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JP2005517411A - Bio-cell chip - Google Patents

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JP2005517411A
JP2005517411A JP2003568093A JP2003568093A JP2005517411A JP 2005517411 A JP2005517411 A JP 2005517411A JP 2003568093 A JP2003568093 A JP 2003568093A JP 2003568093 A JP2003568093 A JP 2003568093A JP 2005517411 A JP2005517411 A JP 2005517411A
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cell chip
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ドン スン イ
ジ ヒョン イ
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Seoul National University Hospital
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Abstract

本発明はバイオ-セルチップおよびその製造方法に関するものである。The present invention relates to a bio-cell chip and a method for manufacturing the same.

Description

技術分野
本発明は、バイオ-セルチップおよびその製造方法に関するものである。 TECHNICAL FIELD The present invention relates to a bio-cell chip and a method for manufacturing the same.

背景技術
近年、遺伝子の詳細情報が大量に公開されており、そのため遺伝子の大量スクリーニング方法に対する要求が増加している。広く知られた例として、高密度cDNAアレイ(high density cDNA array)またはDNAチップは、同時に数千個の遺伝子を分析することを可能にした。このように強力な方法が開発されると共に、臨床的診断または予後評価などの臨床分野におけるゲノム発見(genomic discovery)からの知見を用いる、患者個人への個別医療（personalized medicine）、および疾病関連遺伝子を有する者に対する予防療法の開発が急務となっている。組織学的に同じ形態を有する癌でも、癌の誘発や進行に関与した遺伝子変化は異なり、各遺伝子変化の場合により疾病の予後、抗癌剤に対する応答等が異なる。そのため、臨床的応答を予測するためには、患者から一次的に得られた癌細胞や癌組織の大量の遺伝子を、同時に分析することが必須となる。 Background Art In recent years, detailed information on genes has been released in large quantities, and thus there has been an increasing demand for gene screening methods. As a well-known example, high density cDNA arrays or DNA chips have made it possible to analyze thousands of genes simultaneously. In addition to the development of such powerful methods, personalized medicine to patients and disease-related genes using findings from genomic discovery in clinical fields such as clinical diagnosis or prognostic evaluation There is an urgent need to develop preventive therapies for people with the disease. Even in cancers having the same histological form, the genetic changes involved in the induction and progression of the cancer are different, and the prognosis of the disease, the response to anticancer agents, etc. differ depending on the case of each genetic change. Therefore, in order to predict a clinical response, it is essential to simultaneously analyze a large number of genes of cancer cells and cancer tissues obtained primarily from patients.

現在全ての生命工学研究は、細胞を破壊した後に、DNA、RNA、蛋白質等が細胞から分離された状態で検出されているため、細胞内でのこれらの生物試料の位置的および生理学的有意性は無視されている。細胞を破壊せずに、細胞内に維持されたDNA、RNA、蛋白質等の変化を検出することができれば、結果は生物学的有意性を有すると考えられる。しかし、細胞を損傷無く完全な形で保存するのは容易でなく、様々な種類の分析には数多くの細胞が必要なため、研究に制約があった。   Currently, all biotechnology research has detected the presence of DNA, RNA, proteins, etc. isolated from the cell after the cell has been destroyed, so the positional and physiological significance of these biological samples within the cell Is ignored. If changes in DNA, RNA, protein, etc. maintained in the cell can be detected without destroying the cell, the result is considered to have biological significance. However, it was not easy to store cells in intact form without damage, and many types of analysis required many cells, which limited research.

患者から得られる試料、例えば血液、腫瘍標本、または細胞学標本を遺伝子検出に用いる場合、標本の量が、様々なアッセイ法を行うには少なすぎるため、それが制限要因となりうる。試験に必要な最小の数の細胞で細胞アレイを製作できれば、試料量不足の問題も解決できると同時に、数多くの遺伝子を安価で同時に分析できる。   When using samples from patients, such as blood, tumor specimens, or cytology specimens, for gene detection, the amount of specimen is too small to perform various assays, which can be a limiting factor. If a cell array can be produced with the minimum number of cells required for the test, the problem of insufficient sample volume can be solved, and many genes can be analyzed simultaneously at a low cost.

発明の概要
本発明は、細胞が整列されたバイオ-セルチップ、および細胞をバイオチップ基板上の小さい空間にスポット噴射（spot-spraying）することにより、バイオ-セルチップを製作する方法を提供する。本発明のバイオ-セルチップは、少量の試料で数百種類のアッセイ法を行い、かつアッセイの実施中に数百個の実験対象を同一の条件下におくのに適している。 SUMMARY OF THE INVENTION The present invention provides a bio-cell chip in which cells are aligned and a method for fabricating a bio-cell chip by spot-spraying cells into a small space on a biochip substrate. The bio-cell chip of the present invention is suitable for conducting hundreds of kinds of assay methods with a small amount of sample and keeping hundreds of experimental subjects under the same conditions during the performance of the assay.

本発明は、数千個の細胞を遺伝子発現または蛋白質発現に関して同時に検出可能にすることを目的とする。   The object of the present invention is to make it possible to detect several thousand cells simultaneously with respect to gene expression or protein expression.

発明の開示
本発明はバイオ-セルチップおよびその製造方法に関するものである。 DISCLOSURE OF THE INVENTION The present invention relates to a bio-cell chip and a method for producing the same.

本発明は、既存のDNAチップのDNAの代わりに細胞を整列して固定させたバイオ-セルチップを提供する。即ち、数多くの細胞を狭い空間に整列して固定させたバイオチップを提供する。本発明に係るバイオ-セルチップには、例えば70mm×30mmの狭い空間に100〜2000個、好ましくは100〜500個の細胞を整列させて固定することができる。   The present invention provides a bio-cell chip in which cells are aligned and fixed instead of DNA of an existing DNA chip. That is, a biochip in which a large number of cells are aligned and fixed in a narrow space is provided. For example, 100 to 2000 cells, preferably 100 to 500 cells, can be aligned and fixed in a narrow space of 70 mm × 30 mm, for example, on the bio-cell chip according to the present invention.

細胞を整列させ固定させて本発明のバイオ-セルチップが構築される支持材料、即ちチップ基板は、既存のバイオチップで用いられるものであってよい。例えば、背景蛍光が低いスライドガラス、プラスチック、シリコン等を挙げることができる。   The supporting material on which the cells are aligned and fixed to construct the bio-cell chip of the present invention, that is, the chip substrate, may be used in existing biochips. For example, a glass slide, plastic, silicon or the like having a low background fluorescence can be used.

本発明のバイオ-セルチップは、DNAチップ等の既存のバイオチップの確立された製造方法に従い製造することができる。既存のDNA、RNA、蛋白質等を利用した製造と大きく異なるのは、細胞の場合、狭い空間に数多くの細胞を整列して固定するため、固定剤内に保存された細胞懸濁液が広がってはならない点である。このため、特別な措置が要求される。   The bio-cell chip of the present invention can be manufactured according to an established manufacturing method of an existing biochip such as a DNA chip. The major difference from the production using existing DNA, RNA, protein, etc. is that in the case of cells, a large number of cells are aligned and fixed in a narrow space, so that the cell suspension stored in the fixative spreads. It is a point that must not be. For this reason, special measures are required.

本発明者らは、各細胞が互いに離れて固定されるように別々の部屋を提供することにより、上記の問題を解決した。別々の部屋を形成させる方法としては、
(a) 試料間に隔膜(septum)を設置する方法、
(b) チップを形成する支持材料の表面、即ちチップ基板の表面を一定の形に切断して、試料を分注できるウェルを形成する方法、または
(c) チップを形成する支持材料表面、即ちチップ基板の表面を刻んで溝を作製する方法がある。 The inventors have solved the above problem by providing separate rooms so that each cell is fixed away from each other. As a method of forming separate rooms,
(a) a method of installing a septum between samples;
(b) a method of forming a well into which a sample can be dispensed by cutting the surface of the support material forming the chip, that is, the surface of the chip substrate into a certain shape, or
(c) There is a method in which a groove is formed by cutting the surface of a support material forming a chip, that is, the surface of a chip substrate.

ウェルと溝の違いは、ウェルは深さを所望するだけ深く形成するのは不可能であるため、溝は穴の横幅を切断して長いくぼみを作ることによって作製される点にある。よって一試料当り、相対的に多量の細胞が適用される必要がある場合は、溝を作製することが好ましい。隔膜を設置するための物質としては、ゴムセメント(rubber cement)のようなセメント、ステッカー(sticker)等が挙げられる。これらの物質は、バイオ-セルチップ製作後に難なく完全に除去されなければならず、インサイチューハイブリダイゼーション染色や細胞の形に何ら影響を及ぼしてはならない。   The difference between a well and a groove is that the groove cannot be formed as deep as desired, so that the groove is made by cutting the lateral width of the hole to make a long recess. Therefore, when a relatively large amount of cells needs to be applied per sample, it is preferable to create a groove. Examples of the material for installing the diaphragm include cement such as rubber cement, sticker and the like. These substances must be removed completely without difficulty after bio-cell chip fabrication and should not affect in situ hybridization staining or cell shape.

前記のように部屋を形成させる方法以外にも、支持材料の表面、即ちチップを構成するチップ基板の表面に細胞懸濁溶液が広がらないように、表面の特性を変更させる特殊処理を施した後、試料を噴射する別の方法も可能である。このときの特殊処理は、例えばポリマーのような粘着性材料での表面処理が含まれる。   In addition to the method of forming the chamber as described above, after performing a special treatment to change the surface characteristics so that the cell suspension solution does not spread on the surface of the support material, that is, the surface of the chip substrate constituting the chip. Other methods of jetting the sample are possible. The special treatment at this time includes, for example, a surface treatment with an adhesive material such as a polymer.

よって、本発明のバイオ-セルチップにおいて、細胞は
a) 別々の部屋に配置され固定されるか、または
b) 細胞懸濁液をスポットもしくは噴射することで整列され、かつチップ基板の表面に処理された粘着性材料により、別々に固定されうる。 Therefore, in the bio-cell chip of the present invention, the cells are
a) placed and fixed in a separate room, or
b) Can be fixed separately by an adhesive material that is aligned by spotting or spraying the cell suspension and treated on the surface of the chip substrate.

より具体的には、前記の部屋は、隔膜、ウェル、またはチップ基板の表面に刻まれた溝により形成されてもよく、前記の隔膜はセメントまたはスティッカーで構成されてもよい。   More specifically, the chamber may be formed by a groove, a well, or a groove cut in the surface of the chip substrate, and the diaphragm may be made of cement or sticker.

本発明のバイオ-セルチップ製造において、細胞試料の噴射方法には、少なくとも500個または平均2000個の細胞を同時に充填(loading)することが可能な自動化ロボットシステムを作動させることを含む。前記のロボットシステムは、次のような二つの異なる方式で動作させることができる。一つ目は、スライドに少なくとも500個または平均2000個の異なる試料を充填し、一種類のインサイチューハイブリダイゼーション試薬を適用するものである。また他の方法は、最小量自動化システムを利用して、異なるインサイチューハイブリダイゼーション反応試薬を一種類の試料と反応させる方法である。   In the production of the bio-cell chip of the present invention, the cell sample injection method includes operating an automated robotic system capable of simultaneously loading at least 500 cells or an average of 2000 cells. The robot system can be operated in two different ways: The first is to load a slide with at least 500 or an average of 2000 different samples and apply one in situ hybridization reagent. Another method is a method in which different in situ hybridization reaction reagents are reacted with one type of sample using a minimum amount automation system.

それぞれの試料は固有の番号を持っており、判読顕微鏡（reading microscope）と連結されたプログラムを作動させ、自動で標的試料を探すことを可能にする。結果の判定は、検出データが入力されたプログラムを利用して、スライド全体の全試料に対し、自動で実行することができる。   Each sample has a unique number, which activates a program linked to a reading microscope and makes it possible to automatically find a target sample. The determination of the result can be automatically executed for all samples on the entire slide using a program in which detection data is input.

従って本発明は、インサイチューハイブリダイゼーション法で最も問題になるスライド間の偏差(slide to slide variation)を生じない。実際のアッセイ法または臨床検査において、各スライド間の条件が同一でない可能性があり、結果評価を難しくしている。一つのスライドに約100〜5000個の試料を適用したバイオ-セルチップを利用すれば、標準化(standardization)を実現できる。本発明のバイオ-セルチップは、数千個の細胞に対して遺伝子発現(gene expression)の同時検出を提供する。   Thus, the present invention does not produce slide to slide variation, which is the most problematic in in situ hybridization method. In actual assays or clinical tests, the conditions between slides may not be identical, making it difficult to evaluate results. Standardization can be realized by using a bio-cell chip in which about 100 to 5000 samples are applied to one slide. The bio-cell chip of the present invention provides simultaneous detection of gene expression for thousands of cells.

本発明のバイオ-セルチップは、非常に少ない試料量と少ない試薬量で、大量の試料を対象に遺伝子発現検出を可能にし、これにより癌または遺伝病の疾病診断および薬効評価の分野で費用、時間および労力の面で革新をもたらしただけでなく、その結果に対する信頼性を大きく向上させた。   The bio-cell chip of the present invention enables detection of gene expression in a large amount of samples with a very small amount of sample and a small amount of reagent. Not only brought about innovation in terms of labor and effort, but also greatly improved the reliability of the results.

また、本発明のバイオ-セルチップを利用することにより、次のような応用が可能になる：
a)人体から非侵襲的方法で細胞を容易く採取できる腫瘍の大量スクリーニングに、バイオ-セルチップを利用する。例えば肺癌または膀胱癌の場合、喀痰または尿から採取した細胞を染色し、特異的遺伝子が変化したかまたは特異抗原を有する癌細胞を、自動分析システムを用いて高い正確度で検出することができる。
b)各種腫瘍細胞株を一箇所に敷いた腫瘍細胞株アレイチップを製作でき、アッセイの標準化(standardization)が実現される。
c)患者の腫瘍から採取して分離した腫瘍細胞を含むバイオ-セルチップを製作することができ、さらに基礎生物学研究者に提供することもできる。
d)単一細胞PCR(single cell-PCR)またはインサイチューハイブリダイゼーション(ISH)の標準化(standardization)が実現可能である。
e)微小残存白血病細胞の検出が可能である。
f)抗癌剤感受性を予測するシステムの開発が実現可能である。
g)バイオ-セルチップは、薬物感受性評価に有用である。 In addition, the following applications are possible by using the bio-cell chip of the present invention:
a) Use bio-cell chips for mass screening of tumors that can be easily harvested from the human body by non-invasive methods. For example, in the case of lung cancer or bladder cancer, cells collected from sputum or urine can be stained, and cancer cells with altered specific genes or with specific antigens can be detected with high accuracy using an automated analysis system .
b) Tumor cell line array chips with various tumor cell lines spread in one place can be manufactured, and standardization of the assay is realized.
c) Bio-cell chips containing tumor cells collected and separated from the patient's tumor can be fabricated and further provided to basic biologists.
d) Standardization of single cell PCR or in situ hybridization (ISH) is feasible.
e) Detection of minimal residual leukemia cells is possible.
f) It is feasible to develop a system that predicts anticancer drug sensitivity.
g) Bio-cell chips are useful for drug sensitivity assessment.

発明を実施するための最良の形態
本発明を下記の実施例および実験例でさらに詳細に説明するが、これらの実施例に本発明が限定されないことが理解される。 BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail with reference to the following examples and experimental examples, but it will be understood that the present invention is not limited to these examples.

実施例1
手作業で、または少量の細胞懸濁液を分注できるマルチディスペンサー(multi-dispensor)を用いて、一つのスライドに100個の試料を適用し、分子細胞遺伝学調査および遺伝子変化の検出を実施した。少量の試料と少量の試薬での染色に成功した。 Example 1
Apply 100 samples to a single slide for molecular cytogenetic studies and detection of genetic changes, either manually or using a multi-dispensor that can dispense small amounts of cell suspension did. We succeeded in staining with a small amount of sample and a small amount of reagent.

ゴムセメントを使用してスライドガラスに100個の隔膜を形成し、10分間乾燥させた。各ウェルに細胞懸濁液を1μlずつ分注した。20分後、分注した試料が完全に乾いたらゴムセメントを完全に取り除き、インサイチューハイブリダイゼーション染色を行った。   100 diaphragms were formed on a glass slide using rubber cement and dried for 10 minutes. 1 μl of cell suspension was dispensed into each well. After 20 minutes, when the dispensed sample was completely dried, the rubber cement was completely removed, and in situ hybridization staining was performed.

通常用いる試薬量はスライド一つ当り10μlである。一試料に必要とされる同量の試薬を用いて、100個の試料の遺伝子変化の検出を実施した。   The amount of reagent normally used is 10 μl per slide. Detection of genetic changes in 100 samples was performed using the same amount of reagent required for one sample.

実験例1
多くの患者から得られた細胞を利用した癌診断に、バイオ-セルチップを利用することができる。肺癌診断や追跡調査のため喀痰の細胞を検査する。大抵はPAP染色(PAP staining)をして細胞を一つ一つ判読するが、形態学的に癌細胞と異形成細胞(dysplastic cell)を区別することが難しいことが多い。韓国人の肺癌細胞の特異的な遺伝子変化を検出することができれば、診断の特異性を高めることができる。患者らの喀痰から分離した細胞を含むバイオ-セルチップを製作して、特異的な遺伝子変化をインサイチューハイブリダイゼーション法で検出する。手作業の必要なく、大量の患者群に対して肺癌の診断が可能である。医学的な措置をせずに人体から容易く得ることができる細胞としては、尿に混ざって自然に排出される膀胱癌細胞が挙げられる。尿試料から分離した細胞でバイオ-セルチップを製作して、膀胱癌特異的遺伝子の変化をインサイチューハイブリダイゼーション法で検出する。調査のために患者を病院に呼ばなくても、特別な操作なく膀胱癌の大量診断および治療後の追跡が可能である。 Example 1
Bio-cell chips can be used for cancer diagnosis using cells obtained from many patients. Examining sputum cells for lung cancer diagnosis and follow-up. Usually, PAP staining (PAP staining) is used to read cells one by one, but it is often difficult to distinguish cancer cells from dysplastic cells morphologically. If a specific genetic change in Korean lung cancer cells can be detected, the specificity of diagnosis can be increased. Bio-cell chips containing cells isolated from patient's sputum are fabricated and specific genetic changes are detected by in situ hybridization. Lung cancer can be diagnosed for a large group of patients without the need for manual work. Examples of cells that can be easily obtained from the human body without taking medical measures include bladder cancer cells that are naturally excreted in urine. Bio-cell chips are made from cells separated from urine samples, and changes in bladder cancer-specific genes are detected by in situ hybridization. Large-scale diagnosis and follow-up of bladder cancer can be performed without any special operation without sending the patient to the hospital for investigation.

下記の公開された参照文献は、本発明のバイオ-セルチップを利用して様々なアッセイ法を実施した例を発表した本発明者らの論文である。   The following published references are our papers that have published examples of performing various assays utilizing the bio-cell chips of the present invention.

1．Kyoung Un Park, Cha Ja She, Hee Young Shin, Hyo Seop Ahn, Chong Jai Kim, Byung Kyu Cho, Han Ik Cho, Dong soon Lee. The Low Incidences of TEL/AML1 fusion and TEL Deletion in Korean hildhood Acute Leukemia by Extra-signal Fluorescence In Situ Hybridization. Cancer Genetics and Cytogenetics The Low Incidences of TEL/AML1 fusion and TEL Deletion in Korean hildhood Acute Leukemia by Extra-signal Fluorescence In Situ Hybridization. Cancer Genetics and Cytogenetics, 2001, 126;73-77. 1． Kyoung Un Park, Cha Ja She, Hee Young Shin, Hyo Seop Ahn, Chong Jai Kim, Byung Kyu Cho, Han Ik Cho, Dong soon Lee .The Low Incidences of TEL / AML1 fusion and TEL Deletion in Korean hildhood Acute Leukemia by Extra -signal Fluorescence In Situ Hybridization.Cancer Genetics and Cytogenetics The Low Incidences of TEL / AML1 fusion and TEL Deletion in Korean hildhood Acute Leukemia by Extra-signal Fluorescence In Situ Hybridization.Cancer Genetics and Cytogenetics, 2001, 126; 73-77.

2．Dong Soon Lee, Sunny Kim, Eul Ju Seo, Chan Jung Park, Hyun Sook Chi, Byoung Hak Yoon, Wo Ho Kim, Han Ik Cho. Predominance of trisomy 1q in myelodysplastic syndromes in Korea; Is it a ethnic difference? - 3 year-experience of multi-center study - Genetics and Cytogenetics, 2001, 129;73-77. 2． Dong Soon Lee , Sunny Kim, Eul Ju Seo, Chan Jung Park, Hyun Sook Chi, Byoung Hak Yoon, Wo Ho Kim, Han Ik Cho.Predominance of trisomy 1q in myelodysplastic syndromes in Korea; Is it a ethnic difference? -experience of multi-center study-Genetics and Cytogenetics, 2001, 129; 73-77.

3．Dong Soon Lee, Eu Chong Kim, Byoung Hak Yoon, Eun Kyung Ko, Sun Yang Park, Woo Hoo Kim, Jong Hyun Yoon, Han Ik Cho. Can Minor bcr/abl Translocation in Acute Leukemia be Discriminated from Major bcr/abl by Extra-Signal FISH Analysis Haematologica 2001 86;991-992. 3． Dong Soon Lee , Eu Chong Kim, Byoung Hak Yoon, Eun Kyung Ko, Sun Yang Park, Woo Hoo Kim, Jong Hyun Yoon, Han Ik Cho. Can Minor bcr / abl Translocation in Acute Leukemia be Discriminated from Major bcr / abl by Extra -Signal FISH Analysis Haematologica 2001 86; 991-992.

4．Kyoung Un Park, Dong Soon Lee, Hye Seung Lee, Chong Jai Kim, and Han Ik Cho. Granulocytic Sarcoma in MLL-positive Infant Acute Myelogenous Leukemia: Fluorescence In Situ Hybridization Study of Childhood Acute Myelogenous Leukemia for Detecting MLL Rearrangement. American Journal of Pathology. 2001:159;2001-2016. Four. Kyoung Un Park, Dong Soon Lee , Hye Seung Lee, Chong Jai Kim, and Han Ik Cho. Pathology. 2001: 159; 2001-2016.

Claims (8)

チップ基板上に細胞が整列され固定されたバイオ-セルチップ。   A bio-cell chip in which cells are aligned and fixed on a chip substrate. 細胞がそれぞれ別々の部屋に配置され固定された、請求項1記載のバイオ-セルチップ。   2. The bio-cell chip according to claim 1, wherein the cells are arranged and fixed in separate rooms. 部屋が隔膜、ウェル、またはチップ基板の表面に刻まれた溝により形成される、請求項2記載のバイオ-セルチップ。   The bio-cell chip according to claim 2, wherein the chamber is formed by a groove, a well or a groove carved in the surface of the chip substrate. 隔膜がセメントまたはステッカーで構成される、請求項3記載のバイオ-セルチップ。   The bio-cell chip according to claim 3, wherein the diaphragm is composed of cement or a sticker. セメントがゴムセメントである、請求項4記載のバイオ-セルチップ。   The bio-cell chip according to claim 4, wherein the cement is rubber cement. 細胞が、チップ基板表面に処理された粘着性材料により別々に整列され固定される、請求項1記載のバイオ-セルチップ。   The bio-cell chip according to claim 1, wherein the cells are separately aligned and fixed by the adhesive material treated on the chip substrate surface. 自動化ロボットシステムを利用して、それぞれの細胞を充填した後、一種類のインサイチューハイブリダイゼーション試薬を適用する段階を含む、請求項1記載のバイオ-セルチップの製造方法。   2. The method of producing a bio-cell chip according to claim 1, further comprising the step of applying one kind of in situ hybridization reagent after filling each cell using an automated robot system. 最小量自動化システムを利用し、かつ自動化ロボットシステムを利用して、それぞれの細胞を充填した後、異なる種類のインサイチューハイブリダイゼーション試薬を細胞の標的試料と反応させる段階を含む、請求項1記載のバイオ-セルチップの製造方法。   2. The bio of claim 1, comprising utilizing a minimum volume automation system and utilizing an automated robotic system to react each cell with a different type of in situ hybridization reagent after filling each cell. -Cell chip manufacturing method.
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