JP2003052365A - Separation of mesenchymal stem cell from mammalian animal and method for using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for separating and collecting mesenchymal stem cells to utilize them for the regenerative treatment of a tissue, etc., and for the preparation of mammalian cells used for the regeneration and/or restoration of the tissue, safe for a collected parent body and also easy for collection, and also a practical method for preparing cells for transplant by using the collected mesenchymal mast cells. SOLUTION: This method for collecting mesenchymal stem cells capable of collecting the cells by using a method for separating and collecting the cells from an oral cavity tissue by a simple operation of requiring a minimum exfoliation and incision of the skin and muscle, not imposing a burden to the collected parent body, safely and simply collecting the necessary amount of the cells maintaining an excellent differentiation potency. Further, the use of the mesenchymal stem cells is provided by culturing and differentiation-inducing the separated and collected mesenchymal stem cells to prepare cells used for the regeneration and/or restoration of a tissue having high differentiation potency, and transplanting the prepared cells to the defective part and/or part for restoration such as the bone, cartilage, periodontal tissue, etc., to regenerate and/or restore the tissues.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for separating and collecting mesenchymal stem cells from mammals, and preparing cells for tissue regeneration and / or repair by inducing culture and differentiation of the separated and collected mesenchymal stem cells. And further relates to a method of regenerating mammalian tissue using the cells.

[0002]

BACKGROUND OF THE INVENTION Mesenchymal stem cells are known as pluripotent stem cells existing in the bone marrow of mammals and differentiated into adipocytes, chondrocytes and osteocytes. Due to their pluripotency, mesenchymal stem cells have attracted attention as a transplant material for regenerative medicine of many tissues such as bone, cartilage, tendon, muscle, fat and periodontal tissue (Gene Medicine, Vol. .4, No.2 (2000) p58-6
1). Recently, a review article on the current state and prospects of mesenchymal stem cell research has been published, and reports on the collection and culture of mesenchymal stem cells have been made (Experimental Medicine, Vol. 19, No. 3 (February)) 2
001, p350-356). Furthermore, it was recently reported that mesenchymal stem cells are also present in adipose tissue (Tissue Engineeri
ng, PA Zuk et al., Multilineage cells from human
adipose tissue: implications for cell-based ther
apies. 7: 211-228, 2001).

On the other hand, in recent years, several patent applications have been published regarding the culture and differentiation of mesenchymal stem cells. For example,
Japanese Patent Publication No. 11-506610 discloses a composition and method for maintaining the survival of human mesenchymal progenitor cells in a serum-free environment, and Japanese Patent Publication No. 10-512756 discloses induction of differentiation of mesenchymal stem cells. In order to achieve this, a method comprising contacting with a bioactive factor such as an osteoinductive factor consisting of prostaglandin, ascorbic acid, collagen extracellular matrix, a factor associated with differentiation, a cartilage inducing factor, etc. is disclosed in JP-A-2000-2.
Japanese Patent No. 17576 discloses inventions of methods for culturing pluripotent mesenchymal stem cells in the coexistence of prolactin or its equivalent, and differentiating the mesenchymal stem cells into adipocytes.

In order to utilize mesenchymal stem cells for tissue regenerative medicine, first, these stem cells are collected from living tissue,
It is necessary to grow it, and further differentiate and grow it to prepare a tissue. Mesenchymal stem cells exist in bone marrow, periosteum, etc., but for practical application to tissue regeneration medicine, developing a time-consuming method for collecting mesenchymal stem cells from these tissues, and To develop a method for obtaining sufficient amount of lobular stem cells,
It is an important issue to develop a problem-free method for pain. Conventionally, mesenchymal stem cells for transplantation used for regenerative medicine of bone, cartilage, etc. are separated from the bone marrow and / or periosteum of the pelvis (iliac bone) and long bones of the limbs (femur, tibia). However, large-scale surgery is required to separate cells / tissues from these tissues, which causes great damage to the collected mother (patient, etc.). Furthermore, there are problems with the time and effort required to collect mesenchymal stem cells from bone marrow, obtaining sufficient amounts of mesenchymal stem cells, and safety in collecting stem cells. It is a barrier to practical use when using. Therefore, for the purpose of utilizing mesenchymal stem cells for regenerative medicine, etc., it is necessary to develop a safe and easy method for separating and collecting the stem cells, and a practical method for preparing transplant cells using the collected cells. Is required to be developed.

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to prepare a mammalian cell for tissue regeneration and / or repair for use in tissue regenerative medicine, etc. Another object of the present invention is to provide a method for separating and collecting simple mesenchymal stem cells and a practical method for preparing cells for transplantation using the collected mesenchymal stem cells.

[0006]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that the method of separating and collecting mesenchymal stem cells from the oral tissue minimizes the incision of skin and muscle. It is possible to obtain mesenchymal stem cells by a simple operation, and therefore, it is possible to safely and easily maintain the excellent mesenchymal stem cells without imposing a severe burden on the collected mother body. The present invention has been made based on the finding that it is possible to obtain Furthermore, in the present invention,
The separated and collected mesenchymal stem cells are cultivated and differentiation-induced to prepare cells for regeneration and / or repair of a tissue having a high differentiation potential, and the cells are used for a defective portion of tissue such as bone, cartilage, or periodontal tissue and And / or transplanting to a repair site to regenerate and / or repair tissue.

[0007] That is, the present invention provides a method for separating and collecting mesenchymal stem cells from a mammalian oral tissue (claim 1), characterized in that the oral tissue is alveolar bone. The method for separating and collecting mammalian mesenchymal stem cells according to claim 1 (claim 2), which is the bone marrow, palate or periosteum of an alveolar bone, or a cell for collecting tissue from a cell collected from oral tissue. And a method for separating and collecting mammalian mesenchymal stem cells (claim 3), which comprises inoculating with a culture container, culturing, and separating and collecting cells adhered to the culture container. The method for culturing mammalian mesenchymal stem cells, wherein the mammalian mesenchymal stem cells separated and collected by the method described above are seeded in a mesenchymal stem cell culture medium, and primary culture and subculture are performed ( Claim 4)
Alternatively, the mesenchymal stem cells cultured by the culture method according to claim 4 are seeded in a mesenchymal stem cell differentiation inducing medium to carry out the differentiation inducing culture, and the differentiation inducing culture of mammalian mesenchymal stem cells is performed. The method (claim 5) or the culture of mesenchymal stem cells is carried out in a medium to which fibroblast growth factor (FGF) is added, wherein the culture of mammalian mesenchymal stem cells according to claim 4 or 5. A method (claim 6).

The present invention also provides mammalian cells (claim 7) for tissue regeneration and / or repair prepared by the method for inducing differentiation according to claim 5, and the cells are derived from human oral tissue. The mammalian cell for regenerating and / or repairing tissue according to claim 7 (claim 8), or the mammalian cell for regenerating and / or repairing tissue according to claim 7 or 8 together with a single substance, if necessary. A method for regenerating mammalian tissue, which comprises transplanting to a defective portion and / or a repaired portion (claim 9).
It consists of

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention comprises separating and collecting mesenchymal stem cells from the oral tissues of mammals. In the present invention, the mesenchymal stem cells are derived from oral tissues and are cells capable of differentiating into cells such as osteoblasts, chondrocytes, adipocytes, periodontal ligament and cementum, or promoting their repair. In the present invention, examples of oral tissues that are sources for separating and collecting mesenchymal stem cells include bone marrow such as alveolar bone, periosteum of palate or alveolar bone, and the like. Further, in the present invention, the collection of stem cells from a tissue can be carried out by a simple operation that requires minimal peeling and incision of the skin and muscle, and the device is also a device usually used for such a simple operation. You can The collected cells are cultured in a culture container such as a culture dish for tissue culture, and the stem cells adhered to the culture container are separated and collected.

In the present invention, the isolated and collected mesenchymal stem cells are subjected to primary culture, subculture, and further differentiation-inducing culture to prepare cells for tissue regeneration and / or repair. As a medium used for culturing cells, an appropriate medium for cell culture can be used, but a DMEM medium for cell culture containing fetal bovine serum (FBS) can be preferably used. For induction of differentiation of cells into tissue cells, an appropriate differentiation induction medium can be used according to each purpose. By adding fibroblast growth factor (FGF) to the medium, the culturing effect can be remarkably enhanced. The prepared cells for regenerating and / or repairing the tissue are mixed with a carrier such as collagen and transplanted into the defect and / or repair site of the tissue. The cells for regenerating and / or repairing the tissue collected and prepared from the stem cell collection source of the present invention have a high level of differentiability and have a remarkable regenerating ability as cells for regenerating and repairing the tissue.
Hereinafter, embodiments of the present invention will be described in more detail.

(Method for Separation and Collection of Oral Bone Marrow-Derived Mesenchymal Stem Cells) The mucous membrane of the human or experimental animal's upper or lower jaw-free region without roots and avoiding neurovasculature is peeled off by about several millimeters square, and 1 Use a dental drill with a diameter of about a millimeter to make a hole until the bone marrow fluid exudes from the alveolar bone. Then, the injection needle (21 gauge) is carefully inserted so as not to penetrate the tooth root, maxillary sinus, etc., and about 0.5 ml to 1 ml of bone marrow fluid is collected. Suitable medium (eg DMEM containing 10% FBS)
10 cm diameter tissue culture dish with (medium) is seeded (about 2 × 10 ⁸ cells), and after 3 days, only the cells adhered to the culture dish are cultured. Floating cells are washed and removed. Adherent cells were collected from 0.5 ml of bone marrow fluid before growth by 10 ³
-10 ⁴ can be obtained.

(Method for Separation and Collection of Oral Periosteum-Derived Mesenchymal Stem Cells) The alveolar mucosa of the palate or upper or lower jaw of a human or experimental animal is peeled off to expose the periosteum on the alveolar bone.
Collect about 5 mm. The collected periosteum is minced and then incubated at 37 ° C. with collagenase. Then, the cells are dispersed by, for example, pipetting, and the cells are collected by filtration or centrifugation. The obtained cells are counted (about 10 ⁴ to 10 ⁵ cells are obtained) and seeded in a tissue culture dish with an appropriate medium (for example, DMEM medium containing 10% FBS).

(Primary Culture Method of Oral Tissue-Derived Mesenchymal Stem Cells) The oral bone marrow-derived or oral periosteum-derived stem cells obtained as described above are added to bF in any medium suitable for culturing the cells.
GF is 0.01 to 100 ng / ml, preferably 0.0
4 to 10 ng / ml, more preferably 0.1 to 1 ng
/ Ml, for example, 1 ng / ml can be added to effect effective culture. FGFs other than bFGF are also effective. It should be noted that oral tissue-derived mesenchymal stem cell culture can be carried out by any culturing method without FGF.
The culture can be carried out under any conditions suitable for culturing mammals, but it is generally preferable to carry out the culture at 37 ° C. in the presence of 5% carbon dioxide gas, and for example, the following culture can be carried out. The cells collected and separated as described above are placed in an appropriate medium (for example, DMEM medium containing 10% FBS) for 10c.
Seed in a m-diameter tissue culture dish (about 2 × 10 ^{8 for} bone marrow; about 10 ⁴ to 10 ^{5 for} periosteum). The medium is changed on the 3rd day (excluding non-adherent cells), and then the medium is changed once every 3 days. In addition, bFGF was 1 ng / from the 5th day
Add to medium in ml.

(Subculture Method of Oral Tissue-Derived Mesenchymal Stem Cells) The subculture of the stem cells can be performed by a suitable method known in the field of the cell culture. For example, cells are collected from a plate of a primary culture which has become close to confluent using a trypsin-EDTA solution, and the cells are seeded in an appropriate medium containing bFGF, and the conditions are the same as those of the primary culture. Culture at. Then, before the cells proliferate and become confluent again, they are passaged by the following method, and this is repeated several times to ten or more times. The primary culture of the above-mentioned stem cells becomes close to confluent around 10 days. Try this plate with trypsin (eg 0.05%) + EDTA (eg 0.2
(mM), the cells are recovered from the plate, and the number of cells obtained is counted. 5 × 10 ³ cultured stem cells /
Seed in a medium containing 10% serum and bFGF (1 ng / ml) at a density of cm ² (4 × 10 ⁵ cells / 10 cm diameter culture dish) and subculture before the cells become intensive. . Further, the above operation is repeated to carry out subculture. In addition, when other soft tissue is mixed in the separated periosteum,
Only stem cell colonies (which can be morphologically distinguished from contaminated fibroblast colonies such as calcification in the center) are isolated from the primary culture system of the above-mentioned stem cells by trypsin. In this case, it is easier to separate the stem cells by seeding the details at a lower density and separating the colonies with trypsin before the cell colonies are fused.

(Method for Inducing Differentiation-Inducing Culture of Oral Tissue-Derived Mesenchymal Stem Cells) In order to obtain, for example, osteoblasts from the cultured stem cells, the cells are trypsinized and then isolated by centrifugation or the like, followed by bone differentiation. A medium suitable for induction (for example, a medium described in Literature: Science 284, 143-147, 1999) can be used to induce differentiation into osteoblasts. To give a specific example of the case of inducing differentiation into osteoblasts, bone marrow-derived mesenchymal stem cells of the 4th to 6th generations are collected to induce differentiation into osteoblasts, and a bone differentiation induction medium having the following composition Move to. Bone differentiation induction medium αMEM 10% FBS 100 nM dexamethasone 10 mM β-glycerol phosphate 50 μg / ml Ascorbic acid-2-phosphate mesenchymal cells were cultured in the above bone differentiation induction medium at 37 ° C.
The cells are cultured in the presence of 5% carbon dioxide gas, and the medium is replaced every two days, and the cells are cultured for 4 to 28 days. The cultured cells show a high level of alkaline phosphatase activity characteristic of osteoblasts, a level of deposited calcium, and a staining property by alizarin red indicating calcification. Furthermore, the expression of osteocalcin mRNA specific to osteoblasts is shown.

[0016]

The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these examples. Example 1 (Collecting Bone Marrow Fluid from Oral Tissue (Alveolar Bone) of Beagle Dog) After scaling of the whole jaw under anesthesia, local anesthesia was performed and the buccal gingiva of the lower molar or the anterior lower lip of the lower jaw was performed. The lateral gingiva was peeled off with a full-thickness valve. Furthermore, after exposing the alveolar bone,
A hole with a diameter of about 1 mm was made in the alveolar bone with a dental round bar, and bone marrow fluid was leached and collected with an injection needle (about 0.5 m
l). 10 ml of DMEM medium (32 units / ml)
Containing penicillin, 50 μg / ml streptomycin, and 6000 units / ml heparin), 30
The cells were separated by centrifugation at 0 × g for 5 minutes, and about 10 ⁹ cells were obtained from the bone marrow.

(Culture of bone marrow-derived mesenchymal stem cells) Stem cells collected from bone marrow were diluted with 10% FBS-containing DMEM medium, and then seeded in a 10 cm diameter culture dish at about 2 × 10 ⁸ cells, 37 Culture was performed at 5 ° C in the presence of 5% carbon dioxide gas. Then, the medium was exchanged on the third day, and thereafter, the medium was exchanged once every three days. In addition, bFGF was 1 ng / from the 5th day
Added to medium in ml. The mesenchymal stem cells proliferated to be almost confluent around 10 days. Trypsin (0.05%) + EDTA (0.2 mM) was added to these culture dishes, and then 5
Cells were isolated by incubation for minutes. Further, the number of cells was counted with a Coulter counter (Z1 single, manufactured by Coulter), and the cells were seeded at a density of 5,000 cells / cm ² . This operation was repeated, and cells obtained from the confluent third passage culture dish were used as transplanted cells.

(Preparation of Alveolar Bone Defect Model) In advance, a healthy periodontal tissue of an experimental animal was established by stone removal and brushing, and under full anesthesia, from the left and right lower first premolars to the first molars, A gingival sulcus incision was made and the buccal gingiva was peeled off with a full-thickness flap. Further, a 3 × 5 mm dehiscence-shaped bone defect was created in the mesial roots of the left, right, lower, second, third, and fourth premolars and the mesial root of the first molar, and root planing of the root surface was performed to prepare a transplant receiving bed. did.

(Transplantation of Autologous Oral Bone Marrow-Derived Mesenchymal Cells to Beagle Dog Alveolar Bone Defects) The autologous oral bone marrow-derived mesenchymal cells previously collected, separated and cultured by the above-mentioned method are used as the experimental alveolar bone. It was transplanted to the defect. Autologous oral bone marrow-derived mesenchymal cells are mixed with a carrier immediately before transplantation and transplanted (500,000 cells / defective portion). As a carrier, collagen (Atelocollagen, manufactured by Koken Co., final concentration 2%, 5
(Dissolved in DMEM at 0,000 cells / 10 μl) was used.
One month after the operation, histological evaluation revealed that regeneration of alveolar bone and cementum was not observed in the untreated group or the group containing only the collagen carrier, as shown in FIG. 1 (see Reference Photo 1). However, in the group in which autologous cells were transplanted together with collagen, marked regeneration of alveolar bone and cementum was observed.

Example 2 (Isolation of Human Oral Bone Marrow-Derived Mesenchymal Cells) At the time of surgery (mandibular osteotomy), 0.5 ml of exuded bone marrow fluid was collected (with the consent of the patient). . This is 10 ml of D
MEM medium (32 units / ml penicillin, 50 μg / m
1 streptomycin and 6000 units / ml heparin) and were centrifuged at 300 xg for 5 minutes to separate the cells. About 10 ⁹ cells were obtained from the bone marrow. Stem cells collected from bone marrow containing 10% FBS in DM
After dilution with EM medium, the cells were seeded in a 10 cm diameter culture dish so that the number of cells was about 2 × 10 ⁸ cells, and the cells were cultured at 37 ° C. in the presence of 5% carbon dioxide gas. The medium was exchanged on the 3rd day, and thereafter, the medium was exchanged once every 3 days. In addition, bFGF is 1 from the 5th day
ng × ml was added to the medium. After about 10 days, they grew to be almost confluent. Add these dishes to trypsin (0.0
Cells were isolated by incubation with 5%) + EDTA (0.2 mM) for 5 minutes. The number of cells was counted with a Coulter counter (Z1 single, manufactured by Coulter), and the cells were seeded at a density of 5000 cells / cm ² .
By repeating this operation, cells obtained from the 4th or 6th passage culture dish were used for inducing differentiation into osteoblasts.

(Induction of Differentiation into Osteoblasts) In order to induce differentiation into osteoblasts, human oral bone marrow-derived mesenchymal stem cells (ABMC) of the 4th or 6th generation were collected and used in an osteodifferentiation induction medium having the following composition. Moved. Bone differentiation inducing medium αMEM 10% FBS 100 nM dexamethasone 10 mM β-glycerol phosphate 50 μg / ml ascorbic acid-2-phosphate Human oral bone marrow-derived mesenchymal cells were mixed in the medium 3
The cells were cultured at 7 ° C in the presence of 5% carbon dioxide gas. The medium was replaced every 2 days and the cells were cultured for 4 to 20 days (see FIG. 2; reference photograph 2). This cell exhibited a high level of alkaline phosphatase activity characteristic of osteoblasts (Fig. 2; Reference photograph 2).
), Deposited calcium level (see FIG. 2; reference photograph 2), and staining with alizarin red indicating calcification (see FIG. 2; reference photograph 2). Furthermore, the expression of osteocalcin mRNA specific for osteoblasts was shown (see FIG. 2; reference photograph 2).

Example 3 (Isolation of Human Oral Periosteum-Derived Mesenchymal Stem Cells) At the time of surgery (with the consent of the patient), the maxillary premolar part palate gingiva was locally anesthetized, and the palate gingiva was partially flapped. After peeling, the periosteal connective tissue (5 × 5 mm) immediately below was collected. 1 collected periosteum
After cutting into mm or less, serum-free D for 1 hour at 37 ℃
It was incubated with collagenase (2.5 mg / ml, manufactured by Wako Pure Chemical Industries, for cell dispersion) dissolved in MEM. The cells were then dispersed by pipetting and harvested by centrifugation. The obtained cells were counted (about 10 ⁴ to 10 ⁵ were obtained), and DM containing 10% FBS was used.
The tissue culture dish was seeded with the EM medium.

Oral periosteum-derived mesenchymal stem cells were treated with 10% FBS
After diluting with the containing DMEM medium, the cells were seeded on a 10 cm diameter culture dish so that the number of cells was about 2 × 10 ⁴ cells, and the cells were incubated at 37 ° C. for 5 hours.
Culture was performed in the presence of% carbon dioxide. Change the medium on the 3rd day,
Thereafter, the medium was changed once every 3 days. In addition, bFGF was added to the medium every 2 days from the 5th day at 1 ng / ml. Oral periosteum-derived mesenchymal stem cells proliferated almost confluently around 10 days. Trypsin (0.05%) +
Cells were isolated by adding EDTA (0.2 mM) and incubating for 5 minutes. The number of cells was counted with a Coulter counter (Z1 single, manufactured by Coulter), and the cells were seeded at a density of 5,000 cells / cm ² . This operation was repeated, and the cells obtained from the 4th passage culture dish were used for inducing differentiation into osteoblasts.

(Induction of Differentiation of Oral Bone Marrow-Derived Mesenchymal Cells into Osteoblasts) Fourth generation human oral bone marrow-derived mesenchymal cells were collected and transferred to an osteodifferentiation induction medium having the following composition. Bone differentiation inducing medium 10% FBS-containing αMEM 100 nM dexamethasone 10 mM β-glycerol phosphate 50 μg / ml ascorbic acid-2-phosphate

Human oral bone marrow-derived mesenchymal stem cells were cultured in the above medium at 37 ° C. in the presence of 5% carbon dioxide gas. The medium was replaced every 2 days, and the cells were cultured for 28 days (see FIG. 2; reference photograph 2). Oral periosteum-derived stem cells (OPC)
Shows a high level of alkaline phosphatase activity, which is lower than that of bone marrow-derived stem cells (ABMC), but is characteristic of osteoblasts (Fig. 2; see Reference Photo 2), and deposited calcium level (Fig. 2; see Reference Photo 2). , And showed a dyeing property with alizarin red indicating calcification (see FIG. 2; reference photograph 2).
Furthermore, in the presence of active vitamin D, osteocalcin mRNA-specific expression of osteocalcin was shown (Fig. 2; see reference photo 2). It should be noted that, although the proliferation was promoted by the addition of FGF, a high level of calcification ability was shown (Fig. 2; see Reference Photo 2).

Example 4 Induction of differentiation into chondrocytes (culture for inducing differentiation into chondrocytes) In order to induce differentiation into chondrocytes, 10% FBS alone, 2F, on an EMC petri dish.
BS + bFGF + ITS or 5% FBS + bFG
1 with F + ITS or on a regular plastic petri dish
The cells were subcultured up to the 4th generation only with 0% FBS, and the 4th generation bone marrow-derived human mesenchymal stem cells (basement membrane extracellular matrix (EC
M) Petri dishes or those grown on ordinary plastic petri dishes) were collected and transferred to a cartilage differentiation inducing medium having the following composition. 200,000 cells were placed in a centrifuge tube (for 15 ml) and incubated in 0.5-1 ml of the following medium. High glucose αMEM medium 10 ng / ml TGF-β1 100 nM dexamethasone 50 μg / ml ascorbic acid-2-phosphate 100 μg / ml sodium pyruvate ITS-plus 6.25 μg / ml transferrin 6.25 μg / ml insulin 6.25 ng / ml selenate 5.33 μg / ml linoleic acid 1.25 mg / ml bovine plasma albumin human mesenchymal stem cells in the above medium at 37 ° C., 5%
Culture was performed in the presence of CO ₂ . 24 hours after the start of the culture, the cells formed a spherical pellet. The medium was replaced every two days, and the cells were cultured for 28 days.

(Differentiation into chondrocytes) The preparations of the pellets after culturing were adjusted and stained with toluidine blue. As a result, in cells (10% FBS added) grown on an ordinary plastic petri dish, only 50% of the cells differentiated into chondrocytes forming a toluidine blue-staining matrix (plastic, 10% P4). E
8 cells from CM petri dish (10% FBS added)
Although differentiated into 0% cartilage, toluidine blue had low staining properties (low cartilage matrix production). On the other hand, stem cells grown with 2 or 5% FBS, bFGF and ITS became 80-90% cartilage by switching to cartilage inducing medium, and the cartilage matrix production level was also higher than 10% FBS (Fig. 3; Reference). (See Photo 3).

Example 5 Adipocyte Differentiation Induction (Adipocyte Differentiation Induction Culture) As in Example 4, various fourth-generation mesenchymal stem cells cultured in the following medium were collected. Plastic culture dish 10% FBS + bFGF (control group) ECM-coated culture dish 10% FBS only ECM-coated culture dish 2% FBS + bFGF + ITS ECM-coated culture dish 5% FBS + bFGF + ITS Collected mesenchymal stem cells in a 9 mm diameter dish, 4 × 10
After cultivating ⁴ seeds, the cells were cultured in a DMEM medium containing 10% FBS for 3 days, and then transferred to an adipose differentiation induction medium having the following composition. (Fat differentiation induction medium) DMEM (high glucose) 10 μg / ml insulin 0.2 mM indomethacin 1 μM dexamethasone 0.5 mM 3-isobutyl-1-methylxanthine 10% FBS

(Differentiation into adipocytes) After culturing for 25 days, fat was stained with Oil Red-O (see FIG. 4; reference photograph 4). All the mesenchymal stem cells cultured by the above method retained high adipose differentiation ability.

[0030]

EFFECTS OF THE INVENTION By the method of separating and collecting mesenchymal stem cells from oral tissues according to the present invention, it is possible to obtain mesenchymal stem cells by a simple operation that requires minimal peeling and incision of skin and muscle. Therefore, it is possible to safely and easily obtain the required amount of mesenchymal stem cells that maintain excellent differentiation ability without imposing a severe burden on the collected mother body. Furthermore, according to the present invention, mesenchymal stem cells that have been separated and collected can be cultured and differentiation-induced to prepare cells for regeneration and / or repair of a tissue having a high differentiation potential. It is possible to regenerate and / or repair the tissue by implanting it in the defect portion and / or the repair portion of the tissue such as periodontal tissue. Therefore, the present invention provides a practical method in regenerative medicine using mesenchymal stem cells.

[Brief description of drawings]

FIG. 1 is a view showing a histological image one month after transplantation to a beak dog alveolar bone defect site. Stem cells + collagen carrier (C) compared to transplantation of untreated (A) and collagen carrier alone (B)
In transplantation, prominent bone regeneration and cementitious regeneration are recognized. The following figure shows an enlarged image of each defect.

FIG. 2 is a diagram showing the results of proliferative ability and bone differentiation ability of human oral periosteal cells and human alveolar bone marrow mesenchymal cells.

FIG. 3 is a diagram showing the results of enhancing the chondrogenic potential of human mesenchymal stem cells grown in an ECM petri dish with low-concentration serum and ITS.

FIG. 4 is a diagram showing the results of adipose differentiation ability of human mesenchymal stem cells grown on ECM petri dishes with low-concentration serum and ITS.

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[Procedure amendment]

[Submission date] September 6, 2001 (2001.9.6)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction content]

(Subculture Method of Oral Tissue-Derived Mesenchymal Stem Cells) The subculture of the stem cells can be performed by a suitable method known in the field of the cell culture. For example, cells are collected from a plate of a primary culture which has become close to confluent using a trypsin-EDTA solution, and the cells are seeded in an appropriate medium containing bFGF, and the conditions are the same as those of the primary culture. Culture at. Then, before the cells proliferate and become confluent again, they are passaged by the following method, and this is repeated several times to ten or more times. The primary culture of the above-mentioned stem cells becomes close to confluent around 10 days. Try this plate with trypsin (eg 0.05%) + EDTA (eg 0.2
(mM), the cells are recovered from the plate, and the number of cells obtained is counted. 5 × 10 ³ cultured stem cells /
Seed in a medium containing 10% serum and bFGF (1 ng / ml) at a density of cm ² (4 × 10 ⁵ cells / 10 cm diameter culture dish) and subculture before the cells become intensive. . Further, the above operation is repeated to carry out subculture. In addition, when other soft tissue is mixed in the separated periosteum,
Only stem cell colonies (which can be morphologically distinguished from contaminated fibroblast colonies such as calcification in the center) are isolated from the primary culture system of the above-mentioned stem cells by trypsin. In this case, it is easier to separate the stem cells by seeding the cells at a low density and separating the colonies with trypsin before the cell colonies are fused.

Continued front page    (72) Inventor Shinichi Tsutsumi             1-16-5, Shinonomehoncho, Minami-ku, Hiroshima City, Hiroshima Prefecture             601 (72) Inventor Kazuko Miyazaki             Hiroshima Prefecture Hiroshima City Minami-ku Midori 3-11-30-301 (72) Inventor Mayoko Hara             Hiroshima Prefecture Hiroshima City Minami-ku Minami-cho 4-1-15, 2F (72) Inventor Hiroyuki Kawaguchi             1-17-9 Deshio, Minami-ku, Hiroshima-shi, Hiroshima (72) Inventor Hidemi Kurihara             2-27-31 Koihigashi, Nishi-ku, Hiroshima City, Hiroshima Prefecture F-term (reference) 4B065 AA93X BA30 BB25 BB40                       BD15 CA44                 4C081 AB02 AB04 AB06 AB11 AB18                       BA12 CD29 CD34 EA01 EA11

Claims (9)

[Claims] 1. A method for separating and collecting mesenchymal stem cells, which comprises collecting and collecting mesenchymal stem cells from the oral tissue of a mammal. 2. The method for separating and collecting mammalian mesenchymal stem cells according to claim 1, wherein the oral tissue is bone marrow of alveolar bone, palate or periosteum of alveolar bone. 3. A mammalian mesenchymal stem cell characterized in that cells collected from oral tissue are seeded in a culture container together with a tissue culture medium, cultured, and cells adhered to the culture container are separated and collected. Separation and collection method. 4. A method of seeding mammalian mesenchymal stem cells separated and collected by the method according to any one of claims 1 to 3 in a mesenchymal stem cell culture medium to perform primary culture and subculture. And a method for culturing mammalian mesenchymal stem cells. 5. A mesenchymal stem cell, which is characterized in that the mesenchymal stem cell cultivated by the culturing method according to claim 4 is seeded in a mesenchymal stem cell differentiation-inducing medium to carry out differentiation-inducing culture. Differentiation induction culture method. 6. The method for culturing mammalian mesenchymal stem cells according to claim 4, wherein the mesenchymal stem cells are cultured in a medium supplemented with fibroblast growth factor (FGF). 7. A mammalian cell for tissue regeneration and / or repair prepared by the method for inducing differentiation according to claim 5. 8. The mammalian cell for tissue regeneration and / or repair according to claim 7, wherein the cell is derived from human oral tissue. 9. A mammalian tissue, wherein the mammalian cell for tissue regeneration and / or repair according to claim 7 or 8 is transplanted together with a single cell into a defective portion and / or a repaired portion of the tissue, if necessary. How to play.