JP6905234B2 - Method for culturing a cell population containing Tie2-positive stem / progenitor cells and its utilization - Google Patents

Description

The present invention relates to the cell surface marker Tie2 (tyrosine kinase with Ig and EGF homology d).
Stem cells and / or progenitor cells that are positive for omain-2) expression ("Ti" herein.
It is called "e2-positive stem / progenitor cell". ) Containing a method for culturing a cell population. For more details,
The present invention relates to a step of amplifying Tie2-positive stem / progenitor cells (for example, nucleus pulposus stem / progenitor cells) in a cell population, and cells having predetermined traits from Tie2-positive stems / progenitor cells (for example, type II collagen-expressing nucleus pulposus). The present invention relates to a method for culturing a cell population containing Tie2-positive stem / progenitor cells, which can be used in the step of inducing differentiation into cells).

Back pain ranks second in the rate of complaints and is a common illness once experienced by two-thirds of the adult population, contributing to work-related injuries and social problems in the health economics. Disc damage, which is said to be 20% of the causes of low back pain, is a serious problem that can induce disc herniated disk, degenerative spondylosis, spinal canal stenosis, spondylolisthesis, etc. Physically, it is a condition called intervertebral disc degeneration. The intervertebral disc is a donut-shaped cartilage organ, the central nucleus pulposus (NP) and the annulus, the fibrous cartilage that surrounds it many times.
s Fibrosus (AF), and Cartilaginous Endpla, which connects the adjacent vertebrae up and down.
It is formed by te; EP). Gelatin-like NP is an avascular organ and contains a large amount of extracellular matrix (ECM) composed of large proteoglycan and collagen secreted from notochord-derived nucleus pulposus cells contained in NP. .. Notochord-derived nucleus pulposus cells have been reported to disappear early in life in some vertebrates, including humans, and after their disappearance, their origin has not yet been determined, but morphologically chondrocytes. Chondrocytes similar to are forming the nucleus pulposus. It is considered that such conversion of cell traits affects the ECM composition, causes aging and degeneration of the intervertebral disc such as decrease in water content and fibrosis, and is ultimately greatly related to low back pain and lumbar degenerative diseases. Many animal species such as mice, rats, rabbits, and pigs retain notochord-derived nucleus pulposus cells for life, and almost no intervertebral disc degeneration is observed. Therefore, the control mechanism of notochord-derived nucleus pulposus cells and other nucleus pulposus cells. Is considered to be different from humans.

As an example of a method for preventing or treating intervertebral disc degeneration, research and development of an allogeneic disc cell preparation, that is, a cell preparation containing allogeneic nucleus pulposus cells, ECM, etc. for administration to intervertebral disc tissue is underway. A certain amount of allogeneic nucleus pulposus cells is required for the production of such a cell preparation. For example, the spinal cord nucleus tissue excised by surgery in a patient with herniated disk disk can be used as a source of nucleus pulposus cells, but the amount of nucleus pulposus tissue that can be collected in this way, that is, the nucleus pulposus cells contained therein. The number is small. However, in order to secure the number of nucleus pulposus cells, it is not possible to mix and use nucleus pulposus cells derived from multiple herniated disc patients (donors).
The risk of viral infection cannot be completely ruled out and should be avoided. Therefore, rare stem or progenitor cells that are contained in a small amount of disc tissue (nucleus, annulus fibrosus, etc.) derived from a single donor and capable of inducing differentiation into mature nucleus pulposus cells are cultured and treated. It is important to prepare a cell population containing a sufficient number of nucleus pulposus cells for this purpose.

Patent Document 1 states that a nucleus pulposus cell (a cell population derived from the nucleus pulposus of the intervertebral disc) is cultured under "conditions that interfere with cell attachment" (preferably cultured in a serum-free medium) to obtain the cell population. It is disclosed to make a "discosphere" composed of contained stem cells and progenitor cells. That is, Patent Document 1 describes a step of growing a nucleus pulposus cell in a medium under "conditions that interfere with cell attachment", and (b) a disc stem cell and a disc progenitor cell. Alternatively, a "method for producing a disk stem cell population" including a step of concentrating for a combination thereof and (c) a step of producing a disk sphere containing nucleus pulposus cells and thereby producing a disk stem cell population is described. (Claim 3 etc.). Regarding the "disc sphere", a single disc stem cell, which is an in vitro free floating circular-spherical structure containing a disc stem cell, a disc progenitor cell, or a combination thereof, is used. A nucleus pulposus containing a disk sphere, including floating nucleus pulposus stem cells and nucleus pulposus progenitor cells arranged in a circular-spherical structure, which is the cell sphere that gives rise to its own clones and progenitor cells. It is explained that the cells are attached to each other, etc. (paragraph 002).
4,0039). Patent Document 1 further states that the cells were cultured under "conditions that interfere with cell adhesion".
"Isolated disk stem cell population" enriched for disk stem cells, disk precursor cells or combinations thereof (eg, claim 1); disk stem cells, disk precursor cells, or disk precursor cells enriched from nucleus pulposus cells, or An "isolated disc sphere" (such as claim 10) that contains the mixture and is an in vitro floating sphere structure; enriched for disc scaffolds and disc stem cells, disc precursor cells or combinations thereof. "Artificial disk replacement device" including a disk sphere containing the medullary nucleus cells (claim 1)
1st grade); A method for producing a disc artificial replacement device, which comprises growing a disc sphere containing disc stem cells, disc precursor cells or a mixture thereof enriched from nucleus pulposus cells in a disc scaffold. (Claim 12 et al.); In vitro containing disc stem cells, disc precursor cells or a mixture thereof, and a step of culturing nucleus pulposus cells plated at a predetermined low density under "conditions that interfere with cell attachment". "Methods for producing enriched cell populations" (eg, claim 17), comprising the step of selecting floating sphere structures and thereby producing enriched cell populations; disc stem cells, disc precursor cells or them. The step of dissociating a disc sphere containing the combination of the above into one or more dissociated disc sphere cells, and the one or more dissociated disc sphere cells interfering with cell attachment to a predetermined value. Also described is "a method of expanding a population of concentrated disc stem cells, disc precursor cells or a combination thereof" including a step of culturing in a medium containing additives (eg, FGF2, EGF) (claim 18 et al.). Has been done. The "disk" in Patent Document 1 is a literal translation of the original word "disc", but is considered to mean "intervertebral disc".

Patent Document 1 relates to a culture vessel or medium for culturing an inhomogeneous cell population (medullary nucleus-derived cell population) including disc stem cells, disc progenitor cells, disc cells, etc. derived from disc nucleus pulposus tissue. Regarding the matters, the following can be said.

In Patent Document 1, as a culture under "conditions that interfere with cell adhesion", the cells are plated and cultured at a low cell density in a serum-free medium containing a substance (methyl cellulose) that interferes with cell adhesion, or are ultra-low. An embodiment is disclosed in which a disc sphere, which is a floating sphere structure, is formed from a nucleus pulposus-derived cell population (stem cells and the like contained therein) by culturing in an attachment plate (paragraph 0156, Example). 1: See paragraphs 0170 to 0181, corresponding to the inventions of claims 3, 17 and the like). However, Patent Document 1 describes a nucleus pulposus-derived cell population (eg, in a medium containing a substance that degrades extracellular matrix (eg, collagenase), or on a cell-adherent culture surface.
There is no description or suggestion of culturing (such as stem cells contained therein) to proliferate or induce differentiation of disc stem cells without forming disc spheres.

In Patent Document 1, as a method for expanding a cell population containing concentrated disc stem cells and the like, first, one or a plurality of disc spheres (floating sphere structures) are formed by incubation in a medium supplemented with collagenase. An embodiment of dissociating into disc stem cells and then replating the dissociated cells in a methylcellulose-containing medium is disclosed (paragraph 015).
7. Example 2: See paragraphs 0182 to 0184, corresponding to the invention of claim 18 and the like). However, the culture in the medium supplemented with collagenase in the embodiment is only a temporary treatment for dissociating the once formed disc spheres into individual disc stem cells and the like, and induces differentiation of the disc stem cells and the like. The dissociated disc stem cells and the like are again cultured under "conditions that interfere with cell adhesion" (methyl cellulose-containing medium, etc.). In the state where the disk ball is not formed (
Start culturing in a medium supplemented with collagenase using disc stem cells (before formation) to expand (proliferate) or induce differentiation of the disc stem cells, or to induce the disc stem cells to differentiate from each other. After dissociation, continue culturing in a medium supplemented with collagenase to expand (proliferate) and differentiate disc stem cells, etc., while maintaining the state in which disc spheres, which are floating sphere structures, are not formed. No description or suggestion is made in Patent Document 1 about making the cell.

In Patent Document 1, "a compound that inhibits cell maturation" (for example, FGF) or "a compound that maintains cell immaturity" (for example, TGF-β superfamily member or BMP)
, IL-6, LIF, etc.), although it has been described to grow disc stem cells in a serum-free medium (paragraphs 0035 to 0037), but in a medium supplemented with a substance that promotes the activation of Tie2. There is no mention of culturing plate stem cells.

In addition, Patent Document 1 describes a method for preparing a nucleus pulposus tissue for obtaining a nucleus pulposus-derived cell population by fragmenting the nucleus pulposus (surgically obtained human disk material or biopsy specimen) and collagenase. Only the general method of fragmenting the nucleus pulposus tissue and dissociating individual cells (making a single cell suspension) by treatment with II, crostridium collagenase, etc. is described (paragraph). 0026, 0029, 0030, Example 1: Paragraphs 0171 to 0174, etc.)
..

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

As a more specific embodiment, Patent Document 2 and Non-Patent Document 1 describe spherical colonies (along with adherent colonies) by suspending culture of cell populations contained in intervertebral disc tissue (medullary nucleus) in a methylcellulose medium. Was formed, such spherical colonies were T
It is described that it is derived from ie2-positive (and GD2-positive) cells, and that type II collagen and proteoglycan are expressed in spherical colonies (some cells thereof) (for example, Examples of Patent Document 2). , Paragraphs 0067, 0070, etc.). However, Patent Document 2 and Non-Patent Document 1 also use a medium containing a substance that decomposes extracellular matrix (for example, collagenase) or on a cell-adherent culture surface (using a medium to which methyl cellulose is not added). ), Nucleus stem / progenitor cells (Tie2) without forming spherical colonies
And / or GD2-positive cells) are not described or suggested for proliferation or differentiation induction.

According to Patent Document 2 and Non-Patent Document 1, as a method for preparing a cell population derived from the nucleus pulposus tissue of the intervertebral disc, the tissue is fragmented with scissors and then digested with a protein digestive enzyme (Triple Express, Collagenase P). , Only general techniques are described (Example: Paragraph 0048).

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

By the way, Tie2 is also expressed in vascular endothelial cells, and activation of Tie2 brings about maturation, normalization or stabilization of blood vessels, for example, tumor, rheumatoid arthritis, diabetic retinopathy, hypertension. It is known that it can suppress the disordered amplification of blood vessels (angiogenesis) observed in blood vessels, hypertension, etc., and prevent and improve wrinkles. Examples of the Tie2 activator having such an action include an extract derived from a plant of the genus Cinnamomum (so-called cinnamon powder, Patent Document 3), an olive fruit extract (Patent Document 4), and the like.
In addition, Kiraya, twilight, ginkgo, oyster, turmeric, chrysanthemum, jujube, kuko, chamomile, butcher bloom, sanzashi, star fruit, shell ginger, hass, louis boss, Indian dates, karin, shijuum guaba, long pepper, siberian carrot, mango Ginger, Koryo carrot, Akigumi, Okahijiki, Harigiri, Ryobu, Yabukanzo, Hasuimo, Mitsubautsugi, Kusagi, Mube, Sanshosou, Konara, Kunugi, Akinonogeshi, Suishogaki, Oobako, Nobir, Yamamomo, Nikkei Various plant-derived extracts have been proposed, such as carrot vulture (see Patent Document 4: Background Technology).

In Patent Document 3, as a test (example) relating to "Tie2 activator", "blood cell Baf3 cells forcibly expressing Tie2" or "normal human diatomose vein vascular endothelial cells" in a medium supplemented with Keihi hot water extract. (HUVEC) ”, T expressed in those cells
It has been confirmed by Western blotting that the amount of phosphorylated ie2 protein is higher than that of the control (paragraphs 0024 to 0027, FIGS. 1 to 3 and the like).

However, in Patent Documents 3 and 4, etc., the use of a Tie2 activator in culturing a nucleus pulposus-derived cell population (Tie2-positive stem / progenitor cells contained therein) obtained from an intervertebral disc, and thereby, which It is not described whether such effects are brought about.

Japanese Patent No. 5509073 (corresponding to WO2009 / 00902020) Japanese Patent No. 5863639 (corresponding to WO2011 / 12261) WO2009 / 123211 WO2016 / 060249

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

As mentioned above, in order to produce allogeneic disc cell preparations for the prevention or treatment of degenerative disc disease, functional nucleus pulposus cells that produce extracellular matrix such as type II collagen and proteoglycan are gathered to some extent. Needed in quantity. For this purpose, for example, Tie2-positive cells, which are considered to be stem cells and / or progenitor cells of nucleus pulposus cells, contained in the intervertebral disc tissue (such as the nucleus pulposus) excised from the affected part of a patient with herniated disk are efficiently used. It is necessary to proliferate and differentiate into a large amount of functional nucleus pulposus cells. In particular, in order to enhance the therapeutic effect when a cell preparation is administered to a patient with disc degeneration, etc., when culturing Tie2-positive cells (marrow nucleus stem / precursor cells) contained in the disc and inducing differentiation, simply It is important not to differentiate into nucleus pulposus cells, but to differentiate into functional nucleus pulposus cells that produce a large amount of extracellular matrix such as type II collagen as efficiently as possible. At the same time, Tie2-positive cells (nucleoside stem / progenitor cells) in the cell population obtained from the tissue in advance before inducing differentiation as described above.
It is also important to efficiently amplify the cells in order to increase the number of functional nucleus pulposus cells finally obtained. That is, from a cell population containing a certain number of Tie2-positive cells (nucleus stem / progenitor cells), functional nucleus pulposus cells in the cell population finally prepared and administered by efficient amplification and differentiation induction There is a need for practical means to enrich it.

Further, in a typical embodiment of the prior art as described in Patent Documents 1 and 2 described above, methyl cellulose was added to a cell population containing a nucleus pulposus stem / progenitor cell contained in the intervertebral disc tissue. By culturing in a medium, spherical colonies (disc spheres, spheroids) were formed and then differentiated into nucleus pulposus cells. However, since methyl cellulose is a highly viscous substance, it is difficult or a great deal to recover the cell population (useful functional nucleus pulposus cells contained therein) generated from the medium to which it is added without waste. It required labor and was a hindrance to the efficient production of cell preparations.

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

The present inventors are Tie2-positive cells (nucleoside stem / progenitor cells) contained in intervertebral disc nucleoside tissue
As a result of conducting research focusing on the state of the cell population to be cultured and the culture conditions when culturing the cell population containing the above, we found several characteristic technical matters that can contribute to the solution of the above problems. .. In addition, some of the culture methods having these technical features are in the culture step of the stage (amplification culture stage) in which the main purpose is to amplify the nucleus pulposus stem / precursor cells, and some are the nucleus pulposus stem. / In the culture step of the stage (differentiation culture stage) whose main purpose is to induce differentiation from precursor cells to functional nucleus pulposus cells, they can be used in combination sequentially or simultaneously, and in particular, such a culture method is "fused". It has been found that the action and effect of the present invention can be synergistically produced by performing the culturing steps simultaneously in this manner.

That is, in one aspect, the present invention is combined (preferably) as a method for culturing a cell population containing Tie2-positive stem / progenitor cells represented by Tie2-positive cells (medullary nucleus stem / progenitor cells) contained in the intervertebral disc tissue. Provides the following first to fourth culture methods capable of fusion).

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

Conventionally, when trying to culture a cell population contained in an intervertebral disc nucleus pulposus tissue, after chopping the collected intervertebral disc nucleus pulposus tissue, first, a proteolytic enzyme (protease) such as collagenase is used.
It was common practice to perform a treatment (digestion treatment) to digest the tissue, collect the cell population separated from the nucleus pulposus tissue by the treatment, and start culturing. However, the present inventors do not perform digestion treatment after shredding the nucleus pulposus tissue, suspend the shredded nucleus pulposus tissue in a culture medium, and culture the cells for a certain period of time while keeping the cell population in the tissue. In this case, the ratio of Tie2-positive stem / progenitor cells (medullary nucleus stem / progenitor cells) in the cell population is improved, and the expression of Tie2 in individual cells is also enhanced as compared with the case of conventional digestion treatment. Found to be done. Such an action effect is preferable for the nucleus pulposus stem / progenitor cells by not performing digestion treatment of the nucleus pulposus tissue, that is, growth factors such as angiopoetin-1 (Ang-1) and VEGF-A are present. The microenvironment (nitch) in the tissue that maintains the Tie2-positive cells is not destroyed (in the absence of Ang-1 etc., the Tie2-positive cells eventually lead to apoptosis), and the state of being retained in such a niche. By initiating culture using a cell population containing the nucleus pulposus stem / progenitor cells of Tie2, the activity of Tie2 is maintained (the expression of Tie2 is enhanced as compared with the conventional method of isolating cells from the niche. ) Since the nucleus pulposus stem / progenitor cells can start proliferating rapidly, it is considered that this reflects the improvement in the positive rate and the increase in the expression level as described above in the cell population obtained after culturing.

Moreover, by not performing the work of separating and collecting the cell population from the nucleus pulposus tissue,
Valuable Tie2-positive stem / progenitor cells contained in the nucleus pulposus tissue can be utilized without waste. For example, the amount of nucleus pulposus tissue contained in the hernia portion removed from a herniated disc patient is about 1 to 2 g, and the Tie2-positive stem / progenitor cells contained in 1 g of the nucleus pulposus tissue are (
Although it varies depending on the age of the patient, for example, it is about 50,000. Number of white blood cells contained in the umbilical cord blood 1cc is 10 ⁶ orders, the number of cancer cells contained in cancerous tissue 1g compares the like is 10 ⁸ orders, Tie2 positive stem / progenitor of the nucleus pulposus tissues You can see how precious cells are. In addition to preventing such Tie2-positive stem / progenitor cells from being lost by the work of separating and recovering the cell population from the nucleus pulposus tissue, T
It is extremely beneficial to be favorable for the amplified culture of ie2-positive stem / progenitor cells.

In the "second culture method" of a cell population containing Tie2-positive stem / progenitor cells according to the present invention, Tie2-positive stem / progenitor cells are cultivated in a medium to which at least one Tie2 expression enhancer other than a growth factor has been added. It is a method of culturing a cell population containing the cells.

Tie2-positive stem / progenitor cells originally activate Tie2 by binding to angiopoietin-1 (Ang-1), which is a ligand for Tie2 (receptor tyrosine kinase).
Phosphorylation) is known to be enhanced, and a method of culturing Tie2-positive stem / progenitor cells (enhancing Tie2 expression) in a medium supplemented with Ang-1 is based on prior art (prior art documents, etc.). It is known. Similarly, FGF2 (bFGF) is also known as a growth factor having an action of enhancing the expression of Tie2, and Tie in a medium supplemented with FGF2.
Methods for culturing 2-positive stem / progenitor cells are also known.

However, the present inventors have different types of Ti from growth factors such as Ang-1, FGF2 and the like.
Tie, a plant-derived extract such as an e2 expression enhancer, eg, an extract of cinnamon powder
2 When the expression enhancer is used for culturing a cell population containing Tie2-positive stem / progenitor cells derived from nucleus pulposus tissue, which has not been reported so far, the Tie2 expression enhancer, which is a plant-derived extract, is particularly used as FGF2 or the like. It has been found that when used in combination with a growth factor, there is an effect such as an improvement in the ratio of Tie2-positive stem / progenitor cells (medullary nucleus stem / progenitor cells) in the cell population obtained by culturing.

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

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

Conventionally, like many other stem / progenitor cells, a cell population containing Tie2-positive cells (medullary nucleus stem / progenitor cells) derived from the nucleus pulposus tissue of the intervertebral disc is also described in Patent Document 1 above. It was cultivated so as to form floating spherical colonies under "conditions that interfere with cell adhesion", that is, by using a culture vessel having low adhesion or using a methyl cellulose medium. However, the present inventors have a cell population containing a nucleus pulposus stem / precursor cell, preferably a nucleus pulposus stem / precursor cell in which the expression of Tie2 is enhanced by the first culture method and / or the second culture method as described above. Is not "a condition that interferes with cell adhesion", but conversely, "a culture vessel having a culture surface that has been treated to enhance cell adhesion (cell adhesion treatment)", for example, a coating agent containing polylysine. In a two-dimensional culture environment using a coated culture vessel (without using methyl cellulose or the like), the nucleus pulposus stem / precursor cells can be cultured by adhering to the culture surface without forming spherical colonies. I found it.

By carrying out such a third culture method in the step of the differentiation culture stage, a culture vessel in which the culture surface was not treated (or conversely, a treatment for inhibiting cell adhesion (low adhesion treatment) was performed. Compared with the case of using a culture vessel), the efficiency of differentiation from nucleus pulposus stem / precursor cells into functional nucleus pulposus cells (Col2-positive cells, etc.) can be improved, and Col2 in the cell population can be improved.
A cell population with a high number or proportion of positive cells can be prepared.

The third culturing method can also be carried out in the culturing step of the amplification culturing stage as a method fused with the above-mentioned second culturing method. That is, if Tie2-positive stem / progenitor cells are cultured in a cell-adherent culture vessel and a medium to which a Tie2 activator has been added, Tie2-positive (without using methyl cellulose or the like) in a two-dimensional culture environment. The stem / progenitor cells can be amplified.

In the "fourth culture method" of a cell population containing Tie2-positive stem / progenitor cells according to the present invention, Tie while suppressing the formation of spherical colonies in a medium to which an extracellular matrix degrading agent is added
It is a method of culturing a cell population containing 2 positive stem / progenitor cells.

Conventionally, a substance having an action of decomposing extracellular matrix (collagen, proteoglycan, etc.) such as collagenase or other protease separates cells from the collected tissue as described above in relation to the first culture method. Or, it was usually used temporarily in a culture method in which spherical colonies formed by stem / proteoglycans were dissected once, the medium was replaced, and spherical colonies were formed again.

However, the present inventors have found that a substance (extracellular matrix degrading agent) having an action of degrading extracellular matrix such as collagenase can be used for a completely different purpose (for use). That is, the present inventors have Tie2-positive stem / progenitor cells such as nucleus pulposus stem / progenitor cells capable of differentiating into nucleus pulposus cells, preferably the first culture method and / or the second culture method as described above. Tie2-positive stems whose expression of Tie2 is enhanced by
In the case of progenitor cells, surprisingly, even in a medium supplemented with an extracellular matrix degrading agent, that is, in a situation where the extracellular matrix cannot form spherical colonies in which Tie2-positive stems / progenitor cells are bound to each other. Even so, they have found that Tie2-positive stem / precursor cells can be cultured, and that differentiation can be induced into cells that are positive in the expression of extracellular matrix such as type II collagen and proteoglycan while preferably proliferating.

The present inventors further combine the above-mentioned "third culture method" and "fourth culture method", preferably the type and concentration of the extracellular matrix degrading agent in the medium and the type of the coating agent on the surface of the culture vessel. Properly combine and fuse with to significantly improve the efficiency of differentiation of nucleus pulposus stem / precursor cells into functional nucleus pulposus cells such as type II collagen (Col2) positive cells. It was also found that a cell population in which the number or ratio of Col2-positive cells and the like is significantly higher than before can be prepared.

Moreover, by using the third culture method and / or the fourth culture method, when the number or ratio of Col2-positive cells and the like reaches a certain level, the Tie2-positive stem / progenitor cells are not completely eliminated. It is also possible to obtain a cell population that retains a certain level of number or ratio.
The present inventors have found. By including a certain level of number or ratio of Tie2-positive stem / progenitor cells, such a cell population has the utility of having a more excellent therapeutic effect on the intervertebral disc nucleus pulposus when administered.

Based on each of the above culture methods, the present inventors have differentiated the target cells (C) from the Tie2-positive stem / progenitor cells from the cell population containing the Tie2-positive stems / progenitor cells (medullary nucleus stem / progenitor cells, etc.).
A method for preparing into a cell population containing ol2-positive nucleus pulposus cells, etc.) was constructed. This method for preparing a cell population is a culture step (amplification culture step) for amplifying Tie2-positive stems / progenitor cells in a cell population by proliferating while enhancing the expression of Tie2 in Tie2-positive stems / progenitor cells. And at least one of the culture steps (differentiation culture step) for inducing differentiation of Tie2-positive stem / progenitor cells into target cells, preferably both. The amplification culture step includes a step of carrying out at least one of the above-mentioned first culture method and second culture method, preferably both (which may be a fusion method). The differentiation culture step includes a step of carrying out at least one of the above-mentioned third culture method and fourth culture method, preferably both (these may be a fused method). An amplification culture step that includes the step of performing both the first and second culture methods (preferably as fused methods), and both the third and fourth culture methods (preferably as fused methods). The method for preparing a cell population including a differentiation culture step including a step to carry out is a particularly excellent embodiment in the present invention, and the number or ratio of target cells having a predetermined functionality as compared with a conventionally known preparation method. Can be prepared for a dramatically improved cell population. In some conventional preparation methods, the number of cells in the entire cell population or the number of cells of the nucleus pulposus meets a certain level, but among them, the nucleus pulposus cells having functionality such as positive expression of Col2 Not so many, another, the proportion of Col2-positive cells in the cell population and the expression level of individual cells meet certain levels, but the absolute number of Col2-positive cells is insufficient (
There is a limit to the number of Col2-positive cells that can be increased from the nucleus pulposus tissue that can be collected from one donor)
It was like that. As described above, it was difficult to achieve both the number of Col2-positive cells and the expression level (intensity of expression) in the cell population related to the nucleus pulposus, but the preparation method of the present invention succeeded in achieving both. It can be said that this is an epoch-making preparation method that has not been proposed in.

From another point of view, in order to take advantage of the property that conventionally (Tie2-positive) stem / progenitor cells have scaffold-independent proliferative ability and can form spherical colonies, the stem / progenitor cells are also used. In order to avoid losing the desired functionality by culturing the cells differentiated from the cells on the culture surface (scaffold), the cell population contained in the collected tissue is subjected to a methylcellulose medium or low adhesion. A method of differentiating the stem / progenitor cells into target cells having a predetermined functionality while culturing on the culture surface has been adopted. The present inventors have described each of the above culture methods (
In particular, by the third culture method and the fourth culture method), without using methyl cellulose, which is highly viscous and makes it difficult to collect cells, and in an environment where cells are allowed to adhere to the culture surface ( Tie2-positive) The efficiency of proliferation of stem / progenitor cells and differentiation of the stem / progenitor cells into target cells having predetermined functionality is remarkably enhanced, and the produced cell population can be easily and efficiently recovered from the medium. It can be said that we have found a timely method.

In connection with the above culture methods and preparation methods, especially in relation to the first culture method and the amplification culture step, we present Tie2-positive stems / precursors in the presence in undigested tissue. By cryopreserving a cell population containing cells, a Tie2-positive stem that can maintain the activated and / or expressed state of Tie2 or suppress the decrease of Tie2-positive stem / progenitor cells in the cell population. / We have found a preferred method for preserving a cell population containing progenitor cells. Conventionally, a cell population containing Tie2-positive stem / progenitor cells contained in the collected intervertebral disc nucleus pulposus tissue is separated from the tissue by digestion treatment using collagenase or the like, which takes a relatively long time, and then only the cell population is frozen. Preservation was done. However, immediate cryopreservation of the collected disc nucleus pulposus improves work process convenience and maintains a good niche in the tissue (especially from young donors). The cell population can be maintained as it is. After thawing such cryopreserved tissue, the thawed tissue is placed in a medium and the cell population is cultured by the first culture method described above, and T.
ie2-positive stem / progenitor cells can be efficiently amplified.

If the above-mentioned technical idea is embodied in combination with a (preferably) embodiment described in detail later, the present invention can be expressed as, for example, an invention including at least the following matters.

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

[6]
A method for 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, the T
A method of culturing a cell population containing ie2-positive stem / progenitor cells (hereinafter referred to as "second culture method".
).
[7]
Item 2. The second item according to Item 6, wherein the Tie2 expression enhancer other than the growth factor is a plant-derived extract.
Culture method.
[8]
Item 2. The second culture method according to Item 7, wherein the plant is a plant belonging to the genus Cinnamomum.
[9]
Item 2. The second culture method according to any one of Items 6 to 8, which is carried out when amplifying the Tie2-positive stem / progenitor cells in a cell population.

[10]
A method for culturing a cell population containing Tie2-positive stem / progenitor cells.
A method of culturing a cell population containing the Tie2-positive stem / progenitor cell in a culture vessel having a culture surface treated to enhance cell adhesion (hereinafter referred to as "third culture method").
[11]
The Tie2-positive stem / progenitor cells have been subjected to the Tie2 expression enhancing treatment.
Item 3. The third culture method according to Item 10.
[12]
Item 3. The third culture method according to Item 10 or 11, wherein the treatment for enhancing the adhesion of cells is a treatment for applying a coating agent containing an extracellular matrix or other bio-related molecules.
[13]
Item 3. The third culture method according to any one of Items 10 to 12, which is carried out when differentiating the Tie2-positive stem / progenitor cells in a cell population into target cells.
[14]
Item 12 using at least one selected from the group consisting of type IV collagen, fibronectin and polylysine as the extracellular matrix or other bio-related molecule.
Alternatively, the third culture method according to 13.
[15]
Item 3. The third culture method according to any one of Items 10 to 14, which is carried out when amplifying the Tie2-positive stem / progenitor cells in a cell population.
[16]
Item 3. The third culture method according to any one of Items 12 to 15, wherein gelatin is used as the extracellular matrix.

[17]
A method for culturing a cell population containing Tie2-positive stem / progenitor cells.
A method of culturing a cell population containing the Tie2-positive stem / progenitor cell in a medium to which an extracellular matrix-degrading agent has been added while suppressing the formation of spherical colonies (hereinafter, "fourth culture method").
Called. ).
[18]
The Tie2-positive stem / progenitor cells have been subjected to the Tie2 expression enhancing treatment.
Item 4. The fourth culture method according to Item 17.
[19]
Item 4. The fourth culture method according to Item 17 or 18, wherein the extracellular matrix degrading agent contains a protease having at least degrading activity for type II collagen.
[20]
Item 4. The fourth culture method according to any one of Items 17 to 19, which is carried out when differentiating the Tie2-positive stem / progenitor cells in a cell population into target cells.

[21]
A method for preparing a cell population containing Tie2-positive stem / progenitor cells.
The expression of Tie2 in Tie2-positive stem / progenitor cells is enhanced and T in the cell population includes the step of carrying out the first culturing method according to Item 5 and / or the second culturing method according to Item 9.
Culture stage for amplifying ie2-positive stem / progenitor cells (hereinafter referred to as "amplification culture stage")
Preparation method including.
[22]
Item 2. The preparation method according to Item 21, wherein the step carried out in the amplification culture step is a step of simultaneously carrying out the first culture method and the second culture method.
[23]
Item 21 or 22, wherein the amplification culture step further comprises a step of culturing a cell population containing Tie2-positive stem / progenitor cells in a medium to which only a growth factor having a Tie2 expression-enhancing effect is added as a Tie2 expression-enhancing agent. The preparation method described.

[24]
A method for preparing a cell population containing target cells differentiated from the Tie2-positive stem / progenitor cell from a cell population containing the Tie2-positive stem / progenitor cell.
A culturing step for inducing differentiation of Tie2-positive stem / progenitor cells into target cells, which comprises the step of carrying out the third culturing method according to Item 13 or 14 and / or the fourth culturing method according to Item 20. Called the "differentiation culture stage")
Preparation method including.
[25]
Item 2. The preparation method according to Item 24, wherein the step carried out in the differentiation culture step is a step of simultaneously carrying out the third culture method and the fourth culture method.
[26]
Item 24 or 25, wherein the target cell is a cell expressing at least type II collagen.
The preparation method described in.
[27]
Item 2. The preparation method according to Item 26, wherein the cell expressing at least type II collagen is a nucleus pulposus cell.
[28]
Item 8. The preparation method according to any one of Items 24 to 27, wherein a cell population in which Tie2-positive stem / progenitor cells also remain is obtained by the differentiation culture step.
[29]
A method for preparing a cell population containing Tie2-positive stem / progenitor cells to a cell population containing target cells differentiated from the Tie2-positive stem / progenitor cells.
A preparation method comprising the amplification culture step according to any one of Items 21 to 23 and the differentiation culture step according to any one of Items 24 to 28.

[30]
A cell population obtained by the culture method according to any one of Items 1 to 20.
[31]
A culture containing the medium in the culturing method according to any one of Items 1 to 20 and the cultivated or obtained cell population used in the culturing method.
[32]
A cell population obtained by an amplification culture step or a differentiation culture step in the preparation method according to any one of Items 21 to 29.
[33]
Item 2. The medium for the amplification culture stage or the medium for the differentiation culture stage in the preparation method according to any one of Items 21 to 29, and the culture medium to be subjected to the amplification culture stage or the differentiation culture stage, or obtained A culture containing a cell population.

[34]
A composition for cell therapy, which comprises the cell population according to item 30 or 32.
[35]
34. The cell therapeutic composition according to Item 34, which is for treating or preventing a disease in which a disorder, degeneration or hernia of an intervertebral disc appears as a symptomatology.

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

According to the method for culturing Tie2-positive stem / progenitor cells of the present invention, an amplification culture step mainly aimed at amplifying Tie2-positive stems / progenitor cells, and mature cells having predetermined traits from Tie2-positive stems / progenitor cells. A cell population rich in target cells can be prepared by a culture method including a differentiation culture step whose main purpose is to induce differentiation into. By utilizing the cell population obtained based on such a culture method of the present invention, it becomes possible to efficiently produce an effective cell preparation for the treatment or prevention of a predetermined disease.

Further, in the present invention, it is not necessary to add a highly viscous component such as methyl cellulose used in the prior art described in the above-mentioned Patent Documents 1 and 2 to the medium.
It is possible to recover a cell population containing Tie2-positive stem / progenitor cells or target cells induced to differentiate from the Tie2-positive stem / progenitor cells from the medium without waste.

According to a typical embodiment of the present invention, a Tie2-positive stem / progenitor cell (nuclear nucleus stem cell) contained therein is used by using an intervertebral disc (nucleus pulposus) that can be collected only in a small amount by surgery of a herniated disk patient. Etc.), which are expected to have a high therapeutic effect when transplanted by efficiently proliferating and differentiating), and are rich in (and somewhat) functional nucleus pulposus cells with high extracellular matrix-producing ability such as type II collagen. A large number of cell populations (where Tie2-positive stem / progenitor cells also remain) can be easily and reproducibly obtained. Conventionally, it has been impossible to prepare such a suitable cell population, but since it is possible by the present invention, it is dramatically easier to perform intervertebral disc regeneration therapy by administration of (cell preparation containing) the cell population. Therefore, industrialization becomes realistic.

It is expected that the reason why the fourth culture method of the present invention exerts its action and effect is that, for example, the principle shown in FIG. 1 is working. However, this conjecture is intended to assist the understanding of the present invention and does not unnecessarily bind the present invention. Even if it is found after the fact that a part or all of the action and effect of the present invention is performed based on a principle and a mechanism of action different from those shown in FIG. The action and effect of the invention and the constituent requirements of the present invention for that purpose are not denied by the explanation based on FIG. 1 below.

FIG. 1 [A] shows the state of cultured cells when a cell population containing Tie2-positive stem / progenitor cells (for example, nucleus pulposus stem / progenitor cells) is proliferated and differentiated by two-dimensional culture (monolayer static culture). Represents. The culture surface of the culture vessel (flask, etc.) is prepared in advance by extracellular matrix (ECM).
In some cases, surface treatment is applied so that cells can adhere, such as by applying a coating agent containing. The stem / progenitor cells attach to the culture surface of the culture vessel and differentiate while extending and proliferating on the culture surface. In such two-dimensional culture, the ECM applied on the culture surface or the ECM secreted from the cultured cells is eventually affected by the interaction between the binding protein (integrin, etc.) expressed on the surface of the cultured cells. Integrin transmission occurs, which stops production and secretion. For example, in the case of two-dimensional culture of a nucleus pulposus-derived cell population, the nucleus pulposus stem / progenitor cells contained in the cell population proliferate and differentiate together with the mature nucleus pulposus cells originally contained in the cell population. ECMs such as type II collagen and proteoglycan are actively secreted from the mature nucleus pulposus cells produced in the above process. However, with the lapse of the culture period, the production and secretion of type II collagen and the like are stopped (instead of that) due to the intracellular signal transduction as described above.
Increased production and secretion of type I collagen), leading to dedifferentiation of mature nucleus pulposus cells, including a fibroblast-like phenotype. Therefore, in general two-dimensional culture, it is considered difficult to achieve both an increase in the number of cells in a cell population and an increase in the proportion of cells that retain a specific trait (not dedifferentiated). In the third culture method of the present invention, preferably, by using Tie2-positive stem / progenitor cells in which the expression of Tie2 is enhanced, a certain amount of nucleus pulposus cells expressing type II collagen or the like can be obtained even in two-dimensional culture. It is possible to relatively easily prepare a cell population containing a proportion.

1 [B] and [C] show a cell population containing Tie2-positive stem / progenitor cells (eg, nucleus pulposus stem / progenitor cells) in a methylcellulose-containing medium (extracellular matrix (ECM) degrading agent not added). Alternatively, it shows the state of cultured cells when they are proliferated and differentiated by culturing using a low-adsorption culture vessel. Unlike the two-dimensional culture shown in FIG. 1 [A], in the culture shown in FIG. 1 [B], the ECM or the like on the culture surface of the culture vessel does not interact with the cultured cells, and the ECM due to the interaction does not occur. There is also no intracellular signaling that stops the production and secretion of. Therefore, in the early stages of culture, Tie2-positive stem / progenitor cells proliferate while producing and secreting ECM, eventually forming spherical colonies. However, in the formed spherical colonies, Tie2-positive stem / progenitor cells or cells differentiated from them (for example, nucleus pulposus cells) come into contact with each other via secreted ECM. Therefore, FIG. 1 [C]
As shown in FIG. 1, the interaction between the ECM and the cultured cells began to occur with the lapse of the culture period, and the interaction generated an ECM production stop signal, which caused the same dedifferentiation as in FIG. 1 [A]. .. Therefore, in the culture method as shown in FIGS. 1 [B] and 1 [C], a predetermined ECM (for example, type II collagen) is used in the cell population collected as spherical colonies.
It is difficult to increase the proportion of cells that are positive for expression above a certain level.

FIG. 1 [D] shows a cell population containing Tie2-positive stem / precursor cells in a medium supplemented with an extracellular matrix (ECM) degrading agent (does not contain methyl cellulose or the like) according to the fourth culture method of the present invention. The state of Tie2-positive cultured cells when proliferated and differentiated by culturing using. Also in such a culture method, ECM is secreted from stem / progenitor cells or cells differentiated from them, as in FIG. 1 [B]. However, since the ECM-degrading agent added to the medium constantly decomposes the ECM secreted extracellularly, spherical colonies are not formed, and the cells are displayed on the culture surface without using a low-adsorption culture vessel. Does not adhere. Further, in the third and fourth culture methods of the present invention, even if a coating agent containing ECM is previously applied to the culture surface of the culture container as in the two-dimensional culture of FIG. 1 [A], the culture surface. The adhesion of cells to the cells remains weak. Therefore, the ECM production cessation signal due to the interaction between ECM and cultured cells is suppressed, and FIGS. 1 [A], 1 [B] and [C]
], Since dedifferentiation as in the case of [] is less likely to occur, it becomes possible to prepare a cell population in which the ratio of cells positive for the expression of a predetermined ECM (for example, type II collagen) is higher than that of the prior art.

The ECM secreted extracellularly by the ECM degrading agent in the medium is decomposed, but the intracellular ECM is not decomposed and the accumulation proceeds. Therefore, if the fourth culture method of the present invention is carried out in the step of the differentiation culture stage, and then the obtained cell population is recovered from the medium to produce a cell preparation, the cells in the tissue to which the cell preparation is administered are treated. The ECM accumulated inside can be rapidly secreted extracellularly to create an environment suitable for the survival of the cell population, and the subsequent production and secretion of ECM from the administered cells (that is, treatment with a cell preparation) It is expected that the effect) can be promoted.

FIG. 1 shows a normal two-dimensional culture (single-layer static culture), a suspension culture using a medium containing no conventional extracellular matrix (ECM) degrading agent, and an ECM degrading agent according to the fourth culture method of the present invention. A diagram schematically showing the interaction between the production or degradation of ECM and the cultured cells in each of the suspension cultures using the medium containing the above is shown, and a photograph of each cultured cell is shown. A (normal two-dimensional culture): ECM production stop signal is transmitted by adhering nucleus pulposus (NP) cells to the culture surface of a culture flask via adherent molecules. B (suspended culture in a low-adsorption flask, methylcellulose medium, etc.): There is no surface contact with the flask culture, and the ECM production stop signal is not transmitted (x mark and dotted arrow). C (no enzyme in the medium): The ECM produced by itself shows the same action as the culture surface (arrow), and the ECM production stop signal is transmitted into the cell. D (enzyme in the medium): The extracellular ECM produced by itself is decomposed and the ECM production stop signal is not transmitted into the cell (x mark and dotted arrow), but the ECM is accumulated in the cell. FIG. 2 is a graph showing the result of the Tie2 positive rate in Test Example 1 (amplification culture step: first culture step). FIG. 3 is a graph showing the result of Tie2 average luminescence intensity (MFI) in Test Example 1 (amplification culture step: first culture step). FIG. 4 is a graph showing the result of the Tie2 positive rate in Test Example 2 (amplification culture step (2 steps): 1st and 2nd culture steps + additional steps). FIG. 5 is a graph showing the result of the number of Tie2-positive cells produced per 1 g of nucleus pulposus tissue in Test Example 2 (amplification culture step (2 steps): 1st and 2nd culture steps + additional steps). FIG. 6 shows the results of the type II collagen (Col2) positive rate in Test Example 3 (amplification culture step (2 steps): 1st and 2nd culture steps + additional step → differentiation culture step: 3rd culture step). It is a graph. FIG. 7 shows type II collagen produced per 1 g of nucleus pulposus tissue in Test Example 3 (amplification culture step (2 steps): 1st and 2nd culture steps + additional step → differentiation culture step: 3rd culture step). (Col2) is a graph showing the result of the number of positive cells. FIG. 8 shows the results of the proteoglycan (PG) positive rate in Test Example 4 (amplification culture step (2 steps): 1st and 2nd culture steps + additional step → differentiation culture step: 3rd and 4th culture steps). It is a graph showing. FIG. 9 shows the positive rate of type II collagen (Col2) in Test Example 4 (amplification culture stage (2 stages): 1st and 2nd culture steps + additional step → differentiation culture stage: 3rd and 4th culture steps). It is a graph showing the result. FIG. 10 shows proteoglycan (PG) and type II in Test Example 5 (amplification culture step (2 steps): 1st and 2nd culture steps + additional step → differentiation culture step: 3rd and 4th culture steps 2). It is a graph which shows the result of the positive rate of each collagen (Col2). GEL: gelatin, Col1: type I collagen, Col4: type IV collagen, FN: fibronectin, PLL: poly-L-lysine (same for FIG. 11). FIG. 11 shows proteoglycan (PG) and type II in Test Example 6 (amplification culture step (2 steps): 1st and 2nd culture steps + additional step → differentiation culture step: 3rd and 4th culture steps 3). It is a graph which shows the result of the positive rate of each collagen (Col2). FIG. 12 is an optical micrograph of the cell population in Test Example 5-12.

− Terminology −
"Stem cells" are self-renewal and differentiation (totipotent), pluripoten
t), a term that refers to cells with multipotent or unipotent). "Progenitor cells" do not have self-renewal ability in a strict sense because they eventually become all terminally differentiated cells, but they have the ability to differentiate into predetermined cells while proliferating relatively actively. It is a term that refers to a cell having. Cells commonly understood (specified) by those skilled in the art by names including "stem cells" or "progenitor cells" fall under "stem cells" or "progenitor cells" herein.

In the present specification, "stem cell and / or progenitor cell" is a notation including stem cell, progenitor cell or both, and may be referred to as "stem / progenitor cell". Further, in the present specification, a cell population containing stem cells and / or progenitor cells is referred to as "stem / progenitor cell population", and a cell population containing mature cells (terminal differentiated cells) differentiated from stem cells and / or progenitor cells. May be referred to as "mature cell population".

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

Whether the expression of a marker gene (cell marker) is "positive" or "negative" depends on the general method of mRNA transcribed from the gene (genome) or its mRN.
The expression level of the protein translated from A is measured quantitatively or qualitatively, and when the expression level is above a certain level (or exceeds a certain level), it is positive and below a certain level (or meets a certain level). If there is no such thing, it can be judged as negative. The expression level of a protein can be measured quantitatively or qualitatively by an immunological assay using an antibody and a labeling agent specific to the protein, such as flow cytometry, immunostaining, and ELISA. The Tie2 protein is a protein expressed on the cell surface, and C
ol2 is a protein expressed inside the cell, and an appropriate method (immunofluorescence staining method, etc.) for detecting the protein existing on the cell surface and inside the cell may be used.
The expression level of mRNA is quantitatively or qualitatively measured by an assay using (complementary) nucleic acid specific to the mRNA such as RT-PCR, microarray, biochip, and a labeling agent or nucleic acid amplification method (means). Can be measured. The ratio (positive rate or negative rate) of cells in the cell population in which the expression of a predetermined marker gene (cell marker) is positive or negative is determined by the number of all cells in the cell population and the method as described above. Alternatively, the number of cells determined to be negative can be calculated by various methods as described above, for example, by measurement in flow cytometry.

In the present specification, "stem cells and / or progenitor cells positive for Tie2 expression", that is, "Tie2-positive stem / progenitor cells" is known as one of the cell markers.
2 (tyrosine kinase with Ig and EGF homology domain-2) refers to a cell having a trait as a stem cell and / or a progenitor cell whose expression as a protein is determined to be positive by, for example, flow cytometry. .. Typical Tie2-positive stem / progenitor cells in the present invention are Tie2-positive stem / progenitor cells "derived from the nucleus pulposus tissue of the intervertebral disc", that is, Tie2-positive (recoverable from the nucleus pulposus) present in the nucleus pulposus of the intervertebral disc. It is a Tie2-positive stem / progenitor cell obtained by subculturing a stem / progenitor cell or its Tie2-positive stem / progenitor cell, and is a cell corresponding to the "nucleus-nucleus stem / progenitor cell" described below.

In the present specification, the "target cell" is a cell having functionality according to a use, which is obtained from a Tie2-positive stem / progenitor cell by a predetermined differentiation induction, and more specifically, a predetermined gene (cell marker). Expression, for example, expression as a protein by flow cytometry,
Refers to cells that are determined to be positive or negative. A typical target cell in the present invention is an extracellular matrix (ECM) such as Col2 and aggrecan among the “medullary nucleus cells” described below.
) Gene expression is positive.

In the present invention, "nucleus cell" refers to a mature cell that has reached terminal differentiation, or a cultured cell having a trait equivalent thereto, which occupies the majority of the cell population in the intervertebral disc (nucleus nucleus). Specifically, nucleus pulposus cells are negative for Tie2 and GD2 (and usually positive for CD24) as marker genes, and are positive for at least type II collagen in the extracellular matrix (more usually). It can be defined as a cell (which is also positive for proteoglycan (aglycan)). For example, a cell determined by flow cytometry to be negative for Tie2 and GD2 (and positive for CD24) and positive for type II collagen (and positive for aggrecan) as proteins (cell markers) is the present invention. Corresponds to the nucleus pulposus cells in. For extracellular matrix such as type II collagen and aggrecan, the amount of protein produced is measured by flow cytometry and their mRN.
The expression level of A may be measured by real-time PCR or the like.

In the present invention, the "medullary nucleus stem / progenitor cell" is a precursor cell (nucleus nucleus progenitor cell) that occupies a part of the cell population in the nucleus pulposus tissue of the intervertebral disc and has at least the ability to differentiate into the nucleus pulposus cell. Stem cells (nucleus stem cells) having the ability to differentiate into and self-renewal, or cultured cells having equivalent traits are collectively referred to. Nucleoside stem / progenitor cells can be specifically defined as Tie2 and / or GD2 positive cells as marker genes. For example, by flow cytometry, as proteins (cell markers), Tie2 is positive and GD2 is negative, Tie2 is positive and GD2 is positive, or Tie2 is negative and GD2.
A cell determined to be either positive or positive corresponds to the nucleoside stem / progenitor cell in the present invention.

In the above-mentioned Patent Document 2, Tie2 and GD2 are used as cell markers for nucleus pulposus-derived cells.
Tie2-positive cells are referred to as "intervertebral disc nucleus pulposus stem cells" (of which G
D2 negative cells are dormant, GD2 positive cells are active), Tie2 negative and GD2 positive cells are "disc progenitor cells", Tie2 negative and GD2 are Negative cells are classified as "differentiated, mature disc nucleus pulposus cells" (paragraphs 0024, 0025, 0032). Further, in Patent Document 2, the cells appearing in the differentiation hierarchy of nucleus pulposus cells are (i) Tie2-positive and GD2-negative (furthermore, CD24-negative, CD44-positive / negative, CD271-positive, Flt1-positive) cells, (ii).
) Tie2 positive and GD2 positive (further CD24 negative, CD44 positive, CD271 positive,
Flt1 positive) cells, (iii) Tie2 negative and GD2 positive (and CD24 negative, CD44 positive, CD271 positive / negative, Flt1 positive / negative) cells, (iv) Ti
e2 negative and GD2 positive (further CD24 positive, CD44 positive, CD271 negative, Flt
1 negative) cells, (v) Tie2 negative and GD2 negative (further CD24 positive, CD4)
The cells are classified into 4 positive, CD271 negative, and Flt1 negative), and are classified into the above (i) to (i).
For ii), "NP stem / progenitor cells", above (iii)
The notation "NP committed cells" is used for ~ (v) (see Fig. 7-2). Although the notation is different, the “medullary nucleus medullary stem cells” and “intervertebral plate medullary nucleus progenitor cells” of Patent Document 2, that is, the cells (i) to (iV) above are the “medullary nucleus stem / progenitor cells” in the present invention. Corresponding to "cells", Patent Document 2 "Mature intervertebral plate nucleus pulposus cells that have completed differentiation",
That is, the cell of (v) above corresponds to the "maturated nucleus pulposus cell" in the present invention. If necessary, the cell in the present invention is defined as the definition described in Patent Document 2 (particularly, CD24, etc., Tie2.
And the definition of positive or negative for one or more cell markers other than GD2)
It is possible to replace the cells according to.

In the present invention, a "spherical colony" is a spherical cell aggregate that includes stem cells and / or progenitor cells, and may further include cells differentiated from them. "Spherical colony"
Is an object generally called "sphere", "spheroid", etc. by those skilled in the art, and is a "discosphere" or a "free floating circular structure" (free floating circular) in Patent Document 2 above. -spherical structure) is also an object corresponding to a "spherical colony".

In the present invention, "Tie2 expression is enhanced" (Tie2 expression enhancement) means that the expression of the Tie2 gene is enhanced in individual stem / progenitor cells, that is, the expression is enhanced more than usual, and mRNA or mRNA or It means that the expression level as a protein is increasing. Even under conditions in which the expression of the Tie2 gene is normally almost eliminated, the expression does not disappear and the expression level is maintained at a constant level, that is, the expression of Tie2 is maintained.
It corresponds to "the expression of Tie2 is enhanced". In addition, as a result of such enhanced expression of Tie2 in individual stem / progenitor cells, the mRN of Tie2 in the cell population
Increasing the number of cells determined to be positive for A or protein expression, that is, the proportion of Tie2-positive cells in the cell population being higher than usual, is also "Tie2 expression enhancement".
It can be understood that it is a manifestation of.

More specifically, for example, a cell population (Tie) that has been previously subjected to a Tie2 expression enhancing treatment.
2 T on the cell surface for the cell population (control group) that has not been subjected to the expression enhancement treatment group)
Cells that are treated to fluorescently label the ie2 protein and are judged to have higher fluorescence intensity than a predetermined level and positive expression in the Tie2 expression-enhancing treatment group compared to the control group by measurement by flow cytometry. When the ratio of Tie2 is high and / or the average fluorescence intensity per cell is high, the expression of Tie2 is enhanced in the cell population (Tie2-expressing cells contained in) of the Tie2 expression-enhancing treatment group (in other words, For example, the Tie2 expression enhancing treatment plays a predetermined role). Furthermore, in morphological observation, cells with enhanced Tie2 expression can be distinguished by having a spindle shape (cells that do not have a nearly spherical shape).

In the present invention, an agent that exerts the above-mentioned action and effect of "Tie2 expression enhancer" is referred to as "Tie2 expression enhancer" in the present invention. In addition, some growth factors (FGF2, etc.) have a Tie2 expression enhancing action and can be said to correspond to a kind of "Tie2 expression enhancing agent". Therefore, when such a growth factor is excluded, "other than the growth factor" It is called "Tie2 activation expression enhancer".

In the present invention, carrying out the first culture method and / or the second culture method is Tie.
It corresponds to performing "Tie2 expression enhancement treatment" on a cell population containing 2 positive stem / progenitor cells.

-Culture method-
The first to fourth culture methods of a cell population containing Tie2-positive stem / progenitor cells according to the present invention are as follows:
First culture method: A method of culturing a cell population containing Tie2-positive stem / progenitor cells in a state of being present in undigested tissue;
Second culture method: A method of culturing a cell population containing Tie2-positive stem / progenitor cells in a medium supplemented with at least one Tie2 expression enhancer other than a growth factor;
Third culture method: A culture container having a culture surface that has been treated to enhance cell adhesion.
Method of culturing a cell population containing ie2-positive stem / progenitor cells;
Fourth culture method: A method of culturing a cell population containing Tie2-positive stem / progenitor cells in a medium to which an extracellular matrix-degrading agent has been added, while suppressing the formation of spherical colonies.

The first to fourth culture methods of the present invention may be carried out individually, or may be carried out sequentially or simultaneously in combination of two or more. To combine and simultaneously carry out a plurality of culture methods selected from the first to fourth culture methods means to fuse the selected culture methods, that is, a culture method that satisfies all the technical matters related to the selected culture method. Means to carry out. For example, the first culture method and the second culture method can be carried out sequentially or simultaneously (fused) in combination (the method in which these are fused may be referred to as "first and second culture methods". )
.. The third culture method and the fourth culture method can also be carried out sequentially or simultaneously (fused) in combination (the method in which these are fused may be referred to as "third and fourth culture methods").

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

In the present specification, the descriptions regarding the first to fourth culture methods (further, the first to fourth culture steps for carrying out them) are cases where each is carried out as an independent method (step) unless otherwise specified. Not only that, it can be appropriately read as a description when it is carried out as a method (process) fused with another method (process).

The Tie2-positive stem / progenitor cells contained in the cell population to which the fourth culture method of the present invention is applied and the cells differentiated from the stem / progenitor cells are subjected to normal medium containing no extracellular matrix degrading agent. Cells that bind to each other by an extracellular matrix secreted extracellularly to form spherical colonies (spheroids). When an extracellular matrix degrading agent is added to a medium according to the present invention, spherical colonies (spheroids) are formed. The type of cell is not particularly limited as long as it is a cell that exerts the action and effect of being suppressed.

In a representative embodiment of the present invention, cells differentiated from Tie2-positive stem / progenitor cells are
A cell that produces and secretes more extracellular matrix than a general cell, for example, a nucleus pulposus cell that is responsible for 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,
In addition, it expresses extracellular matrix such as proteoglycan (aglycan). In a preferred embodiment of the present invention, cells expressing extracellular matrix such as type II collagen and proteoglycan (aglycan) from Tie2-positive stem / precursor cells, particularly type II collagen, are expressed not only as mRNA but also as protein. It makes it possible to differentiate functional collagen cells with excellent amount (production amount).

-Preparation method (culture process)-
The method for preparing a cell population containing Tie2-positive stem / progenitor cells to a cell population containing target cells differentiated from Tie2-positive stem / progenitor cells according to the present invention is the following amplification culture step and / or differentiation culture step. Includes both the amplification and differentiation stages (preferably the amplification and culture stages first, the differentiation and culture stages later):
Amplification culture stage: A culture stage for enhancing Tie2 expression in Tie2-positive stem / progenitor cells and amplifying Tie2-positive stem / progenitor cells in a cell population;
Differentiation culture stage: A culture stage for inducing differentiation of Tie2-positive stem / progenitor cells into target cells.

-Steps Related to Amplification Culture Stage In a preferred embodiment of the present invention, the first culture method and the second culture method are carried out in the step of the amplification culture stage. As the first culture method and the second culture method, only one of them may be carried out, or both may be carried out. When both the first culture method and the second culture method are carried out, the step of carrying out the first culture method in the amplification culture step (in the present specification, "
It is called "first culture step". ) And the step of carrying out the second culturing method (referred to as "the second culturing step" in the present specification) may be a separate step performed sequentially (the first culturing step comes first, the second culturing step comes first). A single step (in the present specification, "first and second culturing steps") in which the two culturing methods are carried out at the same time (the first and second culturing methods in which they are fused are carried out).
It is called. ), That is, a step of culturing a cell population containing Tie2-positive stem / progenitor cells in a medium present in a tissue that has not been digested and in which a Tie2 expression enhancer has been added.

The main purpose of the "step of amplification culture stage" is to amplify Tie2-positive stem / progenitor cells by culturing according to predetermined conditions, and the action and effect for that purpose (relatively stronger than other action and effects). ) Means the process to be performed. That is, if the number and / or ratio of Tie2-positive stem / progenitor cells is higher in the post-cultured cell population than in the pre-cultured cell population, the culturing step is the "amplification culturing step". It is permissible for Tie2-positive stem / progenitor cells to differentiate into other cells (target cells) within that limit.

In the present invention, T in individual Tie2-positive stem / progenitor cells (eg, nucleoside stem / progenitor cells)
Along with enhancing the expression of ie2 (including maintaining the state in which Tie2 is expressed),
Since the action and effect such as increasing the number and / or ratio of Tie2-positive stem / progenitor cells contained in the cell population can be synergistically enhanced, the first and second culture methods are carried out as the steps of the amplification culture step. (That is, the first and second culture steps are carried out) is particularly preferable.

-Steps Related to Differentiation and Culture Stages In a preferred embodiment of the present invention, the third culture method and the fourth culture method are carried out in the steps of the differentiation culture stage. As the third culture method and the fourth culture method, only one of them may be carried out, or both may be carried out. When both the third culture method and the fourth culture method are carried out, the step of carrying out the third culture method in the differentiation culture stage (in the present specification, "
It is called "third culture step". ) And the step of carrying out the fourth culturing step (referred to as the "fourth culturing step" in the present specification) may be a separate step carried out sequentially, or two culturing methods are carried out simultaneously (they are fused). A single step (referred to as "third and fourth culture step" in the present specification), that is, a cell population containing Tie2-positive stem / precursor cells, is defined as cells. It may be a step of culturing while suppressing the formation of spherical colonies in a culture vessel having a culture surface treated to enhance the adhesion of the cells and in a medium to which an extracellular matrix degrading agent is added.

The main purpose of the "step of differentiation culture stage" is to differentiate Tie2-positive stem / progenitor cells into predetermined cells by culturing according to predetermined conditions, and the action and effect for that purpose (more than other action and effects). It means a process that is played (relatively strongly). That is, if the number and / or ratio of the target cells is higher in the post-cultured cell population than in the pre-cultured cell population, the culturing step can be said to be a “differentiation culture step”.

As described above, the spherical colonies (spheroids, spheroids) described in Patent Document 1.
The step of temporarily treating in a medium to which collagenase is added in order to dissociate (disk sphere, floating sphere structure) is the fourth culture method of the present invention or the differentiation culture step defined as described above. It does not correspond to the fourth culture step as a step. In addition, a method (step) of treating with collagenase or the like to isolate the cell population contained in the collected tissue, or trypsin for subculturing the cells proliferated in general two-dimensional culture. The method (step) of treating and dissociating cells from the culture surface also does not correspond to the fourth culture method of the present invention defined as described above or the fourth culture step as a step of the differentiation culture step.

In the present invention, the number and / or ratio of cells having predetermined functionality (for example, Col2-positive nucleus pulposus cells) differentiated from Tie2-positive stem / progenitor cells (for example, nucleus pulposus stem / progenitor cells) contained in the cell population is determined. On the other hand, since it is possible to synergistically enhance the action and effect such as maintaining a constant level of the number and / or ratio of Tie2-positive stem / progenitor cells, the third and fourth culture methods are carried out as a step of the differentiation culture stage. (That is, the third and fourth culture steps are carried out) is particularly preferable.

If necessary, the amplification culture step may further include steps other than the first culture step and / or the second culture step, which meet the purpose of the step of amplifying Tie-positive stem / progenitor cells. As such a step, for example, a step of culturing a cell population containing Tie2-positive stem / progenitor cells in a medium to which only a growth factor having a Tie2 expression-enhancing effect as a Tie2 expression-enhancing agent is added (in the present specification, " It is referred to as "additional amplification culture step"). Examples of growth factors having a Tie2 expression-enhancing effect in the additional amplification culture step include, for example.
FGF and / or EGF can be mentioned. The additional amplification culture step includes the first culture step and /
Alternatively, it is preferably performed after the second culturing step, particularly the first culturing step or the first and second culturing steps. Further, in the additional amplification step, the first culture method is not carried out, that is, Tie2.
It is appropriate that the cell population containing positive stem / progenitor cells should be separated from the cells by digestion rather than being present in undigested tissue. In the first culture method of the present invention carried out in the first culture step or the first and second culture steps, a cell population containing Tie2-positive stem / progenitor cells is cultured in a state of being present in undigested tissue. However, when it reaches a certain level, it is thought that it is due to the existence in the organization, but Ti
The amplification of e2-positive stem / progenitor cells is suppressed (the Tie2-positive stem / progenitor cells are not amplified even if the culture period is extended). Therefore, after the first culture step or the first and second culture steps, the tissue is digested, the separated cell population is collected, and an additional amplification step is performed to further amplify the Tie2-positive stem / progenitor cells. can do.

<Cell population>
In a cell population containing Tie2-positive stems / progenitor cells (collectively referred to as "pre-cultured cell population" in the present specification) used in each culture method or each culture step of the present invention, Tie2-positive stems /
The ratio and / or number of progenitor cells and other cells (cells differentiated from Tie2-positive stem / progenitor cells, etc.) is basically arbitrary, and the ratio of Tie2-positive stem cells to Tie2-positive progenitor cells is also basic. Is optional. The composition of the pre-cultured cell population can be appropriately adjusted according to the embodiment of the invention, taking into consideration the action and effect in each culture method or each culture step.

The pre-cultured cell population can be prepared or prepared according to a conventional method, except when it is subjected to the first culture method or the first culture step. For example, when using a cell population contained in the intervertebral disc nucleus pulposus tissue collected from the body as a pre-cultured cell population, the nucleus pulposus tissue is first chopped to an appropriate size using an instrument such as a scissors (eg: After mincing to a few millimeters square), the cells are then treated with a proteolytic enzyme such as collagenase to disperse the cells, and if necessary, treatments such as filtration, centrifugation, and washing are performed to form the nucleus pulposus tissue. The contained cell population can be isolated and recovered. The cell population thus obtained can be used as a pre-culture cell population other than the first culture method or the first culture step.

On the other hand, in the first culturing method or the first culturing step of the present invention, in the above procedure, the nucleus pulposus tissue was stopped at the stage of being shredded (without treatment with a proteolytic enzyme) and shredded. The cell population contained in the nucleus pulposus tissue is used as the pre-cultured cell population.

The cell population separated from the tissue or the cell population contained in the tissue (tissue containing the cell population) prepared as described above is subjected to the next culturing method or culturing step. Can be cryopreserved according to the conventional method. Cryopreserved cell populations or tissues
When starting the next culturing method or culturing step, thawing can be carried out according to a conventional method. Treatments preferred for the cell population or tissue may be combined during cryopreservation and thawing. For example, a cryoprotectant (DMSO or the like) may be added during cryopreservation, in which case the cryoprotectant may be removed under appropriate conditions during thawing.

In a cell population containing Tie2-positive stem / progenitor cells obtained by each culture method or each culture step of the present invention (collectively referred to as "post-culture cell population" in the present specification), Tie2-positive stem / progenitor cells and the same. The ratio and / or number of each cell other than (Tie2-positive stem / cells differentiated from progenitor cells, etc.) is basically arbitrary, and Tie2-positive stem cells and Tie
The ratio of 2-positive progenitor cells is also basically arbitrary. The composition of the cell population after culturing can be appropriately adjusted according to the embodiment of the invention, taking into consideration the use of the cell population obtained by each culturing method or each culturing step.

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

In a cell population containing Tie2-positive stem / progenitor cells in the middle of each culturing method or each culturing step of the present invention (collectively referred to as "in-cultured cell population" in the present specification), Tie2-positive stem / progenitor cells and the same. The ratio of cells other than (Tie2-positive stem / cells differentiated from progenitor cells, etc.) is basically arbitrary, and the ratio of Tie2-positive stem cells and Tie2-positive progenitor cells is also basically arbitrary. The composition of the culturing cell population is a composition in the process of transition from the pre-cultured cell population to the post-culturing cell population. For example, the ratio of Tie2-positive stem / progenitor cells in the culturing cell population (in the present specification, "Tie2-positive stem /". The "progenitor cell rate") is usually Ti in the pre-cultured cell population.
The value is included in the range between the e2-positive stem / progenitor cell ratio and the Tie2-positive stem / progenitor cell ratio of the cultured cell population, but it is permissible that the value temporarily deviates from the range. The composition of the cell population during culturing varies depending on the embodiment of the invention, the number of days in each culturing method or each culturing step, the number of passages, and the like.

The "human or other animal" (donor) from which each of the above cell populations is derived is the Ti of the present invention.
It can be selected in consideration of the use of the cell population finally obtained by the method for culturing e2-positive stem / progenitor cells, the use of the cell population obtained by each culture method or each culture step included in the method, and the like. .. In a typical embodiment of the present invention, when preparing a cell population for producing a cell preparation for preventing or treating a predetermined disease, symptom, etc., "human or other animal" is the cell preparation thereof. It is an organism of the same species as the administration target (recipient) of, preferably human.

-Cell population related to the amplification culture stage In the present invention, the cell population used in the first culture method and / or the second culture method, or the cells used in the first culture step and / or the second culture step in the amplification culture stage. Populations (collectively referred to herein as "pre-amplification culture cell populations") are typically cell populations (primary cultures) contained in tissues (intervertebral discs) taken from the body of humans or other animals. Cell population) or a cell population obtained by subculturing the primary cultured cell population (passaged cultured cell population)
Is.

When a cell population contained in an intervertebral disc collected from a human is used as the pre-amplification culture cell population, the Tie2-positive stem / progenitor cell ratio generally tends to be high and the niche tends to be good.
It is preferably a cell population contained in an intervertebral disc collected from a human in his teens or twenties. Further, the pre-amplification culture cell population is preferably a cell population having a Tie2-positive stem / progenitor cell ratio as high as possible, for example, 30% or more, 40% or more, 50% or more, 60% or more.

In addition, as the cell population before amplification culture, depending on the embodiment, a cell population other than the cell population contained in the tissue collected from the body of a human or other animal, for example, a cell of a human or other animal is used. It may be a cell population containing Tie2-positive stem / progenitor cells obtained by inducing differentiation of pluripotent or pluripotent cells such as iPS cells or ES cells prepared in the above.

In the present invention, the cell population obtained by the first culture method and / or the second culture method, or the cell population obtained by the first culture step and / or the second culture step in the amplification culture step (in the present specification, "amplification culture"). The use of "post-cell population") is not particularly limited, and the composition of the obtained cell population and the like can be appropriately adjusted according to the use.

In a typical embodiment of the present invention, the post-amplification culture cell population is the cell population subjected to the third culture method and / or the fourth culture method, or the third culture step and / or the fourth culture in the differentiation culture stage. It is used as a cell population to be used in the process. The cell population after amplification culture in such an embodiment (use) preferably has a Tie2-positive stem / progenitor cell ratio and / or a cell number as high as possible. The Tie2-positive stem / progenitor cell ratio in the post-amplification culture cell population varies depending on individual differences in the pre-amplification culture cell population and the nucleus pulposus tissue from which it is derived. Is 7% or more, 9% or more, 1
It is 1% or more, 13% or more, and 15% or more. The number of Tie2-positive stems / progenitor cells in the post-amplification culture cell population varies depending on individual differences in the pre-amplification culture cell population and the nucleus pulposus tissue from which it is derived. Compared to the number of cells, for example, 5 times or more, preferably 10 times or more, 15 times or more, 20 times or more, 25 times or more, 30
More than double.

-Cell population related to the differentiation culture stage In the present invention, the cell population used in the third culture method and / or the fourth culture method, or the cells used in the third culture step and / or the fourth culture step in the differentiation culture stage. The population (referred to as "pre-differential culture cell population" in the present specification) is preferably a cell population enriched with Tie2-positive stem / precursor cells in advance. The Tie2-positive stem / progenitor cell ratio in the pre-differentiation culture stage cell population varies depending on the individual differences in the pre-amplification culture or post-amplification culture cell population and the nucleus pulposus tissue from which it is derived. 5
% Or more, preferably 7% or more, 9% or more, 11% or more, 13% or more, 15% or more.

In a typical embodiment of the present invention, the pre-differentiation culture cell population is a cell population obtained by the amplification culture step of the present invention (post-amplification culture cell population), for example, an amplified Tie2-positive stem /.
It is a cell population obtained by dividing a cell population containing progenitor cells into an appropriate number of cells according to an embodiment (type and size of a culture vessel) of the differentiation culture stage. The cell population obtained by the amplification culture step of the present invention contains the Tie2-positive stem / progenitor cells having the ratio and / or the number of cells as described above, and the Tie2 expression of the Tie2-positive stem / progenitor cells is enhanced. Yes (Tie
Since the expression of 2 is maintained), it is preferable as a pre-differentiation culture cell population from the viewpoint of enhancing the action and effect at the differentiation culture stage.

In addition, the cell population before differentiation culture is a cell population, for example, human or other animal, which is not obtained by the amplification culture step (first culture step and / or second culture step) of the present invention depending on the embodiment. Induces differentiation of pluripotent or pluripotent cells such as cell populations contained in tissues collected from the body of the body and iPS cells or ES cells prepared using human or other animal cells. It may be a cell population containing the target cells obtained (via Tie2-positive stem / precursor cells).

In the present invention, the cell population obtained by the third culture method and / or the fourth culture method, or the cell population obtained by the third culture step and / or the fourth culture step in the differentiation culture step (in the present specification, "differentiation culture". The use of "post-cell population") is not particularly limited, and the composition of the obtained cell population and the like can be appropriately adjusted according to the use. For example, for a cell population used to produce a cell preparation for transplantation, a target cell having functionality useful for achieving a therapeutic or prophylactic effect by transplantation (for example, a nucleus pulposus cell producing II collagen: Col2). It is preferable that the cell population contains as much positive medullary nuclei cells as possible, and at the same time, contains some Tie2-positive stem / progenitor cells (for example, medullary nucleus stem / progenitor cells) in which the ability to produce such target cells remains. ..

The Col2-positive (medullary nucleus) cell rate in the post-differentiation culture cell population varies depending on the individual difference in the pre-differentiation culture cell population and the nucleus pulposus tissue from which it is derived, so it cannot be said unconditionally.
For example, 5% or more, preferably 10% or more, 15% or more, 20% or more, 25% or more, 30%
That is all.

The Tie2-positive (medullary nucleus) stem / progenitor cell ratio in the post-differentiation culture cell population varies depending on the individual difference in the pre-differentiation culture cell population and the nucleus pulposus tissue from which it is derived. % Or more, preferably 2% or more, 4% or more, 6% or more, 8% or more, 10%
That is all.

The number of cells contained in the cell population usually increases even in the differentiation culture step. Number of cells in the cell population after differentiation and culture (Col2-positive cells, Tie2-positive stem / progenitor cells, etc.)
Can not be said unconditionally because it varies depending on the individual difference of the pre-differentiation culture cell population and the nucleus pulposus tissue from which it is derived, but it is, for example, more than twice or more than the number of cells in the pre-differentiation culture cell population. It is double or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more.

<Medium>
The medium used in each culturing method or each culturing step of the present invention may be any medium suitable for culturing Tie2-positive stem / precursor cells and cells differentiating from the Tie2-positive stem / precursor cells, taking into consideration the purpose of the culturing method or culturing step. Appropriate basal medium and additive components can be selected. The additive component is an additive component suitable for amplification culture of Tie2-positive stem / progenitor cells if the culturing method is performed when the Tie2-positive stem / progenitor cells are amplified, that is, if the culturing step is in the amplification culture stage. If the culturing method is performed when inducing differentiation of Tie2-positive stem / precursor cells, that is, if the culturing step is in the differentiation culture stage, those suitable for inducing differentiation of Tie2-positive stem / precursor cells into target cells are suitable. Be selected.

In the third culture method and the fourth culture method of the present invention, and in the third culture step and the fourth culture step including the step of carrying out those methods, Tie2-positive stem / progenitor cells and cells differentiated from them are cultured. It is not necessary to add components such as methylcellulose to the medium that prevent them from adhering to the culture surface of the container. That is, the medium of the third culture step and the fourth culture method of the present invention of the present invention, and the third culture step and the fourth culture step including the step of carrying out those methods is usually a culture of a culture vessel such as methyl cellulose. Contains no ingredients to prevent cell adhesion to the surface.

In a typical embodiment of the present invention, when the nucleus pulposus stem / precursor cells and the nucleus pulposus cells differentiated from them are cultured, the medium for each step of the amplification culture step and the differentiation culture step is based on, for example, as follows. It can be prepared by using an appropriate amount of each of the medium, additive components, growth factors, and other components.

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

As an additive component for amplification culture or differentiation culture, for example, FBS (fetal bovine serum),
BSA (bovine serum albumin), L-ascorbic acid (as L-ascorbic acid magnesium phosphate, etc.), selenite (as insulin-transferrin-sodium selenite (ITS), etc.) and 2 -Mercaptoinsulin can be mentioned. If necessary, antibiotics such as penicillin and streptomycin and other components may be added to the medium.

Examples of the growth factor include FGF (fibroblast growth factor), EGF (Epidermal Growth Factor), and Ang-1 (angiopoietin-1). In one embodiment of the present invention, it is preferable to use at least FGF as the growth factor to be added to the medium, more preferably to use both FGF and EGF, and to use Ang-1 in addition to them as necessary. It is also preferable.

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

Ang-1 is preferably added in a serum-free medium. Further, as Ang-1, those solubilized in water (Soluble Ang-1, Recombinant Ang-1) are preferable. The concentration of Ang-1 (preferably soluble Ang-1) in the medium is usually 100-10.
It can be in the range of 00 ng / mL, for example about 500 ng / mL.

The above-mentioned growth factors such as FGF, EGF, and Ang-1 are "growth factors having a Tie2 expression-enhancing effect" and can be understood to correspond to "Tie2 expression-enhancing agents" in a broad sense. How to handle these growth factors will be described separately herein.

<Tie2 expression enhancer>
In the second culture method of the present invention, at least one "Tie2 expression enhancer" other than a growth factor having a Tie2 expression enhancer action is added to the medium. In particular, when the second culture method is carried out in the step of the amplification culture stage, the addition of the Tie2 expression enhancer has the effect of increasing the number of cells while maintaining the immaturity of the Tie2-positive stem / precursor cells, and further. When the cell population obtained in the amplification culture step is subjected to the differentiation culture step, the effect of improving the cell growth rate of the cell population obtained after the differentiation culture step and the ratio of Tie2-positive stem / precursor cells and functional target cells, etc. And so on. Any one of the Tie2 expression enhancers may be used, or two or more of them may be used in combination, and the Tie2 expression enhancer may be added to the medium in an amount in which the above-mentioned Tie2 activity is observed.

Examples of the "growth factor having a Tie2 expression enhancing effect" include angiopoietin-1.
(Ang-1), FGF2 (bFGF) and the like can be mentioned. In the second culture method of the present invention,
At least one kind of "Tie" other than such "growth factor having Tie2 expression enhancing action"
2 Expression enhancer ”is used, but if necessary, a growth factor having a Tie2 expression enhancer action can also be used in combination. In particular, when the second culture method is carried out in the step of the amplification culture step, a growth factor having a Tie2 expression enhancing action and other Tie2 expression enhancing agents, for example, animal and plant-derived extracts as described below, preferably. A synergistic effect can be achieved by using it in combination with a plant-derived extract. As a step in the amplification culture stage,
At least, it is essential to use a Tie2 expression enhancer other than a growth factor (a growth factor having a Tie2 expression enhancer action may be used in combination therewith as an optional component). A step may be carried out using only a growth factor having a Tie2 expression-enhancing effect (substantially no Tie2 expression-enhancing agent other than the growth factor is used).

As a Tie2 expression enhancer other than a growth factor, in the prior art, "Tie2 activator"
Various animal and plant derived extracts known as can be used. Such animal and plant-derived extracts include, for example, Akigumi, Akinonogeshi, Indian Dates, Turmeric, Yellow Pepper, Ousei, Obako, Okahijiki, Olive Fruit, Oyster, Chamomile, Karin, Karonin Hagekite, Chrysanthemum, Gyokuchiku, Kiraya, Ginkgo, Kusagi. , Kuco, Kunugi, Shell Ginger, Koryo Carrot, Konara, Sanzashi, Sanshosou, Shijuum Guaba, Siberian Carrot, Suishogaki, Star Fruit, Soukakushi, Jujube, Nikkei, Nobile, Hass, Bayberry, Harigiri, Long Pepper, Butcher Bloom, Mango Extracts such as ginger, long pepper, mube, shell ginger, bayberry, ryobu, and louis boss can be mentioned. Those skilled in the art can appropriately set the amount of each extract to be used that enhances the expression of Tie2, the site (material) of animals and plants suitable for preparing each extract, and the extraction method.

From an industrial point of view, it is cheaper than growth factors such as Ang-1 and FGF2, preferably has a better Tie2 expression-enhancing effect than those growth factors, and more preferably synergistically when used in combination with those growth factors. One or two or more selected from the above-mentioned animal and plant-derived extracts, preferably one or two or more selected from the above-mentioned plant-derived extracts, which are effective, are used in the second culturing step of the present invention for Tie2. It is advantageous to use it as an expression enhancer.

-Cinnamomum plant-derived extract In one preferred embodiment of the present invention, a Cinnamomum plant-derived extract can be used as the Tie2 expression enhancer. In the genus Cinnamomum, Cinnamomumcass
ia Blume), Camphora (C.camphora), Malvanikei (C.daphnoides), Shibanikei (C.doederleinii), Yabunikei (C.japonicum), Ogasawara Yabunikei (C.pseud)
o-pedunculatum), Nikkei (C.sieboldii), Shibayabu Nikkei Ceylon Nikkei (Cv)
It contains more than 300 species, including erum) and cinnamon (C. zeylanicum). For example, an extract of a product manufactured and sold as cinnamon powder, which is a young branch of cinnamon or cinnamon bark, or cinnamon powder obtained by processing them into powder, is derived from a plant of the genus Cinnamomum in the present invention. It can be used as an extract.

An extract derived from a plant of the genus Cinnamomum can be obtained by a conventional method. Alternatively, it can be prepared by filtering the filtrate and concentrating if necessary. As the extraction solvent, a solvent usually used for extraction, for example, an aqueous solvent, for example, water, physiological saline, a phosphate buffer, a borate buffer, or an organic solvent, for example, ethanol, propylene glycol, 1,3-butylene glycol, etc. , Alcohols such as glycerin, mixed water alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane and the like can be used alone or in combination, respectively. Preferably, water is used as the solvent. The extract obtained by extracting with the above solvent may be used as it is in the form of an extract, but from the viewpoint of convenience, it is solidified (powdered) by drying or freeze-drying, stored, and used. Sometimes diluted or redissolved (redispersed) with the appropriate solvent as needed
Then, if necessary, it can be used after being subjected to a treatment such as filtration. The extract derived from a plant of the genus Cinnamomum is a product (purified product) from which impurities have been removed by an adsorption method using an ion exchange resin (for example, a porous polymer such as Amberlite XAD-2), if necessary. May be good.

The concentration of the Cinnamomum plant-derived extract in the medium can be appropriately adjusted according to the properties of the extract to be used and the degree of action and effect as a Tie2 expression enhancer. For example, when an extract obtained by extracting 1 mg of cinnamon powder with 1 mL of physiological saline is used as an extract derived from a plant of the genus Cinnamomum, the extract is applied to a medium at 1 to 50 v /.
It can be added in an amount of about v%, for example, about 20 v / v%. Even when the extraction and addition embodiments are changed, the active ingredient as the Tie2 expression enhancer can be made to be comparable to the extraction and addition embodiments described above.

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

Generally, extracellular matrix (ECM) secreted from stem / precursor cells or cells differentiated from them includes, for example, collagen, proteoglycan, fibronectin, laminin, tenascin, entactin, elastin, fibrillin, hyaluronic acid and the like. Collagen includes type I, type II, type III, type IV, type IX (a2), and other types of collagen. Proteoglycans include aggrecan, versican, permacan (above, classification based on core protein size and number of sugar chains), chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratane sulfate proteoglycan, dermatan sulfate proteoglycan (above, binding to core protein). Classification based on glycosaminoglycan) and the like are included.

Therefore, the ECM degrading agent in the present invention corresponds to the embodiment of the fourth culture method, that is, the ECM secreted from the Tie2 stem / progenitor cells to be cultured or the cells differentiated from the ECM, as exemplified above. A substance (agent) that has an activity of degrading the cells and can suppress the formation of spherical colonies may be used. As the ECM decomposing agent, any one type may be used, or two or more types may be used in combination.

Typical ECM degrading agents include proteases having an activity of degrading protein moieties constituting ECM, for example, collagenase which is a protease having degrading activity against collagen. Collagenase includes class I collagenase showing high activity against high molecular weight collagen and class II collagenase showing high activity against low molecular weight collagen fragments. Also, vertebrate-derived collagenase cleaves collagen in the natural triple-helical region (on a very limited α-chain), while bacterial-derived collagenase acts on almost all types of collagen, resulting in triple helix. Collagen can be cleaved at multiple points within the spiral region. In addition to collagenase (collagenase I, collagenase II), collagenase preparations obtained by concentrating from the culture supernatant of bacteria include proteases other than collagenase (neutral protease, closttripain, trypsin, elastase, etc.).
Aminopeptidase, etc.) and non-proteolytic enzymes are included, and preparations excluding specific components by purification or the like are also manufactured. In the present invention, various known collagenases (preparations)
, Proteases and the like can be selected and used as an ECM decomposing agent.

In a typical embodiment of the fourth culture method of the present invention, the Tie2-positive stem / progenitor cell is a nucleus pulposus stem / progenitor cell, and the cell (target cell) generated by inducing differentiation from the Tie2-positive stem / progenitor cell is Nucleus cells. The nucleus pulposus cells express type II collagen, type IX collagen, type XI collagen, proteoglycan and the like as ECM. Therefore, as the ECM decomposing agent in this embodiment, one having a decomposing activity for those ECMs, for example, collagenase having a degrading activity for type II collagen or the like (or a preparation containing the same) may be selected. Examples of such collagenase (preparation) include "collagenase P".
(Derived from Roche, Clostridium histolyticum), "Liberase" (Roche, a mixture of collagenases I and II and neutral proteases) and the like.

As the ECM degrading agent, an enzyme (protein) such as a protease having a specific degrading activity for a protein contained in ECM but having low cytotoxicity is typical, but the action and effect of the present invention should be exhibited. Degradation activity above a certain level for ECM,
Substances other than enzymes (proteins) having cytotoxicity below a certain level, for example, low molecular weight compounds may also be used as ECM degrading agents.

The concentration of the ECM degrading agent in the medium may be any concentration that can suppress the formation of spherical colonies from the cell population containing Tie2 stem / progenitor cells, depending on the type of ECM degrading agent used, and the fourth culture. When the method is carried out in a step in the differentiation culture step (performed as the fourth culture step), the effect on the increase rate of target cells and the expression level or positive rate of a predetermined gene (marker gene) is taken into consideration. , Can be adjusted as appropriate. For example, if the concentration of the ECM decomposing agent is too high, the advantageous effect due to the above action may not be sufficiently recognized (conversely, it becomes a disadvantageous effect). It is preferable to adjust the concentration within.

In the present invention, a method in which the third culture method and the fourth culture method are fused (third and fourth culture methods).
Alternatively, in the step of fusing the third culture step and the fourth culture step (third and fourth culture steps), the target cell depends on the combination of the type and concentration of the ECM degrading agent in the medium and the type of the coating agent on the culture surface. The effect on the rate of increase, the expression level of a predetermined gene (marker gene), or the positive rate may fluctuate. Those skilled in the art will consider the properties of the pre-differentiation cell population and other embodiments depending on the viewpoint from which the action and effect are expected.
Each of the above conditions suitable for carrying out the present invention can be set through preliminary tests and the like.

As described above, the concentration of the ECM decomposing agent in the medium is not unconditionally determined and depends on the combination with the type of coating agent on the culture surface, for example, 0.0025 to 5.0.
It can be adjusted in the range of% by weight, 0.005 to 2.0% by weight, 0.01 to 1.0% by weight, and the like. In one embodiment of the present invention, when "collagenase P" is used as the ECM decomposing agent, the concentrations in the medium are 0.005 to 0.05% by weight and 0.0125% to 0.0.
It can be adjusted within the range of 25% by weight, for example, about 0.0125% by weight. In one embodiment of the present invention, when "Liberase" is used as the ECM decomposing agent, the concentration in the medium is within the range of 0.25% to 2.0% by weight, 0.5 to 1.0% by weight, or the like. It can be adjusted with, for example, about 1.0% by weight.

<Culture period and other conditions>
Each culturing method of the present invention, the duration of each culturing step and other conditions (eg pH, CO ₂₎
Concentration, O ₂ concentration, etc.) is basically in accordance with the purpose of the culturing process (culturing step includes), so that the cell population with the desired cell composition (type and number-ratio) is obtained, suitably adjusted can do. The pH can be weakly alkaline (eg, about 7.15). C
The O ₂ concentration can be, for example, about 5%. O ₂ concentration is 5% or less (for example, about 2%)
Can be. During each culturing method and each culturing step (step), if necessary, the medium should be replaced with a fresh medium every predetermined number of days, or an ingredient should be added after the lapse of a predetermined number of days, or the concentration of the ingredient may be adjusted. The medium may be changed or the atmosphere may be changed by increasing or decreasing the pH.

In the amplification culture step of the present invention, the period of the first culture step, the second culture step, or the first and second culture steps in which they are fused is usually about 1 to 3 weeks, for example, about 2 weeks, respectively.
Further, the period of other steps that can be optionally included in the amplification culture step of the present invention is also the same, for example, the period of the culture step using the FGF-added medium is about one week. The amplification culture step may be terminated when the desired post-amplification culture cell population is obtained.

In the differentiation culture step of the present invention, the period of the third culture step, the fourth culture step, or the third and fourth culture steps in which they are fused is usually about 1 to 3 weeks, for example, about 10 days, respectively.
Further, the period of other steps that can be optionally included in the growth culture step of the present invention is also the same, for example, the period of the culture step using the FGF-added medium is about 1 week (about 7 days). also,
The duration of other steps that the differentiation culture step of the present invention can optionally include is similar. The differentiation culture step may be terminated when the desired post-differentiation culture cell population is obtained.

<Culture container>
The culturing method of the present invention and the culturing vessel, culturing apparatus, etc. used in each culturing step basically have a desired cell composition (type and number) according to the purpose of the culturing method and the culturing step (including the culturing step). -It can be appropriately selected so as to obtain a cell population having a ratio).

As the culture vessel, a flask, a dish, a plate, a bag, or the like having a general shape can be used, and a well having a well for accommodating cells may be formed.
As the culture container, one made of a general material such as glass, plastic, or resin can be used. The surface of the culture vessel (culture surface) may be untreated, or may have been subjected to a treatment related to cell adhesion or other treatment. The size (area, volume) of the culture vessel, and if the culture vessel has wells, the size of the wells (caliber, volume)
Depth) and number can also be selected as appropriate. If necessary, the cell population may be cultured while shaking or rotating the culture vessel and stirring the medium.

In the third culture method (step) and the fourth culture method (step) of the present invention, the culture vessel and the culture device can be in the embodiment according to the two-dimensional culture (plane culture). Further, the first culturing method (step) of the present invention can be said to be a three-dimensional culture in that the cell population is cultivated in the tissue, and the tissue (fine pieces) containing the cell population is cultivated. It is placed in a floating state in the liquid.
The second culturing method (step) of the present invention may be an embodiment similar to three-dimensional culturing when it is carried out alone, but it is fused with the first culturing method (step) for the first and second culturing. When it is carried out as a method (step), it is placed in a floating state in the culture solution in the same manner as the above-mentioned first culture method (step). In these methods (steps), a culture vessel that has been surface-treated to enhance cell adhesion as in the third culture method (step) may be used, but a normal culture vessel that has not been surface-treated may be used. There is no problem even if it is used.

<Cell adhesion treatment>
In the third culture method (step) of the present invention, a culture vessel subjected to surface treatment (sometimes referred to as "cell adhesion treatment" in the present specification) for enhancing cell adhesion is used. A typical example of the cell adhesion treatment is a treatment of applying a coating agent containing extracellular matrix (ECM) or other bio-related substances to the culture surface. In addition, materials with low cell adhesion,
For example, modifying a culture vessel made of polystyrene, which has strong hydrophobicity, into hydrophilicity by plasma treatment is also an example of cell adhesion treatment.

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

Here, in the third and fourth culture methods (steps), as described above, the present invention depends on the combination of the type of the coating agent for cell adhesion treatment and the type and concentration of the ECM decomposing agent added to the medium. The action and effect (cell growth rate of cell population, ratio of target cells, etc.) may fluctuate. One reason for this is that the ECM or other bio-related substances contained in the coating agent for cell adhesion treatment may be affected by the degrading activity of the ECM degrading agent added to the medium. However, as long as the action and effect of the present invention are exerted to a certain extent (not completely inhibited), a coating agent for cell adhesion treatment and E may have such an effect.
Embodiments that are used in combination with a CM degrading agent are also acceptable. For example, as an ECM decomposing agent
When collagenase (preparation) having collagen-degrading activity of type II is added to the medium at a predetermined concentration, the coating agent for cell adhesion treatment is not easily affected by the type and concentration of the ECM-degrading agent, or the purpose. Those in which differentiation induction into cells (for example, Col2-positive cells) is achieved at a certain level, for example, those containing non-collagen polylysine (poly-L-lysine or poly-D-lysine) or fibronectin, or type IV collagen are preferable. ..

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

-Cell therapy composition-
The cell therapeutic composition of the present invention contains the cell population obtained by the culture method or preparation method of the present invention as described above, and optionally contains other pharmaceutically acceptable components. Can be done.

In a representative embodiment of the present invention, the cell therapeutic composition comprises Col2-positive nucleus pulposus cells differentiated from the nucleus pulposus stem / progenitor cells (preferably also including Tie2-positive stems / progenitor cells). Is. The target of application of the cell therapy composition in the embodiment, that is, the disease that can be prevented or treated by administering the composition is the intervertebral disc (nucleus).
Disorders, degeneration, herniated disks, etc., such as lumbar or cervical spine disc disease, herniated disk, cervical spondylotic myelopathy, radiculopathy, spondylolysis / spondylolysis, lumbar spinal canal stenosis, lumbar degeneration Examples include spondylolysis and lumbar degenerative scoliosis.

The dosage form of the cell therapeutic composition of the present invention may be any one that can be transplanted or delivered to a site targeting a cell population (for example, the nucleus pulposus of an intervertebral disc), and for example, an injection, preferably an intervertebral disc (the nucleus pulposus). Alternatively, it can be an injection for local administration to the vicinity thereof, or an injection for vascular administration that can be targeted.

Pharmaceutically acceptable components include, for example, water for injection or physiological saline when prepared as an injection, a culture solution for a cell population, other suitable solvent / dispersion medium, other additives, and the like. ..

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

-How to save-
The method for preserving a cell population containing Tie2-positive stems / progenitor cells of the present invention is to cryopreserve the cell population containing Tie2-positive stems / progenitor cells in a state of being present in undigested tissue to obtain Tie2. Maintains activation and / or expression, or suppresses the loss of Tie2-positive stem / progenitor cells in the cell population.

As for the technical matters concerning the preservation method of the present invention, the same technical matters as those described above can be applied in relation to the first culture method. For tissues that have not been digested containing Tie2-positive stems / progenitor cells, cryopreservation procedures and cryoprotectants used as needed include conventional Tie2-positive stems / that have been separated from the tissues by digestion treatment. Basically similar to those for cell populations containing progenitor cells can be applied.

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

As the medium in each step of the "differentiation culture stage" of this example (referred to as "medium for the differentiation culture stage" in the following examples), DMEM (no Glucose, wako) 60 mL and F1
1 μL of 2-mercaptoethanol, 6 μL of selenous acid (0.01%), 1.5 mL of ascorbic acid (5 mg / mL) and 30% BSA in a mixed medium of 0 (gibco) 40 mL.
5 mL was added, and 30% of FBS was further added immediately before use to prepare and use the ones to which the additional components shown in each table in the examples were added (+) or not (-).

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

The nucleus pulposus tissue of the intervertebral disc excised from the affected part of a herniated disc patient (32-year-old woman, 28-year-old woman and 20-year-old man) was cut into small pieces with scissors or the like to a size of several millimeters square. After suspending 0.1 to 0.5 g of shredded nucleus pulposus tissue containing the cell population in 3 mL of the medium prepared as shown in Table 1 for the additional components to the medium for the amplification culture stage, 6 holes. Culture dish (culture surface is untreated)
It was injected into one hole of No. 1 and cultured for 7 days (WTC method). As a control, instead of culturing the shredded nucleus pulposus tissue as it is, the cell population is digested with collagenase according to a conventional method to collect the isolated cell population, and otherwise the cell population is prepared in the same manner as the WTC method. Cultured (two-dimensional culture method).

After culturing, the cell population is collected and T on the cell surface by the flow cytometry (FCM) method.
The cell number and fluorescence intensity of cells positive for ie2 expression were measured, and the ratio to the cell number of the entire cell population (Tie2 positive rate) and the average fluorescence intensity (MFI) were calculated. In the FMC method, a fluorescent labeling agent (R &) which is a complex of an anti-human Tie2 antibody and a fluorescent dye allophycocyanin.
Company D, Anti-Tie-2, Human, Mouse-Mono (87315), Allophicocyanin, Catalog No. FAB
3131A) was used.

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

[Test Example 2] Amplification culture step (2 steps): 1st and 2nd culture steps + additional steps

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

16-year-old women, 28-year-old women and 38 patients with herniated discs from which the nucleus pulposus tissue of the intervertebral disc was collected
Except for being a year-old woman, using the medium prepared as shown in Table 2 for the additional components to the culture medium for the amplification culture stage as the first stage of the amplification culture stage, and setting the culture period to 14 days. , [Test Example 1] (Test Examples 1-1 and 1-2) was subjected to the same culture step.

After the first-stage culture step, Roche's "collagenase-P" (final concentration 0.025%) was added to the medium to disperse the nucleus pulposus tissue. Collect the cell population isolated from the nucleus pulposus tissue and collect 20%
After suspended at a density of 1.0 × ¹⁰ 4 / 3mL in FBS-supplemented MEM alpha, 6-well culture dish (culture surface untreated) was injected into one well of, after addition of 10 ng / ml bFGF, further 7 days (
Incubated for a total of 21 days).

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

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

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

After culturing, the cell population was collected, and the number of cells in which the expression of intracellular type II collagen (Col2) was positive was measured by the flow cytometry (FCM) method, and the ratio to the total number of cells in the cell population ( Col2 positive rate) was calculated. The cell population was previously treated with the membrane permeation reagent "IntraPrep" (Beckman Coulter) so that intracellular Col2 could be fluorescently labeled. As a fluorescent labeling method for Col2, a mouse anti-human Col2 antibody (Kyowa Pharma Chemical Co., Ltd. (formerly Daiichi Fine Chemical Co., Ltd.,) was used as the primary antibody.
Anti-hCL (II) (purified IgG), Catalog No. F-57) as a secondary antibody, a complex of goat anti-mouse IgG antibody and fluorescent dye FITC (BD, Goat Anti-Mouse Ig FITC, Catalog No. 349031) Was used. The results are shown in FIG. In addition, the number of Col2-positive cells was calculated assuming that all 1 g of nucleus pulposus-derived cells were amplified and cultured according to Test Example 2 and then differentiated and cultured according to Test Example 3. The results are shown in FIG. When the third culture step was applied (Test 3-1), the number of Col2-positive cells increased about 3 times as compared with the case where it was not applied (Test 3-2).

The number of cells in which the expression of proteoglycan (PG) in the cells was positive was also measured by the FCM method, and the ratio to the number of cells in the entire cell population (PG positive rate) was calculated. As a fluorescent labeling method for PG, mouse anti-human PG antibody (EMD milliporee, An) is used as the primary antibody.
ti-Cartilage Proteoglycan Antibody, adult, clone EFG-4, catalog number MAB201
5) as a secondary antibody, a complex of goat anti-mouse IgG antibody and fluorescent dye FITC (BD, Inc.)
, Goat Anti-Mouse Ig FITC, Catalog No. 349031). As a result, regardless of the application of the third culture step (PLL coating), the PG positive rates were both close to 100%, and no significant difference was observed (not shown). Unlike proteoglycans, functional nucleus pulposus cells expressing type II collagen have been difficult to increase the number of cells in the final cell population by the conventional method. This was made possible by combining the second culture step and the third culture process, demonstrating its superiority as a culture method.

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

In the same procedure as in Test Example 2, a two-step amplification culture step was performed for a total of 21 days.
The cell population after culturing is collected, and as a step of the differentiation culturing stage, a medium coated with poly-L-lysine (PLL) and to which additional components as shown in Table 4 are applied to the medium for the differentiation culturing stage. Was cultured for 14 days.

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

Six more samples were prepared for each of Test Examples 4-1 to 4-3 (the herniated disc patients from whom the nucleus pulposus tissue of the intervertebral disc was collected were 32-year-old female, 28-year-old female, 20-year-old male, 16-year-old female, 28. (Aged female and 38 year old female), the Col2 positive rate was calculated in the same manner as in [Test Example 3]. The results are shown in FIG. Although there were differences depending on the sample (individual differences in collecting the intervertebral disc nucleus pulposus tissue), in 4 out of 6 samples, either or both of the two types of collagenase were added to the medium. Compared to the case without addition
An improvement in the Col2 positive rate was observed.

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

Regarding the differentiation culture stage, the procedure of the amplification culture stage and the differentiation culture stage was the same as that of Test Example 4 except that the coating agent of the culture vessel and the additional component (collagenase P) to the medium were changed as shown in Table 5. The process was performed and the PG positive rate and the Col2 positive rate were measured. The results are shown in FIG. For example, when "collagenase P" is added to the medium, a coating agent containing Col4 (type IV collagen), FN (fibronectin) or PLL (poly-L-lysine) as a coating agent, particularly PLL, depends on the concentration. It was found that it is preferable to use a coating agent containing the above for improving the Col2 positive rate.

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

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

Claims (4)

Stem cells and / or progenitor cells derived from the nucleus pulposus tissue of the intervertebral disc and positive for the expression of Tie2 (tyrosine kinase with Ig and EGF homology domain-2) (hereinafter referred to as "nucleus-derived Tie2-positive stem / progenitor cells". ) Is cultured in a state of being retained in the niche of the nucleus pulposus tissue (except when the culture time is 24 hours or less) to amplify the nucleus pulposus-derived Tie2-positive stem / progenitor cells. (Hereinafter referred to as "first A culture method") , the expression of Tie2 in the nucleus pulposus-derived Tie2-positive stem / progenitor cell is enhanced, and the nucleus pulposus-derived Tie2-positive stem / progenitor cell in the cell population is included. culture stage for amplifying (hereinafter referred to as "amplification culture stage a".) only contains,
After the amplification culture step A, the nucleus pulposus-derived Tie2-positive stem / progenitor cell further comprises a culture step for inducing differentiation of the nucleus pulposus-derived Tie2-positive stem / progenitor cell into a nucleus pulposus cell expressing at least type II collagen. A method for preparing a cell population containing cells. By making the nucleus pulposus tissue thawed in the first A culture method, the state in which Tie2 is activated and / or expressed is maintained, or the number of nucleus pulposus-derived Tie2-positive stems / progenitor cells in the cell population is reduced. The preparation method according to claim 1, wherein the method is suppressed. The preparation method according to claim 1 or 2, wherein the culture period in the first A culture method is one week or longer. The amplification culture step A is further medullary nuclei in a medium supplemented with at least one selected from the group consisting of FGF (fibroblast growth factor), EGF (epithelial growth factor) and Ang-1 (angiopoietin-1). The preparation method according to any one of claims 1 to 3, which comprises a step of culturing a cell population containing a derived Tie2-positive stem / precursor cell.

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