WO2018097189A1 - Stem cell isolation method and use of nonwoven fabric formed of norbornene-based polymer - Google Patents

Abstract

The present invention selectively isolates and cultures a specific stem cell with ease by means of a simple culturing operation. Provided is a method for isolating stem cells, wherein a group of tissue-derived cells including stem cells is cultured on a nonwoven fabric in which at least a culturing surface thereof is formed of a norbornene-based polymer.

Description

Method for separating stem cells and use of non-woven fabric composed of norbornene polymer

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

In recent years, the development of stem cell therapy that repairs or regenerates cells and tissues of damaged patients using stem cells or cells derived from stem cells has been progressing. For example, Patent Document 1 proposes a closed treatment system that obtains adipose tissue from a patient and administers stem cells concentrated by concentration treatment to the patient.

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

International Publication No. 03/053346 International Publication No. 2005/042730 JP 2013-034436 A JP 2008-199897 A

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

There are many examples of using a nonwoven fabric for the separation of stem cells, and as described in Patent Document 3 and the like, the nonwoven fabric is usually used as a filtration substrate. 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 not using a cell suspension, Patent Document 4 proposes a method of culturing cells using a nonwoven fabric whose surface is coated with hydroxyapatite. However, this method has a problem that it is difficult to ensure that the hydroxyapatite is uniformly coated on the surface of the nonwoven fabric, 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 thereof is to easily selectively isolate and culture a specific stem cell by a simple culture operation.

In order to solve the above-mentioned problems, the present inventors diligently studied a method for separating stem cells from a tissue-derived cell group.
As a result, when the adipose tissue-derived cell group is cultured on a nonwoven fabric in which at least the cell culture surface (culture surface) is composed of a norbornene polymer, only the adipose stem cells are exposed to the nonwoven fabric surface without any surface coating treatment. As a result, it was found that adipose stem cells can be selectively separated 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, there are provided the method for separating stem cells described in (1) and (2) below, and the use of a nonwoven fabric composed of the norbornene polymer in (3).
(1) A method for separating stem cells, wherein a tissue-derived cell group containing stem cells is cultured on a nonwoven fabric having at least a culture surface made of a norbornene polymer.
(2) The method for separating stem cells according to (1), wherein the tissue-derived cell group is a group of cells derived from adipose tissue.
(3) Use of a nonwoven fabric in which at least the culture surface is composed of a norbornene polymer for separating stem cells from a tissue-derived cell group containing stem cells.

According to the present invention, specific stem cells can be easily selectively separated 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 a ZEONOR nonwoven fabric in Example 1. FIG. 2 is a phase contrast micrograph of ASC 18 days after the start of culture growing on the Zeonex nonwoven fabric 2 in Example 1. 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 Zeonore nonwoven 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. 6 is a phase contrast micrograph of BMSC subcultured in a flask for 4 days in Example 2. FIG. 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 photomicrograph of adipocytes stained in Example 3. FIG. 10 is a photomicrograph of stained osteoblasts in Example 3. FIG. 11 is a photomicrograph of the chondrocytes stained in Example 3. FIG. 12 is a fluorescence micrograph of nerve cells stained in Example 3.

Hereinafter, embodiments of the present invention will be described in detail.
A nonwoven fabric composed of a norbornene polymer used in the present invention (hereinafter sometimes simply referred to as “nonwoven fabric used in the present invention” or “nonwoven fabric”) is composed of a norbornene polymer having at least a cultured surface. Is.
In the nonwoven fabric of the present invention, “at least the culture surface is composed of a norbornene-based polymer” means that at least the surface on which cells are cultured (the portion in contact with the cells, for example, the fibers constituting one surface of the nonwoven fabric) is norbornene-based. Means including a polymer. The culture surface may be composed of only a norbornene polymer.

The nonwoven fabric used in the present invention can be used usually in various containers used for culturing cells. The nonwoven fabric may float from the bottom surface of the container or may sink to the bottom surface of the container. In addition, the cells may adhere to only one side of the nonwoven fabric or both sides, but from the viewpoint of cell retention, the nonwoven fabric covers the entire bottom surface of the container and adheres the cells to only one side of the nonwoven fabric. It is desirable to let it grow.
The material of the container in which the nonwoven fabric is put is not particularly limited, and a conventionally known container such as a polystyrene container or a glass container for cell culture can be used.

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

Examples of the ring-opening polymer obtained by ring-opening polymerization include ring-opening polymers obtained by ring-opening polymerization of one or a mixture of two or more norbornene-based monomers, and norbornene-based monomers and ring-opening copolymers thereof. Examples thereof include ring-opening polymers obtained by ring-opening polymerization with other monomers, and hydrides thereof.
Examples of the polymers obtained by addition polymerization include addition polymers obtained by addition polymerization of one or a mixture of two or more norbornene monomers and other monomers copolymerizable with the norbornene monomers. And addition polymers obtained by addition polymerization.
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 or a mixture of two or more norbornene-based monomers, or a norbornene-based single monomer. A hydride of a ring-opening polymer obtained by ring-opening polymerization of a monomer and this and another monomer capable of ring-opening copolymerization is preferred, and one or a mixture of two or more norbornene monomers is opened. A hydride of a ring-opening polymer obtained by ring polymerization is more preferable.

Examples of norbornene monomers that can be used for the synthesis of norbornene polymers include bicyclo [2.2.1] hept-2-ene (commonly known as norbornene) and 5-methyl-bicyclo [2.2.1] hepta. -2-ene, 5,5-dimethyl-bicyclo [2.2.1] hept-2-ene, 5-ethyl-bicyclo [2.2.1] hept-2-ene, 5-ethylidene-bicyclo [2 2.1] hept-2-ene, 5-vinyl-bicyclo [2.2.1] hept-2-ene, 5-propenylbicyclo [2.2.1] hept-2-ene, 5-methoxycarbonyl -Bicyclo [2.2.1] hept-2-ene, 5-cyanobicyclo [2.2.1] hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo [2.2.1] hepta -Bicyclic monomers such as 2-ene;
Tricyclo [4.3.0 ^1,6 . 1 ^2,5 ] deca-3,7-diene (common name dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, 2,3-dihydroxydicyclopentadiene, etc. Mer;
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 . 1 ^7,10 ] -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 ] dec-3-ene (common name methanotetrahydrofluorene: also called 1,4-methano-1,4,4a, 9a-tetrahydrofluorene) 1,4-methano-8-methyl-1,4,4a, 9a-tetrahydrofluorene, 1,4-methano-8-chloro-1,4,4a, 9a-tetrahydrofluorene, 1,4-methano-8 -Tetracyclic monomers such as bromo-1,4,4a, 9a-tetrahydrofluorene;
These norbornene 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, and an alkylidene group.

Other monomers capable of ring-opening copolymerization with norbornene monomers include cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, And monocyclic cycloolefin monomers such as 1,5,9-cyclododecatriene and 1,5,9,13-cyclohexadecatetraene.

Other monomers capable of addition copolymerization with norbornene monomers include α-olefin 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.} Cycloolefin monomers such as 0 ^3,8 ] tetradeca-3,5,7,12-tetraene (also referred to as 3a, 5,6,7a-tetrahydro-4,7-methano-1H-indene); Non-conjugated diene monomers such as 4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, 1,7-octadiene, and the like.
Among these, as other monomers capable of addition copolymerization with norbornene monomers, α-olefin monomers are 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, and an alkylidene group.

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 with a monomer component is a known ring-opening polymerization. It can be obtained by polymerization in the presence of a catalyst.
Examples of the ring-opening polymerization catalyst include a catalyst comprising a metal halide such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a metal halide or acetylacetone such as titanium, zirconium, tungsten, or molybdenum. A catalyst comprising a compound and an organoaluminum compound can be used.
The 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 and then adding a carbon-carbon unsaturated bond. Can be obtained by hydrogenation.

An addition polymer of a norbornene monomer, or an addition polymer of a norbornene monomer and another monomer copolymerizable with the norbornene monomer, 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 restriction on the molecular weight of the norbornene polymer, but it is usually a weight average molecular weight in terms of polystyrene measured by gel permeation chromatography (GPC) of a cyclohexane solution (or a 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, which is preferable.

The glass transition temperature of the norbornene polymer may be appropriately selected depending on the purpose of use, but is usually 50 to 300 ° C., preferably 100 to 280 ° C., particularly preferably 115 to 250 ° C., and more preferably 130 to 200 ° C. ° C. 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 K7121.

The norbornene polymers can be used alone or in combination of two or more.
In addition to the norbornene-based polymer as a resin component constituting the nonwoven fabric, optionally, a compounding agent usually used in thermoplastic resin materials, for example, a soft polymer, an antioxidant, an ultraviolet absorber, a light stabilizer Additives, near-infrared absorbers, release agents, colorants such as dyes and pigments, plasticizers, antistatic agents, fluorescent whitening agents, and other compounding agents can be added in the amounts usually employed. Here, when a soft polymer is used in combination with a norbornene polymer, it is usually 0.01 to 20 parts by mass with respect to 100 parts by mass of the alicyclic structure-containing polymer that is a norbornene polymer. The amount is preferably 0.05 to 10 parts by mass, more preferably 0.05 to 5 parts by mass.

In addition to the norbornene polymer and the soft polymer that is one of the above-mentioned compounding agents, other polymers (hereinafter simply referred to as “other polymers”) are mixed as the resin component constituting the nonwoven fabric. May be. The amount of the other polymer mixed with the norbornene 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 polymer.
If the proportion of various compounding agents and other polymers to be blended with respect to the norbornene polymer is too large, the cells are difficult to float. Therefore, it is preferable to blend them in a range that does not impair the properties of the norbornene polymer.

The mixing method of the norbornene polymer and the compounding agent or other polymer is not particularly limited as long as the compounding agent is sufficiently dispersed in the polymer. Moreover, there is no special restriction | limiting in the order of a mixing | blending. As a blending method, for example, a method of kneading a resin in a molten state using a mixer, a uniaxial kneader, a biaxial kneader, a roll, a Brabender, an extruder, etc., after dissolving and dispersing in a suitable solvent, Examples thereof include a method of removing the solvent by a coagulation method, a casting method, or a direct drying method.
When a biaxial kneader is used, after kneading, it is usually extruded in a rod shape in a molten state, cut into an appropriate length with a strand cutter, and pelletized in many cases.

There is no special restriction | limiting in the method of manufacturing the nonwoven fabric used for this invention, The manufacturing method of a general nonwoven fabric can be employ | adopted.
Especially, when manufacturing the nonwoven fabric which has the preferable fiber diameter mentioned later, a fabric weight, and a surface coverage, the method using a melt blow method is employ | adopted suitably.

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 nonwoven fabric using a digital microscope VHX-1000 (manufactured by Keyence Corporation).
The basis weight of the nonwoven 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 3 cm square nonwoven fabric.
The surface coverage of the nonwoven fabric is preferably 50% to 95%. Here, the surface coverage is the area of the entire image obtained by removing the voids where no fibers are present from the entire area of the image taken with the digital microscope using image analysis software (Image J). It is a value converted by dividing from.

The nonwoven fabric used in the present invention is usually used after sterilization. There are no particular restrictions on the method of sterilization, heating methods such as the high-pressure steam method and dry heat method; radiation methods that irradiate radiation such as gamma rays and electron beams; irradiation methods that irradiate high frequencies; ethylene oxide gas (EOG) The method can be arbitrarily selected from methods generally employed in the medical field, such as a gas method in which a gas is brought into contact, a filtration method using a sterilizing 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. Examples thereof include a mesenchymal vascular cell group, a fat-derived cell group, and a bone marrow-derived cell group that are used for cell therapy. In addition, some cells may be already separated from these cell groups.

The medium for separating the stem cells from the tissue-derived cell group using such a non-woven fabric may be a medium capable of growing the stem cells to be separated, and can be grown particularly while maintaining undifferentiation. It is preferable to use a medium. Such a medium is commercially available. For example, if it is an adipose-derived stem cell, the KBM ADSC series manufactured by Kojin Bio Co., Ltd. can be used. If it is a bone marrow-derived stem cell, Stemline, a mesenchymal system manufactured by SIGMA-ALDRICH Stem cell growth medium series etc. are mentioned.

An additive can also be mix | blended with a culture medium. Examples of additives include inducers 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, the cells can be cultured using a humidified thermostat 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 nonwoven fabric having at least a culture surface made of a norbornene polymer, only adipose stem cells can be adhered and cultured, and as a result, adipose stem cells are separated. It can be done.

The isolated stem cells can be used for stem cell therapy, and further cultured in a differentiation-inducing medium to obtain differentiated cells, which can be used for drug discovery and medical applications.

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

Additives for inducing differentiation include ligands, agonists and antagonists acting on cell surface receptors; nuclear receptors, ligands, agonists and antagonists; extracellular matrix such as collagen and fivenectin; extracellular matrix A compound that acts on a protein involved in an intracellular signal transduction pathway; a component that acts on an enzyme of primary or secondary metabolism in a cell; a gene in a nucleus or mitochondrion in a cell Examples include components that affect expression; DNA and RNA that can be introduced into cells in combination with viral vectors and the like.
These additives can be used alone or in combination of two or more.
Examples of the commercially available differentiation induction 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, the cells can be cultured using a humidified thermostat 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 polymer>
Norbornene-based polymer (Zeonor (registered trademark) 1060R, manufactured by Nippon Zeon Co., Ltd .; norbornene-based ring-opening polymer hydride), and norbornene-based polymer (Zeonex (registered trademark) 5000, manufactured by Nippon Zeon Co., Ltd .; norbornene-based ring opening Polymer hydride) is used in a melt-blowing method under the following conditions: a nonwoven fabric made of ZEONOR (registered trademark) 1060R (hereinafter referred to as "ZEONOR nonwoven fabric") and two types of products made of ZEONEX (registered trademark) 5000 Nonwoven fabrics (hereinafter referred to as “ZEONEX nonwoven fabric 1” and “ZEONEX nonwoven fabric 2”) were produced.

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

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

<ZEONEX nonwoven fabric 2>
Resin: Zeonex (registered trademark) 5000
・ Nozzle temperature: 298 ℃
-Die temperature: 290 ° C
・ Rotation speed: 100rpm
・ Conveyor speed: 14.2 m / min
DCD: 52mm

The fiber diameter, basis weight, and surface coverage of the obtained nonwoven fabric are as follows.
・ Zeonoa nonwoven fabric: Fiber diameter 10.05 μm, basis weight 0.98 mg / cm ² , surface coverage 91%
ZEONEX nonwoven fabric 1: Fiber diameter 15.05 μm, basis weight 0.94 mg / cm ² , surface coverage 58%
ZEONEX nonwoven 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>
A fat tissue with a diameter of about 3 mm extracted from a human is divided into four parts, and this is placed on a ZEONOR nonwoven fabric and a ZEONEX nonwoven fabric 2 cut into a circular shape with a diameter of 2 cm. It was put in a polystyrene dish (FALCON (registered trademark), Corning, TCPS having a diameter of 35 mm).
Thereafter, a mesenchymal stem cell growth medium (Stemline (registered trademark), manufactured by Sigma-Aldrich) supplemented with 2% fetal bovine serum (FBS) was added to the dish, and cultured at 37 ° C. in a 5% CO ₂ atmosphere. .
It was confirmed that adipose tissue-derived stem cells (ASC) began to separate and proliferate after 7 to 10 days on any nonwoven fabric. After confirming the ASC separation and growth, the medium was changed, and the culture was continued. After 18 days from the start of the culture, the ASC was peeled off from the nonwoven fabric with trypsin-EDTA, and then a 279.8 mL flask for cell culture (model number “T-75”). And then subcultured for 4 days in the same medium, it became 90% confluent.
A phase contrast micrograph (magnification 64 times) of ASC 18 days after the start of culture growing on the Zeonore nonwoven fabric and the Zeonex nonwoven fabric 2 is shown in FIGS. It is confirmed that ASC is proliferating on any nonwoven fabric.
Further, FIG. 3 shows a phase contrast micrograph (magnification 64 times) when 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 stirred well to prepare a diluted bone marrow fluid. The ZEONOR nonwoven fabric and ZEONEX nonwoven fabric 1 cut into a circle with a diameter of 2 cm were respectively set on a 100 μm cell strainer (Falcon (registered trademark) model number 352360, manufactured by Corning), and the bone marrow fluid diluted solution previously prepared was passed through the HBSS. After washing twice, the nonwoven fabrics were placed in separate polystyrene dishes for cell culture (FALCON (registered trademark), Corning, TCPS with a diameter of 35 mm). Thereafter, a mesenchymal stem cell growth medium (Stemline (registered trademark); manufactured by Sigma-Aldrich) supplemented with 2% fetal bovine serum (FBS) was added to the dish, and cultured under conditions of 37 ° C. in a 5% CO ₂ atmosphere. .
In any nonwoven fabric, it was confirmed that bone marrow-derived stem cells (BMSC) began to separate and proliferate after 7 to 10 days. After confirming the isolated growth of BMSC, the medium was changed, and the culture was further continued. 18 days after the start of culture, BMSC was peeled off from the nonwoven fabric with trypsin-EDTA, transferred to a 279.8 mL flask for cell culture (model number “T-75”, manufactured by Eppendorf), and subcultured for 4 days in the same medium. It was 90% confluent.
4 and 5 show phase contrast micrographs (magnification 64 times) of BMSC grown on the Zeonore nonwoven fabric and the Zeonex nonwoven fabric 1 after 18 days from the start of culture. It is confirmed that BMSC is proliferating on any nonwoven fabric.
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.

[Comparative example]
A 6-well plate was placed in a core polyethylene outer layer polypropylene non-woven fabric (10 mm × 32 mm), and a suspension of BMSC, 3 mL (0.45 × 10 ⁶ cells / mL) was added thereto and cultured for 3 days. The medium was a mesenchymal stem cell growth medium (Sigma Aldrich, Stemline (registered trademark)) supplemented with 2% FBS.
As a result, MSC was only slightly adhered on the core polyethylene outer layer polypropylene non-woven fabric (FIG. 7) and adhered to the bottom surface of the plate in a 100% confluent state (FIG. 8). The effect was not obtained.

[Example 3]
In Example 1, the differentiation induction experiment of ASC separated on the ZEONOR nonwoven fabric and the ZEONEX nonwoven fabric 2 and then passaged (hereinafter referred to as “passage ASC”) was performed as follows.
(1) Differentiation into adipocytes Passage ASC was seeded on a 24-well multiplate (FALCON (registered trademark) model No. 353047, manufactured by Corning) at 1.9 × 10 ⁵ cells / well, and 2% fetal bovine serum ( A mesenchymal stem cell growth medium (Stemline (registered trademark), manufactured by Sigma-Aldrich) supplemented with FBS) was added and cultured under conditions of 37 ° C. in a 5% CO ₂ atmosphere. After 24 hours, the medium was discarded, washed twice with a phosphate buffer (PBS), replaced with an adipocyte differentiation medium (model number BBDM2, DS Pharma Biomedical), and cultured for 1 week. Thereafter, the medium was replaced with an adipocyte culture medium (model number BBAM1, manufactured by DS Pharma Biomedical Co., Ltd.), cultured for 1 week, and then stained with Oil-Red. A photomicrograph of stained adipocytes is shown in FIG.
From FIG. 9, fat cells with 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 Passage ASC was seeded on a 24-well multiplate (FALCON (registered trademark) model No. 353047, manufactured by Corning) at 1.5 × 10 ⁴ cells / well, and 2% fetal bovine serum A mesenchymal stem cell growth medium (Stemline (registered trademark), manufactured by Sigma-Aldrich Co.) supplemented with (FBS) was added and cultured under conditions of 37 ° C. in a 5% CO ₂ atmosphere. Thereafter, the medium was replaced with an osteoblast differentiation medium (model number BBOB1, manufactured by DS Pharma Biomedical Co., Ltd.), and cultured at a cell density of about 60% (about 60% confluency) for 2 weeks. Thereafter, the cells were stained with Alizarin Red. FIG. 10 shows a photomicrograph of stained osteoblasts.
From FIG. 10, since the cells and their surroundings were stained with Alizarin Red in light red, calcium deposition was observed, and the passage ASC obtained in Example 1 also has 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 dropped on a 24-well multiplate (FALCON (registered trademark) model number 353047, manufactured by Corning) for 2 hours. Later, a chondrocyte differentiation medium (StemPro Chronogenesis differentiation Kit; model number A10070-01, manufactured by Corning) was added, and the cells were cultured under conditions of 37 ° C. in a 5% CO ₂ atmosphere. Thereafter, the cells were cultured for 2 weeks while changing the medium at intervals of 4 to 5 days. The obtained cells were stained with Alcian Blue. A photomicrograph in which chondrocytes are stained is shown in FIG.
From FIG. 11, since the cells and their surroundings were stained blue with Alcian Blue, the deposition of acidic mucopolysaccharides abundant in cartilage was observed, and the passage ASC obtained in Example 1 differentiated into chondrocytes. It was confirmed that it also has a function.

(4) Differentiation into nerve cells Passage ASC was seeded on a 24-well multiplate (FALCON (registered trademark) model No. 353047, manufactured by Corning) at 4.8 × 10 ³ cells / well, and 2% fetal bovine serum ( A mesenchymal stem cell growth medium (Stemline (registered trademark), manufactured by Sigma-Aldrich) supplemented with FBS) was added and cultured under conditions of 37 ° C. in a 5% CO ₂ atmosphere. 48 hours after the start of the culture, the medium was discarded and washed twice with a phosphate buffer (PBS), and then a neuronal differentiation medium (HyClone Advance STEM Neural Differentiation Kit, manufactured by Thermo Fisher Scientific) When the cells were cultured at a cell density of about 30% (about 30% confluency) for 2 days, neurites could be confirmed. Therefore, the cells were fluorescently stained with mouse anti-β-tubulin class III antibody (obtained from Funakoshi) labeled with Alexa Fluor (registered trademark) 488 (manufactured by Thermo Fisher Scientific). FIG. 12 shows a fluorescence micrograph in which nerve cells are stained.
From FIG. 12, since the cell bodies and processes are stained green, it is recognized that the cells are mature mammalian neurons, and the passage ASC obtained in Example 1 also has 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 nonwoven fabric having at least a culture surface made of a norbornene polymer, and that the proliferated stem cells have high differentiation ability. Recognize.

Claims (3)

A method for separating stem cells, wherein a tissue-derived cell group containing stem cells is cultured on a nonwoven fabric having at least a culture surface made of a norbornene polymer. The method for separating stem cells according to claim 1, wherein the tissue-derived cell group is derived from adipose tissue. Use of a nonwoven fabric in which at least the culture surface is composed of a norbornene polymer for separating stem cells from a tissue-derived cell group containing stem cells.

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