JP2007175028A - Closed system container for cell culture, kit for closed system cell culture and method for producing closed system container for cell culture - Google Patents

Closed system container for cell culture, kit for closed system cell culture and method for producing closed system container for cell culture Download PDF

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JP2007175028A
JP2007175028A JP2005380115A JP2005380115A JP2007175028A JP 2007175028 A JP2007175028 A JP 2007175028A JP 2005380115 A JP2005380115 A JP 2005380115A JP 2005380115 A JP2005380115 A JP 2005380115A JP 2007175028 A JP2007175028 A JP 2007175028A
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container
cell culture
antibody
auxiliary
flexible film
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Shinichi Yamada
進一 山田
Noritsugu Yabe
則次 矢部
Hiroko Taguchi
裕子 田口
Takao Yoshida
孝夫 吉田
Takashi Morimura
孝史 森村
Kenji Sakai
賢志 酒井
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KOOJIN BIO KK
Fukoku Co Ltd
Fukoku KK
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Fukoku Co Ltd
Fukoku KK
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a closed system container for cell culture, easily housing a housed assistant material in a wide range of the inside surface of a container wall and also suppressing the wastefulness of the housed assistant material. <P>SOLUTION: This closed system container for the cell culture is provided by having at least one of ports 17, 19, being equipped with a container main body of which at least one of opposing flat surface-formed container walls 11, 12 consists of a flexible film, having a closely sealed state housing part 13 between the opposing flat surface-formed container walls 11, 12 and the housed assistant material sealed in the housing part 13 and containing a protein becoming a scaffolding of cells introduced into the housing part 13 and/or a protein activating the cells introduced into the housing part, and presenting the housed assistant material as a plane form in the housing part. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

本発明は、細胞を無菌状態のまま簡易に培養するのに好適な閉鎖系細胞培養容器、閉鎖系細胞培養用キット、及び閉鎖系細胞培養容器の製造方法に関する。   The present invention relates to a closed-system cell culture container, a closed-system cell culture kit, and a method for producing a closed-system cell culture container suitable for easily culturing cells in a sterile state.

近年、生体から採取した細胞を体外で培養し、増殖した細胞を再び体内に戻す養子免疫治療において、細胞の伸展、増殖及び分化を促進させる為に、各種タンパク質を予めフラスコやシャーレ等の硬質容器に固相化したものが用いられている。   In recent years, in adoptive immunotherapy in which cells collected from a living body are cultured outside the body and the proliferated cells are returned to the body again, various proteins are preliminarily placed in a rigid container such as a flask or petri dish in order to promote cell expansion, proliferation and differentiation. The solid phase is used.

具体的には、活性化リンパ球療法において、活性化Ｔリンパ球の誘導は、抗ＣＤ３抗体を固相化した培養容器を用いられているが、固相化の方法は、用事、濃度調整された抗ＣＤ３をフラスコ等の容器に封入し、所定時間放置する事で固相化した後、固相化されていない抗ＣＤ３抗体を容器外へ除去し、さらに容器内部をリン酸緩衝生理食塩水溶液（ＰＢＳ）等で洗浄する処理が一般的に行われている。   Specifically, in activated lymphocyte therapy, activated T lymphocytes are induced by using a culture vessel in which an anti-CD3 antibody is immobilized, but the method of immobilization is adjusted for usage and concentration. After sealing the anti-CD3 in a container such as a flask and allowing it to stand for a predetermined time, the anti-CD3 antibody that has not been immobilized is removed from the container, and the inside of the container is further phosphate-buffered saline solution. A process of washing with (PBS) or the like is generally performed.

これらの操作に用いられるフラスコ等の容器は酸素等の通気性を有していない事から、細胞培養時に必要な通気性を確保する為に、容器本体とキャップに隙間をもたせる、若しくは、キャップの一部に通気性フィルターを具備したベント機能を付与する等により通気性を確保している。これらフラスコ等の硬質容器で細胞培養を行う際は、抗ＣＤ３抗体の固相化等、補助収容物を収容した後に、患者から採取した細胞や細胞培地を注入する操作や細胞培養状態を確認するサンプリング等の目的で、フラスコのキャップやシャーレの蓋を開放し、細胞の採取、培地の添加等をピペッティングにより行う必要がある。そのため、細菌やマイコプラズマ等の微生物（以降、細菌等という。）のコンタミネーションを招く危険性があり、また、ベント機構は完全密封系では無い為、該隙間から液漏れを生じることもあり、閉鎖系で処理できる細胞培養容器が求められていた。   Since containers such as flasks used in these operations do not have oxygen or other air permeability, a gap is provided between the container body and the cap in order to ensure the required air permeability during cell culture, or the cap Breathability is ensured by providing a vent function that is partially equipped with a breathable filter. When cell culture is performed in a hard container such as a flask, an operation for injecting cells or a cell culture medium collected from a patient after confirming an auxiliary container, such as immobilization of an anti-CD3 antibody, and a cell culture state are confirmed. For sampling and other purposes, it is necessary to open the cap of the flask and the lid of the petri dish, and collect cells, add a medium, etc. by pipetting. Therefore, there is a risk of causing contamination of microorganisms such as bacteria and mycoplasma (hereinafter referred to as bacteria), and the vent mechanism is not a completely sealed system. There has been a need for a cell culture vessel that can be treated in a system.

上記問題を解決する為に、フラスコを用いた細胞培養を閉鎖系の培養容器としたものが、特開２００５−５８１０３により提案されている。この提案では、ポアサイズ０．０１〜１．２μｍの孔を有している気体透過性シートが保形性を有する材料から構成されたフラスコ等の立体的な容器に配設され、この気体透過性シートを通して細胞の増殖に必要な酸素、二酸化炭素等の気体透過を確保した閉鎖系フラスコが提案されている。更に、該閉鎖系フラスコのネジ蓋を貫通する中空チューブにより、他の培地供給ユニットを繋ぐキット製品も提案されている。
特開２００５−５８１０３号公報 In order to solve the above problem, Japanese Patent Application Laid-Open No. 2005-58103 proposes a closed culture vessel in which cell culture using a flask is performed. In this proposal, a gas permeable sheet having pores having a pore size of 0.01 to 1.2 μm is disposed in a three-dimensional container such as a flask made of a material having a shape retaining property. There has been proposed a closed-type flask in which gas such as oxygen and carbon dioxide necessary for cell growth is secured through the sheet. Furthermore, a kit product is also proposed in which another medium supply unit is connected by a hollow tube that passes through the screw cap of the closed flask.
JP 2005-58103 A

しかしながら、閉鎖系の培養容器では、容器内壁面を露出させることができないため、抗ＣＤ３抗体の固相化など、各種の補助収容物を容器内壁面に付着させるような処理を行う場合、補助収容物を容器内に注入して容器壁内面に接触させることにより行なわなければならない。その際、上記特許文献１のような立体的な硬質容器では、容器壁内面のより広い範囲に処理を施すには、付着させる量に比べて大過剰の補助収容物を注入して行なわなければならなかった。そのため補助収容物の無駄が多いと共に、補助収容物の収容に手間を要するという問題点があった。
更に、実際には、気体透過性フィルムのポアサイズ制御や、容器本体と気体透過性シートの異材より成形された部材のシール性を維持する事は難しく、また、実施例に記載のキャップとカテーテル等に用いられるシリコーン製チューブの溶着は製造が非常に難しいなど、容器の製造に著しく手間を要するという問題点も有していた。 However, in a closed culture container, the inner wall surface of the container cannot be exposed. Therefore, when processing such as immobilization of anti-CD3 antibody is performed to attach various auxiliary containers to the inner wall surface of the container, auxiliary container is used. This must be done by pouring the object into the container and bringing it into contact with the inner surface of the container wall. At that time, in a three-dimensional rigid container such as the above-mentioned Patent Document 1, in order to perform the treatment on a wider range of the inner surface of the container wall, a large excess of auxiliary container must be injected compared to the amount to be adhered. did not become. For this reason, there is a problem that there is a lot of waste of the auxiliary storage and that it takes time to store the auxiliary storage.
Further, in practice, it is difficult to control the pore size of the gas permeable film and to maintain the sealing performance of the member formed from the different material of the container main body and the gas permeable sheet, and the cap and catheter described in the examples, etc. In addition, it is very difficult to manufacture the silicone tube used in the manufacturing process, and there is a problem that it takes much time and labor to manufacture the container.

そこで、本発明では、容器壁内面の広い範囲に補助収容物を容易に収容し易いと共に補助収容物の無駄を抑えることができる製造容易な閉鎖系細胞培養容器を提供することを課題とすると共に、そのような閉鎖系細胞容器を用いた閉鎖系細胞培養用キットを提供することを他の課題とする。
また、補助収容物を容器壁内面の広い範囲に無駄を抑えて容易に収容できる閉鎖系細胞容器の製造方法を提供することを別の課題とする。 In view of the above, the present invention has an object to provide an easily manufactured closed cell culture container that can easily accommodate the auxiliary container in a wide range of the inner surface of the container wall and can suppress waste of the auxiliary container. Another object is to provide a closed cell culture kit using such a closed cell container.
Another object of the present invention is to provide a method for manufacturing a closed cell container that can easily accommodate auxiliary containers in a wide area on the inner surface of the container wall with little waste.

上記課題を解決する請求項１に記載の発明は、少なくとも１つのポートを有するとともに、対向する平面状容器壁の少なくとも一方が可撓性フィルムからなり、前記対向する平面状容器壁の間に密封状の収容部を有する容器本体と、前記収容部に封入され、前記収容部に導入される細胞の足場となるタンパク質及び／又は前記収容部に導入される細胞を活性化するタンパク質を含む補助収容物とを備え、前記補助収容物が前記収容部に面状に存在することを特徴とする。   The invention according to claim 1, which solves the above problem, has at least one port, and at least one of the opposed planar container walls is made of a flexible film, and is sealed between the opposed planar container walls. A container body having a container-like storage part, and an auxiliary storage containing a protein that is enclosed in the storage part and serves as a scaffold for cells introduced into the storage part and / or a protein that activates cells introduced into the storage part The auxiliary container is present in a planar shape in the container.

ここで、可撓性フィルムとは、収容部内に収容される収容物量に対応して容器壁が変形できる程度の可撓性を有するフィルムである。例えば、可撓性の評価として、フィルムを１％伸ばした時の応力（１％モジュラス）が挙げられる。
可撓性フィルムが単層フィルムの場合は、平行部分の幅が５ｍｍ、標線間距離２０ｍｍのダンベル型の試験片とし、引張速度５０ｍｍ／ｍｉｎの条件で引張試験を行った際の１％モジュラスが２０ＭＰａ以下であり、より好ましくは、１０ＭＰａ以下のものが好適である。
１％モジュラスが２０ＭＰａより大きいと、収容部の容積変化が容易に行なえず、対応する平面状容器壁を外側から厚さ方向に押圧したり、収容部内の気体を吸引して、収容部を平坦化することにより、補助収容物を均一な面状にし難くなる。
また、可撓性フィルムが多層フィルムの場合は、先に記載した単層フィルムと同様の曲げやすさを有しているものが挙げられるが、１％モジュラスは２０ＭＰａ以下に限定されるものではない。
この理由として、多層フィルムはフィルム層を自由に構成する事ができる為、例えば、１％モジュラスが高いフィルムの層間に、柔軟なフィルム層を設ける事で、柔軟なフィルム層の厚さ分だけ変形自由度が増し、曲げやすさを改善する事が出来る事による。
このような可撓性フィルムとしては、低密度ポリエチレン、直鎖状低密度ポリエチレン、ポリプロピレン、エチレン酢酸ビニル共重合体、メチルペンテン樹脂、ポリブタジエン樹脂、スチレンブタジエンスチレンブロック共重合体、塩化ビニル、ＰＥＴ樹脂等の熱可塑性樹脂より成るフィルムが挙げられ、単層フィルムに限定されるものではなく、多層構造のフィルムでも構わない。また、このフィルムは、Tダイやインフレーションフィルム等により形することができる。 Here, a flexible film is a film which has the flexibility which can deform | transform a container wall according to the amount of accommodation accommodated in an accommodating part. For example, as an evaluation of flexibility, stress (1% modulus) when the film is stretched by 1% can be mentioned.
When the flexible film is a single layer film, it is a dumbbell-shaped test piece having a parallel part width of 5 mm and a distance between marked lines of 20 mm, and a 1% modulus when a tensile test is performed at a tensile speed of 50 mm / min. Is 20 MPa or less, more preferably 10 MPa or less.
If the 1% modulus is greater than 20 MPa, the volume of the container cannot be easily changed, and the corresponding flat container wall is pressed in the thickness direction from the outside, or the gas in the container is sucked to flatten the container. By making it, it becomes difficult to make the auxiliary accommodation into a uniform surface.
Moreover, when a flexible film is a multilayer film, what has the ease of bending similar to the single layer film described previously is mentioned, However, 1% modulus is not limited to 20 Mpa or less. .
The reason for this is that a multilayer film can be freely configured as a film layer. For example, by providing a flexible film layer between layers of a film having a high 1% modulus, it is deformed by the thickness of the flexible film layer. This is because the degree of freedom is increased and the ease of bending can be improved.
Such flexible films include low density polyethylene, linear low density polyethylene, polypropylene, ethylene vinyl acetate copolymer, methylpentene resin, polybutadiene resin, styrene butadiene styrene block copolymer, vinyl chloride, PET resin. A film made of a thermoplastic resin such as, but not limited to a single layer film, may be a film having a multilayer structure. Further, this film can be formed by a T-die, an inflation film or the like.

また、補助収容物とは、収容部に導入される培養対象の細胞の足場となるタンパク質、収容部に導入される細胞を活性化するタンパク質、若しくはこれらのタンパク質の組合せ、または、これらと他の物質との組合せなどである。
収容部に導入される細胞の足場となるタンパク質としては、例えばコラーゲン、フィブロネクチン、ラミニン、マトリゲル等の線維芽細胞等の細胞接着性因子などが挙げられる。
また、収容部に導入される細胞を活性化するタンパク質としては、例えば抗ＣＤ３抗体、ＩＬ−１、ＩＬ−４、ＩＬ−６、ＩＦＮ−γ、ＧＭ−ＣＳＦ、ＴＮＦα、ＴＮＦβ等の細胞刺激因子などが挙げられる。
この補助収容物は、適宜な状態で収容部に面状に収容可能であるが、液等の流体又はゲルなどの半流動体の状態で収容するのが好適である。変形自在であるため面状に存在させ易いからである。流体又は半流動体の状態で収容する場合、補助収容物を注入して収容することの他、注入後に一部の成分を残留させて残部を排出することにより収容することも可能である。
なお、この発明において、補助収容物が面状に存在するとは、少なくとも補助収容物が平面状容器壁に沿って広く、好ましくは略全面に薄膜状に広がった状態をいい、平面状容器壁が折り畳まれていても構わない。 The auxiliary container is a protein serving as a scaffold for cells to be cultured introduced into the container, a protein that activates cells introduced into the container, a combination of these proteins, or these and other proteins For example, combinations with substances.
Examples of the protein that serves as a scaffold for cells introduced into the housing part include cell adhesion factors such as fibroblasts such as collagen, fibronectin, laminin, and matrigel.
Examples of the protein that activates the cells introduced into the accommodating part include cell stimulating factors such as anti-CD3 antibody, IL-1, IL-4, IL-6, IFN-γ, GM-CSF, TNFα, and TNFβ. Etc.
Although this auxiliary | assistant accommodation thing can be accommodated planarly in an accommodating part in a suitable state, it is suitable to accommodate in the state of semifluids, such as fluids, such as a liquid, or a gel. This is because it is easily deformable and can be present in a planar shape. When accommodating in the state of a fluid or a semi-fluid, in addition to injecting and accommodating the auxiliary contents, it is also possible to accommodate by leaving a part of the components after the injection and discharging the remainder.
In the present invention, the term “auxiliary accommodation” exists in a planar shape means that at least the auxiliary accommodation is wide along the planar container wall, and preferably spreads in a thin film on the entire surface. It does not matter if it is folded.

請求項２に記載の発明は、対向する平面状容器壁の双方が可撓性フィルムからなることを特徴とする。   The invention according to claim 2 is characterized in that both of the opposing planar container walls are made of a flexible film.

請求項３に記載の発明は、面状に存在する前記補助収容物の平均厚さが１．０ｍｍ以下であることを特徴とする。
ここで言う平均厚さとは、面状にされた補助収容物の膜厚の最大と最小厚さの平均である。
ここで、補助収容物を流体又は半流動体の状態で収容する場合には、面状に存在する補助収容物の平均厚さを０．０５ｍｍ以上とするのが好適である。平均厚さが薄すぎると、補助収容物を平面状容器壁の広い範囲に均一に面状に存在させ難く、培養効果にむらが生じ易いからである。
例えば、平面状容器壁の片面が２００ｃｍ２の場合、容器内部へ補助収容物を１ｃｃ〜２０ｃｃ封入し、平面状容器壁に沿って面状とすると、０．０５ｍｍ以上１．０ｍｍ以下の薄膜を形成する事ができる。 The invention according to claim 3 is characterized in that an average thickness of the auxiliary contained in a planar shape is 1.0 mm or less.
The average thickness mentioned here is the average of the maximum thickness and the minimum thickness of the auxiliary storage material formed into a planar shape.
Here, when the auxiliary container is stored in a fluid or semi-fluid state, it is preferable that the average thickness of the auxiliary container existing in a planar shape is 0.05 mm or more. This is because if the average thickness is too thin, it is difficult for the auxiliary contents to be uniformly present in a wide area on the flat container wall, and unevenness in the culture effect tends to occur.
For example, when one side of the flat container wall is 200 cm 2 , 1cc to 20 cc of auxiliary container is enclosed inside the container, and a thin film having a thickness of 0.05 mm or more and 1.0 mm or less is formed along the planar container wall. Can be formed.

請求項４に記載の発明は、前記補助収容物が、前記収容部に液密状に封入されていることを特徴とする。
ここで液密状とは、収容部から気体が完全に脱気された状態や、収容部の概ねの気体が抜かれている状態であり、容器本体を作製後、先に、収容部の気体を脱気してから補助収容物を封入したり、一旦、気体で膨らませた収容部に補助収容物を充填し、シリンジ等で収容部の気体を吸引する事で、収容部を液密状とする事が出来る。 The invention according to claim 4 is characterized in that the auxiliary container is liquid-tightly sealed in the container.
Here, the liquid-tight state refers to a state in which the gas is completely degassed from the housing portion or a state in which the gas in the housing portion is almost exhausted. After the deaeration, the auxiliary container is sealed, or the storage part once inflated with gas is filled with the auxiliary container, and the gas in the storage part is sucked with a syringe or the like to make the storage part liquid-tight. I can do it.

請求項５に記載の発明は、前記収容部に導入される細胞を活性化するタンパク質が、抗ＣＤ３抗体であることを特徴とする。
ここでは、細胞を活性化するタンパク質として、抗ＣＤ３抗体を容器内に封入するものとして挙げているが、抗ＣＤ３抗体と共に他の細胞活性因子を封入する事により、さらに細胞増殖性をあげることも含む。 The invention according to claim 5 is characterized in that the protein that activates the cells introduced into the accommodating portion is an anti-CD3 antibody.
Here, as a protein that activates cells, an anti-CD3 antibody is encapsulated in a container, but by encapsulating other cell activating factors together with the anti-CD3 antibody, cell proliferation can be further increased. Including.

請求項６に記載の発明は、前記抗ＣＤ３抗体の少なくとも一部が、前記可撓性フィルムの内面に固相化していることを特徴とする。
ここで言う固相化とは、浮遊状態の抗ＣＤ３抗体が可撓性フィルムの表面に固定または定着した状態をいい、抗ＣＤ３抗体を溶解した溶解液を収容部に封入し、所定時間静置することで、抗ＣＤ３抗体を可撓性フィルム表面に固定または定着させる事ができる。 The invention according to claim 6 is characterized in that at least a part of the anti-CD3 antibody is solid-phased on the inner surface of the flexible film.
The term “solid phase” as used herein refers to a state in which the anti-CD3 antibody in a floating state is fixed or fixed on the surface of the flexible film. A solution in which the anti-CD3 antibody is dissolved is enclosed in a container and allowed to stand for a predetermined time. Thus, the anti-CD3 antibody can be fixed or fixed on the surface of the flexible film.

請求項７に記載の発明は、前記可撓性フィルムの内面に固相化されていない前記抗ＣＤ３抗体の非固相化分が前記容器本体から除去されていることを特徴とする。   The invention according to claim 7 is characterized in that the non-solid phase component of the anti-CD3 antibody that is not solid-phased on the inner surface of the flexible film is removed from the container body.

請求項８に記載の発明は、前記非固相化分は、前記容器本体に注入した洗浄液とともに排出されていることを特徴とする。 The invention described in claim 8 is characterized in that the non-solid phase component is discharged together with the cleaning liquid injected into the container body.

請求項９に記載の発明は、前記抗ＣＤ３抗体の封入量が、前記収容部を構成する前記可撓性フィルムの単位面積あたり、０．０２μｇ／ｃｍ２〜４．４μｇ／ｃｍ２であることを特徴とする。 The invention according to claim 9, it enclosed amount of said anti-CD3 antibody, per unit area of the flexible film constituting the accommodating portion is 0.02μg / cm 2 ~4.4μg / cm 2 It is characterized by.

請求項１０に記載の発明は、前記抗ＣＤ３抗体の封入量が、前記収容部を構性する前記可撓性フィルムの単位面積あたり、０．１１μｇ／ｃｍ２〜０．４４μｇ／ｃｍ２であることを特徴とする。 The invention according to claim 10, the amount of the enclosed anti-CD3 antibody, per unit area of the flexible film that構性the accommodating portion, is 0.11μg / cm 2 ~0.44μg / cm 2 It is characterized by that.

請求項１１に記載の発明は、前記可撓性フィルムの前記補助収容物と接する面が、ポリエチレンからなることを特徴とする。   According to an eleventh aspect of the present invention, the surface of the flexible film that contacts the auxiliary container is made of polyethylene.

請求項１２に記載の発明は、前記容器本体が、ガスバリア性及び／又は水蒸気バリア性の高い包装材で包装されたことを特徴とする。
ガスバリア性及び／又は水蒸気バリア性の高い包装材としては、酸素透過度が２５℃、８０％ＲＨ環境下において、５ｃｃ以下／ｍ２・ｄａｙ・ＡＴＭのものが挙げられ、より好ましくは、１ｃｃ以下／ｍ２・ｄａｙ・ＡＴＭが好適である。また、水蒸気透過度は、４０℃、９０％環境下において、２ｇ以下／ｍ２・ｄａｙのものが挙げられ、より好ましくは、１ｇ以下／ｍ２・ｄａｙが好適である。
ガスバリア性及び／又は水蒸気バリア性の高い包装材の代表的なものとして、エチレンビニルアルコール共重合体（ＥＶＯＨ）フィルム、または、アルミ箔、シリカ蒸着フィルム、若しくはアルミナ蒸着フィルム等によるバリア性フィルム層と熱可塑性樹脂によるシーラント層とより構成された多層フィルムなどが挙げられる。 The invention according to claim 12 is characterized in that the container body is packaged with a packaging material having high gas barrier properties and / or water vapor barrier properties.
Examples of the packaging material having a high gas barrier property and / or water vapor barrier property include those having an oxygen permeability of 5 cc or less / m 2 · day · ATM in an environment of 25 ° C. and 80% RH, more preferably 1 cc or less. / M 2 · day · ATM is preferred. Further, the water vapor transmission rate is 2 g or less / m 2 · day, more preferably 1 g or less / m 2 · day, in an environment of 40 ° C. and 90%.
As a typical packaging material having a high gas barrier property and / or water vapor barrier property, an ethylene vinyl alcohol copolymer (EVOH) film, or a barrier film layer made of an aluminum foil, a silica-deposited film, an alumina-deposited film, etc. Examples thereof include a multilayer film composed of a sealant layer made of a thermoplastic resin.

特に、アルミ箔を用いたラミネートフィルムは、ガスバリア性及び水蒸気バリア性が極めて高く、包装材の外部から内部への酸素透過や、包装材の内部から外部への水蒸気の蒸散を生じない為、好適である。
さらに、包装材内部が確認できる様、アルミ箔を用いたラミネートフィルムと透明性を有するシリカ蒸着フィルムを片面づつ貼り合せた包装材でも良い。
フィルム構成の例として、アルミ箔を用いたラミネートフィルムは、最内層（シーラント層）を直鎖状低密度ポリエチレン、中間層（バリア層）をアルミ箔、最外層（保護層）をナイロンフィルムより構成することが出来る。
また、シリカ蒸着フィルムを用いた構成の例として、最内層を直鎖状低密度ポリエチレン、中間層をシリカ蒸着PETフィルム、最外層をナイロンフィルムとしたものが挙げられる。
これらのバリア性の高い包材で包装する際、脱酸素材の同封や、真空包装をする事により、酸素による酸化劣化を押える事が出来て、長期的な保管が可能となる。 In particular, a laminate film using aluminum foil is suitable because it has extremely high gas barrier properties and water vapor barrier properties, and does not cause oxygen permeation from the outside to the inside of the packaging material or evaporation of water vapor from the inside to the outside of the packaging material. It is.
Further, a packaging material in which a laminated film using aluminum foil and a transparent silica vapor deposition film are bonded to each other so that the inside of the packaging material can be confirmed may be used.
As an example of film configuration, a laminate film using aluminum foil is composed of linear low density polyethylene for the innermost layer (sealant layer), aluminum foil for the intermediate layer (barrier layer), and nylon film for the outermost layer (protective layer). I can do it.
Moreover, as an example of the structure using a silica vapor deposition film, the innermost layer is a linear low density polyethylene, the intermediate layer is a silica vapor deposition PET film, and the outermost layer is a nylon film.
When packaging with these packaging materials having high barrier properties, it is possible to suppress oxidative deterioration due to oxygen by enclosing a deoxidized material and vacuum packaging, and long-term storage becomes possible.

請求項１３に記載の発明は、前記請求項１乃至１２のいずれか一に記載の閉鎖系細胞培養容器と、細胞培養用培地を封入した培地用容器とを備え、前記細胞培養容器と前記培地用容器とが使用時に連通可能な連通手段で接続されていることを特徴とする。
連通可能な連通手段としては、閉鎖系細胞培養容器のポート部と培地用容器に設けられたポート部とを、柔軟性のあるチューブにより接続し、ポート部或いはチューブを連通可能に閉塞手段で閉塞したものを適宜使用することができる。例えば、ポートの容器内側端部をクリップチップ等で閉塞する閉塞手段を設けたり、柔軟性のあるチューブにクレンメ等で閉塞する閉塞手段を装着したり、両閉塞手段を併用して、連通可能とすることができる。
柔軟性のあるチューブの材質としては、軟質塩ビニル樹脂、ポリブタジエン樹脂、エチレン酢酸ビニル共重合体、塩素化ポリエチレン樹脂、ポリウレタン系熱可塑性エラストマー、ポリエステル系熱可塑性エラストマー等が挙げられる。 A thirteenth aspect of the invention includes the closed cell culture container according to any one of the first to twelfth aspects of the present invention, and a medium container enclosing the cell culture medium, and the cell culture container and the medium. The container for use is connected by a communication means capable of communicating during use.
As the communication means that can communicate, the port part of the closed cell culture container and the port part provided in the medium container are connected by a flexible tube, and the port part or the tube is closed by the closing means so that the communication is possible. What was done can be used suitably. For example, a closing means for closing the container inner end portion of the port with a clip chip or the like, a closing means for closing with a clamp or the like on a flexible tube, or a combination of both closing means can be communicated. can do.
Examples of the material of the flexible tube include soft vinyl chloride resin, polybutadiene resin, ethylene vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomer, polyester-based thermoplastic elastomer, and the like.

請求項１４に記載の発明は、前記閉鎖系細胞培養容器と前記培地用容器とが、ガスバリア性及び／又は水蒸気バリア性の高い包装材で包装されたことを特徴とする。
培養用培地は、細胞培養容器での培養が終了するまで、酸素及び水蒸気バリア性を有する包装材により包装し、培地の酸化劣化や水蒸気の容器外への蒸散を防止させる。
また、細胞培養容器とバリア性包材で包装された培養用培地を接続したキットは、使用時までバリア性包材に同封若しくは、個別包装されていることが好ましい。 The invention according to claim 14 is characterized in that the closed cell culture container and the medium container are packaged with a packaging material having a high gas barrier property and / or a water vapor barrier property.
The culture medium is packaged with a packaging material having oxygen and water vapor barrier properties until the culture in the cell culture container is completed to prevent oxidative degradation of the medium and evaporation of water vapor out of the container.
Moreover, it is preferable that the kit which connected the culture medium packaged with the cell culture container and the barrier packaging material is enclosed in the barrier packaging material or individually packaged until use.

請求項１５に記載の発明は、少なくとも１つのポートを有するとともに、対向する平面状容器壁の少なくとも一方が可撓性フィルムからなり、前記対向する平面状容器壁の間に密封状の収容部を有する容器本体を準備する工程と、前記収容部に導入される細胞の足場となるタンパク質及び／又は前記収容部に導入される細胞を活性化するタンパク質を含む補助収容物を前記収容部に封入する工程と、前記対向する平面状容器壁を外側から厚さ方向に押圧し、及び／又は前記収容部内の気体を吸引して前記収容部を平坦化することにより、前記補助収容物を前記対向する平面状容器壁の少なくとも一方の内面に面状にする工程とを備えたことを特徴とする。   The invention according to claim 15 has at least one port, and at least one of the opposing planar container walls is made of a flexible film, and a sealed container is provided between the opposing planar container walls. Preparing a container main body having an auxiliary container containing a protein serving as a scaffold for cells introduced into the container and / or a protein that activates cells introduced into the container is enclosed in the container. Pressing the opposing planar container wall in the thickness direction from the outside and / or flattening the accommodating part by sucking the gas in the accommodating part to face the auxiliary container And a step of forming a planar shape on at least one inner surface of the planar container wall.

請求項１６に記載の発明は、少なくとも１つのポートを有するとともに、対向する平面状容器壁の少なくとも一方が可撓性フィルムからなり、前記対向する平面状容器壁の間に密封状の収容部を有する容器本体を準備する工程と、前記収容部に抗ＣＤ３抗体を含む補助収容物を前記収容部に封入する工程と、前記対向する平面状容器壁を外側から厚さ方向に押圧し、及び／又は前記収容部の気体を吸引して前記収容部を平坦化することにより、前記補助収容物を前記対向する平面状容器壁の少なくとも一方の内面に面状にすると共に前記抗ＣＤ３抗体を固相化する工程と、前記収容部に洗浄液を注入し、前記補助収容物に接触させて排出することにより、該洗浄液と共に前記抗ＣＤ３抗体の非固相化分を排出する工程とを備えたことを特徴とする。   The invention according to claim 16 has at least one port, and at least one of the opposing planar container walls is made of a flexible film, and a sealed container is provided between the opposing planar container walls. A container main body having a container, an auxiliary container containing an anti-CD3 antibody in the container, the container is sealed in the container, the opposing planar container wall is pressed in the thickness direction from the outside, and / or Alternatively, the auxiliary container is planarized on at least one inner surface of the opposed planar container wall by sucking the gas in the container and flattening the container, and the anti-CD3 antibody is immobilized on the solid phase. And a step of discharging a non-solid phase component of the anti-CD3 antibody together with the cleaning liquid by injecting a cleaning liquid into the storage unit and discharging it in contact with the auxiliary storage. Features and That.

請求項１の記載の発明によれば、少なくとも１つのポートを有する容器本体の密封状の収容部内に、培養される細胞の足場となるタンパク質及び／又は細胞を活性化するタンパク質を含有する補助収容物が封入されているので、大気中に開放する事無く、具備されたポートより培養する細胞を接種することができる為、無菌保障が容易であり、細菌等によるコンタミネーションを生じ難い。   According to the first aspect of the present invention, the auxiliary storage containing the protein serving as a scaffold for the cells to be cultured and / or the protein for activating the cells in the sealed housing of the container body having at least one port. Since an object is enclosed, cells to be cultured can be inoculated from the provided port without being opened to the atmosphere, so that sterility is easily guaranteed and contamination by bacteria or the like is unlikely to occur.

また、その容器本体では、平面状容器壁の少なくとも一方が可撓性フィルムからなるので、容器壁が変形可能であるため、収容部の容積変化が容易である。そのため、各種の補助収容物を収容する際、収容部の容積を小さくして補助収容物を収容したり、平面状容器壁を変形させて収容物を広げれば、より少ない収容量で容器壁内面の広い範囲に容易に接触させることが可能で、補助収容物を収容部に面状に収容させ易い。その結果、無駄を抑えて補助収容物を容易に収容することが可能である。
更に、これにより補助収容物が平面状容器壁に沿って面状にされるため、細胞培養時に収容部に充填される培養混合物の体積に対して補助収容物を広い接触面積で接触させることができ、細胞の培養を促進することが可能である。 Further, in the container body, since at least one of the planar container walls is made of a flexible film, the container wall can be deformed, so that the volume of the storage portion can be easily changed. Therefore, when accommodating various auxiliary containers, if the volume of the storage part is reduced to accommodate the auxiliary containers, or if the container is expanded by deforming the flat container wall, the inner surface of the container wall can be reduced with a smaller capacity. It is possible to easily make contact with a wide range of the above, and it is easy to accommodate the auxiliary storage object in a planar shape in the storage portion. As a result, it is possible to easily accommodate auxiliary storage items while reducing waste.
In addition, since the auxiliary container is planarized along the planar container wall, the auxiliary container can be brought into contact with a large contact area with the volume of the culture mixture filled in the container during cell culture. It is possible to promote cell culture.

請求項２に記載の発明によれば、対向する平面状容器壁の双方を可撓性フィルムとする事で、容積変化がさらに容易となり、無駄を抑えて、より容易に補助収容物を収容することができ、また、大気中の空気を容器内へ入れる事無く、細胞増殖後に行う内容物の排出や他の培養容器への移行が可能である。   According to the invention described in claim 2, by making both of the opposing flat container walls flexible films, the volume change is further facilitated, the waste is suppressed, and the auxiliary accommodation is more easily accommodated. In addition, it is possible to discharge the contents after cell growth and transfer to another culture container without introducing atmospheric air into the container.

請求項３に記載の発明によれば、面状に存在する前記補助収容物の平均厚さを１．０ｍｍ以下とする事で、確実に補助収容物の無駄を抑えることができると共に、過剰な補助収容物による培養への悪影響を抑え易い。
特に、タンパク質を含有する液を用いて固相化させる場合には、その含有量が少量であることから、固相化されずに排出されるタンパク質量を削減できる。また、タンパク質が固相化され易いため、固相化のための時間を短縮できる。 According to the invention described in claim 3, by making the average thickness of the auxiliary storage object existing in a planar shape 1.0 mm or less, waste of the auxiliary storage object can be surely suppressed, and excessively It is easy to suppress adverse effects on the culture caused by the auxiliary contents.
In particular, when a solid phase is formed using a protein-containing solution, the amount of protein discharged without being solid-phased can be reduced because the content is small. In addition, since the protein is easily immobilized, the time for immobilization can be shortened.

請求項４に記載の発明によれば、補助収容物が収容部に液密状に封入されている事から、閉鎖系細胞培養容器の作製時には、収容部に封入された補助収容物が平面状容器壁の両内面に接触することができるため、容易に両内面に補助収容物を面状に存在させることができる。更に、細胞の足場となるタンパク質や、細胞を活性化するタンパク質を対向する可撓性フィルムの両面に接触して固定化等の処理を行う際にも、対向する平面状容器壁の両内面に存在させ易い。
また、使用の際には、両内面に補助収容物を面状に存在するため、収容部に収容されている培養混合物が対向する平面状容器壁の両内面に存在する補助収容物と接触することができる。そのため、フラスコやシャーレ等のように片面で補助収容物に接触させて培養する場合に比べ、細胞の伸展、増殖及び分化の促進効率が良好である。 According to the invention described in claim 4, since the auxiliary container is sealed in a liquid-tight manner in the container, the auxiliary container enclosed in the container is flat when the closed cell culture container is manufactured. Since it can contact both inner surfaces of a container wall, an auxiliary | assistant accommodation thing can be made to exist in planar shape on both inner surfaces easily. Furthermore, even when the protein that becomes the cell scaffold or the protein that activates the cell is brought into contact with both surfaces of the opposing flexible film to perform immobilization, both the inner surfaces of the opposing flat container walls Easy to exist.
In addition, in use, since the auxiliary container is present in a planar shape on both inner surfaces, the culture mixture accommodated in the container comes into contact with the auxiliary containers present on both inner surfaces of the opposing planar container walls. be able to. Therefore, compared with the case where it culture | cultivates by making it contact with an auxiliary | assistant accommodation on one side like a flask or a petri dish, the promotion efficiency of cell extension, proliferation, and differentiation is favorable.

請求項５乃至１０に記載の発明によれば、養子免疫治療の中でも特に頻度が高い活性化リンパ球療法では増殖した細胞を人体へ戻す事から、抗ＣＤ３抗体との接触を含めた一連操作の無菌保証が必須であるが、本発明の容器を使用して収容部に導入される細胞を活性化するタンパク質を抗ＣＤ３抗体とすることにより、無菌保証のための煩雑な操作を解消する事ができ、活性化リンパ球療法に好適に使用することができる。   According to the inventions described in claims 5 to 10, since activated lymphocyte therapy, which is particularly frequent among adoptive immunotherapy, returns the proliferated cells to the human body, a series of operations including contact with an anti-CD3 antibody is performed. Although sterility assurance is indispensable, the troublesome operation for sterility assurance can be eliminated by using an anti-CD3 antibody as a protein that activates cells introduced into the container using the container of the present invention. Can be suitably used for activated lymphocyte therapy.

特に、請求項９及び１０に記載の発明によれば、容器に封入される溶液中の抗ＣＤ３抗体の総量を特定の範囲とするので、抗ＣＤ３抗体の存在量が少なくてＴリンパ球が活性化されないこと防止できると同時に、抗ＣＤ３抗体の存在量が多くて細胞に対し毒性を生じ、細胞増殖が低下することを防止し易い。   In particular, according to the invention described in claims 9 and 10, since the total amount of anti-CD3 antibody in the solution sealed in the container is in a specific range, the amount of anti-CD3 antibody present is small and T lymphocytes are active. At the same time, it is easy to prevent a decrease in cell proliferation due to a large amount of anti-CD3 antibody presenting toxicity to cells.

請求項１１に記載の発明によれば、容器本体の補助収容物と接する面がポリエチレンからなるので、シーラント層である可撓性フィルムの内面をポリエチレンとする事ができ、容器本体のシール強度、例えばフィルムとフィルム、フィルムとポートのシール強度が良好で、耐衝撃強度等を向上できる。特に、抗ＣＤ３抗体を用いる場合、２〜８℃の冷蔵庫保管が必要である為、低温時の耐衝撃性にも優れているポリエチレンを選択することで液漏れしにくい容器とすることができる。更に、添加剤を極力控えたポリマーグレードを選定する事により、細胞に対して安全性の高い容器を作製することができる。   According to the invention described in claim 11, since the surface of the container main body that contacts the auxiliary container is made of polyethylene, the inner surface of the flexible film as the sealant layer can be made of polyethylene, and the sealing strength of the container main body, For example, the sealing strength between the film and the film, the film and the port is good, and the impact strength can be improved. In particular, when an anti-CD3 antibody is used, since it is necessary to store in a refrigerator at 2 to 8 ° C., it is possible to make a container that does not leak easily by selecting polyethylene having excellent impact resistance at low temperatures. Furthermore, by selecting a polymer grade with as few additives as possible, a highly safe container for cells can be produced.

請求項１２に記載の発明によれば、容器本体がガスバリア性及び／又は水蒸気バリア性の高い包装材で包装される事により、容器本体の収容部の酸素濃度や湿度を長期的に安定させる事が出来る。   According to the twelfth aspect of the present invention, the container body is packaged with a packaging material having a high gas barrier property and / or water vapor barrier property, so that the oxygen concentration and humidity of the container body container can be stabilized for a long period of time. I can do it.

請求項１３に記載の発明によれば、細胞培養容器と細胞培養用培地を封入した培地用容器が接続されており、使用時に連通可能である事から、大気に触れる事無く、無菌的な操作で一部の培地を容易に移行することができ、無菌保証を確保し易い。   According to the thirteenth aspect of the present invention, the cell culture container and the culture medium container enclosing the cell culture medium are connected and can communicate with each other at the time of use. It is possible to easily transfer a part of the culture medium, and it is easy to ensure sterility assurance.

請求項１４に記載の発明によれば、閉鎖系細胞培養容器と培地用容器とがガスバリア性及び／又は水蒸気バリア性の高い包装材で包装される事により、容器本体の収容部や培地用容器内の酸素濃度や湿度を長期的に安定させる事が出来る。   According to the invention described in claim 14, the closed cell culture container and the medium container are packaged with a packaging material having a high gas barrier property and / or a water vapor barrier property, so that the container main body container and the medium container are provided. The oxygen concentration and humidity inside can be stabilized for a long time.

請求項１５に記載の発明によれば、容器本体の収容部に補助収容物を封入し、対向する平面状容器壁を外側から厚さ方向に押圧したり、収容部内の気体を吸引して収容部を平坦化することにより、補助収容物を対向する平面状容器壁の少なくとも一方の内面に面状にするので、少ない量の補助収容物で収容部の内面の広い範囲に補助収容物を面状にすることが可能である。そのため、収容部に導入される細胞の足場となるタンパク質や収容部に導入される細胞を活性化するタンパク質などの無駄を十分に抑制することができる。   According to the fifteenth aspect of the present invention, the auxiliary container is enclosed in the container of the container body, and the opposing flat container wall is pressed in the thickness direction from the outside, or the gas in the container is sucked and stored. By flattening the portion, the auxiliary container is planarized on at least one inner surface of the opposing planar container wall, so that the auxiliary container can be applied to a wide area of the inner surface of the container with a small amount of auxiliary container. Can be made. Therefore, it is possible to sufficiently suppress waste such as a protein serving as a scaffold for cells introduced into the accommodating portion and a protein that activates cells introduced into the accommodating portion.

請求項１６に記載の発明によれば、 容器本体の収容部に抗ＣＤ３抗体を含む補助収容物を封入し、対向する平面状容器壁を外側から厚さ方向に押圧したり、収容部内の気体を吸引して収容部を平坦化することにより、補助収容物を対向する平面状容器壁の少なくとも一方の内面に面状にすると共に抗ＣＤ３抗体を固相化し、洗浄液を注入して抗ＣＤ３抗体の非固相化分を排出するので、抗ＣＤ３抗体を含む補助収容物を少ない量で使用して、収容部の内面の広い範囲に抗ＣＤ３抗体を固相化することが可能である。そのため、固相化後に、洗浄液で排出される抗ＣＤ３抗体の量を少なく抑えることができ、抗ＣＤ３抗体の無駄を抑制することができる。   According to the invention of claim 16, an auxiliary container containing an anti-CD3 antibody is sealed in the container main body and the opposing flat container wall is pressed in the thickness direction from the outside, or the gas in the container is To flatten the container, the auxiliary container is planarized on the inner surface of at least one of the opposing planar container walls, the anti-CD3 antibody is solid-phased, and a washing solution is injected to thereby anti-CD3 antibody. Since the non-solid phase component is discharged, it is possible to immobilize the anti-CD3 antibody in a wide area on the inner surface of the housing portion by using a small amount of auxiliary containing material containing the anti-CD3 antibody. Therefore, the amount of the anti-CD3 antibody discharged with the washing solution after the solid phase can be reduced, and the waste of the anti-CD3 antibody can be suppressed.

以下、この発明の実施の形態について説明する。
［実施の形態１］
図１（ａ）、（ｂ）に、本実施の形態１の閉鎖系細胞培養容器としての抗ＣＤ３抗体封入容器（以下、本容器）を示す。
図において、符号１０は容器本体であり、一対の対向する平面状容器壁１１、１２間に密封状の収容部１３が形成され、上端に本容器を吊るす為の懸垂孔１５が設けられると共に、下端に収容部１３の内外を連通可能な２つのポート１７、１９が設けられている。 Embodiments of the present invention will be described below.
[Embodiment 1]
1 (a) and 1 (b) show an anti-CD3 antibody-enclosed container (hereinafter referred to as “this container”) as a closed cell culture container according to the first embodiment.
In the figure, reference numeral 10 denotes a container body, a sealed housing portion 13 is formed between a pair of opposed planar container walls 11 and 12, a suspension hole 15 for hanging the container is provided at the upper end, and Two ports 17 and 19 capable of communicating between the inside and the outside of the accommodating portion 13 are provided at the lower end.

この実施の形態では、収容部１３は可撓性フィルムからなる一対の平面状容器壁１１、１２から形成されており、図１（ｂ）に示されるように、一対の平面状容器壁１１、１２が略全面において接するように対向配置されて、周縁で接合されている。
本容器で使用する可撓性フィルムは、ポリエチレン、ポリプロピレン、塩化ビニル、エチレン―酢酸ビニル共重合体等の熱可塑性樹脂よりなり、内容物の増減により変形できるフィルムであれば任意に使用可能であるが、細胞培養時の酸素を確保する為に、酸素透過度が２５℃環境下において２０００ｃｃ以上／ｍ２・ｄａｙ・ＡＴＭのフィルムが好ましい。本実施の形態では、ガス透過性を有するポリエチレンを用いている。 In this embodiment, the accommodating portion 13 is formed of a pair of planar container walls 11 and 12 made of a flexible film, and as shown in FIG. 12 are arranged so as to be in contact with each other over substantially the entire surface, and are joined at the periphery.
The flexible film used in this container is made of a thermoplastic resin such as polyethylene, polypropylene, vinyl chloride, ethylene-vinyl acetate copolymer, and any film can be used as long as it can be deformed by increasing or decreasing the contents. However, in order to ensure oxygen during cell culture, a film having an oxygen permeability of 2000 cc or more / m 2 · day · ATM in an environment of 25 ° C. is preferable. In this embodiment, polyethylene having gas permeability is used.

一方のポート１７は、充填ポートとして使用されるもので、充填チューブ構成体２１が装着されている。この充填チューブ構成体２１には、ポート１７と接続する為の接続口２３と、培養する細胞及び培地を含む培養混合物を導入したり、他バッグと連結する為の連結チューブ２４及びその端部の連結口２５と、収容部１３へ抗ＣＤ３抗体含有液を注入する為等の注入チューブ２７及びその端部の注入口２９とを有している。連結口２５は使用可能に閉塞されると共に剥離可能なフィルム等の保護部材３１により密封されている。注入チューブ２７は溶断して密封可能な熱可塑性樹脂からなり、連結口２５は密封されていれば、特に制限されず、輸液、血液バッグ等で使用されているものが使用可能である。   One port 17 is used as a filling port, and is equipped with a filling tube structure 21. In this filled tube structure 21, a connection port 23 for connection to the port 17, a culture mixture containing cells to be cultured and a culture medium, or a connection tube 24 for connection to another bag and the end of the connection tube 24 are connected. It has a connection port 25, an injection tube 27 for injecting an anti-CD3 antibody-containing solution into the accommodating portion 13, and an injection port 29 at its end. The connection port 25 is closed to be usable and sealed with a protective member 31 such as a peelable film. The injection tube 27 is made of a thermoplastic resin that can be melted and sealed, and the connection port 25 is not particularly limited as long as the connection port 25 is sealed, and those used in infusion solutions, blood bags, and the like can be used.

他方のポート１９は、培養状態を確認する為のサンプリングポートとして使用される。このサンプリングポート１９は連結口２５と同様に密封されていれば、特に制限されず、輸液、血液バッグ等で使用されているものが使用可能である。例えば、ゴムにより形成された封止栓とキャップとからなり封止栓をポートとキャップで挟み密封状態としたものが挙げられる。
これらのポート１７、１９は、可撓性フィルムと溶着されるため、ポリエチレン、ポリプロピレン等の熱可塑性樹脂により成形されており、該可撓性フィルムと同一素材が好ましく、本実施形態では、ポリエチレンにより成形されたポートを使用している。 The other port 19 is used as a sampling port for confirming the culture state. The sampling port 19 is not particularly limited as long as it is sealed in the same manner as the connection port 25, and those used for infusion, blood bags, and the like can be used. For example, a sealing plug made of rubber and a cap is used, and the sealing plug is sandwiched between a port and a cap and sealed.
Since these ports 17 and 19 are welded to a flexible film, they are molded from a thermoplastic resin such as polyethylene and polypropylene. The same material as the flexible film is preferable. In this embodiment, polyethylene is used. A molded port is used.

本容器は、このような容器本体１０の収容部１３に補助収容物を収容して面状に存在させた状態で構成されている。本実施形態では、補助収容物として、収容部に導入される細胞を活性化するタンパク質である抗ＣＤ３抗体が一対の平面状容器壁１１、１２の両内面に固相化されて面状に収容されている。ここでは、対向する平面状容器壁１１、１２間が全面において略接触して配置されているため、収容部１３から気体が完全に脱気された状態、或いは、収容部１３の概ねの気体が抜かれている状態となっており、抗ＣＤ３抗体含有液は収容部１３に液密状に封入されている。   This container is comprised in the state which accommodated the auxiliary | assistant accommodation thing in the accommodating part 13 of such a container main body 10, and was made to exist in planar shape. In this embodiment, as an auxiliary container, an anti-CD3 antibody, which is a protein that activates cells introduced into the container, is solidified on both inner surfaces of the pair of planar container walls 11 and 12, and is stored in a planar shape. Has been. Here, since the opposing planar container walls 11 and 12 are disposed so as to be substantially in contact with each other over the entire surface, the state in which the gas is completely degassed from the accommodating portion 13 or the substantial gas in the accommodating portion 13 is present. The anti-CD3 antibody-containing solution is sealed in the container 13 in a liquid-tight manner.

このような抗ＣＤ３抗体封入容器を製造するには、上記のような容器本体１０を準備した後、抗ＣＤ３抗体を含有する溶液またはゲルを注入口２９から収容部１３に注入し、必要により、注入口２９から気体を導入して、注入チューブ２７内に残留する抗ＣＤ３抗体含有液を収容部１３内に押出し、その後、対向する平面状容器壁１１、１２を外側から厚さ方向に押圧したり、収容部１３内の気体を吸引するなどにより、収容部１３を平坦化する。これにより、抗ＣＤ３抗体を含有する溶液またはゲルを対向する平面状容器壁１１、１２の内面に面状にする。必要により静置して抗ＣＤ３抗体の固相化を促進する。
そして、注入口２９から収容部１３に、リン酸緩衝生理食塩水溶液（ＰＢＳ）等の洗浄液を注入し、抗ＣＤ３抗体を含有する溶液と接触、混合して注入口２９から排出することにより、収容部１３内に固相化されずに浮遊している抗ＣＤ３抗体の非固相化分を排出して除去する。その後、必要により収容部１３内の気体を吸引し、注入チューブ２７を溶断部３０で溶断密封することにより、抗ＣＤ３抗体を封入して本容器を製造する。
この製造においては、抗ＣＤ３抗体を含有する溶液またはゲルを対向する平面状容器壁１１、１２の内面に面状にすることにより、抗ＣＤ３抗体の固相化のための静置や非固相化分の排出の処理を行うことなく、抗ＣＤ３抗体封入容器を製造してもよい。その場合、抗ＣＤ３抗体封入容器の製造後から使用時までの間に抗ＣＤ３抗体が固相化されることができ、製造を容易にできる。必要により使用前に上記のような洗浄を行うことも可能である。 In order to manufacture such an anti-CD3 antibody-enclosed container, after preparing the container body 10 as described above, a solution or gel containing the anti-CD3 antibody is injected into the accommodating portion 13 from the injection port 29, and if necessary, A gas is introduced from the injection port 29, and the anti-CD3 antibody-containing liquid remaining in the injection tube 27 is extruded into the accommodating portion 13, and then the opposing planar container walls 11 and 12 are pressed in the thickness direction from the outside. The housing portion 13 is flattened by sucking the gas in the housing portion 13. As a result, the solution or gel containing the anti-CD3 antibody is made planar on the inner surfaces of the opposed planar container walls 11 and 12. If necessary, it is allowed to stand to promote immobilization of the anti-CD3 antibody.
Then, a cleaning solution such as a phosphate buffered saline solution (PBS) is injected from the injection port 29 into the storage unit 13, contacted with a solution containing an anti-CD3 antibody, mixed, and discharged from the injection port 29 to be stored. The non-solid phase component of the anti-CD3 antibody that is not immobilized on the inside of the unit 13 and is floating is discharged and removed. Thereafter, if necessary, the gas in the housing portion 13 is sucked, and the injection tube 27 is fused and sealed with the fusing portion 30 to enclose the anti-CD3 antibody and produce this container.
In this production, a solution or gel containing an anti-CD3 antibody is formed into a planar shape on the inner surfaces of the opposing planar container walls 11 and 12, thereby allowing the anti-CD3 antibody to stand stationary or non-solid phase. You may manufacture an anti-CD3 antibody enclosure container, without performing the process of discharge | evaporating a chemical component. In that case, the anti-CD3 antibody can be solid-phased after the production of the anti-CD3 antibody-encapsulated container until the time of use, and the production can be facilitated. If necessary, the above-described cleaning can be performed before use.

このような抗ＣＤ３抗体封入容器を使用して細胞を培養するには、抗ＣＤ３抗体が固相化された状態で、培地にＴリンパ球等の培養対象となる細胞が混合された培養混合物を連結口２５から収容部１３内に導入し、その培養混合物を収容部１３に収容した状態のまま所定の培養条件下で静置することにより行う。
このとき、この実施形態では、収容部１３には気体が実質的に存在せず、対向する平面状容器壁１１、１２の両内面に培養混合物が接触した状態で培養が行われる。また、培養期間中には、適宜連結口等から培地が補充されてもよい。そして、所定期間経過後に培養を完了する。 In order to culture cells using such an anti-CD3 antibody-enclosed container, a culture mixture in which cells to be cultured such as T lymphocytes are mixed with a medium in a state where the anti-CD3 antibody is immobilized. It introduce | transduces in the accommodating part 13 from the connection port 25, and it carries out by leaving still under predetermined culture conditions in the state accommodated in the accommodating part 13. FIG.
At this time, in this embodiment, the container 13 is substantially free of gas, and the culture is performed in a state in which the culture mixture is in contact with both inner surfaces of the opposed planar container walls 11 and 12. Further, during the culture period, the medium may be supplemented from the connection port or the like as appropriate. And culture | cultivation is completed after progress for a predetermined period.

以上のような本容器では、ポート１７、１９を有する容器本体１０の密封状の収容部１３内に、抗ＣＤ３抗体が封入されているので、大気中に開放する事無く、ポート１７より培養する細胞を接種することができ、無菌保障が容易であり、細菌等によるコンタミネーションを生じ難い。
また、容器本体１０が、平面状容器壁１１、１２が可撓性フィルムからなるので、容器壁１１、１２が変形自在であり、収容部１３の容積変化が容易である。そのため、抗ＣＤ３抗体を固相化する際、容器壁１１、１２を押圧して収容部１３の容積を小さくするように変形させれば、収容部１３内の抗ＣＤ３抗体を含有する溶液を移動させて、平面状容器壁１１、１２の広い範囲に接触させることができ、より少ない収容量で平面状容器壁１１、１２内面の広い範囲に容易に接触させることが可能で、面状に収容させることが容易である。その結果、使用する抗ＣＤ３抗体の量、特に、洗浄液と共に排出される抗ＣＤ３抗体の量を少なく抑えることができ、無駄を抑えて容易に収容することが可能である。 In this container as described above, since the anti-CD3 antibody is sealed in the sealed housing portion 13 of the container body 10 having the ports 17 and 19, it is cultured from the port 17 without being opened to the atmosphere. Cells can be inoculated, sterility assurance is easy, and contamination by bacteria is difficult to occur.
In addition, since the container body 10 includes the flat container walls 11 and 12 made of a flexible film, the container walls 11 and 12 can be deformed, and the volume of the accommodating portion 13 can be easily changed. Therefore, when the anti-CD3 antibody is solid-phased, if the container walls 11 and 12 are pressed so as to reduce the volume of the accommodating part 13, the solution containing the anti-CD3 antibody in the accommodating part 13 is moved. Can be brought into contact with a wide range of the planar container walls 11 and 12, and can be easily brought into contact with a wide range of the inner surfaces of the planar container walls 11 and 12 with a smaller accommodation amount, and accommodated in a planar shape. It is easy to make. As a result, the amount of anti-CD3 antibody to be used, in particular, the amount of anti-CD3 antibody discharged together with the washing solution can be suppressed, and can be easily accommodated with less waste.

更に、これにより抗ＣＤ３抗体が平面状容器壁１１、１２に沿って面状に広い範囲に配置されるため、細胞培養時に収容部１３に充填される培養混合物の体積に対して補助収容物を広い接触面積で接触させることが可能で、細胞の培養を促進することができる。
しかも、抗ＣＤ３抗体が収容部１３に液密状に封入されており、対向する平面状容器壁１１、１２の両内面に存在するから、培養時には、培養液等の培養混合物が対向する平面状容器壁１１、１２の両内面に存在する抗ＣＤ３抗体に接触することができ、フラスコやシャーレ等のように片面で補助収容物に接触させて培養する場合に比べ、細胞の伸展、増殖及び分化の促進効率が良好である。 Further, since the anti-CD3 antibody is arranged in a wide area along the planar container walls 11 and 12 by this, the auxiliary container is added to the volume of the culture mixture filled in the container 13 at the time of cell culture. It is possible to make contact with a wide contact area and promote cell culture.
Moreover, since the anti-CD3 antibody is sealed in the container 13 in a liquid-tight manner and is present on both inner surfaces of the opposed planar container walls 11 and 12, the planar mixture in which a culture mixture such as a culture solution is opposed during culture. It can contact the anti-CD3 antibody existing on both inner surfaces of the container walls 11 and 12, and the cell extension, proliferation and differentiation compared to the case of culturing by contacting the auxiliary container on one side such as a flask or petri dish. The promotion efficiency is good.

［実施の形態２］
図２に、抗ＣＤ３抗体封入容器を有する閉鎖系細胞培養用キットを示す。
この細胞培養用キットは、３つのポート１７、１８、１９を有する他は図１と同様の抗ＣＤ３抗体封入容器２０と、細胞培養用培地を封入した培地用容器４０とを備え、抗ＣＤ３抗体封入容器２０と培地用容器４０とが使用時に連通可能な連通チューブ３７で接続されている。
抗ＣＤ３抗体封入容器２０は、図１と同じく充填用のポート１７及びサンプリング用のポート１９の他に、培地を封入した培地用容器４０を連結する為の連結用のポート１８を別に備えている。この連結用のポート１８の収容部１３側の端部には破断により開封可能なクリックチップ１８ａが備えられている。その他は、図１の容器と同一の符号を付している。
培地用容器４０は、可撓性容器壁４１、４２間に密封状の培地収容部４３が形成され、内部に培地が封入されている。また、上端には懸垂孔４５が設けられ、下端には培地収容部４３の内外を連通可能な２つのポート４７、４９が設けられている。
可撓性容器壁４１、４２及びポート４７、４９は、抗ＣＤ３抗体封入容器２０の平面状容器壁１１、１２及びポート１７、１９と同一の材料により形成することができる。 [Embodiment 2]
FIG. 2 shows a closed cell culture kit having an anti-CD3 antibody-enclosed container.
This cell culture kit includes an anti-CD3 antibody-encapsulating container 20 similar to that shown in FIG. 1 except that it has three ports 17, 18, and 19, and a medium container 40 enclosing a cell culture medium, and the anti-CD3 antibody The enclosing container 20 and the medium container 40 are connected by a communication tube 37 that can communicate with each other during use.
The anti-CD3 antibody-enclosed container 20 has a connection port 18 for connecting a medium container 40 enclosing a medium in addition to the filling port 17 and the sampling port 19 as in FIG. . A click chip 18a that can be opened by breakage is provided at the end of the connecting port 18 on the accommodating portion 13 side. Others are denoted by the same reference numerals as the container of FIG.
In the medium container 40, a sealed medium container 43 is formed between the flexible container walls 41 and 42, and the medium is sealed inside. In addition, a suspension hole 45 is provided at the upper end, and two ports 47 and 49 capable of communicating the inside and outside of the culture medium storage portion 43 are provided at the lower end.
The flexible container walls 41 and 42 and the ports 47 and 49 can be formed of the same material as the planar container walls 11 and 12 and the ports 17 and 19 of the anti-CD3 antibody-sealed container 20.

ポート４７は抗ＣＤ３抗体封入容器２０と連結する連結ポートであり、ポート４９は必要により使用されるポートである。連結ポート４７には、収容部４３内の端部に破断により開封可能なクリックチップ４７ａが設けられている。また、この連結ポート４７と抗ＣＤ３抗体封入容器２０の連結ポート１８との間は、連結チューブ３７により無菌的に連結されて、一時的に流路を閉塞することが可能なクレンメ６３が取付けられている。   The port 47 is a connection port that is connected to the anti-CD3 antibody enclosure 20, and the port 49 is a port that is used if necessary. The connection port 47 is provided with a click chip 47 a that can be opened by breaking at an end in the accommodating portion 43. In addition, a clamp 63 is attached between the connection port 47 and the connection port 18 of the anti-CD3 antibody enclosure 20 aseptically by a connection tube 37 so that the flow path can be temporarily blocked. ing.

更に、この実施形態では、抗ＣＤ３抗体封入容器２０と培地用容器４０とは、それぞれバリア性包材６５、６７により密封被覆されている。バリア性包材６５及び６７は、酸素バリア性及び水蒸気バリア性の高い包装材からなる。なお、細胞培養用キット全体が、包材６９により密封被覆されていてもよい。   Furthermore, in this embodiment, the anti-CD3 antibody-sealed container 20 and the medium container 40 are hermetically covered with barrier packaging materials 65 and 67, respectively. The barrier packaging materials 65 and 67 are made of a packaging material having high oxygen barrier properties and water vapor barrier properties. The entire cell culture kit may be hermetically covered with the packaging material 69.

このような細胞培養用キットを使用して細胞を培養するには、バリア性包材６７を開封せずに、バリア性包材６５を開封して抗ＣＤ３抗体封入容器２０を露出させる。
その後、クリックチップ１８ａ及び４７ａを開封して、連結チューブ３７により両容器２０、４０間を連通させ、培地用容器４０から所望量の培地を抗ＣＤ３抗体封入容器２０に導入し、クレンメ６３により連結チューブ３７を閉塞する。このとき、実施の形態１と同様に、抗ＣＤ３抗体封入容器２０は既に抗ＣＤ３抗体が固相化された状態となっている。
そして、Ｔリンパ球等の培養対象となる細胞が混合された培養混合物を連結口２５から収容部１３内に導入し、その培養混合物を収容部１３に収容した状態のまま所定の培養条件下で静置することにより培養を行う。このとき、実施の形態１と同様に、対向する平面状容器壁１１、１２の両内面に培養混合物が接触した状態で培養が行われる。
所定期間経過後に、クレンメ６３を開放することにより、培地を補充し、再びクレンメ６３により閉塞して、更に、培養を継続する。
このような操作を繰返して、所定期間経過後に培養を完了する。 In order to culture cells using such a cell culture kit, the barrier packaging material 65 is opened without exposing the barrier packaging material 67 to expose the anti-CD3 antibody-sealed container 20.
Thereafter, the click chips 18a and 47a are opened, and the containers 20 and 40 are communicated with each other by the connection tube 37. A desired amount of medium is introduced from the medium container 40 into the anti-CD3 antibody-enclosed container 20 and connected by the clamp 63. The tube 37 is closed. At this time, as in the first embodiment, the anti-CD3 antibody-sealed container 20 is already in a state where the anti-CD3 antibody is immobilized.
Then, a culture mixture in which cells to be cultured such as T lymphocytes are mixed is introduced into the accommodating part 13 through the connection port 25, and the culture mixture is accommodated in the accommodating part 13 under predetermined culture conditions. Cultivation is performed by allowing to stand. At this time, similarly to Embodiment 1, the culture is performed in a state where the culture mixture is in contact with both inner surfaces of the opposing planar container walls 11 and 12.
After the elapse of a predetermined period, the clamp 63 is opened to replenish the medium, closed again with the clamp 63, and the culture is continued.
Such an operation is repeated to complete the culture after a predetermined period.

このような細胞培養用キットでは、図１と同様の抗ＣＤ３抗体封入容器２０を用いているので、同様の効果が得られる。
それと共に、抗ＣＤ３抗体封入容器２０と培地用容器４０が接続されており、使用時にクレンメ６３を外せば連通可能である事から、大気に触れる事無く、無菌的な操作で一部の培地を容易に移行することができ、確実な無菌保証を実現できる。
その際、抗ＣＤ３抗体封入容器２０の収容部１３に抗ＣＤ３抗体が密封状に封入されていると共に、培地用容器４０に培地が密封状に収容されていると、培地を移行させても、抗ＣＤ３抗体封入容器２０の収容部１３内で、培養混合物を平行状容器壁１１、１２の両内面に接触させておくことができ、抗ＣＤ３抗体との接触面積を維持することができる。
また、各容器２０、４０がバリア性包材６５、６７で包装されているので、抗ＣＤ３抗体封入容器２０収容部１３や培地用容器４０の収容部４３内の抗ＣＤ３抗体や培地を酸素や湿度による劣化から長期的に安定に保持する事が出来る。
特に、各容器２０、４０毎にバリア性包材６５、６７で包装していると、抗ＣＤ３抗体封入容器２０を使用して培養開始後にも、培地用容器４０に収容された培地を安定に保持することが可能である。 In such a cell culture kit, since the same anti-CD3 antibody-encapsulating container 20 as in FIG. 1 is used, the same effect can be obtained.
At the same time, the anti-CD3 antibody-enclosed container 20 and the medium container 40 are connected, and can be communicated by removing the clamp 63 at the time of use. It can be easily transferred and a reliable sterility guarantee can be realized.
At that time, when the anti-CD3 antibody is sealed in the housing portion 13 of the anti-CD3 antibody-sealed container 20 and the medium is sealed in the medium container 40, even if the medium is transferred, The culture mixture can be kept in contact with both inner surfaces of the parallel container walls 11 and 12 in the accommodating portion 13 of the anti-CD3 antibody-sealed container 20, and the contact area with the anti-CD3 antibody can be maintained.
Further, since the containers 20 and 40 are packaged by the barrier packaging materials 65 and 67, the anti-CD3 antibody and the medium in the container 13 containing the anti-CD3 antibody-containing container 20 and the container 43 of the medium container 40 can be treated with oxygen or oxygen. It can be held stably for a long time from deterioration due to humidity.
In particular, when the containers 20 and 40 are packaged with the barrier wrapping materials 65 and 67, the medium contained in the medium container 40 can be stabilized even after the culture is started using the anti-CD3 antibody-enclosed container 20. It is possible to hold.

なお、本発明は、上記実施の形態１、２に限定されることなく適宜変更可能であり、例えば、上記では、容器２０の収容部１３に抗ＣＤ３抗体を固相化して封入したが、固相化のための処理を行うことなく、抗ＣＤ３抗体を含有する液、ゲル等を面状に封入してもよい。このようにすると、容器本体１０に液又はゲルが面状に封入された状態で保管されるため、使用時までに静置等の特別な処理を施すことなく平面状容器壁１１、１２の内面に抗ＣＤ３抗体を固相化することができ、好ましい。その一方で、補助収容物を収容部に流体状態又はゲルのような半流動体状態で多量に存在させず、湿潤状態乃至は乾燥状態で存在させることも可能である。
また、収容部１３に導入される細胞を活性化できる他のタンパク質や収容部１３に導入される細胞の足場となるタンパク質を固相化して、或いは固相化することなく面状に封入して用いることも可能である。その際、ゲル状の場合であっても、抗ＣＤ３抗体を含有する溶液を収容部１３に供給した後、平面状容器壁１１、１２の広い範囲に広げたのと同様に、平面状容器壁１１、１２を外側から押圧して容積変化させることにより、容器壁１１、１２のより広い範囲に広げることが可能である。
さらに、タンパク質に限らず、培養する細胞についても、閉鎖系細胞培養容器内で培養できれば、当然に選択可能である。 Note that the present invention is not limited to the first and second embodiments, and can be modified as appropriate. For example, in the above, the anti-CD3 antibody is solid-phased and enclosed in the container 13 of the container 20. A liquid, gel, or the like containing an anti-CD3 antibody may be encapsulated in a planar shape without performing a treatment for phase formation. In this case, since the liquid or gel is stored in the container body 10 in a state of being enclosed in a planar shape, the inner surfaces of the planar container walls 11 and 12 are not subjected to a special treatment such as standing before use. The anti-CD3 antibody can be immobilized on a solid phase, which is preferable. On the other hand, it is possible not to make a large amount of auxiliary storage in the storage part in a fluid state or a semi-fluid state such as a gel, but also in a wet state or a dry state.
In addition, other proteins capable of activating the cells introduced into the accommodating portion 13 and proteins serving as scaffolds for the cells introduced into the accommodating portion 13 are solid-phased or sealed in a planar shape without being solid-phased. It is also possible to use it. At that time, even in the case of a gel, the planar container wall is supplied in the same manner as when the solution containing the anti-CD3 antibody is supplied to the container 13 and then spread over a wide range of the planar container walls 11 and 12. By pressing 11 and 12 from the outside and changing the volume, the container walls 11 and 12 can be spread over a wider range.
Furthermore, not only proteins but also cells to be cultured can be selected as long as they can be cultured in a closed cell culture vessel.

以下、実施例について説明する。
［実施例１］
（閉鎖系容器の準備）
図１に示す抗ＣＤ３抗体封入容器の容器本体１０を用いた。
（抗ＣＤ３抗体溶解液の注入）
オルソクーロンＯＫＴ３注（ヤンセンファーマ株式会社製）をダルベッコりん酸緩衝生理食塩水溶液（以下、ＰＢＳ（−））で２５μｇ／ｍＬに調整し、２ｍＬをシリンジで抜き取り、注入口２９より本容器内部へ注入し、同様に、シリンジを用いて、２０ｃｃの空気を注入口２９より注入し、チューブ２７内に残る抗ＣＤ３抗体を本容器内部へ押し出した。 Examples will be described below.
[Example 1]
(Preparation of closed container)
The container body 10 of the anti-CD3 antibody-enclosed container shown in FIG. 1 was used.
(Injection of anti-CD3 antibody solution)
Orthocoulomb OKT3 injection (manufactured by Janssen Pharma Co., Ltd.) is adjusted to 25 μg / mL with Dulbecco's phosphate buffered saline solution (hereinafter PBS (−)), 2 mL is extracted with a syringe, and injected into the container through the injection port 29 Similarly, 20 cc of air was injected from the injection port 29 using a syringe, and the anti-CD3 antibody remaining in the tube 27 was pushed out into the container.

（抗ＣＤ３抗体の封入）
注入口２９を本容器より高い状態に位置し、シリンジで本容器内部のエアーをゆっくり抜き取り、抜き取った時点で、注入口２９の先端部付近を折り曲げ、一時的に気密状態とした後、シリンジを注入口２９から外し、溶断部３０にて溶断することにより、注入チューブ３０を密封した。 (Encapsulation of anti-CD3 antibody)
The inlet 29 is positioned higher than the main container, and the air inside the main container is slowly extracted with a syringe. When the inlet 29 is extracted, the vicinity of the tip of the injection port 29 is bent and temporarily airtight. The injection tube 30 was sealed by removing from the inlet 29 and fusing at the fusing part 30.

（抗ＣＤ３抗体の固相化）
対向する平面状容器壁１１、１２を数回なぞり、本容器内部へ充填された抗ＣＤ３抗体溶液を本容器内部の全面に延ばし、薄膜とした後、室温で１時間静置した。 (Solid phase of anti-CD3 antibody)
The opposing planar container walls 11 and 12 were traced several times, and the anti-CD3 antibody solution filled into the container was spread over the entire surface of the container to form a thin film, which was then allowed to stand at room temperature for 1 hour.

（リンパ球の培養）
健常者の末梢血から分離した末梢血単核球（５×１０６ｃｅｌｌｓ）に培養培地（コージンバイオ社製、ＫＢＭ５４０、ＩＬ２含有、以下ＫＢＭ５４０という）と１％自己血清とを添加し、５０ｍＬとした後、シリンジを用いて、抗ＣＤ３抗体を固相化した本容器内へ連結口２５より接種し、インキュベータ内で、５％炭酸ガス、３７℃、飽和湿度下で培養を行った。 (Lymphocyte culture)
To a peripheral blood mononuclear cell (5 × 10 6 cells) isolated from the peripheral blood of a healthy person, a culture medium (manufactured by Kojin Bio Inc., containing KBM540, IL2, hereinafter referred to as KBM540) and 1% autologous serum are added, and 50 mL After that, the syringe was inoculated from the connection port 25 into the container in which the anti-CD3 antibody was solid-phased, and cultured in an incubator under 5% carbon dioxide gas, 37 ° C. and saturated humidity.

さらに３日及び５日後に培養培地ＫＢＭ５４０を各５０ｍＬづつ加え、培養を行い、７日後の容器内の細胞数量は、６３×１０６ｃｅｌｌｓであった。また、比較で行った開放系フラスコの培養方法では、５５×１０６ｃｅｌｌｓであった。
この実施例１により、開放系のフラスコに比べて細胞増殖性良好で、且つ、微生物等に汚染される事無い、操作が容易な閉鎖系細胞培養容器を得た。 Further, after 3 days and 5 days, 50 mL each of the culture medium KBM540 was added and cultured, and the cell quantity in the container after 7 days was 63 × 10 6 cells. Moreover, in the culture | cultivation method of the open type flask performed by comparison, it was 55 * 10 < 6 > cells.
According to this Example 1, a closed cell culture vessel having a good cell growth property as compared with an open flask and being easily contaminated without being contaminated by microorganisms or the like was obtained.

［実施例２］
実施例１の（閉鎖系容器の準備）から（抗ＣＤ３抗体の固相化）まで、同様の操作を行い、下記の操作を行った。
（容器内部の洗浄）
処理後の固相化しない抗ＣＤ３抗体溶解液を抜き取った後、ＰＢＳ（―）で２回洗浄した。
（リンパ球の培養）
実施例１の（リンパ球の培養）と同一操作を行い、培養した細胞数量を確認したところ、培養開始後７日後の容器内の細胞数量は、８９×１０６ｃｅｌｌｓであった。比較で行った開放系のフラスコの培養方法では、５５×１０６ｃｅｌｌｓであった。
この実施例２により、抗ＣＤ３抗体を固相化処理した後の抗ＣＤ３抗体溶解液を除去する事で、細胞増殖性がさらに向上する事を確認した。 [Example 2]
The same operation was performed from (preparation of closed system container) to (immobilization of anti-CD3 antibody) in Example 1, and the following operation was performed.
(Cleaning inside the container)
After the treatment, the anti-CD3 antibody lysate that was not immobilized was extracted, and then washed twice with PBS (−).
(Lymphocyte culture)
When the same operation as in Example 1 (Lymphocyte culture) was performed and the number of cultured cells was confirmed, the number of cells in the container 7 days after the start of the culture was 89 × 10 6 cells. In the culture method of the open flask performed in comparison, it was 55 × 10 6 cells.
In Example 2, it was confirmed that the cell proliferation was further improved by removing the anti-CD3 antibody lysate after the anti-CD3 antibody was immobilized.

［実施例３］
次に、図２に示す抗ＣＤ３抗体封入容器２０と培地（ＫＢＭ５４０）が封入された培地用容器４０とを組合せたキット品を準備した。 [Example 3]
Next, a kit product was prepared by combining the anti-CD3 antibody-enclosed container 20 shown in FIG. 2 and a medium container 40 enclosing a medium (KBM540).

健常者の末梢血から分離した末梢血単核球と１％自己血清をシリンジに充填し、包材６９及びバリア性包材６５を脱離した後、抗ＣＤ３抗体封入容器２０のサンプリングポート１９から容器内部へ接種し、また、抗ＣＤ３抗体封入容器２０の閉塞部であるクリックチップ１８ａ及び培地用容器４０の閉塞部であるクリックチップ４７ａを連通した後、クレンメ６３を開口し、培地用容器４０内の培地を培養容器へ約５０ｃｃ移行した後、クレンメ６３を閉塞し、両容器２０、４０を連結させたまま、インキュベータ内で、５％炭酸ガス、３７℃、飽和湿度下で７日間培養を行った。   After filling the peripheral blood mononuclear cells and 1% autologous serum separated from the peripheral blood of a healthy person into the syringe and detaching the packaging material 69 and the barrier packaging material 65, from the sampling port 19 of the anti-CD3 antibody-sealed container 20 After inoculating the inside of the container and communicating the click chip 18a which is the closed part of the anti-CD3 antibody-sealed container 20 and the click chip 47a which is the closed part of the medium container 40, the clamp 63 is opened and the medium container 40 is opened. After about 50 cc of the medium was transferred to the culture vessel, the clamp 63 was closed, and the vessels 20 and 40 were connected, and the cells were cultured in an incubator for 7 days under 5% carbon dioxide, 37 ° C. and saturated humidity. went.

７日後、培地用容器４０を被覆しているバリア性包材６７を脱離した後、抗ＣＤ３抗体封入容器２０と培地用容器４０を閉塞しているクレンメ６３を開口し、抗ＣＤ３抗体封入容器２０内に封入されている培養して増殖した細胞を含む培養混合物全てを培地用容器４０へ移行させた後、再度クレンメ６３で閉塞し、インキュベータ内で、５％炭酸ガス、３７℃、飽和湿度下で数日間培養を続け、さらに、細胞を増殖させる事が出来た。   Seven days later, after removing the barrier packaging material 67 covering the medium container 40, the anti-CD3 antibody-enclosed container 20 and the clamp 63 closing the medium container 40 are opened, and the anti-CD3 antibody-enclosed container is opened. After all the culture mixture containing cultured and proliferated cells enclosed in 20 is transferred to the medium container 40, it is closed again with a clamp 63, and 5% carbon dioxide, 37 ° C., saturated humidity in the incubator. The culture was continued for several days under this condition, and the cells were able to grow.

［実施例４］
実施例３では、抗ＣＤ３抗体封入容器２０で行う初期の培養に必要な培地を、連結している培地用容器４０より一部移行させた初期培養を行うが、実施例４として、初期培養に必要な培地は、他で準備し、包材６９及びバリア性包材６５を脱離した後、末梢血単核球、１％自己血清、ＫＢＭ５４０の混合液５０ｍＬをシリンジで連結口２５から抗ＣＤ３抗体封入容器２０内へ接種した後、７日間培養後、培地用容器４０のバリア性包材６７を脱離し、培地用容器４０の連結ポート４７の閉塞部のクリックチップ４７ａを開口した。
抗ＣＤ３抗体封入容器２０内部の含む培養混合物全てを培地用容器４０へ移行させた後、クレンメ６３で閉塞し、インキュベータ内で、５％炭酸ガス、３７℃、飽和湿度下で数日間培養を続け、細胞を増殖させる事が出来た。
実施例３及び４では、抗ＣＤ３抗体により活性化した細胞を微生物等で汚染される事無く、無菌状態のまま、培地へ移行する事ができるだけでなく、培地用容器４０をバリア性フィルム６７で被覆する事により、抗ＣＤ３抗体封入容器２０内部で細胞培養を行っている期間の酸素による培地劣化を防止する事ができた。 [Example 4]
In Example 3, the initial culture was performed by partially transferring the medium necessary for the initial culture performed in the anti-CD3 antibody-enclosed container 20 from the connected medium container 40. The necessary medium is prepared elsewhere, and after detaching the packaging material 69 and the barrier packaging material 65, 50 mL of a mixture of peripheral blood mononuclear cells, 1% autologous serum, and KBM540 is anti-CD3 from the connection port 25 using a syringe. After inoculating into the antibody-sealed container 20 and culturing for 7 days, the barrier packaging material 67 of the medium container 40 was detached, and the click chip 47a in the closed portion of the connection port 47 of the medium container 40 was opened.
After all the culture mixture contained in the anti-CD3 antibody-enclosed container 20 was transferred to the medium container 40, the culture mixture was closed with a clamp 63, and the culture was continued in an incubator for several days under 5% carbon dioxide, 37 ° C. and saturated humidity. I was able to grow the cells.
In Examples 3 and 4, the cells activated by the anti-CD3 antibody can be transferred to the culture medium in a sterile state without being contaminated with microorganisms or the like. By covering, deterioration of the medium due to oxygen during the period of cell culture inside the anti-CD3 antibody-enclosed container 20 could be prevented.

本発明の実施の形態１の閉鎖系細胞培養容器としての抗ＣＤ３抗体封入容器を示し、（ａ）は正面図、（ｂ）は（ａ）のＡ−Ａ断面模式図である。The anti-CD3 antibody enclosure container as a closed-system cell culture container of Embodiment 1 of this invention is shown, (a) is a front view, (b) is an AA cross-sectional schematic diagram of (a). 本発明の実施の形態２の閉鎖系細胞培養用キットを示す正面図である。It is a front view which shows the closed system cell culture kit of Embodiment 2 of this invention.

符号の説明Explanation of symbols

１０ 容器本体
１１、１２ 平面状容器壁
１３ 収容部
１７、１８、１９ ポート
２０ 抗ＣＤ３抗体封入容器
３７ 連結チューブ
４０ 培地用容器

DESCRIPTION OF SYMBOLS 10 Container main body 11, 12 Planar container wall 13 Storage part 17, 18, 19 Port 20 Anti-CD3 antibody enclosure 37 Connection tube 40 Medium container

Claims (16)

少なくとも１つのポートを有するとともに、対向する平面状容器壁の少なくとも一方が可撓性フィルムからなり、前記対向する平面状容器壁の間に密封状の収容部を有する容器本体と、
前記収容部に封入され、前記収容部に導入される細胞の足場となるタンパク質及び／又は前記収容部に導入される細胞を活性化するタンパク質を含む補助収容物とを備え、
前記補助収容物が前記収容部に面状に存在することを特徴とする閉鎖系細胞培養容器。 A container main body having at least one port, at least one of the opposed planar container walls made of a flexible film, and having a sealed housing between the opposed planar container walls;
An auxiliary container containing a protein enclosed in the container and serving as a scaffold for cells introduced into the container and / or a protein that activates cells introduced into the container;
The closed cell culture container, wherein the auxiliary container is present in a planar shape in the container. 前記対向する平面状容器壁の双方が可撓性フィルムからなることを特徴とする請求項１に記載の閉鎖系細胞培養容器。   2. The closed cell culture container according to claim 1, wherein both of the opposing planar container walls are made of a flexible film. 面状に存在する前記補助収容物の平均厚さが１．０ｍｍ以下であることを特徴とする請求項１又は２に記載の閉鎖系細胞培養容器。   The closed-system cell culture container according to claim 1 or 2, wherein an average thickness of the auxiliary container existing in a planar shape is 1.0 mm or less. 前記補助収容物が、前記収容部に液密状に封入されていることを特徴とする請求項１乃至３のいずれか一に記載の閉鎖系細胞培養容器。   The closed cell culture container according to any one of claims 1 to 3, wherein the auxiliary container is sealed in a liquid-tight manner in the container. 前記補助収容物には、抗ＣＤ３抗体が含まれていることを特徴とする請求項１乃至４のいずれか一に記載の閉鎖系細胞培養容器。   The closed cell culture container according to any one of claims 1 to 4, wherein the auxiliary container contains an anti-CD3 antibody. 前記抗ＣＤ３抗体の少なくとも一部が、前記可撓性フィルムの内面に固相化されていることを特徴とする請求項５に記載の閉鎖系細胞培養容器。   The closed cell culture container according to claim 5, wherein at least a part of the anti-CD3 antibody is immobilized on the inner surface of the flexible film. 前記可撓性フィルムの内面に固相化されていない前記抗ＣＤ３抗体の非固相化分が前記容器本体から除去されていることを特徴とする請求項６に記載の閉鎖系細胞培養容器。   The closed cell culture vessel according to claim 6, wherein the non-solid phase component of the anti-CD3 antibody not immobilized on the inner surface of the flexible film is removed from the vessel body. 前記非固相化分は、前記容器本体に注入した洗浄液とともに排出されていることを特徴とする請求項７に記載の閉鎖系細胞培養容器。   The closed cell culture container according to claim 7, wherein the non-solid phase component is discharged together with the washing liquid injected into the container main body. 前記抗ＣＤ３抗体の封入量が、前記収容部を構成する前記可撓性フィルムの単位面積あたり、０．０２μｇ／ｃｍ２〜４．４μｇ／ｃｍ２であることを特徴とする請求項５乃至８のいずれか一に記載の閉鎖系細胞培養容器。 The enclosed amount of the anti-CD3 antibody, per unit area of the flexible film constituting the accommodating portion 5 through claim, characterized in that a 0.02μg / cm 2 ~4.4μg / cm 2 8 A closed cell culture vessel according to any one of the above. 前記抗ＣＤ３抗体の封入量が、前記収容部を構成する前記可撓性フィルムの単位面積あたり、０．１１μｇ／ｃｍ２〜０．４４μｇ／ｃｍ２であることを特徴とする請求項５乃至８のいずれか一に記載の閉鎖系細胞培養容器。 The enclosed amount of the anti-CD3 antibody, per unit area of the flexible film constituting the accommodating portion 5 through claim, characterized in that a 0.11μg / cm 2 ~0.44μg / cm 2 8 A closed cell culture vessel according to any one of the above. 前記可撓性フィルムの前記補助収容物と接する面が、ポリエチレンからなることを特徴とする請求項５乃至１０のいずれか一に記載の閉鎖系細胞培養容器。   The closed cell culture container according to any one of claims 5 to 10, wherein a surface of the flexible film that contacts the auxiliary container is made of polyethylene. 前記容器本体が、ガスバリアー性及び／又は水蒸気バリアー性の高い包装材で包装されたことを特徴とする請求項１乃至１１のいずれか一に記載の閉鎖系細胞培養容器。   The closed cell culture container according to any one of claims 1 to 11, wherein the container body is packaged with a packaging material having high gas barrier properties and / or water vapor barrier properties. 前記請求項１乃至１２のいずれか一に記載の閉鎖系細胞培養容器と、細胞培養用培地を封入した培地用容器とを備え、前記細胞培養容器と前記培地用容器とが使用時に連通可能な連通手段で接続されていることを特徴とする閉鎖系細胞培養用キット。   A closed-system cell culture container according to any one of claims 1 to 12 and a medium container enclosing a cell culture medium, wherein the cell culture container and the medium container can communicate with each other during use. A closed cell culture kit characterized by being connected by a communication means. 前記閉鎖系細胞培養容器と前記培地用容器とが、ガスバリアー性及び／又は水蒸気バリアー性の高い包装材で包装されたことを特徴とする請求項１３に記載の閉鎖系細胞培養用キット。   14. The closed system cell culture kit according to claim 13, wherein the closed system cell culture container and the culture medium container are packaged with a packaging material having a high gas barrier property and / or a water vapor barrier property. 少なくとも１つのポートを有するとともに、対向する平面状容器壁の少なくとも一方が可撓性フィルムからなり、前記対向する平面状容器壁の間に密封状の収容部を有する容器本体を準備する工程と、
前記収容部に導入される細胞の足場となるタンパク質及び／又は前記収容部に導入される細胞を活性化するタンパク質を含む補助収容物を前記収容部に封入する工程と、
前記対向する平面状容器壁を外側から厚さ方向に押圧し、及び／又は前記収容部内の気体を吸引して前記収容部を平坦化することにより、前記補助収容物を前記対向する平面状容器壁の少なくとも一方の内面に面状にする工程と、
を備えたことを特徴とする閉鎖系細胞培養容器の製造方法。 Preparing a container body having at least one port, at least one of the opposing planar container walls made of a flexible film, and having a sealed container between the opposed planar container walls;
A step of enclosing an auxiliary container containing a protein serving as a scaffold for cells introduced into the container and / or a protein that activates cells introduced into the container into the container;
By pressing the opposing planar container wall in the thickness direction from the outside and / or sucking the gas in the accommodating part to flatten the accommodating part, the auxiliary container is made to face the opposing container. Forming a surface on at least one inner surface of the wall;
A method for producing a closed cell culture vessel. 少なくとも１つのポートを有するとともに、対向する平面状容器壁の少なくとも一方が可撓性フィルムからなり、前記対向する平面状容器壁の間に密封状の収容部を有する容器本体を準備する工程と、
前記収容部に抗ＣＤ３抗体を含む補助収容物を前記収容部に封入する工程と、
前記対向する平面状容器壁を外側から厚さ方向に押圧し、及び／又は前記収容部内の気体を吸引して前記収容部を平坦化することにより、前記補助収容物を前記対向する平面状容器壁の少なくとも一方の内面に面状にすると共に前記抗ＣＤ３抗体を固相化する工程と、
前記収容部に洗浄液を注入し、前記補助収容物に接触させて排出することにより、該洗浄液と共に前記抗ＣＤ３抗体の非固相化分を排出する工程と
を備えたことを特徴とする閉鎖系細胞培養容器の製造方法。
Preparing a container body having at least one port, at least one of the opposing planar container walls made of a flexible film, and having a sealed container between the opposed planar container walls;
Enclosing an auxiliary container containing an anti-CD3 antibody in the container in the container;
By pressing the opposing planar container wall in the thickness direction from the outside and / or sucking the gas in the accommodating part to flatten the accommodating part, the auxiliary container is made to face the opposing container. Forming a surface on at least one inner surface of the wall and immobilizing the anti-CD3 antibody;
And a step of discharging a non-solid phase of the anti-CD3 antibody together with the cleaning liquid by injecting the cleaning liquid into the storage section and discharging it in contact with the auxiliary storage. A method for producing a cell culture container.
JP2005380115A 2005-12-28 2005-12-28 Closed system container for cell culture, kit for closed system cell culture and method for producing closed system container for cell culture Pending JP2007175028A (en)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013114845A1 (en) * 2012-02-01 2013-08-08 東洋製罐グループホールディングス株式会社 Cell culture kit, and method of using cell culture kit
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WO2024009678A1 (en) * 2022-07-07 2024-01-11 東洋製罐グループホールディングス株式会社 Cell culture system, and cell transport method

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03172169A (en) * 1989-11-30 1991-07-25 Koojin Bio Kk Vessel for storage and tissue culture
JP2004514432A (en) * 2000-11-21 2004-05-20 バイオクリスタル・リミテッド Cell culture device and method of use
JP2005095165A (en) * 2003-08-26 2005-04-14 Medeinetto:Kk Culture vessel, culture apparatus, and method for culturing cell
JP2005287425A (en) * 2004-03-31 2005-10-20 Koojin Bio Kk Culture bag with culture medium bag

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03172169A (en) * 1989-11-30 1991-07-25 Koojin Bio Kk Vessel for storage and tissue culture
JP2004514432A (en) * 2000-11-21 2004-05-20 バイオクリスタル・リミテッド Cell culture device and method of use
JP2005095165A (en) * 2003-08-26 2005-04-14 Medeinetto:Kk Culture vessel, culture apparatus, and method for culturing cell
JP2005287425A (en) * 2004-03-31 2005-10-20 Koojin Bio Kk Culture bag with culture medium bag

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