JP2022109136A - Method for producing cell population containing immortalized mesenchymal cells and cell population containing immortalized mesenchymal cells - Google Patents

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

INDUSTRIAL APPLICABILITY The present invention can be used in fields such as medicine and biochemistry, and a method for producing a cell population containing immortalized mesenchymal cells that can be stably subcultured, and an immortalized mesenchymal cell Regarding cells.

Mesenchymal stem cells contained in stromal cells are known as somatic stem cells present in bone marrow, adipose tissue, dental pulp, and fetal appendages such as placenta, umbilical cord and egg membrane. Somatic stem cells have been particularly applied to regenerative medicine, and in recent years, a new treatment method that promotes regeneration and functional improvement of tissues and organs by administering cells such as mesenchymal cells to patients has been put to practical use. is progressing rapidly. For example, bone marrow mesenchymal cell preparations for patients with acute graft-versus-host disease (GVHD) and spinal cord injury, and skeletal myoblast sheets for patients with severe heart failure have been sold as regenerative medicine products in Japan. are on sale.

Mesenchymal cells usually have the property of adhering to a culture substrate, and when producing the cell preparation, in addition to the step of growing and culturing the cells by adhering them to the culture substrate, the culture substrate A step of peeling off the cells adhered to the surface is required. The above-mentioned bone marrow mesenchymal cells also have the property of adhering to the culture substrate. Collect the suspension. Examples of the method for detaching the cells from the culture substrate include chemical means using a protease such as trypsin or chemicals, and physical means using an instrument such as a cell scraper for physically detaching cells. means. For example, Patent Document 1 discloses a method of adding trypsin to a culture substrate to exfoliate mesenchymal cells. Further, Patent Document 2 discloses a method of detaching cells from a culture substrate by temperature change, using a culture substrate coated with a temperature-responsive polymer whose cell adhesiveness changes with temperature.

Mesenchymal cells usually have a limited number of cell divisions. Techniques for immortalizing mesenchymal cells such as bone marrow mesenchymal cells and amniotic mesenchymal cells are known in order to permanently use mesenchymal cells, particularly mesenchymal stem cells, which are highly useful. Non-Patent Document 1 describes that the cell life span was extended by introducing genes having base sequences encoding bmi-1, hTERT, and human papillomavirus (HPV) E6 and E7 into bone marrow stem cells. It is shown. In addition, US Pat. No. 6,200,001 discloses a method of generating a permanent amniotic cell line comprising introducing into the amniotic cell at least one nucleic acid that causes the expression of gene products of the E1A region and the E2B region. Patent Document 4 describes a step of introducing a nucleic acid molecule containing a nucleotide sequence encoding adenoviral gene functions E1A and E1B into primary human amniotic cells, followed by SV40 large T antigen or Ebbsty-Barr virus (EBV) nuclear antigen 1. A method for producing a permanent human amniotic cell line is disclosed, comprising the step of introducing a nucleic acid molecule containing a nucleotide sequence encoding (EBNA-1).

Many of the recombinant polypeptides or proteins used in pharmaceutical compositions and the like are produced using animal cells, particularly mammalian cells, particularly cells of human origin. Studies are also underway on the production of recombinant peptides or proteins using mammalian, particularly human-derived mesenchymal cells as hosts. In Patent Document 5, animal cells such as A549, HER, HeLa, and CHO were introduced with an expression construct comprising (a) a promoter operable in animal cells, and (b) an adenovirus E1-encoding modified gene sequence. Adenovirus-producing cell lines are disclosed. It is also disclosed that human amniotic cells can be used as the above animal cells.

Japanese Patent No. 5394932 WO 2001/068799 Special table 2003-514526 Special table 2012-516690 Special table 2015-500007

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).

Methods for obtaining immortalized mesenchymal cells that can be permanently subcultured are known, as 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 a known method do not always stably become immortalized cells. Therefore, a method for more stably obtaining immortalized mesenchymal cells is desired.

An object 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.

As a result of intensive studies, the present inventors cultured a cell population containing mesenchymal cells using a medium containing a platelet lysate, and then transferred the E1A region and E1B region of adenovirus to the cells constituting the cell population. A cell population containing immortalized mesenchymal cells can be stably obtained by introducing plasmids having genes encoding each, a gene encoding SV40 large T antigen, and a gene encoding Bcl- _xL . The inventors have found that and completed the present invention.
That is, according to this specification, the following inventions are provided.

(1) a) preparing a cell population containing mesenchymal cells;
b) a step of culturing the cell population of a) using a medium containing a platelet lysate; , the gene encoding the SV40 large T antigen, and the gene encoding the Bcl-x _L protein, respectively;
A method for producing a cell population containing immortalized mesenchymal cells.
(2) In the cell population prepared in step a), the ratio of CD324-positive mesenchymal cells is 70% or more, and the ratio of CD90-positive mesenchymal cells is 90% or more. and comprising a cell expressing the TERT gene.
(3) The method of (1) or (2), wherein the proportion of CD326-positive mesenchymal cells in the cell population prepared in step a) is 10% or less.
(4) In the cell population prepared in step a), the ratio of CD73-positive mesenchymal cells is 80% or more, the ratio of CD166-positive mesenchymal cells is 80% or more, and CD45 The method according to any one of (1) to (3), wherein the ratio of mesenchymal cells exhibiting positivity is 10% or less and the ratio of mesenchymal cells exhibiting CD105 positivity is 70% or more.
(5) The method according to any one of (1) to (4), wherein the mesenchymal cells are derived from fetal appendages.
(6) The method according to any one of (1) to (5), further comprising the step of suspension culture of the cell population in a serum-free medium.
(7) Adeno-associated virus-producing cells, comprising the step of introducing a gene for producing adeno-associated virus into the cells constituting the cell population produced by any of the methods (1) to (6); A method of producing a cell population comprising:
(8) A cell population containing immortalized mesenchymal cells produced by the method of any one of (1) to (6).
(9) A cell population containing immortalized mesenchymal cells, wherein the cell population contains at least one gene encoding adenovirus E1A region, E1B region, SV40 large T antigen and Bcl-x _L protein and cells expressing the TERT gene.

According to the present invention, 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 are provided. It is possible to

FIG. 1 is a photomicrograph (40× magnification) of a cell population containing untransformed amnion-derived mesenchymal cells. The cell morphology of untransformed mesenchymal cells was spindle-shaped. FIG. 2 is a photomicrograph (40× magnification) of a cell population containing amnion-derived mesenchymal cells transformed with an immortalizing plasmid. The cell morphology of the transformed immortalized amniotic mesenchymal cells was cobblestone-like. FIG. 3 is a photomicrograph (40× magnification) of a cell population containing untransformed bone marrow-derived mesenchymal cells. The cell morphology of untransformed bone marrow mesenchymal cells was spindle-shaped. FIG. 4 is a photomicrograph (40× magnification) of a cell population containing bone marrow-derived mesenchymal cells transformed with an immortalizing plasmid. The cell morphology of transformed immortalized bone marrow mesenchymal cells became close to cobblestone-like. Figure 5 shows the AAV2 genome concentration produced in immortalized amniotic mesenchymal cells (amnion cells), immortalized bone marrow mesenchymal cells (bone marrow cells) and HEK293T cells into which an adeno-associated virus (AAV)-producing gene was introduced. is a graph comparing It was confirmed that AAV2 was produced in all cells. FIG. 6 is a micrograph (40× magnification) of a cell population containing amnion-derived immortalized mesenchymal cells cultured in suspension with 293 SFM II. A cell population containing amnion-derived immortalized mesenchymal cells was cultured in suspension with single cells dispersed. FIG. 7 is a micrograph (40x magnification) of HEK293T cells cultured in suspension in FreeStyle F17 Expression Medium. A plurality of cells were aggregated and cultured in suspension. FIG. 8 is a micrograph (40x magnification) of HEK293T cells cultured in suspension in FreeStyle293 Expression Medium. A plurality of cells were aggregated and cultured in suspension.

[1] Explanation of Terms “Mesenchymal cells” as used herein includes “mesenchymal stem cells (MSCs)” and refers to cells that satisfy the following definitions.

As "mesenchymal cells", cells satisfying the following definition can be used among somatic cells (tissue cells) that can be collected from various tissues and organs. Examples of the somatic cells include bone marrow cells, bone marrow stem cells, adipocytes, adipose stem cells, dental pulp cells, dental pulp stem cells, nerve cells, neural stem cells, cardiomyocytes, cardiac muscle stem cells, liver 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, gastrointestinal epithelial cells, gastrointestinal epithelium Stem cells, osteoblasts, chondrocytes, synovial cells, synovial stem cells and the like are included.

Definition of mesenchymal cells i) Adhesive to plastic under culture conditions in standard medium. A standard medium is a medium obtained by adding serum, a serum-replacement reagent, or a growth factor (eg, human platelet lysate, which is a serum-replacement reagent) to a basal medium (eg, αMEM medium).
ii) positive for surface antigens CD73 and CD90, and negative for CD45 and CD326;

The "mesenchymal cells" are not particularly limited as long as they satisfy the definitions of i) and ii) above, and have the ability to differentiate into bone, cartilage, fat, and the like. The term "mesenchymal cells" as used herein also includes cells that have the ability to differentiate into bone, cartilage and fat, such as mesenchymal stem cells. The "mesenchymal cells" also include cells that satisfy 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.

As used herein, "immortalized" mesenchymal cells refer to cells that are capable of stably dividing and proliferating without dying under conditions in which the cells can grow. As used herein, immortalized mesenchymal cells are at least 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 110 times or more, 120 times or more, 130 times or more, 140 times or more, 150 times or more, 160 times or more, 170 times or more, 180 times or more, 190 times or more, 200 times or more are possible.

As used herein, "fetal appendages" refers to the egg membrane, placenta, umbilical cord and amniotic fluid. Furthermore, the "egg membrane" is the gestational sac containing the amniotic fluid of the fetus, consisting from the inside of the amniotic membrane, the chorion and the decidua. Of these, the amnion and chorion originate from the fetus. The amniotic membrane is the innermost layer of the egg membrane, which is a transparent thin membrane with few blood vessels. The inner layer of the amniotic membrane (also called the epithelial cell layer) is covered with a single layer of epithelial cells that have a secretory function and secretes amniotic fluid, while the outer layer of the amniotic membrane (also called the extracellular matrix layer, which corresponds to the stroma) contains mesenchymal cells. include.

The "cell population containing mesenchymal cells" as used herein is not particularly limited in its form. For example, cell pellets, cell aggregates, cell sheets, cell suspensions, cell suspensions, frozen products of these is mentioned.

As used herein, the "proportion of mesenchymal cells positive for a given surface antigen" refers to the ratio of cells positive for a given surface antigen analyzed by flow cytometry, as described in Examples below. indicates As used herein, the ratio of cells that are positive for a given surface antigen is sometimes referred to as the "positive rate," and the ratio of cells that are negative for a given surface antigen is referred to as the "negative rate." There is something.

As used herein, "mesenchymal cells", "cell populations containing mesenchymal cells" and "fetal appendages" are preferably derived from humans.

As used herein, adenoviral "E1A" is the first expressed protein in adenoviral replication, and "E1B" is the second expressed protein. Adenoviruses E1A and E1B are known to bind to and inactivate tumor suppressor genes Rb and p53 in animal cells, thereby making the cells cancerous and allowing them to proliferate. The E1A protein has an amino acid sequence represented by any of SEQ ID NOs: 1 to 3, or a sequence represented by any of SEQ ID NOs: 1 to 3 and 80% or more, 85% or more, 90% or more, 95% It has amino acid sequences with 96% or more, 97% or more, 98% or more, or 99% or more identity. In addition, the gene encoding the E1A protein is represented by any of SEQ ID NOS: 1 to 3, or is 80% or more, 85% or more, 90% or more of the sequence represented by any of SEQ ID NOS: 1 to 3, It has a base sequence encoding a protein having an amino acid sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. The nucleotide sequence of the gene encoding the E1A protein is not limited as long as it encodes these proteins. , 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity. The E1B protein has an amino acid sequence represented by any of SEQ ID NOS: 5-8, or a sequence represented by any of SEQ ID NOS: 5-8 and 80% or more, 85% or more, 90% or more, 95% It has amino acid sequences with 96% or more, 97% or more, 98% or more, or 99% or more identity. In addition, the gene encoding the E1B protein is represented by any of SEQ ID NOS: 5 to 8, or is 80% or more, 85% or more, 90% or more of the sequence represented by any of SEQ ID NOS: 5 to 8, It has a base sequence encoding a protein having an amino acid sequence with 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. The nucleotide sequence of the gene encoding the E1B protein is not limited as long as it encodes these proteins. , 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity.

As used herein, "SV40 (Simian Virus 40)" is a DNA virus belonging to the polyomaviruses, and is known to infect cells of mice and the like and cause some of them to become cancerous. The “SV40 large T antigen” (hereinafter also referred to as “LT Ag”) is known to bind to and inactivate the tumor suppressor genes Rb and p53, like the adenoviral E1A and E1B proteins. LT Ag has the amino acid sequence represented by SEQ ID NO: 10, or is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more of the sequence represented by SEQ ID NO: 10 % or greater or 99% or greater amino acid sequence identity. In addition, the gene encoding LTAg is represented by SEQ ID NO: 10, or 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more of the sequence represented by SEQ ID NO: 10 , has a base sequence encoding a protein having an amino acid sequence with 98% or more or 99% or more identity. The nucleotide sequence of the gene encoding LTAg is not limited as long as it encodes these proteins. Sequences can have greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity.

As used herein, “Bcl-x _L ” is a protein that belongs to the Bcl2 family and suppresses apoptosis, and the gene encoding Bcl-x _L is known as an oncogene. The gene encoding Bcl-x _L may be derived from any animal, but, for example, a chicken-derived gene can be preferably used. The Bcl-x _L protein has the amino acid sequence represented by SEQ ID NO: 12 or is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more of the sequence represented by SEQ ID NO: 12 % or greater, 98% or greater, or 99% or greater amino acid sequence identity. In addition, the gene encoding Bcl-x _L is represented by SEQ ID NO: 12, or is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more of the sequence represented by SEQ ID NO: 12, It has a nucleotide sequence that encodes a protein having an amino acid sequence with 97% or more, 98% or more, or 99% or more identity. The nucleotide sequence of the gene encoding Bcl-x _L is not limited as long as it encodes these proteins. % or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity.

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

The surface antigen "CD90" stands for Cluster of Differentiation 90, a protein also known as Thy-1.

The surface antigen "CD326" stands for Cluster of Differentiation 326, a protein also known as EpCAM.

The surface antigen "CD73" stands for Cluster of Differentiation 73, a protein also known as 5-Nucleotidase, or Ecto-5'-nucleotidase.

The surface antigen "CD166" stands for Cluster of Differentiation 166 and is a protein also known as Activated leukocyte cell adhesion molecule (ALCAM).

The surface antigen "CD105" stands for Cluster of Differentiation 105 and is a protein also known as Endoglin.

The surface antigen "CD45" means differentiation cluster 45, and is a protein also known as PTPRC (Protein tyrosine phosphatase, receptor type, C) or LCA (Leukocyte common antigen).

The surface antigen "CD34" stands for Cluster of Differentiation 34, a protein also known as the Hematopoietic progenitor cell antigen CD34.

As used herein, "telomerase reverse transcriptase (TERT)" is a protein that, together with the telomerase RNA component (TERC), constitutes the telomerase complex. Telomerase binds to the ends of chromosomes and adds telomere repeat sequences to the 3' ends of telomere DNA using TERC as a template. Telomerase is rarely expressed in normal human somatic cells, but is expressed in fetal cells, germ cells, and the like. Cells without telomerase activity have a limited number of cell divisions because telomeres shorten with cell division and undergo cell senescence, whereas cells with telomerase activity maintain telomere length due to telomerase activity. , unlimited cell growth is possible. Herein, TERT gene expression is used as an indicator of cell immortalization.

[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:
a) preparing a cell population comprising mesenchymal cells;
b) a step of culturing the cell population of a) using a medium containing platelet lysate; introducing a plasmid carrying the gene encoding SV40 large T antigen and the gene encoding Bcl- _xL .

[2-1] Step of preparing a cell population containing mesenchymal cells In the method of the present invention, the "cell population containing mesenchymal cells" is particularly limited as long as it is a cell population containing at least mesenchymal cells It may be a population containing other cells instead of cells. Although not particularly limited, the ratio of mesenchymal cells in the cell population containing mesenchymal cells is 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.

In addition, the ratio of other cells in the cell population containing the 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 thereof include blood cells such as lymphocytes, granulocytes, and erythrocytes.

The cell population containing mesenchymal cells used in the method of the present invention is not particularly limited as long as it contains cells that satisfy the above definitions i) and ii), but the following (a) and (b) Preferably, the cell population is prepared to have the properties of
(a) the proportion of CD324-positive mesenchymal cells in the cell population is 70% or more,
(b) In the cell population, the proportion of CD90-positive mesenchymal cells is 90% or more.
By providing the above properties, the cell population containing mesenchymal cells can spontaneously detach from the substrate after culturing. As a result, cultured cells can be efficiently obtained without using enzymes or special equipment as in the methods described in Patent Documents 1 and 2.

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

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% Above, 99% or more may be sufficient, and 100% may be sufficient.

In one or more embodiments of the method of the present invention, the cell population preferably has a percentage of CD326-positive mesenchymal cells of 10% or less.

In the cell population, the ratio of CD326-positive mesenchymal cells 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% above), 2% or less (negative rate of 98% or more), 1% or less (negative rate of 99% or more), or 0% (negative rate of 100%).

According to one aspect of the present invention, the cell population containing mesenchymal cells provided by the present invention preferably has a ratio of CD73-positive mesenchymal cells of 90% or more, while exhibiting CD166-positive The ratio of mesenchymal cells is 80% or more, the ratio of mesenchymal cells that are CD105-positive is 70% or more, the ratio of mesenchymal cells that are CD45-positive is 10% or less, and the cells are CD34-positive. Satisfies one or more of the conditions that the proportion of mesenchymal cells is 10% or less.

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 100%.

The ratio of CD166-positive mesenchymal cells in the cell population is more preferably 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher.

The ratio of CD105-positive mesenchymal cells in the cell population is more preferably 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.

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 0% (negative rate of 100%).

The ratio of CD34-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 0% (negative rate of 100%).

Here, CD324-positive, CD90-positive, CD326-positive, CD73-positive, CD166-positive, CD105-positive, CD45-positive and CD34-positive mesenchymal cells are CD324, CD90, CD326, CD73, CD166, CD105, CD45, respectively. and CD34-positive mesenchymal cells.

For cell populations used in one or more embodiments of the methods of the invention, the indicative expression marker (CD324, CD90, CD326, CD73, CD166, CD105, CD45 or CD34) can be any known in the art. It can be detected by a detection method. Methods of 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 that emits more fluorescence than the negative control (isotype control) is detected, the cell is determined to be "positive" for that marker. Any antibody known in the art can be used as the fluorescence-labeled antibody, and examples thereof include antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and the like. include but are not limited to: In cell staining, when colored or fluorescent cells are observed under a microscope, the cells are determined to be "positive" for the marker. Cell staining may be immune cell staining using antibodies or non-immune cell staining without antibodies. In the present specification, expression marker and surface antigen are synonymous and can be used interchangeably.

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

Cell populations used in one or more embodiments of the methods of the invention can be stored in a frozen state until immediately prior to use. The above cell population may contain any component other than mesenchymal cells. Examples of such components include salts, polysaccharides (e.g., HES, dextran, etc.), proteins (e.g., albumin, etc.), DMSO, medium components (e.g., components contained in RPMI1640 medium, etc.), and the like. , but not limited to.

The cell population containing mesenchymal cells used in the method of the present invention is obtained by culturing commercially available mesenchymal stem cells (e.g., human bone marrow-derived mesenchymal stem cells (hMSC mesenchymal stem cells, manufactured by Lonza)). may be prepared using 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 aspiration, fetal appendages such as amnion, and the like.

The raw material for producing the cell population containing the mesenchymal cells of the present invention can be the aforementioned bone marrow fluid or fetal appendages, but is not limited thereto. It is preferred to use fetal appendages to obtain cell populations having the characteristics of (a) and (b) above. An embodiment of preparing a cell population containing mesenchymal cells from fetal appendages as a raw material will be exemplified below.

The amniotic membrane consists of an epithelial cell layer and an extracellular matrix layer, the latter containing amniotic MSCs. The step of obtaining a cell population may further include a step of obtaining an amniotic membrane by caesarean section.

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

Enzymatic treatment of a sample taken from a fetal appendage (preferably a sample comprising an epithelial cell layer and a mesenchymal cell layer) preferably liberates mesenchymal cells contained in the extracellular matrix layer of the fetal appendage. treatment with an enzyme (or a combination thereof) that is capable of removing the epithelial cell layer and does not degrade the epithelial cell layer. Examples of such enzymes include, but are not limited to, collagenase and/or metalloproteinase. Metalloproteinases include, but are not limited to, thermolysin and/or dispase, which are metalloproteinases that cleave the N-terminal side of nonpolar amino acids.

The activity concentration of collagenase is preferably 50 PU/ml or more, more preferably 100 PU/ml or more, still more preferably 200 PU/ml or more. The activity concentration of collagenase is not particularly limited, but is, 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.

The activity concentration of the metalloproteinase (eg, thermolysin and/or dispase) is preferably 50 PU/ml or more, more preferably 100 PU/ml or more, still more preferably 150 PU/ml or more, still more preferably 190 PU/ml or more. In addition, the activity concentration of the metalloproteinase is preferably 1000 PU/ml or less, more preferably 900 PU/ml or less, still more preferably 800 PU/ml or less, still more preferably 700 PU/ml or less, still more preferably 600 PU/ml or less, still more preferably 600 PU/ml or less. is 500 PU/ml or less, more preferably 300 PU/ml or less. Here, in the embodiment using dispase as the metalloproteinase, PU (Protease Unit) is defined as the amount of enzyme that releases an amino acid equivalent to 1 μg of tyrosine per minute from lactic casein at pH 7.5 and 30°C. be. Within the above enzyme concentration range, it is possible to efficiently liberate mesenchymal cells contained in the extracellular matrix layer while preventing epithelial cells contained in the epithelial cell layer of fetal appendages from being mixed. Preferred concentration combinations of collagenase and/or metalloproteinases can be determined by microscopic observation of fetal appendages after enzymatic treatment and by flow cytometry of the obtained cells.

From the viewpoint of efficiently collecting living cells, it is preferable to treat fetal appendages with a combination of collagenase and metalloproteinase. More preferably, the combination treats fetal appendages simultaneously. As the metalloproteinase in this case, thermolysin and/or dispase can be used, but not limited thereto. Mesenchymal cells can be conveniently obtained by treating fetal appendages only once with an enzyme solution containing collagenase and metalloproteinase. In addition, the simultaneous batch processing can reduce the risk of contamination with bacteria, viruses, and the like.

The enzymatic treatment of fetal appendages is preferably carried out by immersing the amniotic membrane, which has been washed with a washing solution such as physiological saline or Hank's balanced salt solution, in the enzyme solution and stirring the enzyme solution with a stirring means. From the viewpoint of efficiently liberating the mesenchymal cells contained in the extracellular matrix layer of the fetal appendage, the stirring means may be, for example, a stirrer or shaker, but is not limited thereto. The stirring speed is not particularly limited, but when a stirrer or shaker is used, it is, 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. The stirring speed is not particularly limited, but when using a stirrer or shaker, it is, 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 is, for example, 10 minutes or longer, 20 minutes or longer, 30 minutes or longer, 40 minutes or longer, 50 minutes or longer, 60 minutes or longer, 70 minutes or longer, 80 minutes or longer, or 90 minutes or longer. The enzyme treatment time is not particularly limited, but is, 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 is, for example, 15°C or higher, 16°C or higher, 17°C or higher, 18°C or higher, 19°C or higher, 20°C or higher, 21°C or higher, 22°C or higher, 23°C or higher, 24°C. 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, or 36°C or higher be. Moreover, although the enzyme treatment temperature is not particularly limited, it is, for example, 40° C. or lower, 39° C. or lower, 38° C. or lower, or 37° C. or lower.

If desired, the released mesenchymal cells can be separated and/or recovered from the enzyme solution containing the released mesenchymal cells by known means such as a filter, centrifugation, hollow fiber separation membrane, cell sorter, and the like. Preferably, the enzyme solution containing mesenchymal cells released by a filter is filtered. In the embodiment in which the enzyme solution is filtered through a filter, only the released cells pass through the filter, and the undegraded epithelial cell layer remains on the filter without passing through the filter. Not only can it be separated and/or recovered easily, but also the risk of contamination with bacteria, viruses, etc. can be reduced. Examples of filters include, but are not limited to, mesh filters. 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 is, 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. . The filtration rate is not particularly limited, but by setting the pore size of the mesh filter within the above range, the enzyme solution containing mesenchymal cells can be filtered by gravity, thereby preventing a decrease in cell viability. be able to.

Nylon is preferably used as the material of the mesh filter. Tubes containing 40 μm, 70 μm, 95 μm or 100 μm nylon mesh filters are available, such as the Falcon cell strainer commonly used for research. In addition, medical mesh cloths (nylon and polyester) used in hemodialysis and the like can be used. Furthermore, an arterial filter (polyester mesh filter, pore size: 40 μm or more and 120 μm or less) used for extracorporeal circulation can also be used. Other materials such as stainless steel mesh filters can also be used.

When passing mesenchymal cells through a filter, natural fall (free fall) is preferred. Forced passage through the filter, such as suction using a pump or the like, is also possible, but it is desirable to use as weak a pressure as possible in order to avoid damaging the cells.

A cell population containing mesenchymal cells that has passed through the filter can be recovered by centrifugation after diluting the filtrate with a double or more volume of medium or balanced salt buffer. Examples of balanced salt buffers that can be used include, but are not limited to, Dulbecco's phosphate buffer (DPBS), Earle's balanced salt solution (EBSS), Hank's balanced salt solution (HBSS), phosphate buffer (PBS), and the like.

[2-2] Step of culturing a cell population containing mesenchymal cells using a medium containing a platelet lysate The method of the present invention comprises mesenchymal cells prepared by the method described in [2-1]. culturing a cell population containing with a medium containing a platelet lysate.

When the present inventors immortalize and subculture a cell population containing mesenchymal cells, the culture conditions of the cell population before the immortalization step affect the stability of the subsequent subculture. I found out.

The “platelet lysate (PL)” in the method of the present invention is preferably mammalian (eg, 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 stromal stem cells. In the method of the present invention, a cell population containing mesenchymal cells is cultured in a medium supplemented with PL (especially hPL) (hereinafter simply referred to as "PL medium" or "hPL medium"), for example, Compared to a medium supplemented with other components such as fetal bovine serum (FBS), the survival rate of cells after the subsequent immortalization step during subculturing can be remarkably increased. This makes it possible to stably obtain a cell population containing immortalized mesenchymal cells.

The cell seeding density in the step of culturing the cell population is not particularly limited, but the cells can be seeded at a density of, for example, 500 to 10,000 cells/cm ² . The lower limit of the seeding density is, for example, 500 cells/cm ² or more, 1,000 cells/cm ² or more, 2,000 cells/cm ² or more, 3,000 cells/cm ² or more, 4,000 cells/cm ² or more. Above, 5,000 cells/cm ² or above is preferable. The upper limit of the seeding density is preferably 10,000 cells/cm ² or less, 9,000 cells/cm ² or less, 8,000 cells/cm ² or less, or 7,000 cells/cm ² or less.

In addition, the step of culturing may include a passage step. The culture period of the above single culture can be, for example, 4 to 10 days, more specifically, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days. can be mentioned.

The medium used for the above culture is prepared by using any liquid medium for animal cell culture as a basal medium, and adding PL, preferably hPL, thereto. Examples of the lower limit of the hPL concentration in the medium include 1% by weight or more, 2% by weight or more, and 3% by weight or more as a final concentration. Moreover, the upper limit of the concentration of hPL in the medium is preferably 20% by weight or less, 10% by weight or less, or 7% by weight or less.

If necessary, other components (serum, growth factors, etc.) may be added to the basal medium. In the embodiment in which the growth factor is added to the basal medium, a reagent (such as heparin) for stabilizing the growth factor in the medium may be added in addition to the growth factor. Alternatively, the growth factor may be stabilized in advance with gel or polysaccharide, and then the stabilized growth factor may be added to the basal medium. Thus, a medium obtained by adding PL, preferably hPL to a basal medium for culturing a cell population containing mesenchymal cells is defined as a standard medium.

The basal medium includes 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 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 medium thereof (e.g., DMEM/F12 medium ( A medium such as Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham)) can be used, but is not particularly limited. αMEM medium can be exemplified as a preferable basal medium.

Other components that can be added to the basal medium include, for example, albumin, bovine serum, or growth factors. Among others, it is preferable to culture in a basal medium that does not contain albumin, bovine serum and growth factors.

Cultivation of a cell population containing mesenchymal cells can be performed, 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 using a medium in a 37° C. environment with a CO ₂ concentration of 3% or more and 5% or less. The cells obtained by culturing as described above are cells that have been cultured once.

For example, the cells that have been cultured once can be further subcultured and cultured as follows. First, cells cultured until the confluency rate reaches 95% or less in the first culture are treated with ethylenediaminetetraacetic acid (EDTA) and then treated with trypsin to detach from the plastic culture vessel. Next, the resulting cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in medium. Finally, the cells are seeded in a plastic culture vessel and cultured using a medium in a 37° C. environment with a CO ₂ concentration of 3% or more and 5% or less. Cells obtained by passage and culture as described above are cells that have been passaged once. By performing similar passages and cultures, cells that have been passaged n times can be obtained (n is an integer of 1 or more). When performing subculture, the cells are cultured until the confluency rate becomes 95% or less, and the cells are detached and collected by the above procedure, and can be used for the next generation culture. From the viewpoint of mass production of cells, the lower limit of the number of passages n is, for example, 1 or more, preferably 2 or more, more preferably 3 or more, still more preferably 4 or more, and still more preferably 5 or more. be. Moreover, the upper limit of the passage number n is preferably, for example, 50 times or less, 45 times or less, 40 times or less, 35 times or less, or 30 times or less from the viewpoint of suppressing cell aging.

[2-3] Step of Selecting Cell Population The method of the present invention may optionally include a step of selecting a cell population having the following properties (a) and (b).
(a) the ratio of mesenchymal cells exhibiting CD324 positive is 70% or more,
(b) The proportion of mesenchymal cells exhibiting CD90 positivity is 90% or more.

Means for selecting a cell population using the above characteristics as an index include, for example, physical means such as FACS, cell sorter, magnetic bead fractionation, and selection of cells that do not meet the index by appropriate culture conditions so that the above index is satisfied. Chemical means for purifying a cell population containing mesenchymal cells can be mentioned, but the means is not particularly limited and may be appropriately selected according to culture conditions and the like. Also in the selection step, culture is preferably performed in a medium containing platelet lysates.

The timing of selecting a cell population having the above characteristics (a) and (b) is not particularly limited, but for example, before culturing, during culturing, after culturing, before collecting the cell population, after collecting the cell population, before preparing a cryopreserved stock of cells, after thawing a cryopreserved stock of cells, and the like.

The procedure for selecting cell populations having the properties (a) and (b) above will be described in more detail.
For example, by seeding a cell population containing mesenchymal cells on a culture substrate and growing and culturing on the culture substrate, mesenchymal cells exhibiting CD324-positive and CD90-positive are positively selected, while exhibiting CD324-positive Means for exfoliating and collecting a cell population having a ratio of mesenchymal cells of 70% or more and a ratio of CD90-positive mesenchymal cells of 90% or more can be mentioned. At this time, 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 culturing is not particularly limited, and can be selected in any subculture. It is preferable to select CD324-positive and CD90-positive mesenchymal cells from the cell population containing mesenchymal cells in .

In addition, CD324-positive and CD90-positive mesenchymal cells can also be selected from a cell population containing mesenchymal cells by a cell separation method using flow cytometry or magnetic beads.

Selection of the cell population is further preferably performed by selecting and fractionating a cell population in which the ratio of mesenchymal cells exhibiting CD326 positive is 10% or less, and further, the ratio of mesenchymal cells exhibiting CD73 positive 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 It is preferable to select and fractionate a cell population that satisfies one or more of 10% or less and a ratio of CD34-positive mesenchymal cells of 10% or less. The sorted cell population may be further cultured by the means described above.

[2-4] A step of introducing an immortalizing plasmid into cells constituting a cell population containing mesenchymal cells. A step of introducing a plasmid having a gene encoding Bcl-x _L (hereinafter also referred to as an “immortalizing plasmid”) into cells constituting a cell population containing mesenchymal cells. Plasmids used in the methods of the present invention may comprise all the genes encoding E1A, E1B, LT Ag and Bcl-x _L in one plasmid, or encode E1A, E1B, LT Ag and Bcl-x _L. The gene may be provided separately in two or more plasmids. Preferably, one plasmid contains all the genes encoding E1A, _E1B , LT Ag and Bcl-xL.

The immortalizing plasmid used in the method of the present invention contains, in addition to genes encoding E1A, E1B, LT Ag and Bcl-x _L , promoters such as CMV promoter, CAG promoter, RSV promoter, SV40 poly A, IRES (internal ribosome entry site) and/or a nucleic acid having a base sequence of a resistance gene such as neomycin.

The nucleotide sequence of the CMV promoter is not particularly limited as long as it is a nucleotide sequence having promoter activity. Sequences can have greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% identity.

The nucleotide sequence of SV40 poly A is not particularly limited as long as it is the nucleotide sequence of poly A that constitutes SV40. Sequences can have 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity.

The IRES sequence is not particularly limited as long as it functions as a translation initiation site. As described above, sequences having 96% or more, 97% or more, 98% or more, or 99% or more identity can be used.

The nucleotide sequence of the neomycin resistance gene (NeoR) is not particularly limited as long as the gene product has neomycin resistance activity. As described above, sequences having 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity can be used.

The means for introducing the plasmid into the cells constituting the cell population containing the mesenchymal cells is not particularly limited, and calcium phosphate method, electroporation, lipofection, microinjection, and combinations of these means are all used. can. Alternatively, it may be carried out using a commercially available kit (for example, Nucleofector (registered trademark) 2/2b (Lonza)).

The method of the present invention may optionally include a step of confirming whether the cells constituting the cell population have been transfected with the gene of interest. Specifically, for example, a method of extracting a genome from a cell population and performing PCR of a transfected gene using the extracted genome as a template can be mentioned. For example, using the genome as a template, primers designed to amplify the E1 region of adenovirus (e.g., having the nucleotide sequences of SEQ ID NOS: 18 and 19) are used to observe the presence or absence of nucleic acid amplification in PCR. , it is possible to confirm the introduction.

Moreover, the method of the present invention may optionally comprise a step of confirming whether the cell population contains immortalized mesenchymal cells. 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 a cell population, and cDNA is prepared using reverse transcriptase. Nucleic acid amplification in real-time PCR using cDNA as a template and primers and probes designed to amplify and detect the TERT gene (e.g., Thermo Fisher Scientific TaqMan (registered trademark) Gene Expression Assays, No: Hs00972650_m1) By measuring the rate, the expression level of the TERT gene can be measured. In the method of the invention, the TERT gene is preferably the human TERT (hTERT) gene.

[2-5] Step of suspension culture of cell population The method of the present invention may include a step of suspension culture of a cell population containing immortalized mesenchymal cells. The cell population to be subjected to suspension culture may be a cell population containing immortalized mesenchymal cells prepared in the above [2-4] section, or a virus-producing gene in the following [2-6] section. It may be a cell population containing immortalized mesenchymal cells introduced with.

Mesenchymal cells are usually cultured in adherent culture, but in some embodiments, cell populations containing immortalized mesenchymal cells produced by the method of the present invention are cultured in suspension culture in serum-free medium. It is possible to As the serum-free medium, an existing liquid medium that is suitable for animal cell suspension culture and does not contain serum components or serum-substitute components can be used. In particular, a medium suitable for suspension culture of human cells can be preferably used. Examples of such serum-free media include, but are not limited to, CD 293 Medium, CD 293 AGT (trademark) Medium, 293 SFM II and FreeStyle (trademark) 293 Expression Medium, CD CHO Medium, CD CHO AGT (trademark). Medium, CD OptiCHO™ Medium, CHO-S-SFM II (both from 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 CHO™ Power Ｓｅｒｕｍ－ｆｒｅｅ ＭｅｄｉｕｍＰｏｗｅｒＣＨＯ（商標） ＧＳ Ｓｅｒｕｍ－ｆｒｅｅ ＣＨＯ ＭｅｄｉｕｍＰｏｗｅｒＣＨＯ Ａｄｖａｎｃｅ（商標） Ｓｅｒｕｍ－ｆｒｅｅ Ｍｅｄｉｕｍ（いずれもＬｏｎｚａ社）、ＣＤＭ４ＨＥＫ２９３、ＳＦＭ４ＨＥＫ２９３、ＳＦＭ４Ｔｒａｎｓｆｘ－２９３、ＨｙＣｅｌｌ ＴｒａｎｓＦｘ－Ｈ、ＣＤＭ４ＰＥＲＭＡｂ、ＨｙＣｅｌｌ ＣＨＯ、ＡｃｔｉＰｒｏ、ＡｃｔｉＳＭ（ Cytiva). In particular, 293 SFM II can be preferably used.

As described above, the cell population containing immortalized mesenchymal cells produced by the method of the present invention can be cultured in suspension, so unlike adherent culture, it does not require a culture substrate. No detachment of cells from is required. Therefore, subculturing can be performed more easily. Also, more efficient cell growth is possible compared to adherent culture.

[2-6] Step of introducing an adeno-associated virus-producing gene A gene for producing an adeno-associated virus is introduced into the constituent cells of the cell population containing the immortalized mesenchymal cells produced by the above method. As a result, a cell population containing cells that produce adeno-associated virus can be produced.

Since the cell population produced by the above method contains immortalized cells, it has become possible to introduce an AAV-producing gene and produce AAV.

AAV is a non-pathogenic virus belonging to parvovirus, lacks self-replicating ability, cannot propagate autonomously, and usually requires co-infection with a helper virus such as adenovirus or herpes virus. Because of its low infectivity and immunogenicity, the virus is highly safe and has a wide host range. Due to such characteristics, AAV is a virus that is expected to be widely applied to gene therapy and the like as a vector for gene transfer.

The genome of AAV is a single-stranded DNA of about 4.7 kb, with a T-shaped hairpin structure called ITR (Inverted Terminal Repeat) at both ends, between which Rep (related to replication and transcription), Cap (external There are three ORFs, encoding coat proteins) and AAP (virus particle formation). There are multiple serotypes of AAV. AAV2, AAV3, AAV5 and AAV6 are known as serotypes discovered from human cells.

The gene for producing AAV is not particularly limited as long as AAV is produced by introduction into immortalized mesenchymal cells, and may be composed of, for example, the following three plasmid vectors.
・pAAV vector: vector plasmid containing expression cassette of target gene and two ITRs ・pRC vector: plasmid containing Rep gene of AAV2 and Cap gene of each serotype ・pHelper vector: containing E2A, E4 and VA derived from adenovirus plasmid

In the embodiment using the above plasmid vector as a gene for AAV production, any known means may be used to introduce the plasmid vector into cells. For example, the polyethylenimine (PEI) transfection method may be used to introduce plasmids into cells of interest. Detailed conditions of the PEI method are described, for example, in Y.K. Durocher, et al. , Nuc. Acid. Res. , Vol. 30, No. 2, e9 (2002).

[2-7] Step of cryopreserving a cell population The method of the present invention can 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 virus-producing gene. In the aspect including the step of cryopreserving the cell population, after thawing the cell population, the cell population may be separated, collected and/or cultured as necessary. Alternatively, the cell population may be used as it is after being thawed.

Means for cryopreserving the cell population containing the mesenchymal cells are not particularly limited, but examples thereof include program freezers, deep freezers, immersion in liquid nitrogen, and the like. The freezing temperature is preferably -30°C or less, -40°C or less, -50°C or less, -60°C or less, -70°C or less, -80°C or less, -90°C or less, -100°C or less, - 110°C or less, -120°C or less, -130°C or less, -140°C or less, -150°C or less, -160°C or less, -170°C or less, -180°C or less, -190°C or less, or -196°C (liquid nitrogen temperature). Preferred freezing rates for freezing are, 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 is. When a program freezer is used as such freezing means, for example, the temperature is reduced to a temperature between -50°C and -30°C (e.g., -40°C) at a freezing rate of -2°C/min or more and -1°C/min or less. and further lower the temperature to -100°C or more and -80°C or less (e.g., -90°C) at a freezing rate of -11°C/min or more and -9°C/min or less (e.g., -10°C/min). be able to.

When freezing by the above freezing means, the above cell population may be frozen in any storage container. Examples of such storage containers include, but are not limited to, cryotubes, cryovials, freezing bags, transfusion bags, and the like.

The cryopreservation solution preferably contains a predetermined concentration of albumin greater than 0% by mass from the viewpoint of increasing the survival rate of MSCs, which have a relatively high proliferative ability. A preferable albumin concentration 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 It is 8% by mass or more. Further, 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. be. Examples of albumin include, but are not limited to, bovine serum albumin, mouse albumin, and human albumin.

[3] A cell population containing mesenchymal cells The cell population of the present invention is an immortalized cell produced by the method described in the section "[2] Method for producing a cell population containing immortalized mesenchymal cells" A cell population containing leaf cells. Alternatively, the cell population of the present invention contains cells containing at least one of the genes encoding adenovirus E1A, E1B, SV40 large T antigen and Bcl-x _L , and cells expressing the TERT gene. A cell population containing immortalized mesenchymal cells, comprising:

The cell population of the present invention has features in common with the cell population described in the section "[2] Method for producing cell population containing immortalized mesenchymal cells" unless otherwise specified below.

In the cell population of the present invention, the "cells containing at least one of the genes encoding adenovirus E1A, E1B, SV40 large T antigen and Bcl-x _L " specifically means that the genomic DNA contains Cells in which genes encoding adenoviral E1A, E1B, SV40 large T antigen or Bcl- _xL are present at levels detectable by existing methods are shown. In the cell population of the present invention, the ratio of cells containing at least one of the genes encoding adenovirus E1A, E1B, SV40 large T antigen and Bcl-x _L is 5% or more, 10% or more, 20% 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 Well, it may be 100% or more.

In the cell population of the present invention, "cells expressing the TERT gene" specifically means that mRNA having a base sequence encoding TERT is at a level at which it can be detected by any existing method such as quantitative real-time PCR. indicates the cells present in . In the cell population of the present invention, the ratio of cells expressing the TERT gene is 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.

<Comparative Example 1: Immortalization of amnion-derived mesenchymal cells (FBS medium culture system)>
(Step 1-1: Collection of amniotic membrane)
Fetal appendages, the egg membrane and placenta, were aseptically harvested from a pregnant woman (donor #1) who had an elective caesarean section with informed consent. The resulting egg membrane and placenta were placed in a sterile vat containing physiological saline, and the amniotic membrane was manually stripped from the egg membrane stump. The amniotic membrane was washed with Hank's balanced salt solution (Ca-Mg free) to remove adhering blood and clots.

(Step 1-2: enzymatic treatment of amniotic membrane and collection of mesenchymal cells)
The amniotic membrane containing the epithelial cell layer and the mesenchymal cell layer was immersed in a Hank's balanced salt solution (containing Ca and Mg) containing 240 PU/mL collagenase and 200 PU/mL dispase I, at 37°C for 90 minutes at 50 rpm. The amniotic membrane was enzymatically treated by shaking and stirring at . The enzyme-treated solution was filtered through a nylon mesh with an opening of 95 μm to remove undigested amniotic membrane, and a cell suspension containing mesenchymal cells was recovered.

(Step 1-3: Culture of mesenchymal cells)
The cell population containing mesenchymal cells obtained in the above "Step 1-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells" was seeded in a culture vessel CellSTACK (registered trademark) (manufactured by Corning). . The seeding density was 6,000 cells/cm ² . After seeding the cells, the cells become sub-confluent in αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing fetal bovine serum (FBS) at a final concentration of 10% and basic fibroblast growth factor (bFGF) at 10 ng/mL. Adherent culture was carried out until After culturing, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack and incubated at 37°C for 3 minutes. remained in Therefore, it was incubated for an additional 5 minutes (8 minutes total) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is the cell population at passage 0. Then, 1/5 of the cell population was seeded on CellSTACK (registered trademark) of the same scale as the previous culture, in αMEM containing 10% FBS and 10 ng/mL bFGF at a final concentration. Subculture was performed. Medium exchange was performed once every 2 to 4 days. When subconfluency was reached, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack and incubated at 37°C for 3 minutes. It remained in the culture vessel as it was. Therefore, it was incubated for an additional 5 minutes (8 minutes total) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is the first passage cell population.

After that, RPMI1640 was added so that the cell concentration was 2×10 ⁷ cells/mL. An equal amount of CP-1 (registered trademark) solution (CP-1 (registered trademark): 25% human serum albumin = 34:16 mixed solution) was added to this, and after transferring 1 mL each to a cryovial, It was slowly frozen to -80°C and then stored frozen for 1 day under liquid nitrogen. Thereafter, the cryopreserved cell population is thawed, and the first passage cell population is seeded on CellSTACK (registered trademark) at a density of about 15,000 to 18,000 cells/cm ² , and the final concentration is The cells were adherently 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 subconfluent. After culturing, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack and incubated at 37°C for 3 minutes. remained in Therefore, it was incubated for an additional 5 minutes (8 minutes total) 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.

Then, 1/5 of the cell population was seeded on CellSTACK (registered trademark) of the same scale as the previous culture, in αMEM containing 10% FBS and 10 ng/mL bFGF at a final concentration. Subculture was performed. Medium exchange was performed once every 2 to 4 days. When subconfluency was reached, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack and incubated at 37°C for 3 minutes. It remained in the culture vessel as it was. Therefore, it was incubated for an additional 5 minutes (8 minutes total) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is the cell population of the 3rd passage. RPMI1640 was added to the cell population so that the cell concentration was 4×10 ⁶ cells/mL. An equal amount of CP-1 (registered trademark) solution (CP-1 (registered trademark): 25% human serum albumin = 34:16 mixed solution) was added to this, and after transferring 1 mL each to a cryovial, It was slowly frozen to -80°C and then stored frozen for 1 day under liquid nitrogen. Thereafter, the cryopreserved cell population was thawed, and the third passage cell population was seeded on CellSTACK (registered trademark) at a density of about 6,000 cells/cm ² , and the final concentration was 10% fetal bovine serum. (FBS) and 10 ng/mL basic fibroblast growth factor (bFGF)-containing αMEM (Alpha Modification of Minimum Essential Medium Eagle) was cultured until subconfluent. After culturing, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack and incubated at 37°C for 3 minutes. remained in Therefore, it was incubated for an additional 5 minutes (8 minutes total) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is the 4th passage cell population.

Then, 1/5 of the cell population was seeded on CellSTACK (registered trademark) of the same scale as the previous culture, in αMEM containing 10% FBS and 10 ng/mL bFGF at a final concentration. Subculture was performed. Medium exchange was performed once every 2 to 4 days. When subconfluent was reached, 15 mL of TrypLE Select was added per CellSTACK (registered trademark) stack and incubated at 37°C for 3 minutes. It remained in the culture vessel as it was. Therefore, it was incubated for an additional 5 minutes (8 minutes total) to completely detach the cell population, and the remaining cell population was also collected. The cell population obtained here is the 5th passage cell population. After that, RPMI1640 was added so that the cell concentration was 4×10 ⁶ cells/mL. An equal amount of CP-1 (registered trademark) solution (CP-1 (registered trademark): 25% human serum albumin = 34:16 mixed solution) was added to this, and 1 mL each was transferred to a cryovial and -80 It was slowly frozen to °C and then cryopreserved under liquid nitrogen.

(Step 1-4: Surface antigen analysis of mesenchymal cells)
Regarding the 5th passage cell population cultured by the above culture method, various surface antigens (positive rate of CD324, positive rate of CD73, positive rate of CD90, positive rate of CD105, positive rate of CD166, CD45 negative rate, CD326 negative rate) were analyzed. As a result, the positive rate of CD324 is less than 70% (specifically 33%), the positive rate of CD105 is 70% or more (specifically 93%), and the positive rate of CD73, CD90, and CD166 is 90%. It was above (specifically, CD73: 99%, CD90: 93%, CD166: 97%). Both CD45 and CD326 negative rates were 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. In addition, although the cell population at passage 5 of Comparative Example 1 satisfies the condition that the ratio of CD90-positive mesenchymal cells is 90% or more, the ratio of CD324-positive mesenchymal cells is 70% or more. It turned out that it is a cell population that does not satisfy the condition.

In this measurement, REA Control (S) APC (Miltenyi Biotec, clone: REA293, model number: 130-113-434) was used as an isotype control antibody, and CD324-APC and Human were used as antibodies against the CD324 antigen. (Miltenyi Biotec, clone: REA811, model number: 130-111-840) as an antibody against the CD73 antigen, CD73-APC, Human (Miltenyi Biotec, clone: REA804, model number: 130-111-909), CD90 CD90-APC, Human (Miltenyi Biotec, clone: REA897, model number: 130-114-861) as an antibody against the antigen, and CD105-APC, Human (Miltenyi Biotec, clone: REA794, model number) as an antibody against the CD105 antigen. : 130-112-166) as an antibody against the CD166 antigen, CD166-APC, Human (Miltenyi Biotec, clone: REA442, model number: 130-106-576) as an antibody against the CD45 antigen, CD45-APC, Human (Miltenyi Biotec, clone: REA747, model number: 130-110-633) was used as an antibody against the CD326 antigen, CD326-APC, Human (Miltenyi Biotec, clone: REA764, model number: 130-111-000). . Surface antigen analysis was performed using Merck's Guava (registered trademark) easyCyte, and measurement conditions were as follows: number of cells analyzed: 30,000 cells; flow rate setting: 35.4 μL/min). In addition, the ratio of positive cells (positive rate) for each antigen was calculated by the following procedure.

(1) The isotype control measurement results were developed in a dot plot with SSC on the vertical axis and FSC on the horizontal axis.
(2) A gate was set for a cell population corresponding to mesenchymal cells, and the cell population was developed in a histogram with the vertical axis representing the count number and the horizontal axis representing the tendency intensity of APC.
(3) In the histogram of (2), all regions (gates) where the cell population with the higher fluorescence intensity is 0.5% or less of the total cells measured with the isotype control antibody were selected.
(4) The percentage of cells included in the gate selected in (2) out of the total cells measured with the antibody corresponding to the surface antigen marker was calculated.

(Step 1-5: Immortalization of mesenchymal cells)
Nucleofector (registered trademark) 2/2b (Lonza) was applied to a cell population isolated and cultured according to the culture method according to Comparative Example 1, and 2.5 μg of an immortalizing plasmid (E1A gene (SEQ ID NO: 4), E1B gene (SEQ ID NO: 9), chicken Bcl-x _L gene (SEQ ID NO: 13), SV40 large T antigen gene (SEQ ID NO: 11), neomycin resistance gene (SEQ ID NO: 17)) were transfected (cell suspension Buffer: Nucleofector® Solution L, program: X-001). The transfected cells were seeded in a 6-well petri dish and cultured in αMEM containing 5% hPL until subconfluent. After reaching subconfluence, the cells 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.

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

(Step 2-2: enzymatic treatment of amniotic membrane and collection of mesenchymal cells)
A cell population containing mesenchymal cells was obtained in the same manner as in Comparative Example 1.

(Step 2-3: Culture of mesenchymal cells)
The cell population containing mesenchymal cells obtained in the above “Step 2-2: Enzymatic treatment of amniotic membrane and recovery of mesenchymal cells” was seeded in a culture vessel CellSTACK (registered trademark) (manufactured by Corning). . The seeding density was 6,000 cells/cm ² . After seeding the cells, the cells were adherently cultured in αMEM containing human platelet lysate (hPL) at a final concentration of 5% until subconfluent. After culturing, 15 mL of TrypLE Select was added per CellSTACK® stack and incubated at 37° C. for 3 minutes to allow complete detachment of the cell population. The cell population obtained here is the cell population at passage 0. After that, subculture was performed in αMEM containing hPL at a final concentration of 5% by inoculating 1/5 of the cell population on CellSTACK (registered trademark) of the same scale as the previous culture. . Medium exchange was performed once every 2 to 4 days. When sub-confluency was reached, 15 mL of TrypLE Select was added per CellSTACK® stack and incubated at 37° C. for 3 minutes to allow complete detachment of the cell population. The cell population obtained here is the first passage cell population.

After that, RPMI1640 was added so that the cell concentration was 2×10 ⁷ cells/mL. An equal amount of CP-1 (registered trademark) solution (CP-1 (registered trademark): 25% human serum albumin = 34:16 mixed solution) was added to this, and after transferring 1 mL each to a cryovial, It was slowly frozen to -80°C and then stored frozen for 1 day under liquid nitrogen. Thereafter, the cryopreserved cell population is thawed, and the first passage cell population is seeded on CellSTACK (registered trademark) at a density of about 15,000 to 18,000 cells/cm ² , and the final concentration is 5. % human platelet lysate (hPL) and adherent culture was performed until subconfluency was achieved. Then, 15 mL of TrypLE Select was added per CellSTACK® stack and incubated at 37° C. for 3 minutes to allow complete detachment of the cell population. The cell population obtained here is the second passage cell population.

Then, subculture was performed in αMEM containing hPL at a final concentration of 5% by inoculating 1/5 of the cell population on CellSTACK (registered trademark) of the same scale as the previous culture. . Medium exchange was performed once every 2 to 4 days. When sub-confluency was reached, 15 mL of TrypLE Select was added per CellSTACK® stack and incubated at 37° C. for 3 minutes to allow complete detachment of the cell population. The cell population obtained here is the 3rd passage cell population.

After that, RPMI1640 was added so that the cell concentration was 4×10 ⁶ cells/mL. An equal amount of CP-1 solution (registered trademark) (CP-1 (registered trademark): 25% human serum albumin = 34:16 mixed solution) was added to this, and after transferring 1 mL each to a cryovial, It was slowly frozen to -80°C and then stored frozen for 1 day under liquid nitrogen. Thereafter, the cryopreserved cell population is thawed, and the third passage cell population is seeded on CellSTACK (registered trademark) at a density of about 6,000 cells/cm ² to a final concentration of 5% human platelets. The cells were adherently cultured in αMEM containing lysate (hPL) until subconfluence. Then, 15 mL of TrypLE Select was added per CellSTACK® stack and incubated at 37° C. for 3 minutes to allow complete detachment of the cell population. The cell population obtained here is the 4th passage cell population.

Then, subculture was performed in αMEM containing hPL at a final concentration of 5% by inoculating 1/5 of the cell population on CellSTACK (registered trademark) of the same scale as the previous culture. . Medium exchange was performed once every 2 to 4 days. When sub-confluency was reached, 15 mL of TrypLE Select was added per CellSTACK® stack and incubated at 37° C. for 3 minutes to allow complete detachment of the cell population. The cell population obtained here is the 5th passage cell population. RPMI1640 was added to the cell population so that the cell concentration was 4×10 ⁶ cells/mL. An equal amount of CP-1 (registered trademark) solution (CP-1 (registered trademark): 25% human serum albumin = 34:16 mixed solution) was added to this, and after transferring 1 mL each to a cryovial, It was slowly frozen to -80°C and then cryopreserved under liquid nitrogen.

(Step 2-4: Surface antigen analysis of mesenchymal cells)
Regarding the 5th passage mesenchymal cell population cultured by the above culture method, various surface antigens (positive rate of CD324, positive rate of CD73 known as MSC marker, positive rate of CD90, CD105 positive rate, CD166 positive rate, CD45 negative rate, CD326 negative rate) were analyzed. As a result, the positive rate of CD324 and CD105 was 70% or more (specifically CD324: 91%, CD105: 95%), and the positive rate of CD73, CD90, and CD166 was all 90% or higher (specifically CD73: 100%, CD90: 100%, CD166: 99%). Both CD45 and CD326 negative rates were 95% or more (specifically, CD45: 100%, CD326: 99%). From the above results, it was found that the cells cultured by the above culture method are cell populations containing mesenchymal cells. In addition, the cell population described in Example 1 has a ratio of mesenchymal cells exhibiting CD324 positivity of 70% or more and a ratio of mesenchymal cells exhibiting CD90 positivity of 90% or more. was confirmed.
The method and reagent for this measurement are the same as in Comparative Example 1.

(Step 2-5: Immortalization of mesenchymal cells)
Nucleofector (registered trademark) 2/2b (Lonza) was applied to mesenchymal cells isolated and cultured according to the culture method according to Example 1, and 2.5 μg of an immortalizing plasmid (E1A gene (SEQ ID NO: 4) , E1B gene (SEQ ID NO: 9), chicken Bcl-x _L gene (SEQ ID NO: 13), SV40 large T antigen gene (SEQ ID NO: 11), neomycin resistance gene (SEQ ID NO: 17)) were transfected (cell suspension Turbidity buffer: Nucleofector® Solution L, program: X-001). The transfected cells were seeded in a 6-well petri dish and cultured in αMEM containing 5% hPL until subconfluent. After reaching subconfluence, subculturing was repeated in αMEM containing 20 μg/mL G418 and 5% hPL, and only transformed mesenchymal cells were selected. The selected mesenchymal cells are hereinafter also referred to as "immortalized amniotic mesenchymal cells". The cell morphology of untransformed mesenchymal cells was spindle-shaped, while the cell morphology of transformed immortalized amniotic mesenchymal cells was cobblestone-shaped. Microscopic photographs of each cell are shown in FIGS. 1 and 2 (40× magnification).

(Step 2-6: Confirmation of plasmid incorporation into immortalized amniotic mesenchymal cells)
The genome was extracted from immortalized amniotic mesenchymal cells using DNeasy (registered trademark) Blood & Tissue Kit (QIAGEN). Real-time PCR was performed using the extracted genome as a template and primers designed for the E1 gene region of the immortalized plasmid used for transfection. The base sequences of the two primers used are represented by SEQ ID NOs: 18 and 19, respectively. PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific) was used as the PCR reaction solution (mixed reaction solution of enzyme and fluorescent dye), and the StepOnePlus real-time PCR system (Thermo Fisher Scientific) was used as the real-time PCR device. 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 StepOnePlus standard conditions for the real-time PCR system). As a result, nucleic acid amplification using SYBR (registered trademark) Green as an index was detected, and the melting curve results confirmed that it was specific amplification. Further, as a result of agarose electrophoresis of the PCR product, it was found that the length of the amplified product was equivalent to that obtained when the target region was amplified with the primers used. From this result, it was confirmed that the immortalizing plasmid was incorporated into the genome of the immortalized amniotic mesenchymal cells.

(Step 2-7: Confirmation of hTERT gene expression in immortalized amniotic mesenchymal cells)
Total RNA was extracted from immortalized amniotic mesenchymal cells using RNeasy (registered trademark) Mini Plus Kit (QIAGEN). Subsequently, cDNA was synthesized from the extracted total RNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio Inc.). Using this cDNA as a template, quantitative real-time PCR was performed using hTERT-targeted primers and TaqMan (registered trademark) probes (TaqMan (registered trademark) Gene Expression Assays from Thermo Fisher Scientific, No: Hs00972650_m1). TaqMan (registered trademark) FAST Advanced MasterMix (Thermo Fisher Scientific) was used as the PCR reaction solution (enzyme-containing mixed reaction solution), and the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) was used as the real-time PCR apparatus. 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, using FAM (fluorescein) as an index, nucleic acid amplification specific to the hTERT gene was detected. These results confirmed that the immortalized amniotic mesenchymal cells expressed hTERT and were immortalized.

<Example 2: Immortalization of bone marrow-derived mesenchymal cells>
(Step 3-1: Culture of bone marrow-derived mesenchymal cells)
Bone marrow fluid collected from healthy donors by bone marrow aspiration is diluted 6-fold with αMEM containing 10% FBS or αMEM containing 5% hPL, and culture vessels for adherent culture (e.g. adherent cell culture petri dish 150 (Sumitomo Bakelite)). ). The medium was changed every 3 to 4 days, and cultured until adherent cells proliferated. After the adherent cells proliferated and reached subconfluence, they were subcultured and expanded. After expansion culture, the cells were suspended in Bambanker (registered trademark) (Nippon Genetics) and cryopreserved. Hereinafter, bone marrow-derived mesenchymal cells obtained by culturing in αMEM containing 10% FBS are referred to as “bone marrow mesenchymal cells (FBS),” and bone marrow-derived mesenchymal cells obtained by culturing in αMEM containing 5% hPL. Mesenchymal cells are also referred to as "bone marrow mesenchymal cells (hPL)".

(Step 3-2: Immortalization of bone marrow mesenchymal cells)
Bone marrow mesenchymal cells (FBS) and bone marrow mesenchymal cells (hPL) separated and cultured according to the culture method according to step 3-1 were treated with Nucleofector (registered trademark) 2/2b (Lonza) to obtain 2 .5 μg of immortalizing plasmid (E1A gene (SEQ ID NO: 4), E1B gene (SEQ ID NO: 9), chicken Bcl-x _L gene (SEQ ID NO: 13), SV40 large T antigen gene (SEQ ID NO: 11), neomycin resistance gene ( SEQ ID NO: 17)) was transfected (cell suspension buffer: Nucleofector® Solution L, program: X-001). The cells after transfection were seeded in a 6-well petri dish and cultured in the same culture medium (αMEM containing 10% FBS or αMEM containing 5% hPL) as before transfection until subconfluent. After reaching subconfluence, subculture was repeated in αMEM containing 20 μg/mL G418 and 10% FBS or αMEM containing 20 μg/mL G418 and 5% hPL. All cells cultured in αMEM containing 20 μg/mL G418 and 10% FBS died, and cells cultured in αMEM containing 20 μg/mL G418 and 5% hPL proliferated to obtain transformed bone marrow-derived mesenchymal cells. done. The transformed bone marrow-derived mesenchymal cells are hereinafter also referred to as "immortalized bone marrow mesenchymal cells". The cell morphology of untransformed bone marrow mesenchymal cells (hPL) was spindle-shaped, whereas the cell morphology of transformed immortalized bone marrow mesenchymal cells became closer to cobblestone. Microscopic photographs of each cell are shown in FIGS. 3 and 4 (40× magnification).

(Step 3-3: Confirmation of plasmid incorporation into immortalized bone marrow mesenchymal cells)
Genomes were extracted from immortalized bone marrow mesenchymal cells using DNeasy (registered trademark) Blood & Tissue Kit (QIAGEN). Using the extracted genome as a template, real-time PCR was performed using primers (SEQ ID NOS: 13 and 14) designed for the E1 gene region of the immortalized plasmid used for transfection. PowerUp SYBR (registered trademark) Green Master Mix (Thermo Fisher Scientific) was used as the PCR reaction solution (mixed reaction solution of enzyme and fluorescent dye), and the StepOnePlus real-time PCR system (Thermo Fisher Scientific) was used as the real-time PCR device. 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 StepOnePlus standard conditions for the real-time PCR system). As a result, nucleic acid amplification using SYBR (registered trademark) Green as an index was detected, and the melting curve results confirmed that it was specific amplification. From this result, it was confirmed that the immortalization plasmid was incorporated into the genome of the immortalized bone marrow mesenchymal cells.

(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 (registered trademark) Mini Plus Kit (QIAGEN). Subsequently, cDNA was synthesized from the extracted total RNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio Inc.). Using this cDNA as a template, quantitative real-time PCR was performed using hTERT-targeted primers and TaqMan (registered trademark) probes (TaqMan (registered trademark) Gene Expression Assays from Thermo Fisher Scientific, No: Hs00972650_m1). TaqMan (registered trademark) FAST Advanced MasterMix (Thermo Fisher Scientific) was used as the PCR reaction solution (enzyme-containing mixed reaction solution), and the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) was used as the real-time PCR apparatus. 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, using FAM as an index, nucleic acid amplification specific to the hTERT gene was detected. These results confirmed that the immortalized bone marrow mesenchymal cells expressed hTERT and were immortalized.

<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, HEK293T (set as a control) were transfected with pAAV vector (containing AAV ITR sequence and Akaluc gene), pRC2-mi342 vector by PEI transfection method. (containing Rep and Cap genes of serotype 2 AAV (AAV2)), pHhelper vectors (containing E2A, E4, VA derived from adenovirus) were transfected. Cultured in serum-free medium for 72 hours after transfection. Subsequently, AAV particles were precipitated by adding polyethylene glycol (PEG) to the culture supernatant, and AAV was collected by surfactant treatment. AAV was also collected from the cells by lysing the cells with a detergent. Real-time PCR was performed using the AAV collected from the culture supernatant and cells as templates and primers designed for the sequences in the pAAV plasmid. The base sequences of the two primers used are represented by SEQ ID NOs: 18 and 19, respectively. Powerup SYBR (registered trademark) Green PCR Master Mix (Thermo Fisher Scientific) was used as a PCR reaction solution (mixed reaction solution of an enzyme and a fluorescent dye), and a QuantStudio3 real-time PCR system was used as a real-time PCR device. 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 QuantStudio3 standard conditions for the 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 AAV2 recovered from culture supernatants and cells of immortalized amniotic mesenchymal cells, immortalized bone marrow mesenchymal cells, and HEK293T were used as templates, SYBR (registered trademark) Green nucleic acid was used as an indicator. Amplification was detected and melting curve results confirmed specific amplification. These results confirmed that AAV was produced from immortalized amniotic mesenchymal cells, immortalized bone marrow mesenchymal cells and HEK293T. The AAV2 genome concentration of each template is shown in FIG.

<Example 4: Suspension culture of immortalized amniotic mesenchymal cells>
(Step 5-1: Suspension of immortalized amniotic mesenchymal cells in serum-free medium)
FreeStyle F17 Expression Medium (Thermo Fisher Scientific), which is a serum-free medium, was used as a medium for HEK293T, an immortalized amniotic mesenchymal cell being adherently cultured in a culture vessel for adherent cells (e.g., adherent cell culture petri dish 150 (Sumitomo Bakelite)). , FreeStyle 293 Expression Medium (Thermo Fisher Scientific), and 293 SFMII (Thermo Fisher Scientific). After continuing the maintenance culture and confirming that the adherent cells were naturally detached, the culture supernatant was collected and the cells were subcultured. Thereafter, passage was repeated in a floating state. As a result, immortalized amniotic mesenchymal cells could be cultured in suspension with 293 SFM II. HEK293T could be cultured in suspension in FreeStyle F17 Expression Medium and FreeStyle293 Expression Medium. Thereafter, the suspended amniotic mesenchymal cells were cultured with agitation in an Erlenmeyer flask with a vent cap (for example, a polycarbonate Erlenmeyer flask with a vent cap of 125 mL (Corning). Single immortalized amniotic mesenchymal cells were dispersed. HEK293T was cultured in suspension in a state in which a plurality of cells aggregated, and microscopic photographs of each cell in suspension culture are shown in FIGS.

Claims (9)

a) preparing a cell population comprising mesenchymal cells;
b) a step of culturing the cell population of a) using a medium containing a platelet lysate; , the gene encoding the SV40 large T antigen, and the gene encoding the Bcl-x _L protein, respectively;
A method for producing a cell population containing immortalized mesenchymal cells. In the cell population prepared in step a), the ratio of CD324-positive mesenchymal cells is 70% or more, the ratio of CD90-positive mesenchymal cells is 90% or more, and the TERT gene 2. The method of claim 1, comprising a cell that expresses 3. The method according to claim 1 or 2, wherein the proportion of CD326-positive mesenchymal cells in the cell population prepared in step a) is 10% or less. In the cell population prepared in step a), the ratio of CD73-positive mesenchymal cells is 80% or more, the ratio of CD166-positive mesenchymal cells is 80% or more, and the cell population is CD45-positive. The method according to any one of claims 1 to 3, wherein the ratio of mesenchymal cells is 10% or less and the ratio of CD105-positive mesenchymal cells is 70% or more. The method of any one of claims 1-4, wherein the mesenchymal cells are derived from fetal appendages. The method according to any one of claims 1 to 5, further comprising the step of suspension culture of the cell population in serum-free medium. Adeno-associated virus-producing cells, comprising the step of introducing a gene for producing adeno-associated virus into the cells constituting the cell population produced by the method according to any one of claims 1 to 6. A method of producing a cell population. A cell population containing immortalized mesenchymal cells produced by the method according to any one of claims 1 to 6. A cell population comprising immortalized mesenchymal cells,
The cell population comprises cells containing at least one of the genes encoding adenovirus E1A region, E1B region, SV40 large T antigen, and Bcl-x _L protein, and
A cell population comprising cells expressing the TERT gene.

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