JP7626993B2 - Method for culturing cell populations containing Tie2-positive stem/progenitor cells using culture substrate and use thereof - Google Patents

Description

The present invention relates to a method for culturing a cell population containing stem cells and/or progenitor cells (referred to as "Tie2-positive stem/progenitor cells" in this specification) that are positive for the expression of the cell surface marker Tie2 (tyrosine kinase with Ig and EGF homology domain-2). More specifically, the present invention relates to a method for culturing a cell population containing Tie2-positive stem/progenitor cells, which is carried out using a specific culture substrate and can be utilized in a process of inducing differentiation of Tie2-positive stem/progenitor cells into cells having a predetermined trait (e.g., type II collagen-expressing nucleus pulposus cells).

Low back pain is the second most common cause of complaints, with two-thirds of the adult population experiencing it at least once, and is one of the causes of work-related disorders and social problems in the medical economy. Disc disorders, which are said to account for 20% of low back pain, are a serious problem that can lead to herniated discs, degenerative spondylosis, spinal canal stenosis, and spondylolisthesis. They are irreversible changes in the intervertebral disc tissue, and pathologically, are a condition called disc degeneration. The intervertebral disc is a donut-shaped cartilaginous organ, composed of the nucleus pulposus (NP) in the center, the annulus fibrosus (AF) which is a fibrous cartilage that surrounds it several times, and the cartilage endplates (EP) which connect the adjacent vertebrae above and below. Gelatinous NPs are avascular organs and contain a large amount of extracellular matrix (ECM) composed of large proteoglycans and collagen secreted by notochord-derived nucleus pulposus cells contained in NPs. It has been reported that notochord-derived nucleus pulposus cells disappear early in life in some vertebrates, including humans, and after their disappearance, chondrocytes morphologically similar to chondrocytes form the nucleus pulposus, although their origin has not yet been determined. Such cell transformation affects the ECM composition, leading to aging and degeneration of the intervertebral disc, such as reduced water content and fibrosis, and is ultimately thought to be closely related to lower back pain and lumbar degenerative diseases. In addition, many animal species, such as mice, rats, rabbits, and pigs, retain notochord-derived nucleus pulposus cells throughout their lives and rarely show intervertebral disc degeneration, suggesting that the control mechanisms of notochord-derived nucleus pulposus cells and other nucleus pulposus cells are different from those in humans.

As an example of a method for preventing or treating intervertebral disc degeneration, research and development is being conducted on allogeneic intervertebral disc cell preparations, i.e., cell preparations containing allogeneic nucleus pulposus cells, ECM, etc., to be administered to intervertebral disc tissue. To produce such cell preparations, a certain amount of allogeneic nucleus pulposus cells is required. For example, intervertebral disc nucleus pulposus tissue excised by surgery from a patient with herniated disc can be used as a source of nucleus pulposus cells, but the amount of nucleus pulposus tissue that can be collected in this way, i.e., the number of nucleus pulposus cells contained therein, is small. However, it is desirable to avoid mixing nucleus pulposus cells derived from multiple herniated disc patients (donors) in order to ensure the number of nucleus pulposus cells, since the risk of viral infection cannot be completely denied. Therefore, it is important to culture rare stem cells or progenitor cells that can be induced to differentiate into mature nucleus pulposus cells and are contained in a small amount of intervertebral disc tissue (nucleus pulposus, annulus fibrosus, etc.) derived from a single donor, and prepare a cell population containing a sufficient number of nucleus pulposus cells for treatment.

Patent Document 1 discloses that nucleus pulposus cells (a cell population derived from the intervertebral disc nucleus pulposus) are cultured (preferably in a serum-free medium) under "conditions that interfere with cell attachment" to produce "discospheres" composed of stem cells and progenitor cells contained in the cell population. That is, Patent Document 1 describes a "method of producing a disc stem cell population" that includes the steps of (a) growing nucleus pulposus cells in a medium under "conditions that interfere with cell attachment", (b) enriching for disc stem cells, disc progenitor cells, or a combination thereof, and (c) producing disc spheres containing nucleus pulposus cells, thereby producing a disc stem cell population (see claim 3, etc.). In addition, the "discosphere" is described as being a free floating circular-spherical structure in vitro comprising disc stem cells, disc progenitor cells or a combination thereof; being a sphere of cells in which a single disc stem cell gives rise to its own clones and progenitor cells; comprising free floating nucleus pulposus stem cells and nucleus pulposus progenitor cells arranged in a circular-spherical structure; and the nucleus pulposus cells comprising the discosphere are attached to each other (paragraphs 0024 and 0039). Patent Document 1 further describes an "isolated disc stem cell population" (claim 1, etc.) enriched for disc stem cells, disc progenitor cells, or a combination thereof, cultured "under conditions that interfere with cell attachment"; an "isolated discosphere" (claim 10, etc.) which is an in vitro floating spherical structure comprising disc stem cells, disc progenitor cells, or a mixture thereof enriched from nucleus pulposus cells; an "artificial disc replacement device" (claim 11, etc.) comprising a disc scaffold and a discosphere comprising nucleus pulposus cells enriched for disc stem cells, disc progenitor cells, or a combination thereof; a method of making an artificial disc replacement device comprising growing a discosphere comprising disc stem cells, disc progenitor cells, or a mixture thereof enriched from nucleus pulposus cells, in a disc scaffold (claim 12, etc.); Also described are "methods for producing enriched cell populations" (claim 17, etc.) including the steps of culturing densely plated nucleus pulposus cells "under conditions that interfere with cell attachment" and selecting in vitro floating sphere structures containing disc stem cells, disc progenitor cells, or a mixture thereof, thereby producing an enriched cell population; and "methods for expanding enriched populations of disc stem cells, disc progenitor cells, or a combination thereof" (claim 18, etc.) including the steps of dissociating disc spheres containing disc stem cells, disc progenitor cells, or a combination thereof into one or more dissociated disc sphere cells, and culturing the one or more dissociated disc sphere cells in a medium that interferes with cell attachment and contains a predetermined additive (e.g., FGF2, EGF). Note that "disc" in Patent Document 1 is a direct translation of the original word "disc," but is thought to mean "intervertebral disc."

Patent Document 1 describes the following regarding culture vessels or culture media for culturing a heterogeneous cell population (nucleus pulposus-derived cell population) derived from intervertebral disc nucleus pulposus tissue and including disc stem cells, disc progenitor cells, disc cells, etc.

Patent document 1 discloses an embodiment in which, as a culture under "conditions that interfere with cell attachment," a discosphere, which is a floating spherical structure, is formed from a nucleus pulposus-derived cell population (such as stem cells contained therein) by plating and culturing at a low cell density in a serum-free medium containing a substance that interferes with cell attachment (methylcellulose) or by culturing on an ultra-low attachment plate (paragraph 0156, Example 1: see paragraphs 0170-0181, corresponding to the inventions of claims 3, 17, etc.). More specifically, an example of Patent document 1 describes the combined use of a cell/medium suspension containing methylcellulose and a 6-well plate precoated with an anti-adhesion substance (poly 2-hydroxyethyl methacrylate) as an ultra-low attachment plate (paragraph 0179). Also, an example of Patent document 1 describes the use of a 35 mm diameter culture dish (paragraph 0050). However, Patent Document 1 does not mention or suggest the use of an ultra-low attachment plate having a substrate surface (well bottom) with a distinctive geometric feature, the use of a medium without added methylcellulose, or the use of a substrate surface (well bottom) with a distinctive geometric feature.

Furthermore, Patent Document 2 and Non-Patent Document 1 describe that, among the cells contained in the intervertebral disc tissue (nucleus pulposus), cells that are positive for the cell surface markers Tie2 and/or GD2 are cells that can be called stem or precursor cells of nucleus pulposus cells, and that in particular, cells that are positive for both Tie2 and GD2 (nucleus pulposus stem cells in an active state) form spherical colonies and have the ability to differentiate into mature nucleus pulposus cells through a series of differentiation cascades (they also have the ability to differentiate into fat cells, bone cells, chondrocytes and nerve cells), and further describe that transplanting nucleus pulposus stem/progenitor cells into an intervertebral disc (nucleus pulposus) can produce extracellular matrix such as type II collagen in the tissue, thereby maintaining or reconstructing intervertebral disc tissue and potentially preventing or treating intervertebral disc degeneration.

As a more specific embodiment, Patent Document 2 and Non-Patent Document 1 describe that spherical colonies (together with adherent colonies) are formed by suspension culture of a cell population contained in intervertebral disc tissue (nucleus pulposus) in a methylcellulose medium, that such spherical colonies are derived from the above-mentioned Tie2-positive (and GD2-positive) cells, and that type II collagen and proteoglycan are expressed in the spherical colonies (some of the cells) (see, for example, the Examples of Patent Document 2, paragraphs 0067 and 0070, etc.). However, Patent Document 2 and Non-Patent Document 1 do not describe or suggest the use of a substrate surface (well bottom surface) with morphological characteristics.

On the other hand, Patent Document 3 proposes a culture substrate that has a high probability of forming three-dimensionally uniformly aggregated spheroids from non-cultured materials such as cells and tissue fragments, and that can efficiently culture spheroids, and that has a plurality of depressions forming compartments in which the cultured materials are cultured, and banks interposed between adjacent depressions on the upper surface of the plate-shaped culture substrate, and the adjacent banks and depressions are continuous curved surfaces. Patent Document 3 also describes that a plurality of such depressions may be formed on the upper surface of the culture substrate and may be densely arranged, and that at least the inner surface of the depressions may be coated with a cell adhesion inhibitor.

However, Patent Document 3 does not describe culturing Tie2-positive nucleus pulposus cells (intervertebral disc nucleus pulposus stem/progenitor cells) on the culture substrate, nor does it describe the possibility of using a culture medium without added methylcellulose for this purpose.

Patent No. 5509073 (corresponding to WO2009/009020) Patent No. 5863639 (corresponding to WO2011/122601) Patent No. 5921437 (corresponding to WO2002/036011)

Sakai D et al., Nat Commun. 2012;3:1264

As described above, in order to produce an allogeneic intervertebral disc cell preparation for preventing or treating intervertebral disc degeneration, etc., a certain amount of functional nucleus pulposus cells that produce extracellular matrices such as type II collagen and proteoglycan are required. For this purpose, it is necessary to efficiently proliferate and differentiate Tie2-positive cells, which are considered to be stem cells and/or precursor cells of nucleus pulposus cells and are contained in intervertebral disc tissue (nucleus pulposus, etc.) excised from the affected area of a patient with herniated disc, to produce a large amount of functional nucleus pulposus cells. In particular, in order to improve the therapeutic effect when a cell preparation is administered to a patient with intervertebral disc degeneration, etc., when culturing and inducing differentiation of Tie2-positive cells (nucleus pulposus stem/progenitor cells) contained in the intervertebral disc, it is important to differentiate as efficiently as possible into functional nucleus pulposus cells that produce a large amount of extracellular matrices such as type II collagen, rather than simply differentiating them into nucleus pulposus cells. In other words, there is a need for a practical means for efficiently expanding and inducing differentiation from a cell population containing a certain number of Tie2-positive cells (nucleus pulposus stem/progenitor cells) to enrich functional nucleus pulposus cells in the cell population that is ultimately prepared and administered.

In addition, in typical embodiments of the prior art described in Patent Documents 1 and 2, a cell population containing nucleus pulposus stem/progenitor cells contained in intervertebral disc tissue was cultured in a medium containing methylcellulose to form spherical colonies (discospheres, spheroids), which were then differentiated into nucleus pulposus cells. However, since methylcellulose is a highly viscous substance, it is difficult or requires a great deal of effort to efficiently recover the cell population (useful functional nucleus pulposus cells contained therein) from the medium to which it is added, and commercially available methylcellulose products are expensive, which has been an obstacle to the efficient production of cell preparations.

The objective of the present invention is to provide a means for efficiently preparing a cell population that is rich in cells having a specific trait according to the application (e.g., functional nucleus pulposus cells that produce extracellular matrix such as type II collagen) from a cell population containing stem cells and/or progenitor cells that are positive for Tie2 expression (e.g., a cell population containing nucleus pulposus stem/progenitor cells derived from an intervertebral disc).

The inventors have discovered that when culturing a cell population containing Tie2-positive cells (nucleus pulposus stem/progenitor cells) contained in intervertebral disc nucleus pulposus tissue, by using a culture substrate having depressions and banks as described in Patent Document 3, in which the opening and depth of the depressions are within a specific range, it is possible to efficiently amplify nucleus pulposus stem/progenitor cells without incorporating methylcellulose into the culture medium, and to obtain a large amount of functional nucleus pulposus cells by inducing differentiation of the expanded cells.

In other words, the present invention can be expressed as an invention that includes at least the following items, for example.

[Item 1]
A culture substrate having a plate-shaped upper surface on which a plurality of depressions forming compartments in which a cultured substance is cultured and banks interposed between adjacent depressions, the adjacent banks and depressions having continuous curved surfaces, a plurality of depressions formed on the upper surface of the culture substrate and densely arranged, and at least the inner surface of the depressions being coated with a cell adhesion inhibitor,
A method for culturing a cell population containing stem cells and/or progenitor cells positive for Tie2 (tyrosine kinase with Ig and EGF homology domain-2) expression (hereinafter referred to as "Tie2-positive stem/progenitor cells"), comprising:
A culture method, wherein the opening of the well has a diameter of 400 to 1000 μm and the depth of the well is 50 to 500 μm.
[Item 2]
Item 2. The culture method according to Item 1, wherein the opening of the well has a diameter of 600 to 1000 μm and the depth of the well is 350 to 450 μm.
[Item 3]
Item 2. The culture method according to Item 1, wherein the opening of the well has a diameter of 400 to 600 μm and the depth of the well is 150 to 250 μm.
[Item 4]
Item 4. The culture method according to any one of Items 1 to 3, wherein the culture medium for the cell population does not contain any substance that interferes with cell attachment.
[Item 5]
Item 5. The culture method according to Item 4, wherein the substance that interferes with cell adhesion is methylcellulose.
[Item 6]
Item 6. The culture method according to any one of Items 1 to 5, wherein the Tie2-positive stem/progenitor cells are Tie2-positive stem/progenitor cells derived from nucleus pulposus tissue of an intervertebral disc.
[Item 7]
A method for preparing a cell population containing target cells differentiated from Tie2-positive stem/progenitor cells from a cell population containing Tie2-positive stem/progenitor cells, comprising:
A culture step for inducing differentiation of Tie2-positive stem/progenitor cells into target cells, comprising a step of carrying out the culture method according to any one of items 1 to 6 (hereinafter referred to as the "differentiation culture step").
A preparation method comprising:
[Item 8]
Item 8. The method according to Item 7, wherein the target cells are cells that express at least type II collagen.
[Item 9]
Item 9. The method according to Item 8, wherein the cells expressing at least type II collagen are nucleus pulposus cells.
[Item 10]
The preparation method according to any one of Items 7 to 9, further comprising a culture step for amplifying Tie2-positive stem/progenitor cells in the cell population (hereinafter referred to as an "amplification culture step") prior to the differentiation culture step.
[Item 11]
Item 11. The preparation method according to any one of Items 7 to 10, wherein a cell population in which Tie2-positive stem/progenitor cells also remain is obtained by the differentiation culture step.
[Item 12]
A cell population obtained by the culture method according to any one of Items 1 to 6 or the preparation method according to any one of Items 7 to 11.
[Item 13]
A culture system comprising a culture substrate defined in the culture method according to any one of items 1 to 6, and a cell population accommodated in a well of the culture substrate.
[Item 14]
A composition for cell therapy comprising the cell population according to item 13.
[Item 15]
Item 15. The cell therapy composition according to Item 14, which is for treating or preventing a disease whose symptoms include damage, degeneration or herniation of the intervertebral disc.

The Tie2-positive stem/progenitor cell culture method of the present invention is preferably a preparation method that includes a combination of a differentiation culture step, the main purpose of which is to induce differentiation of the Tie2-positive stem/progenitor cells subjected to the culture method into mature cells having a predetermined trait, and an amplification culture step performed prior to the differentiation process, the main purpose of which is to amplify the Tie2-positive stem/progenitor cells, thereby making it possible to prepare a cell population rich in the target cells. By utilizing the cell population obtained based on such a culture method and preparation method of the present invention, it becomes possible to efficiently produce a cell preparation effective for the treatment or prevention of a predetermined disease.

Furthermore, in the present invention, there is no need to add highly viscous components such as methylcellulose to the culture medium, which are used in the prior art described in the above-mentioned Patent Documents 1 and 2, and therefore it is possible to recover cell populations containing Tie2-positive stem/progenitor cells or target cells induced to differentiate therefrom from the culture medium without waste.

According to a representative embodiment of the present invention, by using an intervertebral disc (nucleus pulposus) that can only be harvested in small amounts by surgery from a patient with herniated disc, it is possible to efficiently proliferate and differentiate the Tie2-positive stem/progenitor cells (nucleus pulposus stem cells, etc.) contained therein, and to obtain a large amount of a cell population that is rich in functional nucleus pulposus cells with high production capacity of extracellular matrix such as type II collagen (and also has some Tie2-positive stem/progenitor cells remaining), which is expected to have a high therapeutic effect when transplanted, simply, inexpensively, efficiently, and reproducibly. Conventionally, it was impossible to produce such a suitable cell population from an intervertebral disc (nucleus pulposus) derived from a living human, but the present invention makes this possible, making it dramatically easier to perform regenerative therapy for intervertebral discs by administering a cell population (a cell preparation containing the cell population), making industrialization a realistic possibility.

FIG. 1 is an optical microscope photograph of the surface of the culture substrate on the 7th day of culture using the culture vessel 4. FIG. 2 is a graph showing the results of spherical colony formation rate (A) and cell proliferation rate (B) in Example 1. FIG. 3 is a graph showing the results of expression levels of mRNA of aggrecan (Acan), angiopoietin 1 (AngPT1), type I collagen A2 (COL1A2), and type II collagen A1 (COL2A1) in Example 1. FIG. 4 is a graph showing the results of ΔDHI (DHI 3 months after transplantation−DHI 1 month after transplantation) in Example 2.

-term-
"Stem cell" is a term referring to a cell that has the ability to self-renew and the ability to differentiate (totipotent, pluripotent, multipotent, or unipotent). "Progenitor cell" is a term referring to a cell that does not have the ability to self-renew in the strict sense because it will eventually become a terminally differentiated cell, but has the ability to differentiate into a specified cell while proliferating relatively actively. Cells that are generally understood (specified) by those skilled in the art as names including "stem cell" or "progenitor cell" correspond to "stem cell" or "progenitor cell" in this specification.

As used herein, "stem cells and/or progenitor cells" refers to stem cells, progenitor cells, or both, and may also be referred to as "stem/progenitor cells." Furthermore, as used herein, a cell population containing stem cells and/or progenitor cells may also be referred to as a "stem/progenitor cell population," and a cell population containing cells that have differentiated and matured from stem cells and/or progenitor cells (terminally differentiated cells) may also be referred to as a "mature cell population."

"Stem cells" and "progenitor cells" can generally be distinguished from other cells by the positive or negative expression of one or more specific genes (marker genes, cell markers). In other words, "stem cells" and "progenitor cells" that have the self-renewal and/or differentiation capabilities described above can also be defined as terms that refer to cells that have positive or negative expression of specific marker genes, respectively.

Whether the expression of a marker gene (cell marker) is "positive" or "negative" can be determined by quantitatively or qualitatively measuring the expression amount of the mRNA transcribed from the gene (genome) or the protein translated from the mRNA according to a general method, and determining that the expression amount is positive when it is equal to or higher than a certain level (or exceeds a certain level), and negative when it is equal to or lower than a certain level (or does not meet a certain level). The expression amount of a protein can be quantitatively or qualitatively measured by immunological assays using antibodies and labeling agents specific to the protein, such as flow cytometry, immunostaining, and ELISA. Note that Tie2 protein is a protein expressed on the cell surface, and Col2 is a protein expressed inside the cell, and an appropriate method (such as immunofluorescence staining) for detecting proteins present on the cell surface and inside the cell may be used. The expression amount of mRNA can be quantitatively or qualitatively measured by assays using nucleic acids (complementary) specific to the mRNA, labeling agents, nucleic acid amplification methods (means), and the like, such as RT-PCR, microarrays, and biochips. The ratio of cells in a cell population that are positive or negative for the expression of a specific marker gene (cell marker) (positive rate or negative rate) can be calculated by calculating the total number of cells in the cell population and the number of cells determined to be positive or negative by the methods described above, using various methods such as those described above, for example, by measurement in flow cytometry.

In the present specification, "stem cells and/or progenitor cells positive for Tie2 expression", i.e., "Tie2-positive stem/progenitor cells", refer to cells that have the characteristics of stem cells and/or progenitor cells and that are determined to be positive for the expression of Tie2 (tyrosine kinase with Ig and EGF homology domain-2), a known cell marker, for example, as a protein by flow cytometry. Representative Tie2-positive stem/progenitor cells in the present invention are Tie2-positive stem/progenitor cells "derived from nucleus pulposus tissue of an intervertebral disc", i.e., Tie2-positive stem/progenitor cells present in the nucleus pulposus of an intervertebral disc (which can be harvested from the nucleus pulposus) or Tie2-positive stem/progenitor cells obtained by subculturing these Tie2-positive stem/progenitor cells, which correspond to the "nucleus pulposus stem/progenitor cells" described below.

In this specification, the term "target cells" refers to cells having functionality according to the intended use, obtained by a specific differentiation induction from Tie2-positive stem/progenitor cells, more specifically, cells in which the expression of a specific gene (cell marker), for example, the expression of a protein by flow cytometry, is determined to be positive or negative. Representative target cells in the present invention are "nucleus pulposus cells" described below, which are positive for the expression of extracellular matrix (ECM) genes such as type II collagen (Col2) and aggrecan.

In the present invention, "nucleus pulposus cells" refers to mature cells that have reached terminal differentiation and occupy the majority of the cell population in the intervertebral disc (nucleus pulposus), or cultured cells with equivalent characteristics. Specifically, nucleus pulposus cells can be defined as cells that are negative for the marker genes Tie2 and GD2 (and usually positive for CD24) and positive for at least type II collagen (and usually positive for proteoglycan (aggrecan)) in the extracellular matrix. For example, cells that are determined by flow cytometry to be negative for Tie2 and GD2 (and positive for CD24) and positive for type II collagen (and positive for aggrecan) as proteins (cell markers) correspond to nucleus pulposus cells in the present invention. For extracellular matrices such as type II collagen and aggrecan, the production amounts of these proteins may be measured by flow cytometry, and the expression amounts of their mRNA may be measured by real-time PCR or the like.

In the present invention, "nucleus pulposus stem/progenitor cells" refers collectively to precursor cells (nucleus pulposus precursor cells) that have the ability to differentiate into at least nucleus pulposus cells and stem cells (nucleus pulposus stem cells) that have the ability to differentiate into the precursor cells and the ability to self-replicate, which occupy a portion of the cell population in the nucleus pulposus tissue of the intervertebral disc, or cultured cells that have equivalent characteristics. Specifically, nucleus pulposus stem/progenitor cells can be defined as cells that are positive for Tie2 and/or GD2 as marker genes. For example, cells that are determined by flow cytometry to be either Tie2 positive and GD2 negative, Tie2 positive and GD2 positive, or Tie2 negative and GD2 positive as proteins (cell markers) correspond to nucleus pulposus stem/progenitor cells in the present invention.

In addition, in the aforementioned Patent Document 2, based on the expression state of Tie2 and GD2 as cell markers for nucleus pulposus-derived cells, Tie2-positive cells are classified as "intervertebral disc nucleus pulposus stem cells" (among which, GD2-negative cells are in a dormant state and GD2-positive cells are in an active state), Tie2-negative and GD2-positive cells are "intervertebral disc progenitor cells," and Tie2-negative and GD2-negative cells are "mature intervertebral disc nucleus pulposus cells that have completed differentiation" (paragraphs 0024, 0025, 0032). Furthermore, in Patent Document 2, the cells appearing in the differentiation hierarchy of nucleus pulposus cells are classified as follows: (i) Tie2-positive and GD2-negative (further CD24-negative, CD44-positive/negative, CD271-positive, Flt1-positive) cells, (ii) Tie2-positive and GD2-positive (further CD24-negative, CD44-positive, CD271-positive, Flt1-positive) cells, (iii) Tie2-negative and GD2-positive (further CD24-negative, CD44-positive, CD271-positive, Flt1-positive) cells, and (iv) Tie2-negative and GD2-positive (further CD24-negative, CD44-positive, CD271-positive, Flt1-positive) cells. (i) Tie2-negative and GD2-positive (CD24-positive, CD44-positive, CD271-negative, Flt1-positive/negative) cells, (iv) Tie2-negative and GD2-positive (and CD24-positive, CD44-positive, CD271-negative, Flt1-negative) cells, and (v) Tie2-negative and GD2-negative (and CD24-positive, CD44-positive, CD271-negative, Flt1-negative) cells. The above (i) to (iii) are referred to as "intervertebral disc nucleus pulposus stem/progenitor cells," and the above (iii) to (v) are referred to as "nucleus pulposus committed cells" (see Figure 7-2). Although the notation is different, the "intervertebral disc nucleus pulposus stem cells" and "intervertebral disc nucleus pulposus progenitor cells" of Patent Document 2, i.e., the cells (i) to (iv) above, correspond to the "nucleus pulposus stem/progenitor cells" of the present invention, and the "mature intervertebral disc nucleus pulposus cells that have completed differentiation" of Patent Document 2, i.e., the cells (v) above, correspond to the "mature nucleus pulposus cells" of the present invention. If necessary, the cells of the present invention can be replaced with cells that conform to the definition described in Patent Document 2 (particularly the definition of whether they are positive or negative for one or more cell markers other than Tie2 and GD2, such as CD24).

In the present invention, a "spherical colony" is a spherical cell aggregate that contains stem cells and/or progenitor cells and may further contain cells differentiated therefrom. A "spherical colony" is also generally called a "sphere" or "spheroid" by those skilled in the art, and the "discosphere" or "free floating circular-spherical structure" in the aforementioned Patent Document 2 also corresponds to a "spherical colony."

-Culture method-
The method of the present invention for culturing a cell population containing Tie2-positive stem/progenitor cells is a culture method in "a culture substrate having on the upper surface of a plate-shaped culture substrate a plurality of depressions forming compartments in which the cultured material is cultured and banks interposed between adjacent depressions, the adjacent banks and depressions having continuous curved surfaces, the depressions being formed in plurality on the upper surface of the culture substrate and densely packed, and at least the inner surfaces of the depressions being coated with a cell adhesion inhibitor" (a culture vessel provided therewith), in which the diameter and depth of the openings of the depressions are within predetermined ranges.

In a representative embodiment of the present invention, the cell population containing Tie2-positive stem/progenitor cells is a cell population derived from intervertebral disc nucleus pulposus tissue. In a representative embodiment of the present invention, the cells differentiated from the Tie2-positive stem/progenitor cells are cells that produce and secrete more extracellular matrix than general cells, for example, nucleus pulposus cells that play a role in producing and secreting extracellular matrix in intervertebral disc nucleus pulposus tissue. Mature nucleus pulposus cells express at least type II collagen as an extracellular matrix, and also express extracellular matrices such as proteoglycan (aggrecan). In a preferred embodiment of the present invention, the Tie2-positive stem/progenitor cells are differentiated into functional nucleus pulposus cells that express extracellular matrices such as type II collagen and proteoglycan (aggrecan), particularly type II collagen, and have excellent expression (production) levels not only as mRNA but also as protein.

<Culture substrate>
In the culture method of the present invention or in the amplification culture step of carrying out the method, a predetermined cell population is cultured on a culture substrate having the above-mentioned predetermined depressions and banks, or on the surface of a culture vessel equipped with the culture substrate. Such culture substrates and culture vessels equipped with the same are known, and their details can basically be found in the above-mentioned Patent Document 3, and they can also be purchased as "EZSPHERE (registered trademark)" (AGC Technoglass Co., Ltd.).

In the present invention, the diameter (X) of the opening of the recess is 400 to 1000 μm, and the depth (Y) of the recess is 50 to 500 μm. For example, the combination of X/Y can be about 500 μm/about 100 μm, about 500 μm/about 200 μm, about 800 μm/about 400 μm, about 800 μm/about 300 μm, etc. Note that "about" refers to a range that can accommodate the difference (variation) between the catalog value (standard value) for "EZSPHERE (registered trademark)" (AGC Technoglass Co., Ltd.) and the value in the actual product, for example, ±50 μm, ±25 μm, ±20 μm, ±10 μm, ±5 μm. In addition, when the recess is approximately elliptical, its "major axis" is considered to correspond to the above-mentioned "diameter".

In a preferred embodiment of the present invention, the diameter (X) of the opening of the recess is 400-600 μm and the depth (Y) of the recess is 150-250 μm, for example, the combination of X/Y is about 500 μm/about 200 μm.

In a particularly preferred embodiment of the present invention, the diameter (X) of the opening of the recess is 600 to 1000 μm and the depth (Y) of the recess is 350 to 450 μm, for example, the combination X/Y is about 800 μm/about 400 μm.

In addition, the "opening diameter" and "depth" of the recess in the present invention are synonymous with those in the above-mentioned Patent Document 3, and the "diameter" and "depth" of the "well" shown in the catalogue of EZSPHERE (registered trademark) (AGC Technoglass Co., Ltd.) can be regarded as corresponding thereto.

A culture vessel equipped with a culture substrate having a predetermined depression and bank portion can take the form of a dish, a microplate, etc. A dish has, for example, a diameter of 35 mm, 100 mm, etc. In one aspect of the present invention, the diameter of the dish is preferably 20 to 50 mm, for example 35 mm. A microplate has, for example, 6 wells, 24 wells, 96 wells, etc., and the bottom surface of each well is a culture substrate having a predetermined depression and bank portion (a plurality of depressions arranged densely are formed).

The cell suspension to be added to the culture vessel equipped with the culture substrate can be prepared as a suspension containing cells at an appropriate density, taking into consideration the number of depressions on the culture substrate and the area of the region in which the depressions are formed (the density of the depressions calculated from these), etc. The cell density in the cell suspension can usually be adjusted in the range of 1 x ¹⁰³ to 1 x ¹⁰⁵ cells/ml. For example, when the cell suspension is added to a 35 mm dish equipped with a culture substrate in which depressions having the above-mentioned specified opening diameter and depth are formed, a cell suspension containing about 10,000 cells/ml can be used.

Examples of the cell adhesion inhibitor that coats at least the inner surface of the recess include phospholipid polymer, polyhydroxyethyl methacrylate, polyethylene glycol, etc. Examples of the material for the culture substrate include transparent synthetic resins such as polystyrene.

On the other hand, the process of the amplification culture stage, which is performed as necessary in the preparation method described below, can be performed using a general or known culture vessel (substrate), culture device, etc., depending on the embodiment. The culture vessel can be a flask, dish, plate, bag, etc., having a general shape, and may be formed with a well capable of accommodating cells. The culture vessel can be made of a general material, such as glass, plastic, or resin. The surface of the culture vessel (substrate) may be untreated, or may be treated with a treatment related to the adhesion of cells or other treatments. The size (area, volume) of the culture vessel, and if the culture vessel has wells, the size (diameter, depth) and number of the wells, etc., can be appropriately selected. If necessary, the culture vessel may be shaken or rotated to culture the cell population while stirring the medium.

- Preparation method (culture stage) -
The method for preparing a cell population containing target cells differentiated from Tie2-positive stem/progenitor cells from a cell population containing Tie2-positive stem/progenitor cells according to the present invention includes at least the following differentiation culture step, and preferably includes both an expansion culture step and a differentiation culture step (in the order of the expansion culture step first and the differentiation culture step later):
Expansion culture step: a culture step for enhancing the expression of Tie2 in Tie2-positive stem/progenitor cells and expanding the Tie2-positive stem/progenitor cells in the cell population;
Differentiation culture step: a culture step for inducing differentiation of Tie2-positive stem/progenitor cells into target cells.

- Steps related to the differentiation culture stage In the preparation method of the present invention, the culture method of the present invention is carried out in the step of the differentiation culture stage. The "step of the differentiation culture stage" means a step in which the main purpose is to differentiate Tie2-positive stem/progenitor cells into predetermined cells by culturing according to predetermined conditions, and an action effect therefor is exerted (relatively stronger than other action effects). In other words, if the number and/or ratio of the target cells is higher in the cell population after culture than in the cell population before culture, the culture step can be called a "step of the differentiation culture stage."

Steps related to the amplification culture stage In a preferred embodiment of the present invention, the amplification culture stage is carried out before the differentiation culture stage. The "amplification culture stage" refers to a stage in which the main purpose is to amplify Tie2-positive stem/progenitor cells by culturing under predetermined conditions, and the effect thereof is exerted (relatively stronger than other effects). In other words, if the number and/or ratio of Tie2-positive stem/progenitor cells is higher in the cultured cell population than in the pre-culture cell population, the culture stage can be called the "amplification culture stage", and differentiation of Tie2-positive stem/progenitor cells into other cells (target cells) is permitted within that limit.

An example of the step of the expansion culture stage is a step of culturing a cell population containing Tie2-positive stem/progenitor cells in a medium to which a growth factor having an effect of enhancing Tie2 expression has been added. Examples of the growth factor having an effect of enhancing Tie2 expression include FGF and/or EGF.

<Cell population>
In a cell population containing Tie2-positive stem/progenitor cells (collectively referred to herein as a "pre-culture cell population") subjected to the culture method of the present invention or the culture method (each step included therein), the ratio and/or number of Tie2-positive stem/progenitor cells and other cells (such as cells differentiated from Tie2-positive stem/progenitor cells) are basically arbitrary, and the ratio of Tie2-positive stem cells to Tie2-positive progenitor cells is also basically arbitrary. The composition of the pre-culture cell population can be appropriately adjusted depending on the embodiment of the invention, taking into consideration the action and effect of the culture method of the present invention or each culture step.

The pre-culture cell population can be prepared or prepared according to a conventional method. For example, when using a cell population contained in intervertebral disc nucleus pulposus tissue collected from the body as the pre-culture cell population, the nucleus pulposus tissue is first cut into pieces of an appropriate size using an instrument such as scissors (e.g., after mincing into pieces of about a few millimeters square), then treated with a proteolytic enzyme such as collagenase to disperse the cells, and if necessary, filtered, centrifuged, washed, or other processes can be performed to isolate and recover the cell population contained in the nucleus pulposus tissue.

The cell population prepared as described above and separated from the tissue can be cryopreserved in a conventional manner until it is used in the next culture method or culture step. The cryopreserved cell population can be thawed in a conventional manner when the next culture method or culture step is started. When cryopreserving and thawing, a treatment that is preferable for the cell population or tissue may be combined. For example, a cryoprotectant (DMSO, etc.) may be added during cryopreservation, in which case the cryoprotectant may be removed under appropriate conditions during thawing.

In a cell population (collectively referred to herein as a "post-culture cell population") containing Tie2-positive stem/progenitor cells obtained by the culture method or preparation method of the present invention (each step included therein), the ratio and/or number of Tie2-positive stem/progenitor cells and other cells (such as cells differentiated from Tie2-positive stem/progenitor cells) is basically arbitrary, and the ratio of Tie2-positive stem cells to Tie2-positive progenitor cells is also basically arbitrary. The composition of the post-culture cell population can be appropriately adjusted depending on the embodiment of the invention, taking into consideration the use of the cell population obtained by the culture method or culture step of the present invention.

After culturing, the cell population can be recovered from the medium in a conventional manner and subjected to the next culturing method or process, or to other methods or processes such as the preparation of a cell preparation.

In a cell population (collectively referred to as "cell population in culture" in this specification) containing Tie2-positive stem/progenitor cells during the culture method or preparation method of the present invention (each step included therein), the ratio of Tie2-positive stem/progenitor cells to other cells (such as cells differentiated from Tie2-positive stem/progenitor cells) is basically arbitrary, and the ratio of Tie2-positive stem cells to Tie2-positive progenitor cells is also basically arbitrary. The composition of the cell population in culture is the composition during the transition from the pre-culture cell population to the post-culture cell population, and for example, the ratio of Tie2-positive stem/progenitor cells in the cell population in culture (referred to as "Tie2-positive stem/progenitor cell rate" in this specification) is usually a value included in the range between the Tie2-positive stem/progenitor cell rate of the pre-culture cell population and the Tie2-positive stem/progenitor cell rate of the post-culture cell population, but it is also acceptable for the value to temporarily fall outside that range. The composition of the cultured cell population varies depending on the embodiment of the invention, and also depending on the number of days or number of passages in the culture method of the present invention or each culture step.

The "human or other animal" (donor) from which each of the above cell populations is derived can be selected taking into consideration the use of the cell population finally obtained by the Tie2-positive stem/progenitor cell culture method of the present invention, or the use of the cell population obtained by each culture method or each culture step included in the method. In a typical embodiment of the present invention, when preparing a cell population for producing a cell preparation for the prevention or treatment of a specific disease, symptom, etc., the "human or other animal" is an organism of the same species as the recipient to whom the cell preparation is to be administered, and is preferably a human.

Cell populations for the expansion culture step In the present invention, the cell populations subjected to the steps in the expansion culture step (collectively referred to in this specification as "cell populations before expansion culture") are typically cell populations contained in tissue (intervertebral disc) collected from the bodies of humans or other animals (primary cultured cell populations), or cell populations obtained by subculturing the primary cultured cell populations (subcultured cell populations).

When a cell population contained in an intervertebral disc harvested from a human is used as the pre-expansion culture cell population, it is preferable that the cell population is a cell population contained in an intervertebral disc harvested from a human up to the age of 20, which generally tends to have a high rate of Tie2-positive stem/progenitor cells and a good niche, and more preferably from a human in his/her teens. Furthermore, it is preferable that the pre-expansion culture cell population is a cell population in which the rate of Tie2-positive stem/progenitor cells is as high as possible, for example, 30% or more, 40% or more, 50% or more, or 60% or more.

In some embodiments, the pre-expansion culture cell population may be a cell population that is not contained in tissue taken from the body of a human or other animal, for example, a cell population containing Tie2-positive stem/progenitor cells obtained by inducing differentiation of cells having pluripotency or multipotency, such as iPS cells or ES cells produced using cells from a human or other animal.

In the present invention, the cell population obtained by the process in the expansion culture stage (collectively referred to as the "cell population after expansion culture" in this specification) is used as a cell population to be subjected to the process in the differentiation culture stage (the culture method of the present invention). In such an embodiment (use), the cell population after expansion culture preferably has a high ratio and/or cell number of Tie2-positive stem/progenitor cells. The Tie2-positive stem/progenitor cell rate in the cell population after expansion culture varies depending on the individual differences of the cell population before expansion culture and the nucleus pulposus tissue from which it is derived, and cannot be generally stated, but is, for example, 5% or more, preferably 7% or more, 9% or more, 11% or more, 13% or more, or 15% or more. The number of Tie2-positive stem/progenitor cells in the cell population after expansion culture varies depending on the individual differences of the cell population before expansion culture and the nucleus pulposus tissue from which it is derived, and cannot be generally stated, but is, for example, 5 times or more, preferably 10 times or more, 15 times or more, 20 times or more, 25 times or more, or 30 times or more compared to the number of cells in the cell population before expansion culture.

- Cell population related to differentiation culture stage In the present invention, the cell population (referred to as "pre-differentiation culture cell population" in this specification) to be subjected to the step in the differentiation culture stage (the culture method of the present invention) is preferably a cell population in which Tie2-positive stem/progenitor cells have been enriched in advance. The Tie2-positive stem/progenitor cell rate in the pre-differentiation culture cell population varies depending on the individual differences of the pre-expansion culture or post-expansion culture cell population and the nucleus pulposus tissue from which it is derived, and cannot be generally stated, but is, for example, 5% or more, preferably 7% or more, 9% or more, 11% or more, 13% or more, or 15% or more. In one embodiment of the present invention, the pre-differentiation culture cell population is a cell population obtained by dividing a cell population (post-expansion culture cell population) obtained by the expansion culture stage, for example, a cell population containing amplified Tie2-positive stem/progenitor cells, so as to obtain an appropriate number of cells according to the embodiment of the differentiation culture stage (such as the type and size of the culture vessel).

In some embodiments, the pre-differentiation culture cell population may be a cell population not obtained by the amplification culture step, for example, a cell population contained in tissue (intervertebral disc) taken from the body of a human or other animal (primary culture cell population) or a cell population obtained by subculturing that primary culture cell population (subculture cell population), or a cell population containing Tie2-positive stem/progenitor cells obtained by inducing differentiation (via Tie2-positive stem/progenitor cells) of cells having pluripotency or multipotency, such as iPS cells or ES cells produced using cells from a human or other animal.

In the present invention, the use of the cell population obtained by the process in the differentiation culture stage (collectively referred to in this specification as the "cell population after differentiation culture") is not particularly limited, and the composition of the obtained cell population can be appropriately adjusted depending on the use. For example, it is preferable that the cell population used to produce a cell preparation for transplantation contains as many target cells (e.g., nucleus pulposus cells that produce II collagen: Col2-positive nucleus pulposus cells) as possible that have useful functionality in achieving therapeutic or preventive effects by transplantation, and also contains some Tie2-positive stem/progenitor cells (e.g., nucleus pulposus stem/progenitor cells) that still have the ability to produce such target cells.

The rate of Col2-positive (nucleus pulposus) cells in the cell population after differentiation culture cannot be generalized as it varies depending on individual differences in the cell population before differentiation culture and the nucleus pulposus tissue from which it is derived, but is, for example, 5% or more, preferably 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more.

The rate of Tie2-positive (nucleus pulposus) stem/progenitor cells in the cell population after differentiation culture cannot be generalized as it varies depending on individual differences in the cell population before differentiation culture and the nucleus pulposus tissue from which it is derived, but is, for example, 1% or more, preferably 2% or more, 4% or more, 6% or more, 8% or more, or 10% or more.

In addition, the number of cells contained in the cell population usually increases during the differentiation culture process. The number of cells (Col2-positive cells, Tie2-positive stem/progenitor cells, etc.) in the cell population after differentiation culture cannot be generalized because it varies depending on the individual differences of the cell population before differentiation culture and the nucleus pulposus tissue from which it is derived, but it is, for example, 2 times or more, 5 times or more, 10 times or more, 20 times or more, 50 times or more, or 100 times or more compared to the number of cells in the cell population before differentiation culture.

<Culture medium>
The medium used in the culture method or preparation method of the present invention (each step included therein) may be any medium suitable for culturing Tie2-positive stem/progenitor cells and cells differentiated therefrom, and appropriate basal medium and additive components can be selected while taking into consideration the purpose of the culture method or culture step, etc. As for the additive components, if the culture step is an expansion culture step, additive components suitable for expansion culture of Tie2-positive stem/progenitor cells are selected, and if the culture step is a differentiation culture step, additive components suitable for differentiation induction from Tie2-positive stem/progenitor cells to target cells are selected.

In a representative embodiment of the present invention, when culturing nucleus pulposus stem/progenitor cells and nucleus pulposus cells differentiated therefrom, the culture medium for each step of the expansion culture stage and the differentiation culture stage can be prepared by using appropriate amounts of, for example, the following basal medium, supplementary components, growth factors, and other components.

Examples of basal media include DMEM (Dulbecco's modified Eagle's medium, with or without added glucose), αMEM (α-modified Eagle's minimum essential medium), Ham's F-10 medium, Ham's F-12 medium, or mixtures thereof.

Additional components for amplification culture or differentiation culture include, for example, FBS (fetal bovine serum), BSA (bovine serum albumin), L-ascorbic acid (as magnesium L-ascorbic acid phosphate, etc.), selenite (as insulin-transferrin-selenium (ITS), etc.), and 2-mercaptoethanol. If necessary, antibiotics such as penicillin and streptomycin, and other components may be added to the medium.

Examples of growth factors include FGF (fibroblast growth factor), EGF (epidermal growth factor), PDGF (platelet-derived growth factor), and Ang-1 (angiopoietin-1). In one embodiment of the present invention, the growth factors added to the culture medium are preferably at least FGF, more preferably both FGF and EGF, and also preferably Ang-1 in addition to these, if necessary.

As an FGF, for example, bFGF (basic fibroblast growth factor, sometimes called FGF-2) can be used. The concentration of FGF in the medium is usually in the range of 1 to 50 ng/mL, preferably in the range of 5 to 15 ng/mL, for example, about 10 ng/mL.

Ang-1 is preferably added to a serum-free medium. Ang-1 is preferably solubilized in water (soluble Ang-1, recombinant Ang-1). The concentration of Ang-1 (preferably soluble Ang-1) in the medium is usually in the range of 100 to 1000 ng/mL, for example, about 500 ng/mL.

In one aspect of the present invention, the culture medium used in the culture method of the present invention or the differentiation culture step of carrying out the culture method does not contain any substances that interfere with cell adhesion. In the present invention, by using a culture substrate (or a culture vessel having the same) having a predetermined depression and bank, it is possible to obtain a cell population that is rich in functional nucleus pulposus cells that have an excellent cell growth rate and a high production capacity of extracellular matrix such as type II collagen and aggrecan, even without adding substances that interfere with cell adhesion, such as methylcellulose, to the culture medium.

In one aspect of the present invention, the medium used in the culture method of the present invention or the differentiation culture step of carrying out the method contains Ham's F-10 medium and DMEM (e.g., in a ratio of 40:60) as a basal medium, and contains FBS (e.g., 30%), BSA (e.g., 1%), selenious acid (e.g., 0.01%), 2-mercaptoethanol (e.g., 5×10 ⁻⁵ M) and L-ascorbic acid (e.g., 0.075 mg/ml) as added components, and contains bFGF (e.g., 10 ng/ml) and EGF (e.g., 100 ng/ml) as growth factors. By using a medium with such a composition (not containing substances that interfere with cell attachment, such as methylcellulose) in combination with a culture substrate (a culture vessel having a culture substrate) having predetermined depressions and banks, the cell proliferation rate can be further improved.

<Culture period and other conditions>
The duration and other conditions (e.g., pH, _CO2 concentration, _O2 concentration, etc.) of the culture method and the culture method (each step included therein) of the present invention can basically be appropriately adjusted so as to obtain a cell population having a desired cell composition (type and number/ratio) according to the purpose of the culture method and preparation method. The pH can be weakly alkaline (e.g., about 7.15). The _CO2 concentration can be, for example, about 5%. The _O2 concentration can be 5% or less (e.g., about 2%). During the culture method and the culture step (each step) of the present invention, the medium can be replaced with a fresh one every predetermined number of days as necessary, or the medium can be changed by adding a component or increasing or decreasing the concentration or pH of a component after a predetermined number of days, or the atmosphere can be changed.

The duration of the expansion culture stage is usually about 1 to 3 weeks. For example, the duration of the culture step using FGF-supplemented medium can be about 1 week, and such a culture step can be repeated multiple times, for example twice, in the expansion culture stage. When the desired post-expansion culture cell population is obtained, the expansion culture stage can be terminated.

The duration of the differentiation culture stage (the culture method of the present invention) is usually about 1 to 3 weeks, for example about 2 weeks. The duration of other steps that may be optionally included in the differentiation culture stage of the present invention is also about the same. The differentiation culture stage may be terminated when the desired post-differentiation culture cell population is obtained.

-Cell therapy composition-
The cell therapy composition of the present invention contains a cell population obtained by the culture method or preparation method of the present invention as described above, and may contain other pharma- ceutically acceptable components as necessary.

In a representative embodiment of the present invention, the cell therapy composition is a cell therapy composition containing Col2-positive nucleus pulposus cells (preferably also containing Tie2-positive stem/progenitor cells) differentiated from nucleus pulposus stem/progenitor cells. Targets for the cell therapy composition in this embodiment, that is, diseases that can be prevented or treated by administering the composition, include diseases whose symptoms include damage, degeneration, herniation, etc. of the intervertebral disc (nucleus pulposus), such as lumbar or cervical disc disease, intervertebral disc herniation, cervical spondylotic myelopathy, radiculopathy, spondylolysis/spondylolisthesis, lumbar spinal canal stenosis, lumbar degenerative spondylolisthesis, and lumbar degenerative scoliosis.

The dosage form of the cell therapy composition of the present invention may be any dosage form that allows transplantation or delivery of the cell population to the target site (e.g., the nucleus pulposus of an intervertebral disc), and may be, for example, an injectable formulation, preferably an injectable formulation for local administration to the intervertebral disc (nucleus pulposus) or its vicinity, or an injectable formulation for vascular administration that allows targeting.

Pharmaceutically acceptable components include, for example, water for injection or physiological saline when prepared as an injection, culture medium for cell populations, other suitable solvents/dispersion media, other additives, etc.

The cell therapy composition of the present invention may be administered in an amount effective to achieve the desired therapeutic or preventive effect. Such an effective amount may be appropriately adjusted by the dosage amount per administration, the number of administrations, and the administration interval (number of administrations within a certain period of time), taking into consideration the components of the cell therapy composition, the dosage form, the recipient, the administration route, and other embodiments. The cell therapy composition of the present invention may be administered to humans and non-human vertebrates.

[Example 1]
Primary human nucleus pulposus cells (n=3) were used, which were collected and cryopreserved with the consent of the person undergoing surgery for herniated disc. The nucleus pulposus cells were suspended in 10% FBS-supplemented α-MEM medium (Nacalai Tesque) at 10,000 cells/ml. 2 ml of this cell suspension was seeded into each of the six types of "EZSPHERE" (registered trademark, IWAKI) products shown in Table 1. The cells were cultured for 14 days at 37°C, 5% CO ₂ , and 5% O ₂ , and the spherical colony formation rate (well occupancy rate) and cell increase rate were measured. The spherical colony formation rate was calculated by taking an optical microscope photograph of the surface of the culture substrate and calculating it as the ratio of the number of wells in which spherical colonies were formed to the total number of wells contained in the field of view. An optical microscope photograph of the surface of the culture substrate on the 7th day of culture using the culture vessel 4 is shown in FIG. 1.

In addition, the expression levels of mRNA for extracellular matrix proteins (aggrecan, type I collagen (COL1A2), type II collagen (COL2A1), and angiopoietin 1) in the cells after the above culture were measured by real-time PCR. As a control, primary human nucleus pulposus cells were cultured in the same manner, except that methylcellulose medium ("Methocult" manufactured by Stem Technology) was used instead of the above "10% FBS-supplemented α-MEM medium" and a regular 35 mm dish (without low-adhesion coating) was used as the culture vessel instead of "EZSPHERE", and the expression levels were compared.

The results for the spherical colony formation rate and cell proliferation rate are shown in Figure 2. For the spherical colony formation rate (A), Culture Vessel 4 (903) showed the best results (about 45%), which was significantly higher than the other five types. Culture Vessel 3 (902) and Culture Vessel 5 (904) also showed relatively high results. For the cell proliferation rate (B), Culture Vessel 4 (903) showed excellent results (about 7.5 times), and Culture Vessel 3 (902), Culture Vessel 2 (900), and Culture Vessel 4 (903) also showed similarly excellent results.

Regarding the cell proliferation rate, when primary human nucleus pulposus cells were cultured in culture vessel 4 using a medium supplemented with 100 ng/mL EGF and 100 ng/mL PDGF instead of the "10% FBS-supplemented α-MEM medium," the cell proliferation rate increased to 62.5-fold (not shown).

The results regarding the expression level of the extracellular matrix, etc., are shown in Figure 3. The expression level of mRNA of type II collagen (COL2A1), which is important as the extracellular matrix of nucleus pulposus cells, was extremely high (at least several hundred times) in each set compared to the control (methylcellulose medium/normal dish).

[Example 2]
As the cultured human nucleus pulposus cells for transplantation, a cell population obtained in the same manner as the culture method using the culture vessel 4 and "10% FBS-added α-MEM medium" in Example 1 was used. 1×10 ⁵ of these cultured human nucleus pulposus cells were mixed with 25 μL of hyaluronic acid (the product of the present invention) and transplanted by injection into the caudal intervertebral disc of a rat of an intervertebral disc degeneration model. In addition, PBS alone, a mixture of human fibroblasts and hyaluronic acid, and a mixture of a cell population obtained in the same manner as the culture method using a normal 35 mm dish and methylcellulose medium in Example 1 and hyaluronic acid (conventional product) were also transplanted by injection into the caudal intervertebral disc of a rat of an intervertebral disc degeneration model.

Immediately after transplantation and one, two, and three months after transplantation, X-rays were taken for each of the degenerative models with and without any transplantation, and the intervertebral disc height (DHI) at the transplant site was measured. The method for measuring DHI was as described by Hu et al. (J Orthop Res. 2018 Jan;36(1):202-211. doi: 10.1002/jor.23628. Epub 2017 Jul 9.). The difference between the DHI immediately after transplantation and the DHI one month after transplantation was defined as "DHI1", the difference between the DHI immediately after transplantation and the DHI two months after transplantation was defined as "DHI2", and the difference between the DHI immediately after transplantation and the DHI three months after transplantation was defined as "DHI3". The value of DHI3-DHI1 (called ΔDHI) was used as an index for evaluating the regenerative effect of transplantation.

The results are shown in Figure 4. The ΔDHI of the product of the present invention was significantly higher than the ΔDHI of the degenerated model that had not undergone transplantation surgery (P<0.05, Unpaired t-test), demonstrating the regenerative effect of transplantation.

Claims (11)

A culture substrate having a plate-shaped upper surface on which a plurality of depressions forming compartments in which cultured substances are cultured and banks interposed between adjacent depressions, the adjacent banks and depressions having continuous curved surfaces, a plurality of depressions formed on the upper surface of the culture substrate and densely arranged, and at least the inner surface of the depressions being coated with a cell adhesion inhibitor,
A method for culturing a cell population containing stem cells and/or progenitor cells positive for Tie2 (tyrosine kinase with Ig and EGF homology domain-2) expression (hereinafter referred to as "Tie2-positive stem/progenitor cells"), comprising:
A culture method, wherein the opening of the well has a diameter of 400 to 1000 μm and the depth of the well is 50 to 500 μm, and the medium for the cell population does not contain any substance that interferes with cell attachment. The culture method according to claim 1, wherein the opening of the recess has a diameter of 600 to 1000 μm and the depth of the recess is 350 to 450 μm. The culture method according to claim 1, wherein the opening of the recess has a diameter of 400 to 600 μm and the depth of the recess is 150 to 250 μm. The culture method according to any one of claims 1 to 3, wherein the substance that interferes with cell adhesion is methylcellulose. The culture method according to any one of claims 1 to 4, wherein the Tie2-positive stem/progenitor cells are Tie2-positive stem/progenitor cells derived from nucleus pulposus tissue of an intervertebral disc. A method for preparing a cell population containing target cells differentiated from Tie2-positive stem/progenitor cells from a cell population containing Tie2-positive stem/progenitor cells, comprising:
A culture step for inducing differentiation of Tie2-positive stem/progenitor cells into target cells, comprising a step of carrying out the culture method according to any one of claims 1 to 5 (hereinafter referred to as the "differentiation culture step").
A preparation method comprising: The preparation method according to claim 6, wherein the target cells are cells that express at least type II collagen. The preparation method according to claim 7, wherein the cells expressing at least type II collagen are nucleus pulposus cells. The preparation method according to any one of claims 6 to 8, further comprising a culture step (hereinafter referred to as the "expansion culture step") for amplifying Tie2-positive stem/progenitor cells in the cell population prior to the differentiation culture step. The preparation method according to any one of claims 6 to 9, wherein the differentiation culture step results in a cell population in which Tie2-positive stem/progenitor cells also remain. A culture system comprising a culture substrate defined in the culture method according to any one of claims 1 to 5, and a medium and a cell population defined in the culture method according to any one of claims 1 to 5 contained in a recess of the culture substrate.

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