JP2008220205A - Container for forming neural stem cell aggregate, method for producing the same and method for preparing neural stem cell aggregate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a container for forming a neural stem cell aggregate, capable of efficiently and readily affording the neural stem cell aggregate in a step of forming the neural stem cell aggregate for obtaining neural stem cells from a biological tissue containing the neural stem cells, to provide a method for producing the container, and to provide a method for preparing the neural stem cell aggregate. <P>SOLUTION: The method for producing the container for forming the neural stem cell aggregate is characterized by comprising a water-soluble resin coating step of coating the inner surface of the container with a water-soluble resin and forming a water-soluble resin coating layer and a water-insoluble cured film-modifying step of curing the water-soluble resin coating layer and modifying the coating layer into a water-insoluble cured film layer after the step. The container for forming the neural stem cell aggregate is produced by the method for production. The method for preparing the neural stem cell aggregate is characterized by suspending a tissue containing multifunctional neural stem cells in a culture medium containing at least one kind of stem cell growth factor, carrying out seeding and culturing thereof in the container for forming the neural stem cell aggregate and thereby forming the neural stem cell aggregate. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a container for forming neural stem cell aggregates, a method for producing the same, and a method for producing neural stem cell aggregates.

In the mammalian biological tissue, there are neural stem cells having self-replicating ability and pluripotency to produce various types of neurons and glial cells.
Nervous stem cells are present in actively dividing tissues such as bone marrow from the early stage of development to the living body, and are involved in the construction and maintenance of functions.
However, in most mammals, the pluripotency of neural stem cells is limited in vivo, and the ability to generate new neuronal cells is low, so if neuronal cells are lost due to a disorder or disease, for example, new neural tissue is regenerated. However, so-called natural healing is difficult.

  Recent research has revealed that neural stem cells differentiate into neurons and glial cells by differentiating them under specific conditions in vitro, that is, they exhibit pluripotency. It is expected to provide new possibilities for the treatment of disease and trauma.

If neural stem cells that are slightly contained in cells of the central nervous system from fetuses and living bodies can be isolated and generated in an undifferentiated state and cultured in large quantities, research on differentiation-inducing factors and its application Applications to a wide range of applications such as drug screening, cell transplantation, and gene therapy probes can be expected.
Isolating undifferentiated neural stem cells from biological tissue is the first step in the above-mentioned research, and a selective culture method called neurosphere method is currently known as the most common method for isolating neural stem cells.

The neurosphere method is a culture of central nervous system cells, including neural stem cells, in the presence of EGF or FGF-2 in the presence of growth factors. The neural stem cell aggregate obtained in this way is an aggregate of undifferentiated neural stem cells isolated from the tissue and thus obtained using the property of forming neural stem cell aggregates. The aggregate can be used for the next differentiation induction step.
In the above-described formation of neural stem cell aggregates, non-specific differentiation is induced when neural stem cells adhere to the surface of the culture vessel. Therefore, it is important to culture in a completely floating state.

Currently, the culture vessel is a suspension culture vessel that is used when culturing cells in suspension culture. The polystyrene molded product or the surface of the polystyrene molded product is wettable by oxygen plasma treatment or corona treatment. However, in these containers, it is difficult to completely prevent neural stem cell adhesion, and as the number of days of culture passes, a part of the aggregated stem cell aggregates form on the surface of the culture container. The number of stem cells obtained in one culture, that is, the number and size of stem cell aggregates formed is insufficient, and the self-replication ability of stem cells is used to obtain the required amount of neural stem cells. Thus, it is necessary to separate the stem cell aggregates formed in this manner and repeat the same culturing process again.
On the other hand, a method for generating differentiated neurons by generating / differentiating neural progenitor cells from neural stem cells using differentiation-inducing growth factors is disclosed. (For example, see Patent Document 1)

JP 2007-14352 A

  The present invention has been made in view of the above circumstances, and a nerve stem cell aggregate can be efficiently and easily obtained in a neural stem cell aggregate formation step for obtaining neural stem cells from a biological tissue containing neural stem cells. An object of the present invention is to provide a container for forming a stem cell aggregate, a method for producing the same, and a method for producing a neural stem cell aggregate.

Such an object is achieved by the present invention described in the following (1) to (9).
(1) A method for producing a neural stem cell aggregate formation container,
A water-soluble resin coating step of coating the inner surface of the container with a water-soluble resin to form a water-soluble resin coating layer;
A method for producing a neural stem cell aggregate formation container, comprising: a water-insoluble cured film modifying step of curing the water-soluble resin coating layer after the step to modify the water-insoluble cured film layer.
(2) The method for producing a neural stem cell aggregate formation container according to (1), wherein the water-soluble resin has a functional group selected from radiation reactivity, photosensitivity, and heat reactivity in a side chain. .
(3) The method for producing a neural stem cell aggregate formation container according to (1) or (2), wherein the functional group includes one having an azide group.
(4) The method for curing the water-soluble resin coating layer includes the method for forming a neural stem cell aggregate formation according to (1), including a curing method by light irradiation.
(5) A neural stem cell aggregate formation container,
A water-soluble resin coating step of coating the inner surface of the container with a water-soluble resin to form a water-soluble resin coating layer;
A neural stem cell aggregation characterized by having a water-insoluble cured film on the inner surface by the water-insoluble cured film modifying step of curing the water-soluble resin coating layer after the step to modify the water-insoluble cured film layer Container for lump formation.
(6) The container for forming neural stem cell aggregates according to (5), wherein the water-soluble resin includes one having a photosensitive functional group in a side chain.
(7) The container for neural stem cell aggregate formation according to (6), wherein the functional group includes one having an azide group.
(8) The neural stem cell aggregate formation container according to (5), wherein the method of curing the water-soluble resin coating layer includes a curing method by light irradiation.
(9) A tissue containing pluripotent neural stem cells is suspended in a medium containing at least one kind of stem cell growth factor, and seeded and cultured in the neural stem cell aggregate formation container according to any one of (5) to (8). A method for producing a neural stem cell aggregate, which comprises forming a neural stem cell aggregate.

  According to the present invention, a neural stem cell aggregate formation container capable of efficiently and easily obtaining neural stem cell aggregates in a neural stem cell aggregate formation step for obtaining neural stem cells from a biological tissue containing neural stem cells, and its production A method and a method for producing neural stem cell aggregates can be obtained.

  The present invention includes a water-soluble resin coating step in which a water-soluble resin is coated on the inner surface of the container to form a water-soluble resin coating layer, and after the step, the water-soluble resin coating layer is cured to form a water-insoluble cured coating layer. A method for producing a neural stem cell agglomerate forming vessel characterized by comprising: In this container, a tissue containing pluripotent neural stem cells is suspended in a medium containing at least one kind of stem cell growth factor, and seeded and cultured in a neural stem cell aggregate forming container to form neural stem cell aggregates. This is a method for producing a characteristic neural stem cell aggregate.

First, a method for producing a neural stem cell aggregate formation container according to the present invention (hereinafter sometimes simply referred to as “manufacturing method”) will be described.
The production method of the present invention includes a water-soluble resin coating step in which a water-soluble resin is coated on the inner surface of the container to form a water-soluble resin coating layer.
The water-soluble resin used in the present invention is hydrated by an ion or hydrogen bond with a water molecule, and as a result, dissolves in water. In other words, the water-soluble resin is a molecule that dissolves in water. It is a resin having a necessary and sufficient amount of ionic or polar side chains with respect to the main chain. In addition, water-soluble resin means what can melt | dissolve 1.0g or more with respect to 100g of 25 degreeC water here.

  The average degree of polymerization of the water-soluble resin is not particularly limited, but is preferably 100 or more and 10,000 or less, particularly preferably 200 or more and 5,000 or less. When the average degree of polymerization is 100 or more, a uniform film can be formed, and when the average degree of polymerization is 10,000 or less, a water-soluble viscosity suitable for workability can be obtained.

  Examples of water-soluble resins include saponified polyvinyl acetate, polyvinyl pyrrolidone, polyethylene glycol, polyacrylamide, polymethacrylamide, polyhydroxyethyl methacrylate, polypentaerythritol triacrylate, polypentaerythritol tetraacrylate, polydiethylene glycol diacrylate. And copolymers of monomers constituting them, and copolymers of 2-methacryloyloxyethyl phosphorylcholine with other monomers (for example, butyl methacrylate). Among these, a structure comprising at least one selected from saponified products of polyvinyl acetate, polyvinyl pyrrolidone, and polyethylene glycol and the reactive group is preferable. Thereby, the formation rate and formation rate of neural stem cell aggregates can be improved.

  Here, the saponified product of polyvinyl acetate refers to, for example, polyvinyl alcohol or a copolymer of vinyl alcohol and another compound. Furthermore, for example, saponified products of vinyl alcohol and modified vinyl acetate modified with a reactive group such as hydrophilic group modification, hydrophobic group modification, anion modification, cation modification, amide group modification or acetoacetyl group are included.

  Further, when the saponified product of polyvinyl acetate is used, the saponification degree of the saponified product of polyvinyl acetate is not particularly limited, but is preferably 20 mol% or more and 100 mol% or less of the entire polyvinyl acetate, particularly 50 mol% or more, 95 mol% or less is preferable. When the saponification degree of polyvinyl acetate is within the above range, the formation rate and formation rate of neural stem cell aggregates can be particularly improved.

The water-soluble resin is not particularly limited, but is preferably prepared using a solvent so that the viscosity at 20 ° C. is 1 mPa · s or more and 10 mPa · s or less, and further 2 mPa · s or more and 7 mPa · s or less. It is preferable to use it. The solvent used in that case may be water or a mixture of water and an organic solvent in order to increase solubility. When the viscosity of the water-soluble resin is within the above range, the cell adhesion amount is small, and the effect of forming nerve cell aggregates is particularly excellent. A sufficient neuron agglomerate formation property is obtained due to a sufficient cell adhesion reduction effect, and the growth of adherent confluent cells is suppressed, so that selective culture efficiency is excellent. Although it does not specifically limit as thickness of a coating layer, 100 nm or more and 5,000 nm or less are preferable, and 150 or more and 1,000 nm or less are more preferable.
By making the thickness of the coating layer more than the above lower limit value, the physical stimulation that the cells receive from the base material can be further suppressed, and by making the thickness less than the above upper limit value, the amount of protein taken into the coating layer is reduced. In addition, since cell adhesion through proteins can be suppressed, the cell aggregate formation rate is further improved.

  As a method for coating the inner surface of the container with the water-soluble resin, for example, spin coating, dipping, or a method of dispensing the water-soluble resin solution onto the inner surface of the container and then discharging the solution by tilting the container can be used. After bringing the water-soluble resin into contact with the inner surface of the container by such a method, the water-soluble resin coating layer can be formed by drying the water-soluble resin solution remaining on the inner surface of the container.

  The production method of the present invention is characterized by having a water-insoluble cured film modifying step of curing the water-soluble resin coating layer after the above step to modify the water-insoluble cured film layer. By using the water-soluble resin coating layer as a water-insoluble cured film layer, a surface having a high density ionic or polar side chain can be constructed. The ionic or polar side chain constructed on this surface hydrates with water molecules by electrostatic interaction or hydrogen bonding when in contact with the culture solution, and the container surface is substantially water-dense water molecules. A hydration layer suppresses the stimulation from the surface of the base material to cells, and a neural stem cell aggregate is rapidly formed. By doing so, it is possible to prevent the water-soluble resin coating layer from being dissolved and released when the culture solution is brought into contact, and to obtain water resistance necessary for the container.

  The method for curing the water-soluble film layer is not particularly limited, and a water-soluble resin having a functional group for curing, for example, a radiation-reactive, photosensitive, or heat-reactive functional group, in the side chain is used. be able to. For example, if it is a photosensitive functional group, a diazo group, an azide group, a simmonyl group, etc. may be mentioned, and if it is a thermally reactive and radiation reactive functional group, a vinyl group, an epoxy group, etc. may be mentioned. it can. Among these, a water-soluble resin having a photosensitive functional group that can be cured rapidly and can be cured with simple equipment is particularly preferable.

A light source in the case of curing by light irradiation is not particularly limited, can illuminance using 5.0 mW / cm ² of about ultra-high pressure mercury lamp or 0.1 mW / cm ² about UV lamps. Curing by light irradiation can be controlled by illuminance and irradiation time, so when using a light source with low illuminance, the irradiation time can be lengthened, and when a highly reactive photosensitive group is selected, it is cured under a fluorescent lamp. It is also possible. For example, when using an extra-high pressure mercury lamp with 5.0 mW / cm ² irradiation 1 to 10 seconds, when using a UV lamp 0.1 mW / cm ² is sufficiently cured by irradiation of 3 to 10 minutes be able to.

As the photosensitive functional group, a functional group containing an azide group is particularly preferable. Thereby, it can be made to react with a practical wavelength of 230-500 nm, and the formability of a film | membrane can be improved with the further outstanding resolution. Thus, a coating layer having the above thickness can be obtained by forming a water-soluble resin coating layer on the surface in advance and curing the coating layer to modify it into a water-insoluble cured coating layer.
Another advantage of using a water-soluble resin is that the unreacted resin can be easily washed away by washing the surface with water after curing. If the eluate is confirmed due to poor curing reactivity or the like, the eluate can be reduced by adding a washing step after curing, and a better EB body formation rate can be obtained.

Next, the neural stem cell aggregate formation container of the present invention (hereinafter sometimes simply referred to as “container”) will be described.
The container of the present invention is manufactured by the manufacturing method of the present invention. That is, a water-soluble resin coating step of forming a water-soluble resin coating layer by coating the inner surface of the container with a water-soluble resin, and after the step, the water-soluble resin coating layer is cured to be modified into a water-insoluble cured coating layer. And having a water-insoluble cured film on the inner surface by the water-insoluble cured film modifying step.

  The container of the present invention can be formed of a resin material. In addition to making the container into a disposable type, this resin material can easily form various shapes. Examples of the resin material include polypropylene resins, polyethylene resins, polyolefin resins such as ethylene-propylene copolymers or cyclic polyolefin resins, polystyrene resins such as acrylonitrile-butadiene-styrene resins, polycarbonate resins, polyethylene Methacrylic resins such as terephthalate resin, polymethyl methacrylate resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyethersulfone resin, polyetheretherketone resin, polyetherimide resin, polytetrafluoroethylene And fluorine resin such as polymethylpentene resin and polyacrylonitrile, and acrylic resin such as polyacrylonitrile, and fiber resin such as propionate resin. Among these, a polystyrene resin is particularly preferable in terms of moldability, transparency, and radiation resistance required for the container.

  The weight average molecular weight of the resin material is not particularly limited, but is preferably 10,000 or more and 500,000 or less, particularly preferably 20,000 or more and 100,000 or less. When the weight average molecular weight is within the above range, the moldability of the container is excellent.

The weight average molecular weight can be measured using, for example, a size exclusion chromatography method (Gel Permeation Chromatography system, Shodex KF-800 column, both manufactured by Showa Denko KK, elution solvent: tetrahydrofuran).
For the purpose of improving moldability and weather resistance, the resin material is added within the range not impairing the object of the present invention, for example, hydrocarbon-based, fatty acid amide-based lubricants, phenol-based, amine-based antioxidants, etc. An agent can be added.
When manufacturing the container of this invention from the said resin material, it can manufacture by injection molding, blow molding, injection blow molding, for example.

  Examples of the form of the container of the present invention include containers such as a multiwell plate, a petri dish (dish), and a flask. Furthermore, even in the case of a sheet-shaped molded article, the environment such that cells such as the bottom of the container can be cultured. It can be installed and used. Among these, a multi-well plate or petri dish having 6 to 384 holes, which is used for bioreactor generation or medicinal effect and toxicological evaluation, artificial organ development research, and the like is preferable. Thereby, the accuracy of evaluation and research using neural stem cell aggregates can be improved.

Examples of the sterilization that is an essential condition of the container of the present invention include ethylene oxide gas sterilization, heat-sensitive sterilization, steam sterilization, and radiation sterilization. Among them, radiation sterilization using γ rays or electron beams is preferable, For production, γ-ray sterilization is particularly preferred from the viewpoint of radiolucency.
The absorbed dose of radiation is not particularly limited, but if the absorbed dose is too low, sterility is not ensured, and if it is too high, the cell container and the coating layer may be deteriorated.
The absorbed dose of radiation in the container of the present invention is preferably 1 kGy or more and 50 kGy or less, particularly preferably 5 kGy or more and 30 kGy or less. As a result, sterility can be imparted while sufficiently maintaining the characteristics of the container of the present invention.

Next, a method for producing the neural stem cell aggregate of the present invention using the container of the present invention will be described.
In the method for producing a neural stem cell aggregate of the present invention, a tissue containing pluripotent neural stem cells is suspended in a medium containing at least one stem cell growth factor, and seeded and cultured in the above-mentioned container for forming a neural stem cell aggregate of the present invention. It is characterized by forming neural stem cell aggregates.

  A specific example of the method for producing the neural stem cell aggregate of the present invention will be described. First, cells of the central nervous system including neural stem cells collected from the striatum and the like are added with a growth factor such as EGF or FGF-2. The cell suspension dispersed at an arbitrary concentration in the culture medium is seeded in the container of the present invention and cultured in an environment such as a carbon dioxide incubator, so that a neural stem cell aggregate is usually formed in 1 to 3 days. Can be confirmed.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a comparative example, this invention is not limited to this.

(Example 1)
A 24-well multiwell plate (24-well plate) was formed by injection molding using polystyrene resin (manufactured by PS Japan, HF77) as the resin material. The obtained 24-hole plate was subjected to plasma treatment (oxygen plasma for 5 minutes) using a plasma processing apparatus (SERIES7000 manufactured by BRANSON / IPC) to impart wettability to the 24-hole plate surface.
The well shape of the obtained 24-well plate had a height of 20 mm and an inner diameter of 16 mm.
Next, polyvinyl alcohol having an azide group in the side chain as a water-soluble resin (AWP manufactured by Toyo Gosei Kogyo Co., Ltd., average polymerization degree of water-soluble resin 1600, introduction rate of photosensitive group 0.65 mol%) was shielded with aluminum foil. In a glass container, it was dissolved in a 20 vol% ethanol aqueous solution to prepare a 1.0 wt% solution.
The above-mentioned 24-hole plate was immersed in the glass container shielded with aluminum foil for 1 minute, then taken out, and the 24-hole plate was turned upside down, and the solution was thoroughly discarded. UV light of 0.1 mW / cm ² × 3 minutes to cure the water-soluble resin, then repeatedly washed with pure water three times, dried, and then irradiated with γ rays at an absorbed dose of 10 kGy (Radie Industries, Ltd.) Thus, the culture container (24-well plate) of the present invention was obtained.
On the surface of the obtained 24-hole plate, a layer formed of the water-soluble resin was formed with a thickness of 550 nm. In addition, the thickness of the layer was measured using an electron microscope (Quanta 400F manufactured by FEI Co., Ltd.) on the fracture surface of a 24-hole plate broken in liquid nitrogen.

(Example 2)
A culture container (24-well plate) was obtained in the same manner as in Example 1 except that a cyclic olefin copolymer resin (manufactured by Polyplastics, TOPAS ^R 6013) was used as the resin material.
On the surface of the obtained 24-hole plate, a layer formed of a water-soluble resin on the side chain was formed with a thickness of 550 nm.

(Comparative example)
A culture vessel (similar to Example 1) except for the steps from the step of imparting hydrophilicity by plasma treatment in the step of Example 1 to the steps of immersion in a water-soluble resin, and curing, washing, and drying with a UV lamp. 24-well plate) was obtained.

About the obtained container, the formation method of the aggregate was evaluated with the following method. The results are shown in Table 1.
Nerve cells isolated from rat fetal striatum according to a standard method were added to DMEM / F-12 medium containing 20 ng / mL EGF, 20 ng / mL FGF2 (manufactured by SIGMA) and N-2 additive (manufactured by GIBCO) at 1 × 10 4 cells / Suspended at a concentration of mL, seeded 2 mL each in the containers of Examples 1 and 2 and the comparative example, cultured in a 5% carbon dioxide atmosphere, and inverted the presence or absence and form of adherent cells 5 days after seeding. The images were observed at a magnification of 100 times and 40 times under a scanning microscope (Olympus BX51).

  As is clear from Table 1, Examples 1 and 2 using the container of the present invention obtained by the production method of the present invention formed a larger number of agglomerates than the comparative examples not using this. In addition, since the size thereof is also large, it was confirmed that the use of the container of the present invention resulted in very rapid proliferation of neural stem cells and efficient formation of aggregates.

A micrograph (× 100) after 7 days of Example 1 confirms a large number of large neural stem cell aggregates. A micrograph after 7 days of Example 1 (× 20) A micrograph (× 100) after 7 days of the comparative example, the size of neural stem cell aggregates is small, and the number is also small. Photomicrograph after 7 days of comparative example (× 20)

Claims (9)

A method for producing a neural stem cell aggregate formation container,
A water-soluble resin coating step of coating the inner surface of the container with a water-soluble resin to form a water-soluble resin coating layer;
After the step, the water-soluble resin coating layer is cured to modify the water-insoluble cured film layer to a water-insoluble cured film layer; and
The manufacturing method of the container for neural stem cell aggregate formation characterized by including this.   The method for producing a container for forming a neural stem cell aggregate according to claim 1, wherein the water-soluble resin has a functional group selected from radiation reactivity, photosensitivity, and heat reactivity in a side chain.   The method for producing a neural stem cell aggregate formation container according to claim 1 or 2, wherein the functional group includes an azide group.   The method for producing a neural stem cell aggregate formation container according to claim 1, wherein the method for curing the water-soluble resin coating layer includes a curing method by light irradiation. A container for forming neural stem cell aggregates,
A water-soluble resin coating step of coating the inner surface of the container with a water-soluble resin to form a water-soluble resin coating layer;
After the step, the water-soluble resin coating layer is cured to modify the water-insoluble cured film layer to a water-insoluble cured film layer; and
A neural stem cell aggregate formation container characterized by having a water-insoluble cured film on the inner surface.   The said water-soluble resin is a container for neural stem cell aggregate formation of Claim 5 containing what has a photosensitive functional group in a side chain.   The neural stem cell aggregate formation container according to claim 6, wherein the functional group includes one having an azide group.   The neural stem cell aggregate formation container according to claim 5, wherein the method of curing the water-soluble resin coating layer includes a curing method by light irradiation.   Aggregating neural stem cells by suspending a tissue containing pluripotent neural stem cells in a medium containing at least one kind of stem cell growth factor, and seeding and culturing them in a container for forming neural stem cell aggregates according to any one of claims 5 to 8. A method for producing a neural stem cell aggregate comprising forming a mass.