JP2008220350A - Alternative method of soft agar colony forming test and application method thereof - Google Patents
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本発明は、医学、生物学等の分野における軟寒天コロニー形成試験代替法、それを利用した生化学評価用キットに関する。 The present invention relates to a soft agar colony formation test alternative method in fields such as medicine and biology, and a biochemical evaluation kit using the same.
癌は日本人の死因のトップであり、全人口の約３０％が癌で死亡すると今なお言われている。近年、その治療法として外科的手術、放射線照射等のよる物理療法、抗癌剤投与等の化学療法、さらには自己免疫応答を利用した細胞療法等の技術が開発、提案されてきたが、未だに絶対的な治療法が確立されていないのが現状である。その原因としては、対象の癌細胞が生化学的に多様化しており癌細胞を一括したような技術開発の目標を定め難いこと、そして、その癌細胞の生化学的機能を調べるために癌組織から癌細胞を純度高く単離することができないこと等が挙げられる。従って、この単離技術が開発、確立されれば、癌細胞そのものの生化学的機能の研究が発展し、癌細胞に関する多くの知見が得られるようになり、さらにそれらの細胞を用いた医薬品開発、或いは患者本人の癌細胞に有効な抗癌剤をスクリーニングする手段（抗癌剤感受性スクリーニング）として極めて有用な技術となるものと期待される。特に最後の抗癌剤感受性スクリーニング技術が確立されれば、抗癌剤を投与するとき、副作用を最小限に、癌組織に対する効果を最大限にすることができ、癌の種類、部位及び個人の薬に対する感受性の差等へも対応できるようになり、もっとも効率良く抗癌剤使いこなす方法を示せることとなる。 Cancer is still the top cause of death among Japanese people, and it is still said that about 30% of the total population will die from cancer. In recent years, techniques such as surgery, physical therapy such as radiation irradiation, chemotherapy such as administration of anticancer drugs, and cell therapy using autoimmune response have been developed and proposed as treatment methods. Currently, no cure has been established. The reason for this is that the target cancer cells are biochemically diversified and it is difficult to set the goal of technological development such as batching cancer cells, and in order to investigate the biochemical function of the cancer cells, cancer tissue From that, cancer cells cannot be isolated with high purity. Therefore, if this isolation technology is developed and established, research on the biochemical functions of cancer cells themselves will develop, and many insights about cancer cells will be obtained, and drug development using these cells will be possible. Alternatively, it is expected to be an extremely useful technique as a means for screening an anticancer agent effective for a patient's own cancer cells (anticancer agent sensitivity screening). In particular, if the last anti-cancer drug sensitivity screening technology is established, when anti-cancer drugs are administered, side effects can be minimized, the effect on cancer tissue can be maximized, and the sensitivity to cancer type, site and individual drugs can be improved. It will be possible to deal with differences and the like, and it will show the most efficient way to use anticancer drugs.
癌細胞は正常細胞と異なり、生体内組織、細胞、生体外の器材表面等のいずれにも接着することなく増殖することができる。すなわち癌細胞は足場非依存性細胞であり、以前よりこの特性を利用し癌細胞を単離、精製する方法が注目されてきた。その中で、特に軟寒天コロニー形成試験が良く知られており、この方法とは、測定前に基材表面に半固形の軟寒天培地を塗布し、その軟寒天のような親水性表面に癌細胞が含まれた組織由来の細胞を塗布することで、正常細胞を付着、増殖させないようにするものである。一方、癌細胞は基材表面に付着することなく増殖し、結果として癌細胞を選択的に分離、組織中の癌細胞含有率を測定するものである。具体的には、３５ｍｍもしくは６０ｍｍディッシュ上に半固形軟寒天培地を分注し、細胞を３〜４週間培養し、ディッシュ上のコロニーをカウントするものである。この軟寒天コロニー形成試験法とはすでに手法的に確立されたものであるが、問題点としては、大量のサンプルをテストするには時間と手間がかかり、しかも、コロニーサイズが均一ではないので実験結果にばらつきが出ること、軟寒天が不透明であり軟寒天中に存在する癌細胞コロニーを観察し難いこと、また、その癌細胞コロニーを利用しようとするとき軟寒天を溶解させなくてはならず癌細胞に温度ストレスがかかること、さらにその際に軟寒天が混入してしまうこと等が挙げられ、これらの問題点を全て解決できるような技術が望まれていた。 Unlike normal cells, cancer cells can proliferate without adhering to any of in vivo tissues, cells, in vitro device surfaces, and the like. That is, cancer cells are anchorage-independent cells, and a method for isolating and purifying cancer cells using this characteristic has been attracting attention for some time. Among them, the soft agar colony formation test is particularly well known. This method is a method in which a semi-solid soft agar medium is applied to a substrate surface before measurement, and a hydrophilic surface such as soft agar is treated with cancer. By applying cells derived from tissues containing cells, normal cells are prevented from attaching and growing. On the other hand, cancer cells grow without adhering to the surface of the substrate, and as a result, cancer cells are selectively separated and the content of cancer cells in the tissue is measured. Specifically, a semi-solid soft agar medium is dispensed on a 35 mm or 60 mm dish, the cells are cultured for 3 to 4 weeks, and the colonies on the dish are counted. This soft agar colony formation test method has already been established by the method, but the problem is that it takes time and effort to test a large number of samples, and the experiment is not uniform because the colony size is not uniform. Results vary, soft agar is opaque, it is difficult to observe cancer cell colonies present in soft agar, and soft agar must be dissolved when trying to use the cancer cell colonies There is a need for a technique that can solve all of these problems, such as the fact that cancer cells are subjected to temperature stress and that soft agar is mixed at that time.
このような課題を解決すべく、これまで多くの開発がなされてきた。このことは上述した抗癌剤感受性スクリーニング技術の開発経緯で具体化できる。その一つとして、コラーゲンマトリックス中で培養する抗癌剤感受性試験（ＣＤ−ＤＳＴ法、Ｃｏｌｌａｇｅｎ ｇｅｌ−ｄｒｏｐｌｅｔ−ｅｍｂｅｄｄｅｄ−Ｃｕｌｔｕｒｅ ｄｒｕｇ ｓｅｎｓｉｔｉｖｉｔｙ ｔｅｓｔ）が挙げられる。この方法であるとコラーゲンマトリックス中で培養し、少数の細胞で測定可能であるが、このコラーゲンマトリックスによって癌組織から癌細胞を効率良く単離するものではなく、あらかじめ別の手段で癌細胞を単離する必要性があり、上記課題を解決するような技術ではなかった。また、癌細胞の細胞に約２週間必要であり、基材も高価なものであった。Ｆｕｋａｚａｗａら（Ａｎａｌ Ｂｉｏｃｈｅｍ．（１９９５））は、ポリヒドロキシエチルメタクリル酸（Ｐｏｌｙ（ＨＥＭＡ））をマイクロプレートに被覆し、癌細胞特有のＭＴＴ、または３Ｈ−チミジンの取り込みで増殖を評価し、軟寒天コロニー形成試験代替法の結果と強く相関することを見出した。そして、幾つかの抗癌剤がスクリーニングされており、操作が簡便、迅速、定量的、経済的であることが分かったが、（Ｐｏｌｙ（ＨＥＭＡ））の被覆が用事調整であり、操作が煩雑であった。Many developments have been made so far to solve such problems. This can be realized by the development of the anticancer drug sensitivity screening technique described above. One of them is an anticancer agent sensitivity test (CD-DST method, Collagen gel-droplet-embedded-culture drug sensitivity test) cultured in a collagen matrix. In this method, it is possible to measure in a small number of cells after culturing in a collagen matrix. However, this collagen matrix does not efficiently isolate cancer cells from cancer tissue. There was a need to separate them, and it was not a technique for solving the above problems. Moreover, about 2 weeks were required for cancer cells, and the base material was expensive. Fukazawa et al. (Anal Biochem. (1995)) coated polyhydroxyethyl methacrylic acid (Poly (HEMA)) on microplates, evaluated proliferation by cancer cell-specific MTT, or 3H-thymidine incorporation, and soft agar. We found a strong correlation with the results of the alternative method of colony formation test. Several anticancer agents have been screened, and it has been found that the operation is simple, quick, quantitative, and economical. However, the coating of (Poly (HEMA)) is a matter of adjustment and the operation is complicated. It was.
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、透明な含水ゲルが固定化された基材表面を利用することを特徴とする軟寒天コロニー形成試験代替法を提供することを目的とする。また、本発明は、その製造法、並びに利用方法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 soft agar colony formation test alternative method characterized by using a substrate surface on which a transparent hydrous gel is immobilized. Moreover, an object of this invention is to provide the manufacturing method and its utilization method.
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、特定条件で特定の含水ゲルを基材表面に被覆すると、その基材表面には正常細胞が付着せず、癌細胞だけが選択的に増殖することが見いだした。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, it was found that when a specific water-containing gel was coated on the substrate surface under specific conditions, normal cells did not adhere to the substrate surface, and only cancer cells selectively proliferated. The present invention has been completed based on such findings.
すなわち、本発明は、透明な含水ゲルが固定化された基材表面を利用した軟寒天コロニー形成試験代替法を提供する。
また、本発明は、上記軟寒天コロニー形成試験代替法を利用した評価、スクリーニング用キットを提供するものである。That is, the present invention provides an alternative method for soft agar colony formation test utilizing a substrate surface on which a transparent hydrous gel is immobilized.
The present invention also provides an evaluation and screening kit using the above-described alternative method for soft agar colony formation test.
本発明で得られる軟寒天コロニー形成試験代替法は、使用する基材表面に正常細胞を付着させず、癌細胞だけを極めて選択的に増殖させられ、採取した癌組織から癌細胞だけを効率良く単離させることができるようになる。さらに、この技術を用いると純度の高い癌細胞が得られるため、抗癌剤感受性試験を高効率に実施できるようになる。従って、本発明は医学、生物学等の分野における極めて有用な発明である。 The alternative method of soft agar colony formation test obtained in the present invention is that normal cells are not attached to the surface of the substrate to be used, but only cancer cells can be proliferated very selectively, and only cancer cells are efficiently extracted from the collected cancer tissue. It can be isolated. Furthermore, when this technique is used, high-purity cancer cells can be obtained, so that the anticancer drug sensitivity test can be carried out with high efficiency. Therefore, the present invention is extremely useful in the fields of medicine and biology.
本発明は、透明な含水ゲルが固定化された基材表面を利用した軟寒天コロニー形成試験代替法を提供する。本発明に使用される含水ゲルとは正常細胞を少なくとも１０日間付着させないものであり、含水時に透明であれば良く、好適なものとしてポリアクリルアミド、ポリエチレングリコール、澱粉のいずれか１種、もしくは２種以上の細胞が混合されたものが挙げられるが、その種類は、何ら制約されるものではない。これらの含水ゲルが被覆される基材としては、通常細胞培養に用いられるガラス、改質ガラス、ポリスチレン、ポリメチルメタクリレート等の化合物を初めとして、一般に形態付与が可能である物質、例えば、上記以外の高分子化合物、セラミックス類など全て用いることができる。その際、使用される材質は透明なものが、培養している癌細胞を観察することができ好ましい。 The present invention provides an alternative method of soft agar colony formation test utilizing a substrate surface on which a transparent hydrous gel is immobilized. The water-containing gel used in the present invention is a gel that does not allow normal cells to adhere for at least 10 days, and should be transparent when it contains water, and preferably one or two of polyacrylamide, polyethylene glycol, and starch. Although the thing with which the above cell was mixed is mentioned, The kind is not restrict | limited at all. Substrates on which these hydrogels are coated include substances that can generally be given a form, such as compounds such as glass, modified glass, polystyrene, polymethyl methacrylate, etc., which are usually used for cell culture, for example, other than the above All of these polymer compounds and ceramics can be used. In this case, it is preferable that the material used is transparent because the cultured cancer cells can be observed.
含水ゲルの培養基材への被覆方法は、特に制限されないが、例えば、特開平２−２１１８６５号公報に記載されている方法に従ってよい。すなわち、かかる被覆は、基材と上記モノマーまたはポリマーを、電子線照射（ＥＢ）、γ線照射、紫外線照射、プラズマ処理、コロナ処理、有機重合反応のいずれかにより、または塗布、混練等の物理的吸着等により行うことができる。その際、増殖した癌細胞を回収することを考慮すると含水ゲルは、基材表面上に共有結合で固定されている方が、回収した細胞にゲルの混入を防ぐことができ好ましい。 The method for coating the hydrated gel on the culture substrate is not particularly limited, and for example, the method described in JP-A-2-21865 may be used. That is, such coating is performed by applying a substrate and the above monomer or polymer to one of electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, organic polymerization reaction, or physical application such as coating and kneading. It can be performed by, for example, mechanical adsorption. In that case, considering that the proliferated cancer cells are collected, it is preferable that the water-containing gel is covalently fixed on the surface of the base material because the gel can be prevented from being mixed into the collected cells.
含水ゲルの被覆量は、乾燥時において１．５〜２０．０μｇ／ｃｍ２の範囲が良く、好ましくは２．０〜１０．０μｇ／ｃｍ２であり、さらに好ましくは３．０〜７．０μｇ／ｃｍ２である。１．５μｇ／ｃｍ２より少ない被覆量のとき、正常細胞が基材表面上に付着してしまい、作業効率が著しく悪くなり好ましくない。逆に２０．０μｇ／ｃｍ２以上であると、含水時のゲル表面の凹凸が顕著となり、培養している癌細胞を観察することが困難となり好ましくない。本発明における培養基材の形態は特に制約されるものではないが、例えばディッシュ、マルチプレート、フラスコ、セルインサートなどが挙げられる。The coverage of the water-containing gel may have a range of 1.5~20.0μg / cm 2 at the time of drying is preferably 2.0~10.0μg / cm 2, more preferably 3.0~7.0μg / Cm 2 . When the coating amount is less than 1.5 μg / cm 2 , normal cells adhere to the surface of the substrate, which is not preferable because work efficiency is remarkably deteriorated. On the contrary, if it is 20.0 μg / cm 2 or more, the unevenness of the gel surface becomes remarkable when water is contained, which makes it difficult to observe the cultured cancer cells. The form of the culture substrate in the present invention is not particularly limited, and examples thereof include dishes, multiplates, flasks, and cell inserts.
本発明に使用される細胞は、癌細胞であれば良く、生体組織から直接採取した細胞、患者から直接採取した細胞、或いは例えばＨＢＣ−４、ＢＳＹ−１、ＨＢＣ−５、ＭＣＦ−５、ＭＣＦ−７、ＭＤＡ−ＭＢ−２３１、Ｕ２５１、ＳＦ−２６８、ＳＦ−２９５、ＳＦ−５３９、ＳＮＢ−７５、ＳＮＢ−７８、ＨＣＣ２９９８、ＫＭ−１２、ＨＴ−２９、ＷｉＤｒ、ＨＣＴ−１５、ＨＣＴ−１１６、ＮＣＩ−Ｈ２３、ＮＣＩ−Ｈ２２６、ＮＣＩ−Ｈ５２２、ＮＣＩ−Ｈ４６０、Ａ５４９、ＤＭＳ２７３、ＤＭＳ１１４、ＬＯＸ−ＩＭＶＩ、ＯＶＣＡＲ−３、ＯＶＣＡＲ−４、ＯＶＣＡＲ−５、ＯＶＣＡＲ−８、ＳＫ−ＯＶ−３、ＲＸＦ−６３１Ｌ、ＡＣＨＮ、Ｓｔ−４、ＭＫＮ１、ＭＫＮ７、ＭＫＮ２８、ＭＫＮ４５、ＭＫＮ７４、ＮＲＫ−ｓｒｃ１、ＮＲＫ−ｍｏｓ１などの細胞株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。これら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、サル、イヌ、ネコ、ウサギ、ラット、ブタ、ヒツジなどが挙げられる。 The cell used in the present invention may be a cancer cell, a cell collected directly from a living tissue, a cell collected directly from a patient, or for example, HBC-4, BSY-1, HBC-5, MCF-5, MCF -7, MDA-MB-231, U251, SF-268, SF-295, SF-539, SNB-75, SNB-78, HCC2998, KM-12, HT-29, WiDr, HCT-15, HCT-116 , NCI-H23, NCI-H226, NCI-H522, NCI-H460, A549, DMS273, DMS114, LOX-IMVI, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3, RXF -631L, ACHN, St-4, MKN1, MKN7, MKN28, MKN45, MKN74, NRK-s c1, NRK-MOS1 its type but cell lines and the like, such as, which by no means limited. The origin of these cells is not particularly limited, and examples thereof include humans, monkeys, dogs, cats, rabbits, rats, pigs, and sheep.
本発明における上述した細胞を培養するための培地組成は特に限定されるものではなく、これらの細胞を培養する際に通常使われているもので良く、例えば、α−ＭＥＭ培地、Ｆ−１２培地、ＤＭＥＭ培地、或いはそれらの混合物に５％〜１０％ウシ血清を混合したもの、或いはそのものにさらに５０μｇ／ｍｌ濃度でアスコルビン酸２リン酸を加えたものでも良い。 The medium composition for culturing the above-described cells in the present invention is not particularly limited, and may be those usually used for culturing these cells. For example, α-MEM medium, F-12 medium , DMEM medium, or a mixture thereof containing 5% to 10% bovine serum, or a substance obtained by further adding ascorbic acid diphosphate at a concentration of 50 μg / ml may be used.
本発明における上述した細胞を培養するための播種濃度は特に限定されるものではなく、これらの細胞を培養する際に通常使われている条件で良く、培養される細胞によっても異なるが、１００００個／ｃｍ２以下が良く、好ましくは５０００個／ｃｍ２以下が良く、さらに好ましくは３０００個／ｃｍ２以下が良い。細胞密度が１００００個／ｃｍ２以上であると、培養細胞数が多過ぎることとなり、作業効率が著しく悪くなり好ましくない。The seeding concentration for culturing the above-described cells in the present invention is not particularly limited, and may be the conditions normally used for culturing these cells, and varies depending on the cells to be cultured, but 10,000 cells. / Cm 2 or less, preferably 5000 pieces / cm 2 or less, more preferably 3000 pieces / cm 2 or less. When the cell density is 10,000 cells / cm 2 or more, the number of cultured cells is too large, and the working efficiency is remarkably deteriorated.
本発明におけるその他の培養条件は、上記細胞を培養する際に通常使われている条件で良く特に限定されるものではない。本特許技術によれば、上述した透明な含水ゲルが固定化された基材表面を利用することで、正常細胞を全く増殖させず、癌細胞のみを増殖させることができるようになる。しかも本特許技術であれば、透明な器材表面であるため培養中の癌細胞の顕微鏡観察ができ、また培養後に各種細胞染色を行え、そのまま測定することもできるようになる。また、器材表面の含水ゲル固定化されているため、培地中に混入するような問題もない。 Other culture conditions in the present invention are not particularly limited and may be conditions normally used when culturing the cells. According to this patent technology, by using the base material surface on which the above-described transparent hydrous gel is immobilized, it becomes possible to grow only cancer cells without growing normal cells at all. In addition, with this patented technology, since the surface is transparent, cancer cells in culture can be observed with a microscope, and after cell culture, various cell stains can be performed and measured as they are. Moreover, since the hydrated gel is immobilized on the surface of the equipment, there is no problem of being mixed into the medium.
本発明に示される軟寒天コロニー形成試験代替法の用途は何ら制約されるものではないが、医薬品開発、或いは患者本人の癌細胞に有効な抗癌剤をスクリーニングする手段（抗癌剤感受性スクリーニング）として極めて有用な技術となるものと期待される。特に後者の抗癌剤感受性スクリーニング技術が確立されれば、抗癌剤を投与するとき、副作用を最小限に、癌組織に対する効果を最大限にすることができ、癌の種類、部位及び個人の薬に対する感受性の差等へも対応できるようになり、もっとも効率良く抗癌剤使いこなす有力な手段となる。その方法をキット化した軟寒天コロニー形成試験代替法キット、抗癌剤スクリーニングキットとしても有用な技術である。 The use of the alternative method for soft agar colony formation test shown in the present invention is not limited at all, but it is extremely useful as a means for screening for an anticancer drug effective for drug development or the patient's own cancer cells (anticancer drug sensitivity screening). Expected to be technology. In particular, if the latter anti-cancer drug susceptibility screening technology is established, when anti-cancer drugs are administered, side effects can be minimized and the effect on cancer tissues can be maximized, and the sensitivity to the type, site and individual drug of the cancer can be improved. It will be able to cope with the difference, etc., and will be the most effective way to use the anticancer drug efficiently. This technique is also useful as an alternative method kit for soft agar colony formation test kits and kits for screening anticancer agents.
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.
市販の３．５ｃｍφ培養皿（ベクトン・ディッキンソン・ラブウェア（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ Ｌａｂｗａｒｅ）社製ファルコン（ＦＡＬＣＯＮ）３００１）上に、アクリルアミドモノマーを１０ｗｔ％になるようにイソプロピルアルコールに溶解させたものを０．０７ｍｌ塗布した。０．２５ＭＧｙの強度の電子線を照射し、培養皿表面にアクリルアミドポリマー（ＰＡＡｍ）を固定化した。照射後、イオン交換水により培養皿を洗浄し、残存モノマーおよび培養皿に結合していないＰＡＡｍを取り除き、クリーンベンチ内で乾燥し、エチレンオキサイドガスで滅菌することで本発明の細胞培養器材を得た。基材表面におけるゲル（乾燥状態）を測定したところ、５．２μｇ／ｃｍ２被覆されていることが分かった。上述した含水ゲルを固定化した培養器材表面上に、正常ラット腎由来の線維芽細胞様株細胞であるＮＲＫ細胞、並びにそれを癌化させたＮＲＫ−ｓｒｃ１細胞、及びＮＲＫ−ｍｏｓ１細胞を１×１０３ｃｅｌｌｓ／ｃｍ２となるように播種した。培地としては、５％牛胎児血清を含むＤＭＥＭ培地を使用した。５日間の培養を継続した後、ＮＲＫ細胞、ＮＲＫ−ｓｒｃ１細胞、及びＮＲＫ−ｍｏｓ１細胞それぞれの増殖性を測定した。得られた結果を図１に示す。0.07 ml of a commercially available 3.5 cmφ culture dish (Falcon 3001 manufactured by Becton Dickinson Labware) dissolved in isopropyl alcohol so that the acrylamide monomer is 10 wt% Applied. An electron beam having an intensity of 0.25 MGy was irradiated to immobilize acrylamide polymer (PAAm) on the surface of the culture dish. After irradiation, the culture dish is washed with ion-exchanged water to remove residual monomers and PAAm not bound to the culture dish, dried in a clean bench, and sterilized with ethylene oxide gas to obtain the cell culture equipment of the present invention. It was. When the gel (dried state) on the substrate surface was measured, it was found to be coated with 5.2 μg / cm 2 . On the surface of the culture device on which the above-mentioned hydrogel is immobilized, NRK cells that are fibroblast-like cell lines derived from normal rat kidney, and NRK-src1 cells and NRK-mos1 cells obtained by transforming them into 1 × It seed | inoculated so that it might become 10 < 3 > cells / cm < 2 >. As the medium, DMEM medium containing 5% fetal calf serum was used. After culturing for 5 days, the proliferation of each of NRK cells, NRK-src1 cells, and NRK-mos1 cells was measured. The obtained results are shown in FIG.
培養する器材をコーニング社製細胞低付着性器材（比較例１）、ヌンク社製細胞低付着性器材（比較例２）、ファルコン社製細胞低付着性器材（比較例３）、住友ベークライト社製細胞低付着性器材（比較例４）を用いて行う以外は、実施例１と同様な実験を実施した。得られた結果を図１に示す。 Corning's low cell adhesion equipment (Comparative Example 1), Nunk's low cell adhesion equipment (Comparative Example 2), Falcon's low cell adhesion equipment (Comparative Example 3), Sumitomo Bakelite Co., Ltd. An experiment similar to that of Example 1 was performed except that the low cell adhesion device (Comparative Example 4) was used. The obtained results are shown in FIG.
比較例１〜４の器材表面上では、正常細胞であるＮＲＫの増殖を完全に抑えられないため、軟寒天培地を用いたコロニー形成試験の代替として用いることは難しい。それに対し、実施例１では正常細胞であるＮＲＫの増殖をほぼ完全に抑えたうえ、ＮＲＫ−ｓｒｃ１細胞、及びＮＲＫ−ｍｏｓ１細胞は極めて良好な増殖性を示し、実施例１は軟寒天培地を用いたコロニー形成試験の代替法となることが分かる。また、当初の播種数からの簡便に増殖比率を算出することが可能であり、この増殖比率を参照として、各種抗癌薬候補薬効成分を倍地中に添加した場合の増殖比率とを比較することで、その抗癌薬候補成分による癌細胞の増殖抑制能を算出することが可能である。 Since the growth of NRK, which is a normal cell, cannot be completely suppressed on the surface of the devices of Comparative Examples 1 to 4, it is difficult to use as an alternative to the colony formation test using a soft agar medium. On the other hand, in Example 1, the growth of NRK, which is a normal cell, was almost completely suppressed, and NRK-src1 cell and NRK-mos1 cell showed extremely good growth properties. Example 1 uses a soft agar medium. It turns out to be an alternative to the colony formation test. In addition, it is possible to easily calculate the growth ratio from the initial seeding number, and with reference to this growth ratio, compare the growth ratio when various anticancer drug candidate medicinal ingredients are added to the medium. Thus, it is possible to calculate the cancer cell growth inhibitory ability of the anticancer drug candidate component.
市販の３．５ｃｍφ培養皿（ベクトン・ディッキンソン・ラブウェア（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ Ｌａｂｗａｒｅ）社製ファルコン（ＦＡＬＣＯＮ）３００１）上に、アクリルアミドモノマーを１５ｗｔ％（実施例２）、２０ｗｔ％（実施例３）になるようにイソプロピルアルコールに溶解させたものを０．０７ｍｌ塗布した。０．２５ＭＧｙの強度の電子線を照射し、培養皿表面にアクリルアミドポリマー（ＰＡＡｍ）を固定化した。照射後、イオン交換水により培養皿を洗浄し、残存モノマーおよび培養皿に結合していないＰＡＡｍを取り除き、クリーンベンチ内で乾燥し、エチレンオキサイドガスで滅菌することで本発明の細胞培養器材を得た。基材表面におけるゲル（乾燥状態）を測定したところ、５．８μｇ／ｃｍ２（実施例２）、６．８μｇ／ｃｍ２（実施例３）被覆されていることが分かった。上述した含水ゲルを固定化した培養器材表面上に、実施例１と同様にＮＲＫ細胞、並びにそれを癌化させたＮＲＫ−ｓｒｃ１細胞、及びＮＲＫ−ｍｏｓ１細胞を１×１０３ｃｅｌｌｓ／ｃｍ２となるように播種し、実施例１と同様な評価を行った。その結果、実施例２、３ともに正常細胞であるＮＲＫの増殖をほぼ完全に抑えたうえ、ＮＲＫ−ｓｒｃ１細胞、及びＮＲＫ−ｍｏｓ１細胞は極めて良好な増殖性を示した。On a commercially available 3.5 cm φ culture dish (Falcon 3001 manufactured by Becton Dickinson Labware), the acrylamide monomer is 15 wt% (Example 2) and 20 wt% (Example 3). Thus, 0.07 ml of a solution dissolved in isopropyl alcohol was applied. An electron beam having an intensity of 0.25 MGy was irradiated to immobilize acrylamide polymer (PAAm) on the surface of the culture dish. After irradiation, the culture dish is washed with ion-exchanged water to remove residual monomers and PAAm not bound to the culture dish, dried in a clean bench, and sterilized with ethylene oxide gas to obtain the cell culture equipment of the present invention. It was. When the gel (dry state) on the substrate surface was measured, it was found that 5.8 μg / cm 2 (Example 2) and 6.8 μg / cm 2 (Example 3) were coated. On the surface of the culture equipment on which the above-mentioned hydrogel was immobilized, the NRK cells, NRK-src1 cells and NRK-mos1 cells obtained by transforming them into cancer cells as in Example 1 were 1 × 10 3 cells / cm 2 . So that the same evaluation as in Example 1 was performed. As a result, in both Examples 2 and 3, the growth of NRK, which is a normal cell, was almost completely suppressed, and the NRK-src1 cell and the NRK-mos1 cell showed extremely good growth properties.
本発明で得られる軟寒天コロニー形成試験代替法であれば、使用する基材表面に正常細胞を付着させず、癌細胞だけを極めて選択的に増殖させられ、採取した癌組織から癌細胞だけを効率良く単離させることができるようになる。しかも本特許技術であれば、透明な器材表面であるため培養中の癌細胞の顕微鏡観察ができ、また培養後に各種細胞染色を行え、そのまま測定することもできるようになる。また、器材表面の含水ゲル固定化されているため、培地中に混入するような問題もない。さらに、この技術を用いると純度の高い癌細胞が得られるため、抗癌剤感受性試験を高効率に実施できるようになる。従って、本発明は医学、生物学等の分野における極めて有用な発明である。 With the soft agar colony formation test alternative method obtained in the present invention, normal cells are not attached to the surface of the substrate to be used, and only cancer cells can be proliferated very selectively. It will be possible to isolate efficiently. In addition, with this patented technology, since the surface is transparent, cancer cells in culture can be observed with a microscope, and after cell culture, various cell stains can be performed and measured as they are. Moreover, since the hydrated gel is immobilized on the surface of the equipment, there is no problem of being mixed into the medium. Furthermore, when this technique is used, cancer cells with high purity can be obtained, so that the anticancer drug sensitivity test can be carried out with high efficiency. Therefore, the present invention is extremely useful in the fields of medicine and biology.
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