JP2003265169A - Biotissue-like structure, method for culturing bone marrow stem cell and culture kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for culturing a bone marrow stem cell, by which excellent effects such as the acquisition of such a transplantation material as early designed can be obtained due to the non-contraction of a support during the culture, an expectation for the efficient proliferation and differentiation of the cell, and the non-contraction of the finally obtained cell-support composite product, to provide a culture kit which can be used for the culturing method, and to provide a biotissue-like structure. <P>SOLUTION: This method for culturing the bone marrow stem cell is characterized by using an animal cell culture solution and a support consisting essentially of a composite material where a biodegradable synthetic polymer exists in a fibrous form in a sponge comprising a cross-linked natural polymer, and proliferating and differentiating the bone marrow stem cell on the support. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the regeneration of defective parts of living tissues and organs, and in-vitro pharmacological, histological, physiological and pathological examinations of living tissues and organs.
The present invention relates to a biological tissue-like structure that can be used to perform in a culture system. Further, the present invention is a culture method capable of obtaining a biological tissue-like structure as described above, wherein the bone marrow stem cells are proliferated and differentiated on a support serving as a scaffold for cells, and further, , A culture kit that can be used in this culture method.

[0002]

2. Description of the Related Art In recent years, attempts have been enthusiastically attempted to reproduce a defect in a living tissue based on tissue engineering technology. As one attempt, it is considered to use stem cells having a high proliferation and differentiation potential, and it has become quite possible actually to proliferate and differentiate stem cells under various conditions using cell culture technology. However, tissue regeneration cannot be realized simply by giving cells to the defect, and in order to enable this, it is necessary to culture stem cells with an appropriate scaffold material to obtain a three-dimensional biological tissue-like structure. Becomes Further, these structures can be used as a material for pharmacological, histological, physiological or pathological evaluation in vitro. Among them, bone marrow stem cells are proliferated for the purpose of repairing or regenerating hard tissue parts such as bone-based tissues such as bone and cartilage, and defects of soft tissue parts such as bone marrow, skin, nerve, fat and muscle tissues. Cultures that have been differentiated and used have attracted attention. In such cell culture and tissue culture, it is naturally important to select the type of culture medium and various additive components such as serum and hormonal agents, but how should it be used as a scaffold for cells? Various researches and developments have been made on the basis that it is particularly important to use the one having various physical properties and functions.

As a support material for the scaffolding of the cells, 1) it has affinity with cells, 2) it can maintain sufficient mechanical strength during the period required for tissue regeneration, and 3).
It has biodegradability and absorbability, and can be rapidly decomposed and eliminated according to its progress so as not to interfere with the progress of tissue regeneration.
4) It is porous to make it easy for cells to enter and to supply oxygen and nutrients to the cells that have entered,
Etc. are considered to be required. When bone marrow stem cells are cultivated using a support material satisfying such requirements, there is no contraction of the scaffold during culturing, and oxygen and nutrients are excellently supplied to the cells. Proliferation and differentiation are expected, and since the finally obtained composite of cells and scaffold material does not shrink, it is expected that excellent effects such as obtaining transplant material as originally designed can be expected. To be

As a support material for the scaffold, a sponge-like molded article using collagen, which is a natural polymer, has been conventionally known, but it has a drawback of low mechanical strength and is actually used in vivo. There was a problem that the shape could not be maintained when it was embedded in the cell or immersed in the cell suspension for cell inoculation. On the other hand, as a material for regenerating a biological tissue that solves the problem of mechanical strength, for example, polylactic acid, polyglycolic acid, or a copolymer thereof, or a biodegradable material such as a copolymer of these and ε-caprolactone is used. Although sponge-like moldings and non-woven fabrics using synthetic polymers have been proposed, materials consisting only of biodegradable synthetic polymers have poor affinity for cells as compared with collagen, and at the same time, their degradation disappears. There is a problem that it takes a long time and the remaining material hinders the regeneration of the living tissue. As described above, it was difficult to simultaneously satisfy the above 1) to 3) with a material using collagen or biodegradable synthetic polymer alone.

Therefore, as one method for compensating for the disadvantages of both, a composite material of collagen and a biodegradable synthetic polymer has been developed in recent years. For example, JP-A-7-236
Japanese Patent No. 688 proposes a composite material in which at least a part of collagen sponge material is coated with biodegradable plastic. However, since this composite material has a synthetic polymer on its surface, it has excellent mechanical strength, but its biocompatibility is still low and there is room for improvement. On the other hand, "artificial organs" 29
Volume 2, 463-467 (2000) reports a composite material obtained by immersing a sponge made of a lactic acid / glycolic acid copolymer in a collagen solution and then freeze-drying it. However, the composite material obtained in this way has a high ratio of synthetic polymer to collagen, and it is still not sufficiently satisfactory in terms of affinity with cells, and the material is decomposed and absorbed in the body. There was a problem that the time required for the treatment was too long from the viewpoint of tissue regeneration.

[0006]

Therefore, the problem to be solved by the present invention is that there is no contraction of the scaffold during culturing and the supply of oxygen and nutrients to the cells is excellent, so that more efficient cell growth is possible. , Bone marrow that is expected to undergo differentiation, and does not shrink the finally obtained complex of cells and scaffolding material, so that it is possible to obtain excellent effects such as obtaining the transplantation material as originally designed. Stem cell culture method, culture kit that can be used in this culture method, and
It is intended to provide a biological tissue-like structure.

[0007]

Means for Solving the Problems The inventors of the present invention have made extensive studies to solve the above problems. As a result, we thought that it would be good to use a support material in the method of culturing bone marrow stem cells that has a low synthetic polymer content and that effectively retains mechanical strength. After repeated mistakes and studies, it was found that it was a composite material of a natural polymer such as collagen and a biodegradable and absorbable synthetic polymer. It was confirmed that the above problems can be solved at once by using a crosslinked support material as an essential element, and the present invention has been completed.

That is, the biological tissue-like structure according to the present invention is constituted by using a composite material in which a biodegradable synthetic polymer is present in a fibrous form in a sponge composed of a crosslinked natural polymer as an essential material. The support contains an animal cell culture medium, and supports bone marrow stem cells and / or living tissue cells differentiated from the bone marrow stem cells and / or intermediate cells in the differentiation process. To do. In addition, the method for culturing bone marrow stem cells according to the present invention is a support composed essentially of a composite material in which a biodegradable synthetic polymer is present in a fibrous form in a sponge composed of a crosslinked natural polymer. And an animal cell culture medium are used to grow and / or differentiate bone marrow stem cells on the support.

In addition, the culture kit according to the present invention is a support composed essentially of a composite material in which a biodegradable synthetic polymer is present in a fibrous form in a sponge composed of a crosslinked natural polymer. It is characterized in that the body, the bone marrow stem cells, and / or the living tissue cells differentiated from the bone marrow stem cells, and / or the intermediate cells in the differentiation process, and the animal cell culture medium are independently contained as constituent elements.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. However, the scope of the present invention is not restricted by these explanations, and other than the following examples, the scope of the present invention is not impaired. It can be carried out. The method for culturing bone marrow stem cells according to the present invention (hereinafter sometimes referred to as the culturing method of the present invention) is such that a biodegradable synthetic polymer is present in a fibrous form in a sponge composed of a crosslinked natural polymer. This is a culture method in which a bone marrow stem cell is proliferated and / or differentiated on the support using a support and an animal cell culture solution which are composed of the composite material as an essential material.

In the culture method of the present invention, the support is
The above-mentioned composite material that can also be used for regeneration of biological tissue is an essential element. The above-mentioned composite material is not particularly limited, but specifically, lyophilization is performed in a state where a fibrous biodegradable and absorbable synthetic polymer is impregnated in a solution of a natural polymer, and then a crosslinking treatment is performed. It can be obtained by crosslinking a natural polymer. The natural polymer is not particularly limited, but specifically, for example, collagen,
Proteins such as gelatin, fibrin, gluten, fibroin and their derivatives; polyamino acids and their derivatives; polysaccharides such as chitin, chitosan, hyaluronic acid, alginic acid, starch, dextran and their derivatives; mixtures of two or more thereof. And a complex formed by a chemical bond; and the like. In addition,
Only one natural polymer may be used, or two or more natural polymers may be used in combination.

Among the natural polymers listed above, collagen is more preferred as the natural polymer. The collagen is not particularly limited, and may be any conventionally known collagen obtained from animal bones or skins as raw materials, and specifically, for example, acid-solubilized collagen, enzyme-solubilized collagen Preferable examples include soluble collagen such as alkali-solubilized collagen and oxygen-soluble collagen, chemically modified collagen of the solubilized collagen, and regenerated collagen obtained by regenerating collagen fibers from the solubilized collagen. As the soluble collagen,
Type I collagen is more preferred. The type I collagen is
Since it is the same as collagen found in bone, tendon, dentin, etc., it is excellent in the ability to be replaced at the transplant site, which is preferable. Among the type I collagen, atelocollagen is
Since the telopeptide at the terminal of the molecule that causes bio-toxicity is partially or wholly removed by the enzymatic treatment, it has little or no antigenicity to the living body, which is preferable. More specifically, for example, pig skin-derived type I collagen, pig tendon-derived type I collagen, bovine nose cartilage-derived type II collagen, fish type I collagen, gene recombinant collagen, or a mixture thereof and the like. To be

As described above, when the above-mentioned composite material is obtained, the natural polymer is once treated in the form of a solution. When collagen is used as the natural polymer, the concentration of the collagen solution is not particularly limited. In particular,
It is preferably 0.05 to 10% by weight, and 0.3 to
More preferably, it is 2% by weight. If the concentration of the collagen solution is less than 0.05% by weight, the amount of collagen may be too small to form a sponge-like structure sufficiently, and if it exceeds 10% by weight, the viscosity of the aqueous solution may be high. Therefore, it may be difficult to handle the sponge when it is produced.

When collagen is used as a natural polymer, the collagen solution has an organic property that is incompatible with water for the purpose of adjusting the pore size of the collagen sponge obtained by effective foaming to an optimum range. A solvent can be added. The organic solvent is not particularly limited, but specifically, for example, halogenated hydrocarbons such as chloroform, carbon tetrachloride, methylene chloride; esters such as ethyl acetate, ethyl propionate;
Aromatic hydrocarbons such as benzene and toluene; hexane,
Aliphatic hydrocarbons such as cyclohexane; ethers such as diethyl ether and diisopropyl ether; and the like. These may be used alone or in combination of two or more. In addition, when these organic solvents are added, in order to sufficiently bring out the effect, it is preferable that the concentration of the organic solvent after addition is 2% by weight or more, and more preferably 2 to 20% by weight. , And even more preferably 2 to 10% by weight.

Further, when collagen is used as a natural polymer, various additives conventionally used in the production of collagen sponge are added to the collagen solution as needed, as long as the effects of the present invention are not impaired. You may add it. Specifically, for example, by adding mucopolysaccharides, cell adhesion factors, cell growth factors, cytokines, chemokines, etc., or proteins or peptides having physiological activity possessed by these substances,
The biocompatibility can be further improved. The biodegradable synthetic polymer is not particularly limited, but specifically, for example, polyglycolic acid, polylactic acid, poly-ε-
Caprolactone, polydioxanone, poly-β-malic acid, polyorthoester, polydiaminophosphazene,
And copolymers thereof and the like. Among these, polyglycolic acid, polylactic acid, and poly-ε are particularly preferable because they have biodegradability suitable for the period of general tissue regeneration.
-Caprolactone or a copolymer thereof is preferable. The biodegradable synthetic polymer may be used alone or in combination of two or more.

It is important that the biodegradable synthetic polymer is fibrous. By allowing the biodegradable synthetic polymer to exist in a fibrous form, it is possible to maintain sufficient mechanical strength with a small content. Specifically, the fibers may be long fibers or short fibers, but it is preferable that the fibers are sufficiently entangled with each other, for example, in a state in which a nonwoven fabric is loosened. The fiber diameter is also not particularly limited, and specifically, a fiber diameter of 1 to 100 μm can be preferably used. Regarding the content of the biodegradable synthetic polymer, it is preferable that the biodegradable synthetic polymer / natural polymer (dry weight ratio) = 0.01 to 5, and more preferably 0.05 to 3 , And even more preferably 0.1 to 1. If the above weight ratio is less than 0.01, mechanical strength sufficient to maintain the volume of the collagen sponge may not be obtained, and if it exceeds 5, a large amount of biodegradable synthetic polymer is exposed in the collagen sponge. Then
The cell affinity may be lower than that of the sponge alone. As described above, since the biodegradable synthetic polymer is fibrous, sufficient mechanical strength can be maintained even if the content of the biodegradable synthetic polymer is small as in the above range. Since the content of the biodegradable synthetic polymer is low, excellent affinity with cells can be expressed, and at the same time, the biodegradable synthetic polymer can be rapidly decomposed and eliminated after tissue regeneration. When collagen is used as the natural polymer, the biodegradable synthetic polymer is added to the collagen (when the collagen solution (the state before adding the organic solvent and various additives) is used, the amount of the collagen contained is equal to that of the collagen). The weight ratio (dry weight ratio) is preferably 0.01 to 5, and more preferably 0.1 to 1.

Freeze-drying is not particularly limited, but specifically, for example, a solution of a natural polymer is added to a container containing a fibrous biodegradable synthetic polymer, and the biodegradable synthetic polymer fiber is added. May be impregnated. At this time, the solution of the natural polymer added to the container is preferably sufficiently foamed by using an ordinary homogenizer or the like. The freeze-drying method may be carried out according to a conventional method. Specifically, for example, the temperature at the time of freezing is -196.
To -4 ° C is preferable, and -10 is more preferable.
0 to -4 ° C, and even more preferably -90 to -10 ° C. If the temperature during freeze-drying is less than -196 ° C, the resulting sponge has a small pore size, and cells may not penetrate into the sponge to proliferate or differentiate. In this case, the pore size of the sponge becomes too large and cells cannot stably exist in the sponge.
In addition, exposure of the biodegradable polymer fibers into the sponge pores may impair the cell affinity. In addition,
For example, the collagen sponge has a pore size of 80 to 30 because cells easily enter the sponge and the cells can stably exist and proliferate and differentiate in the sponge.
It is preferably 0 μm.

It is important that the sponge obtained by freeze-drying is subsequently subjected to a crosslinking treatment. That is, natural polymers such as collagen are crosslinked by this crosslinking treatment.
By using a crosslinked natural polymer, the biodegradability of the sponge can be controlled and sufficient mechanical strength can be maintained even if the content of the biodegradable synthetic polymer is low. The method of the cross-linking treatment is not particularly limited, but specifically, a conventionally known chemical cross-linking method, a thermal dehydration cross-linking method under vacuum, a cross-linking method by ultraviolet irradiation, or the like may be adopted.

When chemical cross-linking is performed, a cross-linking agent such as glutaraldehyde, carbodiimide, carbonylimidazole, diepoxy compound, diacid anhydride and epichlorohydrin may be used, and a known cross-linking method may be used. When thermally dehydrating and crosslinking under vacuum, there is no particular limitation, but specifically, the crosslinking temperature is preferably 100 to 170 ° C, more preferably 110 to 160 ° C.
It is more preferably 120 to 160 ° C., and the time required for crosslinking is preferably 2 to 120 hours, more preferably 3 to 100 hours, and even more preferably 6
~ 24 hours. If the cross-linking temperature is less than 100 ° C,
There is a possibility that thermal dehydration cross-linking cannot be sufficiently performed, and if the temperature exceeds 170 ° C., there is a risk of causing thermal decomposition or thermal deterioration of collagen or biodegradable synthetic polymer. Also,
If the time required for crosslinking is less than 2 hours, it may depend on the temperature, but there is a possibility that thermal dehydration crosslinking cannot be sufficiently performed.
If it exceeds 120 hours, the collagen or biodegradable synthetic polymer may be thermally decomposed or deteriorated.

The irradiation with ultraviolet rays is not particularly limited, but specifically, the wavelength of ultraviolet rays is 200 to 400.
It is preferable to irradiate ultraviolet rays of nm, more preferably 200 to 350 nm, and even more preferably 200 to 350 nm.
300 nm, particularly preferably 254 nm, and the irradiation time of ultraviolet rays is preferably 1 minute to 24 hours,
It is more preferably 3 minutes to 18 hours, and even more preferably 3 minutes to 10 hours. When the wavelength of ultraviolet rays is less than 200 nm, collagen or biodegradable synthetic polymer may be decomposed or deteriorated, and when it exceeds 400 nm, sufficient crosslinking may not be possible. Further, if the irradiation time is less than 1 minute, sufficient crosslinking may not be possible, and if it exceeds 24 hours, collagen or biodegradable synthetic polymer may be decomposed or deteriorated.

The thus obtained composite material for regenerating biological tissue is a microporous sponge composed of a crosslinked natural polymer in which fibrous biodegradable and absorbable synthetic polymer is randomly embedded. It has the form Therefore, it has excellent affinity with cells, can maintain sufficient mechanical strength during the period required for tissue regeneration, and has appropriate biodegradability that can be rapidly decomposed and eliminated after tissue regeneration. . Therefore, for example, skin, gingiva, pulp, bone marrow, soft tissue such as muscle and fat, nerve tissue, cartilage tissue, bone tissue, liver, pancreas, testis, endocrine tissue such as ovary and kidney, digestive tract such as esophagus and gastrointestinal tract, Urinary organs such as the trachea, bladder, ureter and urethra, teeth, peripheral tissues of the eye, blood vessels, ductal tissues such as lymphatic vessels and lacrimal ducts, salivary glands, glandular tissues such as sweat glands and sebaceous glands, periodontal tissues, tendons, ligaments It is useful as a scaffold material for cell proliferation and differentiation for regenerating a defective portion such as, but in the culture method of the present invention, the composite material is used for proliferation and differentiation of bone marrow stem cells in vitro. It is effectively used as a scaffold material for culture.

As the support in the culture method of the present invention, not only the above composite material but also other materials may be used in combination. The other material is not particularly limited,
Specifically, for example, hydroxyapatite (hyd
roxyapatite (HAP)), tri-cal
cium phosphate (TPC), Poly
(DL-lactic-co-glycolic ac
id (PLGA) porous material, collagen sponge (col
lagen sponge) and the like. The content ratio of the other material in the whole support is preferably such that the effect of the composite material is not extremely hindered.

The animal cell culture medium used in the method for culturing bone marrow stem cells of the present invention is not particularly limited, and, for example, animals that can be generally used for culturing bone marrow stem cells and various biological tissue cells are generally used. It may be a culture medium for cell culture, but is preferably an animal cell culture medium containing serum and hormone substances. The base medium of the animal cell culture medium may be a natural medium or a synthetic medium, and is not particularly limited, but infection by bacteria or viruses from animal-derived substances, composition depending on the time and place of supply. In consideration of the variation of the above, a synthetic medium is more preferable. The synthetic medium is not particularly limited, and specifically, for example, α-MEM (ALPHA).
-MINIMUM ESSENTIAL MEDIU
M), Eagle MEM, Dulbeco MEM,
RPM ± -1640, CMRC, HAM, DME / F1
2, 199 medium, MCDB medium, IMEM and the like can be mentioned. As the natural medium, a generally known natural medium can be mentioned, and it is not particularly limited. These may be used alone or in combination of two or more.

The serum contained in the animal cell culture medium is
Specific examples thereof include, but are not limited to, fetal bovine serum, human autologous serum, commercially available human serum, and horse serum. These may be used alone or in combination of two or more. Serum is a component that contains cell growth factors and the like to promote the growth of cells, and contains cell adhesion factors and the like to play a role in promoting the adhesion of cells to cell substrates (supports). It is an essential element of liquid. Horse serum can also be used for cell selection. Similarly, the hormone substance contained in the animal cell culture medium is not particularly limited, but specifically, for example,
Insulin, transferrin, dexamethasone, hydrocortisone, thyroxine, 3,3 ′, 5-triiodothyronine, β-mercaptoethanol, dimethyl sulfoxide, 1-methyl-3-butylxanthine, progesterone, butylated hydroxyanisole and the like can be mentioned. it can. These may be used alone or in combination of two or more. Of these, dexamethasone can be used for inducing differentiation into bone cells. The hormonal substance is a component that plays a role of survival of cells, induction of differentiation, maintenance of differentiation state, and the like, and is therefore an essential element of the above-mentioned animal cell culture medium.

The animal cell culture medium in the method for culturing bone marrow stem cells of the present invention may contain components other than the above-mentioned serum and hormonal substances. Other components are not particularly limited, and specifically, for example, ascorbic acid (particularly L-ascorbic acid), β-glycerophosphate, biotin, calcium pantothenate, ascorbic acid diphosphate, vitamin D ₃ , Sodium selenite,
Examples thereof include linoleic acid, retinoic acid, pyruvic acid, putrescine, monothioglycerol and glutamine. These may be used alone or in combination of two or more. The content of the other components in the animal cell culture medium is preferably such that the effects of the serum and hormonal substances are not extremely disturbed.

The culturing method of the present invention is a culturing method for proliferating and / or differentiating bone marrow stem cells. Here, differentiating bone marrow stem cells means not only terminal differentiation into living tissue cells but also living tissue. Differentiation into cells in each stage of differentiation in the differentiation process up to cells, for example, intermediate cells such as progenitor cells and blast cells is also included. In addition, the above-mentioned culturing method may include proliferating bone marrow stem cells themselves, including proliferating together with differentiating bone marrow cells, and further may include morphogenesis to finally obtain a living tissue. To do. Specifically, for example, not only the terminal differentiation of bone marrow stem cells into desired biological tissue cells but also proliferation of the biological tissue cells and further morphogenesis may be included.

The bone marrow stem cells are not particularly limited and include bone marrow mesenchymal stem cells and hematopoietic stem cells. Specifically, for example, bone marrow cells aseptically taken from animal bone marrow tissue; It is preferable that dental pulp cells aseptically taken out from the dental pulp tissue of an animal; various blood cells and the like aseptically taken out from an animal are subjected to primary culture on a collagen gel to isolate cells attached to the collagen gel. In addition,
Even without collagen, it may be attached to a normal culture dish and isolated. More specifically, aseptically removed cells are washed with the above-mentioned culture solution in order to remove hybrids that adversely affect cell growth (to prevent contamination during culture), and EGTA (ethylene glycol). After being treated with bis (2-aminoethyl ether) tetraacetic acid, the product obtained by recovering by an operation such as centrifugation and subjecting it to the above primary culture is preferable. Also,
The primary culture is not particularly limited, but specifically,
For example, it is preferably performed on a collagen gel in an incubator at 37 ° C. with 5% CO ₂ . Primary cultured cells can be prepared by digesting a collagen gel with an enzyme that digests collagen (for example, collagenase).
It is preferably obtained by isolation from the state of being attached to collagen gel and used in the culture method of the present invention.

In the culturing method of the present invention, not only bone marrow stem cells but also living tissue cells terminally differentiated from bone marrow stem cells and various intermediate cells such as progenitor cells and blast cells in the differentiation process are used for culturing. It can also be (proliferated, differentiated). These bone marrow stem cells, biological tissue cells, and intermediate cells are preferably used after cryopreservation or cryopreservation as necessary. The culture method of the present invention comprises culturing the above-mentioned bone marrow stem cells on the above-mentioned support, and culturing on the support specifically means that the above-mentioned bone marrow stem cells are seeded on the surface of the support by a usual method. It may be a so-called two-dimensional culture, or it may be an ordinary method (for example, seeding cells on the surface of the support and then forming a support material on the cells). Method of covering, method of injecting cell suspension into the support, method of placing cell suspension on the surface of the support to allow cells to naturally permeate into the support, and suspension for preparing the support The method may be so-called three-dimensional culture, in which cells are seeded and cultured in such a manner that they are embedded by a method in which cells are also suspended in advance), and there is no particular limitation.

In the culturing method of the present invention, when culturing to proliferate bone marrow stem cells, and / or when differentiating bone marrow stem cells into desired living tissue cells or into various intermediate cells in the differentiation process, In the case of further proliferating living tissue cells and intermediate cells, various culture conditions such as the type and addition amount of serum and hormone substances in the culture medium may be appropriately selected from commonly known conditions. The biological tissue cells obtained by terminally differentiating from bone marrow stem cells are not particularly limited, and any biological tissue cells such as biological hard tissue cells and biological soft tissue cells may be the target cells, Specific examples include osteoblasts, osteoclasts, chondroblasts, periodontal ligament cells, adipocytes, muscle cells, nerve cells, and the like. These living tissue cells may be only one type or two or more types may coexist.

The biological tissue-like structure according to the present invention (hereinafter,
It may be referred to as the biological tissue-like structure of the present invention. ) Is
A support composed of a composite material consisting of a biodegradable synthetic polymer present in a fibrous form in a sponge composed of a crosslinked natural polymer as an essential material contains an animal cell culture solution, and contains bone marrow. A biological tissue-like structure in which cells and / or biological tissue cells differentiated from bone marrow stem cells and / or intermediate cells in the process of differentiation from bone marrow cells to biological tissue cells are supported. The biological tissue-like structure of the present invention is used, for example, as a transplant material for regeneration of a defective portion of a biological tissue or an organ, or in-cell pharmacology, histology, physiological and pathology of cells. Although it is preferably used for conducting a physical examination, its use is not particularly limited.

The biological tissue-like structure of the present invention is preferably obtained by the above-mentioned culture method of the present invention.
Further, in the biological tissue-like structure of the present invention, the support, bone marrow stem cells, differentiated biological tissue cells, medium stem cells in the differentiation process, animal cell culture medium, etc. are those described in the description of the culturing method of the present invention. It is preferably the same as Specifically, the biological tissue cells are biological tissue cells that have been terminally differentiated from bone marrow stem cells, and the intermediate cells, cells in the differentiated state of each stage in the differentiation process from bone marrow stem cells to the biological tissue cells, For example, progenitor cells and blast cells. Further, in the biological tissue-like structure of the present invention, when the biological tissue cells are supported by the support, it may include a state in which the biological tissue is finally morphologically formed.

In the biological tissue-like structure of the present invention, bone marrow stem cells, the biological tissue cells or the intermediate cells are supported by the support, and the biological tissue cells or the intermediate cells are used as the target transplanted portion. Those differentiated as suitable living tissue cells or intermediate cells, and those differentiated as living tissue cells or intermediate cells to be subjected to various tests as described above are preferable, but bone marrow stem cells or these living tissue cells or Not only the intermediate cells but also other cells such as living tissue cells that can be used for another tissue or organ may be supported together. The form supported by the support is not particularly limited, but specifically, even in the form of being two-dimensionally supported by being adhered onto the surface of the support,
The sponge-like support may be in a form of being three-dimensionally supported by adhering to various places inside the support, and may be a combination of both forms. When the biological tissue-like structure of the present invention is used for transplantation, it is preferable that it is three-dimensionally supported inside a support having a sponge-like structure. When two or more types of cells are contained, the cells may be present separately or mixed, and the cells or cell aggregates are distributed with a certain regularity such as a layered or sea-island shape. You may have. In the case of the biological tissue-like structure used for the above various tests, combinations of all kinds of cells are possible.

When the living tissue-like structure of the present invention is used as a material for transplantation, it is used for periodontal disease treatment, periodontal tissue regeneration therapy,
In the regeneration of alveolar bone and periodontal ligament absorbed by periodontal disease, as a hybrid type repairing material such as a bone defect repair material, periodontal ligament cell composite implant material, fractures and congenital bone defects and surgical For bone defects such as bone defects that have occurred in the body, defects of bone structure and periodontal defects (such as periodontal ligament cell defects) that require supplements, etc. It can be implanted using standard surgical procedures well known to physicians such as orthopedics and dentistry (eg, the method used to supply only bone marrow or bone fragments). In addition, for defects such as muscle, skin, and soft tissue of other organisms such as fat, neurosurgery, plastic surgery, ophthalmology, otolaryngology, dermatology, pediatrics, general surgery, cardiovascular department, thoracic surgery, abdomen It can be implanted using standard surgical procedures well known to physicians such as departments, orthopedics, such as those used in supplying skin alone. Prior to transplantation
The need and method of checking for tissue compatibility is similar to that of traditional implantable materials.

As described above, the living tissue-like structure of the present invention may be transplanted to a living body in a state containing a desired animal cell culture solution or used for various tests. The support on which the biological tissue cells are supported may be in the form of being impregnated with the culture fluid (so that the sponge contains water), or the support on which the biological tissue cells are supported in the culture fluid. The body may be dipped (further, it may be packaged in a dipped state), or may be a combination thereof, and is not particularly limited. The culture kit according to the present invention (hereinafter, also referred to as the culture kit of the present invention) has a biodegradable synthetic polymer present in a fibrous form in a sponge composed of a crosslinked natural polymer. The support, the bone marrow stem cells, and / or the living tissue cells differentiated from the bone marrow stem cells, and / or the intermediate cells in the differentiation process, and the animal cell culture medium, which are composed of the composite material A culture kit including as a component,
The culture kit may include at least one of these components, but any of the components may include other components in addition to these components.

The culture kit of the present invention can be used, for example, to carry out the culture method of the present invention. Further, in the culture kit of the present invention, the support, bone marrow stem cells, differentiated biological tissue cells, medium stem cells in the differentiation process, animal cell culture medium and the like are the same as those described in the explanation of the culture method of the present invention. Is preferred. Specifically, the biological tissue cells are biological tissue cells that have been terminally differentiated from bone marrow stem cells, and the intermediate cells, cells in the differentiated state of each stage in the differentiation process from bone marrow stem cells to the biological tissue cells, For example, progenitor cells and blast cells. The culture kit of the present invention contains the bone marrow stem cells, the biological tissue cells, and the intermediate cells as described above, and these cells are, for example, the components of the kit, and can usually maintain the cell viability. It may be stored in a condition of a certain degree, and can be in a state capable of proliferating and differentiating cells or forming morphology of living tissue when culturing using the kit of the present invention. Further, when the above-mentioned living tissue cells or intermediate cells are contained, those differentiated and / or expanded from bone marrow stem cells under desired conditions may be used.

In the culture kit of the present invention, the animal cell culture medium may be in a state of being prepared as a culture medium by adding necessary or desired components, or each component may be in a separate state. It may be provided, or when carrying out culture using the kit of the present invention, for example, necessary components and the like may be provided in a powder state so that the user can prepare a culture solution. There is no particular limitation as long as the animal cell culture solution can be prepared finally. In the culture kit of the present invention, the support can be similarly provided in a desired state.
Specifically, for example, the support itself may be provided as it is, or may be provided by immersing it in some kind of storage solution.

The above-mentioned other components are not particularly limited, but specifically, for example, articles used when culturing bone marrow stem cells and the like using a support which is usually a scaffold material such as a culture container, , Various additives such as antibiotics. These may contain only 1 type and may contain 2 or more types.

[0038]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. In the following, "parts by weight" may be simply referred to as "parts" for convenience. In addition, "wt%" may be simply referred to as "wt%". -Example 1- [Preparation of PGA-collagen sponge] 3 mg of a non-woven fabric made of polyglycolic acid (PGA) was cut out and uniformly loosened with tweezers, and then silicon ("Sigma Coat" S
Aluminum cap coated with IGMA (2)
0 mmφ, 34 mmH). Next, chloroform was added to 1.5 g of a pig skin-derived type I collagen solution of 0.3% by weight so that the chloroform concentration was about 5% by weight, and the mixture was stirred at 12000 rpm for 3 minutes using a homogenizer with a generator shaft. Then, the solution was added to the aluminum cap. Then, the aluminum cap was frozen at −80 ° C. for 12 hours, and then freeze-dried for 24 hours under a vacuum of less than 0.1 Torr. Then 140
Thermal dehydration cross-linking was performed for 12 hours under vacuum at a temperature of less than 0.1 Torr to obtain a PGA-collagen sponge composite material having polyglycolic acid / collagen = 0.67 (weight ratio). [Preparation of bone marrow mesenchymal stem cells] Fischer rat (F
(344, male, 7 weeks old) were sacrificed under ether anesthesia, and the left and right femurs were collected. The collected femur was left to stand on a sterilized petri dish on ice to remove the muscle tissue of the diaphysis, and both ends of the femur were excised. Syringe (1.8G, 2.5ml)
Α-MEM containing 10% fetal bovine serum (manufactured by GIBCO)
1 ml was put and 0.5 ml was injected from both ends of the femur to collect bone marrow. After collecting the obtained bone marrow in a 50 ml tube, use a dish with a diameter of 10.0 cm to obtain 10%.
Fetal bovine α-MEM (penicillin 100 unit / ml,
Streptomycin 0.1 g / ml) at 37 ° C. for 5
Culture was performed under the condition of% CO ₂ . Three days later, the floating cells were removed, and the adherent cells attached to the dish surface were collected as bone marrow mesenchymal stem cells. Recovered cells from subcon
Bone marrow mesenchymal stem cells were prepared by culturing until fluent (a state in which proliferating cells cover about 80% of the dish surface) was subcultured for 2 days. [Three-dimensional culture] The prepared bone marrow mesenchymal stem cells were trypsinized to obtain an α-MEM cell suspension. Diameter 1.8c
m, 3 mm in height of PGA-collagen sponge was used as a culture support, and the bone marrow mesenchymal stem cells prepared therein were seeded using an injection needle (21G) at 5 × 10 ⁶ cells / sponge, and b- Α-MEM containing 10% fetal bovine serum containing FGF (2.5 ng / ml, manufactured by Kaken Pharmaceutical Co., Ltd.) and dexamethasone (10 nM, manufactured by Wako Pure Chemical Industries, Ltd.).
It was cultured for 21 days in a culture medium (manufactured by GIBCO).

During the culture period, the PGA-collagen sponge was visually and histologically observed to measure the alkaline phosphatase activity of the cells supported by the sponge. The observation and measurement will be described in detail later. -Comparative Example 1- [Preparation of Collagen Sponge] To 1.5 g of a 0.3% by weight pig skin-derived type I collagen solution, chloroform was added so that the chloroform concentration was about 5% by weight, and a homogenizer with a generator shaft was added. Using 12000r
After stirring at pm for 3 minutes, the solution was added to an aluminum cap (20 mmφ, 34 mmH) coated with silicon ("Sigma Coat" SIGMA). Then, the aluminum cap was frozen at −80 ° C. for 12 hours, and then freeze-dried for 24 hours under a vacuum of less than 0.1 Torr. Then, thermal dehydration crosslinking was performed for 12 hours at 140 ° C. under a vacuum of less than 0.1 Torr to obtain a collagen-only material for comparison. [Three-dimensional culture] The bone marrow mesenchymal stem cells prepared in Example 1 were trypsinized to obtain an α-MEM cell suspension. A collagen sponge having a diameter of 1.8 cm and a height of 3 mm was used as a culture support, and the prepared bone marrow mesenchymal stem cells were seeded using an injection needle (21G) at 5 × 10 ⁶ cells / sponge, and b Α-MEM containing 10% fetal bovine serum containing FGF (2.5 ng / ml, manufactured by Kaken Pharmaceutical Co., Ltd.) and dexamethasone (10 nM, manufactured by Wako Pure Chemical Industries, Ltd.)
It was cultured for 21 days in a culture medium (manufactured by GIBCO).

As in Example 1, during the culture period, PGA-
The collagen sponge was observed macroscopically and histologically, and the alkaline phosphatase activity of the cells supported by the sponge was measured. <Macroscopic and histological observations> [Observation method] As the macroscopic observation, it may be observed with the naked eye, and if necessary, a magnifying glass such as a magnifying glass is used. It is possible to confirm the shape change and the like.

The method of histological observation was as follows. The sponge after culture was transferred to a 12-well (container), washed with PBS (-) for 10 minutes three times, and then 4% paraformaldehyde (in 0.1M PB). Fixed in. Thin sections were prepared and stained with hematoxylin and eosin according to a conventional method.
As a result, the cells can be stained, and the cell morphology and cell localization can be confirmed. [Observation Results / Discussion] In the macroscopic observation, the collagen sponge used in Comparative Example 1 begins to contract from the first day of culture,
The diameter of the PGA-collagen gel sponge used in Example 1 was maintained at the initial morphology on the 21st day of culture, although the diameter was 1/3 of that on the 9th day of culture.

Histological observation revealed that in Comparative Example 1, most cells were found on the sponge surface after culturing.
Then, not only on the surface of the sponge but also inside the sponge. Therefore, PGA-collagen sponge
It was found that it is more useful as a scaffold material for three-dimensional culture than collagen sponge. Since collagen sponge contracts and deforms during culture, cells do not proliferate even in the sponge, whereas PGA-collagen sponge hardly causes such contraction or deformation, and the sponge remains in the sponge during the culture period. This is because the scaffold for invading and proliferating cells into the cell is optimally secured. << Measurement of alkaline phosphatase activity >> [Measurement method] 12 wells (container) of sponge after culturing
And washed 3 times with PBS (-) for 10 minutes. Then, it was cryopreserved at -30 ° C for 24 hours. During the measurement, after repeated freezing and thawing, 16 mM p-NPP (SI
GMA) was added to each well and added at room temperature for 20
Shake for a minute. 1N NaOH was added to each well to stop the reaction, and MICRO PLATE READER
(Manufactured by Tosoh) was used to measure at 415 to 600 nm. With this, the absorbance of the solution can be measured, and the activity can be determined from the calibration curve of the absorbance and the alkaline phosphatase activity which has been prepared in advance. [Measurement Result / Discussion] The alkaline phosphatase activity measurement value in Example 1 was clearly higher than that in Comparative Example 1. Specifically, the first embodiment is 2.
The value was three times higher.

The reason why the activity was high when the PGA-collagen sponge was used was that there was almost no contraction or deformation of the sponge during the culture period as described above. In the method for culturing bone marrow stem cells, the PGA-collagen sponge is ex-
Even in vivo, it has excellent properties including handling, and is a scaffo for transplantation materials such as biological tissue regeneration.
Very useful as an ld (scaffold, support).

[0044]

INDUSTRIAL APPLICABILITY According to the present invention, there is no contraction of scaffolds during culturing and excellent supply of oxygen and nutrients to cells, so that more efficient cell growth and differentiation are expected, and the final Since the complex of the cells and the scaffold material obtained in 1. does not shrink, a method for culturing bone marrow stem cells that can obtain excellent effects such as obtaining a transplant material as originally designed, and used for this culturing method It is possible to provide a culture kit and a biological tissue-like structure that can be obtained.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yasuhiko Tabata             3-8-16 Biwadai, Uji City, Kyoto Prefecture (72) Inventor Takehiko Kinoshita             1-21-17 Kotsubo, Zushi City, Kanagawa Prefecture (72) Inventor Yosuke Hiraoka             2-22, Futamatata, Yao-shi, Osaka Nitta Zera             Chin Co., Ltd. Osaka factory F-term (reference) 4B033 NA16 NB02 NB13 NB34 NB58                       NB65 NB69 NC06 ND12 NG05                       NH04                 4B065 AA91X BC42 BD39 BD40                       CA44                 4C081 AA12 AA13 AA14 AB04 AB05                       AB06 AB11 AB12 AB13 AB15                       AB16 AB18 AB35 BA12 BA13                       BA16 BB04 BB08 CA161                       CA171 CA191 CC01 CC05                       CD011 CD031 CD041 CD081                       CD091 CD111 CD121 CD151                       CD171 CD26 CD27 CD34                       DA02 DA04 DA05 DA06 DB01                       DC13 DC14

Claims (3)

[Claims] 1. A support composed essentially of a composite material comprising a biodegradable synthetic polymer present in a fibrous form in a sponge composed of a cross-linked natural polymer, containing an animal cell culture solution. And / or bone marrow stem cells and / or
Living tissue cells differentiated from bone marrow stem cells, and / or supporting intermediate cells in the differentiation process,
Biological tissue-like structure. 2. A support and an animal cell culture solution, which are composed of a composite material in which a biodegradable synthetic polymer is present in a fibrous form in a sponge composed of a crosslinked natural polymer as an essential material. A method for culturing bone marrow stem cells, which comprises proliferating and / or differentiating bone marrow stem cells on the support. 3. A support, a bone marrow stem cell, and / or a support composed essentially of a composite material in which a biodegradable synthetic polymer is present in a fibrous form in a sponge composed of a crosslinked natural polymer. A culture kit, which contains, as independent components, living tissue cells differentiated from bone marrow stem cells, and / or intermediate cells in the differentiation process, and an animal cell culture medium, respectively, as constituent elements.