Method for producing cellular structure, cellular structure, and bone regeneration agent

Abstract

The present invention addresses the problem of providing a transplant material suitable for clinical application, without using an artificial material. The problem is solved by providing a method for producing a cellular structure, the method comprising: (a) a step of culturing a mesenchymal stem cell cluster in a cartilage-inducing culture medium; and (b) a step of culturing the culture product obtained in step (a) in a bone differentiation-inducing culture medium.

Description

Method for producing cell structure, cell structure and bone regeneration agent

[0001] The present invention relates to a method for producing a cell structure, a cell structure, and a bone regenerating agent.

[0002] For patients who have undergone osteotomy due to serious fractures, infections, or tumors, the most ideal bone regeneration therapy is autologous bone grafting, in which bone is isolated from the chin or ilium and transplanted. However, there are limitations to the amount of autologous bone that can be harvested, and there are risks of invasiveness at the donor site, postoperative pain, and infection, making it practically unsuitable in many cases.

[0003] Therefore, there has been much research into the development of artificial bones that mimic the properties of bone tissue, and into the transplantation of mesenchymal stem cells (hereinafter sometimes referred to as "MSCs") with bone-forming potential mixed with artificial materials. Furthermore, many methods have been investigated that aim to regenerate bone through membranous ossification by enhancing the properties of osteoblasts through prior induction of osteogenic differentiation of MSCs (Patent Documents 1 and 2, Non-Patent Documents 1 and 2).

[0004] WO 2017 / 187941 WO 2020 / 226043 MIZUHO KITAOKA et al., Clumps of a mesenchymal stromal cell / extracellular matrix complex can be a novel tissue engineering therapy for bone regeneration, CYTOTHERAPY, Vol. 17, No. 7, pp. 860-873, 2015 Souta Motoike et al., Clumps of Mesenchymal Stem Cell / Extracellular Matrix Complexes Generated with Xeno-Free Conditions Facilitate Bone Regeneration via Direct and Indirect Osteogenesis, INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, Vol. 20, No. 16, 2019 Susumu Horikoshi et al., Clumps of Mesenchymal Stem Cells / Extracellular Matrix Complexes Generated with Xeno-Free Chondro-Inductive Medium Induce Bone Regeneration via Endochondral Ossification, BIOMEDICINES, Volume 9, Issue 10, 2021

[0005] However, conventional techniques that combine artificial bone with mesenchymal stem cells, etc., have the disadvantage of using artificial materials that are foreign to the body, which is undesirable. Furthermore, while the present inventors have developed techniques that do not use artificial materials, there is still room for improvement in clinical application, particularly in terms of engraftment after transplantation and application to large defect sites.

[0006] Therefore, an object of the present invention is to provide a transplant material that is more suitable for clinical application without using artificial materials.

[0007] As a result of intensive research to solve the above-mentioned problems, the inventors discovered that a cell structure suitable for transplantation can be provided by subjecting a mesenchymal stem cell cluster to a two-stage process using different media, and thus completed the present invention.

[0008] That is, one aspect of the present invention relates to a method for producing a cell structure, comprising: (a) a step of culturing a mesenchymal stem cell cluster in a cartilage induction medium; and (b) a step of culturing the culture obtained in step (a) in a bone differentiation induction medium.

[0009] Another aspect of the present invention relates to a cell structure having cartilage-like tissue with a bone shell on at least a portion of its surface, and containing no blood vessels.

[0010] Furthermore, another aspect of the present invention relates to a bone regenerating agent comprising a cell structure having cartilage-like tissue with a bone shell on at least a portion of its surface.

[0011] According to the present invention, a cell structure that can be rapidly engrafted after transplantation and can be applied to large defect sites can be provided. That is, according to the present invention, a transplant material that is more suitable for clinical use can be provided without using artificial materials.

[0012] 1 is a schematic diagram showing a method for producing a cell structure according to one embodiment of the present invention.

[0023] FIG. 1 is a diagram showing the histological analysis of cartilage-like tissue obtained by culturing mesenchymal stem cell clumps (clumps of MSCs / ECM complexes, hereinafter sometimes referred to as "C-MSCs") in cartilage induction medium (hereinafter sometimes referred to as "CIM") alone. The top row shows the results of HE staining, the middle row shows the results of alizarin red staining, and the bottom row shows the results of safranin-O staining. Furthermore, the central figures in the top and bottom rows are enlarged views of the square frames above the respective left figures, and the right figures in the top and bottom rows are enlarged views of the square frames below the respective left figures. Furthermore, the central figure in the middle row is an enlarged view of the square frame in the left figure.

[0024] FIG. 1 is a diagram showing the histological analysis of a cell structure according to Example 1. The first row from the top shows the results of HE staining (non-decalcified), the second row from the top shows the results of Alizarin Red staining (non-decalcified), the third row from the top shows the results of HE staining (decalcified), and the fourth row from the top shows the results of Safranin-O staining. The center images in the first, third, and fourth rows are enlarged versions of the square frames at the top of the left images, and the right images in the first, third, and fourth rows are enlarged versions of the square frames at the bottom of the left images. The center image in the second row is an enlarged version of the square frame at the bottom of the left image. This figure shows a microCT image of a cartilage-like tissue obtained by culturing C-MSCs in CIM alone. This figure shows a microCT image of a cell structure according to Example 1. This figure shows photographs of an immunodeficient SCID mouse calvarial defect model, taken with and without transplantation of the cell structure according to Example 1 (immediately after transplantation if transplantation was performed) (the right image is Example 2). 1 shows micro-CT images of a cell structure according to Example 1 after transplantation into an immunodeficient SCID mouse calvarial defect model (Example 2). The upper figure shows an overall image of the cell structure, and the lower figure shows a micro-CT image of a cross section of the straight portion of the upper figure. This figure shows tissue analysis of a cell structure according to Example 1 after transplantation into an immunodeficient SCID mouse calvarial defect model (Example 2). The upper row shows the results of HE staining, and the lower row shows the results of Safranin-O staining. This figure shows photographs taken after transplantation of cartilage-like tissue (immediately after transplantation) into an immunodeficient SCID mouse calvarial defect model (Comparative Example 4).Figure 1 shows microCT images of cartilage-like tissue transplanted into an immunodeficient SCID mouse calvarial defect model (Comparative Example 4). The upper figure shows an overall image of the cartilage-like tissue, and the lower figure shows a microCT image of a cross section of the straight portion of the upper figure. Figure 2 shows histological analysis of cartilage-like tissue transplanted into an immunodeficient SCID mouse calvarial defect model (Comparative Example 4). The upper row shows the results of HE staining, and the lower row shows the results of Safranin-O staining. Figure 3 shows histological analysis of a fibrous cell construct obtained by culturing C-MSCs in osteogenic differentiation-inducing medium (hereinafter sometimes referred to as "OIM") alone (Comparative Example 2). The upper row shows the results of HE staining, and the lower row shows the results of Safranin-O staining. Each center figure is an enlargement of the upper square frame in each left figure, and each right figure is an enlargement of the lower square frame in each left figure. Figure 4 shows histological analysis of a fibrous cell construct obtained by culturing C-MSCs in OIM and then in CIM (Comparative Example 3). The upper panel shows the results of HE staining, and the lower panel shows the results of Safranin-O staining. Each center panel is an enlargement of the upper square frame in each left panel, and each right panel is an enlargement of the lower square frame in each left panel. These figures show histological analysis of cartilage-like tissue obtained by culturing in C-MSCs proliferation medium alone (left panel: Comparative Example 6) or OIM alone (right panel: Comparative Example 2). The upper panel shows the results of HE staining, the middle panel shows the results of Safranin-O staining, and the lower panel shows the results of Alizarin Red staining. In each example, the right panel shows an enlargement of the square frame in the left panel. These figures show histological analysis of the cartilage-like tissue of Comparative Example 1 and the cell construct of Example 1 four weeks after transplantation into an immunodeficient SCID mouse calvarial defect model (upper panel: Comparative Example 4, lower panel: Example 2). In each example, the upper panel shows, from left to right, the results of HE staining, Safranin-O staining, and TRAP staining. In each example, the lower panel shows an enlarged view of the rectangular frame in the upper panel. These figures show tissue analysis 8 weeks after transplantation of the cartilage-like tissue of Comparative Example 1 and the cell construct of Example 1 into an immunodeficient SCID mouse calvarial defect model (upper panel: Comparative Example 4, lower panel: Example 2). In each example, the upper panel shows, from left to right, the results of HE staining, Safranin-O staining, and TRAP staining. In each example, the lower panel shows an enlarged view of the rectangular frame in the upper panel.Figure 1 shows photographs taken 8 weeks after subcutaneous transplantation of the cell clumps of Comparative Example 1 and the cell construct of Example 1 into immunodeficient SCID mice (left: Comparative Example 5, right: Example 3). Figure 2 shows microCT images and tissue analysis 8 weeks after subcutaneous transplantation of the cell clumps of Comparative Example 1 and the cell construct of Example 1 into immunodeficient SCID mice (top: Comparative Example 5, bottom: Example 3). In each example, the two left images are microCT images, and from the left, a 3D image and a tomographic image are shown, respectively. The two right images are tissue analysis images, and from the left, the results of HE staining and Safranin-O staining are shown, respectively. Figure 3 shows tissue analysis of the cell construct obtained when multiple C-MSCs were adhered and cultured in a method for producing a cell construct according to one embodiment of the present invention (Example 4). The three right images are enlarged versions of the left images. The top row shows the results of HE staining, the middle row shows the results of alizarin red staining, and the bottom row shows the results of Safranin-O staining.

[0013] An embodiment of the present invention will be described in detail below. In this specification, unless otherwise specified, "A to B" representing a numerical range means "A or more, B or less." In addition, all documents described in this specification are incorporated herein by reference.

[0014] 1. Overview of the Invention As described above, autologous bone grafting for bone regeneration is often inapplicable due to limitations on the amount of bone that can be harvested, invasiveness to the harvested site, postoperative pain, and the risk of infection.

[0015] Therefore, many studies have been conducted to develop artificial bones that mimic the properties of bone tissue, to transplant MSCs with bone-forming ability mixed with artificial materials, and to induce bone differentiation in MSCs in advance to enhance the properties of osteoblasts and aim for bone regeneration through membranous ossification.However, no definitive treatment has yet been established.

[0016] It has long been known that the development of limb bones and the healing mechanism of fractures in vivo occur through endochondral ossification, in which cartilage tissue is absorbed and replaced by bone tissue. The present inventors believed that if it were possible to produce cartilage primordium-like tissue surrounded by a bone shell, which is the tissue at the stage immediately before endochondral ossification, in vitro, bone regeneration therapy that mimics the developmental and healing processes in vivo could be established, and they previously reported a method for bone regeneration through endochondral ossification (Non-Patent Document 3).

[0017] Furthermore, based on this idea, the inventors conducted extensive research to solve the above-mentioned problems and found that by subjecting mesenchymal stem cell clusters to a two-stage process using different media, it is possible to provide a cell structure suitable for transplantation without using artificial materials. Specifically, the inventors found that by culturing mesenchymal stem cell clusters in a cartilage-inducing medium and then switching the medium to an osteogenic differentiation-inducing medium and further culturing the cells, it is possible to obtain a cell structure that engrafts quickly after transplantation and is applicable to large defect sites.

[0018] The cell structure of the present invention is useful in bone regeneration therapy because it rapidly takes root (endochondral ossification) after transplantation and can be applied to large bone defects. In particular, it makes it possible to provide reliable and effective bone regeneration therapy to patients with large bone loss.

[0019] [2. Method for producing a cell structure] In one embodiment of the present invention, there is provided a method for producing a cell structure (hereinafter referred to as "the present production method"), which comprises the steps of (a) culturing a mesenchymal stem cell clump in a cartilage induction medium, and (b) culturing the culture obtained in step (a) in an osteogenic differentiation-inducing medium. In this specification, the cell structure may also be referred to as "the present cell structure."

[0020] As used herein, the term "cell structure" refers to a three-dimensional tissue mainly composed of cartilage-like tissue and a bone shell (i.e., a three-dimensional tissue mainly composed of cartilage primordia). In one embodiment of the present invention, the cartilage-like tissue is present inside the cell structure, and the bone shell is present outside the cell structure. In other words, this cell structure can be referred to as cartilage-like tissue surrounded by a bone shell, a structure having a bone shell on the surface of cartilage-like tissue, etc.

[0021] In one embodiment of the present invention, the cell structure has cartilage-like tissue with a bone shell on at least a portion of its surface. In one embodiment of the present invention, the surface of the cartilage-like tissue in the cell structure is preferably covered by a bone shell over a certain area or more, from the viewpoint of increasing the survival rate after transplantation. For example, 50% or more, preferably 60% or more, more preferably 70% or more of the surface of the cartilage-like tissue is covered by the bone shell, and it is most preferable that substantially 100% is covered by the bone shell.

[0022] In one embodiment of the present invention, the cell structure may consist of cartilage-like tissue and bone shell, or may include other cells besides cartilage-like tissue and bone shell. The cell structure may include, for example, vascular endothelial cells, fibrous tissue, osteoblasts, osteocytes, chondrocytes, stromal cells, mesenchymal stem cells, etc.

[0023] As used herein, the term "cartilage primordium" refers to a tissue that is a cartilage-like tissue covered with a bony shell and has the ability to differentiate into bone. The cartilage primordium of the present invention can be obtained by carrying out steps (a) and (b) in the production method.

[0024] As used herein, the term "cartilage-like tissue" refers to a group of cells present inside a cartilage primordium and composed of chondrocytes and cartilage matrix.

[0025] As used herein, "bone shell" refers to a bone-like tissue layer composed of bone matrix with deposited minerals and osteoblasts. Osteoblasts are the cells that give rise to bone and can produce bone matrix. Bone matrix is ​​an accumulation of substances produced and secreted extracellularly by cells with bone-forming ability (e.g., osteoblasts), such as calcium phosphate, including proteins such as osteocalcin, osteopontin, osteonectin, and collagen. Minerals include calcium, magnesium, and phosphorus. "Mineral deposition" refers to the deposition of at least calcium in the bone matrix, and in this case, magnesium or phosphorus may also be deposited. The presence or absence of mineral deposition can be confirmed, for example, by staining tissue sections with alizarin red, a calcium-binding dye. However, this is not limiting; any other method that can confirm the presence of minerals in the bone matrix can also be used. In addition to bone matrix and osteoblasts, "bone shell" may also include interstitial cells such as osteocytes and periosteal cells.

[0026] (Step (a)) In step (a), a mesenchymal stem cell cluster is cultured in a cartilage induction medium. By culturing a mesenchymal stem cell cluster in a cartilage induction medium, cartilage-like tissue can be induced in at least a portion of the mesenchymal stem cell cluster.

[0027] As used herein, "mesenchymal stem cell clusters (C-MSCs)" refer to cell clusters composed of at least a plurality of mesenchymal stem cells and extracellular matrix proteins (e.g., type I collagen) produced by the cells themselves, and refer to cell clusters that are not in the form of a two-dimensional flat sheet but are in the form of three-dimensional granules or the like. Normally, when mesenchymal stem cells are cultured in adhesion, they grow two-dimensionally (i.e., grow in a sheet-like form) to form a cell sheet, but mesenchymal stem cell clusters can be obtained by detaching the cell sheet from the culture vessel and then culturing the cell sheet in suspension. Specifically, mesenchymal stem cell clusters can be obtained, for example, by step (a') described below.

[0028] The mesenchymal stem cells that form the mesenchymal stem cell clusters are not particularly limited as long as they are mesenchymal stem cells that have the ability to differentiate into bone and cartilage. Examples of such mesenchymal stem cells include all mesenchymal stem cells present in living organisms, such as bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, periodontal ligament-derived mesenchymal stem cells, and gingival-derived mesenchymal stem cells. Furthermore, mesenchymal stem cells induced from pluripotent stem cells such as iPS cells and ES cells are also included in the mesenchymal stem cells of the present invention.

[0029] In one embodiment of the present invention, the mesenchymal stem cell cluster in step (a) may be a cluster of multiple linked mesenchymal stem cells. By performing steps (a) and (b) using a cluster of multiple linked mesenchymal stem cells as a starting material, a large cell cluster can be obtained. Conventional techniques have been unable to obtain transplant materials larger than a certain size, making them unsuitable for transplantation when the defect site is large. However, the present invention can provide a large cell cluster, making it extremely advantageous for clinical application.

[0030] As used herein, "cartilage induction medium (CIM)" refers to a medium capable of inducing mesenchymal stem cells or cells derived from mesenchymal stem cells (e.g., mesenchymal stem cell clumps) into cartilage. The cartilage induction medium is not particularly limited as long as it falls within the above definition, and various known cartilage induction media can be used. Examples of cartilage induction media include serum-free, xenogeneic animal protein-free cartilage induction medium (XF-CIM) prepared by adding MSCgo Chondrogenic XF Supplement Mix (manufactured by BLG) to MSCgo Chondrogenic XF, Human (manufactured by BLG), and serum-free Mesenchymal Stem Cell Chondrogenic Differentiation Medium (manufactured by Promo Cell). The cartilage induction medium may be prepared based on a known protocol, and for example, a cartilage induction medium prepared by adding 0.1 μM dexamethasone, 50 μg / ml ascorbic acid, 100 μg / ml pyruvic acid, 40 μg / ml proline, 50 mg / ml ITS Premix, and 10 ng / ml TGF-β1 to high-glucose DMEM (manufactured by SIGMA) may be used.

[0031] In one embodiment of the present invention, in step (a), it is preferable to culture the mesenchymal stem cell clusters until at least a portion of them contains cartilage-like tissue, and then perform step (b). In other words, it is preferable that the culture obtained in step (a) contains cartilage-like tissue. This allows for the production of a cell structure that is more suitable for transplantation.

[0032] As used herein, "a state in which at least a portion of a mesenchymal stem cell cluster contains cartilage-like tissue" refers to a state in which at least a portion of a mesenchymal stem cell cluster has been induced to become cartilage-like tissue by culture in a cartilage induction medium. The occupancy rate (induction rate) of cartilage-like tissue in the mesenchymal stem cell cluster is, for example, 30% or more, preferably 50% or more, more preferably 80% or more, and may even be 100%. Whether or not a mesenchymal stem cell cluster contains cartilage-like tissue can be determined, for example, by Safranin-O staining or the like.

[0033] The culture time in step (a) is not particularly limited as long as it is the time required for at least a portion of the mesenchymal stem cell clusters to contain cartilage-like tissue, but is, for example, 1 to 20 days, preferably 1.5 to 10 days, and more preferably 2 to 5 days.

[0034] In one embodiment of the present invention, the method may include the following step before step (a): Step (a'): A step of culturing mesenchymal stem cells in a proliferation medium to obtain a mesenchymal stem cell cluster.

[0035] Specifically, step (a') is carried out by the following method. First, mesenchymal stem cells are cultured in a growth medium containing a factor that induces collagen production, using a culture vessel such as a culture dish. As the mesenchymal stem cells, those described in the above section "step (a)" can be used.

[0036] The growth medium used in step (a') may be any medium capable of growing mesenchymal stem cells, and various known media can be used. Examples of growth media include a medium containing high-glucose DMEM supplemented with 10% FBS and 50 μg / ml ascorbic acid, and a serum-free, xenoprotein-free growth medium (XF-GM) containing PRIME-XV (registered trademark) MSC Expansion XSFM (manufactured by FUJIFILM Corporation) containing 100 U / ml penicillin and 100 μg / ml streptomycin. A preferred growth medium is a serum-free, xenoprotein-free growth medium (XF-GM) containing PRIME-XV (registered trademark) MSC Expansion XSFM (manufactured by FUJIFILM Corporation) containing 100 U / ml penicillin and 100 μg / ml streptomycin. Use of this medium allows cell structures to be produced with higher reproducibility in the subsequent steps (i.e., steps (a) and (b)).

[0037] Furthermore, the factor that causes mesenchymal stem cells to produce collagen is not particularly limited as long as it is a compound, protein, etc. that can ultimately cause mesenchymal stem cells to produce collagen. Examples of such factors include ascorbic acid, steroids such as dexamethasone, cytokines, etc., and ascorbic acid is preferred.

[0038] As mesenchymal stem cells proliferate, a sheet-like cell mass (hereafter referred to as a cell sheet) is formed. The periphery of the cell sheet adheres to the edge of the culture vessel and is allowed to proliferate until it reaches confluence.

[0039] The edge of the cell sheet that is attached to the peripheral wall of the culture vessel is then separated from the vessel. For example, a thin rod can be inserted into the inner wall of the culture vessel where the edge of the cell sheet is attached, and the rod can be moved around the inner wall of the culture vessel, separating the cell sheet from the culture vessel. This causes the cell sheet to float.

[0040] This floating cell sheet wraps up by self-aggregation and becomes a mass of mesenchymal stem cell aggregates using the extracellular matrix (ECM) produced by the mesenchymal stem cells themselves. In this way, granular cell aggregates can be obtained.

[0041] The incubator is 1.5 to 2.5 cm ２ When a cylindrical container with a surface area of ​​2 cm is used, cell aggregates with a particle size of 0.5 to 1.5 mm can be obtained. ２ A 24-well plate or the like may be used.

[0042] (Step (b)) In step (b), the culture obtained in step (a) is cultured in an osteogenic differentiation-inducing medium. After step (a), the culture is cultured in an osteogenic differentiation-inducing medium, thereby producing a cell structure suitable for transplantation in bone regeneration therapy, etc.

[0043] As used herein, "osteoblast differentiation-inducing medium (OIM)" refers to a medium capable of inducing mesenchymal stem cells or mesenchymal stem cell-derived cells (e.g., mesenchymal stem cell clusters) into bone. The osteoblast differentiation-inducing medium is not particularly limited as long as it falls within the above definition, and various known osteoblast differentiation-inducing media can be used. The osteoblast differentiation-inducing medium may be, for example, a serum-free induction medium or a serum-containing induction medium. Examples of serum-free induction media include serum-free and xenogeneic animal protein-free osteoblast induction medium (XF-OIM: MSCgo Osteogenic XF, manufactured by BLG), MesenCult Osteogenic Differentiation kit (manufactured by STEMCELL Technologies), and serum-free osteoblast induction medium STK3 (manufactured by Two Cells). Examples of serum-containing induction media include Osteoblast Growth Medium (manufactured by Takara). The bone differentiation induction medium may be prepared based on a known protocol, and for example, a serum-containing bone differentiation induction medium prepared by adding 10% FBS, 0.1 μM dexamethasone, 50 μg / ml ascorbic acid, and 10 mM glycerophosphate to high-glucose DMEM may be used.

[0044] The culture time in step (b) is not particularly limited as long as it is a time that allows a cell structure having cartilage-like tissue with a bone shell on at least a portion of its surface to be obtained, but is, for example, 1 to 20 days, preferably 1.5 to 10 days, and more preferably 2 to 5 days.

[0045] In step (b), the description in the above section (step (a)) applies to the "mesenchymal stem cell cluster."

[0046] In one embodiment of the present invention, the method may further comprise the following step after step (b): Step (c): a step of cryopreserving the cell structure obtained in step (b) together with a cryopreservation agent.

[0047] By including step (c) in this manufacturing method, it is not necessary to manufacture cell structures within a limited period immediately before transplant surgery, etc., and this prevents the transplant surgery from being postponed.

[0048] In step (c), the cell structure obtained in step (b) is placed in a cryopreservation container such as a cryovial together with a cryopreservative and cryopreserved. In this way, a frozen cell structure for transplantation can be obtained. As the cryopreservation agent, various cell freezing preservation solutions may be used, such as a cryopreservation solution containing 10% DMSO (dimethyl sulfoxide), 20% FBS, and 70% DMEM, as well as commercially available cryopreservation solutions such as Cell Banker (manufactured by Takara Bio Inc.) and Bambanker (manufactured by Wako Pure Chemical Industries, Ltd.).

[0049] Cryopreservation may be carried out at a temperature of, for example, -70 to -90°C, preferably -75 to -85°C. Cryopreservation may be carried out by various methods, such as placing the storage container in a freezer. For long-term storage of one month or more, the storage container may be placed at the above temperature for 24 hours and then transferred to a liquid nitrogen tank (-196°C). Pre-freezing may or may not be performed during cryopreservation.

[0050] [3. Cell Structure] In one embodiment of the present invention, a cell structure is provided that has cartilage-like tissue with a bone shell on at least a portion of its surface and does not contain blood vessels (e.g., capillaries). This cell structure is useful in bone regeneration therapy because it quickly takes root after transplantation and can be applied to large defect sites.

[0051] In vivo, cartilage primordia are formed during bone development. However, cartilage primordia formed in vivo contain blood vessels (e.g., capillaries). In contrast, the cell structure does not contain blood vessels (e.g., capillaries), which clearly distinguishes it from cartilage primordia formed in vivo.

[0052] The size of the cell structure is preferably suitable for transplantation, generally, for example, 0.5 to 1.5 mm in diameter, preferably 0.8 to 1.2 mm in diameter. In one embodiment of the present invention, when the defect site is large, the size of the cell structure can be greater than 2.0 mm in diameter. While conventional techniques have made it difficult to produce transplant materials with a diameter greater than 2.0 mm, the present invention has succeeded in increasing the size of cell masses, which is extremely advantageous for clinical application. When the defect site is large, the size of the cell structure is preferably greater than 2.0 mm in diameter, more preferably greater than 2.5 mm in diameter, even more preferably greater than 3.0 mm in diameter, and particularly preferably greater than 3.5 mm in diameter. Furthermore, in one embodiment of the present invention, the size of the cell structure can be even larger, for example, 5.0 mm or more, preferably 6.0 mm, or even greater than 1 cm. According to the present invention, the size of the cell structure can be appropriately adjusted based on the size of the defect site to which it will be transplanted.

[0053] Methods for increasing the size of the present cell construct are not particularly limited, but include, for example, connecting multiple mesenchymal stem cell clusters in a skewered state and carrying out steps (a) and (b) of the present production method. Larger cell constructs can be produced, for example, by the method described in the Examples.

[0054] In one embodiment of the present invention, the cell structure is produced by the production method.

[0055] In one embodiment of the present invention, the cell structure may be a frozen cell structure (also referred to as a "frozen cell structure"). C-MSCs can be produced from MSCs isolated from a patient, and then a cell structure can be produced, and the function and uniformity of the cell structure can be tested in advance. Only those with a certain quality can then be selected through testing and cryopreserved as frozen cell structures. This makes it possible to reliably supply quality-controlled cell structures for transplantation on the day of transplantation, preventing postponements of transplant surgery.

[0056] Frozen cell structures retain their morphology and do not lose cellular function even after thawing, and therefore, when transplanted into defective tissue, they exhibit tissue regeneration capabilities. The inventors speculate that the reason why frozen cell structures can exhibit tissue regeneration capabilities even after thawing is as follows. Specifically, the inventors previously demonstrated that C-MSCs can maintain their three-dimensional structure and cellular activity even after immersion in a cryopreservation solution and cryopreservation at temperatures below -80°C (WO 2017 / 187941; Motoike et al., 2018, Stem Cell Res Ther). This is because the abundant extracellular matrix proteins, such as collagen, that form C-MSCs exert a protective effect against damage caused by ice crystals during cryopreservation. Here, because the present cell structure contains abundant collagen, similar to C-MSCs, it is speculated that its bone formation ability through endochondral ossification will not be impaired even after cryopreservation. Thawing of frozen cell structures can be carried out in a variety of ways, such as by removing the cryopreservation container in which the frozen cell structures are stored from a freezer or the like and placing it at room temperature or in a warm bath (e.g., 40 to 60°C).

[0057] [4. Bone regeneration agent] In one embodiment of the present invention, a bone regeneration agent (hereinafter referred to as "this bone regeneration agent") is provided, which comprises a cell structure having cartilage-like tissue with a bone shell on at least a portion of its surface. This bone regeneration agent is useful in bone regeneration therapy because it contains a cell structure that quickly engrafts after transplantation and can be applied to large defect sites.

[0058] In one embodiment of the present invention, the bone regeneration agent may contain any additives in addition to the cell structure. Such additives include, for example, pharmaceutically acceptable carriers, diluents, excipients, etc. Pharmaceutically acceptable carriers, diluents, excipients, etc. are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro, ed., 1985). The selection of pharmaceutically acceptable carriers, diluents, and excipients can be easily determined by one skilled in the art according to the intended treatment format and standard pharmaceutical practice.

[0059] In one embodiment of the present invention, the bone regeneration agent may further contain any binder, lubricant, suspending agent, etc., and a substance that promotes the engraftment of the cell structure, such as fibrin glue.

[0060] The amount of the additive contained in the bone regenerating agent is not particularly limited and can be appropriately determined by those skilled in the art.

[0061] The bone regenerating agent is used for the purpose of bone regeneration, and is applicable to any bone without any particular limitation, and is suitably applicable to bone regeneration therapy for, for example, intractable fractures, periodontitis, rheumatoid arthritis, femoral head necrosis, osteogenesis imperfecta, osteoporosis, and cases of extensive bone loss after bone removal.

[0062] In one embodiment of the present invention, the bone regenerating agent can also be applied to sites other than bone defects. Sites other than bone defects include, but are not limited to, subcutaneous tissue. The bone regenerating agent rapidly induces endochondral ossification and bone regeneration not only in bone defects but also in other sites.

[0063] The target species to which the present bone regeneration agent can be applied include, but are not limited to, mammals such as humans, cattle, wild boars, pigs, sheep, goats, horses, mice, rats, hamsters, squirrels, rabbits, dogs, cats, and ferrets, with humans being preferred.

[0064] In this bone regeneration agent, the terms "bone shell," "cartilage-like tissue," and "cell structure" are as described above in the sections [2. Method for producing cell structure] and [3. Cell structure].

[0065] 5. Others In one embodiment of the present invention, there is provided a method for treating a bone disease, which comprises administering the cell structure or the bone regenerating agent to a subject suffering from the bone disease.

[0066] In one embodiment of the present invention, administration of the cell structure or bone regeneration agent is generally intended for transplantation, but includes any method applied in bone regeneration therapy.

[0067] Examples of bone diseases include, but are not limited to, the above-mentioned intractable fractures, periodontitis, rheumatoid arthritis, femoral head necrosis, osteogenesis imperfecta, osteoporosis, and extensive bone loss after bone removal.

[0068] As used herein, the term "treatment" refers to an act of bringing about a therapeutic effect on a subject in need of treatment. The therapeutic effect is a concept that encompasses preventive effects and therapeutic effects, and may be, for example, the following types of effects:

[0069] (1) The onset of one or more symptoms of a disease is prevented or the risk is reduced compared to when the cell structure or bone regeneration agent is not administered.

[0070] (2) Preventing or reducing the risk of recurrence of one or more symptoms associated with the disease compared to when the cell structure or bone regeneration agent is not administered.

[0071] (3) Preventing or reducing the risk of one or more symptoms of a disease compared to when the cell structure or bone regeneration agent is not administered.

[0072] (4) The severity of one or more symptoms associated with the disease is reduced compared to when the cell structure or bone regeneration agent is not administered.

[0073] (5) Preventing an increase in the severity or progression of one or more symptoms of a disease compared to when the cell structure or bone regeneration agent is not administered.

[0074] (6) The rate of increase in severity or the rate of progression of one or more symptoms associated with the disease is reduced compared to when the cell structure or the bone regeneration agent is not administered.

[0075] In one embodiment of the present invention, the dosage of the cell structure or bone regeneration agent can be appropriately determined by a person skilled in the art depending on the extent of the defect site, the severity of the disease, the degree of decline in systemic bone metabolic ability, age, etc.

[0076] In another embodiment of the present invention, there is provided the cell structure or the bone regenerating agent for use in treating a bone disease.

[0077] In another embodiment of the present invention, there is provided use of the cell structure in the manufacture of a medicament for treating a bone disease. <1> A method for producing a cell structure, comprising: (a) culturing a mesenchymal stem cell clump in a cartilage-inducing medium; and (b) culturing the culture obtained in step (a) in an osteogenic differentiation-inducing medium. <2> The method according to <1>, wherein the cell structure has cartilage-like tissue with a bone shell on at least a portion of its surface. <3> The method according to <1> or <2>, wherein the mesenchymal stem cell clump is formed by connecting multiple mesenchymal stem cell clumps. <4> The method according to any of <1> to <3>, wherein in step (a), the mesenchymal stem cell clumps are cultured until at least a portion of them contain cartilage-like tissue, and then step (b) is performed. <5> The method according to any of <1> to <4>, further comprising the following step before step (a): (a') culturing mesenchymal stem cells in a proliferation medium to obtain a mesenchymal stem cell clump. <6> A cell structure having cartilage-like tissue with a bone shell on at least a portion of its surface, and not containing blood vessels. <7> The cell structure according to <6>, wherein the cell structure has a diameter of more than 2.0 mm. <8> A bone regenerating agent comprising a cell structure having cartilage-like tissue with a bone shell on at least a portion of its surface.

[0078] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0079] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0080] 1. Culturing mesenchymal stem cells and preparation of cell constructs (Preparation of C-MSCs) Human bone marrow-derived mesenchymal stem cells (MSCs) purchased from Lonza were cultured in a 48-well culture plate (manufactured by Corning) at a concentration of 1.0 × 10 ５The cells were seeded at a cell density of 100 cells / well. The cells were cultured for 4 days in serum-free, xenoprotein-free growth medium (XF-GM) consisting of PRIME-XV® MSC Expansion XSFM (FUJIFILM Corporation) containing 100 U / ml penicillin and 100 μg / ml streptomycin to produce sufficient ECM. The cells were bluntly detached using a micropipette tip and suspended in a cell sheet composed of ECM and MSCs. The resulting MSCs / ECM complexes were transferred to an ultra-low-binding plate (Iwaki Corporation) and cultured in XF-GM for 3 days to yield spherical cell aggregates (C-MSCs) with a diameter of approximately 800-1000 μm.

[0081] (Induction of Cell Structures from C-MSCs) The obtained C-MSCs were continued in suspension culture in serum-free, xenogeneic animal protein-free cartilage induction medium (XF-CIM) (250 μl / well) prepared by adding MSCgo Chondrogenic XF Human (BLG) to MSCgo Chondrogenic XF Supplement Mix (BLG). After 10 days of culture in XF-CIM, the medium was changed to serum-free, xenogeneic animal protein-free bone induction medium (XF-OIM: MSCgo Osteogenic XF (BLG)) at 250 μl / well, and the cells were cultured for 7 or 14 days. During this culture, the medium was changed every 2 days (Example 1).

[0082] For comparison, the following C-MSCs were used: C-MSCs cultured in XF-CIM medium for 17 or 24 days (Comparative Example 1); C-MSCs cultured in XF-OIM medium for 17 or 24 days (Comparative Example 2); C-MSCs cultured in XF-OIM for 10 days and then cultured in XF-CIM for 7 days (Comparative Example 3); and C-MSCs cultured in suspension in growth medium (XF-GM) (250 μl / well) for 17 days (Comparative Example 6).

[0083] (Histological Evaluation of Cell Structure) HE staining, Alizarin Red staining, Safranin-O staining, and micro-CT photography were performed by the following methods.

[0084] <HE staining, Alizarin Red staining, Safranin-O staining> The obtained samples (cell structures or cartilage-like tissues) were washed with PBS, fixed with 4% paraformaldehyde, and embedded in paraffin. Serial sections of 8 μm were prepared and stained with HE and Alizarin Red. In addition, to observe the histological structure, the obtained samples (cell structures or cartilage-like tissues) were decalcified with 10% ethylenediaminetetraacetic acid, embedded in paraffin, and 8 μm sections were prepared and stained with HE and Safranin-O.

[0085] <Micro-CT Imaging> The obtained samples (cell structures or cartilage-like tissues) were washed with PBS and then photographed using a micro-CT (Skyscan 1176, Bruker, Billerica, MA, USA) under the following conditions: tube voltage 50 kV, tube current 0.5 mA, pixel size 8 μm, step angle 0.5°, and exposure time 230 ms.

[0086] (Result-1) In Comparative Example 1, in which C-MSCs were cultured only in XF-CIM, cartilage-like tissue that stained deeply with Safranin-O was formed (bottom panel of Figure 2), and abundant chondrocytes were observed within it, as shown in Figure 2. However, no mineral deposition that stained with Alizarin Red occurred (middle panel of Figure 2).

[0087] In Comparative Example 2, in which C-MSCs were cultured only in XF-OIM, no mineral deposition occurred on the periphery (lower panel of the right panel in Figure 14). Furthermore, no cartilage-like tissue was formed (lower panel in Figure 12, middle panel in the right panel in Figure 14), and the cells were mainly composed of fibrous structures.

[0088] Furthermore, in Comparative Example 3, in which C-MSCs were cultured in XF-OIM and then in XF-CIM, no peripheral mineral deposition stained with alizarin red occurred (not shown). Furthermore, as shown in the bottom panel of Figure 13, no cartilage-like tissue was formed, and the cells were mainly composed of fibrous structures.

[0089] In addition, in Comparative Example 6, in which C-MSCs were cultured only in the growth medium (XF-GM), the cell masses shrank and remained as fibrous tissues (left panel of FIG. 14).

[0090] On the other hand, in Example 1, which was prepared by culturing C-MSCs in XF-CIM and then in XF-OIM, a cell structure was obtained that had a bone shell on the periphery with mineral deposits that stained dark with alizarin red, and an internal cartilage-like tissue (a cell mass consisting of cartilage matrix and chondrocytes contained therein) that stained dark red with Safranin-O, as shown in Figure 3 (first, second, and fourth rows in Figure 3). Furthermore, when decalcified and observed, no cell death was observed in the outer layer where minerals were deposited, and cells present within the osteoid matrix were observed (third row in Figure 3).

[0091] Furthermore, micro-CT images were taken of the cell clumps of Comparative Example 1 and the cell structure of Example 1. No impermeable material was observed in the cell clumps of Comparative Example 1, confirming that no mineral deposition had occurred (Figure 4). On the other hand, the cell structure of Example 1 had impermeable material forming the outer periphery of the sphere, and the interior appeared hollow (Figure 5).

[0092] These results demonstrated that by culturing C-MSCs in XF-CIM and then in XF-OIM, a cell structure having cartilage-like tissue with a bone shell could be produced.

[0093] 2. Transplantation of Cell Constructs into Calvarial Bone Defects in Immunodeficient Mice To examine the bone regeneration effect of transplantation of the obtained cell constructs, an immunodeficient SCID mouse calvarial defect model was used. Specifically, a 2.4 mm diameter bone defect was created in the calvaria of an immunodeficient SCID mouse using a trephine burr. A single cell construct or cartilage-like tissue was directly transplanted into this defect without using any artificial materials (Example 2: transplantation of the cell construct of Example 1 (FIG. 6); Comparative Example 4: transplantation of the cartilage-like tissue of Comparative Example 1 (FIG. 9)). Four and eight weeks after transplantation, the animals were sacrificed, and the calvariae were removed and photographed using micro-CT. The bone tissue was then observed using HE staining, Safranin-O staining, and TRAP staining. Micro-CT photography, HE staining, Safranin-O staining, and TRAP staining were performed using the following methods.

[0094] The obtained samples (mouse calvaria) were washed with PBS and then photographed using a microCT (Skyscan 1176, Bruker, Billerica, MA, USA) under the following conditions: tube voltage 50 kV, tube current 0.5 mA, pixel size 8 μm, step angle 0.5°, and exposure time 230 ms.

[0095] <HE staining and Safranin-O staining> After microCT imaging, the mouse calvaria was fixed with 4% paraformaldehyde, decalcified with 10% ethylenediaminetetraacetic acid, and then embedded in paraffin. 8 μm sections were prepared and stained with HE and Safranin-O.

[0096] <TRAP staining> After washing the above-mentioned paraffin-embedded thin sections with PBS, a TRAP staining solution (tartaric acid solution / substrate solution for phospholipase A) from a TRAP staining kit (294-67001, manufactured by Wako Co., Ltd.) was added and allowed to react for 10 minutes at 37° C. Note that TRAP staining stains osteoclasts.

[0097] (Result-2) Four weeks after transplantation, when cartilage-like tissue prepared using only XF-CIM and C-MSCs was transplanted (Comparative Example 4), micro-CT imaging revealed impermeable material in the defect (Figure 10). However, HE staining and Safranin-O staining revealed that the interior of the impermeable material was cartilage matrix, and bone formation was incomplete (Figure 11). That is, in Comparative Example 4, tissue consisting mostly of cartilage matrix was observed, and there were almost no osteoclasts positive for TRAP staining (Figure 15). Furthermore, eight weeks after transplantation, the cartilage matrix was absorbed, and vascular invasion and osteoclasts were observed (Figure 16).

[0098] On the other hand, when a cell construct prepared by culturing C-MSCs in XF-CIM and then in XF-OIM was transplanted (Example 2), bone-like tissue was formed in the defect (Figure 7). Furthermore, in Example 2, although some cartilage-like tissue stained with Safranin-O remained, a bone marrow-like structure accompanied by the infiltration of vascular and blood cells was observed, indicating that bone formation occurred via endochondral ossification (Figure 8). Furthermore, in Example 2, the cartilage matrix was absorbed 4 weeks after transplantation, and vascular infiltration and TRAP-positive osteoclasts were observed (Figure 15). Furthermore, 8 weeks after transplantation, the cartilage matrix had completely disappeared, and bone tissue with a bone marrow structure was observed (Figure 16). Comparison with Comparative Example 4 demonstrated that Example 2 exhibited faster endochondral ossification and more reliable bone regeneration.

[0099] 3. Subcutaneous Transplantation of Cell Constructs into Immunodeficient Mice Immunodeficient SCID mice were used to examine the effects of transplanting the obtained cell constructs into areas other than bone defects. Specifically, the dorsal skin of the immunodeficient SCID mice was incised, and the cell construct or cartilage-like tissue was directly transplanted without using artificial materials (Example 3: transplantation of the cell construct of Example 1; Comparative Example 5: transplantation of the cartilage-like tissue of Comparative Example 1). Eight weeks after transplantation, the condition of the transplanted tissue was confirmed by macroscopic observation, micro-CT photography, and HE staining and Safranin-O staining. Micro-CT photography and HE staining and Safranin-O staining were performed using the following methods.

[0100] <Micro-CT Imaging> The obtained samples (subcutaneous implantation sites) were washed with PBS and then photographed using a micro-CT (Skyscan 1176, Bruker, Billerica, MA, USA) under the following conditions: tube voltage 50 kV, tube current 0.5 mA, pixel size 8 μm, step angle 0.5°, and exposure time 230 ms.

[0101] <HE staining and Safranin-O staining> After microCT imaging, the subcutaneous implantation site was fixed with 4% paraformaldehyde, decalcified with 10% ethylenediaminetetraacetic acid, and then embedded in paraffin. 8 μm sections were prepared and subjected to HE staining and Safranin-O staining.

[0102] (Result-3) Eight weeks after transplantation, when a cell construct prepared by culturing C-MSCs in XF-CIM and then in XF-OIM was transplanted (Example 3), the transplanted tissue was macroscopically confirmed to have blood vessels (right panel of Figure 17). Furthermore, the cartilage matrix of the transplanted tissue had been completely absorbed, and the tissue was bone tissue with a bone marrow structure (Figure 18).

[0103] On the other hand, when cartilage-like tissue prepared using only XF-CIM and C-MSCs was transplanted (Comparative Example 5), some bone-like tissue was observed, but the tissue had poor blood flow and appeared to be in the midst of endochondral ossification with residual cartilage matrix (Figures 17 and 18).

[0104] The above results, when compared with Comparative Example 5, show that in Example 3, endochondral ossification occurs more quickly not only in the bone defect area but also in the subcutaneous tissue, resulting in more reliable bone regeneration.

[0105] [4. Enlarging the size of cell structures by cell aggregate composite] From day 3 of the culture process described above in [1. Culture of mesenchymal stem cells and preparation of cell structures] (i.e., after C-MSCs were obtained), four cell aggregates of C-MSCs were skewered on a sterilized stainless steel needle and cultured in a 6 cm dish in 5 ml of medium in a state where they were adhered to each other (Example 4).

[0106] (Result-4) At the end of the 10-day XF-CIM + 7-day XF-OIM culture (day 17), the C-MSC cell aggregates fused with each other, producing large rod-shaped cell structures of 5 mm or more (Figure 19).

[0107] The present invention can be suitably used in the field of regenerative medicine and the like.

Claims

1. (a') culturing mesenchymal stem cells in a proliferation medium and then culturing the resulting cell sheet in suspension to obtain a mesenchymal stem cell cluster; (a) culturing the mesenchymal stem cell cluster in a cartilage induction medium; and (b) culturing the culture obtained in step (a) in a bone differentiation-inducing medium.

2. The method according to claim 1 , wherein the cell structure has a cartilage-like tissue having a bone shell on at least a portion of its surface.

3. The method according to claim 1 or 2, wherein the mesenchymal stem cell cluster is a cluster of multiple mesenchymal stem cells linked together.

4. The method of claim 1 or 2, wherein in the step (a), the mesenchymal stem cell cluster is cultured until at least a portion thereof contains cartilage-like tissue, and then the step (b) is carried out.

5. (delete)

6. having a cartilage-like tissue having a bone shell on at least a portion of its surface; A cellular construct that is free of blood vessels and artificial materials and is greater than 2.0 mm in diameter.

7. (delete)

8. The present invention includes a cell structure having a cartilage-like tissue with a bone shell on at least a part of the surface thereof, A bone regeneration agent, wherein the cell structure does not contain artificial material and has a diameter of greater than 2.0 mm.