JP4652139B2 - Very low proliferation cell line - Google Patents
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本発明は、低増殖性細胞株の樹立方法、特に超低増殖性細胞株の樹立方法、その方法により樹立された超低増殖性細胞株及びこれを用いたライ菌の培養方法に関する。 The present invention relates to a method for establishing a low-proliferation cell line, in particular, a method for establishing an ultra-low-proliferation cell line, an ultra-low-proliferation cell line established by the method, and a method for culturing rye fungi using the same.
ハンセン病はＷＨＯ指定特定伝染病であり、全世界で約１２００万人の患者がいると推定されている。また、年間５０万人以上の患者が新たに発生しているとされる。この感染症の原因菌であるライ菌（Mycobacterium leprae）は細胞内寄生菌であり、マクロファージ、シュワン細胞、血管内皮細胞等の内部で増殖する。
ライ菌は試験管内で培養することはできず、抗ライ薬の効果を調べるためにはマウス足底部でのライ菌培養が用いられている。また、ライ菌はアルマジロに感染することが発見されており、ライ腫型ライ（ＬＬ型：ライ菌が最も増殖する臨床病型）の疾患モデルとしてライ研究に利用され、これを用いてライワクチンをつくることが試みられている。
Leprosy is a WHO designated infectious disease and it is estimated that there are approximately 12 million patients worldwide. In addition, more than 500,000 patients are newly generated annually. Mycobacterium leprae , the causative bacterium of this infection, is an intracellular parasite that grows inside macrophages, Schwann cells, vascular endothelial cells, and the like.
Rye bacteria cannot be cultured in a test tube, and in order to examine the effect of anti-rye drugs, rye bacteria culture at the foot of the mouse is used. In addition, it has been discovered that Rye bacteria are infected with armadillo, and it is used for Rye research as a disease model of Ryeoma-type Rye (LL type: clinical disease type in which Rye bacteria are most proliferating). Attempts to make
しかし、動物を用いた培養は取扱いが不便である。マクロファージ、神経細胞などを感
染細胞として用いることも検討されてきたが、培養には成功していない。このように、ｉｎ ｖｉｔｒｏ培養が不可能であるという事情がハンセン病研究の遅れの大きな原因となっている。また、上述の通り、ハンセン病は現在でも多数の患者を有する感染症である。ハンセン病治療薬としてはサルファ剤や各種抗生物質が用いられており、耐性菌に対しては２〜３剤併用の多剤療法が行なわれているが、これらに対しては耐性菌が発生しており、有用な抗ライ薬のスクリーニング系が望まれている。
However, handling using animals is inconvenient. The use of macrophages, nerve cells and the like as infected cells has also been studied, but has not been successfully cultured. Thus, the fact that in vitro culture is impossible is a major cause of delay in leprosy research. In addition, as described above, leprosy is still an infectious disease having a large number of patients. As leprosy treatment drugs, sulfa drugs and various antibiotics are used, and multi-drug therapy of 2 to 3 drugs is performed against resistant bacteria, but resistant bacteria have occurred against these Therefore, a screening system for useful anti-rai drugs is desired.
本発明は、動物を利用することなく、ライ菌の長期的なｉｎ ｖｉｔｒｏ培養を可能とする培養系の確立を目的とする。 An object of the present invention is to establish a culture system that enables long-term in vitro culture of Rye bacteria without using animals.
ライ菌は倍化時間が１４日以上とされる。このため、ライ菌を細胞に感染させる場合、細胞の倍化時間がライ菌の倍化時間よりさらに長くないと細胞内でライ菌が増殖できない。また、感染する細胞はライ菌が増殖しやすい種類の細胞でなければならない。
本発明者はこれらの点に着目し、生体内においてライ球形成（ライ腫型ライの良好な増殖形態とされる）が認められる血管内皮細胞から、増殖速度が低く、ライ菌の感染増殖に適した細胞株を樹立することを試みた。その結果、特定の方法を採ることにより低増殖性細胞株、特に超低増殖性細胞株が樹立され得ること、及び、この方法により得られた細胞株がライ菌の感染増殖に適していることを見出し、本発明を完成するに至った。
The doubling time of lei bacteria is 14 days or more. For this reason, when a cell is infected with a lye bacterium, the lye bacterium cannot grow in the cell unless the doubling time of the cell is longer than the doubling time of the lye bacterium. In addition, the cells to be infected must be the kind of cells in which the bacterium is easy to grow.
The present inventor pays attention to these points, and from the vascular endothelial cells in which rye sphere formation (which is considered to be a good proliferative form of lyeoma type lye) is observed in the living body, the proliferation rate is low, and the infectious proliferation of lye bacteria An attempt was made to establish a suitable cell line. As a result, by adopting a specific method, it is possible to establish a low-proliferation cell line, especially an ultra-low-proliferation cell line, and that the cell line obtained by this method is suitable for infectious growth of rye fungi As a result, the present invention has been completed.
すなわち、本発明は以下の低増殖性細胞株の樹立方法、特に超低増殖性細胞株の樹立方法、その方法により樹立された超低増殖性細胞株及びこれを用いたライ菌の培養方法を提供する。
１．ＳＣＩＤ（重症複合型免疫不全症）マウス皮下可植性ヒト血管肉腫より得た腫瘍細胞をマウス血管肉腫細胞株ＩＳＯＳ−１培養上清含有培地で培養し、トリプシン−ＥＤＴＡ処理によって剥離しない細胞株を選択し、得られた細胞株を剥離継代培養することを特徴とする低増殖性細胞株の樹立方法。
２．前記１に記載の方法において、マウス血管肉腫細胞株ＩＳＯＳ−１培養上清含有培地におけるマウス血管肉腫細胞株ＩＳＯＳ−１培養上清含有濃度を５０％以下とする前記１に記載の低増殖性細胞株の樹立方法。
３．マウス血管肉腫細胞株ＩＳＯＳ−１培養上清含有濃度を２０％以下とする前記２に記載の超低増殖性細胞株の樹立方法。
４．ＳＣＩＤマウス皮下可植性ヒト血管肉腫に由来し、前記３に記載の方法により樹立された超低増殖性細胞株。
５．倍化時間が１０００時間以上である前記４に記載の超低増殖性細胞株。
６．ＳＣＩＤマウス皮下可植性ヒト血管肉腫ＷＢ−ＳＣＩＤに由来する超低増殖性細胞株ＳＬＧ／ＷＢ−ＳＣＩＤ（平成１７年６月８日付で、独立行政法人産業技術総合研究所特許生物寄託センターに受領番号ＦＥＲＭ−ＡＢＰ−１０３４９号として寄託）。
７．前記４〜６のいずれかに記載の超低増殖性細胞株を宿主とすることを特徴とするライ
菌の培養方法。
That is, the present invention provides the following method for establishing a low-proliferative cell line, in particular, a method for establishing an ultra-low-proliferative cell line, an ultra-low-proliferative cell line established by the method, and a method for culturing rye fungi using the same. provide.
1. Tumor cells obtained from SCID (severe combined immunodeficiency) mouse subcutaneously transplantable human hemangiosarcoma were cultured in mouse hemangiosarcoma cell line ISOS-1 culture supernatant-containing cell line, and a cell line that was not detached by trypsin-EDTA treatment A method for establishing a low-proliferation cell line, comprising selecting and subculturing the obtained cell line.
2. 2. The method according to 1 above, wherein the concentration of the mouse hemangiosarcoma cell line ISOS-1 culture supernatant contained in the medium containing the mouse hemangiosarcoma cell line ISOS-1 culture supernatant is 50% or less. How to establish a stock.
3. 3. The method for establishing an ultra-low proliferative cell line according to 2 above, wherein the concentration of the mouse hemangiosarcoma cell line ISOS-1 culture supernatant is 20% or less.
4). An ultra-low proliferative cell line derived from the SCID mouse subcutaneously transplantable human hemangiosarcoma and established by the method described in 3 above.
5. 5. The ultra-low proliferative cell line as described in 4 above, wherein the doubling time is 1000 hours or more.
6). Ultra-low proliferative cell line SLG / WB-SCID derived from SCID mouse subcutaneous implantable human hemangiosarcoma WB-SCID (Received by National Institute of Advanced Industrial Science and Technology Patent Biology Center on June 8, 2005) No. FERM-ABP-10349).
7). 7. A method for cultivating a rye fungus, comprising using the ultra-low proliferative cell line according to any one of 4 to 6 as a host.
本発明によれば、ライ菌の感染増殖に適した超低増殖性細胞株が得られる。このため、ライ菌の長期的なｉｎ ｖｉｔｒｏ培養が可能となる。 According to the present invention, an ultra-low proliferating cell line suitable for infectious growth of Rye bacteria is obtained. For this reason, long-term in vitro culture of Rye bacteria is possible.
（Ａ）低増殖性細胞株の樹立方法
はじめに、本発明による低増殖性細胞株の樹立方法について説明する。
本発明による低増殖性細胞株の樹立方法は、ＳＣＩＤ（重症複合型免疫不全症）マウス皮下可植性ヒト血管肉腫より得た腫瘍細胞をマウス血管肉腫細胞株ＩＳＯＳ−１培養上清含有培地で培養し、トリプシン−ＥＤＴＡ処理によって剥離しない細胞株を選択し、得られた細胞株を剥離継代培養することを特徴とする。
ＳＣＩＤ（severe combined immunodeficiency disease;重症複合型免疫不全症)マウスは、遺伝子再構成能に異常があるために成熟したＢおよびＴ細胞が先天的に欠損したマウスであり、ヒト新生児でみられる遺伝性の重症複合型免疫不全症のモデル動物である。レシピエントとしてのＳＣＩＤマウスは、種々の系統が知られているが、本発明ではいずれも用いることができる。一方、ヒト血管肉腫は稀ではあるが致死性の高い腫瘍である。
ＳＣＩＤマウス皮下可植性ヒト血管肉腫は、ヒト血管肉腫をＳＣＩＤマウスに移値して細胞樹立したものである。このようなＳＣＩＤマウス皮下可植性ヒト血管肉腫は、本発明者らにより樹立されており(Masuzawa M. et al.,摘valuation of recombinant interleukin-2 immunotherapy for human hemangiosarcoma in a SCID mice model(WB-SCID) J. Dermatol. Sci. 27(2):88-94,2001)、本発明でもこの腫瘍（ＷＢ−ＳＣＩＤ）またはこれに準じて作製したＳＣＩＤマウス皮下可植性ヒト血管肉腫を用いることができる。以下の説明では、便宜上、このようにして得られるＳＣＩＤマウス皮下可植性ヒト血管肉腫をＷＢ−ＳＣＩＤ腫瘍細胞と呼ぶ。
(A) Method for Establishing a Low Proliferative Cell Line First, the method for establishing a low proliferative cell line according to the present invention will be described.
The method for establishing a hypoproliferative cell line according to the present invention is a method in which tumor cells obtained from SCID (severe combined immunodeficiency) mouse subcutaneously transplantable human hemangiosarcoma are used in a medium containing mouse hemangiosarcoma cell line ISOS-1 culture supernatant. Culturing, selecting a cell line that does not detach by trypsin-EDTA treatment, and subjecting the obtained cell line to detachment subculture.
SCID (severe combined immunodeficiency disease) mice are mice in which mature B and T cells are congenitally deficient due to abnormal gene rearrangement ability, and are inherited in newborn humans. It is a model animal of severe combined immunodeficiency. Various strains of SCID mice as recipients are known, and any of them can be used in the present invention. On the other hand, human angiosarcoma is a rare but highly lethal tumor.
SCID mouse subcutaneously transplantable human hemangiosarcoma is a cell established by transferring human hemangiosarcoma to SCID mice. Such SCID mouse subcutaneous implantable human hemangiosarcoma has been established by the present inventors (Masuzawa M. et al., Evaluation of recombinant interleukin-2 immunotherapy for human hemangiosarcoma in a SCID mice model (WB- SCID) J. Dermatol. Sci. 27 (2): 88-94, 2001), also in the present invention, this tumor (WB-SCID) or SCID mouse subcutaneously transplantable human hemangiosarcoma prepared in accordance with this tumor can be used. it can. In the following description, for convenience, the SCID mouse subcutaneously implantable human hemangiosarcoma thus obtained is referred to as WB-SCID tumor cells.
本発明では、ＷＢ−ＳＣＩＤ腫瘍細胞を腫瘍から得た後、マウス血管肉腫細胞株ＩＳＯＳ−１培養上清含有培地で培養する。マウス血管肉腫細胞株ＩＳＯＳ−１培養上清を含まない培地ではＷＢ−ＳＣＩＤ腫瘍細胞は数日間で死滅するため、マウス血管肉腫細胞株ＩＳＯＳ−１培養上清は培養に必須である。マウス血管肉腫細胞株ＩＳＯＳ−１も本発明者らにより樹立されている(Masuzawa M. et al.,摘stablishment of a new murine-phenotypic angiosarcoma cell line(ISOS-1) J. Dermatol. Sci., 16(2): 91-98, 1998)。 In the present invention, WB-SCID tumor cells are obtained from a tumor and then cultured in a medium containing a mouse angiosarcoma cell line ISOS-1 culture supernatant. Since WB-SCID tumor cells die in several days in a medium that does not contain mouse hemangiosarcoma cell line ISOS-1 culture supernatant, mouse hemangiosarcoma cell line ISOS-1 culture supernatant is essential for culture. A mouse hemangiosarcoma cell line ISOS-1 has also been established by the present inventors (Masuzawa M. et al., Excision of a new murine-phenotypic angiosarcoma cell line (ISOS-1) J. Dermatol. Sci., 16 (2): 91-98, 1998).
マウス血管肉腫細胞株ＩＳＯＳ−１培養上清は、上記ＩＳＯＳ−１を培養することにより得られるが、典型的には、ＦＢＳ（ウシ胎児血清）を添加したＤＭＥＭ（ダルベッコ改変イーグル培地）を用いることができる。具体的には(a)培地としてＤＭＥＭ＋１０％ＦＢＳ＋２ｍＭ Ｌ−グルタミン＋１０ｍＭ Ｈｅｐｅｓ＋５０μｇ／ｍｌゲンタマイシンを用い、ＩＳＯＳ−１細胞を１×１０6程度フラスコで培養し、コンフルエントになり細胞数約１×１０8となった状態で培地を回収し、遠心（３０００ｒｐｍ）後、上清を凍結保存し、使用時に３７℃で解凍後、濾過して使用する。ＩＳＯＳ−１培養上清の添加濃度は好ましくは５０％以下である。 The culture supernatant of the mouse angiosarcoma cell line ISOS-1 can be obtained by culturing the above ISOS-1, but typically, DMEM (Dulbecco's modified Eagle medium) supplemented with FBS (fetal bovine serum) is used. Can do. Specifically, (a) DMEM + 10% FBS + 2 mM L-glutamine + 10 mM Hepes + 50 μg / ml gentamicin is used as a medium, and ISOS-1 cells are cultured in about 1 × 10 6 flasks to become confluent and the number of cells becomes about 1 × 10 8. The medium is collected in a fresh state, centrifuged (3000 rpm), the supernatant is stored frozen, thawed at 37 ° C., and filtered before use. The added concentration of the ISOS-1 culture supernatant is preferably 50% or less.
ＷＢ−ＳＣＩＤ腫瘍細胞を培養するための培地は、上記ＩＳＯＳ−１培養上清の添加を除けば通常と同様でよく、例えば、ＦＢＳ（ウシ胎児血清）を添加したＤＭＥＭ（ダルベッコ改変イーグル培地）を用いることができる(Masuzawa M. et al.,摘stablishment of a human hemangiosasarcoma cell line(ISO-HAS) Int. J. Cancer, 81(2):305-308,1999参照）。培養条件は後述の超低増殖性培養株を得る場合以外は通常の温度条件及び湿度条件を採用し得る。培養は接着培養にて行なう。 The medium for culturing WB-SCID tumor cells may be the same as that except for the addition of the above ISOS-1 culture supernatant. For example, DMEM (Dulbecco's modified Eagle medium) supplemented with FBS (fetal bovine serum) is used. (See Masuzawa M. et al., Stablishment of a human hemangiosasarcoma cell line (ISO-HAS) Int. J. Cancer, 81 (2): 305-308, 1999). As the culture conditions, normal temperature conditions and humidity conditions can be employed except for obtaining an ultra-low proliferative culture described below. Culture is performed by adhesion culture.
次いで、トリプシン−ＥＤＴＡ処理を行ない、剥離した細胞を除く処理を行なう。これにより、酵素抵抗性細胞以外の細胞が除去できる。トリプシン−ＥＤＴＡ処理は通常５回以上、好ましくは１０回以上行なう。
もっとも、このようにして得られたＷＢ−ＳＣＩＤ腫瘍細胞は、それ自体低増殖性で、本発明では、かくして得られたＷＢ−ＳＣＩＤ腫瘍細胞についてトリプシン−ＥＤＴＡ処理を行なった後、これを機械的に剥離して培養する手法を繰り返して継代培養を行なう（本願において「剥離継代培養」と呼ぶ。）。これにより、より低増殖性の細胞株を得ることができる。剥離継代培養における機械的剥離操作としては接着細胞を穏やかかつ効果的に剥離するために慣用される剥離方法が利用できるが、典型的にはピペッティング操作が用いられる。剥離継代培養は通常１０代以上、好ましくは１００代以上行なう。
得られた細胞は密度依存性であり、密度が高いほど増殖が活発化する。
Next, trypsin-EDTA treatment is performed to remove the detached cells. Thereby, cells other than enzyme resistant cells can be removed. The trypsin-EDTA treatment is usually performed 5 times or more, preferably 10 times or more.
However, the WB-SCID tumor cells thus obtained are themselves hypoproliferative, and in the present invention, the WB-SCID tumor cells thus obtained are treated with trypsin-EDTA and then mechanically treated. Subculturing is repeated by repeating the method of peeling and culturing (referred to as “peeling subculture” in this application). Thereby, a less proliferative cell line can be obtained. As the mechanical detachment operation in the detachment subculture, a detachment method commonly used for detaching adherent cells gently and effectively can be used, but a pipetting operation is typically used. Stripped subculture is usually performed for 10 generations or more, preferably 100 generations or more.
The obtained cells are density-dependent, and the higher the density, the more active the proliferation.
（Ｂ）超低増殖性細胞株の樹立方法
本発明による超低増殖性細胞株の樹立方法は、基本的には上記の低増殖性細胞株の樹立方法と同様であるが、初代培養時のマウス血管肉腫細胞株ＩＳＯＳ−１培養上清の添加濃度は好ましくは５〜２０％程度とする。５％以下では成育せず死滅する。２０％が最適である。また、インキュベータの設定温度を低くすることでより増殖性の低い細胞株が得られる。通常は３７℃以下、好ましくは３４℃以下とする。
(B) Method for Establishing an Ultralow Proliferative Cell Line The method for establishing an ultralow proliferative cell line according to the present invention is basically the same as the above-described method for establishing a low proliferative cell line. The added concentration of the mouse hemangiosarcoma cell line ISOS-1 culture supernatant is preferably about 5 to 20%. If it is less than 5%, it will not grow and die. 20% is optimal. In addition, a cell line with lower proliferation can be obtained by lowering the set temperature of the incubator. Usually, it is 37 ° C. or lower, preferably 34 ° C. or lower.
（Ｃ）（超）低増殖性細胞株
このようにして得られる（超）低増殖性細胞株は、マウスＣＤ３１、ＧＳＡ−１陽性であり、マウス内皮系細胞である。また、染色体はすべてマウス型で、最頻数は７７である。しかし、ヒト／マウス両クラスＩ抗原を同時発現している。
また、低増殖性細胞株で倍化時間が５０〜１００時間程度、超低増殖性細胞株では、５００時間を超え、１０００時間以上、さらに１５００時間以上の細胞株が得られる。このような細胞株としてＳＬＧ／ＷＢ−ＳＣＩＤが得られ、平成１７年６月８日付で、独立行政法人産業技術総合研究所特許生物寄託センターに受領番号ＦＥＲＭ−ＡＢＰ−１０３４９号として寄託した。
(C) (Ultra) hypoproliferative cell line The (super) hypoproliferative cell line obtained in this way is mouse CD31, GSA-1 positive and is a mouse endothelial cell. All chromosomes are mouse-type, and the most frequent number is 77. However, both human / mouse class I antigens are coexpressed.
In addition, a cell line having a doubling time of about 50 to 100 hours in a low-proliferation cell line and over 500 hours, 1000 hours or more, and further 1500 hours or more is obtained in an ultra-low growth cell line. SLG / WB-SCID was obtained as such a cell line, and deposited on June 8, 2005 as an accession number FERM-ABP-10349 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center.
（Ｄ）ライ菌の培養
上述のように、本発明による、低増殖性細胞株、特に超低増殖性細胞株は培養時間が１５００時間以上であり、しかも、至適温度が３４℃である。このため、倍化時間が約２週間（３３０時間）、至適温度が３１〜３４℃であるライ菌の培養に適している。
本発明による超低増殖性細胞株を用いてライ菌の培養を行なうには、ＳＬＧ／ＷＢ−ＳＣＩＤを３４℃インキュベータでＩＳＯＳ−１培養上清２０％添加の培地にて、７５ｃｍ2培養フラスコでコンフルエントになるまで培養した時点で、ライ菌２〜５×１０7を添加して培養を続ける。
(D) Cultivation of Rye Bacteria As described above, the low-proliferation cell line, particularly the ultra-low-proliferation cell line according to the present invention has a culture time of 1500 hours or more and an optimum temperature of 34 ° C. For this reason, the doubling time is about 2 weeks (330 hours), and it is suitable for cultivation of rye fungi having an optimum temperature of 31 to 34 ° C.
To cultivate Rye bacteria using the ultra-low proliferative cell line according to the present invention, SLG / WB-SCID is added to a culture medium containing 20% ISOS-1 culture supernatant in a 34 ° C. incubator in a 75 cm 2 culture flask. When the cells are cultured until they become confluent, Rye bacteria 2 to 5 × 10 7 are added and the culture is continued.
以下に実施例を挙げて本発明をより具体的に説明するが、これらは本発明を制限するものではない。なお、下記の手技はすべて無菌操作で行なった。 The present invention will be described more specifically with reference to the following examples, but these examples do not limit the present invention. All the following procedures were performed aseptically.
実施例１：低増殖性細胞株の樹立
（１）マウス血管肉腫細胞株ＩＳＯＳ−１培養上清の作成
培地としてＤＭＥＭ＋１０％ＦＢＳ＋２ｍＭ Ｌ−グルタミン＋１０ｍＭ Ｈｅｐｅｓ＋５０μｇ／ｍｌゲンタマイシンを用い、ＩＳＯＳ−１(Masuzawa M. et al., 忍stablishment of a new murine-phenotypic angiosarcoma cell line (ISOS-1) J. Dermatol. Sci., 16(2): 91-98, 1998)を１×１０6を１７５ｃｍ2フラスコで上記培地５０ｍｌ中１週間培養した。フラスコがコンフルエントになり細胞数約１×１０8となった状態で培地を回収し、遠心（３０００ｒｐｍで１０分間）後、上清を−２０℃凍結保存した。これを使用時３７℃で解凍後、０．４５μｍフィルターで濾過して用いる。
Example 1: Establishment of hypoproliferative cell line (1) Preparation of mouse hemangiosarcoma cell line ISOS-1 culture supernatant DMEM + 10% FBS + 2 mM L-glutamine + 10 mM Hepes + 50 μg / ml gentamicin was used as a medium, and ISOS-1 (Masuzawa M. et al., Shinobi stablishment of a new murine-phenotypic angiosarcoma cell line (ISOS-1) J. Dermatol. Sci., 16 (2): 91-98, 1998) 1 × 10 6 in 175 cm 2 flask with the above medium Cultured in 50 ml for 1 week. The culture medium was collected in a state where the flask became confluent and the number of cells became about 1 × 10 8, and after centrifugation (3000 rpm for 10 minutes), the supernatant was stored frozen at −20 ° C. When this is used, it is thawed at 37 ° C. and then filtered through a 0.45 μm filter.
（２）ＷＢ−ＳＣＩＤ細胞
ＳＣＩＤマウス皮下可植性ヒト血管肉腫ＷＢ−ＳＣＩＤ(Masuzawa M, et al., 摘valuation of recombinant interleukin-2 immunotherapy for human hemangiosarcoma in a SCID mice model(WB-SCID) J. Dermatol. Sci. 27(2):88-94,2001)の腫瘍を摘出した。
摘出腫瘍をシャーレに移し、生理食塩水内にて半割し、腫瘍中心半壊死組織より円刃を用いて擦過するようにして非酵素処理にて細胞遊出させた。遊出した細胞を遠心チューブに移し、遠心器にて４℃、１０００ｒｐｍで遠心し、細胞を沈下させ上清を除去し、沈下した細胞層を分離した。
(2) WB-SCID cells SCID mouse subcutaneously transplantable human hemangiosarcoma WB-SCID (Masuzawa M, et al., Evaluation of recombinant interleukin-2 immunotherapy for human hemangiosarcoma in a SCID mice model (WB-SCID) Dermatol. Sci. 27 (2): 88-94,2001) was extracted.
The excised tumor was transferred to a petri dish, divided in half in physiological saline, and allowed to migrate by non-enzymatic treatment by scraping with a circular blade from the tumor center half-necrotic tissue. The migrated cells were transferred to a centrifuge tube, and centrifuged at 4 ° C. and 1000 rpm in a centrifuge. The cells were allowed to settle, the supernatant was removed, and the settled cell layer was separated.
細胞層を上記（１）のマウス血管肉腫細胞株ＩＳＯＳ−１培養上清５０％添加し、ヒト血管肉腫細胞用培地（ＤＭＥＭ＋１０％ＦＢＳ＋マウス血管肉腫細胞株ＩＳＯＳ−１培養上清５０％添加）（以後培地と略す）にて再浮遊させて通常の培養皿に移し、ＣＯ2インキュベーター内にて３７℃、５％ＣＯ2の通常の培養環境で培養を開始した（初代培養）。
以後週に２回培地を交換し、培養開始１週間後、培地を除去し、生理食塩水で１回培養皿底を洗い、０．２５％トリプシン−ＥＤＴＡ（ＧＩＢＣＯ製）（以後、トリプシン−ＥＤＴＡと略す）を適量加え１０分間放置した。放置後、剥離した細胞を除去し、培養皿底に残存した細胞のみを生理食塩水で１回洗い、新たに新鮮な培地を加え継続培養した。上記の操作を週２回、計１０回繰り返し、トリプシン−ＥＤＴＡ処理にて剥離する細胞がないことを確認し、トリプシン−ＥＤＴＡで剥離しなかった細胞のみ培養を続けた。
The cell layer was added with 50% mouse hemangiosarcoma cell line ISOS-1 culture supernatant of (1) above, and medium for human hemangiosarcoma cells (DMEM + 10% FBS + mouse hemangiosarcoma cell line ISOS-1 culture supernatant 50%) ( Thereafter, the suspension was resuspended in a medium and transferred to a normal culture dish, and culture was started in a normal culture environment at 37 ° C. and 5% CO 2 in a CO 2 incubator (primary culture).
Thereafter, the medium was changed twice a week. After one week from the start of the culture, the medium was removed, the bottom of the culture dish was washed once with physiological saline, and 0.25% trypsin-EDTA (GIBCO) (hereinafter, trypsin-EDTA). (Abbreviated) was added for 10 minutes. After leaving, the detached cells were removed, and only the cells remaining on the bottom of the culture dish were washed once with physiological saline, and a fresh medium was newly added to continue culture. The above operation was repeated twice a week for a total of 10 times, and it was confirmed that no cells were detached by trypsin-EDTA treatment. Only cells that were not detached by trypsin-EDTA were continuously cultured.
（３）低増殖細胞株の選択
１カ月後、培養皿底に観察される細胞コロニーをトリプシン−ＥＤＴＡで１時間処理し、その後トランスファーピペットで約１０分間ピペッティングして機械的に細胞を剥離した。剥離細胞を新たな培養皿に移し、培養を継続した。
培養皿内で増殖した細胞について上記の同様にピペッティングして機械的に細胞を剥離、剥離継代培養を繰り返した。
２ケ月後、紐状に長い細胞群が単層敷石状に安定して培養された。この細胞は密度依存性（細胞密度が高いほど増殖は活発化）に増殖した。
径３５ｍｍ培養皿（９．６ｃｍ2）で培地２ｍｌ、培地交換週２回の培養条件で、１０5個の細胞を培養すると最も増殖する７日目から１０日目でも、倍化時間は約７０時間であり、低増殖性であった。
上記の培養条件で１０日目以降に細胞は培養皿をほぼ覆い、唐草模様状に単層に増殖した。コンフルエントの状態に近づくと、細胞の剥離が起こった。
細胞数を減らして培養を繰り返すことで細胞は性状を変化せず、ゆっくり増殖を続けた。
得られた細胞は紐状で長い形態（長さ：約１００〜２５０μｍ）である。また、得られた細胞を自動細胞分析装置（ＦＡＣＳｃａｎ：Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ社製）を用いＣＥＬＬＱｕｅｓｔ解析ソフトでＦＡＣＳ解析したところ、マウスＣＤ３１、ＧＳＡ−１陽性でマウス内皮系細胞であることが確認された。染色体はすべてマウス型で、７１〜７９に及び、最頻数は７７であった。また、ヒト／マウス両クラスＩ抗原を同時発現しており、電顕的には高密度顆粒が多く、きわめて細胞小器官に富んでいた。この細胞はＤＭＳＯ１０％添加培地で凍結保存が可絶であった。
(3) Selection of low-growth cell line One month later, cell colonies observed on the bottom of the culture dish were treated with trypsin-EDTA for 1 hour, and then the cells were mechanically detached by pipetting with a transfer pipette for about 10 minutes. . The detached cells were transferred to a new culture dish and the culture was continued.
The cells grown in the culture dish were pipetted in the same manner as described above to mechanically detach the cells, and the detachment subculture was repeated.
Two months later, a long cell group of cords was stably cultured in a single-layer paving stone shape. The cells proliferated in a density-dependent manner (proliferation increased as the cell density increased).
The doubling time is about 70 even on the 7th to 10th day when 10 5 cells are cultured under the culture conditions of 2 ml of medium in a 35 mm diameter dish (9.6 cm 2 ) and culture conditions of medium exchange twice a week. It was time and hypoproliferative.
From the 10th day onward under the above culture conditions, the cells almost covered the culture dish and grew into a single layer in an arabesque pattern. When approaching confluence, cell detachment occurred.
By reducing the number of cells and repeating the culture, the cells did not change their properties and continued to grow slowly.
The obtained cells are string-like and have a long form (length: about 100 to 250 μm). Further, when the obtained cells were subjected to FACS analysis with CELLQuest analysis software using an automatic cell analyzer (FACScan: manufactured by Becton Dickinson), it was confirmed that the cells were positive for mouse CD31 and GSA-1 and were mouse endothelial cells. All the chromosomes were mouse type, ranging from 71 to 79, and the most frequent number was 77. Furthermore, both human / mouse class I antigens were co-expressed, and electron microscopically, there were many high-density granules, and they were extremely rich in organelles. These cells could be frozen and stored in a medium supplemented with 10% DMSO.
実施例２：超低増殖性細胞株の樹立
実施例１と同様にしてＷＢ−ＳＣＩＤ細胞を得た。（１）培地に添加するＩＳＯＳ−１培養上清の添加比率を５０％から２０％に低下させ、（２）ＣＯ2インキュベーターの温度設定を３７℃から３４℃に低下させた他は実施例１と同様の培養条件でこれを培養した。
この結果、変更前の条件でコンフルエントに達した細胞密度の約５０％まで増殖した時点で細胞は超低増殖性となり、この時点での倍化時間は１５００時間以上であった。
細胞の形状等は実施例１と同様であった。単一株をＳＬＧ／ＷＢ−ＳＣＩＤとして、平成１７年６月８日付で、独立行政法人産業技術総合研究所特許生物寄託センターに寄託した（受領番号ＦＥＲＭ−ＡＢＰ−１０３４９号）。
Example 2: Establishment of an ultra-low proliferative cell line WB-SCID cells were obtained in the same manner as in Example 1. (1) Example 1 except that the addition ratio of the ISOS-1 culture supernatant added to the medium was reduced from 50% to 20%, and (2) the temperature setting of the CO 2 incubator was reduced from 37 ° C. to 34 ° C. This was cultured under the same culture conditions.
As a result, when the cell density grew to about 50% of the cell density that reached confluence under the conditions before the change, the cells became extremely low proliferative, and the doubling time at this point was 1500 hours or more.
The cell shape and the like were the same as in Example 1. A single strain was deposited as SLG / WB-SCID on June 8, 2005 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (reception number FERM-ABP-10349).
実施例３：ライ菌の培養
上記のＳＬＧ／ＷＢ−ＳＣＩＤ細胞株を７５ｃｍ2培養フラスコを用いて３４℃インキュベータでＩＳＯＳ−１培養上清２０％添加の培地にて培養し、コンフルエントになった時点（細胞数約１．５×１０6）で、Ｔｈａｉ５３株ライ菌（国立感染症研究所ハンセン病研究センター）２〜５×１０7個を培地に混入させ、上記と同様の培養条件で培養を継続した。
チール・ネルセン染色、ヒトライ菌ＤＮＡ特異的プライマーによるＰＣＲで得られた細胞中のライ菌を確認し、さらにＡＴＰ測定とヌードマウスの足蹠接種での腫瘤形成で生菌の確認を行なった。培養フラスコあたりの回収細胞を０．５％Ｔｒｉｔｏｎ−Ｘ１００処理し、Ｓｈｅｐａｒｄ法により菌数を算定した。これによりライ菌の長期培養が可能であることが確認された。
Example 3: Cultivation of Rye Bacteria The above SLG / WB-SCID cell line was cultured in a medium supplemented with 20% of ISOS-1 culture supernatant in a 34 ° C. incubator using a 75 cm 2 culture flask and became confluent (Number of cells: about 1.5 × 10 6 ) 2 to 5 × 10 7 Thai 53 strain Rai bacteria (National Institute of Infectious Diseases, Hansen's Disease Research Center) are mixed in the medium, and the culture is continued under the same culture conditions as above. did.
Rye bacteria in cells obtained by PCR with Thiel-Nelsen staining and human-trivial DNA-specific primers were confirmed, and further, viable bacteria were confirmed by ATP measurement and tumor formation by footpad inoculation of nude mice. The collected cells per culture flask were treated with 0.5% Triton-X100, and the number of bacteria was calculated by the Shepard method. Thus, it was confirmed that long-term culture of rye fungi is possible.
本発明によれば、ＳＣＩＤマウス皮下可植性ヒト血管肉腫から、増殖速度が低い（倍化時間で３００時間以上、特に１０００時間以上）細胞株が得られる。これは、ライ菌の増殖速度よりも低増殖性であり、至適温度もライ菌に類以しており、ライ菌の感染する血管内皮細胞由来であることからライ菌の感染増殖に適していると考えられ、実際にその有効性が確認された。この結果、ライ菌のｉｎ ｖｉｔｒｏでの培養が可能になり、ライ菌の研究及び抗ライ薬のスクリーニング等に有用である。また、本発明による細胞株はヒト／マウス両クラスＩ抗原を同時発現しておりヒト血管肉腫の特性解明の上でも有用性が期待される。
According to the present invention, a cell line having a low growth rate (doubling time of 300 hours or more, particularly 1000 hours or more) can be obtained from SCID mouse subcutaneously transplantable human hemangiosarcoma. This is less proliferative than the growth rate of lei bacteria, and the optimum temperature is similar to that of lei bacteria, and since it is derived from vascular endothelial cells infected by lei bacteria, it is suitable for infectious growth of lei bacteria. The effectiveness was actually confirmed. As a result, in vitro culturing of Rye bacteria becomes possible, which is useful for research on Rye bacteria, screening for anti-Rye drugs, and the like. The cell line according to the present invention simultaneously expresses both human / mouse class I antigens, and is expected to be useful for elucidating the characteristics of human hemangiosarcoma.
Claims (7)
A method for cultivating a rye fungus, comprising using the ultra-low proliferative cell line according to any one of claims 4 to 6 as a host.
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