JP2010136655A - Method for producing three-dimensional tissue culture product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a three-dimensional tissue culture product, by which the strength of the three-dimensional tissue culture product can simply be enhanced. <P>SOLUTION: This method for producing the three-dimensional tissue culture product containing stem cells and extracellular substrates comprises a sowing process for sowing the stem cells in a culture solution in an incubator, a culture process for culturing the sowed stem cells to adhere the cultured stem cells to the surface of the incubator and for producing the extracellular substrates from the stem cells to form the three-dimensional tissue culture product in the culture solution, a separation process for separating the culture product from the surface of the incubator after a prescribed period, and a floating culture process for culturing the cells in the separated state for at least three days. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a three-dimensional tissue culture.

In recent years, when a part or all of the tissue is damaged due to severe organ failure or intractable disease, or severe sports or traffic accidents, we make full use of genetic engineering, cell tissue engineering, regenerative medicine, etc. Regenerative treatment that compensates for the deficit has attracted attention.
When cell therapy aiming at tissue repair and regeneration has been performed so far, the majority of researches have been conducted to maintain cell accumulation, stabilize cell proliferation, stabilize differentiation function, and protect against mechanical stress applied to the treatment site. Then, organic polymer compounds and biological scaffolds (Scaffold) have been used (for example, Patent Document 1). However, many of the scaffolds contain biological (animal) materials, biopolymer materials, etc., and the safety of using these materials on the living body has been a problem that cannot be predicted for a long time.

  As a cell transplantation method that does not use a base material such as a scaffold, a cell sheet engineering technique using a temperature-sensitive culture dish is known (for example, Non-Patent Document 1). However, when this cell sheet technology is used, a single sheet is often fragile, and in order to obtain a strength that can withstand surgical operations such as transplantation, it has been necessary to devise operations such as overlapping sheets. .

  In order to solve these problems, a cultured tissue containing no scaffold is known (Patent Document 2 and Non-Patent Document 2). In such a scaffold-free self-organizing three-dimensional artificial tissue (3DBT), stem cells derived from synovial membranes are cultured in a cell culture medium containing an extracellular matrix production promoting factor. It can be obtained by culturing In addition, the production of extracellular matrix is promoted by adding an extracellular matrix production promoting factor, and as a result, the obtained cultured tissue or complex is effective and becomes easy to peel off, and contracts when the culture is peeled off. However, it is described that three-dimensionalization, multi-layering, etc. are promoted. Such a cultured tissue and its complex can solve all the problems caused by contamination of the scaffold itself, and enables excellent treatment after the transplantation despite the absence of the scaffold. It is described that it can.

However, because the scaffold-free self-organizing three-dimensional artificial tissue has not yet developed a structure such as a fibrous tissue, it should be handled with care so as not to be damaged by an operation in a surgical procedure even if the self-supporting property can be maintained to some extent. Necessary. In the conventional manufacturing method, there was a limit to improving the tensile strength beyond this.
Thus, there has been a need for a method for simply increasing the strength of cultured tissue without using a polymer material or the like.
International Publication No. 2002/010349 Pamphlet Special table 2007-528756 gazette J Biomed. Mater. Res., Vol. 45, pp. 355-362 (1999) Biomaterials, Vol. 38 (36), pp.5462-5470 (2007)

  Accordingly, an object of the present invention is to provide a method for producing a three-dimensional tissue culture that can easily increase the strength of the three-dimensional tissue culture.

The present invention is as follows.
[1] A production method for producing a three-dimensional tissue culture containing stem cells and extracellular matrix in a culture solution, the seeding step for seeding the stem cells in the incubator, and the incubator by culturing the stem cells after seeding An adhesion culture step in which an extracellular matrix is produced from the stem cells to form a three-dimensional culture in the culture solution, and the culture is separated from the incubator surface after a predetermined period of time. A method for producing a three-dimensional tissue culture, comprising: a step; and a suspension culture step of suspension culture in the separated state for at least 3 days.
[2] The production method according to [1], wherein the stem cells are cultured cells of passage 4 to passage 7.
[3] The production method according to [1] or [2], wherein the stem cells are mesenchymal stem cells.
[4] The production method according to any one of [1] to [3], wherein the suspension culture step is 3 days or more and 7 days or less.
[5] The production method according to any one of [1] to [4], wherein the stem cells are cultured in the presence of an extracellular matrix production promoting factor.

[6] The production method according to [5], wherein the extracellular matrix production promoting factor is at least one selected from the group consisting of ascorbic acid, ascorbic acid derivatives, TGFβ1 and TGFβ3.
[7] The production method according to any one of [1] to [6], wherein the extracellular matrix is at least one selected from the group consisting of collagen I, collagen III, vitronectin and fibronectin.
[8] The production method according to any one of [1] to [7], wherein the three-dimensional tissue culture obtained by the suspension culture step has a tensile strength of at least 0.1 MPa.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the three-dimensional tissue culture which can raise the intensity | strength of a three-dimensional tissue culture simply can be provided.

  The method for producing a three-dimensional tissue culture of the present invention is a production method for producing a three-dimensional tissue culture containing stem cells and extracellular matrix in a culture solution, a seeding step for seeding stem cells in a culture vessel, and seeding A subsequent culture process for culturing stem cells to adhere to the surface of the incubator and producing an extracellular matrix from the stem cells to form a three-dimensional culture in the culture solution; A method for producing a three-dimensional tissue culture, comprising: a separation step of separating the culture from the above surface; and a suspension culture step of suspension culture in the separated state for at least 3 days.

  According to the present invention, stem cells are cultured to produce an extracellular matrix, and after culturing by attaching to the incubator, floating culture is performed for at least 3 days in a state separated from the incubator and separated, Not only the thickness of the three-dimensional tissue culture is increased, but also the breaking load is improved, so that the strength can be easily and sufficiently increased. This makes it possible to produce a culture useful for handling surgical procedures.

The production method of the present invention produces a three-dimensional tissue culture containing stem cells and extracellular matrix in a culture solution.
In the present invention, a stem cell refers to a cell having self-renewal ability and having pluripotency (ie, pluripotency). Examples of the stem cells in the present invention include embryonic stem (ES) cells and tissue stem cells. As long as the stem cells have the above-mentioned ability, artificially prepared pluripotent stem cells are also used in the present invention. Corresponds to stem cells. An embryonic stem cell refers to a pluripotent stem cell derived from an early embryo, and a tissue stem cell is a cell in which the direction of differentiation is more limited than that of an embryonic stem cell. Inter-tissue cells are classified into, for example, the skin system, digestive system, myeloid system, nervous system and the like. Examples of skin tissue stem cells include epidermal stem cells and hair follicle stem cells. Examples of digestive tissue stem cells include pancreatic (common) stem cells and liver stem cells. Examples of myeloid tissue stem cells include hematopoietic stem cells, mesenchymal stem cells (for example, fat-derived, bone marrow-derived), and the like. Examples of nervous system tissue stem cells include neural stem cells and retinal stem cells. In the present invention, it is preferably used for mesenchymal stem cells.

  Mesenchymal stem cells have the ability to proliferate and differentiate into bone cells, chondrocytes, muscle cells, stromal cells, tendon cells, and adipocytes. In the present invention, stem cells capable of differentiating into tendon cells, bone cells, chondrocytes and the like can be preferably used.

  These mesenchymal stem cells can be obtained from a tissue in which these stem cells can exist. Tissues preferably used for obtaining stem cells in the present invention include bone, cartilage, tendon, ligament, meniscus, intervertebral disc, periosteum, blood vessel, blood vessel-like tissue, heart, heart valve, pericardium, dura mater, etc. Can be mentioned. In particular, when stem cells that can differentiate into tendon cells are used, tissues such as synovium can be used as a supply source, and it is more preferable to use synovial stromal cells.

  The origin of the cell is not particularly limited, but when used as a culture graft or the like, it is preferably selected from mammals in consideration of species compatibility and the like. Examples of such mammals include single pores, marsupials, rodents, wings, wings, carnivores, carnivores, long noses, odd-hoofed animals, even-hoofed animals, tube teeth Can include crustaceans, scales, sea cattle, cetaceans, primates, rodents, rabbits, etc., and examples include animals such as cattle, pigs, horses, chickens, cats, dogs, etc. Can do.

  These cells may be so-called primary cultures collected from living organisms or those that have been passaged. From the viewpoint of improving stem cell density and improving extracellular matrix production ability, these cells have undergone 4 or more passages and 7 passages. It is preferable to use cultured cells of the following generation. In the present invention, “passage” is used in the meaning normally used in the art, and the stem cell in the present invention is represented by the number of passages since the primary culture collected from a living body. If the stem cell in the present invention is a cultured cell of 4 passages or more, a culture with sufficient strength can be obtained, and a cultured cell of 7 passages or less is preferable because the cell proliferation ability is sufficiently high. Moreover, if it is a cultured cell of 5 to 7 passages, the balance between the production of extracellular matrix and the cell density when used as a three-dimensional tissue culture can be improved at a high level. preferable.

The culture solution used for culturing the stem cells may be any one that is generally used for culturing the target cells. For example, DMEM, MEM, F12, DME, RPMI1640, MCDB104, 199, MCDB153, L15, SkBM Basal medium and the like can be used. In addition, various components that can be generally added, such as glucose, FBS (fetal bovine serum) or human serum, and antibiotics (such as penicillin and streptomycin) may be added to these culture solutions. In addition, although the density | concentration in the case of adding serum can be suitably changed according to the culture state at that time, it can usually be 10% (v / v). For cell culture, normal culture conditions, for example, culture in an incubator with a temperature of 37 ° C. and a concentration of 5% CO ₂ are applied.

  The incubator used for the culture may be any one that is usually used for the above-described cell culture, and examples thereof include containers such as glass, plastic (for example, polystyrene, polycarbonate, etc.), and silicone. When the cells are separated in the separation step described later, a treatment for improving the cell adhesion may be applied as long as the cells are not damaged.

The seeding of cells can be appropriately adjusted depending on the type of cells, the number of passages of cells, the culture period, etc., but in general, seeding can be performed at a cell density of 5 × 10 ⁴ to 1 × 10 ⁶ cells / cm ² .

When seeding of the cells is completed, stem cells are cultured in the attachment culture process, and the stem cells are attached to the surface of the incubator, and an extracellular matrix is produced from the stem cells to form a three-dimensional culture in the culture solution. .
The attachment of stem cells to the surface of the incubator is more easily achieved by continuing the culture under normal culture conditions, so that the cells adhere to the surface of the incubator according to the nature of the cells. The stem cells do not need to be firmly attached to the surface of the incubator, and may not be suspended independently due to the nature of the cells, but may be in contact with the bottom surface of the incubator, for example, a separation step described later In this case, it is preferable that the adhesive strength is such that it can be easily separated by a predetermined separating means, since it can suppress damage to cells.

Furthermore, by continuing the culture period, extracellular matrix is produced from the stem cells.
Examples of the extracellular matrix produced from the stem cells include collagen I, collagen III, collagen V, elastin, vitronectin, fibronectin, laminin, thrombospondin, proteoglycans (for example, decorin, biglycan, fibromodulin, lumican, hyaluron). Acid, aggrecan, and the like), but are not limited to these, and any extracellular matrix that is responsible for cell adhesion may be used. More preferably, the extracellular matrix includes all of collagen (type I, type III, etc.), vitronectin and fibronectin. By producing these extracellular matrixes, a three-dimensional culture composed of the cells in which the extracellular matrix network is formed and the extracellular matrix is formed.

In order to promote the production of extracellular matrix from stem cells, cell culture is preferably performed in the presence of an extracellular matrix production promoting factor. For this reason, it is preferable that an extracellular matrix production promoting factor is added to the culture solution.
The extracellular matrix production promoting factor may be added to the culture solution from the beginning of the culture, or may be added to the culture solution after cell seeding. Further, the extracellular matrix production promoting factor only needs to be present in the adhesion culture process, and may be present only in the adhesion culture process or both in the adhesion culture process and the floating cell process.
By culturing cells in the presence of these extracellular matrix production promoting factors, the culture can be more easily separated in the separation step described later.

  The extracellular matrix production promoting factor added to the culture medium is a factor that promotes secretion of the extracellular matrix. For example, TGF-β1, TGF-β3, ascorbic acid, ascorbic acid diphosphate or a derivative thereof Or their salts can be mentioned. From the viewpoint of collagen production, ascorbic acid, ascorbic acid diphosphate or a derivative thereof and a salt thereof (for example, sodium salt, magnesium salt, potassium salt, etc.) can be preferably used. Ascorbic acid in the present invention is preferably L-form, but is not limited thereto.

  For example, in the case of ascorbic acid 2-phosphate, the amount of the extracellular matrix production promoting factor used in the present invention is usually 0.01 mM or more, preferably 0 from the viewpoint of the tensile strength of the three-dimensional tissue culture. 0.05 mM or more, more preferably 0.1 mM or more, more preferably 0.2 mM or more. On the other hand, in the case of ascorbic acid diphosphate, the addition amount may be increased, for example, may be 5.0 mM or less. However, from the viewpoint of the efficiency of improving the tensile strength with respect to the addition amount, it is further 1.0 mM. preferable.

  The adhesion culture period for forming the three-dimensional culture varies depending on the number of seeded cells, the type of cells, etc., but in general, it can be at least 7 days, but from the viewpoint of expecting a large increase in strength. More preferably, it is about 21 days. Thereby, the three-dimensional tissue culture of sufficient size according to the objective can be formed.

In the separation step, the culture being cultured is separated from the surface of the incubator.
The three-dimensional culture formed by the culture process floats in the culture medium by separation from the surface of the incubator. By applying this separation stimulus, self-contraction occurs and the culture stiffens (increased stiffness and strength due to orientation and cross-linking of the fibrous tissue). Here, the surface of the incubator means a surface to which the seeded stem cells are attached, and is determined by the shape of the container, but is generally the bottom surface of the incubator, but may be a side surface.

  Separation means may be any stimulus that causes self-contraction in the cells but does not damage the cells. Examples of such separation means include physical means (for example, pipetting of a medium), chemical means (addition of a substance), and the like. Among these, physical means such as pipetting is preferable from the viewpoint of preventing foreign matters from being mixed into the obtained tissue. Further, peeling can be promoted by applying a physical stimulus (for example, applying physical means such as application of shear stress, pipetting, deformation of the container, etc. with a stick or the like at the corner of the container). Self-shrinking occurs naturally after such debonding when a physical stimulus is applied. In the case of chemical stimulation, self-contraction and peeling occur in parallel. It is thought that the self-contraction caused by such stimulation promotes biological binding, particularly in the third dimension, so that the culture can maintain its form as a three-dimensional structure. .

The culture separated in the separation step is subjected to suspension culture for at least 3 days in the separated state in the suspension culture step. As a result of the separation, the three-dimensional culture naturally becomes a floating state in the culture solution, and this floating culture step is performed by continuing the culture in such a floating state for a predetermined period.
Although not bound by a specific theory, adherent cells generally stabilize and build an environment close to the living body by adhering, so that culture in a floating state is unstable for the cells. It is believed that there is. In the present invention, it is unexpected that the culture strength is improved by continuing the culture in such a floating state for a predetermined period, and self-contraction as an effect of separation in the separation step is clearly understood. It is a different phenomenon. That is, the self-shrinkage occurs in a few hours to a short period after separation, whereas the improvement in the strength of the tissue culture according to the present invention is caused by a floating culture period of 3 days or more.

  In the suspension culture step, the culture in the suspended state is continued for at least 3 days. If it is 3 days or more, it can be made strong enough as a culture. If the period of the suspension culture process is less than 3 days, only autocontraction occurs, and the strength of the obtained three-dimensional tissue culture is insufficient. From the strength of the culture, it is preferably 3 days or more and 7 days or less.

  Here, the strength sufficient as a culture means a strength sufficient not to be destroyed when grasped and operated with tweezers or the like in a surgical technique, in addition to a strength sufficient for self-support. “Self-supporting” in the present invention refers to a property that the tissue is not substantially destroyed when at least one point of the tissue is fixed in space. The three-dimensional tissue culture in the present invention exhibits a strength greater than a strength sufficient for self-supporting, and can obtain a tensile strength approximately twice that of a tissue obtained by a conventional culture method. Is possible.

  The mechanical properties of a tissue that resists pulling can generally be determined by conducting a tensile test and measuring tensile strength, rigidity, Young's modulus, and the like.

Here, the mechanical properties of the cultured tissue, the three-dimensional structure, etc. of the present invention can be determined by measuring stress / strain properties in a mechanical test. Briefly, a load is applied to the sample, for example, 1ch is strain, 2ch is input to each AD converter (eg ELK-5000), stress and strain are measured, and tensile strength and contact coefficient (The hardness of the material) can be obtained.
Mechanical properties can also be determined by testing creep properties. The creep characteristic indentation test is a test for examining how the material expands with time under a certain load. For the indentation test of a minute material, a thin material, etc., an indenter having a radius of about 0.1 to 1 μm, for example, a triangular pyramid indenter is pushed into the test object. First, an indenter is pushed into the test piece to give a load. Then, when the test piece is pushed into the test piece by about several tens nm to several μm, the indenter is returned and unloaded. The hardness, Young's modulus, and the like can be obtained from the load load obtained from the load unloading curve obtained by such a test method and the behavior of the indentation depth.

  The high strength of the three-dimensional tissue culture of the present invention can be evaluated using a numerical value based on tensile strength as an index. The numerical value based on the tensile strength is preferably at least 0.1 MPa or more from the viewpoint of handling of the surgical technique, and the higher the better. This tensile strength can be confirmed by a tensile test. The intensity is defined as measured under the following conditions.

  In the tensile test, each sample is sandwiched between aluminum alloy (A6061) clamps in PBS maintained at a constant temperature of 37 ° C., and both sides are trimmed to a width of 6 mm. The temperature of the PBS is controlled by a silicon rubber heater (general SR: Three High) arranged on the bottom of the bath and a temperature controller. One of the clamps is connected to an actuator (LAH-46-3002-F-PA-B1-SP, Harmonic Drive Systems), and the other is integrated with the load detector. A load detection part makes it possible to detect a micro load correctly also by sticking a strain gauge (KGF-02-120-C1-23, Kyowa Dengyo) two sheets on each side. In order to measure the strain of each sample in a non-contact manner during the test, the marker was prepared using a micro magnet (mass 0.002 g, dimensions 1 × 1 × 1 mm) and expanded polystyrene beads (φ2 mm). I caught it. The cross-sectional area was calculated from the thickness and width measured with a microscope (VHX-100, Keyence). 2 mN is applied as a preload, the state is defined as zero strain, and a tensile load is applied until the sample breaks at a tensile speed of 0.05 mm / s.

  In addition, in order to obtain a culture in which the three-dimensional tissue culture is oriented in a specific direction, and to further improve the strength of the three-dimensional tissue culture, the culture after the separation process is performed in a specific direction in the suspension culture process. It is preferable to culture while stretching. The stretching means is not limited at all, but includes a method of simply pulling with tweezers, a method of repeatedly applying tension with a computer-controlled actuator, and the like. By orienting the culture, the fibrous tissue can be oriented and the rigidity of the culture can be increased.

  The conditions for orienting the culture are not particularly limited as long as they are performed while stretching in a specific direction, although they vary depending on the type of cells, the size of the culture, and the like. For example, when a culture having a size of width 6 mm × thickness 0.1 mm is applied, a repeated stress of about 5% of the tensile strength is applied.

The tensile load varies depending on the cell type, the size of the culture, and the like, but in general, it is preferable that the dynamic load of 4 to 8 mN is applied for 1 hour per day for a total of about 3 days. Such a tensile load is preferably performed in a culture solution.
By applying such a tensile load, the strength can be further increased.

  As described above, the three-dimensional tissue culture obtained by the present invention is a so-called scaffold-free tissue culture that does not contain an artifact, and is not damaged by an operation in a surgical procedure. It has the strength that can secure the properties.

  Examples of the present invention will be described below, but the present invention is not limited thereto. Further,% in the examples is based on weight (mass) unless otherwise specified.

[Example 1]
(1) Maintenance of cultured cells A cell for obtaining a three-dimensional tissue culture was prepared using human synovial stromal cells collected after obtaining consent from the patient.
Synovial cells were collected from the knee joint, treated with collagenase, and cultured in 10% FBS (HyClone) -DMEM medium (GIBCO). All media contains 1% penicillin / streptomycin. Cultured cells were maintained by replacing about 1/3 of the culture solution (20 ml) every 7 days, and when subcultured, the cells were subcultured every 14 days.

(2) Preparation of test piece (Sample A)
The primary cells obtained in (1) above were prepared so that the initial cell density was 4.0 × 10 ⁵ cells / cm ² , DMEM medium (10% FBS, 1% penicillin / streptomycin added, 0.2 mM) Suspended in ascorbic acid)), seeded in a 6-well plate culture dish (BD FALCON), and cultured under conditions of 5% CO ₂ and 37 ° C. The cells attached to the bottom of the culture dish shortly after seeding.

  On day 35 of culture, the culture was peeled off from the bottom of the culture dish by light pipetting and allowed to float. Thereafter, the culture was further continued for 7 days in a floating state. After 7 days, the culture was completed, and the culture was taken out of the culture solution and used as sample A (see FIG. 1). The culture period of sample A is 42 days in total.

(3) Preparation of comparative test piece (Sample B)
The primary cells obtained in the above (1) were suspended in a culture solution in the same manner as in the above (2), and the culture was started. On the 42nd day of culture, the culture was detached from the bottom using the tip of a micropipette, so that the culture was suspended from the bottom of the culture dish and maintained in the suspended state for 1 hour to self-contract. After self-contraction, the culture was removed from the culture medium and used as sample B.
(4) Evaluation of test piece The mechanical characteristics of the samples A and B obtained above were evaluated as follows.
(A) Tensile test Each sample was sandwiched between stainless steel clamps, and both sides were cut to a width of 6 mm. In order to measure the strain of each sample in a non-contact manner, the marker was manufactured using a micro magnet (weight 0.002 g, dimensions 1 × 1 × 1 mm) and polystyrene foam beads (φ2 mm), and each sample was sandwiched from above and below. The cross-sectional area was calculated from the thickness and width measured with a microscope (VHX-100, Keyence). A preload of 2 mN is applied by a linear actuator (LAH-46-3002-F-PA-V1-SP, Harmonic Drive Systems), the state is defined as zero strain, and the sample breaks at a tensile speed of 0.05 mm / s. A tensile load was applied. The results are shown in Table 1.

(B) Strain measurement Strain was measured in a non-contact manner by using an image sensor (CV-750, Keyence) with a marker attached to each sample. In addition, in order to simulate the test environment in a living body, a mechanism of keeping the PBS constant at 37 ° C. using a petri dish and a silicon rubber heater (general SR, Three High) and a temperature controller (ON-DO industry) was adopted.
The nominal stress was obtained by dividing the load output of the gauge attached to the upper end of the chuck by the initial cross-sectional area of the test piece. The tangential coefficient and tensile strength were tested by t-test, and the significance level was 5% for both. The results are shown in Table 1.
Moreover, in order to obtain | require the rigidity of a structure | tissue, the inclination in the distortion 5%-10% of a stress-strain diagram was calculated | required and compared as a tangent coefficient. A stress-strain curve is shown in FIG. In FIG. 2, white squares represent Sample A that has been subjected to suspension culture, and black circles represent Sample B that has not been subjected to suspension culture.

(C) Thickness measurement Each sample was spread on a slide glass and sandwiched between cover glasses. Using a digital microscope, the distance between the slide glass and the cover glass was measured at five locations, and the average of the measured values was calculated as the thickness of each sample. did. The results are shown in Table 1.

  As shown in Table 1 and FIG. 2, the sample A, which was subjected to suspension culture for 7 days after detachment from the dish, was not subjected to such suspension culture, although the entire culture period was almost the same. Compared with Sample B, although there was no significant difference in thickness and rigidity, the tensile strength increased by 66.5% (P <0.05). From this, it was possible not only to increase the thickness but also to easily increase the strength of the culture by a simple method of carrying out suspension culture for 7 days after peeling.

[Example 2]
<Effect of suspension culture period>
Samples C, D, and E (n = 5, respectively) were prepared after suspension on the 35th day of culture in the same manner as in Example 1, followed by suspension culture for 1 day, 3 days, and 7 days. The strength of samples C, D, and E was measured in the same manner as in Example 1. The results are shown in Table 2.
As shown in Table 2, it was found that the increase in strength of Sample D that had been subjected to suspension culture for 3 days was better. On the other hand, the strength of Sample C on the first day of suspension culture was 0.1 MPa or less. The strength of this sample C is insufficient for handling in a surgical procedure.

[Example 3]
<Influence of load culture during suspension culture>
Sample F and sample G (n = 5, respectively) suspended on the 35th day of culture as in Example 1 were adjusted to a shape with a width of 8 mm, and then cultured while applying a tensile load only to sample G Was carried out in the same manner as in Example 1 for 3 days of suspension culture. The load was as follows. That is, both ends in the longitudinal direction of the sample G are placed in a culture medium (same as that used in Example 1) while being gripped by the chuck of the testing machine, and dynamically loaded between 4 mN and 8 mN. The cells were cultured for 1 hour per day for a total of 3 days.
After culturing under load, the tensile strength was measured in the same manner as in Example 1. The results are shown in Table 3. As shown in Table 3, when compared with Sample F that was not cultured under load, Sample G that was cultured under load had almost no difference in thickness but increased in strength by 1.6 times. (P <0.05). Therefore, the strength of the cultured cells is increased about twice by performing floating culture, and the strength can be further increased by performing culture under load.

  From this, according to this invention, the intensity | strength of a three-dimensional tissue culture can be raised easily. A three-dimensional tissue culture with increased strength is useful in handling surgical procedures.

It is the figure which showed typically preparation of the sample A concerning the Example of this invention. It is a stress-strain curve of each sample obtained in Example 1 of this invention.

Claims (8)

A production method for producing a three-dimensional tissue culture containing stem cells and extracellular matrix in a culture solution,
A seeding process for seeding stem cells in an incubator;
An adhesion culture step of culturing the stem cells after seeding and attaching them to the surface of the incubator, producing an extracellular matrix from the stem cells, and forming a three-dimensional culture in the culture solution;
A separation step of separating the culture from the surface of the incubator after a predetermined period;
A suspension culture step of suspension culture for at least 3 days in the separated state;
A method for producing a three-dimensional tissue culture comprising:   The production method according to claim 1, wherein the stem cell is a cultured cell of passage 4 to passage 7.   The production method according to claim 1 or 2, wherein the stem cells are mesenchymal stem cells.   The production method according to any one of claims 1 to 3, wherein the suspension culture step is 3 days or more and 7 days or less.   The manufacturing method according to any one of claims 1 to 4, wherein the stem cells are cultured in the presence of an extracellular matrix production promoting factor.   6. The production method according to claim 5, wherein the extracellular matrix production promoting factor is at least one selected from the group consisting of ascorbic acid, ascorbic acid derivatives, TGFβ1 and TGFβ3.   The production method according to any one of claims 1 to 6, wherein the extracellular matrix is at least one selected from the group consisting of collagen I, collagen III, vitronectin and fibronectin.   The manufacturing method according to any one of claims 1 to 7, wherein a tensile strength of the three-dimensional tissue culture obtained by the suspension culture step is at least 0.1 MPa or more.