JP6945242B2 - New tissue regenerated material and its manufacturing method - Google Patents

Description

The present invention relates to a novel tissue regenerated material and a method for producing the same.

Cartilage and ligament degeneration and damage due to aging and sports are increasing. While the tendons, ligaments, and cartilage present in joints play an important role in maintaining joint stability, they have the property of being difficult to self-heal once damaged. When these tissues are damaged, treatment is often performed by transplantation within the same individual, but there is a problem that the mechanical properties of the repaired tissue are inferior to those of normal tissue. For osteoarthritis, which affects more than 30% of the population, there is an urgent need to improve the qualitative level of tissue repair in order to maintain and improve QOL.

The most promising repair material for this purpose is a stem cell-derived tissue repair material based on stem cells such as iPS cells and mesenchymal stem cells. Stem cells are a general term for cells that have both proliferative ability (ability to increase self) and differentiation ability (ability to become other cells). In theory, all living tissues can be created, so a tissue based on stem cells. Expectations are high for restoration materials. Among stem cells, mesenchymal stem cells are a type of somatic stem cells that exist in the body, can be easily collected from individuals, and there is little concern about rejection, so they are attracting attention as a realistic key to regenerative medicine. There is.

For example, as an approach to solve problems related to repair of tendons, ligaments, and cartilage, mesenchymal stem cells (MSCs) collected from the knee synovium are made to self-generate extracellular matrix (ECM) such as collagen. The produced stem cell-based self-assembled tissue (scSAT) has been developed and basic research is being conducted (Non-Patent Document 1). scSAT is also known in the art as a tissue engineered construct (TEC). Since scSAT does not require a scaffold made of animal-derived collagen or an artificial compound, it has a feature that it is unlikely to cause rejection when implanted in a living body. So far, scSAT has been reported to be effective in cartilage repair (Non-Patent Document 2). However, scSAT is a tissue composed of cells and an extracellular matrix that is self-generated by it, and has the problem that it is difficult to handle in the field of transplant surgery such as being thin and easily torn, and the mechanical properties of repair tissue. Is lower than normal cartilage, and the problem is that the collagen density in the surface layer is not as high as that of normal cartilage (Non-Patent Document 3).

However, including the above technique, the tissue regeneration material derived from mesenchymal stem cells has the following three problems.
a) Due to restrictions on the culture environment, the potential such as the ability of stem cells to differentiate is not fully utilized.
b) The mechanical strength of the tissue regenerating material is low, and the ability to fuse with surrounding tissues is low.
c) Due to the small volume of the tissue regenerating material that can be prepared, it cannot be used for wide area treatment.

In order to increase the mechanical strength of the tissue regeneration material, it is conceivable to create a complex of cells and some scaffold, but in general, in the creation of a complex of cells and scaffold, cells invade the scaffold. It is said that it is difficult to make it. For example, studies have been conducted to allow cells to invade the scaffold by applying negative pressure or recirculating the culture medium, but these methods are less effective and make cells fairly uniform and dense like living tissues. It is not possible to create a state that includes it.

Yokoyama et al. Have succeeded in containing a large amount of stem cells in a substrate composed of low molecular weight collagen by using a collagen gel culture method in which cells are cultured in a collagen gel (Non-Patent Document 4). As for this culture method, there are commercially available products such as Nitta gelatin (Nitta) as an existing cell culture method. In terms of this method is to sufficiently penetrate the cells into the scaffold, but is intended to solve the above problems, 10 ⁶ cells / cm ² or more large amounts on cells is needed in this method, the generated The amount of collagen in the material is small, and most of the fibers have a low molecular weight and a low degree of refibrosis. It was impossible to use a smaller amount of cells and to contain the cells at a high density in a large amount of collagen fibers having a high degree of fibrosis, which is closer to the in vivo environment.

Ando, W., et al., Biomaterials, Vol.28, No.36, pp.5462-5470, 2007 Shimomura, K., et al., Biomaterials, Vol.31, No. 31, pp.8004-8011, 2010 Ando, W., et al., European Cells and Materials, Vol.24, pp.292-307, 2012 Yokoyama, A., et al., Cell Tissue Res., Vol.32, pp.289-298, 2005

The present invention provides a novel tissue regenerated material and a method for producing the same.

In view of the above, the inventor of this case focused on culturing stem cells in a collagen solution and started research. As a result of diligent studies, a collagen solution or collagen dispersion was mixed in a culture medium containing a small amount of stem cells, and centrifugal force was applied by centrifugation to accumulate stem cells and collagen at high density on the culture plane, and the aggregate was cultured again. We have developed a culture method that returns to the environment (Fig. 1). By this method, a novel tissue regeneration material (also referred to as "stem cell / collagen complex" in the present specification), which is based on stem cells and collagen fibers having a high degree of fibrosis and has extremely high density and adhesion between the two materials. Succeeded in developing. Based on this finding, the present invention has been completed.

That is, in one aspect, the present invention may be as follows.

[1] A method for producing a tissue-regenerated material, which comprises the following steps:
(1) Mix a neutral culture medium containing stem cells with a collagen solution or dispersion;
(2) Centrifuge the mixture of (1) to accumulate stem cells and collagen;
(3) Culturing stem cells and collagen aggregates;
The method described above.

[2] The method according to the above [1], wherein the stem cell is a mesenchymal stem cell, an ES cell, or an induced pluripotent stem cell (iPS cell).

[3] The stem cells in step (1) are stem cells ^{cultured to a cell density of 1.0 × 10 5} cells / cm ^{2 to} 6.0 × 10 ⁵ cells / cm ² , as described above [1] or [2]. The method described in.

[4] The method according to any one of [1] to [3] above, wherein the collagen is type I, type II or type III collagen.

[5] The method according to any one of [1] to [4] above, wherein the collagen solution or dispersion in step (1) is a fibrotic dispersion of refibrotic collagen.

[6] The method according to any one of the above [1] to [5], which comprises adding ascorbic acid or an ascorbic acid derivative or a salt thereof in the step (1) or the step (3).

[7] A tissue regenerating material produced by the method according to any one of the above [1] to [6].

The novel tissue regeneration material (stem cell / collagen complex) prepared by the method described in the present application has a high cell content as compared with the conventional TEC, and is obtained as a large tissue. In addition, the structure of refibrotic collagen in the stem cell / collagen complex closely resembles the surrounding environment when the cells are placed in vivo, and from the viewpoint of mechanobiology, stem cells cultured in that environment There is a high possibility that the differentiation ability will be maximized. From these characteristics, it is useful as a tissue regeneration material having preferable properties.

FIG. 1 is a conceptual diagram of a method for producing a stem cell / collagen complex. FIG. 2 is a comparison of TEC and stem cell / collagen complexes by SEM image analysis. (A) and (B) are images observed at a magnification of 500, and are images analyzed for different fields of view. (C) is an image of 1000 times observation. FIG. 3 is a photograph comparing the stem cell / collagen complex on the 14th day of culture, the tissue prepared by the experiment lacking the step of centrifuging the collagen fiber / MSCs solution, and the tissue produced by the culture in the collagen gel. be. FIG. 4 shows the tissue obtained by in-collagen gel culture on the 7th day of culture, the stem cell / collagen complex (without ascorbic acid 2-phosphate), and the stem cell / collagen complex (with ascorbic acid 2-phosphate). It is a comparison of the SEM image analysis results of. (A), (B) and (C) are an image of 200 times observation, an image of 1000 times observation, and an image of 2500 times observation, respectively.

The present invention will be specifically described below, but the present invention is not limited thereto.

Unless otherwise defined herein, scientific and technical terms used in the context of the present invention shall have meanings commonly understood by those skilled in the art.

Method for Producing Tissue Regenerated Material In one aspect, the present invention describes the following steps:
(1) Mix a neutral culture medium containing stem cells with a collagen solution or dispersion;
(2) Centrifuge the mixture of (1) to accumulate stem cells and collagen;
(3) Culturing stem cells and collagen aggregates;
The present invention relates to a method for producing a tissue regenerated material, including.

In the above method, the type of stem cell is not particularly limited, but may be selected from the group consisting of, for example, mesenchymal stem cells, ES cells, and induced pluripotent stem cells (iPS cells). Preferably, the stem cells are mesenchymal stem cells. Stem cells are mammalian-derived cells, preferably human-derived cells.

In the above step (1), the density of stem cells is not particularly limited, but for example, even stem cells cultured to a cell density of ^{1.0 × 10 5} cells / cm ^{2 to} 1.0 × 10 ⁶ cells / cm ^2. Stem cells are often and preferably cultured to a cell density ^{of 1.0 × 10 5} cells / cm ^{2 to} 6.0 × 10 ⁵ cells / cm ^2. More preferably 2.0 × 10 ⁵ cells ^/ cm 2 ^~4.0 × 10 ⁵ stem cells cultured to a cell density of cells / cm ^2.

The culture medium or medium is not particularly limited as long as it is a medium suitable for culturing stem cells, but for example, DMEM or the like can be preferably used. The culture medium or medium may contain additional components such as serum and antibiotics, which can be appropriately selected by those skilled in the art depending on the type of stem cells to be cultured. The pH of the medium is neutral and is prepared, for example, in the range of pH 6.0-8.0 or pH 6.5-7.5.

The collagen solution or dispersion is not particularly limited as long as it is a solution or dispersion containing collagen. The collagen dispersion is preferably a fibrotic dispersion of refibrotic collagen. Collagen refibrosis proceeds by placing the collagen solution in a neutral environment. Therefore, fibrotic dispersions of refibrotic collagen may be prepared by placing the collagen solution under neutral conditions.

The collagen contained in the collagen solution or the dispersion is not particularly limited as long as it is collagen. Preferably, collagen is neutral and can form fibers by incubating at 37 ° C. Examples of such collagen include type I, type II, or type III collagen. More preferably, the collagen may be type I or type II collagen.

The temperature at which the above step (1) is carried out is not particularly limited as long as it is a temperature suitable for culturing stem cells, and can be appropriately selected by those skilled in the art. The preferred temperature is, for example, 35 ° C to 40 ° C, more preferably 37 ° C.

In the above step (2), the centrifugation is not particularly limited as long as it is a condition normally used by those skilled in the art for precipitating and accumulating cells. For example, a condition such as 1000 rpm to 3000 rpm, preferably 1000 rpm to 1500 rpm, and more preferably 1000 rpm for 5 minutes may be used.

In the above step (3), the accumulation of stem cells and collagen can be cultured in the culture medium or medium as described above. The culture temperature is not particularly limited as long as it is a temperature suitable for culturing stem cells, and can be appropriately selected by those skilled in the art. The preferred temperature is, for example, 35 ° C to 40 ° C, more preferably 37 ° C. The culturing time may be, for example, between 6 and 28 days, more preferably between 6 and 14 days.

In one aspect, ascorbic acid or ascorbic acid derivatives or salts thereof may be added in steps (1) or (3) in the above method. These additions are expected to have the effect of improving the production of extracellular matrix containing collagen by cells.

The ascorbic acid derivative is not particularly limited, but the hydroxyl groups at the 2-position, 3-position, 5-position, and / or 6-position of ascorbic acid are sugars such as phosphoric acid, sulfuric acid, and glucose, and fatty acids (for example, stearic acid and palmitic acid). , Hexyldecanoic acid, etc.), derivatives esterified with alkyl groups (eg, C _{1 to} C ₂₀ alkyl), and the like. The ascorbic acid derivative may contain ascorbic acid 2-phosphoric acid, ascorbic acid 2-sulfuric acid, ascorbic-2-glucoside, and ascorbic-6-glucoside.

The salt of ascorbic acid or the salt of the ascorbic acid derivative is not particularly limited, and includes an alkali metal salt (sodium salt, potassium salt, etc.) and an alkaline earth metal salt (magnesium salt, potassium salt, etc.).

Ascorbic acid or ascorbic acid derivatives or salts thereof may be present in the form of hydrates.

Through the above steps (1) to (3), a tissue structure having a three-dimensional structure is produced in which stem cells involve a collagen dispersion. The obtained tissue structure can be used as a tissue regeneration material.

In one aspect of the tissue regenerating material , the present invention describes the following steps:
(1) Mix a neutral culture medium containing stem cells with a collagen solution or dispersion;
(2) Centrifuge the mixture of (1) to accumulate stem cells and collagen;
(3) Culturing stem cells and collagen aggregates;
The present invention relates to a tissue regenerating material produced by a method comprising adding ascorbic acid or an ascorbic acid derivative or a salt thereof in the above steps (1) or (3).

The description of each configuration of the method for producing the tissue regenerated material is as described in the item of the above-mentioned "Method for producing the tissue regenerated material".

The tissue regenerated material has a strong sheet-like tissue morphology. Its thickness is about 600Myuemu～1500myuemu, area also can be created of about 30mm ² ~360mm ^2. This is about 10 times thicker and about 100 times larger in volume than the conventional TEC as described in Non-Patent Document 1.

When the tissue regeneration material produced by the above method was observed with a scanning electron microscope (SEM), it had a highly porous surface, and in addition to the collagen (fibril, subfibril) structure having a submicrometer level in diameter, It was observed that the higher-order structure, the micrometer-level collagen (fascicle) structure, coexisted. The structure of such collagen closely resembles the structure of natural biofibers. It was also observed that the amount of collagen that binds to cells was large and three-dimensional. Therefore, the tissue regeneration material produced by the above method is rich in collagen fibrils in the material, has a high degree of fibrosis, and is close to the surrounding environment when cells are placed in a living body.

The tissue regeneration material of the present invention can be used for repairing tendons, ligaments and cartilage. When the tissue regeneration material of the present invention is used for repairing tendons, ligaments and cartilage, the tissue regeneration material of the present invention is placed on the defect site of the tendon, ligament and cartilage to implant the tissue, and the skin covering the defect site is embedded. Should be sutured. The tissue regeneration material of the present invention is a special procedure for conjugating or adhering a tissue repair material to a defective site of tendon, ligament and cartilage because adhesion factors and the like are secreted because stem cells self-generate extracellular matrix. Is unnecessary.

Further, the tissue regenerated material of the present invention can be obtained as a material having a thickness as compared with the conventional TEC as described above, or as a material having a large size. These properties are broader than traditional TECs, which improve stability during surgical handling of materials and facilitate transplantation into defective affected areas when tendon and cartilage repairs are required. It provides advantages such as being available for transplantation into a defect.

Specific examples of the present invention are shown below. These specific examples are intended to provide an explanation for understanding the present invention, and are not intended to limit the scope of the present invention.

Example 1: Preparation of stem cell / collagen complex and observation of SEM (1) Preparation of stem cell / collagen complex Mesenchymal stem cells (MSCs) collected from human synovial membrane were used in a culture medium (DMEM, 10% fetal bovine serum). FBS), cultured in 1% P / S (penicillin / streptomycin) to 4.0 × 10 ⁵ cells / cm ² or 2.0 × 10 ⁵ cells / cm ^2, and the cultured cells were tubed. In culture medium (DMEM, 10% FBS, 1% P / S, 0.2 mM 2-phosphate ascorbic acid), 5.6 × 10 ⁵ cells / mL or 2.8 × 10 ⁵ cells / mL. Prepared to be a cell suspension.

In a separate tube, add medium (DMEM, 10% FBS, 1% P / S, 0.2 mM ascorbic acid 2-phosphate) and NaHCO _{3 to 2.5 mL of 3.0 mg / mL type I collagen solution.} And made it neutral. Incubation at 37 ° C. for 1 day gave a refibrotic collagen dispersion. This was centrifuged at 37 ° C. at 4000 rpm for 20 minutes to remove the supernatant.

3 mL of the above cell suspension was added to the refibrotic collagen from which the supernatant had been removed, and the suspension was suspended with a Komagome pipette to obtain a collagen fiber / MSCs solution. The collagen fiber / MSCs solution was transferred to a 6-well plate with cell culture inserts. The plate was centrifuged at 1000 rpm for 5 minutes to compress and dehydrate collagen and MSCs fibers. This was cultured in an incubator at 37 ° C. The medium was changed the day after the centrifugation, and every two days thereafter. The culture was carried out until the 14th day, with the day of the above centrifugation being 0 day. Self-organization was observed from about the 6th day of culture, and a strong sheet-like tissue that could be grasped with tweezers was formed. This tissue is referred to herein as a stem cell / collagen complex.

(2) In the SEM observation above stem cells / collagen composite (1), using stem cells / collagen composite produced using the cell suspension 2.8 × 10 ⁵ cells / mL, according to the method of Patent Document 1 Comparison with the prepared TEC was performed by scanning electron microscope (SEM) image analysis. The results are shown in FIG.

Regarding TEC, a surface with low porosity was observed, and the collagen structure could not be confirmed. In TEC, it was observed that cells were embedded in the wall-like structure or that spherical cells were scattered.

On the other hand, for the stem cell / collagen complex, a highly porous surface was observed, and a collagen (fibril, subfibril) structure with a diameter of submicrometer level and a micrometer level collagen (fascicle), which is a higher-order structure thereof. It was observed that the structures coexisted. The structure of such collagen closely resembles the structure of natural biofibers. It was also observed that the cells and collagen structure were highly connected.

Example 2: Effect of Centrifugation In Example 1 (1), the collagen fiber / MSCs solution was centrifuged in a 6-well plate. In Example 2, the tissue obtained by this experiment without the centrifugation step was compared with the stem cell / collagen complex by the method of Example 1 (1). In addition, the tissue produced by the collagen gel culture shown in Example 4 below was compared with the stem cell / collagen complex by the method of Example 1 (1).

As a result, at the 14th day of culturing, in the experiment without the centrifugation step and the culture in the collagen gel, a very brittle and large disk-shaped tissue was obtained, whereas in Example 1 including the centrifugation step (the above-mentioned centrifugation step). In the method of 1), a sheet-like strong structure was obtained. (Fig. 3)

Example 3: Effect of addition of ascorbic acid 2-phosphate In order to examine the effect of addition of ascorbic acid 2-phosphate on the formation of the stem cell / collagen complex, a stem cell / collagen complex was prepared by the following method.

The mesenchymal stem cells methods were collected from human synovial (MSCs), in culture medium (DMEM, 10% FBS, 1 % P / S ( penicillin / Stray script mycin), 2.0 × 10 ⁵ cells / cm ² and until cultured. cultured cells were collected in a tube, in culture medium (DMEM, 10% FBS, 1 % P / S, 0.2mM ascorbic acid 2-phosphate), 2.8 × 10 ⁵ cells / mL Was prepared to be a cell suspension of.

In another tube, medium (DMEM, 10% FBS, 1% P / S) and NaHCO ₃ were added to 2.5 mL of 3.0 mg / mL type I collagen solution to neutralize the solution. Incubation at 37 ° C. for 1 day gave a refibrotic collagen dispersion. This was centrifuged at 37 ° C. at 4000 rpm for 20 minutes to remove the supernatant.

3 mL of the above cell suspension was added to the refibrotic collagen from which the supernatant had been removed, and the suspension was suspended with a Komagome pipette to obtain a collagen fiber / MSCs solution. The collagen fiber / MSCs solution was transferred to a 6-well plate with cell culture inserts. The plate was centrifuged at 1000 rpm for 5 minutes to compress and dehydrate collagen and MSCs fibers. This was cultured in an incubator at 37 ° C. The day of centrifugation was set to day 0, and the group to which ascorbic acid 2-phosphate (0.2 mM) was added on the first day of culture, and the group to which ascorbic acid 2-phosphate (0.2 mM) was added on the fourth day of culture. A group and a group to which ascorbic acid 2-phosphate was not added were prepared. The medium was changed the day after the centrifugation, and every two days thereafter. The culture was carried out until the 14th day, with the day of the above centrifugation being 0 day.

Results The group to which ascorbic acid 2-phosphate (0.2 mM) was added on the first day of the culture, the group to which ascorbic acid 2-phosphate (0.2 mM) was added on the fourth day of the culture, and the ascorbic acid 2-. Both groups without the addition of phosphoric acid formed a sheet-like stem cell / collagen complex.

In the morphological observation with the naked eye, neither the effect of the addition or absence of ascorbic acid 2-phosphate nor the effect of the addition time of ascorbic acid 2-phosphate was confirmed.

In addition, the stem cell / collagen complex (with ascorbic acid 2-phosphate) prepared by the method of Example 1 (1) and the stem cell / prepared as a group to which the ascorbic acid 2-phosphate of this example was not added. SEM images for the collagen complex (without ascorbic acid 2-phosphate) were compared. In each case, in addition to the appearance of having a highly porous surface and a collagen (fibril, subfibril) structure with a diameter of submicrometer level, a micrometer level collagen (fascicle) structure, which is a higher-order structure, coexists. It was observed that collagen was present and that collagen binding to cells was abundant and three-dimensional (Fig. 4). Then, in high-magnification (1500-2000 times) SEM observation, it was observed that cells having a three-dimensional shape and a large amount of collagen binding to the cells were observed in both cases, and this tendency was observed as ascorbic acid. It was more prominent in the stem cell / collagen complex with 2-phosphate.

Example 4: Comparative above within collagen gel culture (1), 2.8 × 10 ⁵ cells / mL cell suspension with using stem cells / collagen composite produced in, the collagen gel in the following Morphological comparison with the tissue prepared by culturing was performed by morphological observation with the naked eye and SEM image analysis.

Culture in collagen gel (comparative example)
Intracollagen gel culture is known as a general method for culturing cells together with collagen fibers. Based on the description of Non-Patent Document 4, the tissue was prepared by the following method.

Under ice-cooling, 7 mL of 3 mg / mL collagen solution, 5 times DMEM 2 mL, and 37 g / L NaHCO ₃ 1 mL were mixed to prepare a 10 mL gel solution. 10% FBS, 1% P / S, 0.2 mM ascorbic acid 2-phosphate was added.

The cell suspension containing MSCs cell numbers 2.0 × 10 ⁵ cells / cm ² was centrifuged, the cells are integrated in the bottom of the centrifuge tube, the supernatant was removed. To the cell aggregate, 3.7 mL of a collagen gel solution containing the above 10% FBS, 1% P / S, and 0.2 mM ascorbic acid 2-phosphate was added, and the mixture was incubated at 37 ° C. to gel. It became. Culturing was carried out from the 7th day to the 14th day.

Results The results are shown in FIG. 4 (images of "collagen gel culture" and "cell / collagen complex (with ascorbic acid diphosphate"). As mentioned in Example 2, although large in collagen gel culture, tweezers A very weak tissue containing a large amount of water, which was likely to be destroyed when grasped with, was formed. On the other hand, the stem cell / collagen complex prepared by the method of Example 1 (1) was a strong sheet-like tissue.

In addition, in the low-magnification (200-fold) SEM observation, the collagen fiber structure was not confirmed in the tissue obtained by culturing in the collagen gel, as in the case of the TEC used for comparison in Example 1 (2), and the tissue was flat. A surface with low porosity was observed. On the other hand, in the stem cell / collagen complex prepared by the method of Example 1 (1), a highly porosity surface was observed at a low magnification (200 times observation), and a collagen (facicle) structure having a micrometer level in diameter was observed. It was confirmed to exist (Fig. 4 (A)).

In medium magnification (1000 times) SEM observation, collagen (fibril, subfibril) structure with a diameter of submicrometer level was observed in the tissue obtained by culturing in collagen gel. On the other hand, regarding the stem cell / collagen complex, in addition to the submicrometer-level collagen (fibril, subfibril) structure, the higher-order structure, the micrometer-level collagen (fascicle) structure, coexists. It was observed. It was also observed that the amount of collagen that binds to cells was large and three-dimensional (Fig. 4 (B)).

In high-magnification (1500 to 2500 times) SEM observation, the presence of flat cells was observed in the tissue obtained by culturing in collagen gel. On the other hand, regarding the stem cell / collagen complex, the existence of cells having a three-dimensional shape was observed. In addition, similar to the observation at medium magnification, it was also observed that the collagen binding to the cells was abundant and three-dimensional (FIG. 4 (C)).

Therefore, it was confirmed that the stem cell / collagen complex has a high porosity and a hierarchical structure of collagen fibers having fibrils and facilitates. Moreover, in the stem cell / collagen complex, since the collagen fibers around the cells are dense, it is presumed that the substrate production of the cells is active.

The novel tissue regeneration material (stem cell / collagen complex) prepared by the method described in the present application is obtained as a large tissue with high cell content, and the structure of refibrotic collagen in the stem cell / collagen complex is It can be used as a tissue regeneration material because of its characteristics that it closely resembles the surrounding environment when cells are placed in a living body.

Claims (7)

A method for producing a tissue-regenerated material, which is the following process:
(1) Mix a neutral culture medium containing stem cells with a collagen solution or dispersion;
(2) Centrifuge the mixture of (1) to accumulate stem cells and collagen;
(3) Culturing stem cells and collagen aggregates;
The method described above. The method of claim 1, wherein the stem cells are mesenchymal stem cells, ES cells, or induced pluripotent stem cells (iPS cells). The method according to claim 1 or 2, wherein the stem cell of the step (1) is a stem cell ^{cultured to a cell density of 1.0 × 10 5} cells / cm ^{2 to} 6.0 × 10 ⁵ cells / cm ^2. The method according to any one of claims 1 to 3, wherein the collagen is type I, type II or type III collagen. The method according to any one of claims 1 to 4, wherein the collagen solution or dispersion in step (1) is a fibrotic dispersion of refibrotic collagen. The method according to any one of claims 1 to 5, which comprises adding ascorbic acid or an ascorbic acid derivative or a salt thereof in the step (1) or the step (3). A tissue regenerated material produced by the method according to any one of claims 1 to 6.

Images