JP2008178367A - Culturing container for developing embryoid, method for producing the same and method for developing the embryoid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culturing container for developing an embryoid (EB), developing the EB having a high quality from an embryonic stem cell (ES cell) easily in a good efficiency, and to provide a method for producing the same and a method for developing the EB. <P>SOLUTION: This method for producing the culturing container for developing the EB comprises the water-soluble resin-covering layer-forming process of forming a water-soluble covering layer by covering the inner surface of the culturing container with the water-soluble resin and after that process, the water-insoluble cured film-modifying process of curing the water-soluble covering layer to modify to the water-insoluble cured film layer, and the produced culturing container for developing the EB is also provided. Further, the method for developing the EB is provided by seeding and culturing non-differentiated embryonic stem cells in the container for developing the EB. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a culture vessel for forming an embryoid body, a method for producing the same, and a method for forming an embryoid body.

Embryonic stem cells (ES cells) are obtained by seeding the inner cell mass of blastocysts on feeder cells and adding leukemia inhibitory factor (LIF) to culture.
ES cells that proliferate indefinitely while maintaining an undifferentiated state have multipotency to differentiate into various tissue cells, so various researches in the field of transplantation treatment using tissues obtained by differentiation, so-called regenerative medicine It is the target of.

  In order to induce differentiation of ES cells into various tissues, a method of forming a pseudo embryo called an embryoid body (EB body) is most widely used, and the formation of an EB body is performed in vitro. This is the first step in inducing differentiation of ES cells. In addition, formation of EB bodies requires that ES cells be cultured in a floating state in which the ES cells are not adhered to the culture vessel. In the adhesion culture using a normal culture vessel, the EB bodies are not formed, but adhere / extend, and are non-specific. Differentiation begins.

  The most widely used method for culturing ES cells in a suspended state is a method called hanging culture (see, for example, Non-Patent Document 1). As its name suggests, hanging drop culture is a method of culturing cells in a culture solution suspended in the form of water droplets. In other words, mineral oil and buffer solution are added to the wells of a multi-well plate, and the culture suspension containing ES cells is spotted into droplets at positions that overlap each well of the multi-well plate lid. And incubate on a multiwell plate. However, the hanging drop method has problems such as a low success rate of EB body formation, microscopic observation, a small amount of EB bodies that can be formed at one time, and complicated operation.

In order to solve the problem, for example, in Patent Document 1, a container is made of polypropylene to prevent adhesion of ES cells, and the shape of the container is conical to make it easier for ES cells to collect at the bottom. It is disclosed to form a body simply and efficiently. However, since polypropylene is not transparent and EB body formation cannot be confirmed by microscopic observation, and only one EB body can be formed in a single container, the amount of EB body that can be formed at one time is equivalent to the hanging drop method. is there.
Furthermore, since the inclination of the ES cell to physically slide down is necessary, the shape of the container is limited to the above-mentioned conical shape, so that it is generally used as a multi-well plate-shaped container with a flat dish or bottom. Cannot be used.

  For example, Patent Document 2 discloses an embryoid body-forming container including a coating layer formed using a compound having a specific phosphorylcholine-like group. The patent document describes that EB bodies are favorably formed by culturing ES cells in a floating state using the biocompatibility of the phosphorylcholine group. However, a compound having a phosphorylcholine-like group which is essential for carrying out the patent document is a very special compound, and it is difficult to synthesize without using the method disclosed in Patent Document 3, for example. It will be limited in implementation.

  In addition, it is known that cell adhesion is suppressed by coating the surface of the culture vessel with a hydrophilic compound, and that the culture can be performed in a floating state, and other attempts to use such a culture vessel as an EB body-forming vessel have been carried out. There are examples. However, although some EB bodies are formed by these methods, the quality and formation rate of the obtained EB bodies are various, and satisfactory results are not always obtained in the differentiation induction step. There are various possible causes for the deterioration in the quality and formation rate of EB bodies in a conventional culture vessel coated with a hydrophilic compound. For example, when a hydrophilic compound is coated using graft polymerization, the density of the hydrophilic compound is low. Since it is sufficient, there is a possibility that the adhesion of ES cells cannot be sufficiently prevented. Moreover, when a hydrophilic compound is coated using a coating, there is a possibility that an eluate derived from the hydrophilic compound is liberated in the medium and inhibits EB body formation.

Keller, J .; Physiol. (Lond) 168: 131-139, 1998 JP 2004-254622 A WO2005 / 001019 PR JP 54-36025 A

  The present invention has been made in view of the above circumstances, an embryoid body-forming culture container capable of efficiently and easily forming a high-quality embryoid body from ES cells, and a method for producing the same. And it is providing the formation method of an embryoid body.

Such an object is achieved by the present invention described in the following (1) to (9).
(1) A method for producing a culture container for embryoid body formation,
A water-soluble resin coating layer forming step of coating the inner surface of the culture vessel with a water-soluble resin to form a water-soluble coating layer;
After the step, a water-insoluble cured film modifying step for curing the water-soluble coating layer and modifying it into a water-insoluble cured film layer;
A method for producing a culture vessel for embryoid body formation, comprising:
(2) The method for producing a culture container for embryoid body formation 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 said functional group is a manufacturing method of the culture container for embryoid body formation as described in (1) or (2) containing what has an azide group.
(4) The method for curing the water-soluble coating layer includes the method for curing an embryoid body formation according to (1), including a curing method by light irradiation.
(5) A culture vessel for embryoid body formation,
A water-soluble resin coating layer forming step of coating the inner surface of the culture vessel with a water-soluble resin to form a water-soluble coating layer;
After the step, a water-insoluble cured film modifying step for curing the water-soluble coating layer and modifying it into a water-insoluble cured film layer;
A culture vessel for forming an embryoid body characterized by having a water-insoluble cured film on the inner surface.
(6) The embryoid body-forming culture container according to (5), wherein the water-soluble resin includes one having a photosensitive functional group in a side chain.
(7) The culture container for forming an embryoid body according to (5) or (6), wherein the functional group includes one having an azide group.
(8) The culture vessel for embryoid body formation according to (5), wherein the water-soluble coating layer is cured by light irradiation.
(9) An embryoid body is cultured by seeding an undifferentiated embryonic stem cell in the culture container for embryoid body formation according to any one of (5) to (8), and further culturing the undifferentiated embryonic stem cell. A method of forming an embryoid body, comprising forming the embryoid body.

  ADVANTAGE OF THE INVENTION According to this invention, the culture container for embryoid body formation which can form a high quality embryoid body efficiently and easily from an ES cell, its manufacturing method, and an embryoid body formation method can be obtained. .

  The present invention includes a water-soluble resin coating layer forming step in which a water-soluble resin is coated on the inner surface of a culture vessel to form a water-soluble coating layer, and after the step, the water-soluble coating layer is cured to form a water-insoluble cured coating layer. A method for producing an embryoid body-forming culture container characterized by comprising a water-insoluble cured film denaturing step that is denatured into an embryoid body-forming culture container, An embryoid body formation method characterized in that an embryoid body is formed by seeding undifferentiated embryonic stem cells in this culture container and further culturing the undifferentiated embryonic stem cells.

First, a method for producing an embryoid body-forming culture container according to the present invention (hereinafter sometimes simply referred to as “manufacturing method”) will be described.
The production method of the present invention is characterized by having a water-soluble resin coating layer forming step of forming a water-soluble coating layer by coating the inner surface of a culture vessel with a water-soluble resin.

  The water-soluble resin used in the production method of 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 dissolves in water. Therefore, it is a resin having a necessary and sufficient amount of ionic or polar side chains with respect to the main chain in the molecule. 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 the water-soluble resin include saponified polyvinyl acetate, polyvinyl pyrrolidone, polyethylene glycol, polyacrylamide, polymethacrylamide, polyhydroxyethyl methacrylate, polypentaerythritol triacrylate, polypentaerythritol tetraacrylate, polydiethylene glycol diester. Examples thereof include copolymers of acrylates and monomers constituting them, and copolymers of 2-methacryloyloxyethyl phosphorylcholine with other monomers (such as 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 stimulation with respect to ES cell can be suppressed and the formation rate of EB body, the formation rate, and the quality of the formed EB body 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, particularly 50 mol% or more of the whole polyvinyl acetate. 95 mol% or less is preferable. When the saponification degree of the polyvinyl acetate is within the above range, the formation rate of the EB body, the formation rate, and the quality of the formed EB body can be particularly improved.

The water-soluble resin 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, preferably 2 mPa · s or more and 7 mPa · s or less. . 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 cell aggregate formation effect is particularly excellent. A sufficient cell aggregate formation property can be obtained due to a sufficient cell adhesion reduction effect. The thickness of the coating layer is preferably from 100 nm to 5,000 nm, and more preferably from 150 nm to 1,000 nm.
By making the thickness of the coating layer equal to or higher than the above lower limit value, it is possible to further suppress physical irritation that the cells receive from the substrate, and by setting the thickness to be equal to or lower than the upper limit value, the amount of protein taken into the coating layer is reduced. In addition, since cell adhesion through protein can be suppressed, the cell aggregate formation rate can be further improved.

  Examples of the method for coating the inner surface of the culture vessel with the water-soluble resin include spin coating, dipping, or dispensing the water-soluble resin solution onto the inner surface of the culture vessel and then discharging the solution by tilting the vessel. Can do. After bringing the water-soluble resin into contact with the inner surface of the culture vessel 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 culture vessel.

The production method of the present invention is characterized by having a water-insoluble cured film modification step of curing the water-soluble coating layer after the step to modify the water-insoluble cured film layer.
By using the water-soluble 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 surface of the culture vessel is substantially dense with water molecules. This becomes a hydrated layer, and this hydrated layer suppresses stimulation from the surface of the substrate to ES cells, and a qualitatively good EB body 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 culture vessel.

  The method for curing the water-soluble film layer is not particularly limited, and a water-soluble resin having a functional group for curing to a side chain, for example, a radiation-reactive, photosensitive, or heat-reactive functional group is used. Can do. 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 a 5.0 mW / cm ² ultra-high pressure mercury lamp is used, irradiation is performed for 1 to 10 seconds, and when a 0.1 mW / cm ² UV lamp is used, it is sufficiently cured by irradiation for 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, curing the coating layer, and modifying 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 embryoid body-forming culture container of the present invention (hereinafter sometimes simply referred to as “culture container”) will be described. The culture container of the present invention is manufactured by the above-described manufacturing method of the present invention.

  The culture container of the present invention can be formed of a resin material. In addition to making the culture vessel into a disposable type, the resin material can be easily molded in 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 a culture vessel.

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 culture vessel 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 culture container of this invention from the said resin material, it can manufacture, for example by injection molding, blow molding, and injection blow molding.

  Examples of the form of the cell culture container of the present invention include containers such as a multi-well plate, a petri dish (dish), and a flask. Furthermore, even in the case of a sheet-like molded product, cells such as the container bottom can be cultured. It can be installed and used in an environment. 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 precision of evaluation and research using a cell aggregate can be improved. In addition, when a multi-well plate with a hemispherical or conical bottom surface called a round bottom or V bottom is used, one EB body is formed with a uniform size per well. it can.

Examples of the sterilization that is an essential condition of the culture container include ethylene oxide gas sterilization, heat-sensitive sterilization, steam sterilization, and radiation sterilization. However, radiation sterilization using γ rays or electron beams is preferable, and mass production is performed. In particular, 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 culture container and the coating layer may deteriorate.

  The absorbed dose of radiation in 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 culture container of the present invention.

Next, the method for forming an embryoid body of the present invention using the culture container of the present invention will be described.
A cell suspension in which undifferentiated ES cells cultured on a feeder such as fibroblasts are dispersed at an arbitrary concentration in a known culture solution to which additives such as serum and growth factor are added as required is used. By seeding in the culture vessel of the invention and culturing in an environment such as a carbon dioxide incubator, the formation of embryoid bodies can be confirmed usually in 2 to 7 days.

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)
Using a polystyrene resin (manufactured by PS Japan Co., Ltd., HF77) as a resin material, a dish was formed by injection molding. The obtained dish was subjected to plasma treatment (oxygen plasma for 5 minutes) using a plasma treatment apparatus (SERIES7000 manufactured by BRANSON / IPC), and wettability was imparted to the dish surface as a pretreatment.
The shape of the obtained dish was 13 mm in height and 35 mm in inner diameter.
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.
After immersing the above-mentioned dish in a glass container shielded with aluminum foil for 1 minute, take it out, turn the dish upside down, discard the solution thoroughly, and dry it primarily at 40 ° C. for 60 minutes. Irradiate 0.1 mW / cm ² × 3 minutes to cure the water-soluble resin, then repeatedly wash with pure water three times, dry, and then irradiate γ-rays with an absorbed dose of 10 kGy (implemented at Raje Industrial Co., Ltd.) The culture container (dish) of the present invention was obtained.
On the surface of the obtained dish, a layer formed of the water-soluble resin was formed with a thickness of 600 nm. The thickness of the layer was measured using an electron microscope (Quanta 400F manufactured by FEI Co., Ltd.) on the fracture surface of the dish broken in liquid nitrogen.

(Example 2)
A culture vessel (dish) 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 dish, a layer formed of a water-soluble resin having the first functional group in the side chain was formed with a thickness of 600 nm.

(Comparative Example 1)
A culture vessel (dish) was obtained in the same manner as in Example 1, except that the steps from Example 1 to immersion in a water-soluble resin and steps of curing, washing and drying with a UV lamp were omitted.

(Comparative Example 2)
Except for the steps of immersion in water-soluble resin and curing by UV lamp, washing, and drying, the dish was immersed in a 3% by weight ethanol solution of polyhydroxyethyl methacrylate copolymer (poly-2hydroxyethyl methacrylate) manufactured by Sigma-Aldrich, A culture vessel (dish) was obtained in the same manner as in Example 1 except that it was dried overnight.

The following evaluation was performed about the obtained culture container (dish). Table 1 shows the evaluation items and the obtained results.
1. Mouse ES cells cultured on a feeder (mouse fibroblast) according to a standard method for evaluating EB body formation using mouse ES cells were suspended in the following medium so as to be 2 × 10 4 cells / mL. In each of Examples 1 and 2, 2 mL was seeded, cultured in a 5% carbon dioxide atmosphere, and the morphology after 5 days was observed.

2. Presence / absence of adherent cells Presence / absence and form of adherent cells were observed at 40 times magnification under an inverted microscope (BX51, Olympus Corporation) 5 days after sowing.

As is clear from Table 1, in Examples 1 and 2 using the culture vessel obtained by the production method of the present invention, a plurality of EB bodies were formed, and no further adhered / extended cells were confirmed. It was.
On the other hand, in Comparative Examples 1 and 2 using a culture vessel not based on the production method of the present invention, no EB body was formed, and adhered and extended cells were observed.
The cell states of Example 1 and Comparative Example 1 are shown in FIGS. 1 and 2, respectively.
The EB bodies obtained in the examples were transferred to a gelatin-coated dish (Sumilon Celtite G Petri dish 35φ manufactured by Sumitomo Bakelite Co., Ltd.), differentiated into myocardium by culturing under predetermined differentiation-inducing conditions, and pulsation was further observed. It was confirmed that the formed EB body had good quality.
Even if the cells that have been extended in adhesion in the comparative example are cultured under the same differentiation-inducing conditions as described above, they are not differentiated into myocardium and are presumed to have differentiated nonspecifically.

A microphotograph after 5 days of Example 1 and formation of a plurality of EB bodies are observed. The micrograph after 5 days of Comparative Example 1 and EB bodies are not formed, and adherent cells are confirmed. The cardiomyocytes differentiated from the EB body obtained in Example 1 are shown.

Claims (9)

A method for producing a culture container for embryoid body formation,
A water-soluble resin coating layer forming step of coating the inner surface of the culture vessel with a water-soluble resin to form a water-soluble coating layer;
After the step, a water-insoluble cured film modifying step for curing the water-soluble coating layer and modifying it into a water-insoluble cured film layer;
A method for producing a culture vessel for embryoid body formation, comprising:   The method for producing a culture container for embryoid body formation 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 said functional group is a manufacturing method of the culture container for embryoid body formation of Claim 1 or 2 containing what has an azide group.   The method for producing a culture container for embryoid body formation according to claim 1, wherein the method for curing the water-soluble coating layer includes a curing method by light irradiation. A culture vessel for embryoid body formation,
A water-soluble resin coating layer forming step of coating the inner surface of the culture vessel with a water-soluble resin to form a water-soluble coating layer;
After the step, a water-insoluble cured film modifying step for curing the water-soluble coating layer and modifying it into a water-insoluble cured film layer;
A culture vessel for forming an embryoid body characterized by having a water-insoluble cured film on the inner surface.   The culture container for forming an embryoid body according to claim 5, wherein the water-soluble resin includes a resin having a photosensitive functional group in a side chain.   The culture container for embryoid body formation according to claim 6, wherein the functional group includes one having an azide group.   The culture vessel for embryoid body formation according to claim 5, wherein the method of curing the water-soluble coating layer includes a curing method by light irradiation. 9. An embryoid body is formed by seeding undifferentiated embryonic stem cells in the embryoid body-forming culture container according to claim 5 and further culturing the undifferentiated embryonic stem cells. Embryoid body formation method.