JP6331524B2 - Standard cell solution - Google Patents
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Description
本発明は、標準細胞液に関する。 The present invention relates to standard cell fluids.
癌は世界各国で死因の上位を占め、わが国においては年間３０万人以上が癌によって死亡しており、その早期発見及び治療が望まれている。癌による人の死亡は、癌の転移再発によるものがほとんどである。癌の転移再発は、癌細胞が原発巣から血管又はリンパ管を経由して、別臓器組織の血管壁に定着、浸潤して微小転移巣を形成することで起こる。このような血管又はリンパ管を通じて人の体内を循環する癌細胞は、血中循環癌細胞（Ｃｉｒｃｕｌａｔｉｎｇ Ｔｕｍｏｒ Ｃｅｌｌ、以下、場合により「ＣＴＣ」という。）と呼ばれている。 Cancer occupies the top cause of death in countries around the world, and more than 300,000 people die from cancer annually in Japan, and early detection and treatment thereof are desired. Most deaths due to cancer are due to recurrence of cancer metastasis. Recurrence of cancer metastasis occurs when cancer cells settle and infiltrate into the blood vessel wall of another organ tissue from the primary lesion via blood vessels or lymphatic vessels to form micrometastasis. Cancer cells circulating in the human body through such blood vessels or lymphatic vessels are called circulating tumor cells (hereinafter referred to as “CTC” in some cases).
血液には赤血球、白血球及び血小板等の血球成分が多く含まれており、血液１ｍＬあたりの血球成分の個数は３．５〜９．０×１０９個ともいわれている。これに対してＣＴＣは僅か数個程度しか存在しないため、血球成分の中からＣＴＣを効率的に捕捉及び検出する必要がある。ＣＴＣを捕捉及び検出する装置について種々検討されており、例えば、特許文献１には、ニッケル基板に微細貫通孔を有するニッケル基板をフィルタとし、その上下に、試料供給口を備えるポリジメチルシロキサン（ＰＤＭＳ）製上部部材と試料排出口を備える下部部材とを備えるマイクロ流体デバイスが開示されている。 Blood contains many blood cell components such as red blood cells, white blood cells, and platelets, and the number of blood cell components per mL of blood is said to be 3.5 to 9.0 × 10 9 . On the other hand, since there are only a few CTCs, it is necessary to efficiently capture and detect CTCs from blood cell components. Various devices for capturing and detecting CTC have been studied. For example, in Patent Document 1, a nickel substrate having a fine through hole in a nickel substrate is used as a filter, and polydimethylsiloxane (PDMS) having sample supply ports above and below it. ) A microfluidic device comprising an upper member made and a lower member comprising a sample outlet is disclosed.
特許文献１に記載のマイクロ流体デバイスをはじめとし、ＣＴＣを捕捉及び検出するデバイス（以下、「ＣＴＣ捕捉デバイス」ともいう。）の多くは、専用の処理液を通液させ、細胞固定処理、細胞膜透過処理及び抗原抗体反応を行うことで、ＣＴＣと白血球との染め分けを行うことでＣＴＣを検出している。該処理液を通液して染色を行い、染色されたＣＴＣを確認できなかった場合、検体中にＣＴＣが存在しない可能性が考えられるが、一方で、該処理液が染色性能を満たしていない可能性も懸念される。後者の場合では、検体中のＣＴＣを見逃す可能性があり、ＣＴＣ捕捉デバイスの性能に重大な影響を及ぼす。しかしながら、これらの処理液の染色性能を確認するための方法は知られていない。 Many of the devices that capture and detect CTC (hereinafter also referred to as “CTC capture device”), including the microfluidic device described in Patent Document 1, are passed through a dedicated treatment solution to allow cell fixation treatment, cell membrane By performing permeabilization and antigen-antibody reaction, CTCs are detected by differentiating CTCs and leukocytes. When staining is performed by passing the treatment liquid and the stained CTC cannot be confirmed, there is a possibility that CTC does not exist in the specimen, but the treatment liquid does not satisfy the staining performance. The possibility is also a concern. In the latter case, the CTC in the specimen can be missed, which has a significant impact on the performance of the CTC capture device. However, a method for confirming the dyeing performance of these treatment liquids is not known.
そこで本発明では、ＣＴＣと白血球とを染め分ける処理液の染色性能を確認するための標準細胞液を提供することを目的とする。 Therefore, an object of the present invention is to provide a standard cell solution for confirming the staining performance of a treatment solution that separates CTC and leukocytes.
本発明は、血中循環癌細胞（ＣＴＣ）と白血球とを染め分ける処理液の染色性能確認用標準細胞液であって、（ａ）ヒト癌細胞に特異的な抗原を有する細胞株と、（ｂ）ヒト白血球に特異的な抗原を含む細胞株と、（ｃ）吸着抑制剤と、を含み、（ａ）及び（ｂ）成分が、細胞固定剤により固定化された細胞株である、標準細胞液を提供する。 The present invention is a standard cell solution for confirming the staining performance of a treatment solution for dyeing circulating blood cancer cells (CTC) and leukocytes, and (a) a cell line having an antigen specific for human cancer cells; b) a cell line containing an antigen specific for human leukocytes, and (c) an adsorption inhibitor, wherein (a) and (b) components are cell lines immobilized by a cell fixative. Provide cell fluid.
本発明に係る標準細胞液に対して、ＣＴＣと白血球とを染め分ける処理液を用いて検出を行い、ＣＴＣと白血球とをそれぞれ確認することが可能であれば、処理液は染色性能を満たしていると確認することができる。また、ＣＴＣ捕捉デバイスにおいて、本発明に係る標準細胞液を通液し、ＣＴＣ捕捉デバイス内のフィルタに（ａ）及び（ｂ）成分を捕捉し、各ＣＴＣ捕捉デバイス専用の処理液を用いて細胞固定処理、細胞膜透過処理及び抗原抗体反応を行い、ＣＴＣと白血球をそれぞれ確認することが可能であれば、処理液は染色性能を満たしていると確認することができる。 If the standard cell solution according to the present invention is detected using a treatment solution that separates CTC and leukocytes, and the CTC and leukocytes can be confirmed, the treatment solution satisfies the staining performance. Can be confirmed. Further, in the CTC capturing device, the standard cell solution according to the present invention is passed, the components (a) and (b) are captured by the filter in the CTC capturing device, and the cells are processed using a processing solution dedicated to each CTC capturing device. If the fixation treatment, the cell membrane permeabilization treatment, and the antigen-antibody reaction can be performed and CTC and leukocytes can be confirmed, the treatment solution can be confirmed to satisfy the staining performance.
上記標準細胞液は、（ａ）成分が、サイトケラチンを発現するヒト癌細胞株であることが好ましい。サイトケラチンは、上皮細胞及び上皮由来細胞に発現する中間径フィラメント構成タンパク質であり、ＣＴＣを検出する際の標的抗原として利用される頻度が高い。このため、汎用性の高い標準細胞液を提供することが可能となる。 The standard cell solution is preferably a human cancer cell line in which component (a) expresses cytokeratin. Cytokeratin is an intermediate filament constituent protein expressed in epithelial cells and epithelial cells, and is frequently used as a target antigen when detecting CTC. For this reason, it becomes possible to provide a highly versatile standard cell solution.
上記標準細胞液は、（ｂ）成分が、ＣＤ４５を発現するヒト白血病細胞株であることが好ましい。ＣＤ４５は、全造血管細胞に発現するタンパク質であり、白血球を検出する際の標的抗原として利用される頻度が高い。このため、汎用性の高い標準細胞液を提供することが可能となる。 The standard cell solution is preferably a human leukemia cell line in which component (b) expresses CD45. CD45 is a protein expressed in all hematopoietic cells, and is frequently used as a target antigen when detecting leukocytes. For this reason, it becomes possible to provide a highly versatile standard cell solution.
上記標準細胞液は、（ｃ）成分が、ウシ血清アルブミン（ＢＳＡ）、ポリメタクリロイルオキシエチルホスファチジルコリン（ＰＭＰＣ）及びポリエチレングリコール（ＰＥＧ）から選択される少なくとも１種を含むことが好ましい。このような吸着抑制剤を用いることで、（ａ）及び（ｂ）成分の安定性がより向上し、標準細胞液の保存安定性がより向上する。 In the standard cell solution, the component (c) preferably contains at least one selected from bovine serum albumin (BSA), polymethacryloyloxyethyl phosphatidylcholine (PMPC), and polyethylene glycol (PEG). By using such an adsorption inhibitor, the stability of the components (a) and (b) is further improved, and the storage stability of the standard cell solution is further improved.
また、上記標準細胞液は、（ｃ）成分が、ＢＳＡであり、ＢＳＡ濃度が標準細胞液全量に対して０．１質量％〜５．０質量％であることが好ましい。これにより、（ａ）及び（ｂ）成分の安定性がより一層向上し、標準細胞液の保存安定性がより一層向上する。 In the standard cell solution, the component (c) is BSA, and the BSA concentration is preferably 0.1% by mass to 5.0% by mass with respect to the total amount of the standard cell solution. Thereby, the stability of the components (a) and (b) is further improved, and the storage stability of the standard cell solution is further improved.
本発明の標準細胞液によれば、ＣＴＣと白血球とを染め分ける処理液の染色性能を確認することが可能になる。処理液の染色性能を確認することで、より高精度の診断が実現し、ＣＴＣの見過ごしなどの誤診を防ぐことが可能になる。 According to the standard cell solution of the present invention, it is possible to confirm the staining performance of the treatment solution that separates CTC and leukocytes. By confirming the dyeing performance of the treatment liquid, more accurate diagnosis can be realized, and misdiagnosis such as oversight of CTC can be prevented.
以下、本発明を実施するための形態（以下、本実施形態という。）について、具体的に説明する。なお、本発明は、以下の実施形態に限定されるものではなく、その要旨の範囲内で種々変形して実施することができる。なお、本明細書中に記載される「〜」で表示される数値範囲は、それぞれの上限値及び下限値を範囲内に含む。 Hereinafter, a mode for carrying out the present invention (hereinafter referred to as the present embodiment) will be specifically described. In addition, this invention is not limited to the following embodiment, It can implement by changing variously within the range of the summary. In addition, the numerical value range displayed by "-" described in this specification includes each upper limit and lower limit within the range.
本実施形態に係る標準細胞液は、ＣＴＣと白血球とを染め分ける処理液の染色性能確認用である。好ましくは、ＣＴＣ捕捉デバイスを用いて、血液中のＣＴＣを分離し、ＣＴＣと残存する白血球とを染め分ける際に使用する処理液の染色性能確認用である。より好ましくは、ＣＴＣ捕捉デバイスを用いて、血液中のＣＴＣを分離し、ＣＴＣに特異的な抗原に対する抗体及び白血球に特異的な抗原に対する抗体で、ＣＴＣと残存する白血球とを染め分ける際に使用する処理液の染色性能確認用である。 The standard cell solution according to this embodiment is used for confirming the staining performance of a treatment solution that separates CTC and leukocytes. Preferably, the CTC capturing device is used to separate CTCs in blood and to check the staining performance of the treatment liquid used when the CTCs and the remaining white blood cells are dyed separately. More preferably, a CTC capture device is used to separate CTCs in blood and used to separate CTCs from remaining leukocytes with antibodies against antigens specific for CTCs and antigens specific for leukocytes. This is for confirming the dyeing performance of the treatment liquid.
ＣＴＣ捕捉デバイスには、（１）フィルタ方式及び（２）遠心分離方式等がある。（１）のフィルタ方式としては、特許文献１に記載されたマイクロキャビティアレイ法などがある。マイクロキャビティアレイ法では、所定の孔径を有するフィルタを保持するカートリッジ内に、癌患者血液を一定の速度で流し、装置内で、細胞固定処理、細胞膜透過処理、抗原抗体反応を半自動化処理で行い、ＣＴＣを検出する。この時に付属の処理液を使用する。しかし、付属の処理液の染色性能を簡便に確認する手段がなく、患者の血液中にＣＴＣが検出されない場合、ＣＴＣが血液中に存在しないのか、あるいは、処理液の劣化によるものなのかが判断できず、誤診につながるおそれがある。 CTC capture devices include (1) a filter system and (2) a centrifugal system. As the filter method of (1), there is a microcavity array method described in Patent Document 1. In the microcavity array method, cancer patient blood is flowed at a constant speed into a cartridge holding a filter having a predetermined pore size, and cell fixation processing, cell membrane permeation processing, and antigen-antibody reaction are performed in the apparatus by semi-automated processing. , CTC is detected. At this time, the attached processing solution is used. However, if there is no means for easily confirming the staining performance of the attached treatment liquid and CTC is not detected in the blood of the patient, it is determined whether CTC is not present in the blood or due to deterioration of the treatment liquid. It may not be possible and may lead to misdiagnosis.
本実施形態に係る標準細胞液は、上記の誤診を防ぐために、処理液の染色性能を確認するために用いられることが好ましい。 The standard cell solution according to the present embodiment is preferably used for confirming the staining performance of the treatment solution in order to prevent the above-mentioned misdiagnosis.
処理液としては、例えば、細胞固定処理用処理液、細胞膜透過処理用処理液、及び抗原抗体反応用処理液が挙げられる。 Examples of the treatment solution include a cell fixation treatment solution, a cell membrane permeabilization treatment solution, and an antigen-antibody reaction treatment solution.
細胞固定処理用処理液とは、細胞の腐敗又は凝集などの劣化を防ぐために、細胞を固定化するための処理液である。細胞固定処理用処理液としては、公知のものを用いることができ、例えば、ホルムアルデヒド、パラホルムアルデヒド（ＰＦＡ）、グルタルアルデヒド（ＧＡ）などの細胞固定剤を緩衝液に溶解したもの、及びメタノールなどが挙げられる。 The treatment solution for cell fixation treatment is a treatment solution for immobilizing cells in order to prevent deterioration such as cell decay or aggregation. As the treatment solution for cell fixation treatment, known ones can be used, for example, a solution in which a cell fixing agent such as formaldehyde, paraformaldehyde (PFA), glutaraldehyde (GA) is dissolved in a buffer solution, or methanol. Can be mentioned.
細胞膜透過処理用処理液とは、抗体などで細胞内の分子を染色するために、細胞膜を部分的に破壊するための処理液である。細胞膜透過処理用処理液としては、公知のものを用いることができ、Ｔｒｉｔｏｎ Ｘ−１００、ＮＰ−４０、Ｔｗｅｅｎ ２０などの界面活性剤、−２０℃ほどに冷却したメタノールなどの有機溶媒が挙げられる。 The cell membrane permeabilization treatment solution is a treatment solution for partially destroying the cell membrane in order to stain intracellular molecules with an antibody or the like. As the treatment solution for cell membrane permeabilization treatment, known ones can be used, and examples include surfactants such as Triton X-100, NP-40, and Tween 20, and organic solvents such as methanol cooled to about −20 ° C. .
抗原抗体反応用処理液とは、ＣＴＣ及び白血球それぞれに特異的な抗原を認識する抗体を含み、ＣＴＣと白血球とにそれぞれ異なる標識（例えば、蛍光波長の異なる蛍光物質）を付して、検出するための処理液である。標識はそれぞれの抗体に直接結合していてもよいし、例えば、それぞれの抗体と特異的に結合する二次抗体に標識が結合していてもよい。 The antigen-antibody reaction treatment solution includes an antibody that recognizes antigens specific to CTC and leukocytes, and detects CTC and leukocytes with different labels (for example, fluorescent substances having different fluorescence wavelengths). For the treatment liquid. The label may be directly bound to each antibody, or for example, the label may be bound to a secondary antibody that specifically binds to each antibody.
本実施形態に係る標準細胞液は、（ａ）ヒト癌細胞に特異的な抗原を有する細胞株と、（ｂ）ヒト白血球に特異的な抗原を有する細胞株と、（ｃ）吸着抑制剤と、を含む。（ａ）及び（ｂ）成分は、細胞固定剤により固定化されたものである。 The standard cell solution according to the present embodiment includes (a) a cell line having an antigen specific for human cancer cells, (b) a cell line having an antigen specific for human leukocytes, and (c) an adsorption inhibitor. ,including. Components (a) and (b) are immobilized by a cell fixing agent.
ヒト癌細胞に特異的な抗原としては、染色性能確認対象となる処理液に含まれるＣＴＣに特異的に結合する抗体が認識する抗原と同じ抗原であることが好ましい。このような抗原の例としては、例えば、ＣＴＣ捕捉デバイスで用いられる、ＣＴＣに特異的に結合する標識抗体が認識する抗原が挙げられる。より具体的には、上皮細胞及び上皮由来細胞に発現する抗原、例えば、サイトケラチン、上皮細胞接着分子（Ｅｐｉｔｈｅｌｉａｌ ｃｅｌｌ ａｄｈｅｓｉｏｎ ｍｏｌｅｃｌｅ：ＥｐＣＡＭ）、ＣＤ１４６、ＣＤ１７６などが挙げられる。ＣＴＣを検出する際の標的抗原として利用される頻度が高いことから、抗原としては、サイトケラチンが好ましい。サイトケラチンは、上皮細胞及び上皮由来細胞に発現する中間径フィラメント構成タンパク質のひとつである。 The antigen specific to human cancer cells is preferably the same antigen as the antigen recognized by the antibody that specifically binds to CTC contained in the treatment liquid to be stained. As an example of such an antigen, for example, an antigen recognized by a labeled antibody that specifically binds to CTC, which is used in a CTC capture device, can be mentioned. More specifically, antigens expressed in epithelial cells and epithelium-derived cells, such as cytokeratin, epithelial cell adhesion molecule (EpCAM), CD146, CD176, and the like can be mentioned. Cytokeratin is preferred as the antigen because it is frequently used as a target antigen in detecting CTC. Cytokeratin is one of the intermediate filament constituent proteins expressed in epithelial cells and epithelial-derived cells.
（ａ）ヒト癌細胞に特異的な抗原を有する細胞株としては、例えば、上述の抗原を発現するヒト癌細胞株、上述の抗原を発現するように遺伝子組み換えされた細胞株を挙げることができ、サイトケラチンを発現するヒト癌細胞株が好ましい。サイトケラチンを発現するヒト癌細胞株としては、特に、上皮系の癌細胞株、より具体的には、肺癌、乳癌、大腸癌、胃癌などの細胞株などが好ましい。このような細胞株としては、例えば非小細胞肺癌由来細胞株ＮＣＩ−Ｈ３５８、ヒト乳癌細胞株ＳＫ−ＢＲ−３などが挙げられる。 (A) Examples of cell lines having antigens specific to human cancer cells include human cancer cell lines that express the above-mentioned antigens, and cell lines that have been genetically modified to express the above-mentioned antigens. A human cancer cell line expressing cytokeratin is preferred. The human cancer cell line expressing cytokeratin is particularly preferably an epithelial cancer cell line, more specifically, a cell line such as lung cancer, breast cancer, colon cancer, and gastric cancer. Examples of such cell lines include non-small cell lung cancer-derived cell line NCI-H358 and human breast cancer cell line SK-BR-3.
血中のＣＴＣを分離する場合、血中に多く含まれる白血球の混入を避けることは困難である。例えば、フィルタ方式のＣＴＣ捕捉デバイスでは、フィルタ上にＣＴＣと細胞径が近い一部の白血球が残存する。そのため、白血球を染め分けて検出する必要がある。ヒト白血球に特異的な抗原としては、染色性能確認対象となる処理液に含まれる白血球に特異的に結合する抗体が認識する抗原と同じ抗原であることが好ましい。このような抗原の例としては、例えば、ＣＴＣ捕捉デバイスで用いられる、白血球に特異的に結合する標識抗体が認識する抗原が挙げられる。白血球を検出する際の標的抗原として利用される頻度が高いことから、抗原としては、ＣＤ４５が好ましい。ＣＤ４５は全造血管細胞に発現する抗原である。 When separating CTCs in blood, it is difficult to avoid contamination of white blood cells that are contained in the blood. For example, in a filter-type CTC capturing device, some white blood cells having a cell diameter close to that of CTC remain on the filter. Therefore, it is necessary to detect leukocytes separately. The antigen specific to human leukocytes is preferably the same antigen as the antigen recognized by the antibody that specifically binds to leukocytes contained in the treatment liquid to be stained. Examples of such antigens include, for example, antigens that are recognized by labeled antibodies that specifically bind to leukocytes, which are used in CTC capture devices. CD45 is preferred as the antigen because it is frequently used as a target antigen when detecting leukocytes. CD45 is an antigen expressed on all hematopoietic cells.
（ｂ）ヒト白血球に特異的な抗原を有する細胞株としては、例えば、上述の抗原を発現するヒト癌細胞株、上述の抗原を発現するように遺伝子組み換えされた細胞株を挙げることができ、ヒト白血病細胞株が好ましく、ＣＤ４５を発現するヒト白血病細胞株がより好ましい。このような細胞株としては、ヒト急性Ｔ細胞性白血病細胞由来細胞株Ｊｕｒｋａｔ、急性リンパ性白血病由来細胞株ＨＡＬ−０１などが挙げられる。 (B) Examples of cell lines having antigens specific for human leukocytes include human cancer cell lines that express the above-mentioned antigens, cell lines that have been genetically modified to express the above-mentioned antigens, Human leukemia cell lines are preferred, and human leukemia cell lines expressing CD45 are more preferred. Examples of such cell lines include human acute T-cell leukemia cell-derived cell line Jurkat, acute lymphoblastic leukemia-derived cell line HAL-01, and the like.
（ａ）及び（ｂ）成分の細胞株は、細胞膜固定化処理を行い、固定化することが好ましい。細胞を固定化しない場合、細胞株の腐敗又は凝集などを引き起こし、試薬の染色性の確認試験が困難になる。 The cell lines of the components (a) and (b) are preferably immobilized by performing a cell membrane immobilization treatment. If the cells are not fixed, cell strains will rot or aggregate, making it difficult to carry out a test for confirming the stainability of the reagent.
（ａ）及び（ｂ）成分の細胞株の固定化処理は、公知の方法により行うことができる。細胞固定剤としては、例えば、ホルムアルデヒド、パラホルムアルデヒド（ＰＦＡ）、グルタルアルデヒド（ＧＡ）、メタノールなどが好ましい。特に、リン酸緩衝液（ＰＢＳ）に溶解したＰＦＡ溶液が好ましく、そのＰＦＡ濃度としては、０．１質量％〜４．０質量％が好ましい。 The immobilization treatment of the cell lines of the components (a) and (b) can be performed by a known method. As the cell fixing agent, for example, formaldehyde, paraformaldehyde (PFA), glutaraldehyde (GA), methanol and the like are preferable. In particular, a PFA solution dissolved in a phosphate buffer solution (PBS) is preferable, and the PFA concentration is preferably 0.1% by mass to 4.0% by mass.
（ａ）及び（ｂ）成分の混合のタイミングとしては、固定化処理後に行うことが好ましい。 The mixing timing of the components (a) and (b) is preferably performed after the immobilization treatment.
本実施形態に係る標準細胞液内の、（ａ）及び（ｂ）成分の細胞数の比率としては、検出感度の観点から、（ａ）成分：（ｂ）成分＝１：１又は（ａ）成分：（ｂ）成分＝１：２が好ましい。 From the viewpoint of detection sensitivity, the ratio of the number of cells of the components (a) and (b) in the standard cell fluid according to this embodiment is (a) component: (b) component = 1: 1 or (a) Component: (b) Component = 1: 2 is preferable.
本実施形態に係る標準細胞液内の細胞濃度としては、（ａ）及び（ｂ）成分共に、１ｃｅｌｌｓ／ｍＬでも処理液の染色性能を確認することは可能である。検出感度の観点から、細胞濃度は、１００ｃｅｌｌｓ／ｍＬ〜１０００００ｃｅｌｌｓ／ｍＬであることが好ましく、５００ｃｅｌｌｓ／ｍＬ〜１００００ｃｅｌｌｓ／ｍＬがより好ましく、１０００ｃｅｌｌｓ／ｍＬ〜２０００ｃｅｌｌｓ／ｍＬが更に好ましい。 As the cell concentration in the standard cell solution according to the present embodiment, the staining performance of the treatment solution can be confirmed even when the components (a) and (b) are both 1 cell / mL. From the viewpoint of detection sensitivity, the cell concentration is preferably 100 cells / mL to 100,000 cells / mL, more preferably 500 cells / mL to 10,000 cells / mL, and even more preferably 1000 cells / mL to 2000 cells / mL.
本実施形態に係る標準細胞液の（ｃ）吸着抑制剤は、（ａ）成分、（ｂ）成分及び標準細胞液を充填する容器間相互の吸着又は凝集を抑制するものである。（ｃ）吸着抑制剤を含むことで、（ａ）及び（ｂ）成分の安定性がより向上し、標準細胞液の保存安定性がより向上する。（ｃ）吸着抑制剤としては、ウシ血清アルブミン（ＢＳＡ）、ポリメタクリロイルオキシエチルホスファチジルコリン（ＰＭＰＣ）及びポリエチレングリコール（ＰＥＧ）などが好ましい。特に、タンパク質吸着抑制の観点からＢＳＡが好ましい。ＢＳＡ濃度としては、標準細胞液全量に対して、０．１質量％〜５．０質量％が好ましく、０．５質量％〜５．０質量％がより好ましく、０．５質量％〜２．０質量％がさらに好ましい。 The (c) adsorption inhibitor of the standard cell fluid according to the present embodiment suppresses mutual adsorption or aggregation between the containers filled with the component (a), the component (b) and the standard cell fluid. (C) By including an adsorption inhibitor, the stability of the components (a) and (b) is further improved, and the storage stability of the standard cell solution is further improved. (C) As an adsorption inhibitor, bovine serum albumin (BSA), polymethacryloyloxyethyl phosphatidylcholine (PMPC), polyethylene glycol (PEG) and the like are preferable. In particular, BSA is preferable from the viewpoint of suppressing protein adsorption. As a BSA density | concentration, 0.1 mass%-5.0 mass% are preferable with respect to standard cell liquid whole quantity, 0.5 mass%-5.0 mass% are more preferable, 0.5 mass%-2. 0% by mass is more preferable.
続いて、本実施形態に係る標準細胞液を用いた、ＣＴＣと白血球とを染め分ける処理液の染色性能の確認方法を、フィルタ方式のＣＴＣ捕捉デバイスを用いる場合を例にとって説明する。まず、ＣＴＣ捕捉デバイス内に本実施形態に係る標準細胞液を通液し、ＣＴＣ捕捉デバイス内のフィルタ上に、（ａ）及び（ｂ）成分の細胞を捕捉する。捕捉された細胞をＰＢＳなどの緩衝液で洗浄処理を行い、細胞固定処理用処理液を用いて、細胞を固定化する。次に、細胞膜透過処理用処理液により、固定化した細胞の透過処理を行う。続いて、透過した細胞を、抗原抗体反応用処理液によって細胞染色を行う。（ａ）及び（ｂ）成分に特異的に結合する抗体は、それぞれ異なる標識（例えば、蛍光波長の異なる蛍光物質）を有することが好ましい。フィルタ上に捕捉された（ａ）及び（ｂ）成分の細胞を蛍光顕微鏡などで観察し、それぞれ染色されていることが確認できれば、用いたＣＴＣと白血球とを染め分ける処理液の染色性能は良好であると確認できる。 Next, a method for confirming the staining performance of the treatment liquid that separates CTC and leukocytes using the standard cell liquid according to the present embodiment will be described by taking as an example the case of using a filter type CTC capturing device. First, the standard cell fluid according to the present embodiment is passed through the CTC capturing device, and the cells of the components (a) and (b) are captured on the filter in the CTC capturing device. The captured cells are washed with a buffer solution such as PBS, and the cells are immobilized using a cell fixing treatment solution. Next, the immobilized cells are permeabilized with a cell membrane permeabilization treatment solution. Subsequently, the permeated cells are stained with an antigen-antibody reaction treatment solution. The antibodies that specifically bind to the components (a) and (b) preferably have different labels (for example, fluorescent substances having different fluorescence wavelengths). If the cells of the components (a) and (b) captured on the filter are observed with a fluorescence microscope or the like and confirmed to be stained, the staining performance of the treatment liquid that separates the used CTC and leukocytes is good. It can be confirmed that
ＣＴＣ捕捉デバイスに供する（ａ）及び（ｂ）成分の合計細胞数は１００ｃｅｌｌｓ〜１０００００ｃｅｌｌｓであることが好ましく、５００ｃｅｌｌｓ〜１００００ｃｅｌｌｓであることがより好ましく、１０００ｃｅｌｌｓ〜２０００ｃｌｌｓであることが更に好ましい。このような範囲に標準細胞液の細胞数があれば、（ａ）及び（ｂ）成分の両方の細胞株を検出できる可能性が高くなる。 The total number of cells (a) and (b) to be provided to the CTC capture device is preferably 100 cells to 100,000 cells, more preferably 500 cells to 10,000 cells, and even more preferably 1000 cells to 2000 clls. If the number of cells in the standard cell solution is within such a range, there is a high possibility that both the cell lines (a) and (b) can be detected.
（実施例１）
＜固定化された細胞株を含む標準細胞液の調製(吸着抑制剤の濃度検討)＞
（ａ）成分としては非小細胞肺癌から由来する細胞株ＮＣＩ−Ｈ３５８を、（ｂ）成分としてはヒト急性Ｔ細胞性白血病細胞から由来する細胞株Ｊｕｒｋａｔを、それぞれ用いた。（ｃ）成分としては、ウシ血清アルブミン（ＢＳＡ）を用いて、ＢＳＡ濃度条件の検討を行った。
Example 1
<Preparation of standard cell solution containing immobilized cell lines (concentration of adsorption inhibitor)>
As the component (a), the cell line NCI-H358 derived from non-small cell lung cancer was used, and as the component (b), the cell line Jurkat derived from human acute T-cell leukemia cells was used. As component (c), bovine serum albumin (BSA) was used to examine the BSA concentration conditions.
フラスコに接着しているＮＣＩ−Ｈ３５８細胞を０．２５質量％トリプシン−ＥＴＤＡ溶液で剥離処理し、遠心機（日立工機製：ＣＴ１５Ｅ）を用いて、遠心（１０００ｒｐｍ（１８０×ｇ）、３分）を行い、リン酸緩衝液（ＰＢＳ）で２回洗浄することで、トリプシン−ＥＤＴＡ溶液を除去した。その後、４質量％ＰＦＡ−ＰＢＳを用いて２時間、固定化処理を行った。反応後にＰＦＡ−ＰＢＳを除去し、ＰＢＳで２回洗浄し、（１）０．５質量％ＢＳＡ−ＰＢＳ、（２）５．０質量％ＢＳＡ−ＰＢＳ、のＢＳＡ濃度を変えた２種類の吸着抑制剤にそれぞれ置換した。 The NCI-H358 cells adhering to the flask were detached with a 0.25% by mass trypsin-ETDA solution, and centrifuged (1000 rpm (180 × g), 3 minutes) using a centrifuge (manufactured by Hitachi Koki: CT15E). And the trypsin-EDTA solution was removed by washing twice with phosphate buffer (PBS). Then, the immobilization process was performed for 2 hours using 4 mass% PFA-PBS. After the reaction, PFA-PBS was removed, washed twice with PBS, and two types of adsorption (1) 0.5% by mass BSA-PBS and (2) 5.0% by mass BSA-PBS with different BSA concentrations. Each was replaced with an inhibitor.
フラスコ内に浮遊しているＪｕｒｋａｔ細胞を遠心（１０００ｒｐｍ（１８０×ｇ）、３分）し、ＰＢＳで２回洗浄した。その後、４質量％ＰＦＡ−ＰＢＳを用いて２時間、固定化処理を行った。反応後にＰＦＡを除去し、ＰＢＳで２回洗浄し、（１）０．５質量％ＢＳＡ−ＰＢＳ、（２）５．０質量％ＢＳＡ−ＰＢＳ、のＢＳＡ濃度を変えた２種類の吸着抑制剤にそれぞれ置換した。 Jurkat cells floating in the flask were centrifuged (1000 rpm (180 × g), 3 minutes) and washed twice with PBS. Then, the immobilization process was performed for 2 hours using 4 mass% PFA-PBS. After the reaction, PFA was removed and washed twice with PBS. (1) 0.5 mass% BSA-PBS, (2) 5.0 mass% BSA-PBS, two types of adsorption inhibitors with different BSA concentrations Respectively.
＜固定化された細胞株の物理的安定性確認：細胞の粒径測定＞
固定化処理後の標準細胞液を室温で一定期間保存し、ＣＡＳＹ Ｃｅｌｌ Ｃｏｕｎｔｅｒ（Ｒｏｃｈｅ）を用いて、細胞の粒径を測定した。ＮＣＩ−Ｈ３５８細胞の粒径測定結果を表１に、Ｊｕｒｋａｔ細胞の粒径測定結果を表２に示す。それぞれの細胞をＤａｙ３、Ｄａｙ２１、Ｄａｙ５１に細胞粒径測定を行った。
測定条件：使用キャピラリー内径１５０μｍ、サンプル細胞濃度５×１０６ｃｅｌｌｓ／ｍＬ
<Physical stability confirmation of immobilized cell lines: measurement of cell particle size>
The standard cell solution after the immobilization treatment was stored for a certain period at room temperature, and the particle size of the cells was measured using a CASY Cell Counter (Roche). Table 1 shows the particle size measurement results of NCI-H358 cells, and Table 2 shows the particle size measurement results of Jurkat cells. The cell size of each cell was measured on Day 3, Day 21, and Day 51.
Measurement conditions: inner diameter of used capillary 150 μm, sample cell concentration 5 × 10 6 cells / mL
表１及び表２より、ＢＳＡ濃度が０．５質量％の方が、細胞粒径の低下は小さかった。これより、より安定的に細胞を保存するためには、吸着抑制剤のＢＳＡ濃度は０．５質量％が好ましいと判断した。 From Tables 1 and 2, the decrease in cell particle size was smaller when the BSA concentration was 0.5% by mass. From this, in order to preserve | save a cell more stably, it was judged that the BSA density | concentration of an adsorption inhibitor has preferable 0.5 mass%.
（実施例２）
＜固定化された標準細胞液を用いた処理液の染色性能の確認＞
固定化したＮＣＩ−Ｈ３５８細胞（Ｄａｙ３）を最終濃度１０００ｃｅｌｌｓ／ｍＬ、Ｊｕｒｋａｔ細胞を最終濃度２０００ｃｅｌｌｓ／ｍＬになるように０．５質量％ＢＳＡ−ＰＢＳ中で混合調製を行い、標準細胞液とした。調製した標準細胞液を、ＣＴＣ捕捉デバイス内で処理することで、標準細胞液の染色性能を確認した。使用したＣＴＣ捕捉デバイスは、ポリメチルメタクリレート（ＰＭＭＡ）製カートリッジ内に一定の孔径（８×３０μｍ）を有する金属フィルタを設け、カートリッジ内出口流路から一定速度で吸引することができるデバイスである。
(Example 2)
<Confirmation of staining performance of treatment solution using immobilized standard cell solution>
The fixed NCI-H358 cells (Day 3) were mixed and prepared in 0.5% by mass BSA-PBS so that the final concentration was 1000 cells / mL and the Jurkat cells were final concentration 2000 cells / mL, and used as a standard cell solution. The prepared standard cell solution was processed in a CTC capture device, thereby confirming the staining performance of the standard cell solution. The CTC capture device used is a device in which a metal filter having a constant pore diameter (8 × 30 μm) is provided in a polymethyl methacrylate (PMMA) cartridge, and suction can be performed at a constant speed from the outlet channel in the cartridge.
ＣＴＣ捕捉デバイス内に、標準細胞液を１．０ｍＬ通液させ、カートリッジ内フィルタ上に固定化細胞を捕捉した。その後、ＰＢＳを用いて洗浄処理、４質量％ＰＦＡ−ＰＢＳで２０分間固定化処理、０．２質量％Ｔｒｉｔｏｎ Ｘ−１００（登録商標、ＳＩＧＭＡ−ＡＬＤＲＩＣＨ社）溶液で透過処理を行った。次に、抗体染色処理を、染色液を用いて２５℃で３０分間行い、フィルタ上に捕捉された細胞の染色性によって試薬の染色性能を確認した。染色液には、抗サイトケラチン抗体としてＡｎｔｉ−Ｐａｎ−Ｃｙｔｏｋｅｒａｔｉｎ（ＡＥ１／ＡＥ３）Ａｌｅｘａ Ｆｌｕｏｒ ４８８を、抗ＣＤ４５抗体としてＡｎｔｉ−Ｈｕｍａｎ ＣＤ４５ ＰＥを、細胞核染色試薬としてＨｏｅｃｈｓｔ３３３４２試薬を使用した。 1.0 mL of standard cell fluid was passed through the CTC capture device, and the immobilized cells were captured on the filter in the cartridge. Thereafter, washing treatment with PBS was performed, immobilization treatment with 4% by mass PFA-PBS for 20 minutes, and permeation treatment were performed with a 0.2% by mass Triton X-100 (registered trademark, SIGMA-ALDRICH) solution. Next, the antibody staining treatment was performed for 30 minutes at 25 ° C. using a staining solution, and the staining performance of the reagent was confirmed by the staining properties of the cells captured on the filter. In the staining solution, Anti-Pan-Cytokeratin (AE1 / AE3) Alexa Fluor 488 was used as an anti-cytokeratin antibody, Anti-Human CD45 PE as an anti-CD45 antibody, and Hoechst 33342 reagent as a cell nucleus staining reagent.
ＮＣＩ−Ｈ３５８細胞は、ＦＩＴＣの波長領域（５２２ｎｍ）で検出され、Ｊｕｒｋａｔ細胞は、ＰＥの波長領域（５７８ｎｍ）で検出され、これらの細胞は、染色を確認する上で十分な細胞であることを確認した。また、細胞核の染色試薬Ｈｏｅｃｈｓｔ３３３４２の波長（４６１ｎｍ）において、ＮＣＩ−Ｈ３５８細胞、Ｊｕｒｋａｔ細胞ともに検出された。さらに、ＮＣＩ−Ｈ３５８細胞は、ＰＥの波長領域では検出されず、Ｊｕｒｋａｔ細胞はＦＩＴＣの波長領域で検出されなかった。これより、本実施形態に係る標準細胞液を使用することで、処理液の染色性能を確認できることが示された。 NCI-H358 cells are detected in the FITC wavelength region (522 nm), Jurkat cells are detected in the PE wavelength region (578 nm), and these cells are sufficient to confirm staining. confirmed. Further, both NCI-H358 cells and Jurkat cells were detected at the wavelength (461 nm) of the cell nucleus staining reagent Hoechst33342. Furthermore, NCI-H358 cells were not detected in the PE wavelength region, and Jurkat cells were not detected in the FITC wavelength region. From this, it was shown that the staining performance of the treatment liquid can be confirmed by using the standard cell liquid according to the present embodiment.
Claims (5)
（ａ）ヒト癌細胞に特異的な抗原を有する細胞株と、
（ｂ）ヒト白血球に特異的な抗原を有する細胞株と、
（ｃ）吸着抑制剤と、を含み、
前記（ａ）及び（ｂ）成分が、細胞固定剤により固定化された細胞株であり、
容器に充填されている、標準細胞液。 A standard cell solution for confirming the staining performance of a treatment solution that separates blood circulating cancer cells (CTC) and leukocytes,
(A) a cell line having an antigen specific for human cancer cells;
(B) a cell line having an antigen specific for human leukocytes;
(C) an adsorption inhibitor,
Wherein the components (a) and (b), Ri cell lines der immobilized by a cell fixative,
That is filled in a container, a standard cell solution.
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