JP2013143955A - Supply system for closed culture medium for culturing small cell - Google Patents
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本発明は、生物学、医学等の分野における密閉系細胞培養容器を利用した閉鎖系小型細胞培養用培地供給システムに関するものである。 The present invention relates to a closed-system small-cell culture medium supply system using a closed-system cell culture container in fields such as biology and medicine.
医療技術の著しい発展により、近年、治療困難となった臓器を他人の臓器と置き換えようとする臓器移植が一般化してきた。対象となる臓器も皮膚、角膜、腎臓、肝臓、心臓等と実に多様で、また、術後の経過も格段に良くなり、医療の一技術としてすでに確立されつつある。一例として角膜移植をあげると、約４０年前に日本にもアイバンクが設立され移植活動が始められた。しかしながら、未だにドナー数が少なく、国内だけでも角膜移植の必要な患者が年間約２万人いるのに対し、実際に移植治療が行える患者は約１／１０の２０００人程度でしかないといわれている。角膜移植というほぼ確立された技術があるにもかかわらず、ドナー不足という問題のため、次なる医療技術が求められているのが現状である。 Due to the remarkable development of medical technology, in recent years, organ transplants that attempt to replace difficult-to-treat organs with other organs have become common. The target organs are very diverse, such as skin, cornea, kidney, liver, heart, etc., and the progress after surgery has improved remarkably, and has already been established as a medical technology. Taking corneal transplantation as an example, an eye bank was established in Japan about 40 years ago and transplantation activities began. However, the number of donors is still small, and there are about 20,000 patients who need corneal transplants in Japan alone, whereas it is said that there are only about 1/10 of the 2000 patients who can actually perform transplantation treatment. Yes. Despite the almost established technology of corneal transplantation, the current situation is that the next medical technology is required due to the shortage of donors.
このような背景のもと、以前より、人工代替物や細胞を培養して組織化させたものをそのまま移植しようという技術が注目されている。その代表的な例として、人工皮膚及び培養皮膚があげられよう。しかしながら、合成高分子を用いた人工皮膚は拒絶反応等が生じる可能性があり、移植用皮膚としては好ましくない。一方、培養皮膚は本人の正常な皮膚の一部を所望の大きさまで培養したものであるため、これを使用しても拒絶反応等の心配がなく、最も自然なマスキング剤と言える。 Against this background, techniques for transplanting artificial substitutes or cells that have been cultured and organized have attracted attention. Representative examples thereof include artificial skin and cultured skin. However, artificial skin using a synthetic polymer may cause rejection and the like, and is not preferable as skin for transplantation. On the other hand, since the cultured skin is obtained by culturing a part of the normal skin of the person to a desired size, even if it is used, there is no concern about rejection or the like, and it can be said to be the most natural masking agent.
その細胞培養には、従来、器材として細胞培養用ディッシュ、マルチプレート、フラスコ等を用いられてきた。その際、例えばディッシュやマルチプレートを使用するときは、細胞培養時に通常、培養容器上部に蓋を乗せて行われる。また、フラスコを使用する場合では細胞培養時に蓋をゆるめて行われる。すなわち、何れの場合でも細胞が培養される空間が基本的に開放状態であり、細胞の代謝に必要な酸素は十分に確保できる反面、細菌汚染するリスクが極めて高い問題点があった。 Conventionally, cell culture dishes, multiplates, flasks, and the like have been used for the cell culture. At that time, for example, when using a dish or a multi-plate, the cell culture is usually carried out by placing a lid on the upper part of the culture vessel. When using a flask, the lid is loosened during cell culture. That is, in any case, the space in which cells are cultured is basically open, and oxygen necessary for cell metabolism can be sufficiently secured, but there is a problem that the risk of bacterial contamination is extremely high.
こうした背景のもと、特表平１０−５０４７１０公報や特開平１０−２６２６４７公報ではガス透過膜を有する密閉型の細胞培養容器が提案されている。この方法によれば、密閉型の培養容器であるため、細胞培養中では容器外からの菌による汚染は抑制でき、また、密閉系容器で問題となる細胞が生存するための酸素もガス透過膜を介して、供給できるような構造を有している。しかしながら、これらの培養容器では細胞を培養するための培養面が１面だけのものであった。また、特開平１１−２８０８２号公報では、酸素透過性透明部材、シリコンゴム枠、分離膜から構成される多層式の細胞培養容器が記載されているが、この培養容器では宇宙空間で使用するため容器に細胞培養用液体培地の管状流路が設けられていないこと、またこの容器においても培養した細胞を剥離させる際には酵素処理を行わなくてはならず、ガス透過性を有し、しかも酵素処理が不要な膜技術は見出されていなかった。さらに、こうした密閉型の培養容器を使用する場合でも、培地交換は作業者によって行われており、必ずしも培養された細胞を最適な条件で培養されていなかった。また、培養細胞の培地交換毎に作業を行わなくてはならず時間的な制約が生じ、改善が求められていた。 Against this background, a closed cell culture vessel having a gas permeable membrane is proposed in Japanese Patent Laid-Open No. 10-504710 and Japanese Patent Laid-Open No. 10-262647. According to this method, since it is a sealed culture container, contamination by bacteria from outside the container can be suppressed during cell culture, and oxygen for the survival of problematic cells in the sealed container is also a gas permeable membrane. It has the structure which can be supplied via. However, these culture vessels have only one culture surface for culturing cells. Japanese Patent Application Laid-Open No. 11-28082 discloses a multilayer cell culture container composed of an oxygen-permeable transparent member, a silicon rubber frame, and a separation membrane. However, this culture container is used in outer space. The vessel is not provided with a tubular channel for the liquid culture medium for cell culture, and also in this vessel, when the cultured cells are peeled off, an enzyme treatment must be performed, and gas permeability is provided. No membrane technology has been found that does not require enzyme treatment. Further, even when such a sealed culture vessel is used, the medium is exchanged by an operator, and the cultured cells are not necessarily cultured under optimum conditions. In addition, work must be performed every time the culture medium is replaced, and there is a need for improvement due to time constraints.
本発明は、上記のような従来技術の問題点を解決することを意図してなされたものである。すなわち、本発明は、従来技術と全く異なった発想からの密閉系細胞培養容器を利用した閉鎖系小型細胞培養用培地供給システムを提供することを目的とする。 The present invention has been made with the intention of solving the problems of the prior art as described above. That is, an object of the present invention is to provide a closed-system small-cell culture medium supply system using a closed-system cell culture container from an idea completely different from the prior art.
本発明者らは、上記課題を解決するために、種々の角度から検討を加えて、研究開発を行った。その結果、ガス透過膜が装着され閉鎖系細胞培養用容器を用い、その容器の培地出入口につなげられた配管を通して送液ポンプにより培地を供給制御できる閉鎖系小型細胞培養用培地供給システムを見出した。本発明はかかる知見に基づいて完成されたものである。 In order to solve the above-mentioned problems, the present inventors have studied and developed from various angles. As a result, a closed-system cell culture medium supply system was found that can use a closed-system cell culture container equipped with a gas permeable membrane and can control the supply of the medium by a liquid feed pump through a pipe connected to the medium inlet / outlet of the container. . The present invention has been completed based on such findings.
すなわち、本発明は、培地中のガス交換を行うためのガス透過膜が装着され培地の循環流路が設けられた閉鎖系細胞培養用容器を装着でき、その容器の培地出入口につなげられた配管を通して送液ポンプにより新鮮な培地の供給制御ができる閉鎖系小型細胞培養用培地供給システムを提供する。 That is, the present invention is capable of mounting a closed cell culture vessel equipped with a gas permeable membrane for exchanging gas in a culture medium and provided with a circulation channel for the culture medium, and a pipe connected to the culture medium inlet / outlet of the container A closed-system small cell culture medium supply system capable of controlling the supply of fresh medium through a liquid feed pump.
本発明に記載される閉鎖系小型細胞培養用培地供給システムであれば、クリーンベンチ内でなくともどのような場所においても細胞を培養させられ、細胞培養を汚染させることなく、簡便に、安全に行えるようになり、さらに培養細胞を培地供給システムごと移動させたい場所へ汚染させることなく、簡便に、安全に移動させられるようになる。 With the closed-system small cell culture medium supply system described in the present invention, cells can be cultured at any place without being in a clean bench, easily and safely without contaminating the cell culture. In addition, the cultured cells can be easily and safely moved without being contaminated to the place where the culture medium supply system is desired to be moved.
本発明は、培地中のガス交換を行うためのガス透過膜が装着され培地の循環流路が設けられた閉鎖系細胞培養用容器を装着でき、その容器の培地出入口につなげられた配管を通して送液ポンプにより新鮮な培地の供給制御ができる閉鎖系小型細胞培養用培地供給システムである。本発明は以下に記載するような閉鎖系細胞培養用容器を装着し、その容器の培地出入口に配管することで閉鎖系のシステムとしていることが特徴である。必要に応じて容器装着部にヒーターを取り付けても良い。容器と培地導入管との接合部は特に限定されるものではなく、その接合部から菌汚染が起こらなければ良く、容器側の止水弁に対しニードル、もしくはシリンジが装着された培地導入管を差し込む方法で良い。 In the present invention, a closed cell culture vessel equipped with a gas permeable membrane for exchanging gas in a culture medium and provided with a circulation channel for the culture medium can be attached and sent through a pipe connected to the culture medium inlet / outlet of the container. This is a closed-system small cell culture medium supply system in which supply of fresh medium can be controlled by a liquid pump. The present invention is characterized in that a closed system is constructed by mounting a closed cell culture container as described below and piping to the medium inlet / outlet of the container. You may attach a heater to a container mounting part as needed. The junction between the container and the medium introduction tube is not particularly limited, and it is sufficient that no bacterial contamination occurs from the junction, and a medium introduction tube with a needle or syringe attached to the water stop valve on the container side. You can plug it in.
本発明で用いる送液ポンプは特に限定されるものではないが、チューブポンプ、ギヤーポンプ、ロータリーポンプ等が挙げられる。本発明の目的が携帯型の細胞培養用培地供給システムであることを考えると、使用後に配管を廃棄できるチューブポンプを利用することが望ましい。また、閉鎖系細胞培養用容器から配管内にある培地を閉鎖する意味では、チューブポンプ、ギヤーポンプであればポンプ部のところで培地が遮断され閉鎖されることになり好ましい。 Although the liquid feed pump used by this invention is not specifically limited, A tube pump, a gear pump, a rotary pump, etc. are mentioned. Considering that the object of the present invention is a portable cell culture medium supply system, it is desirable to use a tube pump capable of discarding piping after use. In the sense of closing the culture medium in the pipe from the closed cell culture vessel, a tube pump or gear pump is preferable because the culture medium is shut off and closed at the pump section.
本発明では、閉鎖系細胞培養用容器に対し閉鎖系小型細胞培養用培地供給システムによって培地交換をする。その培地交換する際は、容器内に気泡が残らないようにすることが好ましく、その方法としては特に限定されないが、培地導入入口側の配管と出口側の配管を一台以上のチューブポンプにつなげて培地の導入、排出を同時に行う方法、２台以上のギヤーポンプを用いて培地の導入、排出を同時に行う方法等が挙げられる。また、そのポンプの駆動電源も特に問われるものではないが、通常使われる交流電源、並びに蓄電池等が挙げられる。 In the present invention, the medium is exchanged by the closed system small cell culture medium supply system for the closed system cell culture container. When exchanging the medium, it is preferable not to leave bubbles in the container. The method is not particularly limited, but the medium introduction inlet side pipe and the outlet side pipe are connected to one or more tube pumps. And a method of simultaneously introducing and discharging the medium, and a method of simultaneously introducing and discharging the medium using two or more gear pumps. Further, the driving power source of the pump is not particularly limited, and examples thereof include a commonly used AC power source and a storage battery.
本発明で供給される培地貯蔵槽は、本閉鎖系小型細胞培養用培地供給システムに備え付けられていても分離されていても良く、特に制約されるものではないが、例えば、本システムをそのままインキュベーター内に装着する場合、培地が常に加温され好ましくなく、目的に応じて適宜システムを変更すれば良い。 The medium storage tank supplied in the present invention may be provided or separated from the closed-system small cell culture medium supply system, and is not particularly limited. For example, the system is used as it is in an incubator. In the case of mounting in the inside, the culture medium is always heated, which is not preferable, and the system may be appropriately changed according to the purpose.
本発明のシステムは、閉鎖系細胞培養用容器に対し培地供給をプログラム管理できるものである。その結果、培養される細胞に最適な条件で培地交換できるようになり、また、それを実施するための人手も不要であり、細胞を培養する上で極めて好都合なシステムである。また、本発明のシステムは、小型装置であるため常用の細胞培養用インキュベーター内で利用することも可能となる。さらに、運搬車両内に設置して培養細胞を移動させる際にも極めて好都合な装置である。 The system of the present invention is capable of program management of medium supply to a closed cell culture vessel. As a result, the medium can be exchanged under the optimum conditions for the cells to be cultured, and no human hands are required to carry out it, which is a very convenient system for culturing the cells. Further, since the system of the present invention is a small device, it can be used in a conventional cell culture incubator. Furthermore, it is a very convenient device when installed in a transport vehicle to move cultured cells.
本発明では、細胞培養用液体培地の管状流路を設けた成型体にガス透過膜を固定することで密閉された細胞培養用空間を設け、必要に応じその空間を１枚以上の物質透過膜により分割した密閉系細胞培養容器を利用する。本発明に示す密閉系細胞培養容器とは、少なくとも細胞培養用液体培地の管状流路を設けた成型体にガス透過膜を固定することで密閉されたものである。そのガス透過膜とは酸素ガス透過性が０．５×１０１０ｃｃ・ｃｍ／ｃｍ２・ｓｅｃ・ｃｍＨｇ以上、好ましくは０．８×１０１０ｃｃ・ｃｍ／ｃｍ２・ｓｅｃ・ｃｍＨｇ以上、さらに好ましくは１．０×１０１０ｃｃ・ｃｍ／ｃｍ２・ｓｅｃ・ｃｍＨｇ以上のものである。０．５×１０１０ｃｃ・ｃｍ／ｃｍ２・ｓｅｃ・ｃｍＨｇ以下であると、閉鎖系細胞培養容器内で培養されている細胞への酸素供給が不十分となりその生存に障害が生じ好ましくない。一方、通常、細胞培養の際には培地のｐＨを一定に保たせるために炭酸ガスが必要である。本閉鎖系細胞培養容器においてもその必要性は同様であり、従って本閉鎖系細胞培養容器に固定されているガス透過膜には炭酸ガスの透過性も必要である。その炭酸ガス透過性は３．０×１０１０ｃｃ・ｃｍ／ｃｍ２・ｓｅｃ・ｃｍＨｇ以上、好ましくは４．５×１０１０ｃｃ・ｃｍ／ｃｍ２・ｓｅｃ・ｃｍＨｇ以上、さらに好ましくは６．０×１０１０ｃｃ・ｃｍ／ｃｍ２・ｓｅｃ・ｃｍＨｇ以上のものである。３．０×１０１０ｃｃ・ｃｍ／ｃｍ２・ｓｅｃ・ｃｍＨｇ以下であると、閉鎖系細胞培養容器内で培養されている細胞への炭酸ガスの供給が不十分となりその生存に障害が生じ好ましくない。本発明で使用されるガス透過膜とは上述したガス透過性を有するものであれば何ら制約されるものではないが、本発明の目的が細胞を培養するためのものであることを考えるとガス透過膜は光学的に透明であることが好ましい。ガス透過膜の材質についても何ら制約されるものではないが、例えばポリカーボネート、ポリスチレン、ポリジメチルシロキサン、シリコーンポリカーボネート等があげられる。ガス透過膜の膜厚についても上述したガス透過性を有すれば何ら制約されるものではないが、例えば３０μｍ以上、好ましくは５０μｍ以上、さらに好ましくは８０μｍ以上が好ましい。３０μｍ以下であると、閉鎖系空間を保たせることが困難となり好ましくない。本発明の閉鎖系細胞培養容器はガス透過膜を固定することで密閉された空間を有するものである。その際、ガス透過膜が固定される壁の形状、透明性、材質等には特に制約されるものはない。しかしながら、ガス透過膜と同じ材質の壁にすることで、ガス透過膜と壁との固定が強固となり好ましい。 In the present invention, a sealed cell culture space is provided by fixing a gas permeable membrane to a molded body provided with a tubular channel of a liquid culture medium for cell culture, and the space is provided with one or more substance-permeable membranes as necessary. A closed cell culture vessel divided by is used. The closed cell culture vessel shown in the present invention is sealed by fixing a gas permeable membrane to a molded body provided with at least a tubular channel of a liquid culture medium for cell culture. The gas permeable membrane has an oxygen gas permeability of 0.5 × 10 10 cc · cm / cm 2 · sec · cmHg or more, preferably 0.8 × 10 10 cc · cm / cm 2 · sec · cmHg or more, Preferably, it is 1.0 × 10 10 cc · cm / cm 2 · sec · cmHg or more. If it is 0.5 × 10 10 cc · cm / cm 2 · sec · cmHg or less, the supply of oxygen to cells cultured in a closed cell culture vessel becomes insufficient, and the survival is impaired, which is not preferable. On the other hand, carbon dioxide gas is usually required for cell culture in order to keep the pH of the medium constant. The necessity is the same in the closed cell culture vessel, and therefore the gas permeable membrane fixed to the closed cell culture vessel needs to be permeable to carbon dioxide gas. The carbon dioxide gas permeability is 3.0 × 10 10 cc · cm / cm 2 · sec · cmHg or more, preferably 4.5 × 10 10 cc · cm / cm 2 · sec · cmHg or more, more preferably 6.0. × 10 10 cc · cm / cm 2 · sec · cmHg or more. If it is 3.0 × 10 10 cc · cm / cm 2 · sec · cmHg or less, the supply of carbon dioxide gas to the cells cultured in the closed cell culture vessel becomes insufficient, resulting in an obstacle to its survival. Absent. The gas permeable membrane used in the present invention is not limited as long as it has the above-mentioned gas permeability, but considering that the object of the present invention is for culturing cells, gas The permeable membrane is preferably optically transparent. The material of the gas permeable membrane is not limited at all, and examples thereof include polycarbonate, polystyrene, polydimethylsiloxane, and silicone polycarbonate. The thickness of the gas permeable membrane is not limited as long as it has the above-described gas permeability, but is preferably 30 μm or more, preferably 50 μm or more, and more preferably 80 μm or more. When it is 30 μm or less, it is difficult to maintain a closed system space, which is not preferable. The closed cell culture vessel of the present invention has a sealed space by fixing a gas permeable membrane. At that time, there are no particular restrictions on the shape, transparency, material and the like of the wall to which the gas permeable membrane is fixed. However, it is preferable to use a wall made of the same material as the gas permeable membrane because the gas permeable membrane and the wall are firmly fixed.
本発明で示す閉鎖系細胞培養容器とは、ガス透過膜を固定することで密閉された細胞培養用空間に細胞培養用液体培地の管状流路を設けたものである。その管状流路は細胞培養用液体培地を注入、取り出しができれば形状や流路の管径等に特に制約されるものではない。しかしながら、閉鎖系細胞培養容器の閉鎖性を考えるとその管の直径は２．５ｍｍ以下、好ましくは２．０ｍｍ以下、さらに好ましくは１．５ｍｍ以下である。２．５ｍｍ以上であると閉鎖系空間からの培地の流出が起こりやすく好ましくない。 The closed cell culture vessel shown in the present invention is a cell culture liquid medium tubular tube channel provided in a cell culture space sealed by fixing a gas permeable membrane. The tubular channel is not particularly limited by the shape, the diameter of the channel, or the like as long as the cell culture liquid medium can be injected and taken out. However, considering the closing properties of the closed cell culture vessel, the diameter of the tube is 2.5 mm or less, preferably 2.0 mm or less, more preferably 1.5 mm or less. If it is 2.5 mm or more, the medium is likely to flow out of the closed space, which is not preferable.
本発明の密閉系細胞培養容器においては、必要に応じその閉鎖された細胞培養用空間を１枚以上の物質透過膜により分割しても良い。その際、使用される物質透過膜の平均孔径は０．２〜３．０μｍの範囲が良く、好ましくは０．３〜２．０μｍの範囲、さらに好ましくは０．４〜１．０μｍの範囲が良い。平均孔径が０．２μｍ以下の場合は物質透過能力が低く、細胞の培養が効率良く行われない。また、３．０μｍ以上の平均孔径の場合にはその孔を介して細胞が物質透過膜を通過する可能性があり、物質透過膜により培養空間を分割するという目的を達成しておらず好ましくない。本発明で使用される物質透過膜とは上述したように多孔性であり物質透過性を有するものであれば何ら制約されるものではないが、本発明の目的が細胞を培養するためのものであることを考えると物質透過膜は光学的に透明であることが好ましい。物質透過膜の材質についても何ら制約されるものではないが、例えばポリカーボネート、ポリスチレン、ポリジメチルシロキサン、シリコーンポリカーボネート、ポリエチレンテレフタレート等があげられる。物質透過膜の膜厚についても上述した物質透過性を有すれば何ら制約されるものではないが、例えば５μｍ以上、好ましくは１０μｍ以上、さらに好ましくは３０μｍ以上が好ましい。５μｍ以下であると、閉鎖系空間を保たせることが困難となり好ましくない。本発明の閉鎖系細胞培養容器は物質透過膜を固定することで密閉された空間を有するものである。その際、物質透過膜が固定される壁の形状、透明性、材質等には特に制約されるものはない。しかしながら、物質透過膜と同じ材質の壁にすることで、物質透過膜と壁との固定が強固となり好ましい。 In the closed cell culture vessel of the present invention, the closed cell culture space may be divided by one or more substance-permeable membranes as necessary. In that case, the average pore diameter of the material-permeable membrane used is preferably in the range of 0.2 to 3.0 μm, preferably in the range of 0.3 to 2.0 μm, more preferably in the range of 0.4 to 1.0 μm. good. When the average pore size is 0.2 μm or less, the substance permeability is low, and cell culture is not efficiently performed. In addition, when the average pore diameter is 3.0 μm or more, there is a possibility that cells pass through the substance-permeable membrane through the pores, which is not preferable because the purpose of dividing the culture space by the substance-permeable membrane has not been achieved. . The substance permeable membrane used in the present invention is not limited as long as it is porous and has substance permeability as described above, but the object of the present invention is to culture cells. In consideration of the fact, it is preferable that the substance-permeable film is optically transparent. The material of the material permeable membrane is not limited at all, and examples thereof include polycarbonate, polystyrene, polydimethylsiloxane, silicone polycarbonate, and polyethylene terephthalate. The thickness of the material permeable membrane is not limited as long as it has the above-described material permeability. For example, it is 5 μm or more, preferably 10 μm or more, and more preferably 30 μm or more. If it is 5 μm or less, it is difficult to maintain a closed system space, which is not preferable. The closed cell culture vessel of the present invention has a sealed space by fixing a substance-permeable membrane. At that time, there are no particular restrictions on the shape, transparency, material, and the like of the wall to which the substance-permeable membrane is fixed. However, it is preferable to use a wall made of the same material as that of the substance-permeable membrane because the substance-permeable film and the wall are firmly fixed.
以上より、細胞培養用液体培地の管状流路を設けた成型体に透過膜を固定することで密閉された細胞培養用空間を有した密閉系細胞培養容器が得られる。その際、必要に応じ１枚以上の物質透過膜により細胞培養用空間を分割しても良い。細胞の培養は、その密閉系細胞培養容器内の１つ以上の細胞培養用空間内で行われる。 As described above, a sealed cell culture container having a sealed cell culture space can be obtained by fixing a permeable membrane to a molded body provided with a tubular channel of a liquid culture medium for cell culture. At this time, the cell culture space may be divided by one or more substance-permeable membranes as necessary. The cells are cultured in one or more cell culture spaces in the closed cell culture vessel.
密閉系細胞培養容器内に物質透過膜を使用した場合、その物質透過膜の両側を異なった細胞で培養すれば、細胞の通過が無く細胞が産生し培養液中に放出する液性因子だけが通過する培養系が構築でき、有用である。具体的には、物質透過膜を介して上層及び下層に培地を満たし、角膜上皮細胞を物質透過膜上で培養し、物質透過膜下層にフィーダー細胞を培養すれば、物質透過膜上の角膜上皮細胞は重層化することとなる。その際、フィーダー細胞は物質透過膜を通過することができず、物質透過膜上で培養される角膜上皮細胞に混合することなく好都合である。その際、使用されるフィーダー細胞としては、例えば線維芽細胞、組織幹細胞、胚性幹細胞などが挙げられるが、特に制約されるものではない。また、上皮系細胞とフィーダー細胞は必ずしも同一種の動物由来のものでなくても良いが、得られた重層化上皮系細胞シートを移植に用いる場合は上皮系細胞とフィーダー細胞を同一種の動物由来のものとした方が望ましい。また、この重層化上皮系細胞シートをヒトの治療に用いる場合は上皮系細胞とフィーダー細胞をヒト由来の細胞を用いる方が望ましい。 When a substance-permeable membrane is used in a closed cell culture vessel, if both sides of the substance-permeable membrane are cultured with different cells, only the liquid factor that the cell produces without being passed through and released into the culture medium A passing culture system can be constructed and is useful. Specifically, if the medium is filled in the upper layer and the lower layer through the substance-permeable membrane, corneal epithelial cells are cultured on the substance-permeable membrane, and feeder cells are cultured on the substance-permeable membrane, the corneal epithelium on the substance-permeable membrane The cells will be stratified. In this case, the feeder cells cannot pass through the substance-permeable membrane, which is advantageous without mixing with the corneal epithelial cells cultured on the substance-permeable membrane. In this case, examples of feeder cells used include fibroblasts, tissue stem cells, embryonic stem cells, and the like, but are not particularly limited. In addition, the epithelial cells and feeder cells do not necessarily have to be derived from the same species of animal, but when the obtained stratified epithelial cell sheet is used for transplantation, the epithelial cells and feeder cells are the same species of animal. It is desirable to have the origin. In addition, when this stratified epithelial cell sheet is used for human therapy, it is desirable to use human-derived cells for epithelial cells and feeder cells.
本発明に使用される細胞は、例えば角膜上皮細胞、表皮角化細胞、口腔粘膜細胞、結膜上皮細胞、軟骨細胞、神経細胞、心筋細胞、線維芽細胞、血管内皮細胞、肝実質細胞、脂肪細胞及びそれらの幹細胞のいずれかもしくは２者以上の混合物が挙げられるが、その種類は、何ら制約されるものではない。また、その細胞の由来は特に制約されるものではないが、たとえばヒト、イヌ、ネコ、ウサギ、ラット、ブタ、ヒツジなどが挙げられるが、本発明の培養細胞をヒトの治療に用いる場合はヒト由来の細胞を用いる方が望ましい。 The cells used in the present invention are, for example, corneal epithelial cells, epidermal keratinocytes, oral mucosal cells, conjunctival epithelial cells, chondrocytes, nerve cells, cardiomyocytes, fibroblasts, vascular endothelial cells, hepatocytes, adipocytes And any one of those stem cells or a mixture of two or more thereof, but the type is not limited. The origin of the cells is not particularly limited, and examples include humans, dogs, cats, rabbits, rats, pigs, sheep, and the like. When the cultured cells of the present invention are used for human therapy, humans are used. It is desirable to use cells derived from them.
本発明における細胞培養のための培地は培養される細胞に対し通常用いられるものを用いれば特に制約されるものではない。例えば、培地中に公知のウシ胎児血清（ＦＣＳ）等の血清が添加されている培地でもよく、また、このような血清が添加されていない無血清培地でもよい。しかしながら、得られた培養細胞をヒトの治療に用いる場合は用いる培地の成分は由来が明確なもの、もしくは医薬品として認められているものが望ましい。 The medium for cell culture in the present invention is not particularly limited as long as it is a commonly used medium for cells to be cultured. For example, a medium in which serum such as known fetal calf serum (FCS) is added to the medium may be used, or a serum-free medium in which such serum is not added may be used. However, when the obtained cultured cells are used for human therapy, it is desirable that the components of the medium to be used have a clear origin or are recognized as pharmaceuticals.
以下に、本発明を実施例に基づいて更に詳しく説明するが、これらは本発明を何ら限定するものではない。 Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.
図１に示すような寸法の各パーツからなる１台のチューブポンプからなる閉鎖系小型細胞培養用培地供給システムを作成した。その装置内に成型体中央は空間となっている閉鎖系細胞培養用容器を装着した。その容器中央の上部、中央部、下部にそれぞれガス透過膜としてのポリカーボネート膜、物質透過膜、ガス透過膜としてのポリカーボネート膜を物質透過膜、ガス透過膜、ガス透過膜の順に成型体に熱融着させ、２層式の密閉系細胞培養容器を作製した。その際、ポリカーボネート膜のガス透過性は、酸素ガス透過性が１．４×１０１０ｃｃ・ｃｍ／ｃｍ２・ｓｅｃ・ｃｍＨｇ、炭酸ガス透過性が８．０×１０１０ｃｃ・ｃｍ／ｃｍ２・ｓｅｃ・ｃｍＨｇであった。物質透過膜にはワットマン製のポアサイズ０．４μｍのサイクロポアメンブレンを使用した。 A closed-system small cell culture medium supply system consisting of a single tube pump made up of parts having dimensions as shown in FIG. 1 was prepared. A closed cell culture vessel having a space at the center of the molded body was mounted in the apparatus. A polycarbonate membrane as a gas permeable membrane, a substance permeable membrane, and a polycarbonate membrane as a gas permeable membrane are heat-fused into a molded body in the order of a substance permeable membrane, a gas permeable membrane, and a gas permeable membrane in the upper, middle, and lower portions of the container center. A two-layer closed cell culture vessel was prepared. At that time, the gas permeability of the polycarbonate membrane is 1.4 × 10 10 cc · cm / cm 2 · sec · cmHg for oxygen gas permeability, and 8.0 × 10 10 cc · cm / cm 2 for carbon dioxide gas permeability. -It was sec-cmHg. As the material permeable membrane, a cyclopore membrane having a pore size of 0.4 μm manufactured by Whatman was used.
密閉系細胞培養容器の下層部にフィーダー細胞としてマイトマイシンＣ処理したＮＩＨ−３Ｔ３細胞を２×１０４ｃｅｌｌｓ／ｃｍ２となるように播種した。密閉系細胞培養容器の上層部は培地のみを満たし、密閉系細胞培養容器を３７℃、５％ＣＯ２インキュベーター内に入れた。翌日、深麻酔下のウサギ角膜輪部から常法に従って角膜上皮組織を採取し、０．０５％トリプシン処理することで得られた細胞を、上記密閉系細胞培養容器の物質透過膜上（密閉系細胞培養容器の上層部）に２×１０４ｃｅｌｌｓ／ｃｍ２となるように播種した。本閉鎖系小型細胞培養用培地供給システム全体をインキュベーター内に入れ、培地としては、上層、下層共に角膜上皮細胞用として通常使われる５％ＦＣＳ含有ＤＭＥＭ系培地を使用した（３７℃、５％ＣＯ２）。培地交換は閉鎖系小型細胞培養用培地供給システムを利用し８時間毎に行った。培養開始１１日後には培養した細胞はコンフルエントの状態になっており、重層化していた。得られた重層化角膜上皮細胞シートは移植に使用できる十分な強度を有しており、本密閉系細胞培養容器が本発明として好ましいものであることが分かった。 NIH-3T3 cells treated with mitomycin C as feeder cells were seeded at 2 × 10 4 cells / cm 2 in the lower layer of the closed cell culture vessel. The upper part of the closed cell culture vessel was filled with only the medium, and the closed cell culture vessel was placed in a 37 ° C., 5% CO 2 incubator. The next day, corneal epithelial tissue is collected from the rabbit corneal ring under deep anesthesia according to a conventional method, and the cells obtained by 0.05% trypsin treatment are placed on the substance-permeable membrane (sealed system) of the above closed cell culture vessel. It seed | inoculated so that it might become 2 * 10 < 4 > cells / cm < 2 > to a cell culture container upper layer part). The entire closed-system small cell culture medium supply system was placed in an incubator, and the medium used was a DMEM medium containing 5% FCS that is usually used for corneal epithelial cells in both the upper and lower layers (37 ° C., 5% CO 2. 2 ). Medium exchange was performed every 8 hours using a medium supply system for closed cell culture. After 11 days from the start of the culture, the cultured cells were confluent and stratified. The obtained stratified corneal epithelial cell sheet has sufficient strength that can be used for transplantation, and it was found that this closed cell culture vessel is preferable as the present invention.
図２に示すような寸法の各パーツからなる２台のチューブポンプからなる閉鎖系小型細胞培養用培地供給システムを使用する以外は、実施例１と同様な実験を行った。実施例１と同様にウサギ角膜上皮細胞を培養したところ、培養開始１１日後には培養した細胞はコンフルエントの状態になっており、重層化していた。得られた重層化角膜上皮細胞シートは移植に使用できる十分な強度を有しており、本密閉系細胞培養容器が本発明として好ましいものであることが分かった。 Experiments similar to those in Example 1 were performed, except that a closed-system small cell culture medium supply system composed of two tube pumps each having dimensions as shown in FIG. 2 was used. When rabbit corneal epithelial cells were cultured in the same manner as in Example 1, the cultured cells were in a confluent state 11 days after the start of the culture, and were stratified. The obtained stratified corneal epithelial cell sheet has sufficient strength that can be used for transplantation, and it was found that this closed cell culture vessel is preferable as the present invention.
本発明に記載される閉鎖系小型細胞培養用培地供給システムであれば、クリーンベンチ内でなくともどのような場所においても細胞を培養させられ、細胞培養を汚染させることなく、簡便に、安全に行えるようになり、さらに培養細胞を培地供給システムごと移動させたい場所へ汚染させることなく、簡便に、安全に移動させられるようになる。この方法で得られる培養細胞シートは、たとえば角膜移植、軟骨移植、皮膚移植、虚血性心疾患治療、角膜疾患治療、近視治療等の臨床応用が強く期待される。したがって、本発明は細胞工学、医用工学、などの医学、生物学等の分野における極めて有用な発明である。 With the closed-system small cell culture medium supply system described in the present invention, cells can be cultured at any place without being in a clean bench, easily and safely without contaminating the cell culture. In addition, the cultured cells can be easily and safely moved without being contaminated to the place where the culture medium supply system is desired to be moved. The cultured cell sheet obtained by this method is strongly expected to have clinical applications such as corneal transplantation, cartilage transplantation, skin transplantation, ischemic heart disease treatment, corneal disease treatment, myopia treatment and the like. Therefore, the present invention is extremely useful in the fields of medicine, biology, etc., such as cell engineering and medical engineering.
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