DEPArray™ NxT 單細胞分離篩選系統

隨著DEPArray™技術的發展,基因的異質性研究可達單顆細胞層級。可探索每顆細胞基因中隱藏的故事,例如整倍體的基因變異。DEPArray™技術可依細胞表型分選,以及運用不同參數組合與影像系統的雙重確認,可確保分選出純度100%的標的細胞,採用高純度細胞故可觀察出等位基因表現。

Menarini Silicon Biosystems

技術原理

DEPArray NxT Recovery Workflow

DEPArray™獨特技術建立在晶片內形成介電場,使細胞在電場中懸浮,並排列整齊。

富集過後的細胞,經過螢光染色後可注入晶片。晶片置入DEPArray™ NxT機台後,細胞會自動進入主要晶片區,進行分選。

晶片中的電場活化後形成電籠包裹細胞懸浮,儀器有6個螢光通道可供選擇,搭配影像系統可用來分析細胞表現,篩選出目標細胞。高質量影像系統搭配電極控制電籠移動,精確地進行細胞分選和回收。

每一顆被選取的細胞會自動移動到「暫存區」,一旦完成,便可將細胞單顆/多顆單管回收到微管,進行後續分子層級的分析。

系統介紹

DEPArray NxT icon DEPArray

  • 桌上型系統平台,節省空間
  • 精確分離目標細胞,純度100%,獲得後續二代測序準確信息
  • 多樣化樣品上樣,活細胞,固定細胞皆可
  • 自選標定用抗體種類

DEPArray NxT sorting

  • 全新晶片設計,可回收高達96管單顆細胞
  • 電籠包裹細胞移動,對細胞傷害性小,更適合活細胞分選
  • 已設定標準化分選程序,統一篩選條件,目前應用在CTC、FFPE、法醫檢體方面
  • 可根據實驗設定,自定義篩選條件

DEPArray NxT icon monitor

  • 高質量成像,圖像式操作界面,高度自動化
  • 一部電腦可控制多台儀器,擴充方便

挑選與收集細胞

DEPArray NxT Select Recover Cells

DEPArrayTM 晶片

  1. 注入樣品,擷取細胞影像
  2. 移動目標細胞到暫存區
  3. 回收目標細胞

高質量成像

DEPArray NxT High definition

DEPArray NxT High resolution

  • 每個細胞都拍攝高質量影像
  • 同時掃描,同時分析影像,自動將不同螢光影像選圖
  • 每個細胞分析後給予獨立ID,並記錄相關參數

人性化設計

  • 操作者、樣品、芯片、回收盤,皆可使用掃描條形碼方式輸入,節省時間
  • 自動化整理實驗報告,以PDF檔輸出
  • CTC、FFPE、法醫檢體方面,默認篩選模組,統一分選標準。
  • 觸控面板,圖像化設計,方便操作

DEPArray NxT Barcode Reader

DEPArray NxT Report 01

CTC應用

DEPArray™ NxT於CTC應用上的優勢

腫瘤是基因高度異質性的疾病,可能由基因突變引起,不論是遺傳或後天都有可能。大部分腫瘤都有特殊的分子改變,了解分子層級的變化有助於診斷和藥物開發。

  • 捕獲單顆腫瘤細胞,甚至是循環性腫瘤細胞(CTC)
  • 確認有上皮-間質轉化現象的相關亞型,或發掘新的亞群
  • 分辨腫瘤亞型特徵
  • 各種腫瘤細胞來源皆可分析 :石蠟包埋組織,血液,細針活穿樣品等。

隨著DEPArray™技術的發展,基因的異質性研究可達單顆細胞層級。可探索每顆細胞基因中隱藏的故事,例如整倍體的基因變異。DEPArray™技術可依細胞表型分選,以及運用不同參數組合與影像系統的雙重確認,可確保分選出純度100%的標的細胞,採用高純度細胞故可觀察出等位基因表現。

完整流程規劃

DEPArray NxT CTC Workflow

後續分析

在腫瘤研究上,從外周血液中經由富集和篩選可純化出循環性腫瘤細胞(CTC),透過全基因組放大,有足夠的DNA產物進行後續不同的分子層級分析。單顆細胞的研究有助於解開腫瘤細胞間的異質性。全面性了解腫瘤轉移的機制。

分離出的單顆細胞,可使用Ampli1™ WGA Kit進行全基因組放大,再以Ampli1™ LowPass Kit處理後,可以進行二代定序,結果可進行CNV分析,可以明顯分辨出腫瘤細胞基因突變位點。

DEPArray NxT app Oncology research CTC cammpo

DEPArray NxT app Oncology research WBC Cammpo

CTC實際應用

2014 Nature medicine

Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer

研究者介紹

Prof. Caroline Dive是英國曼徹斯特大學藥理學教授,也是曼徹斯特癌症研究中心的傑出研究員(Manchester Cancer Research Centre),她與她的研究團隊致力於以非侵入性的方式,透過偵測血液中的循環性腫瘤細胞(Circulating tumor cells),歸納出可以實際應用於疾病診斷以及追踪治療效果的數據庫,達到轉譯醫學的目的。

實驗設計

DEPArray app cancer experiment design

論文摘要

小細胞肺癌(SCLC)在肺癌中佔15~20%,早期就轉移,預後差,多數病人無法以手術方式去除腫瘤。Prof. Caroline Dive將病人周邊血液中的循環性腫瘤細胞(CTC)分離出來,成功在免疫不全的老鼠建立疾病模式(CTC-derived explants,CDXs),與病人相似,可藉由動物模式測試藥物,選取最適合藥物進行治療,邁向個人化醫療。文中並比較CTC與CDXs在基因表現上高度相關,CTC有益於監控病程,CDXs有助於研究抗藥性的作用機制。

2016 Nature medicine

Molecular analysis of circulating tumor cells identifies distinct copy-number profiles in patients with chemosensitive and chemorefractory small-cell lung cancer

論文摘要

Prof. Caroline Dive利用已經建立的實驗流程,以DEPArray分離出已知化療敏感或化療不敏感的病患CTC,進行基因檢測,找出16個基因組可以預測化療反應。之後在病人與老鼠模式上都可以成功預測,辨識正確率達83.3%。

FFPE應用

  • 組織蠟塊除了使用蘇木精-伊紅染色判別外,以DEPArray™ NxT分選,可直接準確分選腫瘤細胞&基質細胞進行二代測序。
  • 精準測得腫瘤序列,有效去除基質細胞干擾

傳統方式 VS DEPArray

DEPArray NxT app FFPE Workflow Different

完整流程規劃

DEPArray NxT app FFPE Workflow

分辨出腫瘤特異突變基因

DEPArray NxT app FFPE Data

相關產品

持續優化的流程

CTC分析優化流程

CELLSEARCH® System
CTC檢測系統

目前唯一通過美國FDA認證的CTCs檢測技術,具有精準性與可重複性;在乳癌、大腸直腸癌與前列腺癌已有臨床檢驗標準,可做為評估指標。利用免疫磁珠與免疫螢光捕捉與標定CTCs,全程自動化,可降低人為影響。一次最多可以同時執行8個樣品,適合應用於定期監測病患狀況,評估藥物療效與復發跡象。於研究方面,也陸續開發不同類型細胞的試劑組,使研究人員能依據需求富集特殊細胞。

Ampli1™ WGA Kit
單顆細胞全基因組擴增套組

單顆細胞中使全基因組擴增優化,單一引子進行PCR,確保相間比例擴增DNA片段,平均且完整擴增DNA片段,擴增長度0.2~2K bp 適用任何種類的細胞,單管操作,無需沉澱DNA,滅少損失,過程快速,手動操作時間1.5小時,單顆細胞最多可獲得4ug DNA 適用於後續的基因分析應用,包括全基因組定序。

Ampli1™ LowPass Kit
染色體非整倍體和拷貝數變異 建庫套組

接續Ampli1™ WGA Kit產物使用Ampli1™ LowPass Kit,可輕鬆完成測序前建庫,免除中間複雜的實驗流程,像是純化,去掉adaptor,再接合等步驟。

此產品目前沒有詳細的產品列表,請來電洽詢。

DEParray™ 在腫瘤學Oncology的應用

Hodara et al, Journal od Clinical Oncology Insights 2019, “Multi-parametric liquid biopsy analysis in metastatic prostate cancer

Dorssers et al, British Journal of Cancer 2019, “Molecular heterogeneity and early metastatic clone selection in testicular germ cell cancer development

Rihawi et al, Translational Oncology 2019, “Amplification as a Potential Mechanism of Primary Resistance to Crizotinib in ALK-Rearranged Non-Small Cell Lung Cancer: A Brief Report

Vishnoi et al, Cancer Research 2018, “Targeting USP7 Identifies a MetastasisCompetent State within Bone Marrow–Resident Melanoma CTCs

De Laere et al, Clinical Cancer Research 2018, “TP53 outperforms other androgen receptor biomarkers to predict abiraterone or enzalutamide outcome in metastatic castration-resistant prostate cancer

Chalopin et al, Journal of Bone Oncology, 2018 “Isolation of circulating tumor cells in a preclinical model of osteosarcoma: effect of chemotherapy

Petrossian et al, Oncotarget 2018, “ERα-mediated cell cycle progression is an important requisite for CDK4/6 inhibitor response in HR+ breast cancer

Ji Won Lee et al, Journal of Applied Genetics 2018, “Identification of novel mutations in FFPE lung adenocarcinomas using DEPArray sorting technology and next-generation sequencing

Ferrarini A et al, PLOS ONE 2018, “A streamlined workflow for single-cells genome-wide copy-number profiling by low-pass sequencing of LM-PCR whole-genome amplification products

Boulding et al, Scientific Reports 2018, “LSD1 activation promotes inducible EMT programs and modulates the tumour microenvironment in breast cancer

Paoletti et al, Cancer Research 2017, “Comprehensive mutation and copy number profiling in archived circulating breast cancer tumor cells documents heterogeneous resistance mechanisms

Boral et al, Nature Communications 2017, “Molecular characterization of breast cancer CTCs associated with brain metastasis

Mesquita et al, Molecular Oncology 2017, “Molecular analysis of single circulating tumour cells following long-term storage of clinical samples

Paolillo et al, Clinical Cancer Research 2017, “Detection of Activating Estrogen Receptor Gene (ESR1) Mutations in Single Circulating Tumor Cells

Palmirotta et al, Cancer Genomics and Proteomics 2017, “Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA)

Rapp C et al, Acta Neuropathol 2017, “Identification of T cell target antigens in glioblastoma stem‑like cells using an integrated proteomics‑based approach in patient specimens

Bingham C et al, Breast Cancer Res Treat 2017, “Mutational studies on single circulating tumor clles isolated form the blood of inflammatory breast cancer patients

Tellez Gabriel M et al, European Journal of Cell Biology 2017, “Analysis of gap junctional intercellular communications using a dielectrophoresis-based microchip

De Laere B et al, JTS 2016, “Patients with metastatic hormone receptor-positive breast cancer express PIK3CA oncogene mutational heterogeneity in circulating tumor cells

Carter L et al, Nature Medicine 2016, “Molecular analysis of circulating tumor cells identifies distinct copy-number profiles in patients with chemo sensitive and chemo refractory small-cell lung cancer

Zhaomei Mu et al, IJMC 2016, “Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer

Shaw J et al, Clinical Cancer Research 2016, “Mutation analysis of cell-free DNA and single circulating tumor cells in metastatic breast cancer patients with high CTC counts

Millner LM et al, Cancer Research Frontiers 2016, “Comprehensive isolation, identification, and nucleic acid analysis of single breast cancer cells: CTC-isoTECH

De Luca F et al, Oncotarget 2016, “Mutational analysis of single circulating tumor cells by next generation sequencing in metastatic breast cancer

Bulfoni M et al, Breast Cancer Research 2016, “In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis

Yee S.S et al, Molecular Genetics and Genomic Medicine 2016, “A novel approach for next-generation sequencing of circulating tumor cells

Bolognesi C et al, Nature Scientific Reports 2016, “Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing

Rothwell G et al, Molecular Oncology 2015, “Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample

Vishnoi M et al, Nature Scientific Reports 2015, “The isolation and characterization of CTC subsets related to breast cancer dormancy

Salvianti et al, Biomolecular Detection and Quantification 2015, “Feasibility of a workflow for the molecular characterization of singlecells by next generation sequencing

Ewelina Krzywinska et al, EBioMedicine 2015, “Identification of Anti-tumor Cells Carrying Natural Killer (NK) Cell Antigens in Patients with Hematological Cancers

Maltoni R et al, Cancer Letters 2015, “Circulating tumor cells in early breast cancer: A connection with vascular invasion

Pestrin M et al, Molecular Oncology 2014, “Heterogeneity of PIK3CA mutational status at the single cell level in circulating tumor cells from metastatic breast cancer patients

Fernandez S.V. et al, Breast Cancer Research 2014, “TP53 mutations detected in circulating tumor cellspresent in the blood of metastatic triple negative breast cancer patients

Carpenter E, et al, Frontiers in Oncology 2014, “Dielectrophoretic capture and genetic analisys of single neuroblastoma tumor cells

Hodgkinson C.L.et al, Nature Medicine 2014, “Tumorigenicity and genetic profiling of Circulating Tumor Cells in Small Cell Lung Cancer

Peeters DJE, et al. British Journal of Cancer 2013, “Semiautomated isolation and molecular characterization of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting

Fabbri F, et al. Cancer Letters 2013, “Detection and recovery of circulating colon cancer cells using a dielectrophoresis-based device: KRAS mutation status in pure CTCs


DEParray™ 在法醫鑑識Forensic的應用

Anslinger K et al, International Journal of Legal Medicine 2018, “Whose blood is it? Application of DEPArray™ technology for the identification of individual/s who contributed blood to a mixed stain

Williamson VR et al, FSI Genetics 2018, “Enhanced DNA Mixture Deconvolution of Sexual Offense Samples Using the DEPArrayTM System

Aslinger K et al, Rechtsmedizin 2017, “Application of DEPArray™ technology for the isolation of white blood cells from cell mixtures in chimerism analysis

Fontana F et al, FSI Genetics 2017, “Isolation and genetic analysis of pure cells from forensic biological mixtures: The precision of a digital approach

Hansson O et al, FSI Genetics 2017, “Characterization of artefacts and drop-in events using STR-validator and single-cell analysis


DEParray™ 在蠟塊包埋FFPE的應用

Dorssers et al, British Journal of Cancer 2019, “Molecular heterogeneity and early metastatic clone selection in testicular germ cell cancer development

Isidori et al, BMC Cancer 2018, “Genomic profiles of primary and metastatic esophageal adenocarcinoma identified via digital sorting of pure cell populations: results from a case report

Ji Won Lee et al, Journal of Applied Genetics 2018, “Identification of novel mutations in FFPE lung adenocarcinomas using DEPArray sorting technology and next-generation sequencing

Bolognesi C et al, Nature Scientific Reports 2016, “Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing


DEParray™ 在分離幹細胞Isolation of Stem Cells的應用

Silvestris et al, Human Reproduction 2018, “In vitro differentiation of human oocytelike cells from oogonial stem cells: single-cell isolation and molecular characterization


DEParray™ 在蠟塊包埋FFPE應用的社論

Kathy Liszewski, GEN 2016, “The Next Next Thing in Sequencing

Farideh Bischoff, Nicolò Manaresi and Chiara Bolognesi, GEN 2015, “Isolation of Pure Tumor Cell Populations


DEParray™技術出版品

以下參考文獻是有關於介電泳技術和移動細胞的原理

Abonnenc M et al, J Immunol 2013, “Lysis-on-Chip of Single Target Cells following Forced Interaction with CTLs or NK Cells on a Dielectrophoresis-Based Array

Abonnenc M et al, Anal. Chem. 2013, “Programmable Interactions of Functionalized Single Bioparticles in a Dielectrophoresis-Based Microarray Chip

Gagnon ZR, Electrophoresis 2011, “Cellular dielectrophoresis: applications to the characterization, manipulation, separation and patterning of cells

Fabbri E et al, J Appl Polymer Sci 2008, “Levitation and movement of tripalmitin-based cationic lipospheres on a dielectrophoresis-based lab-on-a-chip device

Borgatti M et al, Int J Mol Med 2008, “New trends in non-invasive prenatal diagnosis: Applications of dielectrophoresis-based Lab-on-a-chip platforms to the identification and manipulation of rare cells

Vulto P et al, J Micromech Microeng 2006, “Selective sample recovery of DEP-separated cells and particles by phaseguide-controlled laminar flow

Fuchs AB et al, Lab Chip 2005, “Electronic Sorting and Recovery of Single Live Cells From Microlitre Sized Samples

Borgatti M et al, Int J Mol Med 2005, “Separation of White Blood Cells From Erythrocytes on a Dielectrophoresis (DEP) Based ‘Lab-On-A-Chip’ Device

Borgatti M et al, International Journal Oncology 2005, “Dielectrophoresis-based ‘Lab-on-a-chip’ devices for programmable binding of microspheres to target cells

Abonnenc M et al, NanoBiotechnology 2005, “A dielectrophoretic microchip for controlled cell targeting with functionalized microspheres

Medoro G et al, IEEE Sensors J 2003, “A lab-on-a-chip for cell detection and manipulation

Altomare L et al, Biotechnol and Bioeng 2003, “Levitation and movement of human tumor cells using a printed circuit board device based on software-controlled dielectrophoresis

Gascoyne PR et al, Electrophoresis 2002, “Particle separation by dielectrophoresis

Archer S et al, Biochem Biophys Res Commun 1999, “Cell Reactions to Dielectrophoretic Manipulation

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