JP4802875B2 - Cell culture container and cell transfer method - Google Patents

Cell culture container and cell transfer method Download PDF

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JP4802875B2
JP4802875B2 JP2006162489A JP2006162489A JP4802875B2 JP 4802875 B2 JP4802875 B2 JP 4802875B2 JP 2006162489 A JP2006162489 A JP 2006162489A JP 2006162489 A JP2006162489 A JP 2006162489A JP 4802875 B2 JP4802875 B2 JP 4802875B2
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義洋 吉川
直樹 児玉
桂 松尾
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Description

本発明は、閉鎖的に液体連通を可能とする接続部材を含む細胞培養容器に関する。   The present invention relates to a cell culture container including a connection member that enables fluid communication in a closed manner.

従来、哺乳動物の付着細胞や懸濁細胞を生体外で行う培養は、ポリスチレン製容器の中で行われてきた。しかし、空気との接触面積が小さいために培地への通気が十分ではなく、常時培地を交換する必要があり、培地を浪費していた。さらに、培地を交換する際には、雑菌の混入が問題となる。   Conventionally, culturing of mammalian adherent cells and suspension cells in vitro has been performed in polystyrene containers. However, since the contact area with air is small, ventilation to the culture medium is not sufficient, and it is necessary to constantly replace the culture medium, which wastes the culture medium. Furthermore, contamination of germs becomes a problem when the medium is changed.

かかる問題を解決すべく、気体透過性を有するアイオノマー樹脂からなる細胞培養容器が開示されている（特許文献１）。また、エチレンビニルアルコールを主成分とするフィルムからなる細胞培養容器が開示されている（特許文献２）。これらの培養容器には予め使用する培地を容器内に充填し、それをガスバリア性のフィルムで包装した培地保存容器と培養容器を兼用できる形態も考案されている（特許文献３）。   In order to solve this problem, a cell culture container made of ionomer resin having gas permeability is disclosed (Patent Document 1). In addition, a cell culture vessel made of a film containing ethylene vinyl alcohol as a main component is disclosed (Patent Document 2). These culture containers have been devised in such a manner that a culture medium used in advance can be used as a culture medium storage container in which a culture medium to be used is filled in the container and wrapped with a gas barrier film (Patent Document 3).

これらの細胞培養容器を用いる培養においては、一定量まで細胞が増殖すると酸素や栄養源の消費が多くなるため、思うように細胞が増殖しなくなる。したがって、別途培地を充填した細胞培養容器を用意し、それぞれの細胞培養容器に増殖した細胞を２つに分割する作業を行う（スプリット作業）。このスプリット作業は、例えば、図５又は特許文献４に示すような両端にプラスチック針を具備する連結チューブを用いて行う。具体的には、連結チューブの両端に具備するそれぞれのプラスチック針を、それぞれの細胞培養容器に具備する膜チューブに接続して行う。プラスチック針を用いる理由としては、金属針と比較して安全であり、且つ内腔の太さが当該金属針の内腔よりも太いためであり、針刺し事故防止と、スプリット作業を迅速に行うことができるためである。   In the culture using these cell culture vessels, if the cells grow to a certain amount, the consumption of oxygen and nutrient sources increases, so that the cells do not grow as expected. Therefore, a cell culture container filled with a separate medium is prepared, and an operation of dividing the cells grown in each cell culture container into two is performed (split operation). This split operation is performed using, for example, a connecting tube having plastic needles at both ends as shown in FIG. Specifically, each of the plastic needles provided at both ends of the connection tube is connected to the membrane tube provided in each cell culture container. The reason for using a plastic needle is that it is safer than a metal needle and the thickness of the lumen is thicker than the lumen of the metal needle. It is because it can do.

スプリット作業後においては、連結チューブを熱シールし、２バッグを切り離して培養するが、バッグに接続した連結チューブのプラスチック針を取り外すことができない。何故ならば、内腔の薄肉の隔壁が破壊されているため、細胞培養容器はもはや密閉状態とならないためである。そのため、万が一連結チューブのプラスチック針が、膜チューブから外れてしまえば、バッグから培養液が漏れだして、バッグだけでなくインキュベーターなどの培養環境を汚染してしまう。またスプリット作業の後、プラスチック針が刺さったままであるため、次のスプリット操作や細胞を回収するときには、そのポートを繰り返し使用できず、他のポートを使用しなくてはならない。そのため、全てのスプリット作業や回収作業数量のポートが必要となり、バッグの形状や連結チューブの形状が複雑なものとなり、コスト高を招き、培養作業も煩雑であった。   After the split operation, the connecting tube is heat-sealed and the two bags are separated and cultured, but the plastic needle of the connecting tube connected to the bag cannot be removed. This is because the thin-walled septum of the lumen is destroyed, and the cell culture container is no longer sealed. For this reason, if the plastic needle of the connection tube is detached from the membrane tube, the culture solution leaks from the bag and contaminates not only the bag but also the culture environment such as an incubator. In addition, since the plastic needle remains stuck after the split operation, the port cannot be used repeatedly when the next split operation or cell is collected, and another port must be used. Therefore, it is necessary to provide ports for all the split operations and collection operations, and the shape of the bag and the shape of the connecting tube become complicated, resulting in high costs and complicated culture operations.

一方、輸液等で針なし接続の機構をもった混注ポートをもつもものある。例えばシリンジ先端でポート口にあるシリコンゴムを押圧すると流入口が開いたりする機構である。これらを培養バッグに適用すれば、スプリット作業の煩雑性は一見解消できるように考えられる。ところが、これらは圧力によって開閉するため、リークする可能性が高い。培養期間中は、バッグ全体に圧力のかかることはまれであるため、リークの危険性は低い。しかし、培養バッグに予め培養液を充填し保管する場合、保管庫にバッグを積み重ねたりして、バッグに予想以上の圧力がかかることが考えられる。また場合によっては培地が充填されたバッグを異なる施設に搬送することもあり得る。この時、搬送による振動や梱包の形態によってはバッグに圧力がかかることが予想される。このような場合、単なる圧力による開閉機構や逆止弁的なポートは、リークする可能性が極めて高い。従って、これらのポートを培養バッグ等のように液が充填されるような容器に適用するのは容易ではない。   On the other hand, some have a mixed injection port with a needleless connection mechanism for infusion. For example, it is a mechanism in which the inflow port opens when silicone rubber in a port port is pressed at the tip of a syringe. If these are applied to the culture bag, it seems that the complexity of the split operation can be eliminated at a glance. However, since these open and close by pressure, there is a high possibility of leakage. During the incubation period, it is rare that pressure is applied to the entire bag, so the risk of leakage is low. However, when the culture bag is filled with a culture solution in advance and stored, the bag may be stacked in a storage and a pressure higher than expected may be applied to the bag. In some cases, a bag filled with a culture medium may be transported to a different facility. At this time, it is expected that pressure is applied to the bag depending on the vibration caused by the conveyance and the form of packing. In such a case, an opening / closing mechanism based on mere pressure or a check valve port is very likely to leak. Therefore, it is not easy to apply these ports to containers filled with liquid such as culture bags.

また、細胞培養が終了した後は、細胞培養容器はそのまま破棄していたが、膜チューブの隔壁が破壊されている以上、細胞培養容器内に残存した細胞によりバイオハザードが起こるおそれがある。   Further, after the cell culture is completed, the cell culture container is discarded as it is. However, as long as the partition of the membrane tube is destroyed, biohazard may occur due to the cells remaining in the cell culture container.

特開昭６０−１６０８８１号公報JP 60-160881 A 特開２００５−２８７４２５号公報JP 2005-287425 A 特公平０６−０９７９８６号Japanese Patent Publication No. 06-097986 特開平０７−０４７１０５号公報Japanese Patent Application Laid-Open No. 07-047105

本発明は、培養液を予め充填しても、ポートからのリークがなく、かつスプリット作業後における細胞培養が容易であり、細胞培養終了後におけるバイオハザードのおそれがない細胞培養容器を提供することを目的とする。   The present invention provides a cell culture container that does not leak from a port even if it is pre-filled with a culture solution, and that cell culture after splitting work is easy and that there is no risk of biohazard after cell culture is completed. With the goal.

本発明は、
［１］ 閉鎖的に液体連通を可能とする接続部材を具備する細胞培養容器であって、
前記細胞培養容器は、接続部材を具備し、
前記接続部材は、
弾性部材からなり、中心部に上下方向に貫通したスリットを設けた栓体と、
前記栓体に周設し、前記栓体のスリットを圧縮密閉する圧縮部材と、
前記圧縮部材から下方に延出し、内腔が前記細胞培養容器及び前記圧縮部材の内部と連通し、前記内腔に薄肉の隔壁を設けた管状部材
を備えたものである細胞培養容器、
［２］ 前記容器が、バッグである［１］に記載の細胞培養容器、
［３］ 前記接続部材が、前記バッグのポートとして具備するものである［１］に記載の細胞培養容器、
［４］ さらに、前記圧縮部材に前記栓体天面を覆うキャップ部材を付設した［１］に記載の細胞培養容器、
［５］ ２つの細胞培養容器の液体連通を可能とするキットであって、
前記キットは、２つの細胞培養容器と、連結デバイスを含み、
前記細胞培養容器は、接続部材を具備するものであり、
前記接続部材は、
弾性部材からなり、中心部に上下方向に貫通したスリットを設けた栓体と、
前記栓体に周設し、前記栓体のスリットを圧縮密閉する圧縮部材と、
前記圧縮部材から下方向に延出し、内腔が前記細胞培養容器本体内部及び前記圧縮部材の内部と連通し、前記内腔に薄肉の隔壁を設けることにより前記圧縮部材の内部と前記細胞培養とを区画する管状部材を備え、
前記連結デバイスは、
チューブと、
前記チューブの両端に接続したプラスチック針
を備えたものである
細胞培養キット、
［６］ 前記容器が、バッグである［５］に記載の細胞培養キット、
［７］ 前記接続部材が、前記バッグのポートとして具備するものである［５］に記載の細胞培養キット、
［８］ さらに、前記圧縮部材に前記栓体天面を覆うキャップ部材を付設した［５］に記載の細胞培養キット、及び、
［９］ 第１の細胞培養容器で培養された細胞を、第２の細胞培養容器に移す方法であって、
第１の細胞培養容器は、培養された細胞と、当該細胞を懸濁する培地が収容されたものであり、
それぞれの細胞培養容器は、接続部材を具備するものであり、
前記接続部材は、
弾性部材からなり、中心部に上下方向に貫通したスリットを設けた栓体と、
前記栓体に周設し、前記栓体のスリットを圧縮密閉する圧縮部材と、
前記圧縮部材から下方向に延出し、内腔が閉鎖系容器本体内部と前記圧縮部材の内部とを液密に連通し、前記内腔に薄肉の隔壁を設けることにより前記圧縮部材の内部と前記細胞培養とを区画する管状部材を備えたものであって、
１）両端にプラスチック針を具備するチューブを備えた連結デバイスであって、当該デバイスのプラスチック針をそれぞれの細胞培養容器の接続部材のスリットに差し込むことで、第１の細胞培養容器と第２の細胞培養容器とを液体的に流通するステップと、
２）第１の細胞培養容器に収容した細胞の全容量のうち少なくとも一部を第２の細胞培養容器に移注するステップ
を含む細胞移注方法に関する。 The present invention
[1] A cell culture container provided with a connection member that enables liquid communication in a closed manner,
The cell culture container comprises a connecting member,
The connecting member is
A plug body made of an elastic member and provided with a slit penetrating in the vertical direction in the center,
A compression member that is provided around the plug body and compresses and seals the slit of the plug body;
A cell culture container that includes a tubular member that extends downward from the compression member, communicates with the inside of the cell culture container and the compression member, and has a thin partition wall in the lumen;
[2] The cell culture container according to [1], wherein the container is a bag,
[3] The cell culture container according to [1], wherein the connection member is provided as a port of the bag.
[4] The cell culture container according to [1], wherein a cap member that covers the top surface of the stopper is additionally provided on the compression member.
[5] A kit that enables fluid communication between two cell culture containers,
The kit includes two cell culture containers and a coupling device,
The cell culture container comprises a connecting member,
The connecting member is
A plug body made of an elastic member and provided with a slit penetrating in the vertical direction in the center,
A compression member that is provided around the plug body and compresses and seals the slit of the plug body;
Extending downward from the compression member, a lumen communicates with the inside of the cell culture container main body and the compression member, and by providing a thin partition wall in the lumen, the inside of the compression member and the cell culture A tubular member that divides
The coupling device is:
Tubes,
A cell culture kit comprising a plastic needle connected to both ends of the tube;
[6] The cell culture kit according to [5], wherein the container is a bag,
[7] The cell culture kit according to [5], wherein the connection member is provided as a port of the bag,
[8] The cell culture kit according to [5], wherein a cap member that covers the top surface of the stopper is attached to the compression member;
[9] A method for transferring cells cultured in a first cell culture vessel to a second cell culture vessel,
The first cell culture container contains cultured cells and a medium for suspending the cells,
Each cell culture vessel comprises a connecting member,
The connecting member is
A plug body made of an elastic member and provided with a slit penetrating in the vertical direction in the center,
A compression member that is provided around the plug body and compresses and seals the slit of the plug body;
Extending downward from the compression member, a lumen communicates the inside of the closed system container body and the compression member in a fluid-tight manner, and a thin-walled partition is provided in the lumen, thereby providing an inside of the compression member and the A tubular member for partitioning cell culture,
1) A connecting device having a tube having plastic needles at both ends, wherein the plastic needle of the device is inserted into the slit of the connection member of each cell culture container, so that the first cell culture container and the second cell A liquid flow through the cell culture vessel;
2) The present invention relates to a cell transfer method including a step of transferring at least a part of the total volume of cells accommodated in a first cell culture container to a second cell culture container.

本発明の細胞培養容器によれば、培養液を予め充填しても、ポートからのリークがなく、かつスプリット作業後における分割された細胞培養系を独立して取り扱うことができるため、作業非常に簡便となる。また、細胞培養終了後におけるバイオハザードのおそれがない。   According to the cell culture container of the present invention, there is no leak from the port even if the culture solution is filled in advance, and the divided cell culture system after the split operation can be handled independently, It becomes simple. In addition, there is no risk of biohazard after cell culture.

以下、本発明について図面を用いて説明する。   The present invention will be described below with reference to the drawings.

図１は本発明の細胞培養容器を示す図である。本発明の細胞培養容器とは、容器本体１１内部と閉鎖的に液体連通を可能とする接続部材２を具備する容器をいう。容器本体の形状は、バッグ及びボトルなどが挙げられるが、量産が容易であり、軽量である観点からバッグであることが好ましい。   FIG. 1 is a view showing a cell culture container of the present invention. The cell culture container of the present invention refers to a container provided with a connecting member 2 that enables closed liquid communication with the inside of the container body 11. Examples of the shape of the container body include a bag and a bottle, but a bag is preferable from the viewpoint of easy mass production and light weight.

バッグの内容量は、細胞の培養における使用される培地の量に応じて当業者が適宜設定することができるため、特に限定されるものではないが、取り扱うのに適度な大きさである観点から、約１０〜２，０００ｍｌ、好ましくは約５０〜１，０００ｍｌである。   The content of the bag is not particularly limited because it can be appropriately set by a person skilled in the art depending on the amount of medium used in cell culture, but from the viewpoint of an appropriate size for handling. , About 10 to 2,000 ml, preferably about 50 to 1,000 ml.

バッグを構成する可撓性フィルムは、細胞培養時における細胞の代謝活動を補助するために、安定したガス交換を行うことができる通気性材料であれば、特に限定されるものではない。通気性材料は、温度２５℃における酸素の透過係数が約１００〜５，０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍ、好ましくは約１，１００〜３，０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍ、さらに好ましくは約１，２５０〜２，７５０ｃｍ３／ｍ２・２４ｈｒ・ａｔｍであればよい。又は、二酸化炭素透過係数が約１００１〜２０，０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍ、好ましくは約５，０００〜９，０００ｃｍ３／ｍ２・２４ｈｒ・ａｔｍであればよい。これらの要件を満足する適切な材料としては、ポリエチレン、ポリ塩化ビニル、ポリ（エチレン−ビニルアセテート）コポリマー、ポリ（エチレン−エチルアクリレート）コポリマー、ポリ（エチレン−メタアクリレート）コポリマーなどが挙げられる。これらの材料の中でも、工業的な製造が容易であり、ガンマ線滅菌に耐えうるものであり、且つ内部の培地の様子を観察することができる程度の透明性を有する観点からポリエチレンが好ましいが、本発明はこれらに限定されるものではない。 The flexible film constituting the bag is not particularly limited as long as it is a breathable material capable of performing stable gas exchange in order to assist the metabolic activity of cells during cell culture. The breathable material has an oxygen permeability coefficient at a temperature of 25 ° C. of about 100 to 5,000 cm 3 / m 2 · 24 hr · atm, preferably about 1,100 to 3,000 cm 3 / m 2 · 24 hr · atm, more preferably May be about 1,250-2,750 cm 3 / m 2 · 24 hr · atm. Alternatively, the carbon dioxide permeability coefficient may be about 1001 to 20,000 cm 3 / m 2 · 24 hr · atm, preferably about 5,000 to 9,000 cm 3 / m 2 · 24 hr · atm. Suitable materials that meet these requirements include polyethylene, polyvinyl chloride, poly (ethylene-vinyl acetate) copolymers, poly (ethylene-ethyl acrylate) copolymers, poly (ethylene-methacrylate) copolymers, and the like. Among these materials, polyethylene is preferable from the viewpoint that it is easy to manufacture industrially, can withstand gamma sterilization, and has a transparency that allows the state of the internal medium to be observed. The invention is not limited to these examples.

本発明の細胞培養容器１は、接続部材２を含む。接続部材２は、容器本体１に具備するポートから延出するチューブの先端に備えてもよいし、図１に示すように当該接続部材２自身を細胞培養容器１のポートとして備えてもよい。特に、細胞培養容器１を構成する材料が減り、原価を抑えることができる観点から、図１に示すように接続部材２自身を細胞培養容器１のポートとして備えることが好ましい。   The cell culture container 1 of the present invention includes a connection member 2. The connection member 2 may be provided at the tip of a tube extending from the port provided in the container body 1, or the connection member 2 itself may be provided as a port of the cell culture container 1 as shown in FIG. 1. In particular, it is preferable to provide the connection member 2 itself as a port of the cell culture container 1 as shown in FIG. 1 from the viewpoint of reducing the material constituting the cell culture container 1 and reducing the cost.

本発明の接続部材とは、閉鎖的に液体連通を可能とする部材をいう。その構成は、栓体２１と、栓体２１に周設した圧縮部材２２と、圧縮部材２２から下方に延出した管状部材２３を備える。図２は接続部材の拡大断面図を示す図である。   The connection member of the present invention refers to a member that enables liquid communication in a closed manner. The configuration includes a plug body 21, a compression member 22 provided around the plug body 21, and a tubular member 23 extending downward from the compression member 22. FIG. 2 is an enlarged cross-sectional view of the connection member.

栓体２１は、弾性部材からなる一般的なゴム栓をいうが、中心部には上下方向に貫通したスリット２１１が設けられている。   The plug body 21 is a general rubber plug made of an elastic member, and a slit 211 penetrating in the vertical direction is provided at the center.

スリット２１１は、いわゆるプラスチック針３２が刺通するために具備する部分である（図４）。スリット２１１は、後述する圧縮部材２により圧縮密閉されているが、プラスチック針３２を圧入することにより、容易に開くことができる。また、当該プラスチック針３２を引き抜けば、栓体２１自身の弾性力により元の形状に復元し、再びスリット２１１は圧縮密閉される。   The slit 211 is a part provided for the so-called plastic needle 32 to be pierced (FIG. 4). The slit 211 is compressed and sealed by a compression member 2 described later, but can be easily opened by press-fitting the plastic needle 32. When the plastic needle 32 is pulled out, the original shape is restored by the elastic force of the plug 21 itself, and the slit 211 is compressed and sealed again.

したがって、栓体２１を構成する材料は、自身の形状が復元できる程度の弾性力を有するものが選択される。特に、ゴム又はエラストマーが選択される。例えば、ゴムの場合、ブタジエンゴム、スチレンブタジエンゴム、イソプレンゴム、エチレンプロピレンゴム、ブチルゴム、クロロプレンゴム、ニトリルゴム、アクリルゴム、ウレタンゴム、シリコンゴム及び天然ゴムなどが使用できる。特に、プラスチック針の刺通における摺動性がよく、プラスチック針の抜き差しによりスリット２１１からの裂けが生じない観点から、イソプレンゴム、シリコンゴム及び天然ゴムが好ましいが本発明はこれに限定されるものではない。   Therefore, the material constituting the plug body 21 is selected to have an elastic force that can restore its own shape. In particular, rubber or elastomer is selected. For example, in the case of rubber, butadiene rubber, styrene butadiene rubber, isoprene rubber, ethylene propylene rubber, butyl rubber, chloroprene rubber, nitrile rubber, acrylic rubber, urethane rubber, silicon rubber and natural rubber can be used. In particular, isoprene rubber, silicone rubber, and natural rubber are preferable from the viewpoint of good sliding performance in the insertion of a plastic needle and no tearing from the slit 211 due to insertion and removal of the plastic needle, but the present invention is limited to this. is not.

ここで、栓体２１にスリット２１１を設ければ、プラスチック針３２などの針先が鈍い針を刺通することはできない。また、スリット２１１を設けない栓体を用意し、当該栓体を金属針などで穿刺してスプリットを行うことも考えられるが、金属針の内腔は太くて０．９ｍｍ程度（いわゆる１８ゲージの金属針）であり、十分な流量を得ることができない。さらに金属針は、針刺し事故が起こる原因となる。その上、内腔の太い金属針でスリットを具備しない栓体を刺通すると、コアリングが発生し、ゴム片が培地に混入してしまう。コアリングが発生した栓体は、もはや細胞培養容器１を密封することができない。   Here, if the stopper 211 is provided with the slit 211, a needle having a blunt needle such as the plastic needle 32 cannot be pierced. It is also conceivable to prepare a plug body without slits 211 and puncture the plug body with a metal needle or the like to perform splitting. However, the lumen of the metal needle is thick and about 0.9 mm (so-called 18 gauge Metal needle), and a sufficient flow rate cannot be obtained. Furthermore, metal needles can cause needle stick accidents. In addition, when a plug that does not have a slit is pierced with a metal needle having a thick lumen, coring occurs and rubber pieces are mixed into the medium. The plug body in which the coring has occurred can no longer seal the cell culture container 1.

スリット２１１の幅は、後述する圧縮部材２２により圧縮密閉可能である程度の幅であれば特に限定されるものではない。但し、必要以上に太くする必要性はなく、例えば、刃物などにより切れ込んだ程度であれば十分である。この場合におけるスリット２１１の幅は限りなく０に近いと言える。   The width of the slit 211 is not particularly limited as long as it can be compressed and sealed by the compression member 22 described later. However, it is not necessary to make it thicker than necessary. For example, it is sufficient if it is cut by a blade or the like. In this case, it can be said that the width of the slit 211 is as close to 0 as possible.

さらに、スリット２１１の形状も、後述する圧縮部材２２により圧縮密閉可能であれば、特に限定されるものではない。但し、必要以上に特殊な形状にする必要はなく、例えば、刃物などにより切れ込みにより生じる一文字形状又は十文字形状であれば十分である。   Further, the shape of the slit 211 is not particularly limited as long as it can be compressed and sealed by the compression member 22 described later. However, it is not necessary to use a special shape more than necessary. For example, a one-letter shape or a ten-letter shape generated by cutting with a blade or the like is sufficient.

例えば、スリット２１１の形状が一文字形状又は十文字形状である場合、その長さは、プラスチック針３２が刺通できる程度の長さであれば特に限定されるものではない。例えば、３〜５ｍｍ、好ましくは３．５〜４．５ｍｍである。   For example, when the shape of the slit 211 is a single character shape or a cross shape, the length is not particularly limited as long as the plastic needle 32 can be pierced. For example, it is 3 to 5 mm, preferably 3.5 to 4.5 mm.

また、栓体２１の水平断面形状は、当該栓体２１にて生じる圧を均等にする観点から通常は円形であるが、本発明はこれらに限定されるものではない。栓体２１の形状が円系である場合、その外径はプラスチック針３２が刺通できる程度の大きさであればよく、約３〜５ｍｍ、好ましくは約３．５〜４．５ｍｍである。   The horizontal cross-sectional shape of the plug body 21 is usually circular from the viewpoint of equalizing the pressure generated in the plug body 21, but the present invention is not limited to these. When the shape of the plug 21 is a circular system, the outer diameter may be a size that allows the plastic needle 32 to be pierced, and is about 3 to 5 mm, preferably about 3.5 to 4.5 mm.

上述した栓体２１には、圧縮部材２２が周設される。本発明の圧縮部材とは、栓体２１に周設し、栓体２１の水平面方向において締付圧を発生させ、当該栓体２１のスリット２１１を圧縮密閉するものをいう。栓体２１のスリット２１１の圧縮密閉は、例えば、栓体２１の水平断面形状よりも若干小さめの断面形状を有する部材を用意し、当該部材に栓体２１を圧入すれば、栓体２１の水平面方向において締付圧が発生し、達成される。より詳細には、例えば、栓体２１の水平断面形状が円形である場合は、当該円より若干径の小さい円筒状の部材に、当該栓体２１を圧入すれば、中心方向に向かって締付圧が発生し、栓体２１のスリット２１１を圧縮密閉する。例えば、栓体２１の形状が円形である場合は、当該栓体２１を周設する圧縮部材２２の内径は、当該栓体の外径よりも約０〜１ｍｍ、好ましくは約０〜０．５ｍｍ小さい部材を用いることができるが、これに限定するものではない。この締付圧により、内部の培地が外部に漏れることがない。よって、培養収容後も安全に破棄することができる。   A compression member 22 is provided around the plug body 21 described above. The compression member of the present invention refers to a member that is provided around the plug body 21, generates a tightening pressure in the horizontal plane direction of the plug body 21, and compresses and seals the slit 211 of the plug body 21. For compression sealing of the slit 211 of the plug body 21, for example, a member having a cross-sectional shape slightly smaller than the horizontal cross-sectional shape of the plug body 21 is prepared, and the plug body 21 is press-fitted into the member. A clamping pressure is generated in the direction and is achieved. More specifically, for example, when the horizontal cross-sectional shape of the plug body 21 is circular, if the plug body 21 is press-fitted into a cylindrical member having a diameter slightly smaller than the circle, the plug body 21 is tightened toward the center. A pressure is generated, and the slit 211 of the plug body 21 is compressed and sealed. For example, when the shape of the plug body 21 is circular, the inner diameter of the compression member 22 that surrounds the plug body 21 is about 0 to 1 mm, preferably about 0 to 0.5 mm, than the outer diameter of the plug body. Although a small member can be used, it is not limited to this. This tightening pressure prevents the internal culture medium from leaking outside. Therefore, it can be safely discarded after the culture is accommodated.

圧縮部材２２の素材は、栓体２１に常に締付圧を発生させる程度の形状維持能力を有する材料であれば特に限定されるものではない。例えば、ポリプロピレン、ポリカーボネート、ポリテトラフロオロエチレン、ポリアクリルアミド及びポリエチレンテレフタレート等のプラスチックや、ガラス並びに金属類などが挙げられる。特に安価かつ軽量であるポリプロピレン及びポリカーボネートが好ましい。   The material of the compression member 22 is not particularly limited as long as it is a material having a shape maintaining ability that always generates a tightening pressure in the plug body 21. Examples thereof include plastics such as polypropylene, polycarbonate, polytetrafluoroethylene, polyacrylamide and polyethylene terephthalate, glass and metals. In particular, polypropylene and polycarbonate that are inexpensive and lightweight are preferred.

また、図２に示すように圧縮部材２２に栓体２１天面を覆うキャップ部材２２１を付設することが好ましい。キャップ部材２２１により、栓体２１が圧縮部材２２から外れることがなく、さらに、二重に封をすることができ、培養における汚染（コンタミ）のリスクを低減することができる。その素材は、圧縮部材２２と同様のものが使用される。   Moreover, it is preferable to attach the cap member 221 which covers the stopper 21 top surface to the compression member 22 as shown in FIG. The cap member 221 prevents the plug body 21 from being detached from the compression member 22 and can be sealed twice, thereby reducing the risk of contamination (contamination) in culture. The same material as the compression member 22 is used.

そして、上述した圧縮部材２２の下方には管状部材２３が延出して備える。本発明の管状部材とは、内腔が細胞培養容器１及び圧縮部材２２の内部と液密に連通するものをいう。環状部材３の形状は、円柱形状、四角柱形状及び三角柱形状など特に限定されるものではない。また、管状部材２３は、いわゆるチューブ状であってもよいし、ポート状であってもよい。   A tubular member 23 is provided below the compression member 22 described above. The tubular member of the present invention refers to a member whose lumen communicates with the inside of the cell culture container 1 and the compression member 22 in a liquid-tight manner. The shape of the annular member 3 is not particularly limited, such as a cylindrical shape, a quadrangular prism shape, or a triangular prism shape. The tubular member 23 may be a so-called tube shape or a port shape.

圧縮部材２２と管状部材２３との接続は、液体が連通する形態であれば特に限定されるものではない。例えば、図２では管状部材２３の内腔に圧縮部材２２から下方向に延出した部材を挿入する形態をとる。一方、管状部材２３をポート状として扱う場合は、図３に示すように圧縮部材２２と管状部材２３を一体で成型してもよい。   The connection between the compression member 22 and the tubular member 23 is not particularly limited as long as the liquid communicates therewith. For example, FIG. 2 takes a form in which a member extending downward from the compression member 22 is inserted into the lumen of the tubular member 23. On the other hand, when the tubular member 23 is handled as a port shape, the compression member 22 and the tubular member 23 may be integrally molded as shown in FIG.

管状部材２３の素材は、当該管状部材２３をチューブ状として用いる場合は、チューブに使用される材料を、バッグのポート状として用いる場合は、ポートに使用される材料をそれぞれ選択することができる。チューブに使用される材料とは、例えば、ポリ塩化ビニル、ポリエチレン、ポリプロピレン及びエチレン酢酸ビニル共重合体等が挙げられる。ポートに使用される材料は、例えば、ポリエチレン、ポリプロピレン、ポリカーボネート、ポリテトラフロオロエチレン、ポリアクリルアミド及びポリエチレンテレフタレート等のプラスチックや、ガラス並びに金属類などが挙げられる。特に安価かつ軽量であるポリエチレン及びポリプロピレンが好ましい。   When the tubular member 23 is used as a tube shape, the material used for the tube can be selected from the material used for the tube. When the tubular member 23 is used as the port shape of the bag, the material used for the port can be selected. Examples of the material used for the tube include polyvinyl chloride, polyethylene, polypropylene, and ethylene vinyl acetate copolymer. Examples of the material used for the port include plastics such as polyethylene, polypropylene, polycarbonate, polytetrafluoroethylene, polyacrylamide, and polyethylene terephthalate, glass, and metals. Particularly preferred are polyethylene and polypropylene which are inexpensive and lightweight.

また、管状部材２３には、細胞を培養する培地を収容する細胞培養容器１の搬送及び使用前における保存の際、栓体２１のスリット２１１からの微量な培地の漏出（スローリーク）を防止するために、内腔に破壊可能な隔壁２３１を設ける。これにより、細胞培養容器１の内部と圧縮部材２２の内部は隔壁２３１により区画される。隔壁２３１は、図２に示すように管状部材２３と一体とする形態、図３に示すように管状部材２３とは別部材として設ける形態が挙げられる。図２に示す構造は、いわゆる膜チューブと呼ばれる構造である。隔壁２３１の素材は、図２に示すように管状部材２３と隔壁２３１を一体とする形態の場合は上述した素材と同一のものが使用される。一方、図３に示すように管状部材２３１とは別部材として設ける形態の場合、隔壁２３１の素材は、例えば、ポリエチレン、ポリプロピレン、ポリエチレンテレフタレート、エチレンビニルアルコール共重合体、シリコンゴム及びブチルゴムなどが選択される。これらの素材の中でも、隔壁２３１をプラスチック針３２で破壊するとき、クラックが入りにくい観点から、ポリエチレン及びポリプロピレンが好ましい。   Further, the tubular member 23 prevents a slight amount of medium from leaking out from the slit 211 of the plug 21 (slow leak) when the cell culture container 1 containing the medium for culturing cells is stored and stored before use. For this purpose, a breakable partition wall 231 is provided in the lumen. Thereby, the inside of the cell culture container 1 and the inside of the compression member 22 are partitioned by the partition 231. As shown in FIG. 2, the partition wall 231 may be integrated with the tubular member 23, and the partition wall 231 may be provided as a separate member from the tubular member 23 as shown in FIG. 3. The structure shown in FIG. 2 is a so-called membrane tube. In the case where the tubular member 23 and the partition wall 231 are integrated as shown in FIG. 2, the same material as that described above is used as the material of the partition wall 231. On the other hand, in the case where the tubular member 231 is provided as a separate member as shown in FIG. 3, for example, polyethylene, polypropylene, polyethylene terephthalate, ethylene vinyl alcohol copolymer, silicon rubber, and butyl rubber are selected as the material of the partition wall 231. Is done. Among these materials, polyethylene and polypropylene are preferable from the viewpoint of preventing cracks when the partition 231 is broken with the plastic needle 32.

隔壁２３１の厚みは、薄肉であればよい。薄肉とは、プラスチック針３２の刺通により容易に破壊できる程度の厚みであれば、特に限定されるものではない。その厚みは、隔壁２３１（管状部材２３）を構成する材料の力学的物性により依存し、一概に述べることはできないが、例えば、隔壁２３１（管状部材２３）を構成する材料がポリエチレンである場合、隔壁２３１の幅は約０．２〜１．０ｍｍ、好ましくは約０．４〜０．６ｍｍである。   The thickness of the partition wall 231 may be thin. The thin wall is not particularly limited as long as it has a thickness that can be easily broken by piercing the plastic needle 32. The thickness depends on the mechanical properties of the material constituting the partition wall 231 (tubular member 23) and cannot be described generally. For example, when the material constituting the partition wall 231 (tubular member 23) is polyethylene, The width of the partition wall 231 is about 0.2 to 1.0 mm, preferably about 0.4 to 0.6 mm.

本発明の細胞培養容器１は、さらに特許文献４や図５に示すような連結デバイス３を含むキットとしてもよい。連結デバイスは、チューブ３１と、チューブ３１の両端に接続したプラスチック針３２を備えたものをいう。   The cell culture container 1 of the present invention may further be a kit including a connection device 3 as shown in Patent Document 4 and FIG. The connecting device refers to a device provided with a tube 31 and a plastic needle 32 connected to both ends of the tube 31.

チューブ３１は、医療用として市販されているチューブと同様の形状であれば特に限定されるものではない。但し、培地が内部を流れていることを目視で確認できる観点から透明性を有するものであることが好ましい。チューブ３１に使用される材料は、上述したように、例えば、ポリ塩化ビニル、エチレン酢酸ビニル共重合体及びポリエチレン等が挙げられる。特に医療用としてその安全性が認められており、高周波シーラーで封止が容易という観点から、ポリ塩化ビニルが好ましい。   The tube 31 will not be specifically limited if it is the same shape as the tube marketed as medical use. However, it is preferable that the medium has transparency from the viewpoint of visually confirming that the medium is flowing inside. As described above, examples of the material used for the tube 31 include polyvinyl chloride, ethylene vinyl acetate copolymer, and polyethylene. In particular, polyvinyl chloride is preferred from the viewpoint of its safety for medical use and easy sealing with a high-frequency sealer.

プラスチック針３２は、栓体２１のスリット２１１を刺通可能なものであり、針軸方向に内腔を具備するものであり、基端の内腔がチューブ３１と液体的に連通しているものをいう。針先の形状などは、当業者が適宜選択して設計できるが、針刺し事故防止のために丸みがあることが好ましく、さらに先端側の側壁に内腔と連通する連通孔を備えた形態であることが好ましい。特にスプリット操作後、針先端に連通孔がある構造では、プラスチック針３２を抜いた際に、液滴が栓体２１の上部に滴状につきやすいため、連通孔は側面にある方がよい。特に本発明の管状体に設けられた隔壁がプラスチック針３２の側部に設けられた連通孔の培地をこそぎ取る機能も期待され、より栓体上部に培地の液滴がつかない構造である。栓体２１の上部に培地が滴状に残った場合、その部分に細菌などが繁殖し、繰り返し使用が出来なくなるので、上記機構は特に有用である。加えて、上述の接続部材の栓体２１に当該プラスチック針３２を刺通した場合、安定して刺通状態を保持するためのロック機構３２１を設けることが好ましい。   The plastic needle 32 can be pierced through the slit 211 of the plug body 21 and has a lumen in the needle axis direction, and the proximal lumen is in fluid communication with the tube 31. Say. The shape of the needle tip and the like can be appropriately selected and designed by those skilled in the art, but it is preferably rounded to prevent needle stick accidents, and further has a communication hole communicating with the lumen on the side wall on the tip side. It is preferable. In particular, in a structure having a communication hole at the tip of the needle after the split operation, when the plastic needle 32 is pulled out, the droplets are likely to drop onto the top of the plug body 21, so the communication hole should be on the side. In particular, the partition provided in the tubular body of the present invention is expected to have a function of scraping the culture medium in the communication hole provided in the side portion of the plastic needle 32, and has a structure in which the medium droplet does not adhere to the upper part of the plug body. . When the culture medium remains in the upper part of the stopper 21, the above mechanism is particularly useful because bacteria or the like propagates in that part and cannot be used repeatedly. In addition, when the plastic needle 32 is pierced through the plug body 21 of the connecting member described above, it is preferable to provide a lock mechanism 321 for stably maintaining the piercing state.

プラスチック針３２に使用する材料は、市販のプラスチック針と同様の材料を使用することができる。例えば、ＡＢＳ、ポリプロピレン及びポリカーボネートなどが挙げられる。特に、刃先の折れにくいＡＢＳが好ましいが、本発明はこれに限定されるものではない。   The material used for the plastic needle 32 can be the same material as a commercially available plastic needle. Examples include ABS, polypropylene, and polycarbonate. In particular, although the ABS is difficult to break, the present invention is not limited to this.

以下、本発明の細胞培養容器の接続部材を利用した細胞の移注方法について説明する。つまり、第１の細胞培養容器に収容した培養細胞を、第２の細胞培養容器にスプリットする方法について説明する。ここで、第１の細胞培養容器には、細胞と、当該細胞を懸濁する培地、言い換えれば細胞懸濁液を収容したものである。そして、少なくとも、
１）両端にプラスチック針を具備するチューブを備えた連結デバイスであって、当該デバイスのプラスチック針をそれぞれの細胞培養容器の接続部材のスリットに差し込むことで、第１の細胞培養容器と第２の細胞培養容器とを液体的に流通するステップと、
２）第１の細胞培養容器に収容した細胞の全容量のうち少なくとも一部を第２の細胞培養容器に移注するステップ
を含む。 Hereinafter, a cell transfer method using the connection member of the cell culture container of the present invention will be described. That is, a method of splitting the cultured cells accommodated in the first cell culture container into the second cell culture container will be described. Here, the first cell culture container contains cells and a medium in which the cells are suspended, in other words, a cell suspension. And at least,
1) A connecting device having a tube having plastic needles at both ends, wherein the plastic needle of the device is inserted into the slit of the connection member of each cell culture container, so that the first cell culture container and the second cell A liquid flow through the cell culture vessel;
2) including a step of transferring at least a part of the total volume of the cells accommodated in the first cell culture container to the second cell culture container.

「１）両端にプラスチック針を備えた連結デバイスであって、当該デバイスのプラスチック針をそれぞれの細胞培養容器の接続部材のスリットに差し込むことで、第１の細胞培養容器と第２の細胞培養容器とを液体的に流通するステップ」では、図４に示すようにそれぞれのプラスチック針３２を、それぞれの細胞培養容器１における接続部材２のスリット２１１に刺通することにより、液体的に流通することができる。   [1] A coupling device having plastic needles at both ends, and the first cell culture container and the second cell culture container by inserting the plastic needles of the device into the slits of the connection member of each cell culture container In the step of “circulating liquid and liquid”, as shown in FIG. 4, the respective plastic needles 32 are circulated through the slits 211 of the connection members 2 in the respective cell culture containers 1 to circulate in a liquid state. Can do.

そして、「２）第１の細胞培養容器に収容した細胞の全容量のうち少なくとも一部を第２の細胞培養容器に移注するステップ」を実施する。細胞を移注は、培養している細胞の種類によって適宜設定して実施することができる。   Then, “2) a step of transferring at least a part of the total volume of the cells accommodated in the first cell culture container to the second cell culture container” is performed. Transfection of cells can be performed by appropriately setting depending on the type of cells being cultured.

例えば、細胞が浮遊系細胞である場合、第１の細胞培養容器に収容した細胞及び培地は、いわゆる細胞懸濁液の状態であるため、そのまま連結デバイスを通じて第２の細胞培養容器に移注することができる。   For example, when the cells are suspension cells, the cells and the medium accommodated in the first cell culture container are in a so-called cell suspension state, and thus transferred directly to the second cell culture container through the connection device. be able to.

また、例えば、細胞が付着性細胞である場合、付着性細胞は第１の細胞培養容器の壁面に付着しながら増殖しているため、当該付着性細胞を壁面から剥離して細胞懸濁液を調製する必要がある。付着性細胞を壁面から剥離するには、例えば、トリプシンなどの細胞を剥離する試薬を用いる。このようにして細胞懸濁液を調製し、当該細胞懸濁液を第２の細胞培養容器に移注する。   In addition, for example, when the cells are adherent cells, the adherent cells grow while adhering to the wall surface of the first cell culture container. Need to be prepared. In order to detach the adherent cells from the wall surface, for example, a reagent for detaching the cells such as trypsin is used. In this way, a cell suspension is prepared, and the cell suspension is transferred to a second cell culture vessel.

第２の細胞培養容器に移注する細胞懸濁液の量は、当業者が適宜設定できるが、均等に培養を行うことを目的とするならば、半分量だけ移注することが通常である。   The amount of the cell suspension to be transferred to the second cell culture vessel can be appropriately set by those skilled in the art. However, if the purpose is to perform the culture evenly, it is usually transferred by a half amount. .

また、第１の細胞培養容器にて培養された細胞は、非常に高濃度となっているために、細胞懸濁液を調製する際に希釈することが好ましい。この際、第２の細胞培養容器に新鮮な培地を収容していれば、細胞を移注する前に、まず第２の細胞培養容器に収容している培地を第１の細胞培養容器に移動することにより希釈することができる。もちろん、希釈後の培地の総容量は、第１の細胞培養容器の内容量を超えてはならない。そして、第１の細胞培養容器内にて細胞を均等に懸濁させた後、その一部を第２の細胞培養容器に移注すればよい。   In addition, since the cells cultured in the first cell culture container have a very high concentration, it is preferable to dilute them when preparing the cell suspension. At this time, if a fresh medium is contained in the second cell culture container, the medium contained in the second cell culture container is first moved to the first cell culture container before the cells are transferred. Can be diluted. Of course, the total volume of the diluted medium should not exceed the content of the first cell culture vessel. Then, after suspending the cells uniformly in the first cell culture container, a part of the cells may be transferred to the second cell culture container.

以下、本発明の実施例を図面を用いて説明するが、本発明はこれらの実施例に限定されるものではない。   Examples of the present invention will be described below with reference to the drawings. However, the present invention is not limited to these examples.

実施例
図１は、本発明の細胞培養容器１を示す図である。図１における細胞培養容器は、バック形状であり、接続部材２をポートとして設けている。図２は、本発明の接続部材２の拡大断面図である。接続部材２は、中心部に上下方向に貫通した十文字形状のスリット２１１を設けたシリコンゴム製の栓体２１と、栓体２１に周設し、栓体２１のスリット２１１を圧縮密閉するポリカーボネート製の圧縮部材２２と、圧縮部材２２から下方に延出し、内腔が圧縮部材の内部と液密に連通するポリ塩化ビニル製の管状部材２３を備える。栓体２１は、外径約１１ｍｍ、中心部の厚み約２ｍｍであり、下面に環状の脚部２１２（高さ約３ｍｍ）を具備する。スリットは長さ５ｍｍの十文字形状であり、栓体２１の中心部において略垂直に貫通している。そして、圧縮部材２２には、栓体２１天面を覆うポリプロピレン製のキャップ部材２２１と、栓体２１の脚部２１２の下面を支持する支持台２２２が付設している。栓体２１を周設する圧縮部材２２の内径は、約６ｍｍであり、栓体２１に締付圧を生じさせ、スリット２１１を密閉閉鎖している。さらに、管状部材２３の内腔には、厚さ０．４ｍｍの隔壁２３１を備える。 Example FIG. 1 is a diagram showing a cell culture container 1 of the present invention. The cell culture container in FIG. 1 has a back shape and is provided with a connecting member 2 as a port. FIG. 2 is an enlarged cross-sectional view of the connection member 2 of the present invention. The connecting member 2 is made of a silicone rubber plug body 21 provided with a cross-shaped slit 211 penetrating in the vertical direction at the center, and made of polycarbonate that is provided around the plug body 21 and compresses and seals the slit 211 of the plug body 21. And a tubular member 23 made of polyvinyl chloride that extends downward from the compression member 22 and has a lumen communicating fluidly with the inside of the compression member. The plug body 21 has an outer diameter of about 11 mm and a thickness of about 2 mm at the center, and has an annular leg 212 (height of about 3 mm) on the lower surface. The slit has a cross shape with a length of 5 mm, and penetrates substantially vertically at the center of the plug body 21. The compression member 22 is provided with a polypropylene cap member 221 that covers the top surface of the plug body 21 and a support base 222 that supports the lower surface of the leg portion 212 of the plug body 21. The compression member 22 that surrounds the plug body 21 has an inner diameter of about 6 mm. A compression pressure is generated on the plug body 21, and the slit 211 is hermetically closed. Furthermore, a partition wall 231 having a thickness of 0.4 mm is provided in the lumen of the tubular member 23.

本発明の細胞培養容器によれば、培養前に予め使用する培地を充填し長期間保存させてもポート部からのリークの心配がなく、またスプリット作業後における分割された細胞培養系を独立して取り扱うことができるため、作業非常に簡便となる。さらに、細胞培養終了後におけるバイオハザードのおそれがない。このため、効率のよい細胞培養を行うことができる。   According to the cell culture container of the present invention, there is no risk of leakage from the port portion even if the medium used in advance is filled before storage and stored for a long time, and the divided cell culture system after the split operation is independent. Therefore, the operation becomes very easy. Furthermore, there is no risk of biohazard after cell culture. For this reason, efficient cell culture can be performed.

本発明の細胞培養容器１を示す図である。It is a figure which shows the cell culture container 1 of this invention. 本発明の接続部材２の拡大断面図を示す図である。It is a figure which shows the expanded sectional view of the connection member 2 of this invention. 本発明の図２とは接続部材２の実施形態の拡大断面図を示す図である。FIG. 2 of the present invention is an enlarged sectional view of an embodiment of the connecting member 2. 本発明の接続部材２にプラスチック針３２を刺通した後の断面図である。It is sectional drawing after piercing the plastic needle 32 to the connection member 2 of this invention. 連結デバイス３を示す図である。It is a figure which shows the connection device.

符号の説明Explanation of symbols

１ 細胞培養容器
２ 接続部材
２１ 栓体
２１１ スリット
２１２ 脚部
２２ 圧縮部材
２２１ キャップ部材
２２２ 支持台
２３ 管状部材
２３１ 隔壁
３ 連結デバイス
３１ チューブ
３２ プラスチック針
３２１ ロック機構

DESCRIPTION OF SYMBOLS 1 Cell culture container 2 Connection member 21 Plug body 211 Slit 212 Leg part 22 Compression member 221 Cap member 222 Support base 23 Tubular member 231 Partition 3 Connection device 31 Tube 32 Plastic needle 321 Lock mechanism

Claims (9)

閉鎖的に液体連通を可能とする接続部材を具備する細胞培養容器であって、
前記細胞培養容器は、接続部材を具備し、
前記接続部材は、
弾性部材からなり、中心部に上下方向に貫通したスリットを設けた栓体と、
前記栓体に周設し、前記栓体のスリットを圧縮密閉する圧縮部材と、
前記圧縮部材から下方に延出し、内腔が前記細胞培養容器及び前記圧縮部材の内部と連通し、前記内腔に薄肉の隔壁を設けた管状部材
を備えたものである細胞培養容器。 A cell culture container having a connection member that enables fluid communication in a closed manner,
The cell culture container comprises a connecting member,
The connecting member is
A plug body made of an elastic member and provided with a slit penetrating in the vertical direction in the center,
A compression member that is provided around the plug body and compresses and seals the slit of the plug body;
A cell culture container comprising a tubular member extending downward from the compression member, having a lumen communicating with the inside of the cell culture container and the compression member, and provided with a thin partition wall in the lumen. 前記容器が、バッグである請求項１に記載の細胞培養容器。   The cell culture container according to claim 1, wherein the container is a bag. 前記接続部材が、前記バッグのポートとして具備するものである請求項１に記載の細胞培養容器。   The cell culture container according to claim 1, wherein the connection member is provided as a port of the bag. さらに、前記圧縮部材に前記栓体天面を覆うキャップ部材を付設した請求項１に記載の細胞培養容器。   Furthermore, the cell culture container of Claim 1 which attached the cap member which covers the said stopper top surface to the said compression member. ２つの細胞培養容器の液体連通を可能とするキットであって、
前記キットは、２つの細胞培養容器と、連結デバイスを含み、
前記細胞培養容器は、接続部材を具備するものであり、
前記接続部材は、
弾性部材からなり、中心部に上下方向に貫通したスリットを設けた栓体と、
前記栓体に周設し、前記栓体のスリットを圧縮密閉する圧縮部材と、
前記圧縮部材から下方向に延出し、内腔が前記細胞培養容器本体内部及び前記圧縮部材の内部と連通し、前記内腔に薄肉の隔壁を設けることにより前記圧縮部材の内部と前記細胞培養とを区画する管状部材を備え、
前記連結デバイスは、
チューブと、
前記チューブの両端に接続したプラスチック針
を備えたものである
細胞培養キット。 A kit that allows fluid communication between two cell culture containers,
The kit includes two cell culture containers and a coupling device,
The cell culture container comprises a connecting member,
The connecting member is
A plug body made of an elastic member and provided with a slit penetrating in the vertical direction in the center,
A compression member that is provided around the plug body and compresses and seals the slit of the plug body;
Extending downward from the compression member, a lumen communicates with the inside of the cell culture container main body and the compression member, and by providing a thin partition wall in the lumen, the inside of the compression member and the cell culture A tubular member that divides
The coupling device is:
Tubes,
A cell culture kit comprising a plastic needle connected to both ends of the tube. 前記容器が、バッグである請求項５に記載の細胞培養キット。   The cell culture kit according to claim 5, wherein the container is a bag. 前記接続部材が、前記バッグのポートとして具備するものである請求項５に記載の細胞培養キット。   The cell culture kit according to claim 5, wherein the connecting member is provided as a port of the bag. さらに、前記圧縮部材に前記栓体天面を覆うキャップ部材を付設した請求項５に記載の細胞培養キット。   Furthermore, the cell culture kit of Claim 5 which attached the cap member which covers the said stopper top surface to the said compression member. 第１の細胞培養容器で培養された細胞を、第２の細胞培養容器に移す方法であって、
第１の細胞培養容器は、培養された細胞と、当該細胞を懸濁する培地が収容されたものであり、
それぞれの細胞培養容器は、接続部材を具備するものであり、
前記接続部材は、
弾性部材からなり、中心部に上下方向に貫通したスリットを設けた栓体と、
前記栓体に周設し、前記栓体のスリットを圧縮密閉する圧縮部材と、
前記圧縮部材から下方向に延出し、内腔が閉鎖系容器本体内部と前記圧縮部材の内部とを液密に連通し、前記内腔に薄肉の隔壁を設けることにより前記圧縮部材の内部と前記細胞培養とを区画する管状部材を備えたものであって、
１）両端にプラスチック針を具備するチューブを備えた連結デバイスであって、当該デバイスのプラスチック針をそれぞれの細胞培養容器の接続部材のスリットに差し込むことで、第１の細胞培養容器と第２の細胞培養容器とを液体的に流通するステップと、
２）第１の細胞培養容器に収容した細胞の全容量のうち少なくとも一部を第２の細胞培養容器に移注するステップ
を含む細胞移注方法。


A method of transferring cells cultured in a first cell culture container to a second cell culture container,
The first cell culture container contains cultured cells and a medium for suspending the cells,
Each cell culture vessel comprises a connecting member,
The connecting member is
A plug body made of an elastic member and provided with a slit penetrating in the vertical direction in the center,
A compression member that is provided around the plug body and compresses and seals the slit of the plug body;
Extending downward from the compression member, a lumen communicates the inside of the closed system container body and the compression member in a fluid-tight manner, and a thin-walled partition is provided in the lumen, thereby providing an inside of the compression member and the A tubular member for partitioning cell culture,
1) A connecting device having a tube having plastic needles at both ends, wherein the plastic needle of the device is inserted into the slit of the connection member of each cell culture container, so that the first cell culture container and the second cell A liquid flow through the cell culture vessel;
2) A cell transfusion method comprising the step of transfecting at least a part of the total volume of cells accommodated in the first cell culture container to the second cell culture container.


JP2006162489A 2006-06-12 2006-06-12 Cell culture container and cell transfer method Expired - Fee Related JP4802875B2 (en)

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