JP2013099287A - Bottle type cell culture container and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing a bottle type cell culture container having a functional organic compound layer on the inner peripheral surface of a body.SOLUTION: The method for producing a bottle type cell culture container 100 is characterized by comprising a process for cylindrically processing a sheet-like functional substrate 200 having a substrate layer 202 and a functional organic compound layer 201 so that the functional organic compound layer 201 is disposed on the inner side, and so that one side end portion and the other side end portion of the substrate 200 are arranged at mutually bondable positions, and then bonding one side end portion to the other side end portion to form a cylindrical body member 101, and a process for bonding a bottom member 102 to one end of the cylindrical body member 101 and further bonding a tip member 103 to the other end.

Description

  The present invention relates to a bottle-type cell culture container and a method for producing the same.

  Patent Document 1 discloses a cell culture container such as a petri dish, on which a functional organic compound layer such as a temperature-responsive polymer is coated. By using this cell culture container, it is possible to easily peel off and recover from the cell support without destroying the cultured / proliferated cells simply by changing the temperature. However, as described in Patent Document 1, it is necessary to perform surface treatment separately by batch treatment on a cell culture container such as a petri dish to provide a functional compound layer. There is still room for improvement.

  Therefore, in Patent Document 2 and the like, a technique is proposed in which a functional substrate having a functional compound layer on the surface and having a film-like or plate-like shape is separately prepared and the functional substrate is fixed to a cell culture vessel. ing. According to this technique, workability is enhanced as compared with the case where the functional organic compound layer is provided by batch processing of the cell culture containers individually.

JP-A-2-21865 JP 2010-98799 A

  As described in Patent Document 2, a method of fixing a separately prepared functional substrate to a cell culture container is performed on an open flat container surface such as the bottom of a dish-shaped cell culture container (petri dish). This is a method suitable for providing a functional organic compound layer.

  On the other hand, a bottle-type cell culture container is known as a cell culture container suitable for large-capacity cell culture. Since the bottle-type cell culture container is used for cell culture in a laid state, the inner peripheral surface of the trunk mainly interacts with the cells. For this reason, in a bottle-type cell culture container, it is required to provide a functional compound layer on the inner peripheral surface of the trunk. However, it is not easy to fix a separately prepared functional substrate to the inner peripheral surface of the body of the bottle-type cell culture container. It is particularly difficult to fix the functional substrate over the entire circumference of the inner peripheral surface of the trunk portion.

  In addition, as in Patent Document 1, a method of performing a chemical reaction on the surface of a previously formed container and covalently bonding a functional organic compound layer to the surface is fixed to an open surface such as a petri dish bottom surface. Although suitable for application, it is difficult to apply to the inner surface of a closed room such as the inner peripheral surface of the body of the bottle-type cell culture container.

  Then, an object of this invention is to provide the efficient manufacturing method of the bottle type cell culture container provided with functional organic compound layers, such as a temperature-responsive polymer, in the trunk | drum internal peripheral surface. Another object of the present invention is to provide a bottle-type cell culture container in which a functional organic compound layer such as a temperature-responsive polymer is fixed to the inner peripheral surface of the trunk part through a covalent bond.

  A sheet-like functional substrate comprising at least a base material layer and a functional organic compound layer disposed on the base material layer, the surface provided with the functional organic compound layer being the inside, and one of the functional base materials The side end portion and the other side end portion are processed into a cylindrical shape so as to be arranged at positions where they can be joined to each other, and the one side end portion and the other side end portion are joined together to form an inner circumference. A bottle-shaped cell culture container having a functional organic compound layer on the inner peripheral surface of the body portion by forming a cylindrical body member having a functional organic compound layer on the surface and subsequently joining the bottom member and the top member Has been found to be able to be produced efficiently, and the present invention has been completed. The present invention includes the following inventions.

(1) A cylindrical body member having a functional organic compound layer on the inner peripheral surface;
A bottom member joined to one end of the cylindrical body member;
A top member formed with a through-hole, joined to the other end of the cylindrical body member;
A bottle-shaped cell culture container comprising:
A sheet-like functional substrate comprising at least a base material layer and a functional organic compound layer disposed on the base material layer, the surface provided with the functional organic compound layer being the inside, and one of the functional base materials Processing into a cylindrical shape so that the side end portion and the other side end portion are arranged at positions where they can be joined to each other, and joining the one side end portion and the other side end portion, Forming a cylindrical body member having a functional organic compound layer on the inner peripheral surface;
Joining a bottom member to one end of the cylindrical body member and joining a top member to the other end;
Including methods.
(2) a cylindrical trunk member having a functional organic compound layer on the inner peripheral surface;
A bottom member joined to one end of the cylindrical body member;
A top member formed with a through-hole, joined to the other end of the cylindrical body member;
With
The wall portion constituting the cylindrical body member includes a resin base layer and a functional organic compound layer fixed to the surface of the resin base layer via a covalent bond.
Bottle type cell culture container.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to manufacture efficiently the bottle type cell culture container provided with the functional organic compound layer in the trunk | drum internal peripheral surface.

FIG. 1 shows the overall shape of a bottle-type cell culture container produced in the present invention. FIG. 2 is a cross-sectional view of a functional substrate for forming a cylindrical body member. FIG. 3 is a diagram showing an outline of each step of the method for producing a bottle-type cell culture container of the present invention.

  In the following description, the features of the present invention will be described with reference to the drawings as needed. However, each drawing is a schematic view, and the size and shape of each portion are appropriately exaggerated for easy understanding. It shows.

<Shape of bottle type cell culture container>
The shape of the bottle-type cell culture container targeted by the present invention will be described.
As shown in FIG. 1, the bottle-type cell culture container includes a cylindrical body member 101, a bottom member 102 joined to one end of the body member 101, and a top member 103 joined to the other end of the body member 101. At least. A through hole 104 is formed in a part of the top member 103, and more preferably, a neck portion 105 extending from the periphery of the through hole 104 to the outside of the container portion is provided. That is, the top member 103 connects the other end of the body member 101 and one end of the neck portion 105. More preferably, the neck portion 105 is formed with a locking portion 106 for locking the lid 110, and the lid 110 is detachably mounted via the locking portion 106. In the internal space surrounded by the body member 101, the bottom member 102, and the top member 103, an inner chamber for accommodating cells and a culture medium is formed, and the inner chamber communicates with the outside of the container through a through hole 104.

  Although FIG. 1 shows an example in which the body member 101 has a cylindrical shape with a circular cross section, the present invention is not limited to this, and as long as the body portion is cylindrical, there are many shapes such as a triangle, a quadrangle, a pentagon, and a hexagon. It can be a cylindrical shape having various cross sections such as a square shape.

<Bottle cell culture container materials>
The material forming the base material layer, bottom member, top member, neck, lid, etc. of the bottle-shaped cell culture container is not particularly limited, and materials generally used in cell culture can be used. . For example, polystyrene resin, polyester resin, polyethylene resin, polyethylene terephthalate resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride A resin material such as a resin, a resin material containing at least one of the above-described surface hydrophilized treatment, and an inorganic material such as glass or quartz are preferable, but a resin material is preferable. The resin material is preferably a polystyrene resin or a polyethylene terephthalate resin.

<Manufacture of bottle-type cell culture containers>
The bottle-type cell culture container produced in the present invention is characterized in that a functional organic compound layer is present on the inner peripheral surface of a cylindrical body member 101. And in the present invention, as a method for producing a bottle-type cell culture container having such a configuration,
A sheet-like functional substrate comprising at least a base material layer and a functional organic compound layer disposed on the base material layer, the surface provided with the functional organic compound layer being the inside, and one of the functional base materials Processing into a cylindrical shape so that the side end portion and the other side end portion are arranged at positions where they can be joined to each other, and joining the one side end portion and the other side end portion, Forming a cylindrical body member having a functional organic compound layer on the inner peripheral surface;
Joining a bottom member to one end of the cylindrical body member and joining a top member to the other end;
A method comprising:

  The sheet-like functional substrate 200 for forming the body member includes at least two substrate layers 202 and a functional organic compound layer 201 disposed on the substrate layer, as shown in a cross section in FIG. It has a multilayer structure of layers. Between the base material layer 202 and the functional organic compound layer 201, other layers (for example, a primer layer described later) necessary for joining the two layers may be appropriately interposed.

  The base material layer may be any material as long as it can form the above-described functional organic compound layer on one surface and includes a material that can be formed into a cylindrical shape. The base material layer is preferably made of a resin material, and more preferably made of the resin material described in the section <Material of bottle-type cell culture container>. In particular, as the material of the base material layer, polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE), Resin materials such as medium density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polyethylene naphthalate, polypropylene, and acrylic are preferable.

  The surface of the base material layer on the side on which the functional organic compound layer is formed can be a surface subjected to easy adhesion treatment. “Easy adhesion treatment” refers to treatment with an easy adhesive such as polyester, acrylic ester, polyurethane, polyethyleneimine, silane coupling agent, perfluorooctane sulfonic acid (PFOS), and the like.

  The thickness of the base material layer (when the base material layer includes an easy adhesion layer in addition to the base material layer, the total thickness of the base material layer including the easy adhesion layer) is processed into a cylindrical body. In view of the characteristics, it may be selected as appropriate. In the case of a resin-made base material layer, the thickness can typically be 100 to 1000 μm.

The method for producing the sheet-like functional substrate 200 by forming the functional organic compound layer 201 on the surface of the base material layer 202 is not particularly limited, and can be appropriately selected according to the type of the functional organic compound layer. Preferably, a functional organic compound layer can be formed on the surface of the base material layer 202 using a printing technique. Specific examples of the type of the functional organic compound layer and the formation method on the surface of the base material layer are as described later.
The overall shape of the functional substrate 200 is not particularly limited as long as it can form a cylindrical body.

  In the method of the present invention, as shown in FIG. 3A, a sheet-like functional substrate 200 prepared in advance has a surface on the functional organic compound layer 201 side as an inner side (front surface in FIG. 3A), and a base material layer With the surface on the 202 side as the outside, it is processed into a cylindrical shape so that one side end and the other side end of the functional base body 200 are disposed at positions where they can be joined to each other. The cylindrical body member 101 including the functional organic compound layer 201 on the inner peripheral surface is formed by joining one side end of the functional base 200 and the other side end. The method for joining the side ends is not particularly limited as long as it is a method that allows liquid-tight joining so that cells and culture medium do not leak. When the base material layer is made of a resin material, the side ends of the functional substrate 200 can be melted and integrated using an appropriate means such as ultrasonic welding or laser welding. You may join the side edge parts of the functional base | substrate 200 using a suitable adhesive agent. When joining the side edge parts of the functional substrate 200, the edge surfaces of one side edge part of the base material layer 202 may be joined so that the edge surface of the other side edge part faces each other. The vicinity region of one side edge of the functional organic compound layer 201 side surface or the base material layer 202 side surface of the substrate 200 and the other of the functional organic compound layer 201 side surface or the base material layer 202 side surface You may join so that the vicinity area | region of a side edge part may face.

  Subsequently, as shown in FIG. 3B, a bottom member 102 is joined to one open end of the cylindrical body member 101 obtained in the above step, and a top member in which a through hole 104 is formed at the other open end. 103 is joined. The method for joining the members to each other is not particularly limited as long as it is a method in which cells and a medium can be joined in a liquid-tight manner so as not to leak out. When the base material layer 201, the bottom member 102, and the top member 103 of the body member 101 are each made of a resin material, the members to be joined are melted and integrated using an appropriate means such as ultrasonic welding or laser welding. It is possible. Moreover, you may join members using a suitable adhesive agent. As shown in FIG. 3B, the neck 105 can be integrated with the top member 103 in advance.

  Subsequently, other members such as the lid 110 are appropriately combined, and a scale indicating the volume of the contents is added to the surface of the container as necessary using a printing technique or the like to complete the bottle-type cell culture container.

  In the method of the present invention, by using a functional substrate provided in advance with a resin base layer and a functional organic compound layer fixed to the surface of the resin base layer via a covalent bond By manufacturing the barrel member, the wall portion constituting the cylindrical barrel member, which has been difficult to manufacture by the prior art of Patent Document 1 or the like, includes a resin base layer and the resin base layer. A bottle-type cell culture vessel provided with a functional organic compound layer immobilized on the surface of the material layer via a covalent bond can be produced. Examples of such a functional organic compound layer include a stimulus-responsive polymer layer containing a stimulus-responsive polymer grafted on a resin base layer, which will be described later.

<Functional organic compound layer>
In the present invention, the organic compound constituting the functional organic compound layer is not particularly limited as long as it has a desired function, but more preferably, it changes from cell adhesion to cell non-adhesion by a predetermined stimulus. And a hydrophilic compound such as an ethylene glycol chain composed of one or more ethylene glycol units (CH ₂ —CH ₂ —O).

  The film thickness of the functional organic compound layer may be, for example, in the range of 0.5 nm to 300 nm, and preferably in the range of 1 nm to 100 nm.

Hereinafter, preferred embodiments of the “stimulus responsive polymer layer” and the “hydrophilic compound layer” will be described.
<Stimulus responsive polymer layer>
The functional organic compound layer is particularly preferably a stimulus-responsive polymer layer. The stimulus-responsive polymer layer is a layer containing a polymer in which the degree of cell adhesion on the surface is changed by a predetermined stimulus. Examples of the stimulus responsive polymer include a temperature responsive polymer, a pH responsive polymer, an ion responsive polymer, and a photoresponsive polymer. Among these, a temperature-responsive polymer is preferable because it is easy to give a stimulus.

  As the temperature-responsive polymer, for example, a polymer that exhibits cell adhesion at a temperature at which cells are cultured and exhibits cell non-adhesion at a temperature at which the produced cell sheet is peeled may be used. For example, a temperature-responsive polymer has improved affinity to surrounding water at temperatures below the critical dissolution temperature, and the polymer takes up water and swells to make it difficult for cells to adhere to the surface (cell non-adhesiveness). It is preferable to use a material exhibiting a property (cell adhesiveness) that makes the polymer shrink and easily adheres cells to the surface when water is desorbed from the polymer at a temperature equal to or higher than the same temperature. Such a critical solution temperature is called a lower critical solution temperature. A temperature-responsive polymer having a lower critical solution temperature T of 0 ° C. to 80 ° C., more preferably 0 ° C. to 50 ° C. may be used. This is because the cells can be stably cultured when T is 0 ° C to 80 ° C.

  Suitable temperature-responsive polymers include acrylic polymers or methacrylic polymers. Specific examples of suitable temperature-responsive polymers include poly-N-isopropylacrylamide (T = 32 ° C.), poly-Nn-propyl acrylamide (T = 21 ° C.), and poly-Nn-propyl methacrylamide. (T = 32 ° C.), poly-N-ethoxyethyl acrylamide (T = about 35 ° C.), poly-N-tetrahydrofurfuryl acrylamide (T = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T = About 35 ° C.), and poly-N, N-diethylacrylamide (T = 32 ° C.).

  As a monomer for forming these polymers, a monomer that can be polymerized by irradiation with radiation can be used. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, and vinyl ether derivatives. More than seeds may be used. When a single monomer is used alone, the polymer formed on the substrate is a homopolymer, and when multiple monomers are used together, the polymer formed on the substrate is a heteropolymer. Both forms are encompassed by the present invention.

  In addition, when it is necessary to adjust T depending on the type of proliferating cell, when it is necessary to enhance the interaction between the coating substance and the cell culture support, and the balance between the hydrophilicity and hydrophobicity of the cell support is adjusted. If necessary, other monomers other than those described above may be further added for copolymerization. Further, a graft or block copolymer of the above-mentioned polymer used in the present invention and another polymer, or a mixture of the polymer of the present invention and another polymer may be used. Moreover, it is also possible to crosslink within a range where the original properties of the polymer are not impaired.

  As the pH responsive polymer and the ion responsive polymer, those suitable for the cell sheet to be prepared can be appropriately selected.

  The stimulus-responsive polymer layer is prepared by preparing a coating composition containing a monomer that is polymerized to form a target stimulus-responsive polymer and an organic solvent that can dissolve the monomer. A coating film is formed by coating on the surface of the base material layer, and then a polymer is formed on the coating film by polymerizing monomers in the coating film by an appropriate means such as radiation irradiation. It can be formed by causing a grafting reaction between the surface and the polymer. The stimulus-responsive polymer layer thus formed is fixed to the surface of the resin base material layer via a covalent bond.

<Hydrophilic compound layer>
As another embodiment of the functional organic compound layer, hydrophilicity such as an ethylene glycol chain composed of one or more ethylene glycol units (an ethylene glycol chain composed of a plurality of ethylene glycol units can be referred to as a “polyethylene glycol chain”). A layer of a functional compound.

  The terminal of the ethylene glycol chain may be in a form blocked with a hydroxyl group, or the terminal of the ethylene glycol chain may be in a form in which another substance such as a biological substance is linked by a covalent bond.

  A layer containing an ethylene glycol chain whose end is blocked with a hydroxyl group can provide a hydrophilic surface to which cells are difficult to adhere.

  Bio-related substances that can be covalently bonded to the end of ethylene glycol chain include antigens, antibodies, DNA, RNA, peptides, hormones, enzymes, cytokines, sugar chains, lipids, coenzymes, enzyme inhibitors, cells, and other functions. The protein which has is included. Furthermore, the low molecular weight compound which has affinity with such a biological substance, and a high molecular compound are also contained in the range of a biological substance.

  In order to immobilize a layer of a hydrophilic compound such as an ethylene glycol chain on the surface of a resin base material layer, it can be physically adsorbed on the surface of the resin base material layer in advance. Then, a primer layer containing polysiloxane containing a functional group capable of forming a covalent bond by reacting with the terminal hydroxyl group of the ethylene glycol chain is provided. The functional group on the side chain of the polysiloxane is preferably a glycidyl group or an epoxy group. The primer layer can be formed by applying a silanol compound having a desired side chain to the surface of the base material layer, and performing condensation polymerization on the surface to convert it into polysiloxane.

Next, a functional group of the primer layer is reacted with a hydroxyl group of polyethylene glycol in which two or more ethylene glycol or ethylene glycol units are repeated to form a covalent bond, thereby immobilizing the ethylene glycol chain. At this time, ethylene glycol or polyethylene glycol containing a catalytic amount of concentrated sulfuric acid is brought into contact with the primer layer.
A layer containing ethylene glycol chains whose ends are blocked with hydroxyl groups is formed in this way.

  Furthermore, if necessary, at least one functional group capable of forming a covalent bond with another substance is directly or indirectly linked to one end of the ethylene glycol chain. The method for introducing the functional group is not particularly limited.

DESCRIPTION OF SYMBOLS 100 ... Bottle type cell culture container 101 ... Cylindrical trunk | drum member 102 ... Bottom member 103 ... Top member 200 ... Sheet-like functional base | substrate 202 ... Base material layer 201 ...・ Functional organic compound layer

Claims (2)

A cylindrical body member provided with a functional organic compound layer on the inner peripheral surface;
A bottom member joined to one end of the cylindrical body member;
A top member formed with a through-hole, joined to the other end of the cylindrical body member;
A bottle-shaped cell culture container comprising:
A sheet-like functional substrate comprising at least a base material layer and a functional organic compound layer disposed on the base material layer, the surface provided with the functional organic compound layer being the inside, and one of the functional base materials Processing into a cylindrical shape so that the side end portion and the other side end portion are arranged at positions where they can be joined to each other, and joining the one side end portion and the other side end portion, Forming a cylindrical body member having a functional organic compound layer on the inner peripheral surface;
Joining a bottom member to one end of the cylindrical body member and joining a top member to the other end;
Including methods. A cylindrical body member provided with a functional organic compound layer on the inner peripheral surface;
A bottom member joined to one end of the cylindrical body member;
A top member formed with a through-hole, joined to the other end of the cylindrical body member;
With
The wall portion constituting the cylindrical body member includes a resin base layer and a functional organic compound layer fixed to the surface of the resin base layer via a covalent bond.
Bottle type cell culture container.

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