JP2004073195A - Method for culturing tissue cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To make it possible to differentiate and induce marrow cells to arbitrary tissue cells. <P>SOLUTION: A method for culturing the tissue cells 1 comprises mixing the tissue cells 1 which are collected from a patient and to be cultured with other cells 2 which are collected from the same patient and contain stem cells 4, then culturing the mixed cells. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to a method for culturing tissue cells.

In recent years, it has become possible to regenerate bone and repair the defect by replenishing the bone defect caused by removal of a bone tumor or trauma with a bone substitute. Hydroxyapatite (HAP) and tricalcium phosphate (TCP) are known as bone replacement materials. However, from the viewpoint that no foreign substance is left in the body, for example, a calcium phosphate porous material such as β-TCP is used. Scaffolding is used. When β-TCP is kept in contact with the bone tissue of a bone defect, so-called remodeling is performed in which osteoclasts eat β-TCP and osteoblasts form new bone. That is, the bone replacement material that has been repaired in the bone defect part is replaced with autologous bone over time.

On the other hand, it has been proposed to use a cultured bone produced by culturing bone marrow mesenchymal stem cells collected from a patient together with a bone filling material in order to increase the repair rate of a bone defect after surgery. In this case, it is known that a differentiation-inducing factor such as dexamethasone is effective for differentiating mesenchymal stem cells into osteoblasts. Therefore, by culturing with such a differentiation-inducing factor, osteoblasts replenish the cultured bone in a state where the osteoblasts have proliferated using the bone-reinforcing material as a scaffold. As compared with, the number of days until replacement with autologous bone can be greatly reduced (for example, see Non-Patent Document 1).
Uemura et al., “Tissue Engineering in Bone Using Biodegradable β-TCP Porous Material-Ospherion, a New Material that Increases Strength in the Living Body”, Medical Asahi, Asahi Shimbun, October 1, 2001, No. 30 Vol. 10, No. 10, p. 46-49

However, in many other tissue cells other than bone tissue, it is not clear what kind of differentiation inducer can be used to differentiate mesenchymal stem cells into desired tissue cells. For example, it is difficult to differentiate mesenchymal stem cells cultured from bone marrow cells into chondrocytes.

The present invention has been made in view of the above-described circumstances, and provides a method for culturing tissue cells, which can induce differentiation from bone marrow cells into any tissue cells and can shorten the culture period. It is an object.

In order to achieve the above object, the present invention provides the following means.
The invention according to claim 1 provides a method for culturing tissue cells in which tissue cells to be cultured collected from a patient are mixed with cells containing stem cells collected from the same patient and cultured.
According to the present invention, by co-culturing tissue cells collected from the same patient with cells containing stem cells, it becomes possible to induce the differentiation of stem cells into the tissue cells by the tissue cells.

According to a second aspect of the present invention, in the method for culturing a tissue cell according to the first aspect, the tissue cells and the cells including the stem cells are mixed and primary cultured in a predetermined medium, and then mixed with a biological tissue supplement. To provide a culture method for secondary culturing.
According to the present invention, a stem cell that has been induced to differentiate into a tissue cell by being co-cultured is mixed with a living tissue filling material and then subcultured, whereby a supplement having tissue cells grown using the living tissue filling material as a scaffold is provided. The body can be manufactured.

The invention according to claim 3 is the method for culturing tissue cells according to claim 1, wherein the cells including stem cells and the tissue cells are separately primary-cultured in a predetermined medium, and then the cells including primary-cultured stem cells are used. Provided is a culture method in which tissue cells are mixed, and further, a living tissue replacement material is mixed, followed by secondary culture.
According to the present invention, the cells including the stem cells and the tissue cells are grown separately without being differentiated by being separately primary-cultured. After that, by mixing the two, and further mixing the living tissue replacement material and performing a secondary culture, a sufficient amount of tissue cells differentiated from a sufficient amount of stem cells have grown using the living tissue replacement material as a scaffold. Can be obtained.

According to a fourth aspect of the present invention, in the method for culturing tissue cells according to the first aspect, cells including stem cells are primarily cultured in a predetermined medium, and the tissue cells are primarily cultured in a predetermined medium together with a living tissue supplement. After that, the present invention provides a culture method of mixing cells containing primary cultured stem cells and tissue cells attached to a living tissue filling material to perform secondary culture.

According to the present invention, stem cells to be differentiated into tissue cells can be cultured by primary culture until a sufficient amount is obtained. In addition, by primary culturing the tissue cells together with the living tissue filling material, it is possible to produce a living tissue filling material having an amount of tissue cells capable of sufficiently inducing the differentiation of stem cells. Then, by mixing the two and then performing secondary culture, it becomes possible to differentiate the stem cells into desired tissue cells and attach them to the living tissue filling material.

The invention according to claim 5 provides a method for culturing cells in which cells containing stem cells and human fibroblasts are mixed and cultured.
According to the present invention, the proliferation of stem cells can be promoted by the interaction between cytokines (bFGF, VEGF, PDGF, HGF, KGF, GM-CFC) secreted by human fibroblasts and factors such as matrix (collagen). It becomes possible.

According to a sixth aspect of the present invention, in the method for culturing tissue cells according to the fifth aspect, after performing a treatment for losing the proliferation ability using radiation (for example, X-ray or γ-ray) on the human fibroblast, A culture method for mixing with cells including stem cells is provided.
According to the present invention, since the proliferation ability of human fibroblasts is lost by treatment with radiation, in a culture step in a state of being mixed with stem cells, human fibroblasts proliferate and proliferate stem cells to be cultured. Can be prevented from being suppressed.

The invention according to claim 7 provides a method for culturing cells in which, after sowing human fibroblasts in a culture vessel, cells containing stem cells are sowed and cultured on human fibroblasts sowed on the culture vessel. .
According to the present invention, it is possible to improve the efficiency of adhesion of stem cells to the bottom surface of a culture container by the action of human fibroblasts initially seeded in the culture container. As a result, the number of stem cells that can be immediately expanded at the start of culture can be increased, and the culture efficiency can be improved.

According to the method for culturing tissue cells according to the present invention, the desired tissue cells are mixed and co-cultured together with the stem cells capable of differentiating into various living tissue cells, so that the differentiation of the stem cells into the tissue cells is induced. This has the effect of efficiently producing a complement of the tissue cell. In particular, it is effective for producing a complement of tissue cells in which the presence of a differentiation inducing factor is not known.
Further, the interaction between cells to be co-cultured can shorten the culture period and improve the culture efficiency.

The method for culturing tissue cells according to the first embodiment of the present invention will be described below with reference to the drawings.
The culture method according to the present embodiment provides a method of culturing tissue cells in which tissue cells to be cultured collected from a patient are mixed with cells including stem cells collected from the same patient and cultured.
Here, the cells containing stem cells are, for example, bone marrow cells.

More specifically, the culture method according to the present embodiment, as shown in FIG. 1, a primary culture step of culturing predetermined tissue cells 1 and bone marrow cells 2 collected from a patient in a predetermined medium A, A secondary culturing step of further culturing the tissue cells 3 and the mesenchymal stem cells 4 grown by culturing in a predetermined medium B.
In order to carry out this culture method, first, bone marrow cells 2 are extracted by applying, for example, a bone marrow fluid collected from a patient to a centrifuge. In addition, normal tissue cells 1 are collected from the same patient.

In the primary culture step, first, the prepared bone marrow cells 2 and tissue cells 1 are placed in a predetermined medium A, for example, MEM (Minimal Essential Medium), FBS (Fetal Bovine Serum: fetal bovine serum) and antibiotics. The substance is introduced into a medium A constituted by mixing the substances in an appropriate ratio. Subsequently, the cells are cultured for about 10 days in an incubator set under predetermined culture conditions, for example, 37 ± 0.5 ° C. and a CO ₂ concentration of 5%. During the culture, the medium A is changed periodically.

Thus, in the primary culture step, the mesenchymal stem cells 4 in the bone marrow cells 2 adhere to and grow with the tissue cells 3 on the bottom surface of the culture vessel. At this time, the tissue cells 3 come into contact with the mesenchymal stem cells 4 to start inducing the differentiation of the mesenchymal stem cells 4 into the tissue cells 3.
In this state, for example, the mesenchymal stem cells 4 and the tissue cells 3 are detached from the bottom surface of the culture vessel using a protease such as trypsin, and then collected by centrifugation.

Next, in the secondary culture step, the collected mesenchymal stem cells 4 and tissue cells 3 are put into a culture vessel together with the supplementary material 5, and, unlike a predetermined medium B, for example, a MEM different from the medium used for the primary culture, , A nutrient such as vitamin C, a growth factor and the like in addition to FBS and antibiotics. The filler 5 is, for example, a mixture of β-tricalcium phosphate and polylactic acid. Culture conditions are, for example, in an incubator set at 37 ± 0.5 ° C. and a CO ₂ concentration of 5% for about 2 weeks, for example, similarly to the primary culture step described above. During the culturing, the medium B is changed periodically as in the primary culturing step.

In the secondary culture step, the mesenchymal stem cells 4 are induced by the coexisting tissue cells 3 and differentiated into the tissue cells 3, and adhere to the filling material 5, whereby the filling material 5 is scaffolded. To grow as. As a result, the living tissue replacement 6 in which the replacement material 5 is partially replaced by the tissue cells 3 is manufactured. By filling such a defect in the living tissue in the living body, the living tissue filling body 6 can regenerate the living tissue very quickly and repair the defect. Therefore, the postoperative repair speed can be improved, and the burden on the patient can be reduced.

In the culture method according to the present embodiment, the predetermined tissue cells 1 are co-cultured together with the mesenchymal stem cells 4 contained in the bone marrow cells 2 from the primary culture step. Therefore, as the tissue cells 1 to which the culture method of the present embodiment can be applied, the mesenchymal stem cells 4 are allowed to grow under predetermined conditions while suppressing the differentiation of the mesenchymal stem cells 4 into the tissue cells 1. Suitable tissue cells 1, for example chondrocytes, are suitable.

Next, a method for culturing tissue cells according to a second embodiment of the present invention will be described below with reference to FIG.
In the description of the culture method according to the present embodiment, the same components as those in the description of the culture method according to the above-described first embodiment will be denoted by the same reference numerals, and the description will be simplified.
The culture method according to the present embodiment is different from the culture method according to the first embodiment in a primary culture step. In the culture method according to the present embodiment, bone marrow cells 2 and predetermined tissue cells 1 are mixed and cultured with a predetermined medium A in separate culture vessels. The culture conditions are the same as in the culture method according to the first embodiment.

According to the culture method according to the present embodiment, the mesenchymal stem cells 4 and the tissue cells 1 extracted from the bone marrow cells 2 are each cultured purely without being affected by other cells. In this case, the mesenchymal stem cells 4 and the tissue cells 3 can be obtained at an early stage in amounts sufficient to obtain the complement 6 of the present invention. Further, since the cells are cultured without being affected by other cells, there is an advantage that the culture state can be easily managed.

The mesenchymal stem cells 4 and the tissue cells 3 thus produced are separated from the culture vessel by the action of a protease such as trypsin in the same manner as in the first embodiment, and then centrifuged. Each is collected separately in a separator. Thereafter, the mesenchymal stem cells 4 and the tissue cells 3 obtained in the primary culture step are put into the culture vessel together with the predetermined culture medium B and the predetermined supplementary material 5, and the secondary culture step is performed. As a result, a complement 6 similar to that of the culture method according to the first embodiment is obtained.

In the culture method according to the present embodiment, the primary culture step is performed separately from the mesenchymal stem cells 4 in which the predetermined tissue cells are contained in the bone marrow cells 2. Therefore, as the tissue cell 1 to which the culture method of the present embodiment can be applied, a tissue cell 1 having a high differentiation inducing action of the mesenchymal stem cell 4 into the tissue cell 1, for example, a chondrocyte is suitable.

Next, a method for culturing tissue cells according to a third embodiment of the present invention will be described below with reference to FIG.
In the culture method according to the present embodiment, the same primary culture step as described above is performed for only the bone marrow cells 2, and for the predetermined tissue cells 1, the tissue cells 1 are mixed together with the predetermined filler 5 from the primary culture step. Do.

According to this method, similarly to the culture method according to the second embodiment, the mesenchymal stem cells 4 that are cultured alone can grow without being affected by other cells, and the required amount can be rapidly increased. Can reach the mesenchymal stem cells 4. In addition, the predetermined tissue cells 1 can be sufficiently permeated into the filling material 5 by being cultured together with the filling material 5 from the beginning.

{Circle around (4)} When the mesenchymal stem cells 4 have grown to a sufficient amount, the mesenchymal stem cells 4 are mixed with the tissue cells 1 already cultured together with the supplementary material 5 to perform a secondary culture step. As a result, the mesenchymal stem cells 4 are induced to differentiate into the contacted tissue cells 1, and the growth into the tissue cells 1 is promoted using the filler 5 as a scaffold. In this case, since the tissue cells 1 for inducing differentiation are growing so as to penetrate the filling material 5, the differentiation of the mesenchymal stem cells 4 is promoted even inside the filling material 5, and the tissue cells are sufficiently introduced into the inside. It becomes possible to manufacture the complement 6 in which the substitution to 1 has been performed.

In the culturing method according to the present embodiment, similarly to the culturing method according to the second embodiment, as the tissue cells 1, the tissue cells 1 having a high differentiation inducing action of the mesenchymal stem cells 4 into the tissue cells 1 are used. For example, chondrocytes are suitable.

As described above, according to the culture method according to each of the above-described embodiments, no suitable differentiation inducing factor is known, and the complement of tissue cells 1 in which it has been difficult to artificially induce differentiation from mesenchymal stem cells 4 6 can be easily and quickly manufactured.
In each of the above embodiments, the mesenchymal stem cells 4 extracted from the bone marrow cells 2 are exemplified as the stem cells. Alternatively, the mesenchymal stem cells collected from other bodily fluids such as umbilical cord blood or peripheral blood, Alternatively, somatic stem cells may be used.
In addition, chondrocytes have been described as examples of tissue cells having a high differentiation inducing action from mesenchymal stem cells, but nerve cells, bone cells, skin, and the like may be used instead.

Further, as a component to be added to the medium 4, a growth factor such as a cytokine, a concentrated platelet, a substance contributing to the growth such as BMP, FGF, TGF-β, IGF, PDGF, VEGF, HGF, or a combination thereof is mixed. You may decide to do so. Further, a hormonal agent such as estrogen and other nutrients may be mixed.

In this case, depending on the activity of the mesenchymal stem cells 4 and the tissue cells 2 to be cultured, the mixing ratio of these growth factors, hormonal agents or nutrients may be determined. As the antibiotic, any antibiotic such as cephem, macrolide, tetracycline, fosfomycin, aminoglycoside, and new quinolone can be used in addition to the penicillin antibiotic.

In addition, although a mixture of β-tricalcium phosphate and polylactic acid was used as the filling material 5, other porous ceramics, collagen, polylactic acid, or a combination thereof may be used instead. . Beta tricalcium phosphate, collagen, polylactic acid, and the like are biodegradable and need not be removed because they are absorbed by the living body, and apatite and the like have high strength. A person skilled in the art can select an appropriate type of the supplement 5 according to the site to be transplanted or the like.

Next, a method for culturing cells according to a fourth embodiment of the present invention will be described below.
The culture method according to the present embodiment is a culture method in which cells including stem cells and human fibroblasts are mixed and cultured. Specifically, in the culture method according to the present embodiment, cells containing stem cells and human fibroblasts are put into a culture vessel and cultured under the same culture conditions as described above.
According to this culture method, stem cells can be efficiently transformed by the interaction of growth factors such as cytokines (bFGF, VEGF, PDGF, HGF, KGF, GM-CFC) and matrix (collagen) secreted from human fibroblasts. Can be propagated.

In this case, it is preferable that the proliferation ability of human fibroblasts mixed with cells including stem cells be suppressed by irradiation with 15 to 50 Gy of γ-rays. This is because when human fibroblasts have a high proliferative ability, growth factors secreted from cells including stem cells have the disadvantage that human fibroblasts are proliferated instead of stem cells to be cultured originally.

Also, instead of the method of simultaneously introducing and mixing cells containing stem cells and human fibroblasts into a culture container, first, after seeding the human fibroblasts in the culture container, It is effective if cells containing stem cells are seeded from. That is, human fibroblasts have the effect of promoting the adhesion of stem cells to the bottom of the culture vessel. The stem cells adhere to the bottom surface of the culture vessel and grow while forming predetermined colonies. Therefore, when culturing, it is necessary to adhere the stem cells to the bottom face of the culture vessel at an early stage. At present, it is considered that 2800 stem cells are present in 1 ml of bone marrow fluid, but the number of cells adhering to the bottom surface of the culture vessel was very small if the bone marrow fluid was simply poured into the medium as it was.

According to the culture method according to the present embodiment, the number of stem cells adhering to the bottom of the culture vessel can be significantly increased early from the start of culture, and as a result, a large number of cells can be obtained in a short period of time. There is an effect that can be. That is, the culture efficiency can be improved and the culture period can be shortened.

It is a flowchart which shows the culture method of the tissue cell concerning 1st Embodiment of this invention. It is a flow chart which shows the culture method of the tissue cell concerning a 2nd embodiment of this invention. It is a flowchart which shows the culture method of the tissue cell concerning 3rd Embodiment of this invention.

Explanation of reference numerals

1 tissue cells 2 bone marrow cells (cells including stem cells)
4 Mesenchymal stem cells (stem cells)
5 Filling material (living material for living tissue)
A, B medium

Claims (7)

(4) A method for culturing tissue cells in which tissue cells to be cultured collected from a patient are mixed and cultured with cells containing stem cells collected from the same patient. 2. The method for culturing tissue cells according to claim 1, wherein the tissue cells and the cells including the stem cells are mixed and primary-cultured in a predetermined medium, and then mixed with a biological tissue-supplementing material and subjected to secondary culture. After separately culturing the cells containing the stem cells and the tissue cells in a predetermined medium separately, mixing the cells containing the primary cultured stem cells and the tissue cells, and further mixing the living tissue-supplementing material for the secondary culture. Item 4. The method for culturing tissue cells according to Item 1. The cells containing stem cells are primarily cultured in a predetermined medium, and the tissue cells are primarily cultured in a predetermined medium together with a biological tissue filler, and then the cells containing the primary cultured stem cells and the tissue cells attached to the biological tissue filler. 2. The method for culturing tissue cells according to claim 1, wherein the mixture is subjected to secondary culture. (4) A method for culturing cells in which cells containing stem cells and human fibroblasts are mixed and cultured. (6) The method for culturing cells according to claim 5, wherein the cells are treated with cells containing stem cells after the human fibroblasts are subjected to treatment for losing the proliferation ability using radiation. (4) A method for culturing cells in which human fibroblasts are seeded in a culture vessel, and then cells containing stem cells are seeded and cultured on the human fibroblasts seeded on the culture vessel.

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