Method for culturing cell populations and use thereof

Abstract

The present application provides a method for efficiently producing a cell population enriched in cells having a predetermined property corresponding to the use (e.g., a type II collagen-positive nucleus pulposus cell) from a cell population containing Tie2-positive stem / progenitor cells (e.g., a nucleus pulposus stem / progenitor cell). The culture method of the present application is a culture method in which a cell population containing Tie2-positive stem / progenitor cells is cultured while inhibiting the formation of a spherical colony in (1) a state present in a tissue that has not been subjected to a digestion treatment, (2) a culture medium to which at least one Tie2 expression enhancer other than a growth factor has been added, (3) a culture vessel having a culture surface subjected to a treatment for improving the adhesion of cells, or (4) a culture medium to which an extracellular matrix degrading agent has been added.

Description

Technical Field

[0001] This invention relates to a method for culturing a cell population containing a positive expression of the cell surface marker Tie2 (tyrosine kinase with Ig and EGF homology domain-2, referred to herein as "Tie2-positive stem / progenitor cells"). More specifically, this invention relates to a method for culturing a cell population containing Tie2-positive stem / progenitor cells that can be used in the process of expanding a cell population of Tie2-positive stem / progenitor cells (e.g., nucleus pulposus stem / progenitor cells) and in the process of differentiating and inducing Tie2-positive stem / progenitor cells into cells with specified traits (e.g., nucleus pulposus cells expressing type II collagen). Background Technology

[0002] In Japan, lower back pain ranks second in terms of complaint rate and is a common ailment that two-thirds of adults will experience at least once in their lifetime, making it a contributing factor to workplace injuries and the medical economy. It is said that 20% of lower back pain cases are caused by intervertebral disc injuries, which can lead to serious problems such as disc herniation, osteoarthritis of the spine, spinal stenosis, and spondylolisthesis. These are caused by irreversible changes in the intervertebral disc tissue, a pathological condition known as disc degeneration. The intervertebral disc is a ring-shaped cartilaginous organ composed of the nucleus pulposus (NP) at the center, several rings of fibrous cartilage surrounding it (Annulus Fibrosus (AF), and cartilaginous endplates (EP) connecting to adjacent vertebrae. The gelatinous nucleus pulposus (NP) is an avascular organ containing a large amount of extracellular matrix (ECM) composed of large proteoglycans and collagen, secreted by notochord-derived nucleus pulposus cells within the NP. It has been reported that in some vertebrates, including humans, notochord-derived nucleus pulposus cells disappear early in life. After their disappearance, chondrocyte-like cells of uncertain origin, morphologically similar to chondrocytes, form the nucleus pulposus. It is believed that this transformation of cellular traits affects ECM composition, leading to decreased water content, fibrosis, and other signs of intervertebral disc aging and degeneration, ultimately closely related to low back pain and lumbar degenerative diseases. It should be noted that because mice, rats, rabbits, pigs, and other animal species retain notochord-derived nucleus pulposus cells throughout their lives, and intervertebral disc degeneration is almost never observed, it is believed that the regulatory mechanisms of notochord-derived nucleus pulposus cells and other nucleus pulposus cells differ from those in humans.

[0003] As an example of a method for preventing or treating intervertebral disc degeneration, research and development are underway on allogeneic intervertebral disc cell preparations, i.e., cell preparations containing allogeneic nucleus pulposus cells, ECM, etc., for administration to intervertebral disc tissue. To manufacture such cell preparations, a sufficient quantity of allogeneic nucleus pulposus cells is required. For example, while the nucleus pulposus tissue surgically removed from a patient with a herniated disc can be used as a source of nucleus pulposus cells for such cell preparations, the amount of nucleus pulposus tissue that can be collected in this way, i.e., the number of nucleus pulposus cells contained therein, is limited. Nevertheless, considering the risks of viral infections, it is desirable to avoid the practice of mixing nucleus pulposus cells from multiple patients with herniated discs (donors) to ensure a sufficient number of nucleus pulposus cells. Therefore, it is important to establish a technique that prepares a cell population containing a sufficient number of nucleus pulposus cells for treatment by culturing rare stem cells or progenitor cells contained in a small amount of intervertebral disc tissue (nucleus pulposus, annulus fibrosus, etc.) from a single donor that can differentiate into mature nucleus pulposus cells.

[0004] Patent Document 1 discloses the creation of a "discosphere" composed of stem cells and progenitor cells contained within a cell population by culturing nucleus pulposus cells (a cell population derived from the nucleus pulposus of the intervertebral disc) under conditions that interfere with cell adhesion (preferably in a serum-free medium). Specifically, Patent Document 1 describes a "method for generating a population of stem cells" (claim 3, etc.), the method comprising the steps of: (b) growing nucleus pulposus cells in a culture medium under conditions that interfere with cell adhesion; (c) concentrating intervertebral disc stem cells, intervertebral disc progenitor cells, or a combination thereof; and (d) generating an intervertebral disc sphere containing nucleus pulposus cells, thereby generating a population of intervertebral disc stem cells. It should be noted that, as a description of the aforementioned "intervertebral disc ball," the following explanations are provided: it refers to an in vitro freefloating circular-spherical structure containing intervertebral disc stem cells, intervertebral disc progenitor cells, or combinations thereof; a cell ball obtained by a single intervertebral disc stem cell generating its own clones and progenitor cells; including suspended nucleus pulposus stem cells and nucleus pulposus progenitor cells configured in a circular-spherical structure; or nucleus pulposus cells containing an intervertebral disc ball adhering to each other, etc. (paragraphs

[0024] and

[0039] ).Patent document 1 also describes: an "isolated intervertebral disc stem cell population" obtained by concentrating intervertebral disc stem cells, intervertebral disc progenitor cells, or combinations thereof cultured under "conditions that interfere with cell adhesion" (claim 1, etc.); an "isolated intervertebral disc sphere" comprising intervertebral disc stem cells, intervertebral disc progenitor cells, or mixtures thereof obtained by concentrating nucleus pulposus cells, and serving as an "in vitro suspended spherical structure" (claim 10, etc.); an "artificial intervertebral disc replacement device" comprising an intervertebral disc scaffold and an intervertebral disc sphere containing nucleus pulposus cells obtained by concentrating intervertebral disc stem cells, intervertebral disc progenitor cells, or combinations thereof (claim 11, etc.); and a method for manufacturing an artificial intervertebral disc replacement device, comprising growing an intervertebral disc sphere comprising intervertebral disc stem cells, intervertebral disc progenitor cells, or mixtures thereof obtained by concentrating nucleus pulposus cells within an intervertebral disc scaffold (claim 11, etc.). Claim 12, etc.; A method for "generating a concentrated cell population", comprising the steps of: culturing nucleus pulposus cells seeded at a prescribed low density under "conditions that interfere with cell adhesion", selecting an in vitro "suspension spherical structure" containing intervertebral disc stem cells, intervertebral disc progenitor cells, or a mixture thereof, thereby generating a concentrated cell population (claim 17, etc.); A method for "expanding a population of concentrated intervertebral disc stem cells, intervertebral disc progenitor cells, or combinations thereof", comprising the steps of: dispersing an intervertebral disc sphere containing intervertebral disc stem cells, intervertebral disc progenitor cells, or combinations thereof into one or more dispersed intervertebral disc sphere cells, and culturing the one or more dispersed intervertebral disc sphere cells in a culture medium that "interferes with cell adhesion" and contains prescribed additives (e.g., FGF2, EGF) (claim 18, etc.). It should be noted that "disc" in Patent Document 1 can be considered a direct translation of the original term "disc", referring to "intervertebral disc".

[0005] Regarding the culture container or culture medium disclosed in Patent Document 1 for culturing a heterogeneous cell population (nucleus pulposus-derived cell population) containing intervertebral disc stem cells, intervertebral disc progenitor cells, intervertebral disc cells, etc., derived from intervertebral disc nucleus pulposus tissue, the following can be explained.

[0006] Patent Document 1 discloses an embodiment of culture under "conditions that interfere with cell adhesion": intervertebral disc spheres are formed from a population of nucleus pulposus-derived cells (including stem cells, etc.) as suspended spherical structures by seeding and culturing at low cell density in a serum-free culture medium containing a substance that interferes with cell adhesion (specifically, methylcellulose), or by culturing in ultra-low adhesion plates (see paragraphs

[0156] hereinafter, Example 1: paragraphs

[0170] to

[0181] , corresponding to the inventions of claims 3, 17, etc.). However, Patent Document 1 neither describes nor suggests culturing a population of nucleus pulposus-derived cells (including stem cells, etc.) in a culture medium containing a substance that decomposes the extracellular matrix (e.g., collagenase) or on a cell-adhesive culture surface, thereby causing intervertebral disc stem cells to proliferate or be induced to differentiate without forming intervertebral disc spheres (suspended spherical structures).

[0007] Furthermore, Patent Document 1 discloses a method for amplifying a concentrated cell population containing intervertebral disc stem cells, etc., as follows: First, intervertebral disc spheres (suspended spherical structures) are dispersed into one or more intervertebral disc stem cells by incubation in a culture medium supplemented with collagenase. Then, the dispersed cells are seeded again in a culture medium containing methylcellulose (see paragraph

[0157] , Example 2: paragraphs

[0182] to

[0184] , corresponding to the invention of claim 18, etc.). However, the culture in the culture medium supplemented with collagenase in this embodiment is only a temporary treatment for dispersing temporarily formed intervertebral disc spheres into individual intervertebral disc stem cells, etc., and is not a treatment for inducing differentiation of intervertebral disc stem cells, etc. The dispersed intervertebral disc stem cells, etc., are cultured again under "conditions that interfere with cell adhesion" (culture medium containing methylcellulose, etc.). Patent document 1 neither records nor suggests the following: using intervertebral disc stem cells (before they form) in a state where they have not formed a disc ball to begin culturing them in a culture medium supplemented with collagenase, and then expanding (proliferating) or inducing differentiation of these intervertebral disc stem cells; after dispersing the intervertebral disc stem cells, they continue to be cultured in a culture medium supplemented with collagenase, and expanding (proliferating) and differentiating the intervertebral disc stem cells while maintaining a state where they have not formed a disc ball as a suspended spherical structure.

[0008] It should be noted that although Patent Document 1 describes growing intervertebral disc stem cells in a serum-free culture medium containing "compounds that inhibit cell maturation" (e.g., FGF) or "compounds that maintain cell immaturity" (e.g., TGF-β superfamily members, BMP, IL-6, LIF, etc.) (paragraphs

[0035] to

[0037] ), it neither describes nor suggests culturing intervertebral disc stem cells in a culture medium containing substances that promote the activation of Tie2.

[0009] In addition, in Patent Document 1, the method for preparing nucleus pulposus tissue for obtaining a population of nucleus pulposus-derived cells is only described as follows: fragmenting the nucleus pulposus (human intervertebral disc material obtained through surgery, or biopsy specimen), and treating it with collagenase II, Clostridium collagenase, etc., thereby fragmenting the nucleus pulposus tissue and dispersing each cell (preparing a single cell suspension) (paragraphs

[0026] ,

[0029] ,

[0030] , Example 1: paragraphs

[0171] to

[0174] , etc.).

[0010] On the other hand, Patent Document 2 and Non-Patent Document 1 describe the following: Cells in the intervertebral disc tissue (nucleus pulposus) that are positive for Tie2 and / or GD2 as cell surface markers are stem cells or progenitor cells that should be called nucleus pulposus cells; in particular, cells that are positive for both Tie2 and GD2 (nucleus pulposus stem cells in an active state) have the ability to form spherical colonies and eventually differentiate into mature nucleus pulposus cells through a series of differentiation cascades (in addition, they also have the ability to differentiate into adipocytes, osteocytes, chondrocytes and nerve cells); and by transplanting nucleus pulposus stem / progenitor cells into the intervertebral disc (nucleus pulposus), it is possible to produce extracellular matrix such as type II collagen in the tissue, and there is a possibility of maintaining or reconstructing intervertebral disc tissue and preventing or treating intervertebral disc degenerative diseases; etc.

[0011] As a more specific implementation, Patent Document 2 and Non-Patent Document 1 describe the following: forming spherical colonies (along with adhesive colonies) by suspending and culturing cell populations contained in intervertebral disc tissue (nucleus pulposus) in a methylcellulose medium; such spherical colonies are derived from the aforementioned Tie2-positive (and GD2-positive) cells; type II collagen and proteoglycans are expressed in the spherical colonies (a portion of which are cells); etc. (for example, see the embodiments of Patent Document 2, paragraphs

[0067] ,

[0070] , etc.). However, neither Patent Document 2 nor Non-Patent Document 1 describes or suggests the following: proliferating or inducing differentiation of nucleus pulposus stem / progenitor cells (Tie2 and / or GD2-positive cells) in a medium containing substances that decompose the extracellular matrix (e.g., collagenase) or on a cell-adhesive culture surface (using a medium without added methylcellulose) without forming spherical colonies.

[0012] In addition, in Patent Document 2 and Non-Patent Document 1, the method for preparing cell populations derived from intervertebral disc nucleus pulposus tissue only describes the following general method: after cutting the tissue into pieces with scissors or the like, digesting it with a protein digesting enzyme (TrypLEExpress, collagenase P) (Example: Paragraph

[0048] ).

[0013] It should be noted that Patent Document 2 and Non-Patent Document 1 describe that in order to maintain Tie2-positive nucleus pulposus cells (intervertebral disc nucleus pulposus stem / progenitor cells), a signal transduction mechanism between Tie2 (receptor) and Ang-1 (angiopoietin-1, ligand) is required. Tie2-positive cells can be expanded by culturing in the presence of Ang-1 (co-culturing with AHESS5 that forcibly expresses Ang-1). Therefore, Ang-1 is considered to be a niche factor that controls the differentiation level of nucleus pulposus cells (Examples: paragraphs

[0049] ,

[0069] ,

[0075] , etc.).

[0014] However, Tie2 is also expressed in vascular endothelial cells, and activating Tie2 can also achieve the maturation, normalization or stabilization of blood vessels. It is known to inhibit the disordered expansion of blood vessels (angiogenesis) observed in diseases such as tumors, rheumatoid arthritis, diabetic retinopathy, hyperlipidemia, and hypertension, or prevent or improve wrinkles. As Tie2 activators with this effect, for example, extracts from plants of the genus Cinnamomum (so-called cinnamon powder, Patent Document 3), olive fruit extract (Patent Document 4), and extracts from various plants and animals such as soapberry, wolfberry, ginkgo, oyster, turmeric, chrysanthemum, jujube, wolfberry, chamomile, butcher's broom, hawthorn, star fruit, ginger, lotus, asparagus linergus, tamarind, papaya, guava, long pepper, eleutherococcus senticosus, mango ginger, red ginseng, milkweed, wingless pigweed, prickly ash, mountain willow, double-flowered daylily, wild taro, sage oil, datura, five-finger vine, small red clover, oak, hemp oak, winged chrysanthemum, golden star fruit, plantain, allium macrostemon, bayberry, soapberry thorn, polygonatum, polygonatum odoratum, trichosanthes kirilowii, morinda officinalis, etc. (Patent Documents 5-11) have been proposed. Furthermore, as components that bring about Tie2 activation, for example, ursolic acid, corosolic acid, 3-O-galloyl proanthocyanidin B-1, linolenic acid, 13-hydroxy-9Z,11E,15E-octadecanoic acid, proanthocyanidin B-2, epicatechin-(4β-6)-epicatechin-(4β-8)-epicatechin, proanthocyanidin C-1, astragaloside VIII, soybean saponin I, 3'-O-methylgallocatechin, piperidine nonadiene, eugenol, 2-methoxycinnamonaldehyde, eleutheroside E, eleutheroside E1, sesamin, eudistomins, forsythinol, pinoresinol, liriope resin phenol B dimethyl ether, forsythinol, coumarin, etc. (Patent documents 6, 12-14).

[0015] For example, in Patent Document 3, as an experiment (example) related to "Tie2 activator", Western blotting confirmed that when "Baf3 cells with forced expression of Tie2" or "normal human umbilical vein endothelial cells (HUVEC)" were cultured in a medium supplemented with hot water extract of cinnamon, the phosphorylated Tie2 protein expressed in these cells was higher than that in the control (paragraphs

[0024] to

[0027] ). Figures 1-3 wait).

[0016] However, patent documents 3-14 neither describe nor suggest the use of Tie2 activators in culturing nucleus pulposus-derived cell populations (including Tie2-positive stem / progenitor cells) obtained from intervertebral discs, nor what effects this may bring.

[0017] Existing technical documents

[0018] Patent documents

[0019] Patent Document 1: Japanese Patent No. 5509073 (corresponding to WO2009 / 009020)

[0020] Patent Document 2: Japanese Patent No. 5863639 (corresponding to WO2011 / 122601)

[0021] Patent Document 3: Japanese Patent Application Publication No. 2009-263358 (related to WO2009 / 123211)

[0022] Patent Document 4: WO2016 / 060249

[0023] Patent Document 5: WO2012 / 073627

[0024] Patent Document 6: Japanese Patent Application Publication No. 2012-236795

[0025] Patent Document 7: Japanese Patent Publication No. 2009-154237

[0026] Patent Document 8: Japanese Patent Application Publication No. 2011-201811

[0027] Patent Document 9: Japanese Patent Application Publication No. 2011-102275

[0028] Patent Document 10: Japanese Patent Application Publication No. 2011-102274

[0029] Patent Document 11: Japanese Patent Application Publication No. 2011-102273

[0030] Patent Document 12: Japanese Patent Application Publication No. 2014-97977

[0031] Patent Document 13: Japanese Patent Application Publication No. 2013-241356

[0032] Patent Document 14: Japanese Patent Application Publication No. 2011-102272

[0033] Non-patent literature

[0034] Non-patent literature 1: Sakai D et al., Nat Commun. 2012; 3:1264 Summary of the Invention

[0035] The problem the invention aims to solve

[0036] As mentioned earlier, in order to manufacture allogeneic disc cell preparations for the prevention or treatment of intervertebral disc degeneration diseases, it is necessary to achieve a certain level of functional nucleus pulposus cells that produce extracellular matrix such as type II collagen and proteoglycans. For this purpose, for example, it is necessary to efficiently proliferate and differentiate Tie2-positive cells, which are considered nucleus pulposus cells, contained in the intervertebral disc tissue (nucleus pulposus, etc.) removed from the affected area of ​​a herniated disc patient, to generate a large number of functional nucleus pulposus cells. In particular, to improve the therapeutic effect of cell preparations administered to patients with intervertebral disc degeneration diseases, it is important to differentiate the Tie2-positive cells (nucleus pulposus stem / progenitor cells) contained in the intervertebral disc into functional nucleus pulposus cells that produce a large amount of extracellular matrix such as type II collagen as efficiently as possible during the culture and differentiation induction, rather than simply differentiating them into nucleus pulposus cells. At the same time, as mentioned above, it is also important to efficiently expand the Tie2-positive cells (nucleus pulposus stem / progenitor cells) in the tissue-derived cell population before differentiation induction to increase the final number of functional nucleus pulposus cells. That is, there is a need for a practical means that can efficiently expand and differentiate Tie2-positive cells (nucleus pulposus stem / progenitor cells) by a cell population containing a certain number of Tie2-positive cells (nucleus pulposus stem / progenitor cells), thereby enriching the final prepared and administered cell preparation (cell population) with functional nucleus pulposus cells.

[0037] Furthermore, in typical embodiments of the prior art described in Patent Documents 1 and 2, a cell population containing nucleus pulposus stem / progenitor cells from intervertebral disc tissue is cultured in a medium supplemented with methylcellulose, thereby differentiating into nucleus pulposus cells after forming spherical colonies (disc spheres, spheroids). However, since methylcellulose is a highly viscous substance, it is difficult or labor-intensive to recover the generated cell population (containing useful functional nucleus pulposus cells) from the medium supplemented with this substance without waste, hindering the efficient production and practical application of cell preparations.

[0038] The subject of this invention is to provide a means for efficiently preparing a cell population rich in cells with specified traits appropriate to the intended use (e.g., functional nucleus pulposus cells that produce extracellular matrix such as type II collagen) from a cell population containing positive Tie2-expressing stem cells and / or progenitor cells (e.g., a cell population containing intervertebral disc-derived nucleus pulposus stem / progenitor cells).

[0039] Solution for solving the problem

[0040] The inventors conducted in-depth research on the state and culture conditions of cell populations containing Tie2-positive cells (nucleus pulposus stem / progenitor cells) from intervertebral disc nucleus pulposus tissue, and discovered several characteristic technical aspects that can help solve the aforementioned problems. Among the culture methods possessing these technical characteristics, some are very useful in the culture process where the primary purpose is to expand nucleus pulposus stem / progenitor cells (expansion culture stage), and others are very useful in the culture process where the primary purpose is to induce differentiation of nucleus pulposus stem / progenitor cells into functional nucleus pulposus cells (differentiation culture stage). Furthermore, it was found that by combining these methods and using them sequentially or simultaneously, especially in culture processes implemented simultaneously to "integrate" these culture methods, the synergistic effect of the invention can be achieved.

[0041] That is, in one aspect of the present invention, as a method for culturing a cell population containing Tie2-positive stem / progenitor cells, represented by Tie2-positive cells (nucleus pulposus stem / progenitor cells) contained in intervertebral disc tissue, the following first to fourth culture methods are provided, which can be combined (preferably fused).

[0042] Culture Method 1

[0043] The "first culture method" based on the present invention, which describes a cell population containing Tie2-positive stem / progenitor cells, is a method of culturing a cell population containing Tie2-positive stem / progenitor cells in a state present in undigested tissue.

[0044] Currently, the usual procedure for culturing cell populations from intervertebral disc nucleus pulposus tissue is as follows: the collected nucleus pulposus tissue is minced and then digested with proteases such as collagenase (digestion treatment). The cell population isolated from the nucleus pulposus tissue after this treatment is then recovered and cultured. However, the inventors have discovered that when the nucleus pulposus tissue is minced and then directly suspended in a culture medium without digestion treatment, and cultured for a certain period of time with the cell population remaining in the tissue, the ratio of Tie2-positive stem / progenitor cells (nucleus pulposus stem / progenitor cells) in the cell population is improved compared to the previous method of digestion treatment; the expression of Tie2 in each cell is also enhanced. This effect can be attributed to the fact that by not digesting the nucleus pulposus tissue, the microenvironment (niche) of nucleus pulposus stem / progenitor cells, which is preferred for these cells and contains growth factors such as angiopoietin-1 (Ang-1) and VEGF-A, remains intact (without Ang-1, Tie2-positive cells would eventually apoptosis). Culture of these cells, containing a population of nucleus pulposus stem / progenitor cells maintained in this niche, allows the nucleus pulposus stem / progenitor cells to rapidly proliferate, thus maintaining Tie2 activity (compared to existing methods that isolate cells from their niche, Tie2 expression is enhanced). This results in the aforementioned improvement in positivity and increased expression levels in the resulting cell population.

[0045] Furthermore, since no cell population is separated and recycled from the nucleus pulposus tissue, the valuable Tie2-positive stem / progenitor cells contained within the nucleus pulposus tissue can be utilized without waste. For example, the amount of nucleus pulposus tissue contained in the herniated portion harvested from a herniated disc patient is at most about 1-2g, and the number of Tie2-positive stem / progenitor cells per gram of this nucleus pulposus tissue varies depending on factors such as the patient's age, but is, for example, approximately 50,000. This is comparable to the number of white blood cells in 1cc of umbilical cord blood. 6 Grade 1, 1g of cancerous tissue contains 10 cancer cells. 8 Compared to other methods, Tie2-positive stem / progenitor cells in nucleus pulposus tissue are extremely valuable. Considering the need to prevent the reduction of such Tie2-positive stem / progenitor cells through the separation and recovery of cell populations from nucleus pulposus tissue, and considering that the first culture method is preferred for the expansion and culture of Tie2-positive stem / progenitor cells, it is highly advantageous.

[0046] Second cultivation method

[0047] The "second culture method" based on the present invention for a cell population containing Tie2-positive stem / progenitor cells is a method of culturing a cell population containing Tie2-positive stem / progenitor cells in a culture medium supplemented with at least one Tie2 expression enhancer other than a growth factor.

[0048] It is known that Tie2-positive stem / progenitor cells normally exhibit heightened Tie2 activation (phosphorylation) through binding to angiopoietin-1 (Ang-1), a ligand for Tie2 (receptor tyrosine kinase). The method of culturing Tie2-positive stem / progenitor cells in a medium supplemented with Ang-1 (to enhance Tie2 expression) is well-known in the prior art (existing art literature, etc.). Furthermore, FGF2 (bFGF) is also known to enhance Tie2 expression, and the method of culturing Tie2-positive stem / progenitor cells in a medium supplemented with FGF2 is also well-known.

[0049] However, the inventors have discovered that when Tie2 expression enhancers of different types than growth factors such as Ang-1 and FGF2, such as extracts of cinnamon powder, are used as plant-derived Tie2 expression enhancers to culture previously unreported cell populations containing Tie2-positive stem / progenitor cells derived from nucleus pulposus tissue, especially when Tie2 expression enhancers of plant-derived extracts are used in combination with growth factors such as FGF2, there is a significant effect such as an increase in the ratio of Tie2-positive stem / progenitor cells (nucleus pulposus stem / progenitor cells) in the cell population obtained through culture.

[0050] The inventors have also discovered that by combining (especially fusing) the above-mentioned "first culture method" and "second culture method", a cell population with a high number or ratio of Tie2-positive stem / progenitor cells (nucleus pulposus stem / progenitor cells) can be obtained synergistically and efficiently from a cell population containing Tie2-positive stem / progenitor cells (nucleus pulposus stem / progenitor cells) in the nucleus pulposus tissue.

[0051] Third cultivation method

[0052] On the other hand, the "third culture method" based on the present invention, which involves culturing a cell population containing Tie2 positive stem / progenitor cells in a culture vessel having a culture surface treated to enhance cell adhesion, 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.

[0053] Currently, similar to many other stem / progenitor cells, cell populations derived from intervertebral disc nucleus pulposus tissue containing Tie2-positive cells (nucleus pulposus stem / progenitor cells) are cultured under "conditions that interfere with cell adhesion," i.e., using low-adhesion culture containers or using methylcellulose culture medium to form suspended spherical colonies, as described in Patent Document 1 above. However, the inventors have discovered that for cell populations containing nucleus pulposus stem / progenitor cells, preferably containing nucleus pulposus stem / progenitor cells whose Tie2 expression is enhanced using the first and / or second culture methods described above, it is possible to culture them without "conditions that interfere with cell adhesion," but rather, by using a "culture container with a culture surface that has undergone a treatment to enhance cell adhesion (cell adhesion treatment)," such as a culture container coated with a polylysine-containing coating agent (without using methylcellulose), in a two-dimensional culture environment (without using methylcellulose, etc.), without forming spherical colonies of nucleus pulposus stem / progenitor cells.

[0054] By implementing this third culture method in the differentiation culture stage, compared with the use of culture containers with untreated culture surfaces (or culture containers that have undergone treatment to inhibit cell adhesion (low adhesion treatment)), the efficiency of differentiating nucleus pulposus stem / progenitor cells into functional nucleus pulposus cells (Col2 positive cells, etc.) can be improved, and a cell population with a high number or ratio of Col2 positive cells can be prepared.

[0055] It should be noted that the third culture method can also be integrated with the second culture method described above and implemented in the culture process of the expansion culture stage. That is, if Tie2-positive stem / progenitor cells are cultured in a culture container that has undergone cell adhesion treatment and in a culture medium containing Tie2 activator, Tie2-positive stem / progenitor cells can be efficiently expanded in a two-dimensional culture environment (without using methylcellulose, etc.).

[0056] Fourth cultivation method

[0057] The "fourth culture method" based on the present invention, which involves culturing a cell population containing Tie2-positive stem / progenitor cells in a culture medium supplemented with an extracellular matrix degrading agent while inhibiting the formation of spherical colonies, is a method for culturing a cell population containing Tie2-positive stem / progenitor cells.

[0058] Previously, substances such as collagenase or other proteases that can break down the extracellular matrix (collagen, proteoglycans, etc.), as described in the relation to the first culture method, were typically used to isolate cells from collected tissues; or temporarily used in culture methods such as temporarily dispersing spherical colonies formed by stem / progenitor cells, changing the culture medium, and forming spherical colonies again.

[0059] However, the inventors have discovered that substances such as collagenase, which have the function of decomposing the extracellular matrix (extracellular matrix degrading agents), can be used for completely different purposes. That is, the inventors have discovered that Tie2-positive stem / progenitor cells, such as nucleus pulposus stem / progenitor cells capable of differentiating into nucleus pulposus cells (more preferably, those whose Tie2 expression has been enhanced using the first culture method and / or the second culture method), can be surprisingly cultured in a culture medium containing extracellular matrix degrading agents, even when spherical colonies formed by Tie2-positive stem / progenitor cells binding to each other through the extracellular matrix cannot be formed; and thus, they can be induced to differentiate into cells that express type II collagen, proteoglycans, and other extracellular matrix substances while simultaneously proliferating.

[0060] The inventors have also discovered that by combining the above-mentioned "third culture method" and "fourth culture method", preferably by fusing them based on an appropriate combination of the types and concentrations of extracellular matrix degrading agents in the culture medium and the types of coating agents on the surface of the culture container, the efficiency of differentiating functional nucleus pulposus cells such as type II collagen (Col2) positive cells from nucleus pulposus stem / progenitor cells can be significantly improved synergistically and efficiently, and the number or ratio of Col2 positive cells can be significantly higher than that of existing cell populations.

[0061] Furthermore, the inventors have discovered that by using a third culture method and / or a fourth culture method, a cell population in which Tie2-positive stem / progenitor cells are not completely lost and are retained at a certain level when the number or ratio of Col2-positive cells reaches a certain level. Such a cell population, containing a certain number or ratio of Tie2-positive stem / progenitor cells, has the added advantage of improved therapeutic effects on the intervertebral disc nucleus pulposus during drug administration.

[0062] Based on the above-described culture methods, the inventors have constructed a method for preparing a cell population containing target cells (Col2-positive nucleus pulposus cells, etc.) differentiated from Tie2-positive stem / progenitor cells (nucleus pulposus stem / progenitor cells, etc.) from a cell population containing Tie2-positive stem / progenitor cells (nucleus pulposus stem / progenitor cells, etc.). This cell population preparation method, by simultaneously enhancing the expression of Tie2 in Tie2-positive stem / progenitor cells and promoting their proliferation, includes at least one of a culture phase for expanding the Tie2-positive stem / progenitor cells in the cell population (expansion culture phase) and a culture phase for inducing the differentiation of Tie2-positive stem / progenitor cells into target cells (differentiation culture phase), preferably both. The expansion culture phase includes the steps of implementing at least one of the first and second culture methods described above, preferably both (a fusion of these methods). The differentiation culture phase includes the steps of implementing at least one of the third and fourth culture methods described above, preferably both (a fusion of these methods). The method for preparing a cell population comprising an amplification culture stage (preferably a fusion of the first and second culture methods) and a differentiation culture stage (preferably a fusion of the third and fourth culture methods) is a particularly superior embodiment of the present invention. Compared with existing known preparation methods, it is possible to prepare a cell population with a significantly improved number or ratio of target cells possessing specified functionalities. Existing preparation methods have the following drawbacks: while the overall cell count or the number of nucleus pulposus cells in some cell populations may reach a certain level, the number of functional nucleus pulposus cells, such as those expressing Col2 positively, is not high. Furthermore, while the ratio of Col2 positive cells and the expression level per cell may reach a certain level, the absolute number of Col2 positive cells is insufficient (there is a limit to the number of Col2 positive cells that can be amplified from nucleus pulposus tissue collected from a single donor). Thus, it is difficult to simultaneously achieve both the number and expression level (intensity) of Col2 positive cells in nucleus pulposus-related cell populations. However, it can be said that the preparation method of the present invention is a groundbreaking preparation method that successfully achieves both of these goals.

[0063] From another perspective, in order to utilize the scaffold-independent proliferation ability and the ability to form spherical colonies of (Tie2-positive) stem / progenitor cells, and to avoid the loss of desired functionality due to the culture of cells differentiated from these stem / progenitor cells on the culture surface (scaffold), methods have been employed to simultaneously culture cell populations contained in collected tissues in methylcellulose medium or on low-adhesion culture surfaces, and to differentiate these stem / progenitor cells into target cells with specified functions. It can be said that the inventors have discovered a revolutionary method that, using the aforementioned culture methods (especially the third and fourth methods), significantly improves the proliferation of (Tie2-positive) stem / progenitor cells and the efficiency of their differentiation into target cells with specified functions without using highly viscous methylcellulose that makes cell recovery difficult, and in an environment that allows cell adhesion to the culture surface. Furthermore, the resulting cell populations can be easily and without wastefully recovered from the culture medium.

[0064] In relation to the aforementioned culture and preparation methods, particularly the first culture method and the amplification culture step, the inventors have discovered a preferred preservation method for cell populations containing Tie2-positive stem / progenitor cells. This method involves cryopreserving a cell population containing Tie2-positive stem / progenitor cells while it exists in undigested tissue, thereby maintaining the state of Tie2 activation and / or expression, or inhibiting the reduction of Tie2-positive stem / progenitor cells in the cell population. Currently, for cell populations containing Tie2-positive stem / progenitor cells contained in collected intervertebral disc nucleus pulposus tissue, the process involves isolating the cell population from the tissue using a relatively time-consuming digestion process with collagenases, etc., and then cryopreserving only the cell population. However, by immediately cryopreserving the collected intervertebral disc nucleus pulposus tissue, the ease of operation can be improved, and the cell population can be maintained in a favorable ecological niche in the tissue (especially when collected from young donors). After thawing such cryopreserved tissue, the cell population can be cultured in a culture medium using the first culture method described above, enabling efficient amplification of Tie2-positive stem / progenitor cells.

[0065] If the above-described technical concept is combined with and embodied in the (preferred) embodiments described below, the present invention may be described as an invention that includes at least the following: [1]

[0067] A method for culturing cell populations (hereinafter referred to as "the first culture method"), which is a method for culturing cell populations containing Tie2-positive stem cells and / or progenitor cells (hereinafter referred to as "Tie2-positive stem / progenitor cells") expressing Ig and EGF homologous domain-2 tyrosine kinases.

[0068] The cell population is cultured in a state where the cell population containing the Tie2 positive stem / progenitor cells exists in undigested tissue. [2]

[0070] According to the first culture method described in Project 1, the Tie2-positive stem / progenitor cells are Tie2-positive stem / progenitor cells derived from the nucleus pulposus tissue of the intervertebral disc. [3]

[0072] According to the first culture method described in Project 1 or 2, the undigested tissue is the nucleus pulposus tissue of the intervertebral disc. [4]

[0074] According to the first culture method of any one of items 1 to 3, wherein the undigested tissue is obtained by thawing frozen tissue. [5]

[0076] The first culture method according to any one of items 1 to 4 is carried out when expanding the Tie2 positive stem / progenitor cells in the cell population. [6]

[0078] A method for culturing cell populations (hereinafter referred to as "Second Culture Method"), which is a method for culturing cell populations containing Tie2-positive stem / progenitor cells.

[0079] The cell population is cultured in a medium containing Tie2-positive stem / progenitor cells in which at least one Tie2 expression enhancer other than a growth factor has been added. [7]

[0081] According to the second culture method described in Project 6, the Tie2 expression enhancer other than growth factors is an extract from an animal or plant. [8]

[0083] According to the second cultivation method described in Project 7, the plant is a plant of the genus Cinnamomum. [9]

[0085] The second culture method according to any one of items 6 to 8 is carried out when expanding the Tie2 positive stem / progenitor cells in the cell population.

[10]

[0087] A method for culturing cell populations (hereinafter referred to as "the third culture method"), which is a method for culturing cell populations containing Tie2-positive stem / progenitor cells.

[0088] The cell population is cultured in a culture vessel containing the Tie2 positive stem / progenitor cells in a culture vessel with a culture surface treated to enhance cell adhesion.

[11]

[0090] According to the third culture method described in Project 10, the Tie2-positive stem / progenitor cells were subjected to Tie2 expression enhancement treatment.

[12]

[0092] According to the third culture method described in Project 10 or 11, the treatment to improve cell adhesion is a treatment of applying a coating agent containing an extracellular matrix and / or polyamino acids.

[13]

[0094] The third culture method according to any one of items 10 to 12 is carried out when the Tie2 positive stem / progenitor cells in the cell population are differentiated into target cells.

[14]

[0096] According to the third culture method described in Project 12 or 13, the extracellular matrix and / or polyamino acids are at least one selected from the group consisting of type IV collagen, fibronectin and polylysine.

[15]

[0098] The third culture method according to any one of items 10 to 14 is carried out when expanding the Tie2 positive stem / progenitor cells in the cell population.

[16]

[0100] According to the third culture method of any one of items 12 to 15, wherein the extracellular matrix is ​​gelatin.

[17]

[0102] A method for culturing cell populations (hereinafter referred to as "the fourth culture method"), which is a method for culturing cell populations containing Tie2-positive stem / progenitor cells.

[0103] The cell population is cultured in a culture medium supplemented with an extracellular matrix degrading agent while inhibiting the formation of spherical colonies.

[18]

[0105] According to the fourth culture method described in Project 17, the Tie2-positive stem / progenitor cells were subjected to Tie2 expression enhancement treatment.

[19]

[0107] According to the fourth culture method described in Project 17 or 18, the extracellular matrix degrading agent contains at least a protease with degrading activity against type II collagen.

[20]

[0109] The fourth culture method according to any one of items 17 to 19 is carried out when the Tie2 positive stem / progenitor cells in the cell population are differentiated into target cells. [twenty one]

[0111] A preparation method comprising a cell population containing Tie2-positive stem / progenitor cells, the preparation method comprising: a culture stage (hereinafter referred to as "amplification culture stage") for enhancing Tie2 expression in Tie2-positive stem / progenitor cells and amplifying the Tie2-positive stem / progenitor cells in the cell population by including the steps of the first culture method described in item 5 and / or the second culture method described in item 9. [twenty two]

[0113] According to the preparation method described in Project 21, the step of performing the amplification culture is a step of simultaneously performing the first culture method and the second culture method. [twenty three]

[0115] According to the preparation method described in Project 21 or 22, the amplification culture stage further includes a step of culturing a cell population containing Tie2-positive stem / progenitor cells in a culture medium in which only growth factors with Tie2 expression-enhancing effects are added as Tie2 expression enhancers. [twenty four]

[0117] A method for preparing a cell population comprising target cells differentiated from a cell population containing Tie2-positive stem / progenitor cells.

[0118] The preparation method includes a culture stage (hereinafter referred to as the "differentiation culture stage") for inducing Tie2 positive stem / progenitor cells to differentiate into target cells, comprising the steps of implementing the third culture method described in item 13 or 14 and / or the fourth culture method described in item 20.

[25]

[0120] According to the preparation method described in Project 24, the step performed in the differentiation culture stage is a step in which the third culture method and the fourth culture method are performed simultaneously.

[26]

[0122] According to the preparation method described in item 24 or 25, the target cells are cells that express at least type II collagen.

[27]

[0124] According to the preparation method described in Project 26, the cells expressing at least type II collagen are nucleus pulposus cells.

[28]

[0126] According to any one of items 24 to 27, the preparation method wherein, through the differentiation culture stage, a cell population containing residual Tie2 positive stem / progenitor cells is obtained.

[29]

[0128] A method for preparing a cell population comprising target cells differentiated from a cell population containing Tie2-positive stem / progenitor cells.

[0129] The preparation method includes:

[0130] The amplification culture stage described in any one of items 21 to 23, and

[0131] The differentiation and culture stage as described in any one of items 24 to 28.

[30]

[0133] A cell population obtained by any one of the culture methods described in items 1 to 20.

[31]

[0135] A culture comprising: a culture medium as described in any one of items 1 to 20; and a cultured or obtained cell population supplied for the culture method.

[32]

[0137] A cell population obtained by means of the amplification culture stage and / or differentiation culture stage in any one of items 21 to 29.

[33]

[0139] A culture comprising: a culture medium for the amplification culture stage or a culture medium for the differentiation culture stage in any one of the preparation methods described in items 21 to 29; and various cultured or obtained cell populations supplied for the amplification culture stage or the differentiation culture stage.

[34]

[0141] A cell therapy composition comprising the cell population described in item 30 or 32.

[35]

[0143] The cell therapy composition according to item 34 is used to treat or prevent diseases manifested as damage, degeneration, or protrusion of the intervertebral disc.

[36]

[0145] A preservation method for a cell population containing Tie2-positive stem / progenitor cells.

[0146] The preservation method maintains the activation and / or expression of Tie2 or inhibits the reduction of Tie2-positive stem / progenitor cells in the cell population by cryopreserving a cell population containing the Tie2-positive stem / progenitor cells in a state present in undigested tissue.

[0147] The effects of the invention

[0148] The method for culturing Tie2-positive stem / progenitor cells according to the present invention preferably includes an expansion culture step primarily aimed at expanding Tie2-positive stem / progenitor cells, and a differentiation culture step primarily aimed at inducing mature cells with specified traits through differentiation from Tie2-positive stem / progenitor cells. This method enables the preparation of cell populations rich in target cells. By utilizing cell populations obtained based on such a culture method of the present invention, cell preparations effective for the treatment or prevention of specified diseases can be efficiently manufactured.

[0149] Furthermore, since the present invention does not require the addition of highly viscous components such as methylcellulose used in the prior art as described in Patent Documents 1 and 2, it is possible to recover cell populations containing Tie2-positive stem / progenitor cells or target cells induced by their differentiation from the culture medium without waste.

[0150] According to a representative embodiment of the present invention, using the intervertebral disc (nucleus pulposus) that can only be harvested in small quantities during surgery on patients with herniated discs, and by efficiently proliferating and differentiating the Tie2-positive stem / progenitor cells (nucleus pulposus stem cells, etc.) contained therein, a suitable cell population that is expected to have a high therapeutic effect upon transplantation can be obtained simply, efficiently, reproducibly, and in large quantities. This cell population consists of functional nucleus pulposus cells rich in extracellular matrix production capacity such as type II collagen (and also contains a small number of residual Tie2-positive stem / progenitor cells). Currently, it is impossible or difficult to produce such a suitable cell population, but this invention enables it. Therefore, by administering the above-mentioned cell population (a cell preparation containing the above-mentioned cell population), intervertebral disc regeneration therapy becomes very easy to perform, and industrialization is imminent.

[0151] It should be noted that the reason for the effectiveness of the fourth cultivation method of the present invention, for example, is as follows: Figure 1 The principle shown is at work. However, this prediction is intended to aid in understanding the invention, not to unnecessarily limit it. Even if it is subsequently proven that the principle based on... Figure 1 The different principles and mechanisms of action shown demonstrate that the present invention achieves some or all of its effects, through the following... Figure 1The description also cannot negate the effects of the invention that can be confirmed in reality, or the structural features of the invention for this purpose.

[0152] Figure 1 [A] indicates the state of cultured cells when a cell population containing Tie2-positive stem / progenitor cells (e.g., nucleus pulposus stem / progenitor cells) proliferates and differentiates through two-dimensional culture (monolayer static culture). The culture vessel (flask, etc.) is sometimes pre-treated with a coating agent containing extracellular matrix (ECM) to promote cell adhesion. Stem / progenitor cells adhere to the culture surface of the culture vessel, extending, proliferating, and differentiating on this surface. In such two-dimensional culture, the interaction between the ECM coated on the culture surface or the ECM secreted by the cultured cells and binding proteins (integrins, etc.) expressed on the surface of the cultured cells leads to intracellular signal transduction that ultimately terminates ECM production and secretion. For example, in two-dimensional culture of a nucleus pulposus-derived cell population, mature nucleus pulposus cells originally contained in the cell population, and mature nucleus pulposus cells generated from the proliferation and differentiation of nucleus pulposus stem / progenitor cells within the cell population, vigorously secrete ECM such as type II collagen and proteoglycans. However, as the culture time progresses, the production and secretion of type II collagen eventually cease through intracellular signal transduction as described above (instead, the production and secretion of type I collagen increases), leading to dedifferentiation of mature nucleus pulposus cells, such as those exhibiting a fibroblast-like phenotype. Therefore, it is considered difficult to simultaneously increase the number of cells in a cell population and increase the proportion of cells maintaining specific traits (undifferentiated) in conventional two-dimensional culture. It should be noted that in the third culture method of the present invention, Tie2-positive stem / progenitor cells with enhanced Tie2 expression are preferably used, thereby enabling the relatively easy preparation of a cell population containing a certain proportion of nucleus pulposus cells expressing type II collagen, etc., even in two-dimensional culture.

[0153] Figure 1 [B] and [C] indicate the state of cultured cells during proliferation and differentiation of a cell population containing Tie2-positive stem / progenitor cells (e.g., nucleus pulposus stem / progenitor cells) using a medium containing methylcellulose (without added extracellular matrix (ECM) degrading agents) or a low-adhesion culture vessel. (As described above) Figure 1 The two-dimensional culture of [A] is different, in such Figure 1In the culture shown in [B], no interaction occurs between the ECM on the culture surface of the culture vessel and the cultured cells, nor does the intracellular signal transduction that would terminate ECM production and secretion due to this interaction occur. Therefore, in the early stages of culture, Tie2-positive stem / progenitor cells proliferate while producing and secreting ECM, eventually forming spherical colonies. However, within these spherical colonies, Tie2-positive stem / progenitor cells, or cells differentiated from them (e.g., nucleus pulposus cells), come into contact with each other through the secreted ECM. Therefore, as... Figure 1 As shown in [C], as the culture time progresses, the ECM interacts with the cultured cells, and through this interaction, the ECM generates a termination signal, causing [the cells to react with the ECM]. Figure 1 The same dedifferentiation occurs in [A]. Therefore, in Figure 1 In culture methods as shown in [B] and [C], it is difficult to increase the proportion of cells expressing a specified ECM (e.g., type II collagen) positively in the cell population recovered as spherical colonies to a level above a certain level.

[0154] Figure 1 [D] indicates: According to the fourth culture method of the present invention, the state of Tie2-positive cultured cells during the proliferation and differentiation of a cell population containing Tie2-positive stem / progenitor cells is achieved by using a culture medium (excluding methylcellulose, etc.) supplemented with an extracellular matrix (ECM) degrading agent. In such a culture method, also... Figure 1 [B] Similarly, ECM is secreted from stem / progenitor cells or cells differentiated from them. However, the ECM secreted extracellularly is continuously broken down by the ECM-degrading agent added to the culture medium, thus preventing the formation of spherical colonies, and the cells do not adhere to the culture surface even without using a low-adhesion culture vessel. Furthermore, in the third / fourth culture method of the present invention, even with... Figure 1 In the two-dimensional culture of [A], a coating agent containing ECM was pre-coated onto the culture surface of the culture vessel, resulting in weaker cell adhesion to this surface. Therefore, the ECM termination signaling arising from the interaction between ECM and cultured cells was suppressed and became less likely to occur. Figure 1 [A] Figure 1 The dedifferentiation of cells in [B] and [C] is such that the proportion of cells expressing a specified ECM (e.g., type II collagen) is increased compared to existing technologies.

[0155] It should be noted that extracellular ECM is broken down by ECM-degrading agents in the culture medium, but intracellular ECM accumulates without being broken down. Therefore, after implementing the fourth culture method of the present invention in the differentiation culture stage, the cell population recovered from the culture medium and a cell preparation is manufactured. In tissues treated with this cell preparation, the intracellular ECM accumulated in the cells is rapidly secreted extracellularly, creating an environment suitable for the survival of the cell population. This can be expected to promote the subsequent production and secretion of ECM from the treated cells (i.e., based on the therapeutic effect of the cell preparation). Attached Figure Description

[0156] Figure 1 The diagram illustrates the interaction between ECM generation and degradation and cultured cells in three cultures: conventional two-dimensional culture (monolayer static culture), suspension culture using conventional media without extracellular matrix (ECM) degrading agents, and suspension culture using media containing ECM degrading agents according to the fourth culture method of the present invention. Photographs of the cultured cells are also provided for each. A (Conventional two-dimensional culture): Nucleus pulposus (NP) cells adhere to the culture surface of the culture flask via adhesion molecules, thereby transducing ECM generation termination signals. B (Suspension culture using low-adhesion flasks, methylcellulose medium, etc.): No contact with the culture surface of the flask, and no transduction of ECM generation termination signals (× and dashed arrows). C (No enzymes in the culture medium): Self-generated ECM exhibits the same effect as the culture surface (arrow), and the ECM generation termination signal is transduced into the cells. D (Enzymes in the culture medium): Self-generated extracellular ECM is degraded, and the ECM generation termination signal is not transduced into the cells (× and dashed arrows), but ECM accumulation occurs within the cells.

[0157] Figure 2 This is a graph showing the Tie2 positivity rate in Experiment Example 1 (Amplification Culture Stage: First Culture Step).

[0158] Figure 3 This is a graph showing the results of the Tie2 mean fluorescence intensity (MFI) in Experiment Example 1 (amplification culture stage: first culture step).

[0159] Figure 4 This is a graph showing the Tie2 positivity rate in Experiment Example 2 (amplification culture stage (two stages): first / second culture step + additional step).

[0160] Figure 5 This is a graph showing the number of Tie2-positive cells produced per 1g of nucleus pulposus tissue in Experiment Example 2 (amplification culture stage (two stages): first / second culture step + additional step).

[0161] Figure 6 This is a graph showing the positive rate of type II collagen (Col2) in Experiment Example 3 (amplification culture stage (two stages): first / second culture step + additional step → differentiation culture stage: third culture step).

[0162] Figure 7 This is a graph showing the number of type II collagen (Col2) positive cells produced per 1g of nucleus pulposus tissue in Experiment Example 3 (amplification culture stage (two stages): first / second culture step + additional step → differentiation culture stage: third culture step).

[0163] Figure 8 This is a graph showing the results of proteoglycan (PG) positivity in Experiment Example 4 (amplification culture stage (two stages): first / second culture step + additional step → differentiation culture stage: third / fourth culture step).

[0164] Figure 9 This is a graph showing the positive rate of type II collagen (Col2) in Experiment Example 4 (amplification culture stage (two stages): first / second culture step + additional step → differentiation culture stage: third / fourth culture step).

[0165] Figure 10 This is a graph showing the positive rates of proteoglycans (PG) and type II collagen (Col2) in Experiment Example 5 (amplification culture stage (two stages): culture step 1 / 2 + additional step → differentiation culture stage: culture step 3 / 4, step 2). GEL: gelatin, Col1: type I collagen, Col4: type IV collagen, FN: fibronectin, PLL: poly-L-lysine ( Figure 11 The same applies to Naka.

[0166] Figure 11 This is a graph showing the positive rates of proteoglycan (PG) and type II collagen (Col2) in Experiment Example 6 (amplification culture stage (two stages): first / second culture step + additional step → differentiation culture stage: third / fourth culture step 3).

[0167] Figure 12 These are optical microscope photographs of the cell populations in Experiment 5-12.

[0168] Figure 13 This is a graph showing the Tie2 positivity rate in Experiment Example 7 (differentiation culture stage: 3rd culture step).

[0169] Figure 14 This is a graph showing the total number of Tie2 positive cells in Experiment Example 7 (differentiation culture stage: 3rd culture step).

[0170] Figure 15 This is a graph showing the Col2 positivity rate in Experiment Example 7 (differentiation culture stage: 3rd culture step). Detailed Implementation

[0171] -the term-

[0172] "Stem cell" refers to a cell possessing the ability to self-replicate and differentiate (totipotent, pluripotent, multipotent, or unipotent). "Progenitor cell" refers to a cell that, while eventually becoming terminally differentiated and lacking strict self-replication capacity, possesses the ability to proliferate relatively actively while gradually differentiating into a defined cell type. The (specific) cells commonly understood by those skilled in the art as including the terms "stem cell" or "progenitor cell" are equivalent to "stem cell" or "progenitor cell" in this specification.

[0173] In this specification, "stem cells and / or progenitor cells" is a term that includes stem cells, progenitor cells, or both, and is sometimes referred to as "stem / progenitor cells". In addition, in this specification, a cell population containing stem cells and / or progenitor cells is sometimes referred to as a "stem / progenitor cell population", and a cell population containing cells differentiated and matured from stem cells and / or progenitor cells (terminally differentiated cells) is referred to as a "mature cell population".

[0174] "Stem cells" and "progenitor cells" are typically distinguished from other cells by the positive or negative expression of one or more specific genes (marker genes, cell markers). That is, "stem cells" and "progenitor cells" with the aforementioned self-replication and / or differentiation capabilities are sometimes also defined as cells whose specific marker genes are expressed positively or negatively.

[0175] To determine whether the expression of a biomarker gene (cell biomarker) is "positive" or "negative," the expression level of the mRNA transcribed from that gene (genome) or the protein translated from that mRNA is typically measured quantitatively or qualitatively. A positive result is defined as an expression level above a certain threshold, while a negative result is defined as an expression level below a certain threshold. Protein expression levels can be quantitatively or qualitatively measured using immunological methods such as flow cytometry, immunostaining, or ELISA, which utilize protein-specific antibodies and labeling agents. It should be noted that Tie2 protein is expressed on the cell surface, while Col2 is expressed inside the cell; appropriate methods for detecting proteins present on the cell surface and inside the cell (such as immunofluorescence staining) can be used respectively. mRNA expression levels can be quantitatively or qualitatively measured using methods such as RT-PCR, microarrays, and biochips, which utilize mRNA-specific (complementary) nucleic acids and labeling agents, and nucleic acid amplification methods. The ratio (positive rate or negative rate) of cells expressing a specified marker gene (cell marker) in a cell population can be calculated using various methods as described above, such as flow cytometry, to determine the total number of cells in the cell population and the number of cells identified as positive or negative using the methods described above.

[0176] In this specification, "Tie2-positive stem cells and / or progenitor cells" or "Tie2-positive stem / progenitor cells" refers to cells that, according to flow cytometry, express Tie2 (a tyrosine kinase containing Ig and EGF homologous domain-2), a known cell marker, and possess characteristics of stem cells and / or progenitor cells, in terms of protein expression. Representative Tie2-positive stem / progenitor cells in this invention are Tie2-positive stem / progenitor cells derived from the nucleus pulposus tissue of the intervertebral disc, i.e., Tie2-positive stem / progenitor cells present in the nucleus pulposus of the intervertebral disc (that can be harvested from the nucleus pulposus), or Tie2-positive stem / progenitor cells obtained by passage of such cells; these are equivalent to "nucleus pulposus stem / progenitor cells" as described below.

[0177] In this specification, "target cell" refers to a cell obtained from Tie2-positive stem / progenitor cells through prescribed differentiation induction, possessing functionality corresponding to its intended use. More specifically, it refers to a cell, for example, that, in terms of protein expression, is determined to be positive or negative by flow cytometry, based on the expression of specified genes (cell markers). A representative target cell in this invention is a cell described below as a "nucleus pulposus cell" that expresses extracellular matrix (ECM) genes such as Col2 and agglutinin.

[0178] In this invention, "nucleus pulposus cells" refers to mature, terminally differentiated cells that constitute the majority of the cell population in the intervertebral disc (nucleus pulposus), or cultured cells with equivalent characteristics. Specifically, nucleus pulposus cells can be defined as cells that are negative for the marker genes Tie2 and GD2 (and, typically, positive for CD24), and positive for at least type II collagen in the extracellular matrix (and, typically, proteoglycans). For example, cells that are negative for the proteins (cell markers) Tie2 and GD2 (and positive for CD24), and positive for type II collagen (and also positive for proteoglycans) are identified by flow cytometry as equivalent to nucleus pulposus cells in this invention. It should be noted that the production levels of these proteins, such as type II collagen and proteoglycans, in the extracellular matrix can be measured using flow cytometry, and their mRNA expression levels can be measured using real-time PCR or similar methods.

[0179] In this invention, "nucleus pulposus stem / progenitor cells" refers to progenitor cells (nucleus pulposus progenitor cells) that occupy a portion of the cell population in the nucleus pulposus tissue of the intervertebral disc and possess the differentiation capacity to differentiate into nucleus pulposus cells, and stem cells (nucleus pulposus stem cells) that possess the differentiation capacity to differentiate into such progenitor cells and the ability to self-replicate, or cultured cells with equivalent characteristics. Specifically, nucleus pulposus stem / progenitor cells can be defined as cells that are positive for Tie2 and / or GD2 as marker genes. For example, cells that, as determined by flow cytometry, are positive for Tie2 as a protein (cell marker) and negative for GD2, positive for Tie2 and positive for GD2, or negative for Tie2 and positive for GD2, are equivalent to nucleus pulposus stem / progenitor cells in this invention.

[0180] It should be noted that in the aforementioned patent document 2, based on the expression status of Tie2 and GD2, which are cell markers for nucleus pulposus-derived cells, cells that are positive for Tie2 are classified as "intervertebral disc nucleus pulposus stem cells" (here, cells that are negative for GD2 are in a dormant state, and cells that are positive for GD2 are in an active state), cells that are negative for Tie2 and positive for GD2 are classified as "intervertebral disc progenitor cells", and cells that are negative for Tie2 and negative for GD2 are classified as "mature intervertebral disc nucleus pulposus cells that have completed differentiation" (paragraphs

[0024] ,

[0025] , and

[0032] ). Furthermore, in Patent Document 2, cells appearing in the differentiation levels of nucleus pulposus cells are classified as: (i) Tie2-positive and GD2-negative (and consequently CD24-negative, CD44-positive / negative, CD271-positive, Flt1-positive) cells; (ii) Tie2-positive and GD2-positive (and consequently CD24-negative, CD44-positive, CD271-positive, Flt1-positive) cells; (iii) Tie2-negative and GD2-positive (and consequently CD24-negative, CD44-positive, CD271-positive / negative, Flt1-positive / negative) cells; (iv) Tie2-negative and GD2-positive (and consequently CD24-positive, CD44-positive, CD271-negative, Flt1-negative) cells; and (v) Tie2-negative and GD2-negative (and consequently CD24-positive, CD44-positive, CD271-negative, Flt1-negative) cells. For the above (i) to (iii), "intervertebral disc nucleus pulposus stem / progenitor cells" (NP stem / progenitor) were used. The expression "nucleus pulposus committed cells" was used for (iii) to (v) above (see reference). Figure 7-2 Although the wording differs, the "intervertebral disc nucleus pulposus stem cells" and "intervertebral disc nucleus pulposus progenitor cells" in Patent Document 2, i.e., the cells mentioned in (i) to (iv) above, are equivalent to the "nucleus pulposus stem / progenitor cells" in this invention. The "mature intervertebral disc nucleus pulposus cells that have completed differentiation" in Patent Document 2, i.e., the cells mentioned in (v) above, are equivalent to the "mature nucleus pulposus cells" in this invention. As needed, the cells in this invention can be replaced with cells defined according to the definitions described in Patent Document 2 (in particular, the definition of whether one or more cell markers other than Tie2 and GD2, such as CD24, are positive or negative).

[0181] In this invention, a "spherical colony" is a spherical cell aggregate containing stem cells and / or progenitor cells, and may further contain cells differentiated from those cells. A "spherical colony" is also commonly referred to by those skilled in the art as a "sphere," "sphere-like body," etc., and the "discosphere" or "freefloating circular-spherical structure" in the aforementioned Patent Document 2 are also equivalent to a "spherical colony."

[0182] In this invention, "enhanced Tie2 expression" refers to the enhanced expression of the Tie2 gene in each stem / progenitor cell, meaning that the expression is more hyperactive than usual, with an increased level of expression as mRNA or protein. Even under conditions where Tie2 gene expression is almost absent, a certain level of expression is maintained, meaning Tie2 expression is preserved; this is also equivalent to "enhanced Tie2 expression." Furthermore, the increased expression of Tie2 in each stem / progenitor cell, resulting in an increase in the number of cells in the cell population that are positive for Tie2 mRNA or protein expression—that is, a higher proportion of Tie2-positive cells in the cell population than usual—can also be understood as "enhanced Tie2 expression."

[0183] More specifically, for example, in a cell population that has undergone pre-treatment to enhance Tie2 expression (Tie2 expression enhancement group) and a cell population that has not undergone this treatment (control group), the cell surface Tie2 protein is fluorescently labeled and measured using flow cytometry. If, compared to the control group, the fluorescence intensity of the Tie2 expression enhancement group is higher than the prescribed level, resulting in a higher percentage of cells judged to be positive for Tie2 expression, and / or a higher average fluorescence intensity per cell, then it can be said that Tie2 expression in the cell population (containing Tie2-expressing cells) is enhanced (in other words, the Tie2 expression enhancement treatment has played its prescribed role). Furthermore, in morphological observation, cells with enhanced Tie2 expression can be distinguished by their spindle-shaped shape (otherwise, the cells are nearly spherical).

[0184] In this invention, the agent that exerts the aforementioned "Tie2 expression enhancement" effect is referred to as a "Tie2 expression enhancer". It should be noted that some growth factors (such as FGF2) have a Tie2 expression enhancement effect, which can be considered equivalent to a "Tie2 expression enhancer". Therefore, those other than such growth factors are referred to as "Tie2 expression enhancers other than growth factors".

[0185] In this invention, implementing the first culture method and / or the second culture method is equivalent to performing "Tie2 expression enhancement treatment" on a cell population containing Tie2-positive stem / progenitor cells.

[0186] -Cultivation Methods-

[0187] The first to fourth culture methods based on the cell population containing Tie2-positive stem / progenitor cells of the present invention are as follows:

[0188] Culture Method 1: A method for culturing cell populations containing Tie2-positive stem / progenitor cells in the presence of undigested tissue;

[0189] The 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 a growth factor;

[0190] The third culture method: a method of culturing cell populations containing Tie2-positive stem / progenitor cells in culture containers with culture surfaces treated to enhance cell adhesion;

[0191] Fourth culture method: A method of culturing cell populations containing Tie2-positive stem / progenitor cells in a culture medium supplemented with extracellular matrix degrading agents while inhibiting the formation of spherical colonies.

[0192] The first to fourth cultivation methods of this invention can be implemented individually or in combination, either sequentially or simultaneously. Simultaneous implementation of multiple cultivation methods selected from the first to fourth cultivation methods means fusing the selected cultivation methods, i.e., implementing a cultivation method that satisfies all the technical requirements of the selected methods. For example, the first and second cultivation methods can be combined and implemented sequentially or simultaneously (integrated) (sometimes these integrated methods are referred to as "the first / second cultivation method"). The third and fourth cultivation methods can also be combined and implemented sequentially or simultaneously (integrated) (sometimes the integrated method is referred to as "the third / fourth cultivation method").

[0193] The purpose of implementing the first to fourth culture methods of the present invention is not particularly limited. The first to fourth culture methods can also be implemented at any stage of the amplification culture stage (or equivalent stage), the differentiation culture stage (or equivalent stage), or other stages of the present invention.

[0194] In this specification, the descriptions of the first to fourth culture methods (and the first to fourth culture steps for performing these methods) can be appropriately replaced, unless otherwise stated, not only as descriptions of their implementation as separate methods (steps), but also as descriptions of their implementation as methods (steps) combined with other methods (steps).

[0195] The cell population to which the fourth culture method of the present invention is applicable includes Tie2-positive stem / progenitor cells and cells differentiated from such stem / progenitor cells, as long as the cells exert the following effect: in a conventional culture medium without an extracellular matrix degrading agent, cells that would bind together to form spherical colonies (spheroids) due to the secretion of extracellular matrix are present, 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.

[0196] In a representative embodiment of the present invention, the cells differentiated from Tie2-positive stem / progenitor cells are cells that produce and secrete more extracellular matrix than ordinary cells, for example, nucleus pulposus cells that have the function of producing and secreting extracellular matrix in the intervertebral disc nucleus pulposus tissue. Mature nucleus pulposus cells express at least type II collagen as an extracellular matrix, and also express extracellular matrix components such as proteoglycans. In a preferred embodiment of the present invention, cells differentiated from Tie2-positive stem / progenitor cells that express extracellular matrix components such as type II collagen and proteoglycans are particularly functional nucleus pulposus cells that exhibit excellent expression levels (production levels) of both type II collagen mRNA and type II collagen protein.

[0197] -Preparation Method (Cultivation Process)-

[0198] The method for preparing a cell population containing target cells differentiated from Tie2-positive stem / progenitor cells based on the present invention includes an expansion culture stage and / or a differentiation culture stage, preferably (in the order of expansion culture stage first, differentiation culture stage later) both expansion culture stage and differentiation culture stage:

[0199] Expansion culture stage: This stage is used to enhance the expression of Tie2 in Tie2-positive stem / progenitor cells and to expand the Tie2-positive stem / progenitor cell population.

[0200] Differentiation culture stage: The culture stage used to induce Tie2-positive stem / progenitor cells to differentiate into target cells.

[0201] • Procedures for the amplification and culture stage

[0202] In a preferred embodiment of the present invention, the first culture method and the second culture method are performed in the amplification culture stage. Either the first culture method or the second culture method may be performed, or both may be performed. When both the first and second culture methods are performed, in the amplification culture stage, the step of performing the first culture method (referred to as the "first culture step" in this specification) and the step of performing the second culture method (referred to as the "second culture step" in this specification) may be set as separate steps performed sequentially (the first culture step first, the second culture step later), or it may be set as a single step (referred to as the "first / second culture step" in this specification) where both culture methods are performed simultaneously (implementing the first / second culture method incorporating these). That is, it may be a step of culturing a cell population containing Tie2-positive stem / progenitor cells in a medium supplemented with a Tie2 expression enhancer, in a state existing in undigested tissue.

[0203] The "expansion culture stage" refers to the process of culturing under specified conditions with the primary purpose of expanding Tie2-positive stem / progenitor cells (to a stronger degree than other cells with the same effect) to achieve this purpose. In other words, if the number and / or ratio of Tie2-positive stem / progenitor cells in the cultured cell population is increased compared to the pre-culture cell population, this culture stage can be called the "expansion culture stage," within which the differentiation of Tie2-positive stem / progenitor cells into other cells (target cells) is permitted.

[0204] In this invention, since the first / second culture method (i.e., the first / second culture step) is particularly preferred as a step in the amplification culture stage because it can synergistically and efficiently enhance the expression of Tie2 in each Tie2-positive stem / progenitor cell (e.g., nucleus pulposus stem / progenitor cell) (including maintaining the state of expressing Tie2) and improve the number and / or ratio of Tie2-positive stem / progenitor cells in the cell population.

[0205] • Procedures related to the differentiation and culture stage

[0206] In a preferred embodiment of the present invention, the third and fourth culture methods are performed during the differentiation culture stage. Either the third or fourth culture method may be performed, or both may be performed. When performing both the third and fourth culture methods, during the differentiation culture stage, the step of performing the third culture method (referred to as the "third culture step" in this specification) and the step of performing the fourth culture step (referred to as the "fourth culture step" in this specification) may be performed sequentially as separate steps, or as a single step (referred to as the "third / fourth culture step" in this specification) simultaneously performing both culture methods (performing a third / fourth culture method incorporating these). That is, it may be a step of culturing a cell population containing Tie2-positive stem / progenitor cells in a culture vessel with a culture surface treated to improve cell adhesion, and in a culture medium supplemented with an extracellular matrix degrading agent, while inhibiting the formation of spherical colonies.

[0207] "The differentiation culture stage" refers to the process of culturing under specified conditions to primarily differentiate Tie2-positive stem / progenitor cells into specified cell types, and to exert this effect (more strongly than other effects). In other words, if the number and / or ratio of target cells in the cultured cell population is higher than the pre-culture cell population, then this culture stage can be called the "differentiation culture stage."

[0208] It should be noted that, as mentioned above, the step described in Patent Document 1, which involves temporarily treating the spherical colonies (spherical bodies, intervertebral disc spheres, suspended spherical structures) in a culture medium supplemented with collagenase, is not equivalent to the fourth culture method of the present invention as described above, or the fourth culture step as a differentiation culture stage. Furthermore, the method (step) of treating the collected tissue with collagenase or the like to separate the cell population, and the method (step) of treating cells with trypsin to detach them from the culture surface for passage culture of cells proliferating in conventional two-dimensional culture, are also not equivalent to the fourth culture method of the present invention as described above, or the fourth culture step as a differentiation culture stage.

[0209] In this invention, the number and / or ratio of cells with specified functionalities (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 are synergistically and efficiently enhanced, while the number and / or ratio of Tie2-positive stem / progenitor cells are also maintained at a certain level. Therefore, it is particularly preferred to implement the third / fourth culture method (i.e., to implement the third / fourth culture step) as a step in the differentiation culture stage.

[0210] The expansion culture stage may, as needed, include steps other than the first and / or second culture stages that are necessary to achieve the purpose of expanding Tie2-positive stem / progenitor cells. Examples of such steps include, for instance, culturing a cell population containing Tie2-positive stem / progenitor cells in a medium supplemented only with a growth factor that enhances Tie2 expression (referred to in this specification as the "additional expansion culture stage"). Examples of growth factors that enhance Tie2 expression in the additional expansion culture stage include, for example, FGF and / or EGF. The additional expansion culture stage is preferably performed after the first and / or second culture stages, particularly after the first culture stage or both. Furthermore, in the additional expansion culture stage, it is suitable that the first culture method is not performed, i.e., the cell population containing Tie2-positive stem / progenitor cells is in a state of separation from cells through digestion rather than in a state present in undigested tissue. In the first culture method of the present invention implemented in the first culture step or the first / second culture step, a cell population containing Tie2-positive stem / progenitor cells is cultured in the presence of undigested tissue. However, once a certain level of culture is reached, the expansion of Tie2-positive stem / progenitor cells is inhibited (even with extended culture time, Tie2-positive stem / progenitor cells no longer expand), which can be attributed to the influence of the presence of tissue. Therefore, after the first culture step or the first / second culture step, the tissue is digested, the isolated cell population is recovered, and an additional expansion step is performed, thereby enabling further expansion of Tie2-positive stem / progenitor cells.

[0211] <Cell population>

[0212] In the cell populations containing Tie2-positive stem / progenitor cells (collectively referred to as "pre-culture cell populations" in this specification) provided for each culture method or culture step of the present invention, the ratio and / or number of Tie2-positive stem / progenitor cells to other cells (such as cells differentiated from Tie2-positive stem / progenitor cells) are substantially arbitrary. Furthermore, the ratio of Tie2-positive stem cells to Tie2-positive progenitor cells is also substantially arbitrary. The composition of the pre-culture cell population can be appropriately adjusted according to the embodiments of the invention and taking into account the effects of each culture method or culture step.

[0213] For pre-culture cell populations, except when used in the first culture method or the first culture step, they can be prepared or processed according to conventional methods. For example, when using cell populations contained in intervertebral disc nucleus pulposus tissue collected in vivo as pre-culture cell populations, the nucleus pulposus tissue is first cut into suitable sizes (e.g., into small pieces of about several square millimeters) using scissors or similar tools. Then, it is treated with proteolytic enzymes such as collagenase to disperse the cells. Filtration, centrifugation, and washing are then performed as needed to separate and recover the cell populations contained in the nucleus pulposus tissue. The cell populations thus obtained can be used as pre-culture cell populations other than those used in the first culture method or the first culture step.

[0214] On the other hand, in the first culture method or the first culture step of the present invention, the stage of cutting up the nucleus pulposus tissue (without proteolytic enzyme treatment) in the steps described above is used as the pre-culture cell population containing the cut nucleus pulposus tissue.

[0215] The cell populations isolated from tissues, or cell populations contained within tissues (tissues containing cell populations), prepared as described above, can be cryopreserved using conventional methods until they are ready for subsequent culture methods or processes. For cryopreserved cell populations or tissues, thawing can be performed using conventional methods before initiating subsequent culture methods or processes. During cryopreservation and thawing, preferred treatments for the cell populations or tissues can be combined. For example, a cryoprotectant (such as DMSO) can be added during cryopreservation; in this case, the cryoprotectant can be removed under suitable conditions during thawing.

[0216] In the cell populations containing Tie2-positive stem / progenitor cells obtained by the various culture methods or culture processes of the present invention (collectively referred to in this specification as "cultured cell populations"), the ratio and / or number of Tie2-positive stem / progenitor cells to other cells (such as cells differentiated from Tie2-positive stem / progenitor cells) are substantially arbitrary. Furthermore, the ratio of Tie2-positive stem cells to Tie2-positive progenitor cells is also substantially arbitrary. The composition of the cultured cell populations can be appropriately adjusted according to the embodiments of the invention and considering the intended use of the cell populations obtained by the various culture methods or culture processes.

[0217] The cultured cell population can be recovered from the culture medium using conventional methods and used in subsequent culture methods or processes, or in other methods or processes such as the preparation of cell preparations.

[0218] In the cell population containing Tie2-positive stem / progenitor cells (collectively referred to as the "cultured cell population" in this specification) during the various culture methods or culture steps of the present invention, the ratio of Tie2-positive stem / progenitor cells to other cells (such as cells differentiated from Tie2-positive stem / progenitor cells) is substantially arbitrary. Furthermore, the ratio of Tie2-positive stem cells to Tie2-positive progenitor cells is also substantially arbitrary. The composition of the cultured cell population is the composition during the process of transforming the pre-cultured cell population into the post-cultured cell population. For example, the ratio of Tie2-positive stem / progenitor cells in the cultured cell population (referred to as the "Tie2-positive stem / progenitor cell rate" in this specification) is generally a value within the range between the Tie2-positive stem / progenitor cell rate of the pre-cultured cell population and the Tie2-positive stem / progenitor cell rate of the post-cultured cell population, and temporary deviations from this range are permissible. The composition of the cultured cell population varies depending on the embodiments of the invention and also on the number of days, passages, etc., in each culture method or culture step.

[0219] Regarding the "human or other animal" (donor) from which the aforementioned cell populations originate, the choice can be made based on the intended use of the cell population ultimately obtained through the Tie2-positive stem / progenitor cell culture method of the present invention, or the intended use of the cell population obtained through the various culture methods or culture steps included in that method. In a typical embodiment of the present invention, when preparing cell populations for the manufacture of cell preparations for the prevention or treatment of specified diseases, symptoms, etc., the "human or other animal" is an organism of the same species as the recipient of the cell preparation, preferably a human.

[0220] • Cell population during the expansion culture phase

[0221] In this invention, the cell population supplied for the first culture method and / or the second culture method, or the cell population supplied for the first culture step and / or the second culture step in the expansion culture stage (collectively referred to in this specification as "pre-expansion culture cell population"), is typically a cell population contained in tissue (intervertebral disc) collected from the body of a human or other animal (primary culture cell population) or a cell population obtained by passage of the primary culture cell population (passaged culture cell population).

[0222] When using cell populations from intervertebral discs collected from humans as the pre-expansion culture cell population, it is preferable to use cell populations from intervertebral discs collected from individuals in their teens or twenties who typically have a high percentage of Tie2-positive stem / progenitor cells and are in a favorable niche. Furthermore, the pre-expansion culture cell population is preferably one with the highest possible percentage of Tie2-positive stem / progenitor cells, for example, 30% or more, 40% or more, 50% or more, or 60% or more.

[0223] It should be noted that, according to the implementation method, the cell population before expansion culture may not be a cell population contained in tissues collected from humans or other animals. For example, it may be a cell population containing Tie2 positive stem / progenitor cells obtained by inducing differentiation of cells with totipotency or pluripotency, such as iPS cells or ES cells prepared using cells from humans or other animals.

[0224] In this invention, the cell populations obtained by the first culture method and / or the second culture method, or the cell populations obtained by the first culture step and / or the second culture step in the amplification culture stage (collectively referred to as "amplified culture cell populations" in this specification) are not particularly limited in their use, and the composition of the obtained cell populations can be appropriately adjusted according to their intended use.

[0225] In a typical embodiment of the present invention, the expanded cultured cell population can be used as a cell population for the third and / or fourth culture methods, or as a cell population for the third and / or fourth culture steps in the differentiation culture stage. In such embodiments (uses), the expanded cultured cell population preferably has a high ratio and / or number of Tie2-positive stem / progenitor cells. The ratio of Tie2-positive stem / progenitor cells in the expanded cultured cell population varies depending on the pre-expansion cultured cell population, individual differences in the nucleus pulposus tissue from which they originate, etc., and therefore cannot be generalized; for example, it is 5% or more, preferably 7% or more, 9% or more, 11% or more, 13% or more, or 15% or more. The number of Tie2-positive stem / progenitor cells in the expanded cultured cell population varies depending on the pre-expansion cultured cell population, individual differences in the nucleus pulposus tissue from which they originate, etc., and therefore cannot be generalized; compared to the number of cells in the pre-expansion cultured cell population, it is, for example, 5 times or more, preferably 10 times or more, 15 times or more, 20 times or more, 25 times or more, or 30 times or more.

[0226] • Cell populations during the differentiation culture stage

[0227] In this invention, the cell population supplied for the third and / or fourth culture methods, or the cell population supplied for the third and / or fourth culture steps in the differentiation culture stage (referred to in this specification as the "pre-differentiation culture cell population"), is preferably a cell population pre-enriched with Tie2-positive stem / progenitor cells. The percentage of Tie2-positive stem / progenitor cells in the pre-differentiation culture cell population varies depending on the cell population before or after expansion culture, individual differences in the nucleus pulposus tissue from which it originates, etc., and therefore cannot be generalized. For example, it is 5% or more, preferably 7% or more, 9% or more, 11% or more, 13% or more, or 15% or more.

[0228] In a typical embodiment of the present invention, the pre-differentiation culture cell population is a cell population obtained through the expansion culture stage of the present invention (post-expansion culture cell population), for example, an expanded cell population containing Tie2-positive stem / progenitor cells, separated in a manner to achieve an appropriate cell number, according to the implementation method of the differentiation culture stage (type and size of culture container, etc.). The cell population obtained through the expansion culture stage of the present invention contains Tie2-positive stem / progenitor cells in the aforementioned ratio and / or cell number, and the expression of Tie2 in these Tie2-positive stem / progenitor cells is enhanced (Tie2 expression is maintained). Therefore, from the viewpoint of enhancing the effect of the differentiation culture stage, it is also preferred as the pre-differentiation culture cell population.

[0229] It should be noted that, according to the embodiments, the predifferentiation culture cell population may not be obtained through the expansion culture stage (first culture step and / or second culture step) of the present invention. For example, it may be a cell population contained in tissues collected from humans or other animals, or a cell population containing target cells obtained by inducing differentiation of totipotent or pluripotent cells such as iPS cells or ES cells made from human or other animal cells (through Tie2 positive stem / progenitor cells).

[0230] In this invention, the cell populations obtained by the third and / or fourth culture methods, or by the third and / or fourth culture steps in the differentiation culture stage (collectively referred to as "differentiated culture cell populations" in this specification), are not particularly limited in their use, and the composition of the obtained cell populations can be appropriately adjusted according to their intended use. For example, for cell populations used to manufacture cell preparations for transplantation, it is preferable to include, as much as possible, target cells that have useful functionalities in exerting therapeutic or preventative effects through transplantation (e.g., type II collagen-producing nucleus pulposus cells: Col2-positive nucleus pulposus cells), and also some Tie2-positive stem / progenitor cells (e.g., nucleus pulposus stem / progenitor cells) that retain the ability to produce such target cells.

[0231] The percentage of Col2-positive (nucleus pulposus) cells in the cell population after differentiation culture varies depending on the cell population before differentiation culture and the individual differences in the nucleus pulposus tissue from which they originated, so it cannot be generalized. For example, it may be 5% or more, preferably 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more.

[0232] The ratio of Tie2-positive (nucleus pulposus) stem / progenitor cells in the cell population after differentiation culture varies depending on the cell population before differentiation culture and the individual differences in the nucleus pulposus tissue from which they originate, so it cannot be generalized. For example, it is 1% or more, preferably 2% or more, 4% or more, 6% or more, 8% or more, or 10% or more.

[0233] It should be noted that the number of cells in the cell population usually increases during the differentiation culture process. The number of cells in the cell population after differentiation culture (Col2-positive cells, Tie2-positive stem / progenitor cells, etc.) varies depending on the cell population before differentiation culture and the individual differences in the nucleus pulposus tissue from which they originate. Therefore, it cannot be generalized that the number of cells in the cell population before differentiation culture is more than 2 times, 5 times, 10 times, 20 times, 50 times, or 100 times that of the cell population before differentiation culture.

[0234] <Culture medium>

[0235] The culture medium used in each culture method or culture step of the present invention can be any medium suitable for the culture of Tie2 positive stem / progenitor cells and cells differentiated from them. Suitable basal culture media and additives can also be selected while considering the purpose of the culture method or culture step. Regarding additives, if the culture method is for expanding Tie2 positive stem / progenitor cells, i.e., the culture step is an expansion culture stage, then additives suitable for expanding Tie2 positive stem / progenitor cells can be selected. If the culture method is for inducing differentiation of Tie2 positive stem / progenitor cells, i.e., the culture step is a differentiation culture stage, then additives suitable for inducing the differentiation of Tie2 positive stem / progenitor cells into target cells can be selected.

[0236] It should be noted that in the third and fourth culture methods of the present invention, as well as in the third and fourth culture steps that include the steps of implementing these methods, it is not necessary to add components such as methylcellulose to the culture medium to prevent Tie2-positive stem / progenitor cells and cells differentiated from these cells from adhering to the culture surface of the culture container. That is, the culture medium in the third and fourth culture methods of the present invention, as well as in the third and fourth culture steps that include the steps of implementing these methods, generally does not contain components such as methylcellulose used to prevent cells from adhering to the culture surface of the culture container.

[0237] In a representative embodiment of the present invention, when culturing nucleus pulposus stem / progenitor cells and differentiated nucleus pulposus cells, the culture media used in each step of the expansion culture stage and the differentiation culture stage can be prepared by using appropriate amounts of the following basal culture medium, additives, growth factors, and other components.

[0238] Examples of basal media include DMEM (Dalberg modified Eagle medium with or without glucose), αMEM (eager basal medium α-modified), Ham's F-10 medium, Ham's F-12 medium, or mixtures thereof.

[0239] Examples of additives used for amplification or differentiation cultures include FBS (fetal bovine serum albumin), BSA (bovine serum albumin), L-ascorbic acid (as L-ascorbic acid magnesium phosphate, etc.), selenite (as insulin-transferrin-selenite (ITS), etc.), and 2-mercaptoethanol. Depending on the needs, antibiotics such as penicillin and streptomycin, as well as other components, can also be added to the culture medium. It should be noted that L-ascorbic acid may not be present in the culture medium used for amplification cultures.

[0240] Examples of growth factors include FGF (fibroblast growth factor), EGF (epidermal growth factor), and Ang-1 (angiogenin-1). In one embodiment of the present invention, the growth factors added to the culture medium preferably include at least FGF, more preferably both FGF and EGF, and preferably Ang-1 is added as needed.

[0241] As an FGF, for example, bFGF (basic fibroblast growth factor, sometimes also called FGF-2) can be used. The concentration of FGF in the culture medium can typically be in the range of 1–50 ng / mL, preferably in the range of 5–15 ng / mL, for example, about 10 ng / mL.

[0242] Ang-1 is preferably added to serum-free culture medium. Furthermore, water-soluble Ang-1 is preferred (soluble Ang-1, recombinant Ang-1). The concentration of Ang-1 (preferably soluble Ang-1) in the culture medium is typically in the range of 100–1000 ng / mL, for example, about 500 ng / mL.

[0243] It should be noted that the aforementioned growth factors such as FGF, EGF, and Ang-1 are "growth factors with Tie2 expression enhancement effects," which can also be broadly understood as equivalent to "Tie2 expression enhancers." However, the processing methods of these growth factors in this invention are described separately in this specification.

[0244] <Tie2 expression enhancer>

[0245] In the second culture method of the present invention, at least one "Tie2 expression enhancer" other than growth factors that enhances Tie2 expression is added to the culture medium. Specifically, when the second culture method is implemented in the amplification culture stage, the addition of the Tie2 expression enhancer has the effect of increasing the cell number while maintaining the immaturity of Tie2-positive stem / progenitor cells. Furthermore, when the cell population obtained in the amplification culture stage is used in the differentiation culture stage, it has the effect of increasing the cell growth rate of the cell population obtained after the differentiation culture stage, the ratio of Tie2-positive stem / progenitor cells to functional target cells, and so on. Any one Tie2 expression enhancer can be used, or two or more can be used in combination, as long as the amount added to the culture medium is sufficient to observe the aforementioned Tie2 activity.

[0246] Examples of "growth factors with Tie2 expression-enhancing effects" include angiopoietin-1 (Ang-1) and FGF2 (bFGF). In the second culture method of the present invention, at least one "Tie2 expression enhancer" other than such a "growth factor with Tie2 expression-enhancing effect" is used, but a combination of growth factors with Tie2 expression-enhancing effects may also be used as needed. In particular, when the second culture method is implemented in the amplification culture stage, a synergistic effect can be achieved by combining growth factors with Tie2 expression-enhancing effects with other Tie2 expression enhancers, such as extracts from plants and animals as described below, and more preferably plant-derived extracts. It should be noted that, as a step in the amplification and culture stage, in addition to the mandatory step of using at least a Tie2 expression enhancer other than growth factors (as an optional component, growth factors with Tie2 expression enhancing effects can be used in combination), a step can also be performed that substantially uses only growth factors with Tie2 expression enhancing effects (substantially not using Tie2 expression enhancers other than growth factors) as Tie2 expression enhancers.

[0247] As a Tie2 expression enhancer other than growth factors, extracts from various source plants and animals known in the prior art as "Tie2 activators" can be used. Examples of such source plant and animal extracts include, for example, extracts of milkweed, winged chrysanthemum, tamarind (Tamarindus indica), turmeric, wolfberry, polygonatum, plantain, wingless pigweed, olive fruit, oyster, chamomile, papaya, trichosanthes seed, morinda root, chrysanthemum, polygonatum rhizome, soapberry, ginkgo, dwarf jasmine, wolfberry, oak, alpinia galanga, red ginseng, oak, hawthorn, small red clover, guava, eleutherococcus senticosus, star fruit, star fruit, soapberry thorn, jujube, camphor, allium macrostemon, lotus, wild taro, prickly ash, long pepper, butcher's broom, mango ginger, sage oil, five-finger vine, double-flowered daylily, myrica, mountain willow, linear asparagus, etc. (see Patent Documents 3-10 above). Alternatively, components contained in such extracts, such as ursolic acid, corosolic acid, 3-O-galloyl proanthocyanidin B-1, linolenic acid, 13-hydroxy-9Z,11E,15E-octadecanoic acid, proanthocyanidin B-2, epicatechin-(4β-6)-epicatechin-(4β-8)-epicatechin, proanthocyanidin C-1, astragaloside VIII, soybean saponin I, 3'-O-methylgallocatechin, piperidine nonadiene, eugenol, 2-methoxycinnamonaldehyde, eleutheroside E, eleutheroside E1, sesamin, eudistomins, forsythinol, pinoresinol, liriope resin phenol B dimethyl ether, forsythinol, coumarin, etc. (refer to the above patent documents 6, 12-14) as Tie2 expression enhancers other than growth factors. For each extract and component, the dosage for which Tie2 expression enhancement is observed, the suitable plant or animal parts (materials) for preparation and extraction methods, and the purification methods for specific components can also be appropriately determined by those skilled in the art based on existing known methods.

[0248] From an industrial point of view, it is advantageous to use one or more extracts selected from the above-mentioned plant-derived plants that are cheaper than growth factors such as Ang-1 and FGF2, preferably have a better Tie2 expression-enhancing effect than these growth factors, and further preferably have a synergistic effect when used in combination with these growth factors, and more preferably have one or more extracts selected from the above-mentioned plant-derived extracts as the Tie2 expression enhancer in the second culture step of the present invention.

[0249] Extracts from Cinnamomum camphora plants

[0250] In a preferred embodiment of the present invention, an extract derived from a plant of the genus *Cinnamomum* can be used as a Tie2 expression enhancer. The genus *Cinnamomum* includes more than 300 species, such as *Cinnamomum cassia* Blume, *Cinnamomum camphora*, *Cinnamomum daphnoides*, *Cinnamomum doederleinii*, *Cinnamomum japonicum*, *Cinnamomum seudo-pedunculatum*, *Cinnamomum sieboldii*, *Cinnamomum verum*, and *Cinnamomum zeylanicum*. For example, extracts from products manufactured and sold as cinnamon twigs (twigs) or bark (bark), or cinnamon powder, can be used as the *Cinnamomum*-derived extract in this invention.

[0251] Extracts derived from *Cinnamomum* species can be obtained using conventional methods. For example, the plant material (e.g., cinnamon powder) is impregnated with an extraction solvent at room temperature or with heat, or refluxed. The supernatant is then recovered, or the filtrate is filtered and concentrated as needed. Extraction solvents can be used individually or in combination with commonly used extraction solvents, such as aqueous solvents (e.g., water, physiological saline, phosphate buffer, borate buffer); or organic solvents (e.g., alcohols such as ethanol, propylene glycol, 1,3-butanediol, glycerol); water-alcohol mixtures; chloroform; dichloroethane; carbon tetrachloride; acetone; ethyl acetate; hexane, etc.). Water is preferred. The extract obtained using the above solvents can be used directly as an extract, but for convenience, it can be solidified (powdered) and stored by drying or freeze-drying. When needed, it can be diluted or redissolved (redispersed) with a suitable solvent, and then filtered as required. Extracts derived from camphor trees can be purified products (purified products) that have had impurities removed as needed, such as by adsorption using ion exchange resins (e.g., porous polymers like Amber Light XAD-2).

[0252] The concentration of the camphor-derived extract in the culture medium can be appropriately adjusted according to the characteristics of the extract used and the degree of its effect as a Tie2 expression enhancer. For example, when using an extract obtained by extracting 1 mg of cinnamon powder with 1 mL of water (distilled water) as the camphor-derived extract, the extract can be added relative to the culture medium at an amount of about 1 to 50 v / v%, for example, about 20 v / v%. By changing the extraction and addition methods, the effective component as a Tie2 expression enhancer can be made to the same extent as in the extraction and addition methods described above.

[0253] <Extracellular matrix degrading agents (ECM degrading agents)>

[0254] In the fourth culture method of the present invention, in order to inhibit the formation of spherical colonies while differentiating Tie2 positive stem / progenitor cells, an "extracellular matrix degrader (ECM degrader)" is added to the culture medium.

[0255] Typically, the extracellular matrix (ECM) secreted by stem / progenitor cells or cells differentiated from them includes, for example, collagen, proteoglycans, fibronectin, laminin, tendinin, nestin, elastin, fibrin, and hyaluronic acid. Collagen includes type I, type II, type III, type IV, type IX (a2), and other types. Proteoglycans include aggregated proteoglycans, multifunctional proteoglycans (versicans), permacans (classified based on the size of the core protein and the number of glycan chains), chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratin sulfate proteoglycans, and dermatan sulfate proteoglycans (classified based on glycosaminoglycans bound to the core protein), etc.

[0256] Therefore, as the ECM decomposing agent in this invention, a substance (agent) that corresponds to the ECM secreted by the cultured Tie2 stem / progenitor cells or their differentiated cells in the fourth culture method embodiment, and has the activity of decomposing the ECM of the above examples and can inhibit the formation of spherical colonies, can be used. Any one type of ECM decomposing agent can be used, or two or more types can be used in combination.

[0257] Typical ECM degrading agents include proteases that are active in breaking down the protein components of ECM, such as collagenases, which are proteases that degrade collagen. Type I collagenases exhibit high activity against high molecular weight collagen, while Type II collagenases exhibit high activity against low molecular weight collagen fragments. Furthermore, vertebrate-derived collagenases cleave collagen in the natural triple-helix region (on a very limited α-chain), while bacterial-derived collagenases act on almost all types of collagen and can cleave collagen at multiple locations within the triple-helix region. Collagenase preparations obtained by concentrating bacterial culture supernatants contain, in addition to collagenases (collagenase I, collagenase II), proteases other than collagenases (neutral proteases, clostridial proteases, trypsin, elastase, aminopeptidase, etc.) and non-proteolytic enzymes. Preparations excluding specific components can also be manufactured through purification, etc. In this invention, suitable enzymes can be selected from various known collagenases (preparations), proteases, etc., to be used as ECM degrading agents.

[0258] In a representative embodiment of the fourth culture method of the present invention, Tie2-positive stem / progenitor cells are nucleus pulposus stem / progenitor cells, and the cells (target cells) generated by differentiation induction of Tie2-positive stem / progenitor cells 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 degrading agent in this embodiment, a collagenase with degrading activity against these ECMs, such as a collagenase (or a preparation containing such a collagenase) with degrading activity against type II collagen, can be selected. Examples of such collagenases (preparations) include "collagenase P" (Roche, derived from Clostridium histolyticum) and "Liberase" (Roche, a mixture of collagenase I and II and a neutral protease).

[0259] It should be noted that, as an ECM degrading agent, a representative example is an enzyme (protein) such as a protease that has specific degradation activity against the proteins contained in ECM and low cytotoxicity. However, substances other than enzymes (proteins) that can exert the effects of the present invention, and have a certain level or higher degradation activity against ECM and a certain level or lower cytotoxicity, such as low molecular weight compounds, may also be used as ECM degrading agents.

[0260] The concentration of the ECM-degrading agent in the culture medium should be sufficient to inhibit the formation of spherical colonies from cell populations containing Tie2 stem / progenitor cells. Depending on the type of ECM-degrading agent used, and considering the implementation of a fourth culture method (as a fourth culture step) during the differentiation culture phase, the concentration can be appropriately adjusted while taking into account the effects on the increase rate of target cells, the expression level of specified genes (marker genes), or the positivity rate. For example, if the concentration of the ECM-degrading agent is too high, the beneficial effects described above may not be fully confirmed (instead becoming detrimental). Therefore, it is preferable to adjust the concentration within a specified range according to the type of ECM-degrading agent.

[0261] In this invention, the effects of a method combining a third and a fourth culture method (third / fourth culture method) or a process combining a third and a fourth culture step (third / fourth culture step) on the increase rate of target cells, the expression level of a specified gene (marker gene), or the positive rate can sometimes vary depending on the combination of the type and concentration of the ECM decomposing agent in the culture medium and the type of coating agent on the culture surface. Those skilled in the art can, based on the desired effect and considering the characteristics of the cell population before differentiation induction and other embodiments, set the above-mentioned conditions suitable for implementing this invention through preparatory experiments, etc.

[0262] As mentioned above, the concentration of the ECM degrading agent in the culture medium cannot be generalized and also depends on the combination with the type of coating agent on the culture surface. For example, it can be adjusted in the range of 0.0025–5.0 wt%, 0.005–2.0 wt%, 0.01–1.0 wt%, etc. In one embodiment of the present invention, when "collagenase P" is used as the ECM degrading agent, the concentration in the culture medium can be adjusted in the range of 0.005–0.05 wt%, 0.0125–0.025 wt%, etc., and for example, it can be set to about 0.0125 wt%. In one embodiment of the present invention, when "Liberase" is used as the ECM degrading agent, the concentration in the culture medium can be adjusted in the range of 0.25 wt%–2.0 wt%, 0.5–1.0 wt%, etc., and for example, it can be set to about 1.0 wt%.

[0263] <Training time and other conditions>

[0264] The time and other conditions (e.g., pH, CO2 concentration, O2 concentration, etc.) of each culture method and culture step of the present invention can be appropriately adjusted according to the purpose of the culture step (including the culture stage of the culture step) to obtain a cell population with the desired cell composition (species and quantity / ratio). The pH can be set to weakly alkaline (e.g., about 7.15). The CO2 concentration can be set to, for example, about 5%. The O2 concentration can be set to less than 5% (e.g., about 2%). During the time of each culture method and each culture step (stage), the culture medium can be appropriately replaced with fresh culture medium every specified number of days as needed, or the culture medium can be changed by adding components or increasing or decreasing the concentration of components, pH, etc., after a specified number of days, or the atmosphere can be changed.

[0265] The time for the first culture step, the second culture step, or the first / second culture step combined with them in the amplification culture stage of the present invention is typically about 1 to 3 weeks, for example, about 2 weeks. Furthermore, the time for other steps that may be arbitrarily included in the amplification culture stage of the present invention is also of the same extent; for example, the time for a culture step using a medium supplemented with FGF is about 1 week. The amplification culture stage is ended when the desired cell population after amplification culture is obtained. It should be noted that culture (treatment) performed for a short period or time (e.g., less than 24 hours) when the purpose of amplification culture cannot be achieved is not equivalent to the steps performed in the amplification culture stage of the present invention.

[0266] The third and fourth culture steps, or a combination of the third and fourth culture steps, in the differentiation culture stage of this invention typically take about 1 to 3 weeks, for example, about 1 to 2 weeks. Furthermore, the other steps that can be arbitrarily included in the proliferation culture stage of this invention also take the same amount of time; for example, the culture step using a medium supplemented with FGF takes about 1 week. The other steps that can be arbitrarily included in the differentiation culture stage of this invention also take the same amount of time. The differentiation culture stage is ended when the desired cell population after differentiation culture is obtained. It should be noted that culture (treatment) performed for a short period or time (e.g., less than 24 hours) that cannot achieve the purpose of differentiation culture is not equivalent to the steps performed in the differentiation culture stage of this invention.

[0267] <Cultivation Container>

[0268] The culture containers, culture devices, etc. used in the various culture methods and culture steps of the present invention can be appropriately selected according to the purpose of the culture method and culture step (including the culture stage of the culture step) to obtain a cell population with a desired cell composition (type and quantity / ratio).

[0269] Culture containers can be flasks, petri dishes, well plates, bags, or other containers with common shapes, and can be formed with pores to accommodate cells. Culture containers can be made of common materials such as glass, plastic, and resin. The surface of the culture container (culture surface) can be untreated or treated in ways related to cell adhesion or other methods. The size (area, volume) of the culture container, and the size (diameter, depth) and number of pores if the container has pores, can also be appropriately selected. Cell populations can be cultured while the culture medium is agitated by shaking or rotating the culture container as needed.

[0270] In the third and fourth culture methods (steps) of the present invention, the culture container and culture apparatus can be implemented based on two-dimensional culture (planar culture). Furthermore, the first culture method (step) of the present invention, which involves culturing in a state where the cell population exists within the tissue, can also be described as a three-dimensional culture, with the tissue (slice) containing the cell population suspended in the culture medium. The second culture method (step) of the present invention can also be implemented based on three-dimensional culture when carried out alone, but when integrated with the first culture method (step) and implemented as the first / second culture method (step), it can be suspended in the culture medium, similar to the first culture method (step) described above. In these methods (steps), culture containers with surface treatments to improve cell adhesion, as in the third culture method (step), can be used, but there is no problem even when using ordinary culture containers without surface treatment.

[0271] <Cell Adhesion Treatment>

[0272] In the third culture method (step) of the present invention, a culture vessel that has undergone a surface treatment to improve cell adhesion (sometimes referred to as "cell adhesion treatment" in this specification) is used. A typical example of cell adhesion treatment is applying a coating agent containing extracellular matrix (ECM) or other biologically related substances to the culture surface. Another example of cell adhesion treatment is modifying a culture vessel made from a raw material with low cell adhesion, such as highly hydrophobic polystyrene, to be hydrophilic through plasma treatment.

[0273] ECMs contained in coating agents used for cell adhesion treatment can include various well-known ECMs, such as collagen (type I, type II, type IV, etc.) or gelatin, chondroitin sulfate A, fibronectin, gelatin, laminin, platelet response proteins, fibronectin, and proteoglycans (aggregate proteoglycans, heparin sulfate proteoglycans, etc.). In addition, as biologically relevant molecules other than ECMs, polyamino acids such as polylysine (poly-L-lysine or poly-D-lysine) can be listed. Other coating agents used for cell adhesion treatment include polyglycolic acid, PLGA (polylactic acid-glycolic acid copolymer), polyhydroxyalkanoic acid (PHA), poly-ε-caprolactone, polyorthoester, polyanhydride, polyphosphazene, polydimethylsiloxane, polyurethane, polytetrafluoroethylene, polyethylene, polysulfone, polymethyl methacrylate, poly(2-hydroxyethyl methacrylate), polyamide, polypropylene, polyvinyl chloride, polystyrene, polyvinylpyrrolidone, and polyornithine, etc. The coating agent for cell adhesion treatment may contain any one of the above-mentioned substances, or may contain two or more of them.

[0274] Here, in the third / fourth culture method (step), as mentioned above, the effects of the present invention (cell increase rate of cell population, target cell ratio, etc.) may sometimes change depending on the combination of the type of coating agent used for cell adhesion treatment and the type and concentration of ECM degrading agent added to the culture medium. One reason for this is that the ECM or other biologically related substances contained in the coating agent used for cell adhesion treatment may be affected by the degradation activity of the ECM degrading agent added to the culture medium. However, embodiments that combine the coating agent used for cell adhesion treatment with the ECM degrading agent, which may have such an effect, are permissible as long as the effects of the present invention can be achieved to a certain extent (not completely suppressed). For example, when collagenase (preparation) with type II collagen degradation activity is added to the culture medium at a specified concentration as an ECM degrading agent, the coating agent used for cell adhesion treatment is preferably a coating agent that is not easily affected by the type and concentration of the ECM degrading agent, or a coating agent that can achieve differentiation induction at a certain level for target cells (e.g., Col2 positive cells). For example, a coating agent containing polylysine (poly-L-lysine or poly-D-lysine) or fibronectin, or type IV collagen, which is not collagen, is preferred.

[0275] The third culture method of the present invention can also be implemented in the amplification culture stage. For example, the third culture method can be implemented in the step (additional amplification culture step) of culturing a cell population containing Tie2-positive stem / progenitor cells in a culture medium in relation to the amplification culture stage, where only a growth factor with Tie2 expression-enhancing effect is added as a Tie2 expression enhancer. Gelatin is preferably a preferred example of the ECM contained in the coating agent used for cell adhesion treatment in such embodiments.

[0276] -Compositions for Cell Therapy-

[0277] The cell therapy composition of the present invention may contain a cell population obtained by the culture or preparation method of the present invention as described above, and may contain other pharmaceutically acceptable ingredients as needed.

[0278] In a representative embodiment of the present invention, the cell therapy composition is a cell therapy composition comprising Col2-positive nucleus pulposus cells differentiated from nucleus pulposus stem / progenitor cells (preferably also comprising Tie2-positive stem / progenitor cells). As for the applicable objects of the cell therapy composition in this embodiment, i.e., diseases that can be prevented or treated by administering the composition, examples include diseases manifested as damage or degeneration, herniation, etc., of the intervertebral disc (nucleus pulposus) in the lumbar or cervical spine, such as intervertebral disc disease, herniated disc, cervical spondylotic myelopathy, radiculopathy, spondylolisthesis / spondylolisthesis, lumbar spinal stenosis, lumbar degenerative spondylolisthesis, and lumbar degenerative scoliosis.

[0279] The dosage form of the cell therapy composition of the present invention can be used to transplant or deliver cell populations to the target site (e.g., the nucleus pulposus of the intervertebral disc), and can be, for example, an injection, preferably an injection for local administration to the intervertebral disc (nucleus pulposus) or its vicinity, or a targeted vascular injection.

[0280] Pharmaceutically acceptable components, such as water for injection or physiological saline, culture medium for cell populations, other suitable solvents / dispersion media, and other additives, can be listed as such when preparing injections.

[0281] The cell therapy composition of the present invention can be administered in an amount effective for achieving the desired therapeutic or preventative effect. This effective amount can be appropriately adjusted based on the composition, dosage form, target population, route of administration, other implementation methods, etc., and according to the dosage, frequency of administration, and dosing interval (number of administrations within a certain period). Treatment using the cell therapy composition of the present invention can be performed on humans and vertebrates other than humans.

[0282] -Saving Method-

[0283] The method for preserving a cell population containing Tie2-positive stem / progenitor cells according to the present invention involves cryopreserving the cell population containing the Tie2-positive stem / progenitor cells in a state existing in undigested tissue, thereby maintaining the state in which Tie2 is activated and / or expressed, or inhibiting the reduction of Tie2-positive stem / progenitor cells in the cell population.

[0284] The technical matters relating to the preservation method of the present invention are applicable to the same technical matters described in relation to the first culture method. The steps for cryopreservation of undigested tissue containing Tie2-positive stem / progenitor cells, the cryoprotectants used as needed, etc., are substantially the same as those for conventional cell populations containing Tie2-positive stem / progenitor cells isolated from tissues through digestion.

[0285] Example

[0286] The culture medium used in each step of the "amplification culture stage" in this embodiment (hereinafter referred to as "amplification culture medium") is prepared in a mixed medium of 60 mL DMEM (no glucose, wako) and 40 mL MEMα (Nacalai Tesque), with 20% FBS added just before use, and then with (+) or without the additional components shown in the tables in the embodiments, and then used.

[0287] The culture medium used in each step of the "differentiation culture stage" in this embodiment (hereinafter referred to as "differentiation culture stage culture medium") is prepared by adding 1 μL of 2-mercaptoethanol, 6 μL of selenite (0.01%), 1.5 mL of ascorbic acid (5 mg / mL), and 5 mL of 30% BSA to a mixed culture medium of 60 mL DMEM (no Glucose, Wako) and 40 mL F10 (Gibco), and then adding 30% FBS just before use, and then adding (+) or not adding (-) the additional components shown in the tables in the embodiments.

[0288] [Experimental Example 1] Amplification and Culture Stage: First Culture Step (WTC Method)

[0289] [Table 1]

[0290]

[0291] Using scissors or similar tools, the nucleus pulposus tissue removed from the affected area of ​​a herniated disc patient (32-year-old female, 28-year-old female, and 20-year-old male) was cut into pieces several square millimeters in size. 0.1–0.5 g of the chopped nucleus pulposus tissue, containing a cell population, was suspended in 3 mL of culture medium prepared according to the methods shown in Table 1 for the expansion culture phase, and then injected into one well of a 6-well culture dish (without surface treatment) and cultured for 7 days (WTC method). As a control, the chopped nucleus pulposus tissue was digested with collagenase using conventional methods instead of direct culture, and the isolated cell population was recovered and cultured in the same manner as the WTC method (two-dimensional culture method).

[0292] After culture, the cell population was recovered, and the number and fluorescence intensity of cells expressing Tie2 on the cell surface were determined by flow cytometry (FCM). The ratio of the number of cells relative to the total cell population (Tie2 positivity rate) and the mean fluorescence intensity (MFI) were calculated. In the FCM method, a fluorescent labeling agent (R&D Corporation, Anti-Tie-2, Human, Mouse-Mono (87315), Allophicocyanin, catalog number FAB3131A) was used as a complex of anti-human Tie2 antibody and the fluorescent pigment allophicocyanin.

[0293] The results are shown in Figure 2 and Figure 3 For example, comparing experimental cases 1-1 and 1-3, experimental case 1-1 showed a significantly higher (in all cases) Figure 2 p < 0.05 Figure 3 (p < 0.01, all results were obtained from t-tests), confirming the enhanced Tie2 expression effect based on the WTC method.

[0294] [Experimental Example 2] Amplification and Culture Stage (Two Stages): First / Second Culture Step + Additional Step

[0295] [Table 2]

[0296]

[0297] 1 mg of commercially available cinnamon powder was suspended in 1 mL of distilled water and extracted overnight at 37°C. The resulting extract (cinnamon extract) was used in this experiment.

[0298] The patients with intervertebral disc herniation who had their nucleus pulposus tissue collected were females aged 16, 28, and 38. As the first stage of the expansion culture phase, the culture medium was prepared according to the method shown in Table 2 for the additional components in the culture medium for the expansion culture phase, and the culture time was set to 14 days. Otherwise, the same culture procedure as in [Experimental Example 1] (Experimental Examples 1-1 and 1-2) was performed.

[0299] Following the first stage of culturing, Roche's collagenase-P (final concentration 0.025%) was added to the culture medium to disperse the nucleus pulposus tissue. The cell population isolated from the nucleus pulposus tissue was then recovered at a concentration of 1.0 × 10⁻⁶ cells / mL. 4 The solution was suspended at a density of 3 mL in MEMα with 20% FBS, then injected into one well of a 6-well culture dish (with untreated culture surface), and 10 ng / mL bFGF was added before culture for 7 days (total 21 days).

[0300] After culturing, the cell population was recovered, and the percentage of cells expressing Tie2 positively on the cell surface and the number of cells derived from 1g of tissue were determined using the same FCM method as in [Experimental Example 1]. The results are presented in... Figure 4 and Figure 5 .

[0301] [Experimental Example 3] Amplification Culture Stage (Two Stages): First / Second Culture Step + Additional Step → Differentiation Culture Stage: Third Culture Step

[0302] [Table 3]

[0303]

[0304] Patients with intervertebral disc herniation who underwent nucleus pulposus tissue sampling were 16-year-old females, 30-year-old males, and 30-year-old females. All other patients underwent a two-stage expansion culture phase totaling 21 days, following the same procedures as in Experiment 2 described above. The cultured cell populations were then harvested and cultured as a differentiation phase on poly-L-lysine (PLL) coated culture dishes (Experiment 3-1) or uncoated culture dishes (Experiment 3-2) for 14 days in a monolayer.

[0305] After culturing, the cell population was recovered, and the number of cells expressing type II collagen (Col2) positively was determined by flow cytometry (FCM). The ratio of the number of cells to the total cell population (Col2 positivity rate) was calculated. The cell population was pretreated with the membrane permeability treatment reagent "IntraPrep" (Beckman Coulter, Inc.) to enable intracellular Col2 fluorescent labeling. For the Col2 fluorescent labeling method, mouse anti-human Col2 antibody (PHARMA CHEMICAL CO.,LTD. (formerly DAIICHI FINE CHEMICAL CO.,LTD., anti-hCL(II) (purified IgG), catalog number F-57) was used as the primary antibody, and a complex of goat anti-mouse IgG antibody and fluorescent dye FITC (BD Biosciences, goat anti-mouse Ig FITC, catalog number 349031) was used as the secondary antibody. The results are shown in... Figure 6 Additionally, the number of Col2-positive cells was calculated assuming that 1g of nucleus pulposus tissue-derived cells were expanded and cultured according to Experimental Example 2, and then differentiated and cultured according to Experimental Example 3. The results are presented below. Figure 7 Compared to the case where the third culture step was applied (Experiment 3-1) and the case where it was not applied (Experiment 3-2), the number of Col2 positive cells increased by approximately 3 times.

[0306] It should be noted that the number of cells expressing proteoglycan (PG) positively was also determined using the FCM method, and the ratio of the number of cells to the total cell population (PG positivity rate) was calculated. As a fluorescent labeling method for PG, mouse anti-human PG antibody (EMD millipore, anti-chondroitin proteoglycan antibody, adult, clone EFG-4, catalog number MAB2015) was used as the primary antibody, and a complex of goat anti-mouse IgG antibody and fluorescent dye FITC (BD Biosciences, goat anti-mouse Ig FITC, catalog number 349031) was used as the secondary antibody. The results showed that the PG positivity rate was close to 100% regardless of whether the third culture step (PLL coating) was applied, and no significant difference was observed (not illustrated). For functional nucleus pulposus cells that express type II collagen, which differs from proteoglycan, it is difficult to increase the number of cells in the final cell population using existing methods, but this can be achieved by combining the first / second and third culture steps of this invention, demonstrating its superiority as a culture method.

[0307] [Experimental Example 4] Amplification Culture Stage (Two Stages): Culture Step 1 / 2 + Additional Step → Differentiation Culture Stage: Culture Step 3 / 4

[0308] [Table 4]

[0309]

[0310] Following the same procedures as in Experimental Example 2 above, a two-stage amplification culture phase was performed for a total of 21 days. The cultured cell population was then recovered and cultured for 14 days in a medium coated with poly-L-lysine (PLL) containing the additional components shown in Table 4 used in the differentiation culture medium for the differentiation phase.

[0311] After culturing, the cell population was recovered, and the PG positivity rate was calculated in the same manner as in [Experimental Example 3]. The results are presented below. Figure 8 When either of the two collagenases was added to the culture medium, the PG positivity rate increased significantly compared to the case where no collagenase was added.

[0312] For Experiments 4-1 to 4-3, six samples were prepared for each (the patients with herniated discs who underwent disc herniation were 32-year-old women, 28-year-old women, 20-year-old men, 16-year-old women, 28-year-old women, and 38-year-old women), and the Col2 positivity rate was calculated in the same manner as in [Experiment 3]. The results are shown below. Figure 9 Although there were differences due to samples (individual differences in the nucleus pulposus tissue collected from the intervertebral disc), in 4 out of 6 samples, an increase in the positive rate of Col2 was observed compared with the absence of collagenase in the culture medium when either or both of the two collagenases were added.

[0313] [Experimental Example 5] Amplification Culture Stage (Two Stages): Culture Step 1 / 2 + Additional Step → Differentiation Culture Stage: Culture Step 3 / 4, Part 2

[0314] [Table 5]

[0315]

[0316] For the differentiation culture stage, the coating agent of the culture vessel and the additional component (collagenase P) in the culture medium were changed as shown in Table 5. Otherwise, the amplification and differentiation culture stages were performed following the same procedures as in Example 4 above, and the PG positivity rate and Col2 positivity rate were measured. The results are shown in... Figure 10 For example, when collagenase P is added to the culture medium, it was observed that, although it also depends on the concentration, using a coating agent containing Col4 (type IV collagen), FN (fibronectin), or PLL (poly-L-lysine), especially a coating agent containing PLL, is preferred for improving the Col2 positivity rate.

[0317] [Experimental Example 6] Amplification Culture Stage (Two Stages): Culture Step 1 / 2 + Additional Step → Differentiation Culture Stage: Culture Step 3 / 4

[0318] [Table 6]

[0319]

[0320] For the differentiation culture stage, the coating agent of the culture vessel and the additional component (Liberase) in the culture medium were changed as shown in Table 6. Otherwise, the amplification and differentiation culture stages were performed following the same procedures as in Example 4 above, and the PG positivity rate and Col2 positivity rate were measured. The results are shown in... Figure 11 For example, when "Liberase" is added to the culture medium, it was observed that, although it also depends on the concentration, using a coating agent containing Col4 (type IV collagen) or PLL (poly-L-lysine) is preferred for improving the Col2 positivity rate.

[0321] [Experimental Example 7] Differentiation Culture Stage: Third Culture Step

[0322] [Table 7]

[0323]

[0324] In this experiment, a cell population isolated from the nucleus pulposus tissue of a patient with a herniated disc was used, similarly to the control of Experimental Example 1 (i.e., without the application of the first culture method of the present invention: WTC method). This cell population was cultured in a medium supplemented with 10 ng / mL bFGF (without the application of the second culture method of the present invention, and without the addition of cinnamon extract as in Experimental Example 2) for 8–9 days (Phase 1) and 6–8 days (Phase 2).

[0325] Next, for the cell population containing Ti2-positive stem / progenitor cells of nucleus pulposus that had undergone amplification culture as described above (the first and / or second culture methods of the present invention were not applied as the amplification culture stage), a step based on the third culture method of the present invention was performed as the differentiation culture stage. In this step, monolayer culture was performed for 6 to 7 days on culture dishes coated with poly-L-lysine, similar to Example 3, etc.

[0326] After culture, the cell population was recovered, and the Tie2 positivity rate and total Tie2-positive cell count were measured in the same manner as in Experiments 1 and 2. Additionally, the Col2 positivity rate was measured in the same manner as in Experiment 3, etc. The results are presented below. Figure 13 (Tie2 positivity rate) Figure 14 (Total number of Tie2-positive cells) and Figure 15(Col2 positivity rate). It can be seen that the third culture method of the present invention, even in embodiments not combined with the first and / or second culture methods of the present invention, can also improve the Tie2 positivity rate, the total number of Tie2 positive cells, and the Col2 positivity rate.

Claims

1. A method for preparing a cell population comprising Tie2-positive stem cells and / or progenitor cells. Tie2 is a tyrosine kinase containing Ig and EGF homologous domain-2; The preparation method includes: This is used to enhance the expression of Tie2 in Tie2-positive stem cells and / or progenitor cells and to expand the culture phase of Tie2-positive stem cells and / or progenitor cells in the cell population. This amplification culture phase includes implementing the following method: the method involves adding at least a portion of Chinese cinnamon (Cinnamomum cassia) to the tissue in a state present in undigested tissue and in an undisturbed tissue microenvironment. Cinnamomum cassia A cell population containing Tie2-positive stem cells and / or progenitor cells was cultured in a medium containing an extract of Blume (as a Tie2 expression enhancer other than growth factors), thereby expanding the Tie2-positive stem cells and / or progenitor cells in the cell population. The extract is an extract obtained by extracting cinnamon powder with water. The undigested tissue is shredded nucleus pulposus tissue from the intervertebral disc. The Tie2-positive stem cells and / or progenitor cells are Tie2-positive stem cells and / or progenitor cells derived from the nucleus pulposus tissue of the intervertebral disc. The expansion culture stage also includes the process of recovering the cultured cell population containing the Tie2 positive stem cells and / or progenitor cells.

2. The preparation method according to claim 1, wherein, The nucleus pulposus tissue of the intervertebral disc is obtained by thawing the nucleus pulposus tissue of the intervertebral disc that has been cryopreserved.

3. The preparation method according to claim 1 or 2, wherein, The expansion culture stage includes the process of culturing a cell population containing Tie2-positive stem cells and / or progenitor cells in a culture medium supplemented with fibroblast growth factor as a Tie2 expression enhancer.

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