sequence of the gene encoding the above is not limited as long as it encodes these proteins, but can be, for example, a sequence represented by SEQ ID NO:13 or a sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence of SEQ ID NO:13.

[0026] The surface antigen "CD324" stands for cluster of differentiation 324, a protein also known as epithelial cadherin (E-cadherin).

[0027] The surface antigen "CD90" stands for cluster of differentiation 90, a protein also known as Thy-1.

[0028] The surface antigen "CD326" stands for cluster of differentiation 326, a protein also known as EpCAM.

[0029] The surface antigen "CD73" stands for cluster of differentiation 73, a protein also known as 5-nucleotidase or Ecto-5'-nucleotidase.

[0030] The surface antigen "CD166" stands for cluster of differentiation 166, a protein also known as activated leukocyte cell adhesion molecule (ALCAM).

[0031] The surface antigen "CD105" stands for cluster of differentiation 105, a protein also known as Endoglin.

[0032] The surface antigen "CD45" stands for cluster of differentiation 45, a protein also known as PTPRC (protein tyrosine phosphatase, receptor type, C) or LCA (leukocyte common antigen).

[0033] The surface antigen "CD34" stands for cluster of differentiation 34, a protein also known as hematopoietic progenitor cell antigen CD34.

[0034] As used herein, "telomerase reverse transcriptase (TERT)" is a protein that constitutes the telomerase complex together with the telomerase RNA component (TERC). Telomerase binds to the end of a chromosome and adds a telomeric repeat sequence to the 3' end of telomeric DNA using TERC as a template. Telomerase is hardly expressed in normal somatic cells in humans, but is expressed in fetal cells, germ cells, etc. Cells that do not have telomerase activity undergo cellular senescence due to shortening of telomeres with cell division, so there is a limit to the number of cell divisions, but cells that have telomerase activity are able to proliferate without limit because telomere length is maintained by telomerase activity. As used herein, expression of the TERT gene is used as an indicator of cell immortalization.

[0035] [2] A method for producing a cell population containing immortalized mesenchymal cells The method for producing a cell population containing immortalized mesenchymal cells of the present invention comprises the steps of: a) preparing a cell population containing mesenchymal cells; b) culturing the cell population of a) with a medium containing a platelet lysate; and c) b) A cell that constitutes a cell population containing mesenchymal cells is transfected with genes encoding adenovirus E1A and E1B, a gene encoding SV40 large T antigen, and Bcl-x L introducing a plasmid carrying a gene encoding the

[0036] [2-1] A step of preparing a cell population containing mesenchymal cells In the method of the present invention, the "cell population containing mesenchymal cells" is not particularly limited as long as it is a cell population containing at least mesenchymal cells, and may be a population containing other cells. Although not particularly limited, the ratio of mesenchymal cells in the cell population containing mesenchymal cells may be 70% or more, 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0037] The ratio of other cells in the cell population containing mesenchymal cells may be 30% or less, 20% or less, 10% or less, or 5% or less. The other cells are not particularly limited as long as they are other than mesenchymal cells, and examples of such other cells include blood cells such as lymphocytes, granulocytes, and red blood cells.

[0038] The cell population containing mesenchymal cells used in the method of the present invention is not particularly limited as long as it is a cell population containing cells that satisfy the above definitions i) and ii), but it is preferable that the cell population is prepared so as to have the following characteristics (a) and (b). (a) the ratio of mesenchymal cells positive for CD324 in the cell population is 70% or more; (b) in the cell population, the ratio of mesenchymal cells positive for CD90 is 90% or more. By providing the above-mentioned properties, the cell population including mesenchymal cells can be spontaneously detached from the substrate after culture, which makes it possible to efficiently obtain the cells after culture without using enzymes or special devices as in the methods described in Patent Documents 1 and 2.

[0039] In the cell population, the ratio of CD324-positive mesenchymal cells may more preferably be 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0040] In the cell population, the ratio of CD90-positive mesenchymal cells is more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or may be 100%.

[0041] In one or more embodiments of the method of the present invention, the cell population preferably has a ratio of mesenchymal cells that are CD326 positive of 10% or less.

[0042] In the cell population, the ratio of mesenchymal cells that are CD326 positive is more preferably 5% or less (negative rate of 95% or more), 4% or less (negative rate of 96% or more), 3% or less (negative rate of 97% or more), 2% or less (negative rate of 98% or more), 1% or less (negative rate of 99% or more), or may be 0% (negative rate of 100%).

[0043] According to one aspect of the present invention, the cell population containing mesenchymal cells provided by the present invention preferably satisfies one or more of the following: the proportion of CD73-positive mesenchymal cells is 90% or more; the proportion of CD166-positive mesenchymal cells is 80% or more; the proportion of CD105-positive mesenchymal cells is 70% or more; the proportion of CD45-positive mesenchymal cells is 10% or less; and the proportion of CD34-positive mesenchymal cells is 10% or less.

[0044] The ratio of CD73-positive mesenchymal cells in the cell population is more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or may be 100%.

[0045] The ratio of CD166-positive mesenchymal cells in the cell population may more preferably be 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0046] The ratio of CD105 positive mesenchymal cells in the cell population may more preferably be 74% or more, 75% or more, 80% or more, 85% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0047] The ratio of CD45 positive mesenchymal cells in the cell population is more preferably 5% or less (negative rate of 95% or more), 4% or less (negative rate of 96% or more), 3% or less (negative rate of 97% or more), 2% or less (negative rate of 98% or more), 1% or less (negative rate of 99% or more), or may be 0% (negative rate of 100%).

[0048] The ratio of CD34 positive mesenchymal cells in the cell population is more preferably 5% or less (negative rate 95% or more), 4% or less (negative rate 96% or more), 3% or less (negative rate 97% or more), 2% or less (negative rate 98% or more), 1% or less (negative rate 99% or more), or may be 0% (negative rate 100%).

[0049] Here, CD324-positive, CD90-positive, CD326-positive, CD73-positive, CD166-positive, CD105-positive, CD45-positive and CD34-positive mesenchymal cells refer to mesenchymal cells that are positive for the expression of CD324, CD90, CD326, CD73, CD166, CD105, CD45 and CD34, respectively.

[0050] For the cell population used in one or more embodiments of the method of the present invention, the expression marker (CD324, CD90, CD326, CD73, CD166, CD105, CD45, or CD34) can be detected by any detection method known in the art. Methods for detecting expression markers include, but are not limited to, flow cytometry or cell staining. In flow cytometry using a fluorescently labeled antibody, if a cell is detected that emits stronger fluorescence compared to a negative control (isotype control), the cell is determined to be "positive" for the marker. The fluorescently labeled antibody can be any antibody known in the art, including, but not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc. In cell staining, if a stained or fluorescent cell is observed under a microscope, the cell is determined to be "positive" for the marker. Cell staining may be immune cell staining using an antibody, or non-immune cell staining using no antibody. In this specification, the expression marker and the surface antigen are synonymous and can be used interchangeably.

[0051] The cell population used in one or more embodiments of the method of the present invention is not particularly limited as to whether or not it has differentiation ability, but preferably has the ability to differentiate into cartilage tissue and has low or no ability to differentiate into adipose tissue, and preferably further has low or no ability to differentiate into bone tissue.

[0052] The cell population used in one or more embodiments of the method of the present invention can be stored in a frozen state until immediately before use. The cell population may contain any component other than mesenchymal cells. Such components include, but are not limited to, salts, polysaccharides (e.g., HES, dextran, etc.), proteins (e.g., albumin, etc.), DMSO, medium components (e.g., components contained in RPMI1640 medium, etc.).

[0053] The cell population containing mesenchymal cells used in the method of the present invention may be prepared by culturing commercially available mesenchymal stem cells (e.g., human bone marrow-derived mesenchymal stem cells (hMSC mesenchymal stem cells, manufactured by Lonza)). Alternatively, the method may further include a cell population obtaining step of obtaining a cell population containing mesenchymal cells from bone marrow fluid collected by bone marrow puncture, fetal appendages such as the amniotic membrane, and the like.

[0054] The raw material for producing the cell population containing mesenchymal cells of the present invention can be, but is not limited to, the above-mentioned bone marrow fluid or fetal appendages. In order to obtain a cell population having the above-mentioned characteristics (a) and (b), it is preferable to use fetal appendages. Hereinafter, an exemplary embodiment for preparing a cell population containing mesenchymal cells using fetal appendages as a raw material will be described.

[0055] The amniotic membrane is composed of an epithelial cell layer and an extracellular matrix layer, the latter of which contains amniotic membrane MSCs. The above-mentioned cell population obtaining step may further include a step of obtaining the amniotic membrane by Caesarean section.

[0056] The cell population containing cells collected from a fetal appendage is preferably a cell population obtained by treating a sample collected from a fetal appendage and containing an epithelial cell layer and a mesenchymal cell layer with at least collagenase.

[0057] The enzymatic treatment of the sample collected from the fetal appendage (preferably a sample containing an epithelial cell layer and a mesenchymal cell layer) is preferably a treatment with an enzyme (or a combination thereof) that can release mesenchymal cells contained in the extracellular matrix layer of the fetal appendage and does not degrade the epithelial cell layer. Such an enzyme is not particularly limited, but may be, for example, collagenase and / or metal proteinase. The metal proteinase may be, but is not particularly limited to, thermolysin and / or dispase, which are metal proteinases that cleave the N-terminal side of nonpolar amino acids.

[0058] The collagenase activity concentration is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, and even more preferably 200 PU / ml or more. The collagenase activity concentration is not particularly limited, but may be, for example, 1000 PU / ml or less, 900 PU / ml or less, 800 PU / ml or less, 700 PU / ml or less, 600 PU / ml or less, or 500 PU / ml or less. Here, PU (Protease Unit) is defined as the amount of enzyme that decomposes 1 ug of FITC-collagen in 1 minute at pH 7.5 and 30°C.

[0059] The activity concentration of the metal proteinase (e.g., thermolysin and / or dispase) is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, even more preferably 150 PU / ml or more, even more preferably 190 PU / ml or more. The activity concentration of the metal proteinase is preferably 1000 PU / ml or less, more preferably 900 PU / ml or less, even more preferably 800 PU / ml or less, even more preferably 700 PU / ml or less, even more preferably 600 PU / ml or less, even more preferably 500 PU / ml or less, even more preferably 300 PU / ml or less. Here, in an embodiment in which dispase is used as the metal proteinase, PU (Protease Unit) is defined as the amount of enzyme that releases amino acids equivalent to 1 ug of tyrosine from lactate casein per minute at pH 7.5 and 30°C. Within the above enzyme concentration range, mesenchymal cells contained in the extracellular matrix layer can be efficiently released while preventing contamination of epithelial cells contained in the epithelial cell layer of the fetal appendage. A preferred combination of collagenase and / or metalloproteinase concentrations can be determined by microscopic observation of the fetal appendages after enzyme treatment or by flow cytometry of the obtained cells.

[0060] From the viewpoint of efficiently recovering living cells, it is preferable to treat the fetal appendages with a combination of collagenase and metal proteinase. More preferably, the fetal appendages are treated simultaneously and in a lump with the combination. In this case, the metal proteinase may be, but is not limited to, thermolysin and / or dispase. By treating the fetal appendages only once with an enzyme solution containing collagenase and metal proteinase, mesenchymal cells can be easily obtained. Furthermore, by treating them simultaneously and in a lump, the risk of contamination with bacteria, viruses, etc. can be reduced.

[0061] The enzyme treatment of the fetal appendages is preferably performed by immersing the amniotic membrane washed with a washing solution such as physiological saline or Hank's balanced salt solution in the enzyme solution and treating the amniotic membrane while stirring with a stirring means. As the stirring means, from the viewpoint of efficiently releasing the mesenchymal cells contained in the extracellular matrix layer of the fetal appendages, for example, a stirrer or a shaker can be used, but is not limited thereto. The stirring speed is not particularly limited, but when a stirrer or a shaker is used, for example, 5 rpm or more, 10 rpm or more, 20 rpm or more, 30 rpm or more, 40 rpm or more, or 50 rpm or more. In addition, the stirring speed is not particularly limited, but when a stirrer or a shaker is used, for example, 100 rpm or less, 90 rpm or less, 80 rpm or less, 70 rpm or less, or 60 rpm or less. The enzyme treatment time is not particularly limited, but for example, 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 60 minutes or more, 70 minutes or more, 80 minutes or more, or 90 minutes or more. The enzyme treatment time is not particularly limited, but may be, for example, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 110 minutes or less, or 100 minutes or less. The enzyme treatment temperature is not particularly limited, but may be, for example, 15°C or more, 16°C or more, 17°C or more, 18°C ​​or more, 19°C or more, 20°C or more, 21°C or more, 22°C or more, 23°C or more, 24°C or more, 25°C or more, 26°C or more, 27°C or more, 28°C or more, 29°C or more, 30°C or more, 31°C or more, 32°C or more, 33°C or more, 34°C or more, 35°C or more, or 36°C or more. The enzyme treatment temperature is not particularly limited, but may be, for example, 40°C or less, 39°C or less, 38°C or less, or 37°C or less.

[0062] If desired, the liberated mesenchymal cells can be separated and / or recovered from the enzyme solution containing the liberated mesenchymal cells by known means such as a filter, centrifugation, a hollow fiber separation membrane, or a cell sorter. Preferably, the enzyme solution containing the liberated mesenchymal cells is filtered through a filter. In an embodiment in which the enzyme solution is filtered through a filter, only the liberated cells pass through the filter, and the epithelial cell layer that has not been decomposed cannot pass through the filter and remains on the filter, so that not only can the liberated mesenchymal cells be easily separated and / or recovered, but also the risk of contamination by bacteria, viruses, etc. can be reduced. The filter is not particularly limited, but for example, a mesh filter can be used. The pore size (mesh size) of the mesh filter is not particularly limited, but is, for example, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, or 90 μm or more. The pore size of the mesh filter is not particularly limited, but may be, for example, 200 μm or less, 190 μm or less, 180 μm or less, 170 μm or less, 160 μm or less, 150 μm or less, 140 μm or less, 130 μm or less, 120 μm or less, 110 μm or less, or 100 μm or less. Although there is no particular limitation regarding the filtration speed, by setting the pore size of the mesh filter in the above range, the enzyme solution containing mesenchymal cells can be filtered by gravity, thereby preventing a decrease in cell viability.

[0063] Nylon is preferably used as the material for the mesh filter. Tubes containing 40 μm, 70 μm, 95 μm, or 100 μm nylon mesh filters, such as Falcon cell strainers, which are widely used for research, can be used. Medical mesh cloths (nylon and polyester) used in hemodialysis can also be used. Furthermore, arterial filters (polyester mesh filters, pore size: 40 μm to 120 μm) used during extracorporeal circulation can also be used. Other materials, such as stainless steel mesh filters, can also be used.

[0064] When passing mesenchymal cells through a filter, natural (free) fall is preferred. Forced passage through a filter, such as by suction using a pump, is also possible, but it is desirable to use as little pressure as possible to avoid damaging the cells.

[0065] The cell population containing the filtered mesenchymal cells can be collected by centrifugation after diluting the filtrate with twice or more of a medium or a balanced salt buffer, such as, but not limited to, Dulbecco's phosphate buffer (DPBS), Earle's balanced salt solution (EBSS), Hank's balanced salt solution (HBSS), and phosphate buffered saline (PBS).

[0066] [2-2] Culturing a cell population containing mesenchymal cells in a medium containing a platelet lysate. The method of the present invention comprises the step of culturing a cell population containing mesenchymal cells prepared by the method described in section [2-1] in a medium containing a platelet lysate.

[0067] The present inventors found that when immortalizing a cell population containing mesenchymal cells and subculturing it, the culture conditions of the cell population prior to the immortalization step affect the stability of the subsequent subculturing.

[0068] The "platelet lysate (PL)" in the method of the present invention is preferably a mammalian (e.g., bovine) platelet lysate, more preferably human platelet lysate (hPL). hPL contains growth factors and various cytokines, and is known to be a component particularly suitable for the growth of mesenchymal stem cells. In the method of the present invention, by culturing a cell population containing mesenchymal cells in a medium supplemented with PL (particularly hPL) (hereinafter also simply referred to as "PL medium" or "hPL medium"), the survival rate of cells during subculture after the subsequent immortalization step can be significantly increased compared to a medium supplemented with other components such as fetal bovine serum (FBS). This makes it possible to stably obtain a cell population containing immortalized mesenchymal cells.

[0069] The seeding density of the cells in the step of culturing the cell population is not particularly limited, but may be, for example, 500 to 10,000 cells / cm. 2 The lower limit of the seeding density is, for example, 500 cells / cm. 2 More than 1,000 cells / cm 2 More than 2,000 cells / cm 2 More than 3,000 cells / cm 2 Over 4,000 cells / cm 2 More than 5,000 cells / cm 2 The upper limit of the seeding density is, for example, 10,000 cells / cm. 2 Below, 9,000 cells / cm 2 Below 8,000 cells / cm 2 Below, 7,000 cells / cm 2 The following is preferred:

[0070] The above-mentioned culturing step may include a subculture step. The culture period for one culture cycle may be, for example, 4 to 10 days, and more specifically, may be 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days.

[0071] The medium used for the above-mentioned culture is prepared by adding PL, preferably hPL, to any liquid medium for animal cell culture as a basal medium. The lower limit of the concentration of hPL in the medium can be, for example, 1% by weight or more, 2% by weight or more, or 3% by weight or more as a final concentration. The upper limit of the concentration of hPL in the medium can be, for example, 20% by weight or less, 10% by weight or less, or 7% by weight or less.

[0072] The basal medium may further contain other components (serum, growth factors, etc.) as necessary. In an embodiment in which a growth factor is added to the basal medium, the basal medium may be prepared by further adding a reagent (heparin, etc.) for stabilizing the growth factor in the medium in addition to the growth factor, or the growth factor may be stabilized in advance with a gel, polysaccharide, etc., and then the stabilized growth factor may be added to the basal medium. Thus, a medium in which PL, preferably hPL, is added to a basal medium in order to culture a cell population containing mesenchymal cells is defined as a standard medium.

[0073] As the basal medium, BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM (Alpha Modification of Minimum Essential Medium Eagle) medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham's F10 medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof (e.g., DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)) can be used, but is not particularly limited. An example of a preferred basal medium is αMEM medium.

[0074] Examples of other components that can be added to the basal medium include albumin, bovine serum, growth factors, etc. Among these, it is preferable to culture in a basal medium that does not contain albumin, bovine serum, or growth factors.

[0075] Culturing of a cell population containing mesenchymal cells can be carried out, for example, by the following steps. First, the cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in a medium. Next, the cells are seeded in a plastic culture vessel and cultured in a medium at a CO2 concentration of 3% to 5% at 37°C. The cells obtained by the above-mentioned culture are cells that have been cultured once.

[0076] The cells cultured once can be further passaged and cultured, for example, as follows. First, the cells cultured in the first culture until the confluence rate is 95% or less are treated with ethylenediaminetetraacetic acid (EDTA) and then with trypsin to detach them from the plastic culture vessel. Next, the obtained cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Finally, the cells are seeded in a plastic culture vessel and cultured using a medium in an environment with a CO2 concentration of 3% or more and 5% or less and at 37°C. The cells obtained by the above-mentioned passage and culture are cells that have been passaged once. By performing the same passage and culture, cells that have been passaged n times can be obtained (n is an integer of 1 or more). When performing passage culture, the cells are cultured until the confluence rate is 95% or less, and the cells are detached and collected by the above procedure and can be used for the next culture. The lower limit of the number of passages n is, for example, 1 time or more, preferably 2 times or more, more preferably 3 times or more, even more preferably 4 times or more, and even more preferably 5 times or more, from the viewpoint of mass production of cells. Moreover, from the viewpoint of suppressing cell aging, the upper limit of the number of passages n is preferably, for example, 50 or less, 45 or less, 40 or less, 35 or less, or 30 or less.

[0077] [2-3] Step of selecting cell populations The method of the present invention may, if necessary, include a step of selecting a cell population having the following characteristics (a) and (b): (a) The ratio of CD324-positive mesenchymal cells is 70% or more; (b) The ratio of mesenchymal cells positive for CD90 is 90% or more.

[0078] The means for selecting the cell population using the above-mentioned characteristics as an index can include, for example, physical means such as FACS, cell sorter, and magnetic bead separation, and chemical means for selecting cells that do not satisfy the index under appropriate culture conditions and purifying the cell population containing mesenchymal cells so that it satisfies the above-mentioned index, but the means is not particularly limited and may be appropriately selected depending on the culture conditions, etc. Note that, even in the selection step, it is preferable to carry out the culture in a medium containing a platelet lysate.

[0079] The timing for selecting a cell population having the above characteristics (a) and (b) is not particularly limited, but examples include before culture, during culture, after culture, before recovery of the cell population, after recovery of the cell population, before preparation of a frozen stock of cells, and after thawing a frozen stock of cells.

[0080] The procedure for selecting a cell population having the above characteristics (a) and (b) will now be described in further detail. For example, a cell population containing mesenchymal cells is seeded on a culture substrate, and the cells are grown and cultured on the culture substrate, whereby CD324-positive and CD90-positive mesenchymal cells are positively selected, and a cell population in which the ratio of CD324-positive mesenchymal cells is 70% or more and the ratio of CD90-positive mesenchymal cells is 90% or more is detached and collected. In this case, the culture may be performed using a culture substrate coated with an anti-CD324 antibody or an anti-CD90 antibody. The timing of selecting CD324-positive and CD90-positive mesenchymal cells from a cell population containing mesenchymal cells by culture is not particularly limited, and the cells can be selected in any subculture, but it is preferable to select CD324-positive and CD90-positive mesenchymal cells from a cell population containing mesenchymal cells in primary culture.

[0081] Furthermore, mesenchymal cells that are both CD324 positive and CD90 positive can also be selected from a cell population containing mesenchymal cells by a cell separation method using flow cytometry or magnetic beads.

[0082] The selection of the cell population is preferably carried out by selecting and separating a cell population in which the ratio of CD326-positive mesenchymal cells is 10% or less, and more preferably by selecting and separating a cell population that satisfies one or more of the following: the ratio of CD73-positive mesenchymal cells is 80% or more, the ratio of CD166-positive mesenchymal cells is 80% or more, the ratio of CD105-positive mesenchymal cells is 70% or more, the ratio of CD45-positive mesenchymal cells is 10% or less, and the ratio of CD34-positive mesenchymal cells is 10% or less. The selected cell population may be further cultured by the above-mentioned means.

[0083] [2-4] A step of introducing an immortalization plasmid into cells constituting a cell population containing mesenchymal cells The method of the present invention comprises the steps of: (1) detecting the adenovirus E1A and E1B genes, the adenovirus LTAg gene, and the adenovirus Bcl-x L The method of the present invention includes a step of introducing a plasmid carrying genes encoding E1A, E1B, LTAg, and Bcl-x (hereinafter also referred to as "immortalization plasmid") into cells constituting a cell population containing mesenchymal cells. L It may also comprise all of the genes encoding E1A, E1B, LTAg and Bcl-x L The genes encoding E1A, E1B, LTAg and Bcl-x may be distributed among two or more plasmids. Preferably, one plasmid contains E1A, E1B, LTAg and Bcl-x. L It contains all the genes encoding

[0084] The immortalization plasmids used in the method of the present invention include E1A, E1B, LTAg and Bcl-x L In addition to the gene encoding the vector, the vector may be provided with a nucleic acid having a promoter such as a CMV promoter, a CAG promoter, or an RSV promoter, an SV40 polyA, an IRES (internal ribosome entry site), and / or a base sequence of a resistance gene such as neomycin.

[0085] The base sequence of the CMV promoter is not particularly limited as long as it is a base sequence having promoter activity, but can be, for example, a sequence represented by SEQ ID NO: 14 or a sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence of SEQ ID NO: 14.

[0086] The base sequence of SV40 polyA is not particularly limited as long as it is the base sequence of polyA that constitutes SV40, but can be, for example, a sequence represented by SEQ ID NO:15 or a sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence of SEQ ID NO:15.

[0087] The IRES sequence is not particularly limited as long as it functions as a translation initiation site, and can be, for example, a sequence represented by SEQ ID NO:16 or a sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence of SEQ ID NO:16.

[0088] The base sequence of the neomycin resistance gene (NeoR) is not particularly limited as long as the gene product has neomycin resistance activity, but can be, for example, a sequence represented by SEQ ID NO: 17 or a sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the sequence of SEQ ID NO: 17.

[0089] The means for introducing the plasmid into the cells constituting the cell population containing mesenchymal cells is not particularly limited, and any of the calcium phosphate method, electroporation, lipofection, microinjection, and a combination of these means can be used. Alternatively, the introduction may be performed using a commercially available kit (e.g., Nucleofector (registered trademark) 2 / 2b (Lonza)).

[0090] The method of the present invention may, if necessary, comprise a step of confirming whether the target gene has been transfected into the cells constituting the cell population. Specifically, for example, a method of extracting genomes from a cell population and performing PCR of the transfected gene using the extracted genome as a template can be mentioned. For example, the introduction can be confirmed by observing the presence or absence of nucleic acid amplification in PCR using primers (e.g., having the nucleotide sequences of SEQ ID NOs: 18 and 19) designed to amplify the E1 region of adenovirus using the genome as a template.

[0091] Furthermore, the method of the present invention may include a step of confirming whether the cell population contains immortalized mesenchymal cells, if necessary. Immortalization can be confirmed by measuring the expression level of the telomerase reverse transcriptase (TERT) gene in the cell population. Specifically, for example, total RNA is extracted from the cell population, and cDNA is prepared using reverse transcriptase. Using cDNA as a template, primers and a probe designed to amplify and detect the TERT gene (for example, Thermo Fisher Scientific's TaqMan (registered trademark) Gene Expression Assays, No: Hs00972650_m1) can be used to measure the expression level of the TERT gene by measuring the nucleic acid amplification rate in real-time PCR. In the method of the present invention, the TERT gene is preferably the human TERT (hTERT) gene.

[0092] [2-5] The process of culturing cell populations in suspension The method of the present invention may include a step of suspension culturing a cell population containing immortalized mesenchymal cells. The cell population to be subjected to suspension culture may be the cell population containing immortalized mesenchymal cells prepared in the above section [2-4], or may be a cell population containing immortalized mesenchymal cells into which a virus production gene has been introduced in the below-mentioned section [2-6].

[0093] Mesenchymal cells are usually cultured in an adherent culture, but in some embodiments, the cell population containing the immortalized mesenchymal cells produced by the method of the present invention can be cultured in suspension in a serum-free medium. As the serum-free medium, an existing liquid medium that is suitable for suspension culture of animal cells and does not contain serum components or serum substitutes can be used. In particular, a medium suitable for suspension culture of human cells can be preferably used. Such serum-free media include, but are not limited to, CD 293 Medium, CD 293 AGT™ Medium, 293 SFM II and FreeStyle™ 293 Expression Medium, CD CHO Medium, CD CHO AGT™ Medium, CD OptiCHO™ Medium, CHO-S-SFM II (all manufactured by Thermo Fisher Scientific), Pro293™a Serum-free Medium, Pro293™s Serum-free Medium, ProCHO™ 4 Protein-free CHO Medium, ProCHO™ 4 Protein-free CHO Medium, ProCHO™ 5 Protein-free CHO Medium, PowerCHO™ 1 Serum-free Medium, PowerCHO™ 2 Serum-free Medium, PowerCHO™ 3 Serum-free Medium, PowerCHO™ GS Serum-free CHO Medium, PowerCHO Advance™ Serum-free Medium (all manufactured by Lonza), CDM4HEK293, SFM4HEK293, SFM4Transfx-293, HyCell TransFx-H, CDM4PERMAb, HyCell CHO, ActiPro, and ActiSM (all manufactured by Cytiva). In particular, 293 SFM II can be preferably used.

[0094] As described above, the cell population containing immortalized mesenchymal cells produced by the method of the present invention can be cultured in suspension, and therefore does not require a culture substrate as in adhesion culture, and therefore does not require detachment of cells from the culture substrate. Therefore, subculture can be performed more easily. Furthermore, more efficient cell proliferation is possible compared to adhesion culture.

[0095] [2-6] Step of introducing adeno-associated virus-producing gene A cell population containing immortalized mesenchymal cells produced by the above-mentioned method can be produced by introducing a gene for producing adeno-associated virus into the cells that constitute the cell population, thereby producing a cell population containing cells that produce adeno-associated virus.

[0096] Since the cell population produced by the above method contains immortalized cells, it is possible to introduce an AAV production gene into the cell population to produce AAV.

[0097] AAV is a non-pathogenic virus belonging to the parvoviruses, and since it lacks the ability for self-replication, it cannot grow autonomously and usually requires co-infection with a helper virus such as adenovirus or herpesvirus. Since it has low infectivity and immunogenicity, it is a highly safe virus with a wide host range. Due to these characteristics, AAV is a virus that is expected to be widely used as a gene transfer vector in gene therapy and other fields.

[0098] The AAV genome is a single-stranded DNA of about 4.7 kb, with T-shaped hairpin structures called ITRs (Inverted Terminal Repeats) at both ends, and three ORFs, Rep (related to replication and transcription), Cap (encoding the outer coat protein), and AAP (viral particle formation), are present between them. There are multiple serotypes of AAV. The serotypes found in human cells are AAV2, AAV3, AAV5, and AAV6.

[0099] The gene for producing AAV is not particularly limited as long as it allows AAV to be produced when introduced into immortalized mesenchymal cells, and may be composed of, for example, the following three plasmid vectors. pAAV vector: A vector plasmid containing an expression cassette of a gene of interest and two ITRs pRC vector: A plasmid containing the Rep gene of AAV2 and the Cap gene of each serotype pHelper vector: a plasmid containing E2A, E4, and VA derived from adenovirus

[0100] In the embodiment in which the above-mentioned plasmid vector is used as a gene for producing AAV, the means for introducing the plasmid vector into a cell may be any known means. For example, the plasmid may be introduced into a target cell using the polyethyleneimine (PEI) transfection method. The detailed conditions of the PEI method may be determined by referring to, for example, the description in Y. Durocher, et al., Nuc. Acid. Res., Vol. 30, No. 2, e9 (2002), etc.

[0101] [2-7] Cryopreservation of cell populations The method of the present invention may also include a step of cryopreserving a cell population containing mesenchymal cells. The cell population to be cryopreserved may be any cell population before culture, after culture, after immortalization, after suspension culture, or after introduction of a virus-producing gene. In an embodiment including a step of cryopreserving a cell population, the cell population may be thawed and then separated, collected, and / or cultured as necessary. The cell population may also be used as is after thawing.

[0102] The means for cryopreserving the cell population containing mesenchymal cells is not particularly limited, and examples thereof include a programmed freezer, a deep freezer, immersion in liquid nitrogen, etc. The temperature during freezing is preferably −30° C. or lower, −40° C. or lower, −50° C. or lower, −60° C. or lower, −70° C. or lower, −80° C. or lower, −90° C. or lower, −100° C. or lower, −110° C. or lower, −120° C. or lower, −130° C. or lower, −140° C. or lower, −150° C. or lower, −160° C. or lower, −170° C. or lower, −180° C. or lower, −190° C. or lower, or −196° C. (liquid nitrogen temperature) or lower. The freezing rate is preferably, for example, -1°C / min, -2°C / min, -3°C / min, -4°C / min, -5°C / min, -6°C / min, -7°C / min, -8°C / min, -9°C / min, -10°C / min, -11°C / min, -12°C / min, -13°C / min, -14°C / min, or -15°C / min. When a programmable freezer is used as the freezing means, the temperature can be lowered to a temperature between -50°C and -30°C (e.g., -40°C) at a freezing rate of -2°C / min to -1°C / min, and further lowered to a temperature between -100°C and -80°C (e.g., -90°C) at a freezing rate of -11°C / min to -9°C / min (e.g., -10°C / min).

[0103] When freezing by the above freezing means, the cell population may be frozen in any storage container, such as, but not limited to, cryotubes, cryovials, freezing bags, infusion bags, etc.

[0104] The cryopreservation solution preferably contains albumin at a predetermined concentration of more than 0% by mass in order to increase the survival rate of MSCs with relatively high proliferation ability. The preferred concentration of albumin is, for example, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, or 8% by mass or more. The preferred concentration of albumin is, for example, 40% by mass or less, 35% by mass or less, 30% by mass or less, 25% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, or 9% by mass or less. Examples of albumin include, but are not limited to, bovine serum albumin, mouse albumin, and human albumin.

[0105] [3] Cell population containing mesenchymal cells The cell population of the present invention is a cell population containing immortalized mesenchymal cells produced by the method described in the section "[2] Method for producing a cell population containing immortalized mesenchymal cells". Alternatively, the cell population of the present invention is a cell population containing immortalized mesenchymal cells, which is ... L The cell population comprises immortalized mesenchymal cells, the immortalized mesenchymal cells including cells containing at least one of the genes encoding the above-mentioned three or more nucleotides and cells expressing the TERT gene.

[0106] Unless otherwise specified below, the cell population of the present invention has characteristics in common with the cell population described in the section "[2] Method for producing a cell population comprising immortalized mesenchymal cells."

[0107] In the cell population of the present invention, "adenovirus E1A, E1B, SV40 large T antigen and Bcl-x L Specifically, the term "cells containing at least one of the genes encoding adenovirus E1A, E1B, SV40 large T antigen, or Bcl-x in their genomic DNA" refers to cells containing at least one of the genes encoding adenovirus E1A, E1B, SV40 large T antigen, or Bcl-x in their genomic DNA. L In the cell population of the present invention, adenovirus E1A, E1B, SV40 large T antigen, and Bcl-x are present at levels detectable by existing methods. LThe ratio of cells containing at least one of the genes encoding the above may be 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or may be 100% or more.

[0108] In the cell population of the present invention, the term "cells expressing the TERT gene" specifically refers to cells in which mRNA having a base sequence encoding TERT is present at a level detectable by any existing method such as quantitative real-time PCR. In the cell population of the present invention, the ratio of cells expressing the TERT gene may be 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or more. EXAMPLES

[0109] Comparative Example 1: Immortalization of amniotic membrane-derived mesenchymal cells (FBS medium culture system) (Step 1-1: Collection of amniotic membrane) Fetal appendages, the amniotic membrane and placenta, were collected aseptically from a pregnant woman (donor #1) undergoing elective cesarean section after obtaining informed consent. The obtained amniotic membrane and placenta were placed in a sterile tray containing physiological saline, and the amniotic membrane was manually peeled off from the stump of the amniotic membrane. The amniotic membrane was washed with Hanks' balanced salt solution (Ca·Mg-free) to remove any attached blood and blood clots.

[0110] (Step 1-2: Enzyme treatment of amniotic membrane and recovery of mesenchymal cells) The amniotic membrane containing the epithelial cell layer and the mesenchymal cell layer was immersed in Hanks' balanced salt solution (containing Ca·Mg) containing 240 PU / mL collagenase and 200 PU / mL dispase I, and the amniotic membrane was enzymatically treated by shaking at 50 rpm for 90 minutes at 37°C. The solution after the enzyme treatment was filtered through a nylon mesh with 95 μm openings to remove undigested material from the amniotic membrane, and a cell suspension containing mesenchymal cells was collected.

[0111] (Step 1-3: Cultivation of mesenchymal cells) The cell population containing mesenchymal cells obtained in the above-mentioned "Step 1-2: Enzyme treatment of amniotic membrane and recovery of mesenchymal cells" was seeded in a culture vessel CellSTACK (registered trademark) (manufactured by Corning Incorporated). The seeding density was 6,000 cells / cm. 2 After seeding, the cells were cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing 10% fetal bovine serum (FBS) and 10 ng / mL basic fibroblast growth factor (bFGF) until the cells became subconfluent. After culturing, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack and incubated at 37°C for 3 minutes, and about 30% of the cell population remained in the culture vessel while adhering to the CellSTACK (registered trademark). Therefore, the cells were incubated for an additional 5 minutes (total 8 minutes) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is the 0th passage cell population. Then, 1 / 5 of the cell population was seeded on CellSTACK (registered trademark) of the same scale as the previous culture, and subcultured in αMEM containing 10% FBS and 10 ng / mL bFGF. The medium was replaced once every 2 to 4 days. When the cells reached subconfluence, 15 mL of TrypLE Select was added per CellSTACK stack and incubated at 37°C for 3 minutes. Approximately 30% of the cell population was not detached and remained attached to the CellSTACK in the culture vessel. The cells were then incubated for an additional 5 minutes (8 minutes in total) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here was the first passage cell population.

[0112] Then, the cell concentration was increased to 2 × 10 7RPMI1640 was added to the medium so that the concentration of cells / mL was approximately 15,000 to 18,000 cells / cm. An equal volume of CP-1 (registered trademark) solution (a solution in which CP-1 (registered trademark):25% human serum albumin was mixed at a ratio of 34:16) was added thereto, and 1 mL of each solution was transferred to a cryovial, slowly frozen to -80°C, and then cryopreserved under liquid nitrogen for one day. The frozen and preserved cell population was then thawed and cultured at approximately 15,000 to 18,000 cells / cm. 2 The first passage cell population was seeded on CellSTACK (registered trademark) at a density of 100 μg / mL and cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing 10% fetal bovine serum (FBS) and 10 ng / mL basic fibroblast growth factor (bFGF) at a final concentration until the cells became subconfluent. After culturing, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack and incubated at 37°C for 3 minutes. Approximately 30% of the cell population remained in the culture vessel without being detached and adhered to the CellSTACK (registered trademark). Therefore, the cells were incubated for an additional 5 minutes (total 8 minutes) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is the second passage cell population.

[0113] Thereafter, 1 / 5 of the cell population was seeded on CellSTACK (registered trademark) of the same scale as the previous culture, and subcultured in αMEM containing 10% FBS and 10 ng / mL bFGF at a final concentration. The medium was replaced once every 2 to 4 days. When the cells reached subconfluence, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack, and incubated at 37°C for 3 minutes. Approximately 30% of the cell population was not detached and remained attached to the CellSTACK (registered trademark) in the culture vessel. Therefore, the cells were incubated for an additional 5 minutes (total 8 minutes) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here was a cell population at the third passage. The cell population had a cell concentration of 4×10 6RPMI1640 was added to the medium so that the cell population reached a density of approximately 6,000 cells / cm. An equal volume of CP-1 (registered trademark) solution (a solution in which CP-1 (registered trademark):25% human serum albumin was mixed at a ratio of 34:16) was then added, and 1 mL of each solution was transferred to a cryovial, which was then slowly frozen to -80°C and then cryopreserved under liquid nitrogen for one day. The frozen and preserved cell population was then thawed, and the cell population reached a density of approximately 6,000 cells / cm. 2 The cell population of the third passage was seeded on CellSTACK (registered trademark) at a density of 100 μg / mL and cultured in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing 10% fetal bovine serum (FBS) and 10 ng / mL basic fibroblast growth factor (bFGF) until the cells became subconfluent. After the culture, 15 mL of TrypLE Select was added per stack of CellSTACK (registered trademark) and incubated at 37°C for 3 minutes. Approximately 30% of the cell population remained in the culture vessel without being detached and adhered to CellSTACK (registered trademark). Therefore, the cells were incubated for an additional 5 minutes (total 8 minutes) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is a cell population of the fourth passage.

[0114] Then, 1 / 5 of the cell population was seeded on CellSTACK (registered trademark) of the same scale as the previous culture, and subcultured in αMEM containing 10% FBS and 10 ng / mL bFGF at a final concentration. The medium was replaced once every 2 to 4 days. When the cells reached subconfluence, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack, and incubated at 37°C for 3 minutes. Approximately 30% of the cell population was not detached and remained attached to the CellSTACK (registered trademark) in the culture vessel. Therefore, the cells were incubated for an additional 5 minutes (total 8 minutes) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here was the 5th passage cell population. After that, when the cell concentration was 4×10 6RPMI1640 was added to the cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution in which CP-1 (registered trademark): 25% human serum albumin was mixed at a ratio of 34:16) was added thereto, and 1 mL of each solution was transferred to a cryovial and slowly frozen to -80°C, and then frozen and stored under liquid nitrogen.

[0115] (Step 1-4: Surface antigen analysis of mesenchymal cells) The cell population at the fifth passage cultured by the above culture method was analyzed for various surface antigens (positive rate of CD324, positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, negative rate of CD326) using a flow cytometer. As a result, the positive rate of CD324 was less than 70% (specifically, 33%), the positive rate of CD105 was 70% or more (specifically, 93%), and the positive rates of CD73, CD90, and CD166 were all 90% or more (specifically, CD73: 99%, CD90: 93%, CD166: 97%). The negative rates of CD45 and CD326 were all 95% or more (specifically, CD45: 100%, CD326: 100%). From the above results, it was found that the cell population cultured by the above culture method was a cell population containing mesenchymal cells. Furthermore, it was found that the cell population at the 5th passage in Comparative Example 1 was a cell population that satisfied the condition that the ratio of CD90-positive mesenchymal cells was 90% or more, but did not satisfy the condition that the ratio of CD324-positive mesenchymal cells was 70% or more.

[0116] In this measurement, the following antibodies were used as isotype controls: REA Control(S)APC (Miltenyi Biotec, clone: ​​REA293, model number: 130-113-434); CD324-APC, Human (Miltenyi Biotec, clone: ​​REA811, model number: 130-111-840); CD73-APC, Human (Miltenyi Biotec, clone: ​​REA804, model number: 130-111-909); CD90-APC, Human (Miltenyi Biotec, clone: ​​REA897, model number: 130-114-861); and CD105-APC, Human (Miltenyi Biotec, clone: ​​REA897, model number: 130-114-861). As an antibody against CD166 antigen, CD166-APC, Human (Miltenyi Biotec, clone: ​​REA442, model number: 130-106-576) was used, as an antibody against CD45 antigen, CD45-APC, Human (Miltenyi Biotec, clone: ​​REA747, model number: 130-110-633) was used, as an antibody against CD326 antigen, CD326-APC, Human (Miltenyi Biotec, clone: ​​REA764, model number: 130-111-000) was used. Surface antigen analysis was performed using Guava (registered trademark) easyCyte from Merck, and the measurement conditions were number of analyzed cells: 30,000 cells, flow rate setting: 35.4 μL / min). The ratio of positive cells for each antigen (positive rate) was calculated by the following procedure.

[0117] (1) The measurement results of the isotype control were plotted in a dot plot with SSC on the vertical axis and FSC on the horizontal axis. (2) A gate was set for the cell population corresponding to mesenchymal cells, and the cell population was plotted in a histogram with the count number on the vertical axis and the APC tendency intensity on the horizontal axis. (3) In the histogram of (2), all regions (gates) in which the cell population with stronger fluorescence intensity accounted for 0.5% or less of the total cells measured with the isotype control antibody were selected. (4) The percentage of cells within the gate selected in (2) among the total cells measured with an antibody corresponding to a surface antigen marker was calculated.

[0118] (Steps 1-5: Immortalization of mesenchymal cells) The cell population isolated and cultured according to the culture method similar to Comparative Example 1 was transfected with 2.5 μg of immortalization plasmid (E1A gene (SEQ ID NO: 4), E1B gene (SEQ ID NO: 9), chicken Bcl-x L The cells were transfected with the gene (SEQ ID NO: 13), the SV40 large T antigen gene (SEQ ID NO: 11), and the neomycin resistance gene (SEQ ID NO: 17) (cell suspension buffer: Nucleofector (registered trademark) Solution L, program: X-001). The transfected cells were seeded in a 6-well dish and cultured in αMEM containing 5% hPL until they became subconfluent. After reaching subconfluence, they were subcultured in αMEM containing 20 μg / mL G418 and 5% hPL. As a result, all the cells died, and no transformed amnion-derived mesenchymal cells could be obtained.

[0119] <Example 1: Immortalization of amnion-derived mesenchymal cells (hPL medium culture system)> (Step 2-1: Collection of amniotic membrane) An amniotic membrane was obtained from the same donor (donor #1) as in Comparative Example 1 by the same method as in Comparative Example 1.

[0120] (Step 2-2: Enzyme treatment of amniotic membrane and recovery of mesenchymal cells) A cell population containing mesenchymal cells was obtained by the same method as in Comparative Example 1.

[0121] (Step 2-3: Cultivation of mesenchymal cells) The cell population containing mesenchymal cells obtained in the above-mentioned "Step 2-2: Enzyme treatment of amniotic membrane and recovery of mesenchymal cells" was seeded in a culture vessel CellSTACK (registered trademark) (manufactured by Corning Incorporated). The seeding density was 6,000 cells / cm. 2 After seeding, the cells were cultured in αMEM containing human platelet lysate (hPL) at a final concentration of 5% until the cells became subconfluent. After the culture, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack, and the cells were incubated at 37°C for 3 minutes, allowing the cell population to be completely detached. The cell population obtained here is a cell population at passage 0. Then, 1 / 5 of the cell population was seeded on CellSTACK (registered trademark) at the same scale as the previous culture, and subcultured in αMEM containing hPL at a final concentration of 5%. The medium was replaced once every 2 to 4 days. When the cells became subconfluent, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack, and the cells were incubated at 37°C for 3 minutes, allowing the cell population to be completely detached. The cell population obtained here is a cell population at passage 1.

[0122] Then, the cell concentration was increased to 2 × 10 7 RPMI1640 was added to the medium so that the concentration of cells / mL was approximately 15,000 to 18,000 cells / cm. An equal volume of CP-1 (registered trademark) solution (a solution in which CP-1 (registered trademark):25% human serum albumin was mixed at a ratio of 34:16) was added thereto, and 1 mL of each solution was transferred to a cryovial, slowly frozen to -80°C, and then cryopreserved under liquid nitrogen for one day. The frozen and preserved cell population was then thawed and cultured at approximately 15,000 to 18,000 cells / cm. 2 The first passage cell population was seeded on CellSTACK® at a density of 100 μg / ml and cultured in αMEM containing 5% human platelet lysate (hPL) until the cells became subconfluent. Then, 15 mL of TrypLE Select was added per CellSTACK® stack and incubated at 37°C for 3 minutes, allowing the cell population to be completely detached. The cell population obtained here is the second passage cell population.

[0123] Next, 1 / 5 of the cell population was seeded on CellSTACK (registered trademark) of the same scale as the previous culture, and subcultured in αMEM containing hPL at a final concentration of 5%. The medium was replaced once every 2 to 4 days. When the cells reached subconfluence, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack, and incubated at 37°C for 3 minutes, allowing the cell population to be completely detached. The cell population obtained here was the third passage cell population.

[0124] Then, the cell concentration was increased to 4 × 10 6 RPMI1640 was added to the cells / mL. An equal amount of CP-1 solution (CP-1:25% human serum albumin mixed at a ratio of 34:16) was added, and 1 mL of each solution was transferred to a cryovial, slowly frozen to -80°C, and then cryopreserved under liquid nitrogen for one day. The frozen cell population was then thawed and cultured at approximately 6,000 cells / cm. 2 The cell population at the third passage was seeded on CellSTACK (registered trademark) at a density of 100 μg / ml and cultured in αMEM containing 5% final concentration of human platelet lysate (hPL) until the cells became subconfluent. Then, 15 mL of TrypLE Select was added per stack of CellSTACK (registered trademark), and the cells were incubated at 37°C for 3 minutes, allowing the cell population to be completely detached. The cell population obtained here is a cell population at the fourth passage.

[0125] Next, 1 / 5 of the cell population was seeded on CellSTACK (registered trademark) of the same scale as the previous culture, and subcultured in αMEM containing hPL at a final concentration of 5%. The medium was replaced once every 2 to 4 days. When the cells reached subconfluence, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack, and incubated at 37°C for 3 minutes, allowing the cell population to be completely detached. The cell population obtained here was a cell population at the 5th passage. The cell population had a cell concentration of 4 x 10 6 RPMI1640 was added to the cells / mL. An equal volume of CP-1 (registered trademark) solution (a solution in which CP-1 (registered trademark):25% human serum albumin was mixed at a ratio of 34:16) was added thereto, and 1 mL of each solution was transferred to a cryovial, slowly frozen to -80°C, and then frozen and stored under liquid nitrogen.

[0126] (Step 2-4: Surface antigen analysis of mesenchymal cells) The mesenchymal cell population at the fifth passage cultured by the above culture method was analyzed for various surface antigens (positive rate of CD324, positive rate of CD73 known as an MSC marker, positive rate of CD90, positive rate of CD105, positive rate of CD166, negative rate of CD45, and negative rate of CD326) using a flow cytometer. As a result, the positive rates of CD324 and CD105 were 70% or more (specifically, CD324: 91%, CD105: 95%), and the positive rates of CD73, CD90, and CD166 were all 90% or more (specifically, CD73: 100%, CD90: 100%, CD166: 99%). The negative rates of CD45 and CD326 were all 95% or more (specifically, CD45: 100%, CD326: 99%). From the above results, it was found that the cells cultured by the above culture method were a cell population containing mesenchymal cells. Furthermore, it was confirmed that the cell population described in Example 1 is a cell population in which the proportion of CD324-positive mesenchymal cells is 70% or more and the proportion of CD90-positive mesenchymal cells is 90% or more. The method and reagents used in this measurement were the same as those in Comparative Example 1.

[0127] (Step 2-5: Immortalization of mesenchymal cells) Mesenchymal cells isolated and cultured according to the culture method similar to Example 1 were transfected with 2.5 μg of immortalization plasmid (E1A gene (SEQ ID NO: 4), E1B gene (SEQ ID NO: 9), chicken Bcl-x L The cells were transfected with the SV40 gene (SEQ ID NO: 13), SV40 large T antigen gene (SEQ ID NO: 11), and neomycin resistance gene (SEQ ID NO: 17) (cell suspension buffer: Nucleofector® Solution L, program: X-001). The transfected cells were seeded in a 6-well dish and cultured in αMEM containing 5% hPL until they became subconfluent. After reaching subconfluence, they were repeatedly subcultured in αMEM containing 20 μg / mL G418 and 5% hPL to select only transformed mesenchymal cells. The selected mesenchymal cells are also referred to as "immortalized amnion mesenchymal cells" below. The cell morphology of the untransformed mesenchymal cells was spindle-shaped, while the cell morphology of the transformed immortalized amnion mesenchymal cells was paving-stone-like. Microscopic photographs of each cell are shown in Figures 1 and 2 (magnification: 40 times).

[0128] (Step 2-6: Confirmation of plasmid integration in immortalized amniotic mesenchymal cells) Genome was extracted from immortalized amniotic mesenchymal cells using DNeasy (registered trademark) Blood & Tissue Kit (QIAGEN). Real-time PCR was performed using primers designed in the E1 gene region of the immortalization plasmid used for transfection, using the extracted genome as a template. The nucleotide sequences of the two primers used are represented by SEQ ID NOs: 18 and 19, respectively. The PCR reaction solution (mixture of enzyme and fluorescent dye) was PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific), and the real-time PCR device was StepOnePlus Real-time PCR System (Thermo Fisher Scientific). The reaction conditions were (50°C, 2 minutes) → (95°C, 20 seconds) → [(95°C, 1 second) → (60°C, 30 seconds)] × 40 cycles (the reaction conditions for the melting curve were the standard conditions for the StepOnePlus Real-time PCR system). As a result, nucleic acid amplification was detected using SYBR (registered trademark) Green as an indicator, and the results of the melting curve confirmed that it was specific amplification. In addition, agarose electrophoresis of the PCR products revealed that the length of the amplified product was equivalent to that of the target region amplified by the primers used. This result confirmed that the immortalization plasmid had been incorporated into the genome of the immortalized amniotic mesenchymal cells.

[0129] (Step 2-7: Confirmation of hTERT gene expression in immortalized amniotic mesenchymal cells) Total RNA was extracted from the immortalized amniotic mesenchymal cells using RNeasy (registered trademark) Mini Plus Kit (QIAGEN). Then, cDNA was synthesized from the extracted total RNA using PrimeScript (trademark) RT reagent Kit with gDNA Eraser (Takara Bio). Quantitative real-time PCR was performed using this cDNA as a template, primers targeting hTERT, and a TaqMan (registered trademark) probe (Thermo Fisher Scientific's TaqMan (registered trademark) Gene Expression Assays, No: Hs00972650_m1). The PCR reaction solution (enzyme-containing mixed reaction solution) was TaqMan (registered trademark) FAST Advanced MasterMix (Thermo Fisher Scientific), and the real-time PCR device was StepOnePlus Real-Time PCR system (Thermo Fisher Scientific). The reaction conditions were (50°C, 2 minutes) → (95°C, 20 seconds) → [(95°C, 1 second) → (60°C, 30 seconds)] × 40 cycles. As a result, specific nucleic acid amplification of the hTERT gene was detected using FAM (fluorescein) as an indicator. From this result, it was confirmed that the immortalized amnion mesenchymal cells expressed hTERT and were immortalized.

[0130] Example 2: Immortalization of bone marrow-derived mesenchymal cells (Step 3-1: Cultivation of bone marrow-derived mesenchymal cells) Bone marrow fluid collected from healthy donors by bone marrow aspiration was diluted 6-fold with αMEM containing 10% FBS or αMEM containing 5% hPL, and seeded in a culture vessel for adhesion culture (for example, Adherent Cell Culture Petri Dish 150 (Sumitomo Bakelite Co., Ltd.)). The medium was changed every 3 to 4 days, and the cells were cultured until the adherent cells proliferated. After the adherent cells proliferated and reached subconfluence, they were subcultured and expanded. After expansion, the cells were suspended in Bambanker (registered trademark) (Japan Genetics Co., Ltd.) and frozen for storage. Hereinafter, bone marrow-derived mesenchymal cells obtained by culturing in αMEM containing 10% FBS are also referred to as "bone marrow mesenchymal cells (FBS)", and bone marrow-derived mesenchymal cells obtained by culturing in αMEM containing 5% hPL are also referred to as "bone marrow mesenchymal cells (hPL)".

[0131] (Step 3-2: Immortalization of bone marrow mesenchymal cells) The bone marrow mesenchymal cells (FBS) and bone marrow mesenchymal cells (hPL) isolated and cultured according to the culture method similar to step 3-1 were transfected with 2.5 μg of immortalization plasmid (E1A gene (SEQ ID NO: 4), E1B gene (SEQ ID NO: 9), chicken Bcl-x LThe cells were transfected with the following genes (SEQ ID NO: 13), SV40 large T antigen gene (SEQ ID NO: 11), and neomycin resistance gene (SEQ ID NO: 17) (cell suspension buffer: Nucleofector® Solution L, program: X-001). The transfected cells were seeded in a 6-well dish and cultured in the same culture medium (αMEM containing 10% FBS or αMEM containing 5% hPL) as before the transfection until they became subconfluent. After reaching subconfluence, they were repeatedly subcultured in αMEM containing 20 μg / mL G418 and 10% FBS, or αMEM containing 20 μg / mL G418 and 5% hPL. All the cells cultured in αMEM containing 20 μg / mL G418 and 10% FBS died, while the cells cultured in αMEM containing 20 μg / mL G418 and 5% hPL proliferated, and transformed bone marrow-derived mesenchymal cells were obtained. The transformed bone marrow-derived mesenchymal cells are hereinafter also referred to as "immortalized bone marrow mesenchymal cells". The cell morphology of non-transformed bone marrow mesenchymal cells (hPL) was spindle-shaped, whereas the cell morphology of transformed immortalized bone marrow mesenchymal cells was closer to paving stone-like. Microscopic photographs of each cell are shown in Figures 3 and 4 (magnification x40).

[0132] (Step 3-3: Confirmation of plasmid integration in immortalized bone marrow mesenchymal cells) Genome was extracted from immortalized bone marrow mesenchymal cells using DNeasy (registered trademark) Blood & Tissue Kit (QIAGEN). Real-time PCR was performed using primers (SEQ ID NOs: 13 and 14) designed in the E1 gene region of the immortalization plasmid used for transfection, using the extracted genome as a template. The PCR reaction solution (mixture of enzyme and fluorescent dye) was PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific), and the real-time PCR device was StepOnePlus Real-time PCR System (Thermo Fisher Scientific). The reaction conditions were (50°C, 2 minutes) → (95°C, 20 seconds) → [(95°C, 1 second) → (60°C, 30 seconds)] × 40 cycles (the reaction conditions for the melting curve were the standard conditions for the StepOnePlus Real-time PCR system). As a result, nucleic acid amplification was detected using SYBR (registered trademark) Green as an indicator, and the results of the melting curve confirmed that it was specific amplification. From these results, it was confirmed that the immortalizing plasmid had been incorporated into the genome of the immortalized bone marrow mesenchymal cells.

[0133] (Step 3-4: Confirmation of hTERT gene expression in immortalized bone marrow mesenchymal cells) Total RNA was extracted from immortalized bone marrow mesenchymal cells using RNeasy Mini Plus Kit (QIAGEN). Then, cDNA was synthesized from the extracted total RNA using PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio). Quantitative real-time PCR was performed using the cDNA as a template, primers targeting hTERT, and a TaqMan probe (Thermo Fisher Scientific TaqMan Gene Expression Assays, No: Hs00972650_m1). The PCR reaction solution (enzyme-containing mixed reaction solution) was TaqMan FAST Advanced MasterMix (Thermo Fisher Scientific), and the real-time PCR device was StepOnePlus Real-Time PCR system (Thermo Fisher Scientific). The reaction conditions were (50°C, 2 minutes) → (95°C, 20 seconds) → [(95°C, 1 second) → (60°C, 30 seconds)] × 40 cycles. As a result, specific nucleic acid amplification of the hTERT gene was detected using FAM as an indicator, confirming that the immortalized bone marrow mesenchymal cells expressed hTERT and were immortalized.

[0134] <Example 3: AAV production by immortalized amniotic mesenchymal cells and immortalized bone marrow mesenchymal cells> (Step 4-1: AAV production by immortalized amniotic mesenchymal cells and immortalized bone marrow mesenchymal cells) Immortalized amniotic mesenchymal cells, immortalized bone marrow mesenchymal cells, and HEK293T (set as a control) were transfected with pAAV vector (containing AAV ITR sequence and Akaluc gene), pRC2-mi342 vector (containing Rep gene and Cap gene of serotype 2 AAV (AAV2)), and pHelper vector (containing E2A, E4, and VA derived from adenovirus) by PEI transfection method. The cells were cultured in serum-free medium for 72 hours after transfection. Then, AAV particles were precipitated by adding polyethylene glycol (PEG) to the culture supernatant, and AAV was recovered by detergent treatment. In addition, cells were lysed using detergent, and AAV was also recovered from the cells. Real-time PCR was performed using primers designed for the sequence in the pAAV plasmid, using the culture supernatant and AAV recovered from the cells as templates. The nucleotide sequences of the two primers used are represented by SEQ ID NOs: 18 and 19, respectively. The PCR reaction solution (mixture of enzyme and fluorescent dye) was Powerup SYBR (registered trademark) Green PCR Master Mix (Thermo Fisher Scientific), and the real-time PCR device was QuantStudio3 real-time PCR system. The reaction conditions were (50 ° C, 2 minutes) → (95 ° C, 20 seconds) → [(95 ° C, 1 second) → (60 ° C, 30 seconds)] × 40 cycles (the reaction conditions for the melting curve were the standard conditions for the QuantStudio3 real-time PCR system). In addition, this time, the AAV2 genome concentration in each template was quantified by simultaneously using a serially diluted sample with a known AAV2 genome concentration as a template. As a result, in all cases where the immortalized amnion mesenchymal cells, immortalized bone marrow mesenchymal cells, and AAV2 recovered from the culture supernatant and cells of HEK293T were used as templates, nucleic acid amplification was detected using SYBR (registered trademark) Green as an index, and it was confirmed from the results of the melting curve that it was a specific amplification. These results confirmed that AAV was produced from immortalized amnion mesenchymal cells, immortalized bone marrow mesenchymal cells, and HEK293T. The AAV2 genome concentration of each template is shown in Figure 5.

[0135] <Example 4: Suspension culture of immortalized amnion mesenchymal cells> (Step 5-1: Suspension of immortalized amniotic mesenchymal cells in serum-free medium) The medium of immortalized amnion mesenchymal cells, HEK293T, which were being cultured in adherent culture vessels (e.g., Adherent Cell Culture Petri Dish 150 (Sumitomo Bakelite)), was replaced with serum-free media, FreeStyleF17 Expression Medium (Thermo Fisher Scientific), FreeStyle293 Expression Medium (Thermo Fisher Scientific), and 293 SFMII (Thermo Fisher Scientific). Maintenance culture was continued, and after confirming that the adherent cells had naturally detached, the culture supernatant was collected and the cells were passaged. After that, all the cells were repeatedly passaged in a suspended state. As a result, the immortalized amnion mesenchymal cells could be cultured in suspension in 293 SFM II. HEK293T could be cultured in suspension in FreeStyleF17 Expression Medium and FreeStyle293 Expression Medium. The suspended amnion mesenchymal cells were then cultured in a vented cap Erlenmeyer flask (e.g., polycarbonate Erlenmeyer flask, vented cap, 125 mL (Corning)) with stirring. The immortalized amnion mesenchymal cells were cultured in a suspended state in which single cells were dispersed, whereas the HEK293T cells were cultured in a suspended state in which multiple cells were aggregated. Microscopic photographs of the suspended cultured cells are shown in Figures 6, 7, and 8 (magnification 40x).

Claims

1. a) preparing a cell population containing mesenchymal cells from a sample collected from a living body; b) culturing the cell population of a) with a medium containing a platelet lysate; and c) A cell constituting the cell population containing mesenchymal cells of b) is transfected with genes encoding the E1A and E1B regions of adenovirus, and Bcl-x Ｌ introducing a plasmid carrying a gene encoding the protein; A method for producing a cell population comprising immortalized mesenchymal cells.

2. The method of claim 1, wherein the plasmid in step c) further comprises a gene encoding the SV40 large T antigen.

3. The method according to claim 1 or 2, wherein the cell population prepared in step a) contains 70% or more of CD324-positive mesenchymal cells, 90% or more of CD90-positive mesenchymal cells, and cells expressing the TERT gene.

4. The method according to any one of claims 1 to 3, wherein the proportion of CD326-positive mesenchymal cells in the cell population prepared in step a) is 10% or less.

5. The method according to any one of claims 1 to 4, wherein in the cell population prepared in the step a), the proportion of CD73-positive mesenchymal cells is 80% or more, the proportion of CD166-positive mesenchymal cells is 80% or more, the proportion of CD45-positive mesenchymal cells is 10% or less, and the proportion of CD105-positive mesenchymal cells is 70% or more.

6. The method according to any one of claims 1 to 5, wherein the mesenchymal cells are derived from a fetal appendage.

7. The method of claim 6 , wherein the mesenchymal cells are derived from amniotic membrane.

8. The method according to any one of claims 1 to 5, wherein the mesenchymal cells are derived from bone marrow.

9. The method according to any one of claims 1 to 8, further comprising a step of culturing the cell population after step c) in suspension in a serum-free medium.

10. A method for producing a cell population containing cells that produce adeno-associated virus, comprising a step of introducing a gene for producing adeno-associated virus into cells that constitute the cell population produced by the method according to any one of claims 1 to 9.

11. A cell population comprising immortalized mesenchymal cells, produced by the method according to any one of claims 1 to 9.

12. A cell population comprising immortalized mesenchymal cells, The cell population is characterized in that it contains the E1A region, E1B region and Bcl-x of adenovirus. Ｌ A cell comprising a gene encoding a protein, A cell population comprising cells expressing the TERT gene.

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