JP7018650B2 - Stem cell separation method and use of non-woven fabric composed of norbornene-based polymer - Google Patents

Description

The present invention relates to a method for separating stem cells from a group of cells including stem cells, and the use of a non-woven fabric composed of a norbornene-based polymer for carrying out this method.

In recent years, the development of stem cell therapy for repairing or regenerating damaged patient cells or tissues using stem cells or cells derived from stem cells has been progressing. For example, Patent Document 1 proposes a closed-system therapeutic system in which adipose tissue is obtained from a patient and stem cells concentrated by a concentration treatment are administered to the patient.

Stem cells used for this purpose are prepared from adipose tissue cell groups obtained by liposuction surgery and the like. In order to obtain stem cells from the collected adipose tissue cell group, usually, treatment with an enzyme such as centrifugation or collagenase is performed, and fat such as adipose stem cells, fibroblasts, vascular endothelial cells, and vascular smooth muscle-like cells is performed. An stromal vascular cell group consisting of tissue-derived cells, blood-derived cells, and other cells is obtained (see, for example, Patent Document 2).

International Publication No. 03/0533346 International Publication No. 2005/042730 Japanese Unexamined Patent Publication No. 2013-034436 Japanese Unexamined Patent Publication No. 2008-199897

The cell group thus obtained also contains various cells, and it is required to efficiently isolate stem cells from these cells.

There are many examples of using a non-woven fabric for separating stem cells, and as described in Patent Document 3 and the like, the non-woven fabric is usually used as a filtration base material. However, in this method, it is necessary to prepare a suspension of cells, and it is also necessary to adjust the concentration of the suspension.
On the other hand, as a method that does not use a cell suspension, Patent Document 4 proposes a method of culturing cells using a non-woven fabric whose surface is coated with hydroxyapatite. However, this method has a problem that it is difficult to ensure that the hydroxyapatite on the surface of the nonwoven fabric is uniformly coated, and that the hydroxyapatite applied to the nonwoven fabric is easily peeled off.

The present invention has been made in view of the actual situation of the prior art, and an object of the present invention is to easily selectively separate and culture specific stem cells by a simple culture operation.

In order to solve the above problems, the present inventors have diligently studied a method for separating stem cells from a tissue-derived cell group.
As a result, when adipose tissue-derived cell groups are cultured on a non-woven fabric in which at least the surface for culturing cells (culture surface) is composed of a norbornene-based polymer, only adipose stem cells are exposed to the non-woven surface without surface coating. It has been found that adipose stem cells can be selectively isolated and cultured, and as a result, adipose stem cells can be isolated, and the present invention has been completed.

Thus, according to the present invention, the following methods (1) and (2) for separating stem cells, and (3) use of a non-woven fabric composed of a norbornene-based polymer are provided.
(1) A method for separating stem cells, in which a tissue-derived cell group containing stem cells is cultured on a non-woven fabric whose culture surface is at least composed of a norbornene-based polymer.
(2) The method for separating stem cells according to (1), wherein the tissue-derived cell group is a cell group derived from adipose tissue.
(3) Use of a non-woven fabric in which at least the culture surface is composed of a norbornene-based polymer for separating stem cells from a group of tissue-derived cells including stem cells.

According to the present invention, specific stem cells can be easily selectively isolated and cultured by a simple culture operation.

FIG. 1 is a phase-contrast micrograph of mesenchymal stem cells (ASC) derived from adipose tissue 18 days after the start of culture growing on the Zeonoa non-woven fabric in Example 1. FIG. 2 is a phase-contrast micrograph of ASC grown on the Zeonex nonwoven fabric 2 in Example 1 18 days after the start of culture. FIG. 3 is a phase-contrast micrograph of ASC subcultured in a flask for 4 days in Example 1. FIG. 4 is a phase-contrast micrograph of bone marrow-derived stem cells (BMSC) 18 days after the start of culture growing on the Zeonoa non-woven fabric in Example 2. FIG. 5 is a phase-contrast micrograph of BMSC 18 days after the start of culture growing on Zeonex nonwoven fabric 1 in Example 2. FIG. 6 is a phase-contrast micrograph of BMSC subcultured in a flask for 4 days in Example 2. FIG. 7 is a phase-contrast micrograph of BMSC in a comparative example. FIG. 8 is a phase-contrast micrograph of BMSC in a comparative example. FIG. 9 is a micrograph of adipocytes stained in Example 3. FIG. 10 is a micrograph of osteoblasts stained in Example 3. FIG. 11 is a micrograph of chondrocytes stained in Example 3. FIG. 12 is a fluorescence micrograph of the nerve cells in Example 3.

Hereinafter, embodiments of the present invention will be described in detail.
The nonwoven fabric composed of the norbornene-based polymer used in the present invention (hereinafter, may be simply referred to as "nonwoven fabric" or "nonwoven fabric" used in the present invention) is composed of at least a fibrous norbornene-based polymer on the culture surface. It is a thing.
In the non-woven fabric of the present invention, "at least the culture surface is composed of a norbornene-based polymer" means that at least the surface for culturing cells (the portion in contact with the cells, for example, the fiber constituting one side of the non-woven fabric) is norbornene-based. It means that it contains a polymer. The culture surface may be composed of only the norbornene-based polymer.

The non-woven fabric used in the present invention can be usually used in various containers used for culturing cells. The non-woven fabric may be floating from the bottom of the container or sunk on the bottom of the container. The cells may be adhered to only one side of the non-woven fabric or to both sides. However, from the viewpoint of cell retention, the non-woven fabric covers the entire bottom surface of the container and adheres the cells to only one side of the non-woven fabric. It is desirable to let it grow and grow.
The material of the container for containing the non-woven fabric is not particularly limited, and conventionally known containers such as polystyrene containers for cell culture and glass containers can be used.

In the norbornene-based polymer constituting the non-woven fabric used in the present invention, the monomer unit having a norbornene skeleton is 50% by mass or more, preferably 60% by mass or more, based on all the monomer units constituting the norbornene-based polymer. It is a polymer containing. More specifically, the norbornene-based polymer is obtained by polymerizing a norbornene-based monomer which is a monomer having a norbornene skeleton, and is obtained by ring-opening polymerization or addition polymerization. It is roughly divided into.

What can be obtained by ring-opening polymerization is a ring-opening polymer obtained by ring-opening polymerization of one kind or a mixture of two or more kinds of norbornene-based monomers, norbornene-based monomers and ring-opening copolymerization thereof. Examples thereof include a ring-opening polymer obtained by ring-opening polymerization with other monomers, and hydrides thereof.
What is obtained by addition polymerization is an addition polymer obtained by addition polymerization of one kind or a mixture of two or more kinds of norbornene-based monomers, norbornene-based monomers and other monomers copolymerizable therewith. Examples thereof include an addition polymer obtained by addition polymerization of and.
Among these, since the effect of the present invention is more easily obtained, a hydride of a ring-opening polymer obtained by ring-opening polymerization of one kind or a mixture of two or more kinds of norbornene-based monomers, or a norbornene-based simple substance. A hydride of a ring-opening polymer obtained by ring-opening polymerization of a weight and this and another monomer capable of ring-opening copolymerization is preferable, and one kind or a mixture of two or more kinds of norbornene-based monomers is opened. A hydride of the ring-opening polymer obtained by ring polymerization is more preferable.

Examples of the norbornene-based monomer that can be used for the synthesis of the norbornene-based polymer include bicyclo [2.2.1] hepta-2-ene (commonly known as norbornene) and 5-methyl-bicyclo [2.2.1] hepta. -2-ene, 5,5-dimethyl-bicyclo [2.2.1] hepta-2-ene, 5-ethyl-bicyclo [2.2.1] hepta-2-ene, 5-ethylidene-bicyclo [2] .2.1] Hepta-2-ene, 5-vinyl-bicyclo [2.2.1] hepta-2-ene, 5-propenylbicyclo [2.2.1] hepta-2-ene, 5-methoxycarbonyl -Vicyclo [2.2.1] hepta-2-ene, 5-cyanobicyclo [2.2.1] hepta-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo [2.2.1] hepta -Bicyclic monomer such as 2-ene;
Tricyclo [4.3.0 ^1,6 . 1 ^2,5 ] Deca-3,7-diene (trivial name dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, 2,3-dihydroxydicyclopentadiene, etc. Quantitative;
Tetracyclo [4.4.0.1 ^2,5 . 1 7, 10] -3-dodecene (tetracyclododecene), tetracyclo [ ^{4.4.0.1 2,5} . 1 7, 10] -3-dodecene, 8-methyltetracyclo [ ^{4.4.0.1 2,5} . 1 ⁷ , 10] -3-dodecene, 8-ethyltetracyclo [4.4.0.1 ^2,5 . 1 7, 10] -3-dodecene, 8-ethylidenetetracyclo [ ^{4.4.0.1 2,5} . ^17,10 ] -3-dodecene, 8,9-dimethyltetracyclo [4.4.0.1 ^2,5 . 1 ⁷ , 10] -3-dodecene, 8-ethyl-9-methyltetracyclo [4.4.0.1 ^2,5 . 1 ⁷ , 10] -3-dodecene, 8-ethylidene-9-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-methyl-8-carboxymethyltetracyclo [4.4.0.1 ^2,5 . 1 ⁷ , 10] -3-Dodecene,
7,8-Benzotricyclo [4.3.0.1 ^2,5 ] Deca-3-ene (commonly known as metanotetrahydrofluorene: 1,4-methano-1,4,4a, 9a-tetrahydrofluorene) , 1,4-Metano-8-methyl-1,4,4a, 9a-tetrahydrofluorene, 1,4-methano-8-chloro-1,4,4a, 9a-tetrahydrofluorene, 1,4-methano-8 -4cyclic monomers such as bromo-1,4,4a, 9a-tetrahydrofluorene; and the like.
These norbornene-based monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, an alkylidene group and the like.

Other monomers that can be ring-opened and copolymerizable with the norbornene-based monomer include cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, and 1,5-cyclodecadien. Examples thereof include monocyclic cycloolefin-based monomers such as 1,5,9-cyclododecatriene and 1,5,9,13-cyclohexadecatetraene.

Other monomers that can be additionally copolymerized with the norbornene-based monomer include α-olefin-based monomers having 2 to 20 carbon atoms such as ethylene, propylene, 1-butene, 1-pentene, and 1-hexene; Cyclobutene, cyclopentene, cyclohexene, cyclooctene, tetracyclo [9.2.1.0 ^2,10 . 0 ^3,8 ] Cycloolefin-based monomers such as tetradeca-3,5,7,12-tetraene (also called 3a, 5,6,7a-tetrahydro-4,7-methano-1H-indene); 1, Examples thereof include non-conjugated diene-based monomers such as 4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, and 1,7-octadene.
Among these, as the other monomer that can be additionally copolymerized with the norbornene-based monomer, an α-olefin-based monomer is preferable, and ethylene is more preferable.
These other monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, an alkylidene group and the like.

A ring-opening polymer of a norbornene-based monomer, or a ring-opening polymer of a norbornene-based monomer and another monomer capable of ring-opening copolymerization thereof, comprises a known ring-opening polymerization of a monomer component. It can be obtained by polymerization in the presence of a catalyst.
Examples of the ring-opening polymerization catalyst include a catalyst composed of a metal halide such as ruthenium and osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a metal halide or acetylacetone such as titanium, zirconium, tungsten and molybdenum. A catalyst composed of a compound and an organoaluminum compound can be used.
A ring-opening polymer hydride of a norbornene-based monomer is usually obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to the polymerization solution of the ring-opening polymer to form a carbon-carbon unsaturated bond. Can be obtained by hydrogenating.

An addition polymer of a norbornene-based monomer, or an addition polymer of a norbornene-based monomer and another monomer copolymerizable therewith, contains a monomer component in the presence of a known addition polymerization catalyst. It can be obtained by polymerization.
As the addition polymerization catalyst, for example, a catalyst composed of a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.

There is no particular limitation on the molecular weight of the norbornene-based polymer, but it is usually a polystyrene-equivalent weight average molecular weight measured by gel permeation chromatography (GPC) of a cyclohexane solution (toluene solution if the polymer does not dissolve). It is 5,000 or more, preferably 5,000 to 500,000, more preferably 8,000 to 200,000, and particularly preferably 10,000 to 100,000. When the weight average molecular weight is within this range, the mechanical strength and the moldability are highly balanced and suitable.

The glass transition temperature of the norbornene-based polymer may be appropriately selected depending on the intended use, but is usually 50 to 300 ° C, preferably 100 to 280 ° C, particularly preferably 115 to 250 ° C, still more preferably 130 to 200. ℃. When the glass transition temperature is within this range, heat resistance and molding processability are highly balanced and suitable.
In the present invention, the glass transition temperature is measured based on JIS K 7121.

The norbornene-based polymers can be used alone or in combination of two or more.
Further, as the resin component constituting the non-woven fabric, in addition to the norbornene-based polymer, optionally, a compounding agent usually used in a thermoplastic resin material, for example, a soft polymer, an antioxidant, an ultraviolet absorber, and a photostabilizing agent. A compounding agent such as an agent, a near-infrared absorber, a mold release agent, a colorant such as a dye or a pigment, a plasticizer, an antistatic agent, and a fluorescent whitening agent can be added in an amount usually adopted. Here, when a soft polymer is mixed with a norbornene-based polymer and used, it is usually 0.01 to 20 parts by mass with respect to 100 parts by mass of the alicyclic structure-containing polymer which is a norbornene-based polymer. It is preferably 0.05 to 10 parts by mass, and more preferably 0.05 to 5 parts by mass.

Further, as a resin component constituting the nonwoven fabric, other polymers (hereinafter, simply referred to as “other polymers”) are mixed in addition to the norbornene-based polymer and the soft polymer which is one of the above-mentioned compounding agents. May be. The amount of the other polymer mixed with the norbornene-based polymer is usually 200 parts by mass or less, preferably 150 parts by mass or less, and more preferably 100 parts by mass or less with respect to 100 parts by mass of the norbornene-based polymer.
If the ratio of various compounding agents and other polymers to be blended with the norbornene-based polymer is too large, it becomes difficult for cells to float. Therefore, it is preferable to blend all of them within a range that does not impair the properties of the norbornene-based polymer.

The method for mixing the norbornene-based polymer with the compounding agent or other polymer is not particularly limited as long as the compounding agent is sufficiently dispersed in the polymer. In addition, there are no particular restrictions on the order of formulation. As a compounding method, for example, a method of kneading the resin in a molten state using a mixer, a uniaxial kneader, a biaxial kneader, a roll, a brabender, an extruder, etc. Examples thereof include a method of removing the solvent by a coagulation method, a casting method, or a direct drying method.
When a twin-screw kneader is used, after kneading, it is usually extruded into a rod shape in a molten state, cut into an appropriate length with a strand cutter, and pelletized.

There are no particular restrictions on the method for producing the nonwoven fabric used in the present invention, and a general method for producing a nonwoven fabric can be adopted.
Among them, when producing a nonwoven fabric having a preferable fiber diameter, basis weight and surface coverage, which will be described later, a method using a melt blow method is preferably adopted.

The fiber diameter of the nonwoven fabric used in the present invention is preferably 10 to 20 μm. Here, the fiber diameter is a value measured by photographing the surface of the non-woven fabric with a digital microscope VHX-1000 (manufactured by KEYENCE CORPORATION).
The basis weight of the non-woven fabric is preferably 0.8 mg / cm ² to 1.0 mg / cm ² . Here, the basis weight is a value obtained by calculating the weight per unit area from the weight of the non-woven fabric of 3 cm square.
The surface coverage of the non-woven fabric is preferably 50% to 95%. Here, the surface coverage is the area of the entire image of the image taken with the digital microscope, excluding the voids where no fibers are present, from the area of the entire image using image analysis software (Image J). It is the value converted by dividing from.

The nonwoven fabric used in the present invention is usually sterilized before use. There are no particular restrictions on the sterilization method, heating methods such as high-pressure steam method and dry heat method; radiation method that irradiates radiation such as γ-rays and electron beams; irradiation method that irradiates high-frequency rays; ethylene oxide gas (EOG) It can be arbitrarily selected from the methods generally adopted in the medical field, such as a gas method in which a gas such as the above is brought into contact; a filtration method using a sterilization filter; and the like.

The tissue-derived cell group containing stem cells cultured on the nonwoven fabric used in the present invention is not particularly limited as long as it is a tissue-derived cell group containing a plurality of types of cells including stem cells. For example, a mesenchymal vascular cell group, an adipose-derived cell group, a bone marrow-derived cell group, etc. to be used for cell therapy can be mentioned. Further, some cells may have already been isolated from these cell groups.

As the medium for separating stem cells from the tissue-derived cell group using such a non-woven fabric, a medium capable of growing the stem cells to be separated may be used, and in particular, the medium can be grown while maintaining undifferentiated state. It is preferable to use a medium. Such a medium is commercially available. For example, in the case of adipose-derived stem cells, KBM ADSC series manufactured by Kojin Bio Co., Ltd. can be mentioned, and in the case of bone marrow-derived stem cells, Stemline manufactured by SIGMA-ALDRICH, mesenchymal system. Examples include the stem cell growth medium series.

Additives can also be added to the medium. Examples of the additive include inducing factors such as proteins, minerals, metals, vitamin components and the like.
These additives can be used alone or in combination of two or more.

The cell culture conditions are not particularly limited and can be appropriately determined according to the cells to be used and the purpose.
For example, cells can be cultured using a humidified incubator having a carbon dioxide concentration of about 5% and a constant temperature in the range of 20 ° C to 37 ° C.

In the present invention, when a tissue-derived cell group is cultured on a non-woven fabric whose culture surface is at least composed of a norbornene-based polymer, only adipose stem cells can be adhered and cultured, and as a result, adipose stem cells are separated. Can be done.

In addition to being used for stem cell therapy, the isolated stem cells can be further cultured in a differentiation-inducing medium to obtain differentiated cells, which can be used for drug discovery and medical purposes.

As the medium used for inducing differentiation of the stem cells isolated by the method of the present invention, a medium suitable for inducing differentiation of stem cells may be used, and a medium in which an additive for inducing differentiation is added to a basal medium or a medium containing an additive for inducing differentiation may be used. A commercially available differentiation-inducing medium can be used.

Additives for inducing differentiation include ligands, agonists, antagonists that act on cell surface receptors; ligands, agonists, antagonists of nuclear receptors; extracellular matrices such as collagen and fivenectin; extracellular matrices. Part of or simulated compound; component that acts on proteins involved in intracellular signal transduction pathways; component that acts on intracellular primary or secondary metabolic enzymes; intracellular nuclear or mitochondrial genes Examples include components that affect expression; DNA and RNA that can be introduced into cells in combination with virus vectors and the like;
These additives can be used alone or in combination of two or more.
Examples of the commercially available differentiation-inducing medium include "Stem Cell Kits" manufactured by R & D Systems.

The cell culture conditions are not particularly limited and can be appropriately determined according to the cells to be used and the purpose.
For example, cells can be cultured using a humidified incubator having a carbon dioxide concentration of about 5% and a constant temperature in the range of 20 ° C to 37 ° C.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited to the following examples.

<Nonwoven fabric composed of norbornene-based polymer>
Norbornene-based polymer (Zeonoa (registered trademark) 1060R, manufactured by Nippon Zeon; norbornene-based ring-opening polymer hydride) and norbornene-based polymer (Zeonex (registered trademark) 5000, manufactured by Nippon Zeon; Norbornene-based ring-opening polymer). Using a polymer hydride) by the melt blow method, under the following conditions, two types of non-woven fabrics made of Zeonoa (registered trademark) 1060R (hereinafter referred to as "Zeonoa non-woven fabric") and Zeonex (registered trademark) 5000. Non-woven fabrics (hereinafter referred to as "Zeonex non-woven fabric 1" and "Zeonex non-woven fabric 2") were produced.

<Zeonoa non-woven fabric>
-Resin: Zeonoa (registered trademark) 1060R
-Nozzle temperature: 293 ° C
・ Dice temperature: 298 ° C
・ Rotation speed: 100 rpm
・ Conveyor speed: 14.5m / min
-Distance from spinneret to collector (hereinafter referred to as "DCD"): 45 mm

<Zeonex Nonwoven Fabric 1>
-Resin: Zeonex (registered trademark) 5000
・ Nozzle temperature: 300 ℃
・ Dice temperature: 290 ° C
・ Rotation speed: 100 rpm
・ Conveyor speed: 14.2m / min
・ DCD: 85mm

<Zeonex Nonwoven Fabric 2>
-Resin: Zeonex (registered trademark) 5000
・ Nozzle temperature: 298 ° C
・ Dice temperature: 290 ° C
・ Rotation speed: 100 rpm
・ Conveyor speed: 14.2m / min
・ DCD: 52 mm

The fiber diameter, basis weight and surface coverage of the obtained nonwoven fabric are as follows.
-Zeonoa non-woven fabric: fiber diameter 10.05 μm, basis weight 0.98 mg / cm ² , surface coverage 91%
Zeonex non-woven fabric 1: Fiber diameter 15.05 μm, basis weight 0.94 mg / cm ² , surface coverage 58%
Zeonex non-woven fabric 2: Fiber diameter 13.33 μm, basis weight 0.81 mg / cm ² , surface coverage 82%

[Example 1]
<Separation of ASC from adipose tissue>
Adipose tissue with a diameter of about 3 mm extracted from a human is divided into four parts, which are placed on a circularly cut Zeonoa non-woven fabric and a Zeonex non-woven fabric 2 having a diameter of 2 cm, and then the non-woven fabric on which this tissue is placed is used for cell culture. It was placed in a polystyrene dish (FALCON (registered trademark), Corning Co., Ltd., TCPS having a diameter of 35 mm).
Then, a mesenchymal stem cell growth medium (Stemline (registered trademark), manufactured by Sigma-Aldrich) to which 2% fetal bovine serum (FBS) was added was placed in the dish and cultured under the condition of 5% CO ₂ atmosphere at 37 ° C. ..
It was confirmed that adipose tissue-derived stem cells (ASC) began to separate and proliferate on any of the non-woven fabrics after 7 to 10 days. After confirming the separation and proliferation of ASC, the medium was replaced, the culture was continued, and 18 days after the start of the culture, the ASC was peeled off from the non-woven fabric with trypsin-EDTA, and then a 279.8 mL cell culture flask (model number "T-75"). , Eppendorf) and subcultured in the same medium for 4 days to become 90% confluent.
FIGS. 1 and 2 show phase-contrast micrographs (magnification of 64 times) of ASCs 18 days after the start of culture growing on the Zeonoa non-woven fabric and the Zeonex non-woven fabric 2. It is confirmed that ASC is grown on all the non-woven fabrics.
In addition, FIG. 3 shows a phase-contrast micrograph (magnification: 64 times) when the cells were transferred to a cell culture flask and subcultured for 4 days. It is confirmed that ASC is proliferating.

[Example 2]
<Separation of BMSC from bone marrow fluid>
An equivalent amount of Hanks Balanced Salt Solution (HBSS) was added to 10 ml of bone marrow fluid collected from humans, and the mixture was stirred well to prepare a diluted bone marrow fluid. Zeonoa non-woven fabric and Zeonex non-woven fabric 1 cut into a circle with a diameter of 2 cm were set in a 100 μm cell strainer (Falcon (registered trademark) model number 352360, manufactured by Corning Inc.), passed through the previously prepared bone marrow fluid diluent, and HBSS. After washing twice with, the non-woven fabric was placed in separate cell culture polystyrene dishes (FALCON (registered trademark), Corning Inc., TCPS having a diameter of 35 mm). Then, a mesenchymal stem cell growth medium (Stemline (registered trademark); manufactured by Sigma-Aldrich) containing 2% fetal bovine serum (FBS) was placed in the dish and cultured under the condition of 5% CO ₂ atmosphere at 37 ° C. ..
It was confirmed that bone marrow-derived stem cells (BMSC) began to separate and proliferate in each of the non-woven fabrics after 7 to 10 days. After confirming the separation and proliferation of BMSC, the medium was replaced and the culture was continued. Eighteen days after the start of culturing, the BMSC was peeled off from the non-woven fabric with trypsin-EDTA, transferred to a 279.8 mL cell culture flask (model number "T-75", manufactured by Eppendorf), and subcultured in the same medium for 4 days. Then it became 90% confluent.
FIG. 4 and FIG. 5 show phase-contrast micrographs (magnification of 64 times) of BMSC 18 days after the start of culture, which are grown on the Zeonoa non-woven fabric and the Zeonex non-woven fabric 1. It is confirmed that BMSC is grown on all the non-woven fabrics.
Further, FIG. 6 shows a phase-contrast micrograph (magnification: 64 times) when this BMSC was transferred to a cell culture flask and subcultured for 4 days. It is confirmed that BMSC is proliferating.

[Comparison example]
A 6-well plate was placed with a core polyethylene outer layer polypropylene non-woven fabric (10 mm × 32 mm), a suspension of BMSC, 3 mL (0.45 × 10 ⁶ cells / mL) was added thereto, and the cells were cultured for 3 days. As the medium, a mesenchymal stem cell growth medium to which 2% FBS was added [Stemmine (registered trademark) manufactured by Sigma-Aldrich Co., Ltd.] was used.
As a result, the MSC was slightly adhered to the core polyethylene outer layer polypropylene non-woven fabric (Fig. 7), and was adhered to the bottom surface of the plate in a 100% confluent state (Fig. 8), and the stem cells were separated. No effect was obtained.

[Example 3]
In Example 1, a differentiation induction experiment of ASC (hereinafter referred to as “passage ASC”) separated on the Zeonoa nonwoven fabric and the Zeonex nonwoven fabric 2 and subsequently subcultured was performed as follows.
(1) Differentiation into adipocytes The passaged ASC was seeded on a 24-well multiplate (FALCON (registered trademark) model number 353047, manufactured by Corning) at 1.9 × 10 ⁵ cells / well, and 2% fetal bovine serum (2% fetal bovine serum). A mesenchymal stem cell proliferation medium (Stemline (registered trademark), manufactured by Sigma Aldrich) to which FBS) was added was placed and cultured under the condition of 5% CO ₂ atmosphere at 37 ° C. After 24 hours, the medium was discarded, washed twice with phosphate buffer (PBS), replaced with an adipocyte differentiation medium (model number BBDM2, manufactured by DS Pharma Biomedical), and cultured for 1 week. Then, the medium was replaced with a medium for adipocyte culture (model number BBAM1, manufactured by DS Pharma Biomedical Co., Ltd.), cultured for 1 week, and then stained with Oil-Red. A micrograph of adipocytes stained is shown in FIG.
From FIG. 9, adipocytes having oil-Red-stained lipid droplets were observed, and it was confirmed that the passage ASC obtained in Example 1 had the ability to differentiate into adipocytes.

(2) Differentiation into osteoblasts Subcultured ASCs were seeded on a 24-well multiplate (FALCON (registered trademark) model number 353047, manufactured by Corning) at 1.5 × 10 ⁴ cells / well, and 2% fetal bovine serum. A mesenchymal stem cell proliferation medium (Stemline (registered trademark), manufactured by Sigma Aldrich) to which (FBS) was added was placed and cultured under the condition of 5% CO ₂ atmosphere at 37 ° C. Then, the medium was replaced with an osteoblast differentiation medium (model number BBOB1, manufactured by DS Pharma Biomedical), and the cells were cultured at a cell density of about 60% (about 60% confluency) for 2 weeks. The cells were then stained with Alizarin Red. A micrograph of osteoblasts stained is shown in FIG.
From FIG. 10, since the cells and their surroundings were stained with Alizarin Red in light red, calcium deposition was observed, and the passaged ASC obtained in Example 1 also had the ability to differentiate into osteoblasts. Was confirmed.

(3) Differentiation into chondrocytes 5 μL of passage ASC prepared at 1.6 × 10 ⁷ cells / mL was added dropwise to a 24-well multiplate (FALCON (registered trademark) model number 353047, manufactured by Corning) for 2 hours. Later, a chondrocyte differentiation medium (StemPro Hondagenesis differentiation Kit; model number A10070-01, manufactured by Corning Co., Ltd.) was added, and the cells were cultured under the condition of 5% CO ₂ atmosphere at 37 ° C. Then, the cells were cultured for 2 weeks while exchanging the medium at intervals of 4 to 5 days. The obtained cells were stained with Alcian Blue. A micrograph of chondrocytes stained is shown in FIG.
From FIG. 11, since the cells and their surroundings were stained blue with Alcian Blue, deposition of acidic mucopolysaccharide abundant in cartilage was observed, and the passage ASC obtained in Example 1 differentiated into chondrocytes. It was confirmed that it also has the ability.

(4) Differentiation into nerve cells Subcultured ASC was seeded on a 24-well multiplate (FALCON (registered trademark) model number 353047, manufactured by Corning) at 4.8 × 10 ³ cells / well, and 2% fetal bovine serum (2% fetal bovine serum). A mesenchymal stem cell growth medium (Stemline (registered trademark), manufactured by Sigma Aldrich) to which FBS) was added was placed and cultured under the condition of 5% CO ₂ atmosphere at 37 ° C. Forty-eight hours after the start of culture, the medium is discarded, washed twice with phosphate buffer (PBS), and then nerve cell differentiation medium (HyClone Advantage STEM Natural Differation Kit, manufactured by Thermo Fisher Scientific). When the cells were cultured at a cell density of about 30% (about 30% confluency) for 2 days, neuronal projections were confirmed. Therefore, it was fluorescently stained with a mouse anti-β-Tubulin Class III antibody (obtained from Funakoshi) labeled with Alexa Fluor (registered trademark) 488 (manufactured by Thermo Fisher Scientific). A fluorescence micrograph of nerve cells stained is shown in FIG.
From FIG. 12, since the cell bodies and protrusions were stained in green, it was confirmed that they were neurons of mature mammals, and the passaged ASC obtained in Example 1 also had the ability to differentiate into neurons. It was confirmed to have.

From the above results, it is possible to easily isolate and proliferate stem cells from tissues by using a non-woven fabric whose culture surface is composed of at least a norbornene-based polymer, and that the proliferated stem cells have high differentiation potential. Recognize.

Claims (3)

A method for separating stem cells, in which a tissue-derived cell group containing stem cells is cultured in a medium on a non-woven fabric whose culture surface is at least composed of a norbornene-based polymer. The method for separating stem cells according to claim 1, wherein the tissue-derived cell group is derived from adipose tissue. In cell culture in a medium, use of a non-woven fabric in which at least the culture surface is composed of a norbornene-based polymer for separating stem cells from a group of tissue-derived cells including stem cells.

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