JP7421182B2 - Method for culturing a cell population containing Tie2-positive stem/progenitor cells and its use - Google Patents

Description

The present invention utilizes the cell surface marker Tie2 (tyrosine kinase with Ig and EGF homology d
omain-2) positive expression of stem cells and/or progenitor cells (herein referred to as “Ti
e2-positive stem/progenitor cells. ). For more details,
The present invention relates to the process of amplifying Tie2-positive stem/progenitor cells (e.g., nucleus pulposus stem/progenitor cells) in a cell population, and the process of amplifying Tie2-positive stem/progenitor cells (e.g., type II collagen-expressing nucleus pulposus) from Tie2-positive stem/progenitor cells to The present invention relates to a method for culturing a cell population containing Tie2-positive stem/progenitor cells, which can be used in the step of inducing differentiation into Tie2-positive stem/progenitor cells.

Low back pain ranks second in the number of people complaining about it, is a common disease that two-thirds of the adult population experiences at least once, and is a contributing factor to social problems in work disability and the medical economy. Intervertebral disc disorders, which are said to account for 20% of the causes of low back pain, are serious problems that can lead to disc herniation, osteoarthritis, spinal canal stenosis, spondylolisthesis, etc.; however, they are irreversible changes in the disc tissue and cannot be cured by disease. Physically, it is a pathological condition called intervertebral disc degeneration. The intervertebral disc is a donut-shaped cartilaginous organ that consists of a central nucleus pulposus (NP) and a fibrous cartilage ring surrounding it.
s Fibrosus; AF), and the endplate cartilage that connects the adjacent vertebrae above and below
te; EP). Gelatinous NPs are avascular organs and contain a large amount of extracellular matrix (ECM) consisting of large proteoglycans and collagen, which is secreted from 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, morphologically they become chondrocytes, although the origin has not yet been determined. Cartilage-like cells similar to the chondrocytes form the nucleus pulposus. It is believed that such transformation of cell traits affects the ECM composition, leading to aging and degeneration of intervertebral discs such as decreased water content and fibrosis, and ultimately is significantly involved in low back pain and degenerative diseases of the lumbar spine. In addition, many animal species such as mice, rats, rabbits, and pigs retain notochord-derived nucleus pulposus cells throughout their lives and almost no disc degeneration is observed, so the control mechanism of notochord-derived nucleus pulposus cells and other nucleus pulposus cells is unknown. is thought to be different from that of humans.

As an example of a method for preventing or treating intervertebral disc degeneration, research and development of allogeneic intervertebral disc cell preparations, that is, cell preparations containing allogeneic nucleus pulposus cells, ECM, etc., for administration to intervertebral disc tissues is underway. In order to manufacture such a cell preparation, a certain amount of allogeneic nucleus pulposus cells is required. For example, intervertebral disc nucleus pulposus tissue that is surgically removed from a patient with a herniated disc can be used as a source of nucleus pulposus cells. There are few. However, using a mixture of nucleus pulposus cells derived from multiple disc herniation patients (donors) in order to ensure the number of nucleus pulposus cells is
It is desirable to avoid it as the risk of viral infection cannot be completely denied. Therefore, rare stem cells or progenitor cells that can be induced to differentiate into mature nucleus pulposus cells, which are contained in a small amount of intervertebral disc tissue (nucleus pulposus, annulus fibrosus, etc.) derived from a single donor, are cultured and treated. It is important to prepare a cell population containing a sufficient number of nucleus pulposus cells for this purpose.

Patent Document 1 describes that by culturing nucleus pulposus cells (a cell population derived from the intervertebral disc nucleus pulposus) under "conditions that interfere with cell adhesion" (preferably in a serum-free medium), the cell population is The creation of a "discosphere" comprised of stem and progenitor cells is disclosed. That is, Patent Document 1 includes a step of growing nucleus pulposus cells in a medium under "conditions that interfere with cell attachment," and (b) disc stem cells, disc progenitor cells. or a combination thereof; and (c) producing discospheres containing nucleus pulposus cells, thereby producing a disc stem cell population. (Claim 3, etc.). The above-mentioned "discosphere" refers to a single disc stem cell, which is an in vitro free floating circular-spherical structure containing disc stem cells, disc progenitor cells, or a combination thereof. The nucleus pulposus contains a discosphere, which is a sphere of cells that gives rise to its own clones and progenitor cells, containing floating nucleus pulposus stem cells and nucleus pulposus progenitor cells arranged in a circular-spherical structure. It is explained that cells are attached to each other (paragraph 002).
4,0039). Patent Document 1 further states that the cells were cultured under "conditions that interfere with cell adhesion,"
"Isolated disc stem cell population" enriched for disc stem cells, disc progenitor cells, or a combination thereof (claim 1, etc.); disc stem cells, disc progenitor cells enriched from nucleus pulposus cells, or "Isolated discospheres" (claim 10, etc.) comprising a mixture thereof and which are floating sphere structures in vitro; enriched for disc scaffolds and disc stem cells, disc progenitor cells or combinations thereof; "Artificial disc replacement device" (Claim 1)
1 etc.); A method for producing a disc prosthetic replacement device comprising growing discospheres containing disc stem cells, disc progenitor cells or a mixture thereof enriched from nucleus pulposus cells within a disc scaffold. (Claim 12, etc.); A step of culturing nucleus pulposus cells plated at a predetermined low density under "conditions that interfere with cell adhesion";"A method for producing an enriched cell population" (claim 17, etc.) comprising the step of selecting a floating spherical structure and thereby producing an enriched cell population; disc stem cells, disc progenitor cells, or the like; dissociating the discosphere into one or more dissociated discosphere cells, and dissociating the one or more dissociated discosphere cells with a combination of: Also described is a "method for expanding a population of enriched disc stem cells, disc progenitor cells, or a combination thereof" (claim 18, etc.), which includes the step of culturing in a medium containing additives (e.g., FGF2, EGF). has been done. Note that "disc" in Patent Document 1 is a literal translation of the original word "disc", but it is thought to mean "intervertebral disc".

Patent Document 1 relates to a culture vessel or medium 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. Regarding matters, the following can be said.

Patent Document 1 describes culturing under "conditions that interfere with cell adhesion" as culturing by plating at low cell density in a serum-free medium containing a substance that interferes with cell adhesion (methylcellulose), or culturing at a low cell density. An embodiment is disclosed in which a discosphere, which is a floating spherical structure, is formed from a nucleus pulposus-derived cell population (stem cells, etc. contained therein) by culturing in an attached plate (Paragraph 0156, Example 1: See paragraphs 0170 to 0181, corresponding to the inventions of claims 3, 17, etc.). However, Patent Document 1 discloses that nucleus pulposus-derived cell populations (
There is no description or suggestion of culturing the stem cells, etc. contained therein, and proliferating or inducing differentiation of the disc stem cells without forming discocytes.

Patent Document 1 describes a method for expanding a cell population containing concentrated disc stem cells, etc., in which discospheres (floating spherical structures) are first grown into one or more circles by incubation in a medium supplemented with collagenase. Embodiments are disclosed that involve dissociating into plate stem cells and then replating the dissociated cells in a methylcellulose-containing medium (paragraph 015).
7. Example 2: See paragraphs 0182 to 0184, corresponding to the invention of claim 18, etc.). However, the culture in a medium supplemented with collagenase in this embodiment is only a temporary treatment for dissociating once formed discospheres into individual disc stem cells, etc., and induces differentiation of disc stem cells, etc. Rather than undergoing treatment to achieve this goal, the dissociated disc stem cells and the like are cultured again under conditions that interfere with cell adhesion (such as a methylcellulose-containing medium). In a state where no disk sphere is formed (
Culture in a medium supplemented with collagenase is started using disc stem cells (before formation), and the disc stem cells, etc. are expanded (proliferated) and differentiated, and the disc stem cells, etc. After dissociation, culture in a medium supplemented with collagenase is continued, and disc stem cells, etc. are expanded (proliferated) and differentiated while maintaining the state in which discospheres, which are floating spherical structures, are not formed. Patent Document 1 neither describes nor suggests this.

Furthermore, Patent Document 1 describes "compounds that inhibit cell maturation" (e.g., FGF) or "compounds that maintain cell youthfulness" (e.g., TGF-β superfamily members, BMP
, IL-6, LIF, etc.) (Paragraphs 0035 to 0037), but it is described that disc stem cells are grown in a serum-free medium containing Tie2 activation. There is no description of culturing plate stem cells.

Further, Patent Document 1 describes a method for preparing nucleus pulposus tissue to obtain a nucleus pulposus-derived cell population, in which the nucleus pulposus (surgically obtained human disc material or biopsy specimen) is fragmented, and collagenase Only the general method of fragmenting nucleus pulposus tissue and dissociating individual cells (single cell suspension) by treating with II, clostridial collagenase, etc. is described (paragraph 0026, 0029, 0030, Example 1: paragraphs 0171 to 0174, etc.)
.

On the other hand, in Patent Document 2 and Non-Patent Document 1, among cells contained in intervertebral disc tissue (nucleus pulposus), cells that are positive for Tie2 and/or GD2 as cell surface markers are stem cells of nucleus pulposus cells or cells that are positive for Tie2 and/or GD2 as cell surface markers. Cells that can be called progenitor cells, especially cells that are positive for both Tie2 and GD2 (nucleus pulposus stem cells in an active state), form spherical colonies and undergo a series of differentiation cascades to finally mature into the nucleus pulposus. By having the ability to differentiate into cells (also having the ability to differentiate into adipocytes, bone cells, chondrocytes, and nerve cells), and by transplanting nucleus pulposus stem/progenitor cells into the intervertebral disc (nucleus pulposus), It has been described that extracellular matrices such as type II collagen can be produced in tissues, and that it may be possible to maintain or reconstruct intervertebral disc tissues and to prevent or treat intervertebral disc degeneration.

As a more specific embodiment, Patent Document 2 and Non-Patent Document 1 disclose that spherical colonies (along with adherent colonies) are obtained by suspending cell populations contained in intervertebral disc tissue (nucleus pulposus) in a methylcellulose medium. was formed, and that such spherical colonies were
It is described that it is derived from ie2-positive (and GD2-positive) cells, and that type II collagen and proteoglycan are expressed in spherical colonies (some of the cells) (for example, Examples of Patent Document 2) , paragraphs 0067, 0070, etc.). However, Patent Document 2 and Non-Patent Document 1 also disclose that the method is performed in a medium containing a substance that degrades the extracellular matrix (e.g. collagenase) or on a cell-adhesive culture surface (using a medium to which methylcellulose is not added). ), nucleus pulposus stem/progenitor cells (Tie2
There is no description or suggestion of proliferating or inducing differentiation of (and/or GD2-positive cells).

Patent Document 2 and Non-Patent Document 1 also state that as a method for preparing a cell population derived from intervertebral disc nucleus pulposus tissue, the tissue is cut into small pieces using scissors, etc., and then digested with protein-digesting enzymes (Triple Express, Collagenase P). , only a general method is described (Example: paragraph 0048).

In addition, Patent Document 2 and Non-Patent Document 1 state that in order to maintain Tie2-positive nucleus pulposus cells (disc nucleus pulposus stem/progenitor cells), Tie2 (receptor) and Ang-1 (angiopoietin-1
, ligand) is required, and cultured in the presence of Ang-1 (
It has been described that Tie2-positive cells can be amplified by co-culturing with AHESS5 in which Ang-1 is forcibly expressed, and that Ang-1 is therefore considered to be a niche factor that controls the differentiation hierarchy of nucleus pulposus cells. (Example: Paragraphs 0049, 0069, 00
75 etc.).

By the way, Tie2 is also expressed in vascular endothelial cells, and activating Tie2 leads to maturation, normalization, or stabilization of blood vessels, such as tumors, rheumatoid arthritis, diabetic retinopathy, and hyperlipidemia. It is known that it can suppress the disordered proliferation of blood vessels (angiogenesis) observed in blood vessels and high blood pressure, and can prevent and improve wrinkles. Examples of Tie2 activators having such an effect include extracts derived from plants of the genus Cinnamomum (so-called cinnamon powder, Patent Document 3), olive fruit extracts (Patent Document 4),
Also available are quillaya, yellow nut, ginkgo, oyster, turmeric, chrysanthemum, jujube, wolfberry, chamomile, butcher bloom, hawthorn, star fruit, algae, lotus, rooibos, Indian date, quince, white guava, blackberry, Siberian ginseng, and mango. Ginger, Korean ginseng, Japanese ginseng, Japanese radish, Japanese daisy, Japanese radish, Yabu daylily, Japanese sweet potato, Mitsuba tsugi, Japanese oak, Mube, salamander, Quercus oak, Sawtooth oak, Japanese poppy, Japanese ginger, plantain, Nobile, Japanese bayberry, Nikkei, Japanese sardine, Japanese sardine, Japanese oak, Various plant-derived extracts have been proposed, such as Karonin Hagekite (see Patent Document 4: Background Art).

In Patent Document 3, as a test (example) regarding a "Tie2 activator", "blood cell type Baf3 cells that forcibly expressed Tie2" or "normal human umbilical vein vascular endothelial cells" were tested in a medium supplemented with a hot water extract of Keihi (HUVEC), the T expressed in those cells
It has been confirmed by Western blotting that more phosphorylated ie2 proteins are present than in the control (paragraphs 0024 to 0027, Figures 1 to 3, etc.).

However, Patent Document 3, Patent Document 4, etc. describe the use of Tie2 activators in culturing nucleus pulposus-derived cell populations (Tie2-positive stem/progenitor cells contained therein) obtained from intervertebral discs, and how There is no description of whether such effects are brought about.

Patent No. 5509073 (corresponding to WO2009/009020) Patent No. 5863639 (corresponding to WO2011/122601) WO2009/123211 WO2016/060249

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

As mentioned above, in order to produce allogeneic disc cell preparations for the prevention or treatment of disc degeneration, etc., it is necessary to collect a certain amount of functional nucleus pulposus cells that produce extracellular matrices such as type II collagen and proteoglycans. required in quantity. To this end, for example, it is necessary to efficiently collect Tie2-positive cells, which are thought to be stem cells and/or progenitor cells of nucleus pulposus cells, contained in intervertebral disc tissue (such as nucleus pulposus) excised from the affected area of a patient with a herniated disc. It is necessary to proliferate and differentiate to produce a large amount of functional nucleus pulposus cells. In particular, in order to increase the therapeutic effect when administering cell preparations to patients with disc degeneration, etc., when culturing and inducing differentiation of Tie2-positive cells (nuclear pulposus stem/progenitor cells) contained in discs, it is necessary to It is important not to differentiate into nucleus pulposus cells, but to differentiate as efficiently as possible into functional nucleus pulposus cells that produce a large amount of extracellular matrix such as type II collagen. At the same time, before inducing differentiation as described above, Tie2-positive cells (nucleus pulposus stem/progenitor cells) in the cell population obtained from the tissue are
It is also important to efficiently amplify the cells to increase the number of functional nucleus pulposus cells that are ultimately obtained. In other words, from a cell population containing a certain number of Tie2-positive cells (nucleus pulposus stem/progenitor cells), functional nucleus pulposus cells in the final prepared and administered cell population are obtained through efficient amplification and differentiation induction. Practical means are needed to make it more abundant.

Further, in typical embodiments of the prior art as described in Patent Documents 1 and 2 mentioned above, a cell population containing nucleus pulposus stem/progenitor cells contained in intervertebral disc tissue is treated by adding methylcellulose. By culturing in a medium, spherical colonies (discospheres, spheroids) were formed and then differentiated into nucleus pulposus cells. However, because methylcellulose is a highly viscous substance, it is difficult or costly to recover the cell population (the useful functional nucleus pulposus cells contained therein) generated from the medium to which it is added. This requires labor and has hindered the efficient production of cell preparations.

The present invention uses cells having predetermined characteristics depending on the intended use ( An object of the present invention is to provide a means for efficiently preparing a cell population rich in functional nucleus pulposus cells that produce an extracellular matrix such as type II collagen.

The present inventors investigated Tie2-positive cells (nucleus pulposus stem/progenitor cells) contained in intervertebral disc nucleus pulposus tissue.
As a result of conducting research focusing on the state of the cell population and culture conditions when culturing cell populations containing . In addition, some of the culture methods with these technical characteristics are used in the culture process at the stage where the main purpose is to amplify nucleus pulposus stem/progenitor cells (amplification culture stage); /In the culture process at the stage where the main purpose is to induce differentiation from progenitor cells to functional nucleus pulposus cells (differentiation culture stage), such culture methods can be used sequentially or simultaneously in combination, especially "fusion" It has been found that the effects of the invention can be synergistically achieved by performing the culturing steps simultaneously in this manner.

That is, in one aspect, the present invention provides a method for culturing a cell population containing Tie2-positive stem/progenitor cells, typified by Tie2-positive cells (nucleus pulposus stem/progenitor cells) contained in intervertebral disc tissue. We provide the following first to fourth culture methods that allow for fusion).

The "first culture method" of a cell population containing Tie2-positive stem/progenitor cells according to the present invention is a method of culturing the cell population containing Tie2-positive stem/progenitor cells in a state that is present in undigested tissue. be.

Conventionally, when attempting to culture a cell population contained in intervertebral disc nucleus pulposus tissue, the collected intervertebral disc nucleus pulposus tissue was cut into small pieces, and then proteolytic enzymes (proteases) such as collagenase were first applied.
It has been common practice to perform a process of digesting the tissue (digestion process), collect a cell population separated from the nucleus pulposus tissue by the process, and start culturing. However, the present inventors did not perform digestion treatment after cutting the nucleus pulposus tissue into pieces, but instead suspended the chopped nucleus pulposus tissue in a culture medium and cultured the cell population for a certain period of time while retaining the cell population in the tissue. When subjected to conventional digestion, the ratio of Tie2-positive stem/progenitor cells (nucleus pulposus stem/progenitor cells) in the cell population was improved, and Tie2 expression in individual cells was also enhanced. I found out that it can be done. Such effects are favorable for nucleus pulposus stem/progenitor cells by not performing digestive processing of nucleus pulposus tissue, that is, growth factors such as angiopoietin-1 (Ang-1) and VEGF-A are present, The microenvironment (niche) in the tissue that maintains Tie2-positive cells is not destroyed (in the absence of Ang-1, etc., Tie2-positive cells eventually turn to apoptosis), and the state maintained in such a niche. By starting the culture with a cell population containing nucleus pulposus stem/progenitor cells, Tie2 activity is maintained (Tie2 expression is enhanced compared to the conventional method of isolating cells from the niche). ) Since nucleus pulposus stem/progenitor cells can rapidly start proliferating, this is thought to reflect the above-mentioned improvement in the positive rate and increase in expression level in the cell population obtained after culture.

Moreover, by not separating and collecting cell populations from nucleus pulposus tissue,
Valuable Tie2-positive stem/progenitor cells contained in nucleus pulposus tissue can be used without waste. For example, the amount of nucleus pulposus tissue contained in the herniated part removed from a patient with intervertebral disc herniation is about 1 to 2 g, and the Tie2-positive stem/progenitor cells contained per 1 g of the nucleus pulposus tissue are (
Although it varies depending on the age of the patient, it is, for example, about 50,000 pieces. The number of white blood cells contained in 1 cc of cord blood is on the order of 10 ⁶ , and the number of cancer cells contained in 1 gram of cancer tissue is on the order of 10 ⁸ . You can see how precious cells are. In addition to preventing such Tie2-positive stem/progenitor cells from being lost through the process of separating and collecting cell populations from nucleus pulposus tissue, Tie2-positive stem/progenitor cells can be
It is highly beneficial that it would be preferred for the expansion culture of ie2 positive stem/progenitor cells.

The "second culture method" of a cell population containing Tie2-positive stem/progenitor cells according to the present invention is to cultivate Tie2-positive stem/progenitor cells in a medium supplemented with at least one Tie2 expression enhancer other than growth factors. This is a method of culturing a cell population containing

Tie2-positive stem/progenitor cells originally activate Tie2 (
It is known that Tie2-positive stem/progenitor cells are cultured in a medium supplemented with Ang-1 (to enhance Tie2 expression), and conventional techniques (prior art documents, etc.) It is publicly known. In addition, FGF2 (bFGF) is also known as a growth factor that has the effect of enhancing Tie2 expression, and Tie2 in a medium supplemented with FGF2.
Methods for culturing 2-positive stem/progenitor cells are also known.

However, the present inventors have discovered that a type of Ti that is different from growth factors such as Ang-1 and FGF2
e2 expression enhancers, such as Tie, which is a plant-derived extract such as cinnamon powder extract;
2 expression enhancer is used to culture a cell population containing Tie2-positive stem/progenitor cells derived from nucleus pulposus tissue, which has not been reported so far. It has been found that when used in combination with a growth factor, there are effects such as improving the ratio of Tie2-positive stem/progenitor cells (nucleus pulposus stem/progenitor cells) in the cell population obtained by culture.

The present inventors further demonstrated that by combining (particularly fusion) the above-mentioned "first culture method" and "second culture method", Tie2-positive stem/progenitor cells in nucleus pulposus tissue (pulp cell It has also been found that a cell population containing a large number of stem/progenitor cells (nuclear stem/progenitor cells) can be obtained from a cell population containing a high number or ratio of stem/progenitor cells.

On the other hand, the "third culture method" of a cell population containing Tie2-positive stem/progenitor cells according to the present invention is as follows:
This is a method of culturing a cell population containing Tie2-positive stem/progenitor cells in a culture vessel having a culture surface treated to enhance cell adhesion.

Conventionally, like many other stem/progenitor cells, a cell population containing Tie2-positive cells (nucleus pulposus stem/progenitor cells) derived from intervertebral disc nucleus pulposus tissue has been used as a cell population as described in Patent Document 1 mentioned above. They have been cultured under conditions that interfere with cell adhesion, that is, using low-adherence culture vessels or methylcellulose media, to form floating spherical colonies. However, the present inventors have developed a cell population comprising nucleus pulposus stem/progenitor cells, preferably nucleus pulposus stem/progenitor cells whose expression of Tie2 is enhanced by the first culture method and/or the second culture method as described above. is not under "conditions that interfere with cell adhesion," but on the contrary, "a culture vessel with a culture surface that has been treated to increase cell adhesion (cell adhesion treatment)," such as a coating agent containing polylysine. In a two-dimensional culture environment using coated culture vessels (without using methylcellulose, etc.), nucleus pulposus stem/progenitor cells can be attached to the culture surface and cultured without forming spherical colonies. I found it.

By implementing such a third culture method in the step of the differentiation culture stage, a culture vessel whose culture surface has not been treated (or conversely, a culture vessel whose culture surface has been treated to inhibit cell adhesion (low adhesion treatment)) The efficiency of differentiation of nucleus pulposus stem/progenitor cells into functional nucleus pulposus cells (Col2-positive cells, etc.) can be improved compared to the case of using a culture vessel), and Col2 in the cell population can be improved.
Cell populations with a high number or proportion of positive cells can be prepared.

In addition, the third culture method can also be implemented in the culture step of the amplification culture stage as a method fused with the second culture method described above. That is, if Tie2-positive stem/progenitor cells are cultured in a culture vessel treated with cell attachment and a medium supplemented with a Tie2 activator, Tie2-positive stem/progenitor cells can be grown in a two-dimensional culture environment (without using methylcellulose, etc.). Stem/progenitor cells can be expanded.

The "fourth culture method" of a cell population containing Tie2-positive stem/progenitor cells according to the present invention is a method for culturing Tie2-positive stem/progenitor cells while suppressing the formation of spherical colonies in a medium supplemented with an extracellular matrix degrading agent.
This is a method of culturing a cell population containing 2-positive stem/progenitor cells.

Conventionally, substances that have the effect of degrading extracellular matrices (collagen, proteoglycans, etc.), such as collagenase or other proteases, have been used to separate cells from collected tissues, as described above in relation to the first culture method. It has usually been used temporarily in culture methods where spherical colonies formed by stem/progenitor cells are once dissociated, the medium is replaced, and spherical colonies are formed again.

However, the present inventors have discovered that substances having the action of degrading extracellular matrix (extracellular matrix degrading agents), such as collagenase, can be used for completely different purposes. That is, the present inventors use Tie2-positive stem/progenitor cells such as nucleus pulposus stem/progenitor cells that have the ability to differentiate into nucleus pulposus cells, preferably the first culture method and/or the second culture method as described above. Tie2-positive stems with enhanced Tie2 expression/
Surprisingly, in the case of progenitor cells, even in a medium supplemented with an extracellular matrix degrading agent, in other words, in a situation where Tie2-positive stem/progenitor cells cannot form spherical colonies in which Tie2-positive stem/progenitor cells are bound together by the extracellular matrix. We have found that even if Tie2-positive stem/progenitor cells are present, it is possible to culture them, and preferably to induce differentiation into cells that are positive for the expression of extracellular matrices such as type II collagen and proteoglycans while being allowed to proliferate.

The present inventors have further determined that by combining the above-mentioned "third culture method" and "fourth culture method," the type and concentration of the extracellular matrix decomposing agent in the medium and the type of coating agent on the surface of the culture vessel can be combined. By appropriately combining and fusing them, synergistically, the efficiency of differentiation from nucleus pulposus stem/progenitor cells to functional nucleus pulposus cells such as type II collagen (Col2) positive cells is significantly improved. It has also been found that it is possible to prepare a cell population in which the number or ratio of Col2-positive cells etc. is significantly higher than conventional methods.

Moreover, by using the third culture method and/or the fourth culture method, at the stage when the number or ratio of Col2-positive cells etc. reaches a certain level, Tie2-positive stem/progenitor cells are not completely eliminated. It is also possible to obtain a cell population that maintains a certain level of number or ratio.
The present inventors discovered this. Such a cell population is useful because it contains a certain number or ratio of Tie2-positive stem/progenitor cells, so that when administered, the therapeutic effect on the intervertebral disc nucleus pulposus becomes more excellent.

Based on each of the above-mentioned culture methods, the present inventors collected target cells (C
We constructed a method for preparing a cell population containing ol2-positive nucleus pulposus cells, etc.). The method for preparing this cell population includes a culture step (amplification culture step) for amplifying Tie2-positive stem/progenitor cells in the cell population by growing the Tie2-positive stem/progenitor cells while enhancing Tie2 expression; and a culture step (differentiation culture step) for inducing differentiation of Tie2-positive stem/progenitor cells into target cells, preferably both. The amplification culture step includes a step of implementing at least one of the first culture method and the second culture method, preferably both (a method in which these methods are combined may be used). The differentiation culture step includes the step of implementing at least one of the third culture method and the fourth culture method, preferably both (a method in which these methods are combined may be used). an amplification culture step comprising carrying out both the first culture method and the second culture method (preferably as a fused method), and the step of carrying out both the third culture method and the fourth culture method (preferably as a fused method); The method for preparing a cell population comprising a differentiation culture step is a particularly advantageous embodiment of the present invention, and is a particularly advantageous embodiment of the present invention. It is possible to prepare a cell population with dramatically improved properties. In some conventional preparation methods, the cell number of the entire cell population or the number of nucleus pulposus cells satisfies a certain level, but among them, the nucleus pulposus cells that have functionality such as positive Col2 expression are Another type of not-so-common case is that the ratio of Col2-positive cells in the cell population and the expression level of individual cells meet a certain level, but the absolute number of Col2-positive cells is insufficient (
There is a limit to the number of Col2-positive cells that can be increased from nucleus pulposus tissue collected from one donor.)
, it seemed. As mentioned above, it has been difficult to achieve both the number and expression level (strength of expression) of Col2-positive cells with respect to the cell population related to the nucleus pulposus, but the preparation method of the present invention has succeeded in achieving both. It can be said that this is an innovative preparation method that has not been proposed before.

From another perspective, in order to take advantage of the properties of (Tie2-positive) stem/progenitor cells that have an anchorage-independent proliferation ability and can form spherical colonies, In order to avoid losing the desired functionality by culturing cells differentiated from A method has been adopted in which the stem/progenitor cells are differentiated into target cells having a predetermined functionality while being cultured on a culture surface. The present inventors have investigated each of the above culture methods (
In particular, by the third culture method and the fourth culture method), without using methylcellulose, which is highly viscous and makes it difficult to recover cells, and in an environment that allows cells to adhere to the culture surface ( Tie2 positive) stem/progenitor cell proliferation and differentiation from the stem/progenitor cells into target cells with a predetermined functionality are significantly increased in efficiency, and the produced cell population can be recovered from the culture medium easily and without waste. It can be said that we have found a promising method.

In connection with the above culture methods and preparation methods, particularly in connection with the first culture method and the amplification culture step, the present inventors discovered that Tie2-positive stems/progenitors are present in undigested tissues. By cryopreserving a cell population containing cells, Tie2-positive stem cells can maintain an activated and/or expressed state of Tie2 or suppress the decrease of Tie2-positive stem/progenitor cells in the cell population. /We have discovered a preferred method for preserving cell populations including progenitor cells. Conventionally, a cell population containing Tie2-positive stem/progenitor cells contained in collected intervertebral disc nucleus pulposus tissue is separated from the tissue by a relatively time-consuming digestion process using collagenase, etc., and then only the cell population is frozen. Saving was done. However, immediate cryopreservation of harvested disc nucleus pulposus tissue improves the convenience of the process and preserves a good niche in the tissue (especially from young donors). Cell populations can be maintained intact. After thawing such cryopreserved tissue, the thawed tissue is placed in a medium to culture a cell population by the first culture method described above, and T
IE2-positive stem/progenitor cells can be efficiently expanded.

By embodying the above-mentioned technical idea in combination with (preferred) embodiments, details of which will be described later, the present invention can be expressed as an invention that includes at least the following matters, for example.

[1]
A method for culturing a cell population containing stem cells and/or progenitor cells (hereinafter referred to as "Tie2-positive stem/progenitor cells") that are positive for Tie2 (tyrosine kinase with Ig and EGF homology domain-2) expression, the method comprising:
A method of culturing a cell population containing the Tie2-positive stem/progenitor cells in a state that exists in undigested tissue (hereinafter referred to as "first culture method").
[2]
Item 2. The first culture method according to item 1, wherein the Tie2-positive stem/progenitor cells are Tie2-positive stem/progenitor cells derived from nucleus pulposus tissue of an intervertebral disc.
[3]
The first item according to item 1 or 2, wherein the undigested tissue is nucleus pulposus tissue of an intervertebral disc.
Culture method.
[4]
Items 1 to 3, wherein the undigested tissue is a tissue obtained by thawing a cryopreserved tissue.
3. The first culture method according to any one of 3.
[5]
Item 5. The first culture method according to any one of Items 1 to 4, which is performed when expanding the Tie2-positive stem/progenitor cells in the cell population.

[6]
A method for culturing a cell population containing Tie2-positive stem/progenitor cells, the method comprising:
In a medium supplemented with at least one Tie2 expression enhancer other than growth factors,
A method of culturing a cell population containing ie2-positive stem/progenitor cells (hereinafter referred to as "second culture method").
).
[7]
Item 6. The second item according to Item 6, wherein the Tie2 expression enhancer other than the growth factor is a plant-derived extract.
Culture method.
[8]
Item 8. The second culturing method according to item 7, wherein the plant is a plant of the genus Cinnamomum.
[9]
Item 9. The second culture method according to any one of Items 6 to 8, which is performed when expanding the Tie2-positive stem/progenitor cells in the cell population.

[10]
A method for culturing a cell population containing Tie2-positive stem/progenitor cells, the method comprising:
A method of culturing a cell population containing the Tie2-positive stem/progenitor cells in a culture vessel having a culture surface treated to enhance cell adhesion (hereinafter referred to as the "third culture method").
[11]
The Tie2-positive stem/progenitor cells have been treated to enhance Tie2 expression,
The third culture method according to item 10.
[12]
Item 12. The third culturing method according to item 10 or 11, wherein the treatment for increasing the adhesion of cells is a treatment of applying a coating agent containing an extracellular matrix or other biologically relevant molecules.
[13]
Item 13. The third culture method according to any one of Items 10 to 12, which is performed when the Tie2-positive stem/progenitor cells in the cell population are differentiated into target cells.
[14]
Item 12, wherein at least one selected from the group consisting of type IV collagen, fibronectin, and polylysine is used as the extracellular matrix or other biologically relevant molecules.
or the third culture method described in 13.
[15]
Item 15. The third culture method according to any one of Items 10 to 14, which is performed when expanding the Tie2-positive stem/progenitor cells in the cell population.
[16]
Item 16. The third culture method according to any one of Items 12 to 15, wherein gelatin is used as the extracellular matrix.

[17]
A method for culturing a cell population containing Tie2-positive stem/progenitor cells, the method comprising:
A method of culturing a cell population containing Tie2-positive stem/progenitor cells in a medium supplemented with an extracellular matrix degrading agent while suppressing the formation of spherical colonies (hereinafter referred to as the "fourth culture method")
It is called. ).
[18]
The Tie2-positive stem/progenitor cells have been treated to enhance Tie2 expression,
The fourth culture method according to item 17.
[19]
Item 19. The fourth culture method according to item 17 or 18, wherein the extracellular matrix degrading agent includes a protease having degrading activity for at least type II collagen.
[20]
Item 20. The fourth culture method according to any one of Items 17 to 19, which is performed when the Tie2-positive stem/progenitor cells in the cell population are differentiated into target cells.

[21]
A method for preparing a cell population containing Tie2-positive stem/progenitor cells, the method comprising:
The step of implementing the first culture method according to item 5 and/or the second culture method according to item 9 enhances Tie2 expression in Tie2-positive stem/progenitor cells, and at the same time enhances Tie2 expression in the cell population.
Culture step for amplifying ie2-positive stem/progenitor cells (hereinafter referred to as "amplification culture step")
Preparation methods, including.
[22]
Item 22. The preparation method according to Item 21, wherein the step performed in the amplification culture step is a step of simultaneously implementing the first culture method and the second culture method.
[23]
Item 21 or 22, wherein the amplification culture step further includes the step of culturing the cell population containing Tie2-positive stem/progenitor cells in a medium to which only a growth factor having an effect of enhancing Tie2 expression is added as a Tie2 expression enhancer. Preparation method as described.

[24]
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, the method comprising:
A culture step for inducing differentiation of Tie2-positive stem/progenitor cells into target cells (hereinafter referred to as a step of culturing for inducing differentiation of Tie2-positive stem/progenitor cells into target cells), including the step of implementing the third culture method described in Item 13 or 14 and/or the fourth culture method described in Item 20. (This is called the “differentiation culture stage.”)
Preparation methods, including.
[25]
25. The preparation method according to item 24, wherein the step performed in the differentiation culture stage is a step of simultaneously implementing the third culture method and the fourth culture method.
[26]
Item 24 or 25, wherein the target cell is a cell expressing at least type II collagen.
Preparation method described in.
[27]
27. The preparation method according to item 26, wherein the cell expressing at least type II collagen is a nucleus pulposus cell.
[28]
28. The preparation method according to any one of Items 24 to 27, wherein the differentiation culture step yields a cell population in which Tie2-positive stem/progenitor cells also remain.
[29]
A method for preparing a cell population containing target cells differentiated from the Tie2-positive stem/progenitor cells from a cell population containing Tie2-positive stem/progenitor cells, the method comprising:
A preparation method comprising: the amplification culture step according to any one of Items 21 to 23; and the differentiation culture step according to any one of Items 24 to 28.

[30]
A cell population obtained by the culture method according to any one of Items 1 to 20.
[31]
Item 21. A culture comprising a culture medium in the culture method according to any one of Items 1 to 20 and a cell population that is cultured or obtained and is subjected to the culture method.
[32]
Item 30. A cell population obtained by the amplification culture step or differentiation culture step in the preparation method according to any one of Items 21 to 29.
[33]
In the preparation method according to any one of Items 21 to 29, the medium for the amplification culture stage or the medium for the differentiation culture stage, and the cultured or obtained medium for the amplification culture stage or the differentiation culture stage. A culture containing a cell population.

[34]
Item 33. A composition for cell therapy containing the cell population according to item 30 or 32.
[35]
Item 35. The composition for cell therapy according to Item 34, which is used for the treatment or prevention of a disease in which intervertebral disc disorder, degeneration, or herniation is a symptom.

[36]
A method for preserving a cell population containing Tie2-positive stem/progenitor cells, the method comprising:
By cryopreserving the cell population containing the Tie2-positive stem/progenitor cells while existing in undigested tissue, Tie2 is maintained in an activated and/or expressed state, or the cell population is maintained in a state in which Tie2 is activated and/or expressed. A preservation method that suppresses the decrease of Tie2-positive stem/progenitor cells in cells.

The method for culturing Tie2-positive stem/progenitor cells of the present invention preferably includes an amplification culture step whose main purpose is to amplify Tie2-positive stem/progenitor cells, and mature cells having predetermined characteristics from the Tie2-positive stem/progenitor cells. A cell population rich in target cells can be prepared by a culture method that includes a differentiation culture step whose main purpose is to induce differentiation into cells of interest. By utilizing the cell population obtained based on the culture method of the present invention, it becomes possible to efficiently produce cell preparations that are effective for treating or preventing a given disease.

Furthermore, in the present invention, there is no need to add highly viscous components such as methylcellulose to the medium, which is used in the conventional techniques described in Patent Documents 1 and 2, etc.
It becomes possible to collect a cell population containing Tie2-positive stem/progenitor cells or target cells differentiated therefrom from the culture medium without waste.

According to a typical embodiment of the present invention, the Tie2-positive stem/progenitor cells (nucleus pulposus stem cells) contained therein are obtained by using intervertebral discs (nucleus pulposus), which can only be collected in small quantities through surgery from patients with disc herniation. By efficiently proliferating and differentiating these cells (such as Tie2-positive stem/progenitor cells (Tie2-positive stem/progenitor cells also remain) can be easily and reproducibly obtained in large quantities. Conventionally, it has been impossible to create such a suitable cell population, but the present invention makes it possible, making it dramatically easier to perform intervertebral disc regeneration therapy by administering the cell population (cell preparation containing it). As a result, industrialization becomes a reality.

Note that the reason why the fourth culture method of the present invention is effective is expected to be due to the principle as shown in FIG. 1 working, for example. However, this prediction is for assisting in understanding the present invention, and is not intended to unnecessarily constrain the present invention. Even if it is later found that some or all of the effects of the present invention are achieved based on a principle or mechanism different from that shown in Figure 1, this book does not allow for actual confirmation. The effects of the invention and the constituent elements of the present invention therefor are not denied by the following explanation based on FIG. 1.

Figure 1 [A] shows the state of cultured cells when a cell population containing Tie2-positive stem/progenitor cells (e.g. nucleus pulposus stem/progenitor cells) is grown and differentiated by two-dimensional culture (monolayer static culture). represents. The culture surface of the culture container (flask, etc.) is coated with extracellular matrix (ECM) in advance.
In some cases, the surface has been treated to allow cells to attach, such as by applying a coating containing . Stem/progenitor cells adhere to the culture surface of the culture container, and differentiate while spreading and proliferating on the culture surface. In such two-dimensional culture, ECM applied on the culture surface or ECM secreted from the cultured cells interacts with binding proteins (integrin, etc.) expressed on the surface of the cultured cells, and eventually the ECM is removed. Intracellular signaling occurs that stops production and secretion. For example, when a nucleus pulposus-derived cell population is two-dimensionally cultured, the nucleus pulposus stem/progenitor cells contained in the cell population proliferate and differentiate together with the mature nucleus pulposus cells originally contained in the cell population. ECM such as type II collagen and proteoglycans are actively secreted from the mature nucleus pulposus cells generated in this process. However, as the culture period progresses, the production and secretion of type II collagen etc. eventually stop due to the intracellular signal transduction described above (instead,
This results in dedifferentiation of mature nucleus pulposus cells, including increased production and secretion of type I collagen) and a fibroblast-like phenotype. Therefore, in general two-dimensional culture, it is considered difficult to simultaneously increase the number of cells in a cell population and increase the proportion of cells that retain specific traits (not dedifferentiated). In addition, in the third culture method of the present invention, by preferably using Tie2-positive stem/progenitor cells in which Tie2 expression is enhanced, nucleus pulposus cells expressing type II collagen etc. can be maintained at a certain level even in two-dimensional culture. It has become possible to relatively easily prepare a cell population containing the same proportions.

Figures 1 [B] and [C] show cell populations containing Tie2-positive stem/progenitor cells (e.g. nucleus pulposus stem/progenitor cells) in methylcellulose-containing medium (no extracellular matrix (ECM) degrading agents were added). Or, it shows the state of cultured cells when they are grown and differentiated by culture using a low-adsorption culture container. Unlike the two-dimensional culture shown in FIG. 1 [A], in the culture shown in FIG. 1 [B], there is no interaction between the ECM, etc. on the culture surface of the culture container and the cultured cells, and the ECM due to the interaction does not occur. Intracellular signaling that stops the production and secretion of is also not occurring. Therefore, in the early stage of culture, Tie2-positive stem/progenitor cells proliferate while producing and secreting ECM, and eventually form spherical colonies. However, in the formed spherical colony, Tie2-positive stem/progenitor cells or cells differentiated from them (for example, nucleus pulposus cells) come into contact with each other via secreted ECM. Therefore, Figure 1[C]
As shown in Figure 1, as the culture period progresses, interaction between ECM and cultured cells begins to occur, and this interaction generates a signal to stop ECM production, causing dedifferentiation similar to Figure 1 [A]. . Therefore, in the culture method shown in FIGS. 1 [B] and [C], in the cell population collected as spherical colonies, a predetermined ECM (for example, type II collagen)
It is difficult to increase the proportion of cells expressing positive expression of

FIG. 1 [D] shows a cell population containing Tie2-positive stem/progenitor cells grown in a medium containing an extracellular matrix (ECM) degrading agent (does not contain methylcellulose, etc.) according to the fourth culture method of the present invention. This figure shows the appearance of Tie2-positive cultured cells when they are grown and differentiated by culture using. Even in such a culture method, ECM is secreted from stem/progenitor cells or cells differentiated from them, as in FIG. 1 [B]. However, ECM secreted to the outside of the cells is constantly degraded by the ECM-degrading agent added to the culture medium, so spherical colonies are not formed, and cells do not remain on the culture surface even without using a low-adsorption culture vessel. Does not stick. In addition, in the third and fourth culture methods of the present invention, even if a coating agent containing ECM is applied to the culture surface of the culture container in advance, as in the two-dimensional culture of FIG. 1 [A], the culture surface Attachment of cells to the membrane remains weak. Therefore, the ECM production stop signal caused by the interaction between ECM and cultured cells is suppressed, and Fig. 1 [A], Fig. 1 [B] and [C
] Since such dedifferentiation as occurs in the case of [1] is less likely to occur, it becomes possible to prepare a cell population in which the proportion of cells positive for expression of a certain ECM (for example, type II collagen) is improved compared to conventional techniques.

Note that, although ECM secreted outside the cells is degraded by an ECM degrading agent in the medium, ECM inside the cells is not degraded and continues to accumulate. Therefore, if a cell preparation is produced by collecting the obtained cell population from the medium after carrying out the fourth culture method of the present invention in the step of the differentiation culture stage, the cells in the tissue to which the cell preparation has been administered are The accumulated ECM can be rapidly secreted to the outside of the cell to create a suitable environment for the survival of the cell population, and the subsequent production and secretion of ECM from the administered cells (i.e., treatment with cell preparations) It is expected that it will be able to promote the

Figure 1 shows a normal two-dimensional culture (monolayer static culture), a conventional suspension culture using a medium that does not contain an extracellular matrix (ECM) degrading agent, and an ECM degrading agent according to the fourth culture method of the present invention. A diagram schematically representing the interaction between ECM production or decomposition and cultured cells in each suspension culture using a medium containing , and photographs of each cultured cell are shown. A (normal two-dimensional culture): When nucleus pulposus (NP) cells adhere to the culture surface of a culture flask via adhesion molecules, an ECM production stop signal is transmitted. B (suspension culture in a low-adsorption flask, methylcellulose medium, etc.): There is no contact with the culture surface of the flask, and no ECM production stop signal is transmitted (x mark and dotted arrow). C (no enzyme in the medium): Self-produced ECM exhibits the same effect as the culture surface (arrow), and an ECM production stop signal is transmitted into the cells. D (enzyme present in medium): Self-produced extracellular ECM is degraded and the ECM production stop signal is not transmitted into the cells (x mark and dotted arrow), but ECM is accumulated inside the cells. FIG. 2 is a graph showing the results of the Tie2 positive rate in Test Example 1 (amplification culture stage: first culture step). FIG. 3 is a graph showing the results of Tie2 average luminescence intensity (MFI) in Test Example 1 (amplification culture stage: first culture step). FIG. 4 is a graph showing the results of the Tie2 positive rate in Test Example 2 (amplification culture stage (2 stages): first and second culture steps + additional step). FIG. 5 is a graph showing the results of the number of Tie2-positive cells produced per 1 g of nucleus pulposus tissue in Test Example 2 (amplification culture stage (2 stages): first and second culture steps + additional step). Figure 6 shows the results of type II collagen (Col2) positive rate in Test Example 3 (amplification culture stage (2 stages): first and second culture steps + additional step → differentiation culture stage: third culture step) It is a graph. Figure 7 shows type II collagen produced from per gram of nucleus pulposus tissue in Test Example 3 (amplification culture stage (2 stages): first and second culture steps + additional step → differentiation culture stage: third culture step). (Col2) is a graph showing the results of the number of positive cells. Figure 8 shows the results of the proteoglycan (PG) positive rate in Test Example 4 (amplification culture stage (2 stages): 1st and 2nd culture process + additional process → differentiation culture stage: 3rd and 4th culture process). This is a graph representing Figure 9 shows the type II collagen (Col2) positive rate in Test Example 4 (amplification culture stage (2 stages): 1st and 2nd culture process + additional process → differentiation culture stage: 3rd and 4th culture process). This is a graph showing the results. Figure 10 shows proteoglycan (PG) and type II It is a graph showing the result of each positive rate of collagen (Col2). GEL: gelatin, Col1: type I collagen, Col4: type IV collagen, FN: fibronectin, PLL: poly-L-lysine (the same applies to FIG. 11). Figure 11 shows proteoglycan (PG) and type II It is a graph showing the result of each positive rate of collagen (Col2). FIG. 12 is an optical micrograph of the cell population in Test Example 5-12.

-term-
“Stem cells” have the ability to self-renew and differentiate (totipotent, pluripotent).
t), multipotent or unipotent). "Progenitor cells" do not have the ability to self-renew in the strict sense because they all eventually become terminally differentiated cells, but they have the ability to differentiate into specific cells while proliferating relatively actively. This term refers to cells that have the following. Cells that are commonly understood (and specified) by those skilled in the art by names including "stem cells" or "progenitor cells" correspond to "stem cells" or "progenitor cells" herein.

As used herein, "stem cells and/or progenitor cells" includes stem cells, progenitor cells, or both, and may also be referred to as "stem/progenitor cells." In addition, in this specification, a cell population containing stem cells and/or progenitor cells is referred to as a "stem/progenitor cell population", and a cell population containing mature cells (terminally differentiated cells) differentiated from stem cells and/or progenitor cells. is sometimes referred to as a "mature cell population."

“Stem cells” and “progenitor cells” generally contain one or more specific genes (
Cells can be distinguished from other cells by positive or negative expression of marker genes, cell markers). That is, "stem cells" and "progenitor cells" having self-renewal ability and/or differentiation ability as described above can also be defined as terms that refer to cells in which the expression of a specific marker gene is positive or negative, respectively.

Whether the expression of a marker gene (cell marker) is "positive" or "negative" is determined by the mRNA transcribed from that gene (genome) or its mRNA, according to a general method.
The expression level of the protein translated from A is measured quantitatively or qualitatively, and if the expression level is above a certain level (or exceeds a certain level), it is positive, and it is below a certain level (or meets a certain level). The test result can be determined as negative if the test result is not present. The expression level of a protein can be measured quantitatively or qualitatively, for example, by an immunoassay using an antibody and a labeling agent specific to the protein, such as flow cytometry, immunostaining, or ELISA. In addition, Tie2 protein is a protein expressed on the cell surface, and C
ol2 is a protein expressed inside cells, and any appropriate method (such as immunofluorescence staining) may be used to detect proteins present on the cell surface and inside cells, respectively.
The expression level of mRNA can be determined quantitatively or qualitatively by an assay using a nucleic acid specific (complementary) to the mRNA, a labeling agent, or a nucleic acid amplification method (means), such as RT-PCR, microarray, or biochip. can be measured accurately. The ratio of cells (positive rate or negative rate) in a cell population that express positive or negative expression of a given marker gene (cell marker) is determined by the number of total cells in the cell population and the number of positive cells expressed by the method described above. Alternatively, the number of cells determined to be negative can be calculated by various methods such as those described above, for example, by measurement using flow cytometry.

As used herein, "stem cells and/or progenitor cells that are positive for Tie2 expression", that is, "Tie2-positive stem/progenitor cells" refer to Tie2-positive stem/progenitor cells, which are known as one of the cell markers.
2 (tyrosine kinase with Ig and EGF homology domain-2) expression as a protein is determined to be positive, for example, by flow cytometry, and refers to cells with characteristics as stem cells and/or progenitor cells. . Typical Tie2-positive stem/progenitor cells in the present invention are Tie2-positive stem/progenitor cells "derived from nucleus pulposus tissue of an intervertebral disc", that is, Tie2-positive stem/progenitor cells that exist in the nucleus pulposus of an intervertebral disc (can be collected from the nucleus pulposus). These are stem/progenitor cells or Tie2-positive stem/progenitor cells obtained by passage of Tie2-positive stem/progenitor cells, and correspond to "nucleus pulposus stem/progenitor cells" described below.

As used herein, "target cells" refer to cells that are obtained from Tie2-positive stem/progenitor cells by predetermined differentiation induction and that have functionality according to the intended use, more specifically, cells that have a predetermined gene (cell marker). The expression is determined as a protein by, for example, flow cytometry.
Refers to cells that are determined to be positive or negative. Representative target cells in the present invention include extracellular matrices (ECM) such as Col2 and aggrecan among the "nucleus pulposus cells" described below.
) gene expression is positive.

In the present invention, "nucleus pulposus cells" refer to cells that have matured and reached terminal differentiation, which account for the majority of the cell population in an intervertebral disc (nucleus pulposus), or cultured cells that have characteristics equivalent thereto. Specifically, nucleus pulposus cells are negative for Tie2 and GD2 as marker genes (and usually positive for CD24), and positive for at least type II collagen in the extracellular matrix (furthermore usually cells that are also positive for proteoglycan (aggrecan). For example, cells that are determined 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) by flow cytometry are subject to the present invention. corresponds to nucleus pulposus cells in Regarding extracellular matrices such as type II collagen and aggrecan, the production amount of these proteins was measured by flow cytometry, and their mRNA
The expression level of A may be measured by real-time PCR or the like.

In the present invention, "nucleus pulposus stem/progenitor cells" refer to progenitor cells having at least the ability to differentiate into nucleus pulposus cells (nucleus pulposus progenitor cells) and the progenitor cells, which occupy a part of the cell population in the nucleus pulposus tissue of an intervertebral disc. Collectively refers to stem cells (nucleus pulposus stem cells) that have the ability to differentiate and self-renew, 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, flow cytometry shows that Tie2 is positive and GD2 is negative, Tie2 is positive and GD2 is positive, or Tie2 is negative and GD2 is negative as a protein (cell marker).
Cells determined to be positive correspond to nucleus pulposus stem/progenitor cells in the present invention.

In addition, in the above-mentioned Patent Document 2, Tie2 and GD2 are used as cell markers for nucleus pulposus-derived cells.
Based on the expression status of
Cells that are negative for D2 are in a dormant state, cells that are positive for GD2 are in an active state), cells that are negative for Tie2 and positive for GD2 are called "disc progenitor cells", and cells that are negative for Tie2 and positive for GD2 are called "disc progenitor cells". Cells that are negative are classified as "mature disc nucleus pulposus cells that have completed differentiation" (paragraphs 0024, 0025, 0032). Furthermore, in Patent Document 2, regarding cells that appear in the differentiation hierarchy of nucleus pulposus cells, (i) cells that are Tie2 positive and GD2 negative (and CD24 negative, CD44 positive/negative, CD271 positive, Flt1 positive), (ii)
) Tie2 positive and GD2 positive (also CD24 negative, CD44 positive, CD271 positive,
(iii) cells that are Tie2 negative and GD2 positive (also CD24 negative, CD44 positive, CD271 positive/negative, Flt1 positive/negative), (iv) Ti
e2 negative and GD2 positive (also CD24 positive, CD44 positive, CD271 negative, Flt
(v) cells that are Tie2 negative and GD2 negative (and CD24 positive, CD4
4 positive, CD271 negative, Flt1 negative), and the cells are classified into (i) to (i) above.
ii), “NP stem/progenitor cells”, and (iii) above.
The expression “NP committed cells” is used for ~(v) (see Figure 7-2). Although the notations are different, the "disc nucleus pulposus stem cells" and "disc nucleus pulposus progenitor cells" of Patent Document 2, that is, the cells (i) to (iV) above, are the "nucleus pulposus stem/progenitor cells" in the present invention. "Mature intervertebral disc nucleus pulposus cells that have completed differentiation" in Patent Document 2,
That is, the cell (v) above corresponds to the "nucleus pulposus mature cell" in the present invention. If necessary, the cells in the present invention may be defined as defined in Patent Document 2 (in particular, cells such as CD24, Tie2
and definition of positive or negative for one or more cell markers other than GD2)
It is possible to replace the cells with cells according to the following.

In the present invention, a "spherical colony" is a spherical cell aggregate that contains stem cells and/or progenitor cells, and may also contain cells differentiated from them. "Spherical colony"
is an object that is commonly referred to as a "sphere" or "spheroid" by those skilled in the art, and is referred to as a "discosphere" or "free floating circular structure" in Patent Document 2 mentioned above. -spherical structure) is also an object that corresponds to a "spherical colony."

In the present invention, "the expression of Tie2 is enhanced" (enhanced Tie2 expression) means that the expression of the Tie2 gene is enhanced in each stem/progenitor cell, that is, the expression is increased more than normal, and the mRNA or This refers to an increase in the amount of protein expressed. Even under conditions where the expression of the Tie2 gene would normally disappear, the expression does not disappear and maintains a constant level of expression, that is, the expression of Tie2 is maintained.
This corresponds to "the expression of Tie2 is enhanced". In addition, as a result of the enhanced expression of Tie2 in individual stem/progenitor cells, Tie2 mRNA in the cell population
An increase in the number of cells determined to be positive for A or protein expression, that is, an increase in the proportion of Tie2-positive cells in the cell population than normal, is also referred to as "enhanced Tie2 expression."
It can be interpreted as an expression of

More specifically, for example, a cell population (Tie2
2 expression enhancement treatment group) and the untreated cell population (control group), cell surface T
Cells that have been treated to fluorescently label the ie2 protein and are determined to be positive for expression, with the Tie2 expression enhancement treatment group having a higher fluorescence intensity than a predetermined level compared to the control group as measured by flow cytometry. and/or the average fluorescence intensity per cell is high, the cell population (Tie2-expressing cells included in the Tie2 expression enhancement treatment group) has enhanced Tie2 expression (in other words, For example, it can be said that the Tie2 expression enhancement treatment plays a predetermined role. Furthermore, in morphological observation, cells with enhanced Tie2 expression can be distinguished from each other based on the fact that they are spindle-shaped (other cells are nearly spherical).

In the present invention, an agent that exhibits the effect of "enhancing Tie2 expression" as described above is referred to as a "Tie2 expression enhancer". Note that some growth factors (such as FGF2) have Tie2 expression enhancing effects and can be said to fall under a type of "Tie2 expression enhancer", so when excluding such growth factors, "other than growth factors" Tie2 activation expression enhancer.

In the present invention, implementing the first culture method and/or the second culture method includes
This corresponds to performing "Tie2 expression enhancement treatment" on a cell population containing 2-positive stem/progenitor cells.

-Culture method-
The first to fourth methods for culturing cell populations containing Tie2-positive stem/progenitor cells according to the present invention are as follows:
First culture method: A method of culturing a cell population containing Tie2-positive stem/progenitor cells in a state that exists in undigested tissue;
Second culture method: a method of culturing a cell population containing Tie2-positive stem/progenitor cells in a medium supplemented with at least one Tie2 expression enhancer other than growth factors;
Third culture method: T
A method for culturing a cell population containing ie2-positive stem/progenitor cells;
Fourth culture method: A method of culturing a cell population containing Tie2-positive stem/progenitor cells while suppressing the formation of spherical colonies in a medium supplemented with an extracellular matrix degrading agent.

The first to fourth culture methods of the present invention may be performed alone, or may be performed in combination, sequentially or simultaneously. Combining and simultaneously implementing multiple culture methods selected from the first to fourth culture methods means merging the selected culture methods, that is, a culture method that satisfies all the technical matters related to the selected culture methods. It means carrying out. For example, the first culture method and the second culture method can be performed in combination, sequentially or simultaneously (in a fusion) (a method in which these are fused may be referred to as a "first and second culture method"). )
. The third culture method and the fourth culture method can also be carried out sequentially or simultaneously (in fusion) in combination (a method in which these are fused may be referred to as a "third and fourth culture method").

The purpose of carrying out the first to fourth culture methods of the present invention is not particularly limited. Each of the first to fourth culture methods can be carried out at any of the amplification culture stage (a stage corresponding to), the differentiation culture stage (a stage corresponding to), and other stages of the present invention.

In this specification, descriptions regarding the first to fourth culture methods (and the first to fourth culture steps that carry them out) refer to cases in which each of them is performed as a separate method (step) unless otherwise specified. In addition, it can be read as appropriate as a description of the case where the method (step) is implemented as a method (step) combined with other methods (steps).

Tie2-positive stem/progenitor cells included in the cell population to which the fourth culture method of the present invention is applied, and cells differentiated from the stem/progenitor cells, in a normal medium that does not contain an extracellular matrix degrading agent, These are cells that bind to each other to form spherical colonies (spheroids) using extracellular matrix secreted outside the cells, and when an extracellular matrix degrading agent is added to the culture medium according to the present invention, the formation of spherical colonies (spheroids) is inhibited. The type of cell is not particularly limited as long as it has the effect of being suppressed.

In an exemplary embodiment of the invention, the cells differentiated from Tie2 positive stem/progenitor cells are
Cells that produce and secrete more extracellular matrix than ordinary cells, such as nucleus pulposus cells, which play the role of producing and secreting extracellular matrix in the nucleus pulposus tissues of intervertebral discs.
Mature nucleus pulposus cells express at least type II collagen as an extracellular matrix,
It also expresses extracellular matrices such as proteoglycans (aggrecan). In a preferred embodiment of the present invention, cells expressing extracellular matrices such as type II collagen, proteoglycans (aggrecan), especially type II collagen, are expressed not only as mRNA but also as protein from Tie2-positive stem/progenitor cells. Differentiate functional nucleus pulposus cells with excellent quantity (production amount).

-Preparation method (cultivation process)-
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 the following expansion culture stage and/or differentiation culture stage. , preferably comprising both an amplification culture step and a differentiation culture step (in order of amplification culture step first and differentiation culture step later):
Amplification culture step: A culture step for enhancing the expression of Tie2 in Tie2-positive stem/progenitor cells and amplifying the Tie2-positive stem/progenitor cells in the cell population;
Differentiation culture stage: A culture stage for inducing differentiation of Tie2-positive stem/progenitor cells into target cells.

- Steps related to the amplification culture stage In a preferred embodiment of the present invention, the first culture method and the second culture method are carried out in the steps of the amplification culture stage. Either one of the first culture method and the second culture method may be practiced, or both may be practiced. When implementing both the first culture method and the second culture method, the step of implementing the first culture method (herein referred to as "
This is called the "first culture step". ) and the step of implementing the second culture method (herein referred to as "second culture step") may be separate steps performed sequentially (the first culture step comes first, the second culture step comes first, etc.). ), a single step in which two culture methods are performed simultaneously (implementing the first and second culture methods in which they are fused) (herein referred to as "first and second culture steps")
It is called. ), that is, the cell population containing Tie2-positive stem/progenitor cells may be cultured in a medium that is present in undigested tissue and added with a Tie2 expression enhancer.

The main purpose of the "amplification culture stage process" is to amplify Tie2-positive stem/progenitor cells by culturing according to predetermined conditions, and the effect for this purpose is (relatively stronger than other effects). ) means the process of playing. In other words, if the number and/or ratio of Tie2-positive stem/progenitor cells is higher in the post-culture cell population than in the pre-culture cell population, the culture process is considered to be an "amplification culture step". Within these limits, differentiation of Tie2-positive stem/progenitor cells into other cells (target cells) is allowed.

In the present invention, T in individual Tie2-positive stem/progenitor cells (e.g. nucleus pulposus stem/progenitor cells)
Enhance the expression of ie2 (including maintaining the state in which Tie2 is expressed),
The first and second culture methods are carried out as steps in the amplification culture stage because they can synergistically enhance effects such as improving the number and/or ratio of Tie2-positive stem/progenitor cells contained in the cell population. (That is, carrying out the first and second culture steps) is particularly preferable.

- Steps related to the differentiation culture stage In a preferred embodiment of the present invention, the third culture method and the fourth culture method are carried out in the differentiation culture stage. Either one of the third culture method and the fourth culture method may be practiced, or both may be practiced. When implementing both the third culture method and the fourth culture method, the step of implementing the third culture method (herein referred to as "
This is called the "third culture step". ) and the step of implementing the fourth culture step (herein referred to as the "fourth culture step") may be separate steps performed sequentially, or the two culture methods may be performed simultaneously (if they are fused). In other words, a cell population containing Tie2-positive stem/progenitor cells is It may also be a step of culturing while suppressing the formation of spherical colonies in a culture container having a culture surface treated to increase the adhesion of the cells and in a medium to which an extracellular matrix decomposing agent has been added.

The "differentiation culture stage process" has the main purpose of differentiating Tie2-positive stem/progenitor cells into predetermined cells by culturing according to predetermined conditions. means a process played (relatively strongly). In other words, if the number and/or ratio of target cells is higher in the post-culture cell population than in the pre-culture cell population, the culture step can be called a "differentiation culture step".

As mentioned above, spherical colonies (spheroids,
The step of temporarily treating the discospheres (discospheres, floating spherical structures) in a medium supplemented with collagenase in order to dissociate them is carried out in the fourth culture method of the present invention or the differentiation culture stage defined as above. This does not correspond to the fourth culture step as a process. In addition, there are methods (processes) of treating with collagenase, etc. to isolate cell populations contained in collected tissues, and trypsin treatment for subculturing cells grown in general two-dimensional culture. The method (step) of dissociating cells from the culture surface by treatment also does not fall under the fourth culture method of the present invention defined above or the fourth culture step as a step of the differentiation culture stage.

In the present invention, the number and/or ratio of cells having a predetermined functionality (e.g., Col2-positive nucleus pulposus cells) differentiated from Tie2-positive stem/progenitor cells (e.g., nucleus pulposus stem/progenitor cells) contained in a cell population is determined. The third and fourth culture methods are implemented as steps in the differentiation culture stage, as they can synergistically enhance the effects of improving Tie2-positive stem/progenitor cells while maintaining a constant level of cell number and/or ratio. It is particularly preferable to carry out the third and fourth culturing steps.

The amplification culture step may further include steps other than the first culture step and/or the second culture step, if necessary, consistent with the purpose of the step to amplify Tie-positive stem/progenitor cells. Such a step includes, for example, a step of culturing a cell population containing Tie2-positive stem/progenitor cells in a medium to which only a growth factor having an effect of enhancing Tie2 expression is added as a Tie2 expression enhancer (herein referred to as " (referred to as "additional amplification culture step"). Examples of growth factors having an effect of enhancing Tie2 expression in the additional amplification culture step include:
FGF and/or EGF may be mentioned. The additional amplification culture step is the first culture step and/or
Alternatively, it is preferable to perform the second culture step, particularly after the first culture step or the first and second culture steps. In addition, in the additional amplification step, the first culture method is not performed, that is, Tie2
It is appropriate that the cell population containing positive stem/progenitor cells be separated from the cells by digestion, rather than being present in undigested tissue. In the first culture method of the present invention, which is carried out in the first culture step or the first and second culture steps, a cell population containing Tie2-positive stem/progenitor cells is cultured in a state that is present in undigested tissue. However, when Ti reaches a certain level, this is thought to be due to its presence in the tissue.
The expansion of e2-positive stem/progenitor cells is suppressed (Tie2-positive stem/progenitor cells are no longer expanded even if the culture period is extended). Therefore, after the first culture step or the first and second culture steps, the tissue is digested, the separated cell population is collected, and an additional amplification step is performed to further amplify Tie2-positive stem/progenitor cells. can do.

<Cell population>
In the cell population containing Tie2-positive stem/progenitor cells (herein collectively referred to as "pre-culture cell population") subjected to each culture method or each culture step of the present invention, Tie2-positive stem/progenitor cells are
The ratio and/or number of progenitor cells and other cells (cells differentiated from Tie2-positive stem/progenitor cells, etc.) is basically arbitrary, and the ratio of Tie2-positive stem cells to Tie2-positive progenitor cells is also basic. It is optional. The composition of the cell population before culturing can be adjusted as appropriate depending on the embodiment of the invention, taking into account the effects of each culture method or each culture step.

The pre-culture cell population can be prepared or prepared according to a conventional method, except when it is subjected to the first culture method or the first culture step. For example, when using a cell population contained in intervertebral disc nucleus pulposus tissue collected from the body as a pre-culture cell population, first cut the nucleus pulposus tissue into appropriate sizes using a device such as scissors (e.g. After mincing the cells into pieces of several millimeters square), the cells are then treated with proteolytic enzymes such as collagenase to disperse the cells, and if necessary, the cells are processed by filtration, centrifugation, washing, etc. to form the nucleus pulposus tissue. The contained cell population can be isolated and recovered. The cell population obtained in this manner can be used as a pre-culture cell population for purposes other than the first culture method or first culture step.

On the other hand, in the first culture method or first culture step of the present invention, among the above steps, the nucleus pulposus tissue is stopped at the stage where it is cut into pieces (no treatment with proteolytic enzymes is performed), and the cut The cell population contained in the nucleus pulposus tissue is used as a pre-culture cell population.

The cell population separated from the tissue or the cell population contained in the tissue (tissue containing the cell population) prepared as described above is subjected to the following culture method or culture step. It can be frozen and preserved according to conventional methods. Cryopreserved cell populations or tissues are
When starting the next culture method or culture step, thawing can be carried out according to a conventional method. During cryopreservation and thawing, treatments preferable for the cell population or tissue may be combined. For example, a cryoprotectant (DMSO, etc.) may be added during cryopreservation, and in that case, the cryoprotectant may be removed under appropriate conditions during thawing.

In the cell population containing Tie2-positive stem/progenitor cells (herein collectively referred to as "cultured cell population") obtained by each culture method or each culture step of the present invention, Tie2-positive stem/progenitor cells and The ratio and/or number of cells other than Tie2-positive stem cells (cells differentiated from Tie2-positive stem/progenitor cells, etc.) is basically arbitrary;
The ratio of 2-positive progenitor cells is also basically arbitrary. The composition of the cultured cell population can be adjusted as appropriate depending on the embodiment of the invention, taking into consideration the use of the cell population obtained by each culture method or each culture step.

The cultured cell population can be recovered from the medium according to a conventional method and subjected to the next culture method or step, or to other methods or steps such as the preparation of cell preparations.

In the cell population containing Tie2-positive stem/progenitor cells in the middle of each culture method or each culture step of the present invention (herein collectively referred to as "cell population in culture"), Tie2-positive stem/progenitor cells and The ratio of 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 cultured cell population is the composition in the process of transition from the pre-cultured cell population to the post-cultured cell population, for example, the ratio of Tie2-positive stem/progenitor cells in the cultured cell population (herein referred to as "Tie2-positive stem/progenitor cells"). (referred to as "progenitor cell rate") is usually the Ti
Although the value is within the range between the e2-positive stem/progenitor cell rate and the Tie2-positive stem/progenitor cell rate of the cultured cell population, it is also acceptable for the value to temporarily deviate from the range. The composition of the cell population during culture varies depending on the embodiment of the invention, the number of days in each culture method or each culture step, the number of passages, etc.

The "human or other animal" (donor) from which each of the above cell populations is derived is the Ti of the present invention.
It can be selected in consideration of the use of the cell population finally obtained by the e2-positive stem/progenitor cell culture method, 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 prevention or treatment of a predetermined disease, symptom, etc., "human or other animal" refers to the cell preparation. An organism of the same species as the recipient (recipient), preferably a human.

- Cell population related to the amplification culture stage In the present invention, the cell population subjected to the first culture method and/or the second culture method, or the cell population subjected to the first culture step and/or the second culture step in the amplification culture stage The population (herein collectively referred to as "cell population before amplification culture") is typically a cell population (primary culture) contained in tissue (intervertebral disc) collected from the body of a human or other animal. cell population) or a cell population obtained by passage of the primary cultured cell population (subcultured cell population)
It is.

When using a cell population contained in an intervertebral disc collected from a human as a pre-expanded cell population, the Tie2-positive stem/progenitor cell rate is generally high and the niche tends to be favorable.
Preferably, the cell population is contained in an intervertebral disc collected from a human in their 10s or 20s. Further, the pre-expanded cell population is preferably a cell population in which the Tie2-positive stem/progenitor cell rate is as high as possible, for example, 30% or more, 40% or more, 50% or more, or 60% or more.

Note that, depending on the embodiment, the pre-expanded cell population may be a cell population that is not included in a tissue collected from the body of a human or other animal, for example, a cell population of a human or other animal. The cell population may also be a cell population containing Tie2-positive stem/progenitor cells obtained by inducing differentiation of cells having pluripotency or pluripotency such as iPS cells or ES cells prepared by the above method.

In the present invention, the cell population obtained by the first culture method and/or the second culture method, or the cell population obtained by the first culture step and/or the second culture step in the amplification culture stage (herein referred to as "amplification culture There are no particular limitations on the use of the cell population (hereinafter collectively referred to as "post-cell population"), and the composition etc. of the resulting cell population can be adjusted as appropriate depending on the use.

In a typical embodiment of the present invention, the amplified cultured cell population is a cell population subjected to a third culture method and/or a fourth culture method, or a third culture step and/or a fourth culture step in a differentiation culture stage. It is used as a cell population for the process. In such embodiments (applications), the cell population after expansion and culture preferably has as high a ratio and/or number of Tie2-positive stem/progenitor cells as possible. The Tie2-positive stem/progenitor cell rate in the cell population after amplification culture cannot be generalized because it varies depending on individual differences in the cell population before amplification culture and the nucleus pulposus tissue from which it is derived, but it is preferably 5% or more, for example. is 7% or more, 9% or more, 1
1% or more, 13% or more, 15% or more. The number of Tie2-positive stem/progenitor cells in the cell population after amplification culture cannot be generalized because it varies depending on individual differences in the cell population before amplification culture and the nucleus pulposus tissue from which it is derived; Compared to the number of cells, for example, 5 times or more, preferably 10 times or more, 15 times or more, 20 times or more, 25 times or more, 30 times or more
That's more than double that.

-Cell population related to the differentiation culture stage In the present invention, the cell population subjected to the third culture method and/or the fourth culture method, or the cell population subjected to the third culture step and/or the fourth culture step in the differentiation culture stage The population (herein referred to as "pre-differentiation culture cell population") is preferably a cell population enriched in Tie2-positive stem/progenitor cells in advance. The Tie2-positive stem/progenitor cell rate in the cell population before the differentiation culture stage cannot be generalized because it varies depending on individual differences in the cell population before or after amplification culture and the nucleus pulposus tissue from which it is derived, but for example, 5
% or more, preferably 7% or more, 9% or more, 11% or more, 13% or more, 15% or more.

In a typical embodiment of the present invention, the pre-differentiation culture cell population is a cell population obtained by the amplification culture step of the present invention (post-expansion culture cell population), for example, an amplified Tie2-positive stem/
A cell population containing progenitor cells is divided into an appropriate number of cells depending on the embodiment of the differentiation culture stage (type and size of culture container, etc.). The cell population obtained by the expansion culture step of the present invention contains Tie2-positive stem/progenitor cells in the ratio and/or number of cells described above, and the Tie2 expression of the Tie2-positive stem/progenitor cells is enhanced. There is (Tie
2), it is preferable as a pre-differentiation culture cell population from the viewpoint of enhancing the action and effect in the differentiation culture stage.

Note that, depending on the embodiment, the pre-differentiation culture cell population is a cell population that is not obtained by the amplification culture step (first culture step and/or second culture step) of the present invention, for example, a human or other animal cell population. Inducing the differentiation of pluripotent or pluripotent cells such as cell populations contained in tissues collected from the body of humans or iPS cells or ES cells produced using human or other animal cells. It may also be a cell population containing target cells obtained by (via Tie2-positive stem/progenitor cells).

In the present invention, the cell population obtained by the third culture method and/or the fourth culture method, or the cell population obtained by the third culture step and/or fourth culture step in the differentiation culture stage (herein referred to as "differentiation culture There are no particular limitations on the use of the cell population (hereinafter collectively referred to as "post-cell population"), and the composition etc. of the resulting cell population can be adjusted as appropriate depending on the use. For example, for cell populations used to manufacture cell preparations for transplantation, target cells with useful functionality for achieving therapeutic or preventive effects through transplantation (e.g., nucleus pulposus cells that produce collagen II: Col2 It is preferable that the cell population contains as many Tie2-positive stem/progenitor cells (e.g., nucleus pulposus stem/progenitor cells) that still have the ability to produce such target cells as possible (for example, nucleus pulposus stem/progenitor cells). .

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

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

Note that the number of cells included in the cell population usually increases during the differentiation culture step as well. Number of cells in cell population after differentiation culture (Col2-positive cells, Tie2-positive stem/progenitor cells, etc.)
Although it cannot be generalized because it varies depending on individual differences in the pre-differentiated cell population and the nucleus pulposus tissue from which it is derived, it is, for example, more than twice the number of cells in the pre-differentiated culture cell population, 10 times or more, 20 times or more, 50 times or more, 100 times or more.

<Medium>
The culture medium used in each culture method or each culture step of the present invention may be any medium suitable for culturing Tie2-positive stem/progenitor cells and cells differentiated therefrom, and while considering the purpose of the culture method or culture step, Appropriate basal media and additional components can be selected. The additive components are those that are carried out when the culture method is used to amplify Tie2-positive stem/progenitor cells, that is, if the culture process is at the amplification culture stage, additive components that are suitable for the amplification culture of Tie2-positive stem/progenitor cells; If the culture method is used to induce differentiation of Tie2-positive stem/progenitor cells, that is, if the culture process is at the differentiation culture stage, a method suitable for inducing differentiation of Tie2-positive stem/progenitor cells into target cells is recommended. selected.

In addition, in the third culture method and fourth culture method of the present invention, and in the third culture step and fourth culture step including the step of implementing these methods, Tie2-positive stem/progenitor cells and cells differentiated therefrom are cultured. There is no need to add components to the medium that prevent it from adhering to the culture surfaces of the vessels, such as methylcellulose. In other words, the medium of the third culture method and fourth culture method of the present invention, and the third culture step and fourth culture step including the steps of carrying out these methods are usually methylcellulose, etc. Contains no ingredients to prevent cell attachment to surfaces.

In a typical embodiment of the present invention, when culturing nucleus pulposus stem/progenitor cells and nucleus pulposus cells differentiated therefrom, the medium for each step of the amplification culture stage and the differentiation culture stage is based on, for example, the following: It can be prepared by using appropriate amounts of the culture medium, additive components, growth factors, and other components.

As the basal medium, for example, DMEM (Dulbecco's modified Eagle's medium, without or with glucose addition), αMEM (Eagle's minimal essential medium α modified type), Ham's F-10 medium, Ham's F-12 medium, or a mixture thereof. can be mentioned.

Additional components for amplification culture or differentiation culture include, for example, FBS (fetal bovine serum),
BSA (bovine serum albumin), L-ascorbic acid (as L-ascorbic acid phosphate magnesium salt, etc.), selenite (as insulin-transferrin-sodium selenite (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), and Ang-1 (angiopoietin-1). In one embodiment of the present invention, the growth factor added to the medium is preferably at least FGF, more preferably both FGF and EGF, and if necessary, Ang-1 is added to them. It is also preferable.

As FGF, for example, bFGF (basic fibroblast growth factor, sometimes referred to as FGF-2) can be used. FG in the medium
The concentration of F can generally be 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 in a serum-free medium. Furthermore, as Ang-1, those solubilized in water (soluble Ang-1, recombinant Ang-1) are preferable. The concentration of Ang-1 (preferably soluble Ang-1) in the medium is usually 100 to 10
00 ng/mL, for example about 500 ng/mL.

The growth factors such as FGF, EGF, and Ang-1 mentioned above are "growth factors that have an effect of enhancing Tie2 expression" and can be understood to fall under "Tie2 expression enhancers" in a broad sense; How to handle these growth factors will be described separately herein.

<Tie2 expression enhancer>
In the second culture method of the present invention, at least one "Tie2 expression enhancer" other than the growth factor having an effect of enhancing Tie2 expression is added to the medium. In particular, when carrying out the second culture method in the step of the amplification culture stage, the addition of the Tie2 expression enhancer has the effect of increasing the cell number while maintaining the juvenile nature of Tie2-positive stem/progenitor cells. Effect of improving the cell increase rate, the ratio of Tie2-positive stem/progenitor cells and functional target cells, etc. of the cell population obtained after the differentiation culture step, when the cell population obtained in the expansion culture step is subjected to the differentiation culture step. etc. Any one type of Tie2 expression enhancer may be used, or two or more types may be used in combination, and the Tie2 expression enhancer may be added to the medium in an amount that allows the Tie2 activation effect as described above to be observed.

Examples of "growth factors having an effect of enhancing Tie2 expression" include angiopoietin-1
(Ang-1), FGF2 (bFGF), etc. In the second culture method of the present invention,
At least one type of "Tie2 expression-enhancing growth factor" other than such "Tie2 expression enhancing effect"
2 expression enhancer, but if necessary, a growth factor having an effect of enhancing Tie2 expression may also be used in combination. In particular, when the second culture method is carried out in the amplification culture step, a growth factor having an effect of enhancing Tie2 expression and another Tie2 expression enhancer, such as an extract derived from an animal or plant as described below, are preferably used. A synergistic effect can be achieved by using it in combination with a plant-derived extract. In addition, as a step in the amplification culture stage,
It is essential to use at least a Tie2 expression enhancer other than a growth factor (as an optional component, a growth factor having an effect of enhancing Tie2 expression may be used in combination). Alternatively, a step using only a growth factor having an effect of enhancing Tie2 expression (substantially no use of a Tie2 expression enhancer other than the growth factor) may be performed.

As a Tie2 expression enhancer other than growth factors, in the prior art, "Tie2 activator"
Extracts derived from various plants and animals known as can be used. Such extracts derived from plants and animals include, for example, Akigumi, Acanthus japonicum, Indian date, Turmeric, Acanthus japonicum, Indian date, Japanese turmeric, Japanese plantain, Okahijiki, olive fruit, oyster, chamomile, Japanese quince, Japanese quince, chrysanthemum, Gyokuchiku, Quiraya, Ginkgo nut, Japanese cypress. , wolfberry, sawtooth oak, algae, Japanese ginseng, Quercus serrata, hawthorn, salamander, white guava, Siberian ginseng, Japanese oyster, star fruit, sour oak, jujube, daylily, nobile, lotus, lotus, yellowtail, yellowtail, butcher bloom, mango Examples include extracts of ginger, honeysuckle, mube, daylily, bayberry, rhubarb, and rooibos. Those skilled in the art can also appropriately determine the amount of each extract at which the Tie2 expression enhancing effect is observed, as well as appropriate animal and plant parts (materials) and extraction methods for preparing each extract.

From an industrial perspective, it is cheaper than growth factors such as Ang-1 and FGF2, preferably has a better Tie2 expression enhancing effect than those growth factors, and is more preferably synergistic when used in combination with those growth factors. In the second culture step of the present invention, one or more kinds selected from the above-mentioned extracts derived from plants and animals, preferably one or more kinds selected from the above-mentioned plant-derived extracts, which exhibit effects, are added to Tie2 in the second culture step of the present invention. Advantageously, it is used as an expression enhancer.

- Extract derived from a plant belonging to the genus Daylifolia In a preferred embodiment of the present invention, an extract derived from a plant belonging to the genus Daylily genus can be used as the Tie2 expression enhancer. The genus Cinnamomum includes the genus Cinnamomumcass.
ia Blume), camphora (C. camphora), C. daphnoides, C. doederleinii, C. japonicum, C. pseud
C. o-pedunculatum), C. sieboldii, Ceylon Nikkei (Cv.
It includes more than 300 species, including C. erum) and cinnamon (C. zeylanicum). For example, in the present invention, an extract of a product manufactured and sold as cinnamon powder, which is the young branch of cinnamon, the bark of cinnamon, or cinnamon powder processed into powder, is used as It can be used as an extract.

Extracts derived from plants of the genus Daylily can be obtained by conventional methods, for example, by soaking or heating refluxing a raw material plant (e.g. cinnamon powder) with an extraction solvent at room temperature or by heating, and then collecting the supernatant. Alternatively, it can be prepared by filtering the filtrate and concentrating if necessary. Extraction solvents include solvents commonly used for extraction, such as aqueous solvents such as water, physiological saline, phosphate buffers, borate buffers, or organic solvents such as ethanol, propylene glycol, 1,3-butylene glycol. , alcohols such as glycerin, hydroalcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc. can be used alone or in combination. Preferably water is used as solvent. The extract obtained by extraction with the above solvent may be used as it is in the form of an extract liquid, but for convenience, it should be solidified (powdered) by drying or freeze-drying, etc., and stored before use. Dilute or redissolve (redisperse) with an appropriate solvent as necessary.
Then, it can be used after further treatment such as filtration if necessary. The extract derived from plants of the genus Daylily is a product (purified product) from which impurities have been removed, if necessary, by an adsorption method using an ion exchange resin (for example, a porous polymer such as Amberlite XAD-2). Good too.

The concentration of the extract derived from a plant of the genus Daylik in the medium can be adjusted as appropriate depending on the properties of the extract used and taking into account the degree of action and effect as a Tie2 expression enhancer. For example, when using an extract obtained by extracting 1 mg of cinnamon powder with 1 mL of physiological saline as an extract derived from a plant of the genus Niki, the extract is added at a rate of 1 to 50 v/v to the culture medium.
It can be added in an amount of about 20% v/v, for example about 20% v/v. Even when the embodiment of extraction and addition is changed, the amount of active ingredient as a Tie2 expression enhancer can be made to be the same as in the embodiment of extraction and addition described above.

<Extracellular matrix degrading agent (ECM degrading agent)>
In the fourth culture method of the present invention, an "extracellular matrix degrading agent (ECM degrading agent)" is added to the medium in order to differentiate Tie2-positive stem/progenitor cells while suppressing the formation of spherical colonies.

In general, the extracellular matrix (ECM) secreted from stem/progenitor cells or cells differentiated therefrom includes, for example, collagen, proteoglycan, fibronectin, laminin, tenascin, entactin, elastin, fibrillin, hyaluronic acid, and the like. Collagen includes type I, type II, type III, type IV, type IX (a2), and other types of collagen. Proteoglycans include aggrecan, versican, and permacan (classified based on the core protein size and number of sugar chains), chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan (all of which are classified based on the size of the core protein and the number of sugar chains). classification based on glycosaminoglycans).

Therefore, as the ECM degrading agent in the present invention, the ECM as exemplified above corresponds to the embodiment of the fourth culture method, that is, the ECM secreted from the cultured Tie2 stem/progenitor cells or cells differentiated therefrom. What is necessary is to use a substance (agent) that has the activity of degrading , and can inhibit the formation of spherical colonies. Any one type of ECM decomposer may be used, or two or more types may be used in combination.

Typical ECM degrading agents include proteases that have the activity of degrading protein moieties that make up ECM, such as collagenase, which is a protease that has degrading activity for collagen. Collagenases include class I collagenase, which shows high activity against high-molecular-weight collagen, and class II collagenase, which shows high activity against low-molecular-weight collagen fragments. In addition, collagenases derived from vertebrates cleave collagen in the natural triple helical region (on a very limited α chain), while collagenases derived from bacteria act on almost all types of collagen and Collagen can be cut at multiple locations within the helical region. Collagenase preparations obtained by concentrating bacterial culture supernatants contain, in addition to collagenase (collagenase I, collagenase II), proteases other than collagenase (neutral protease, clostripain, trypsin, elastase,
aminopeptidase, etc.) and non-proteolytic enzymes, and preparations that exclude specific components through purification are also manufactured. In the present invention, various known collagenases (preparations)
, protease, etc., can be selected and used as an ECM decomposing agent.

In a typical embodiment of the fourth culture method of the present invention, the Tie2-positive stem/progenitor cells are nucleus pulposus stem/progenitor cells, and the cells (target cells) generated by inducing differentiation from the Tie2-positive stem/progenitor cells are These are nucleus pulposus cells. Nucleus pulposus cells express type II collagen, type IX collagen, type XI collagen, proteoglycans, etc. as ECM. Therefore, as the ECM decomposing agent in this embodiment, one having a degrading activity for these ECMs, for example, a collagenase (or a preparation containing the same) having a degrading activity for type II collagen and the like may be selected. As such collagenase (preparation), for example, "Collagenase P"
(Roche, derived from Clostridium histolyticum), "Liberase" (Roche, a mixture of collagenase I and II and neutral protease), and the like.

Incidentally, as an ECM decomposing agent, an enzyme (protein) such as protease, which has a specific degrading activity for proteins contained in ECM but has low cytotoxicity, is typical, but it can achieve the effects of the present invention. has a decomposition activity against ECM that is above a certain level,
Substances other than enzymes (proteins), such as low molecular weight compounds, which have cytotoxicity below a certain level, may also be used as ECM decomposing agents.

The concentration of the ECM decomposing agent in the medium may be any concentration that can inhibit the formation of spherical colonies from the cell population containing Tie2 stem/progenitor cells, and may vary depending on the type of ECM degrading agent used and the concentration during the fourth culture. When the method is carried out at the differentiation culture stage (carried out as the fourth culture step), it is carried out while taking into account the increase rate of target cells and the effect on the expression level or positivity rate of a predetermined gene (marker gene). , can be adjusted as appropriate. For example, if the concentration of the ECM decomposer is too high, the beneficial effects of the above action may not be fully recognized (on the contrary, it may become a disadvantageous effect). It is preferable to adjust the concentration within the range.

In the present invention, a method that combines the third culture method and the fourth culture method (third and fourth culture methods)
Alternatively, in a step that combines the third and fourth culture steps (third and fourth culture steps), target cells can be The effects on the rate of increase, the expression level or positivity rate of a given gene (marker gene), etc. may vary. Those skilled in the art will, depending on what kind of effects are expected, consider the properties of the pre-differentiation induced cell population and other embodiments.
Through preliminary tests and the like, each of the above conditions can be set as appropriate for implementing the present invention.

As mentioned above, the concentration of the ECM decomposing agent in the medium is not determined in general, and depends on the combination with the type of coating agent on the culture surface, but is, for example, 0.0025 to 5.0.
It can be adjusted within the range of 0.005 to 2.0 weight %, 0.01 to 1.0 weight %, etc. In one embodiment of the present invention, when "collagenase P" is used as an ECM degrading agent, its concentration in the medium is 0.005 to 0.05% by weight, 0.0125% to 0.0
It can be adjusted within a range such as 25% by weight, for example about 0.0125% by weight. In one embodiment of the present invention, when "Liberase" is used as an ECM degrading agent, its concentration in the medium is within the range of 0.25% to 2.0% by weight, 0.5 to 1.0% by weight, etc. For example, it can be adjusted to about 1.0% by weight.

<Culture period and other conditions>
The period and other conditions of each culture method and each culture step of the present invention (e.g. pH, _CO2
Basically, the concentration (O2 concentration, _O2 concentration, etc.) should be adjusted as appropriate to obtain a cell population with the desired cell composition (type, number, ratio), depending on the purpose of the culture process (the culture stage it includes). can do. The pH can be slightly alkaline (eg, about 7.15). C
The O ₂ concentration may be approximately 5%, for example. _O2 concentration is 5% or less (e.g. about 2%)
It can be done. During each culture method and each culture step (stage), the medium may be replaced with a fresh one every predetermined number of days, or components may be added after a predetermined number of days, or the concentration of the components may be adjusted as necessary. The culture medium may be changed by increasing or decreasing the pH, or the atmosphere may be changed.

In the amplification culture stage of the present invention, the period of the first culture step, the second culture step, or the first and second culture steps in which they are combined is usually about 1 to 3 weeks, for example about 2 weeks.
Also, the duration of other steps that the amplification culture step of the present invention may optionally include are also comparable, for example, the duration of the culture step using FGF-supplemented medium is about one week. The amplification culture step may be completed at the time when the desired cell population after amplification culture is obtained.

In the differentiation culture stage of the present invention, the period of the third culture step, the fourth culture step, or the third and fourth culture steps in which they are combined is usually about 1 to 3 weeks, for example about 10 days.
Additionally, the duration of other steps that may optionally be included in the propagation culture step of the present invention is also comparable; for example, the duration of the culture step using an FGF-supplemented medium is about 1 week (about 7 days). Also,
The durations of other steps that the differentiation culture stage of the present invention may optionally include are also comparable. The differentiation culture stage may be terminated when the desired cell population after differentiation culture is obtained.

<Culture container>
The culture vessels, culture devices, etc. used in each culture method and each culture process of the present invention are basically designed to achieve the desired cell composition (type and number) depending on the purpose of the culture method and culture process (culture stage including).・Ratio) can be selected as appropriate so as to obtain a cell population having the following ratio.

The culture container may have a general shape such as a flask, dish, plate, or bag, and may have a well formed therein to accommodate cells.
The culture container can be made of common materials such as glass, plastic, and resin. The surface of the culture container (cultivation surface) may be untreated, or may be subjected to treatment related to cell adhesion or other treatment. The size of the culture vessel (area, volume), and if the culture vessel is equipped with a well, the size of the well (caliber,
depth) and number can also be selected as appropriate. If necessary, the culture container may be shaken or rotated to culture the cell population while stirring the medium.

In the third culture method (step) and the fourth culture method (step) of the present invention, the culture container and the culture device can be an embodiment based on two-dimensional culture (plane culture). Furthermore, the first culture method (step) of the present invention can be called a three-dimensional culture in that the cell population is cultured in a state in which it exists in the tissue, and the tissue (strip) containing the cell population is cultured. placed suspended in liquid.
The second culture method (step) of the present invention can be implemented in accordance with three-dimensional culture when carried out alone, but it can be combined with the first culture method (step) to perform the first and second culture. When carried out as a method (step), the cells are placed in a suspended state in a culture solution as in the first culture method (step) described above. In these methods (steps), culture vessels that have been surface-treated to increase cell adhesion may be used as in the third culture method (step), but normal culture vessels that have not been surface-treated may be used. There is no problem in using it.

<Cell attachment treatment>
In the third culture method (step) of the present invention, a culture vessel is used which has been subjected to a surface treatment to enhance cell adhesion (herein sometimes referred to as "cell adhesion treatment"). Typical cell attachment treatments include applying a coating containing extracellular matrix (ECM) or other biologically relevant substances to the culture surface. In addition, materials with low cell adhesion,
For example, modifying a culture container made of highly hydrophobic polystyrene to make it more hydrophilic by plasma treatment is also an example of cell adhesion treatment.

The ECM contained in the coating agent for cell adhesion treatment includes various known ECMs, such as collagen (type I, type II, type IV, etc.) or its heat-treated gelatin, chondroitin sulfate A, fibronectin, gelatin, Examples include laminin, thrombospondin, vitronectin, and proteoglycans (aggrecan, heparin sulfate proteoglycan, etc.). Furthermore, biologically related substances other than ECM include polyamino acids such as polylysine (poly-L-lysine or poly-D-lysine). Other coating agents for cell adhesion treatment include polyglycolic acid, PLGA (polylactic acid/glycolic acid copolymer),
Polyhydroxyalkanoic acid (PHA), poly-ε-caprolactone, polyorthoester, polyacid anhydride, polyphosphazene, polydimethylsiloxane, polyurethane, polytetrafluoroethylene, polyethylene, polysulfone, poly-methyl methacrylate, poly-2 -Hydroxyethyl methacrylate, polyamide, polypropylene, polyvinyl chloride, polystyrene, polyvinylpyrrolidone polyornithine, and the like. The coating agent for cell adhesion treatment may contain any one of the above substances, or may contain two or more of the above substances.

Here, in the third and fourth culture methods (steps), as described above, the method of the present invention is determined by the combination of the type of coating agent for cell adhesion treatment and the type and concentration of the ECM decomposing agent added to the medium. Effects (increase rate of cell population, ratio of target cells, etc.) may vary. One possible reason for this is that the ECM or other biologically related substances contained in the coating agent for cell adhesion treatment may be affected by the decomposition activity of the ECM decomposition agent added to the culture medium. However, as long as the effects of the present invention are achieved to a certain extent (not completely inhibited), coating agents for cell adhesion treatment that may have such effects and E.
Embodiments using combinations with CM decomposers are also acceptable. For example, as an ECM decomposer.
When adding collagenase (preparation) that has type II collagen degrading activity to the culture medium at a predetermined concentration, the coating agent for cell adhesion treatment should be one that is not easily affected by the type and concentration of the ECM degrading agent, or the purpose. Those that can induce differentiation into cells (for example, Col2-positive cells) at a certain level, such as those containing polylysine (poly-L-lysine or poly-D-lysine) that is not collagen, fibronectin, or type IV collagen, are preferable. .

The third culture method of the present invention can also be carried out in the amplification culture stage. for example,
A step of culturing a cell population containing Tie2-positive stem/progenitor cells in a medium to which only a growth factor having an effect of enhancing Tie2 expression is added as a Tie2 expression enhancer (additional amplification culture step), as described above in relation to the amplification culture step. ), it is possible to carry out a third culture method. In such an embodiment, the ECM contained in the coating agent for cell adhesion treatment is preferably gelatin, for example.

-Cell therapy composition-
The composition for cell therapy 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 pharmaceutically acceptable components as necessary. Can be done.

In a typical embodiment of the present invention, the cell therapy composition comprises Col2-positive nucleus pulposus cells differentiated from nucleus pulposus stem/progenitor cells (preferably also includes Tie2-positive stem/progenitor cells). It is. The subject of the cell therapy composition in this embodiment, that is, the disease that can be prevented or treated by administering the composition, is an intervertebral disc (nucleus pulposus).
Diseases that manifest as symptoms such as disorder, degeneration, hernia, etc., such as lumbar or cervical disc disease, herniated disc, cervical spondylotic myelopathy, radiculopathy, spondylolysis/listhesis, lumbar spinal stenosis, lumbar degeneration. Examples include spondylolisthesis and lumbar degenerative scoliosis.

The cell therapy composition of the present invention may be in any dosage form as long as it can be transplanted or delivered to a target site (for example, the nucleus pulposus of an intervertebral disc), but for example, an injection, preferably an intervertebral disc (nucleus pulposus). It can be an injection for local administration to or near the area, or an injection for vascular administration that allows targeting.

Pharmaceutically acceptable ingredients include, for example, water for injection or physiological saline when preparing an injection, a culture medium for cell populations, other appropriate solvents/dispersion media, and other additives. .

The composition for cell therapy of the present invention may be administered in an amount effective to achieve the desired therapeutic or preventive effect. Such an effective amount is determined by taking into account the components of the composition for cell therapy, the dosage form, the administration target, the administration route, and other embodiments, and determines the amount per dose, the number of administrations, and the administration interval (a certain period of time). The dosage can be adjusted as appropriate depending on the number of administrations within the same period. The cell therapy composition of the present invention can be performed on humans and non-human vertebrates.

-Preservation method-
The method for preserving a cell population containing Tie2-positive stem/progenitor cells of the present invention involves cryopreserving the cell population containing Tie2-positive stem/progenitor cells in a state that is present in an undigested tissue. Tie2-positive stem/progenitor cells remain activated and/or expressed, or suppress the decrease of Tie2-positive stem/progenitor cells in the cell population.

As for the technical matters related to the preservation method of the present invention, the same technical matters as those described above in relation to the first culture method can be applied. Cryopreservation procedures and cryoprotectants used as necessary for undigested tissues containing Tie2-positive stem/progenitor cells are different from conventional Tie2-positive stem/progenitor cells separated from tissues by digestion. Basically, the same methods as those for cell populations including progenitor cells can be applied.

The medium used in each step of the "amplification culture stage" of this example (referred to as "amplification culture stage medium" in the following examples) was 60 mL of DMEM (no glucose, wako) and ME
Immediately before use, 20% FBS was added to 40 mL of Mα (Nacalai Tesque) mixed medium, and additional components shown in each table in the examples were added (+) or not added (-). used.

As a medium for each step of the "differentiation culture stage" in this example (referred to as "differentiation culture stage medium" in the following examples), 60 mL of DMEM (no glucose, wako) and F1
0 (gibco) 40 mL mixed medium, 1 μL of 2-mercaptoethanol, 6 μL of selenite (0.01%), 1.5 mL of ascorbic acid (5 mg/mL) and 30% BSA.
5 mL was added thereto, and 30% FBS was added immediately before use, and products with (+) or without (-) the additional components shown in each table in the examples were prepared and used.

[Test Example 1] Amplification culture stage: 1st culture step (WTC method)

Nucleus pulposus tissues of intervertebral discs excised from the affected areas of patients with intervertebral disc herniation (32-year-old female, 28-year-old female, and 20-year-old male) were cut into pieces several millimeters square using scissors or the like. 0.1 to 0.5 g of minced nucleus pulposus tissue still containing the cell population was suspended in 3 mL of medium prepared as shown in Table 1 for additional components to the medium for the amplification culture stage, and then suspended in 6 wells. Culture dish (culture surface not treated)
and cultured for 7 days (WTC method). As a control, instead of culturing the minced nucleus pulposus tissue as it is, it was digested with collagenase according to a conventional method and an isolated cell population was collected. cultured (two-dimensional culture method).

After culturing, the cell population was collected, and the cell surface T was determined by flow cytometry (FCM).
The number and fluorescence intensity of cells positive for ie2 expression were measured, and the ratio of the total cell population to the number of cells (Tie2 positive rate) and mean fluorescence intensity (MFI) were calculated. In the FMC method, a fluorescent labeling agent (R&
Company D, Anti-Tie-2, Human, Mouse-Mono(87315), Allophicocyanin, Catalog number FAB
3131A) was used.

The results are shown in FIGS. 2 and 3. For example, when comparing Test Examples 1-1 and 1-3, both results are significantly higher in Test Example 1-1 (Figure 2: p<0.05, Figure 3: p<0.
01, both t-test), the effect of enhancing Tie2 expression by the WTC method was observed.

[Test Example 2] Amplification culture stage (2 stages): 1st and 2nd culture process + additional process

1 mg of commercially available cinnamon powder was suspended in 1 mL of physiological saline and extracted overnight at 37° C., and the resulting extract (cinnamon extract) was used in this test.

The patients with disc herniation from whom nucleus pulposus tissue was collected were a 16-year-old female, a 28-year-old female, and a 38-year-old female.
The following exceptions were noted: that she was a 2000-year-old female, that as the first stage of the amplification culture stage, a medium prepared as shown in Table 2 was used for the additional components to the culture medium for the amplification culture stage, and that the culture period was 14 days. The same culture process as in [Test Example 1] (Test Examples 1-1 and 1-2) was performed.

After the first culture step, "Collagenase-P" manufactured by Roche (final concentration 0.025%) was added to the medium to disperse the nucleus pulposus tissue. The cell population separated from the nucleus pulposus tissue was collected and 20%
They were suspended in MEMα supplemented with FBS at a density of 1.0×10 ⁴ /3 mL, then injected into one hole of a 6-well culture dish (the culture surface was untreated), and 10 ng/mL bFGF was added, followed by a further 7 days (
The cells were cultured for a total of 21 days).

After culturing, the cell populations were collected, and the ratio of cells positively expressing Tie2 on the cell surface and the number of cells derived from 1 g of tissue were measured by the same FCM method as in [Test Example 1]. The results are shown in FIGS. 4 and 5.

[Test Example 3] Amplification culture stage (2 stages): 1st and 2nd culture process + additional process → differentiation culture stage:
Third culture step

The patients with disc herniation from whom nucleus pulposus tissue was collected were a 16-year-old female, a 30-year-old male, and a 30-year-old male.
A two-step amplification culture step for a total of 21 days was carried out in the same manner as in Test Example 2, except that the test sample became a female. The cell population after culture is collected, and as a step in the differentiation culture stage, it is grown on a culture dish coated with poly-L-lysine (PLL) (Test 3-1) or on an uncoated culture dish (Test Example 3- In 2), monolayer culture was performed for 14 days.

After culturing, the cell population was collected, and the number of cells positive for intracellular type II collagen (Col2) expression was measured by flow cytometry (FCM), and the ratio of the total cell population to the number of cells ( Col2 positive rate) was calculated. The cell population was treated in advance with a membrane permeabilization reagent "IntraPrep" (Beckman Coulter) so that intracellular Col2 could be fluorescently labeled. As a fluorescent labeling method for Col2, mouse anti-human Col2 antibody (Kyowa Pharma Chemical Co., Ltd. (formerly Daiichi Fine Chemical Co., Ltd.,
Anti-hCL(II) (purified IgG), catalog number F-57) was used as a secondary antibody, a complex of goat anti-mouse IgG antibody and fluorescent dye FITC (BD, Goat Anti-Mouse Ig FITC, catalog number 349031). was used. The results are shown in FIG. Furthermore, the number of Col2-positive cells was calculated assuming that all 1 g of nucleus pulposus tissue-derived cells were amplified and cultured according to Test Example 2, and then differentiated and cultured according to Test Example 3. The results are shown in FIG. When the third culture step was applied (Test 3-1), the number of Col2-positive cells increased approximately three times compared to when it was not applied (Test 3-2).

The number of cells positive for intracellular proteoglycan (PG) expression was also measured by the FCM method, and the ratio of the total cell population to the number of cells (PG positive rate) was calculated. As a fluorescent labeling method for PG, mouse anti-human PG antibody (EMD milliporee, An
ti-Cartilage Proteoglycan Antibody, adult, clone EFG-4, catalog number MAB201
5) as a secondary antibody, a complex of goat anti-mouse IgG antibody and fluorescent dye FITC (BD Company,
, Goat Anti-Mouse Ig FITC, catalog number 349031). As a result, regardless of whether or not the third culture step (PLL coating) was applied, the PG positive rate was close to 100%, and no significant difference was observed (not shown). Unlike proteoglycans, it has been difficult to increase the number of functional nucleus pulposus cells that express type II collagen in the final cell population using conventional methods. This became possible by combining the second culture step and the third culture step, demonstrating its superiority as a culture method.

[Test Example 4] Amplification culture stage (2 stages): 1st and 2nd culture process + additional process → differentiation culture stage:
3rd and 4th culture process

A two-step amplification culture step for a total of 21 days was carried out in the same manner as in Test Example 2 above.
The cell population after culture is collected, and as a step in the differentiation culture stage, the culture medium is prepared in a test tube coated with poly-L-lysine (PLL), with the additional components shown in Table 4 applied to the differentiation culture stage medium. Culture was performed for 14 days using.

After culturing, the cell population was collected, and the PG positive rate was calculated in the same manner as in [Test Example 3]. The results are shown in FIG. When either of the two types of collagenase was added to the medium, the PG positive rate significantly increased compared to when no collagenase was added.

Six samples were further prepared for each of Test Examples 4-1 to 4-3 (the disc herniation patients from whom the intervertebral disc nucleus pulposus tissues were collected were a 32-year-old female, a 28-year-old female, a 20-year-old male, a 16-year-old female, and a 28-year-old female). (a 38-year-old woman and a 38-year-old woman), the Col2 positivity rate was calculated in the same manner as in [Test Example 3]. The results are shown in FIG. Although there were differences between samples (individual differences from which disc nucleus pulposus tissue was collected), in 4 out of 6 samples, either or both of the two types of collagenase were effective in stimulating collagenase when added to the culture medium. Compared to when it was not added,
An improvement in Col2 positivity rate was observed.

[Test Example 5] Amplification culture stage (2 stages): 1st and 2nd culture process + additional process → differentiation culture stage:
3rd and 4th culture process part 2

Regarding the differentiation culture stage, the amplification culture stage and differentiation culture stage were carried out in the same manner as in Test Example 4 above, except that the coating agent for the culture container and the additional component (collagenase P) to the medium were changed as shown in Table 5. The PG positive rate and Col2 positive rate were measured. The results are shown in FIG. For example, when adding "collagenase P" to the medium, depending on the concentration, a coating agent containing Col4 (type IV collagen), FN (fibronectin), or PLL (poly-L-lysine), especially PLL. It was found that using a coating agent containing the following was preferable for improving the Col2 positivity rate.

[Test Example 6] Amplification culture stage (2 stages): 1st and 2nd culture process + additional process → differentiation culture stage:
3rd and 4th culture process part 3

Regarding the differentiation culture stage, the steps of the amplification culture stage and differentiation culture stage were carried out in the same manner as in Test Example 4 above, except that the coating agent for the culture container and the additional component (Liberase) to the medium were changed as shown in Table 6. The PG positive rate and Col2 positive rate were measured. The results are shown in FIG. For example, when adding "Liberase" to the culture medium, it is recommended to use a coating agent containing Col4 (type IV collagen) or PLL (poly-L-lysine) as a coating agent, depending on the concentration, to reduce the Col2 positive rate. It was found to be favorable for improvement.

Claims (4)

Stem cells and/or progenitor cells that are positive for expression of Tie2 (tyrosine kinase with Ig and EGF homology domain-2) and are derived from the nucleus pulposus tissue of an intervertebral disc (hereinafter referred to as "nucleus pulposus-derived Tie2-positive stem/progenitor cells"). ) A culture method for amplifying nucleus pulposus-derived Tie2-positive stem/progenitor cells (hereinafter referred to as "1B culture method") by culturing a cell population containing a cell population containing the nucleus pulposus in a niche of the nucleus pulposus tissue. And,
The culture period of the 1B culture method is 7 to 21 days,
A culture method in which the medium used in the 1B culture method is a mixed medium of αMEM medium and DMEM medium . By using the nucleus pulposus tissue in the 1B culture method as a thawed tissue, Tie2 is maintained in an activated and/or expressed state, or the number of nucleus pulposus-derived Tie2-positive stem/progenitor cells in the cell population is reduced. The culture method according to claim 1, wherein the culture method suppresses. A method for preparing a cell population comprising nucleus pulposus cells expressing at least type II collagen , the method comprising:
Enhancement of Tie2 expression in nucleus pulposus-derived Tie2-positive stem/progenitor cells and amplification of nucleus pulposus-derived Tie2-positive stem/progenitor cells in a cell population, comprising the step of implementing the culture method according to claim 1 or 2. (hereinafter referred to as "amplification culture step B"), and after the amplification culture step B, differentiation of nucleus pulposus-derived Tie2-positive stem/progenitor cells into nucleus pulposus cells expressing at least type II collagen. Culture stage for induction (hereinafter referred to as "differentiation culture stage B")
Preparation methods, including. In the amplification culture stage B, nucleus pulposus is grown in a medium supplemented with at least one selected from the group consisting of FGF ( fibroblast growth factor), EGF (epidermal growth factor), and Ang-1 (angiopoietin-1). 4. The preparation method according to claim 3, further comprising the step of culturing a cell population containing derived Tie2-positive stem/progenitor cells.

Images