CellCelector : enabling fast automated image-based isolation of pure single CTCs for personalized therapy

Transcription

1 short version CellCelector : enabling fast automated image-based isolation of pure single CTCs for personalized therapy Titre Dr. Constantin Nelep - September,

2 Who we are ALS Headquarters in Jena, Germany Innovative automation solutions for life sciences and clinical research 1998: AVISO GmbH founded (OEM and custom manufacturing) 2006: CellCelector commercial launch 2011: AVISO restructured and became ALS 26 persons: robotics, cell manipulation, imaging, software 50+ CellCelectors installed in the world (all applications together) Own international service and distributor network Strong automation and customization expertise ISO 9001:2008 certified 2

3 Fully automated platform for isolation of single cells, cell clusters and cell colonies Ultra-precise robotics and pipetting tools for mechanical cell isolation Automated high resolution fluorescent microscope Powerful image analysis software 3

4 CellCelector at a glance Picking module Cooled 14 bit CCD camera Bright field & phase contrast illumination Destination plate(s) Heated/cooled positions Autofocus Source plate (microplates, Petri dishes, slides) High-precision fast XY stage Objectives up to 40x Fluorescence inverted microscope 4

5 CellCelector housings Laminar flow cabinet Sterile conditions (customized Thermo KS12 supplied by ALS with CellCelector or already existing one) ALS Incubator Flowbox Long live cell experiments Laminar flow Temp., CO 2 and humidity control UV-for over-night sterilization Installation at Inserm U935/ESTeam, Paris region, France Installation at Rudbeck Lab, Uppsala University, Sweden 5

6 Single cell picking tool: High precision glass capillary Liquid buffered precise glass capillary Recommended capillary diameter: Single cells/ctcs: 30 µm Cell clusters : 80 µm Other capillaries available 20, 50, 150, 220 µm Tunable aspiration before volume: after e.g. 0.5 µl (low limit: 20 nl) Zoomed original image of the picked clone Very gentle aspiration that doesn t hurt cells allowing recover viable living cells 6

7 General CellCelector workflow Process documentation Scan Detect Revise Pick Analysis Scanning of source plate (image acquisition) Detection and selection of target objects (image analysis) Revision and additional filtering by human Transfer of objects into destination plates Downstream molecular analysis or Culture 7

8 Plate formats SOURCE PLATES Petri-dishes up to 100 mm diameter SBS format microplates (from 1 to 96 wells) Mutli-well or micro-well slides Glass slides or chips Filters Any custom vessel DESTINATION PLATES SBS format microplates (up to 384w) Multi-well slides PCR tubes or plates Petri-dishes Custom vessels No proprietary expensive consumables Example: single cell picking from a 30mm Petri dish to two standard microplates 24w 8

9 User friendly CellCelector software The user is guided through the whole workflow: 9

10 Fast high resolution scanning Dish border Dish installed onto the microscope stage for scanning Definition of the scanning /picking area Defined scanning area The acquired images are stitched automatically; the resulting stitched overview image is used for visualization and navigation; After scanning the software switches to Analyze step 10

11 Fast high resolution scanning Wells or slide are imaged at 10x, 20x or 40x magnification in scanning and documentation modes. Bright field, phase contrast and up to 6 fluo channels Large overview images of the well or slide Fast imaging, e.g. for 1 full well in 96-well plate (6 mm2): Scanning time BF: Scanning time 3 channels : 10x: ~18 sec.; 40x: ~2 min. 10x: ~55 sec.; 20x: ~3 min. 11

12 Flexible workflow Automated cell selection Manual selection of cells directly on screen Target single cells can be automatically detected and fine filtered using fluorescence and morphological parameters Target single cells can be manually selected bu the user directly on the screen using Add multiple objects feature during live imaging With manual selection the first cells might be recovered in minutes. 12

13 Image analysis and target object detection step Single cells, cell colonies or other target objects can be detected automatically based on pre-defined grey-value thresholds. CellCelector automatically calculates a user-defined panel of morphological parameters for detected clones including: Area, Diameter, Sphericity, Convexity, Nearest Neighbor Distance Using these parameters the user can filter out artifacts during Analyze step, as well as narrow the selection and/or rank the selected objects at the next step ( Revise ) Definition of the list of measured intensity and morphological parameters as well as range filters. 13

14 Pick list revision step 14

15 Single cell picking process Sterilization (optional) Pre-filling with buffer Aspiration Deposition Sterilization (optional) in ethanol solution (tank1) Pre-filling of the capillary with buffer solution, 0.1 µm to few µm (tank 2) Aspiration of the target single cell Transfer and deposition of the cell to the destination well ~30 sec.* * While isolating a group of single cells to be deposited into a unique destination well there are one prefilling and one deposition step per cell group. In this case picking takes approx. 5 sec. per single cell. 15

16 The fastest automated system at the market Delivery of one cell per well: recovery of 20 cells in 10 min. Delivery of a group of cells in one well: 20 cells in 2 min. In comparison: DEP cell sorting technology needs ~10 min. per 1 cell recovery. 16

17 Picking QC & documentation Target cell Leukocyte Leukocyte Images obtained in collaboration with Rarecells S.A. (tumor cells enriched using ISET device) 17

18 Documentation step (data and image export) The user can export all numerical data relative to cells, well overview images and all individual QC images before & after picking. QC images Before and After picking Pick status Buttons for image and data export Plate ID Cell ID Destin. well number Cell parameters 18

19 Picking efficiency Sub-cloning of 8 isogenic cell lines (NSCLC) Gallery of Before Picking images corresponding to 96 dest. wells wt mut.1 mut.2 mut.3 mut.4 mut.5 mut.6 mut.7 19

20 Picking efficiency Sub-cloning of 8 isogenic cell lines (NSCLC) Gallery of After Picking images corresponding to to 96 dest. wells wt mut.1 mut.2 mut.3 mut.4 mut.5 mut.6 mut.7 picking accuracy 92/96 = 95,8 % 20

21 example Using CellCelector within single CTC isolation and molecular analysis workflow 1 CTC / 10 7 WBCs 1 CTC / WBCs 100% pure CTCs Blood drawing RBC lysis Enrichment Recovery of pure CTCs Single CTC characterization Immuno-magnetic : CellSearch, Isoflux, ; size-based: ISET, ScreenCell, CellSieve ; µfluidics: CTChip, CellCelector : Image-based single cell picking platform DNA, RNA and protein marker analysis NGS studies Culture Injection into mouse Functional assays CTC : Circulating Tumor Cell 21

22 Examples of compatibility with upstream cell enrichment technologies Immuno-magnetic Isoflux (Fluxion) Filter based technology ISET (Rarecells) Direct slide scanning FASTcell (SRI Intl) Hoechst+ CellTracker+ CD45- Label free (size selection) DAPI+ CK+ CD54-22

23 Example: Combined CellSearch-CellCelector CTC analysis workflow Blood drawing RBC lysis Enrichment Recovery of pure CTCs Single CTC characterization EpCAM+ CTCs DAPI, C11 (CK), CD45 - whole genome amplification (WGA) - comparative genomic hybridization (CGH) or - point mutation analysis via sequencing Cartridge content (300 µl) is pipetted onto 1 or 2 glass slides Slides installed on a magnetic adapter; scanned with 300 ms exposure time; events are filtered; picking w/1 µl buffer uptake; aspiration vol. 10 nl; deposition in PCR tubes Martin Neumann, UKD, unpublished data, June

24 Comparison of imaging and detection of spiked tumor cells using CellSearch cartridge with CellSearch vs CellCelector Selection criteria: Diameter 5-50 µm DAPI + (nucleus) CK+ (EP) CD45+ (APC) Her2 (FITC). Optional Signal structure: different 97% of CellSearch scan events detected by CellCelector (n=6) Martin Neumann, UKD, unpublished data 24

25 Spiked tumor cells detected by CellCelector after transferring CellSerach enriched blood sample onto the slide CellSearch CellCelector Overall recovery rate of spiked cells detected with CellSearch: 89% (n=4) CellSearch CellCelector CellSearch CellCelector CellSearch CellCelector Martin Neumann, UKD, unpublished data 25

26 Using CellCelector special magnetic adapter for glass slides for easy single cell picking Bright field Fluorescence Aspiration volume 10 nl. Striation patterns visible in BF are due to ferrofluid particles aligned in the magnetic field Martin Neumann, UKD, unpublished data 26

27 More picking examples (cropped images for 8 picking events) Before After Aspiration Volume 10 nl Martin Neumann, UKD, unpublished data 27

28 CTC isolation from patient blood samples Overall recovery rate of cells detected with CellSearch: 87% (n=10) CellSearch Merge/BF CK-PE/TRITC Dapi CD45-APC/Cy5 HER-2/neu-FITC CellCelector Pickvorgang Before After CellSearch CellCelector Pickvorgang Before After Courtesy of Martin Neumann, UKD, unpublished data 28

29 Single cell picking after immuno-magnetic enrichment by Isoflux system Learn about Isoflux technology: 29

30 Single cell picking after Isoflux enrichment, BF 20x Before After Aspiration volume 10 nl. Striation patterns visible in BF are due to ferrofluid beads aligned in the magnetic field Martin Neumann, UKD, unpublished data 30

31 Picking after Isoflux enrichment (cropped images) Before After Before After Before After Before After Martin Neumann, UKD, unpublished data 31

32 Cell picking directly from filters (cell size-based CTC enrichment) 32

33 Single cell picking directly from ScreenCell filters Living spiked tumor cells, BF 20x Before picking After picking About ScreenCell technology: 33

34 Single cell picking from ISET filter, BF 20x Before picking After picking Before picking After picking Another cell Zoom About ISET technology: Martin Neumann, UKD, unpublished data 34

35 Picking from VyCap-Filter (Bright field) Before picking After picking CellSearch CTRL (SkBr3 cells) ; Aspiration volume: 4 µl Martin Neumann, UKD, unpublished data About VyCap technology: 35

36 Examples of single cell applications Isolation of pure viable single cell for molecular analysis, single cell sequencing or cloning Fast isolation of cell subpopulation based on expression of fluorescent markers and/or cell morphology Isolation of single cells from colonies Single cell heterogeneity studies Oncology (including CTC picking), immunology, cell line development etc. Cell recovery from any kind of surface: glass, plastic, membranes or filters 37

37 CellCelector summary EASY TO USE VERY FAST FLEXIBLE RELIABLE GENTLE LOW BUFFER VOLUME ANY FORMATS No sample preparation needed : picking cells directly from cell culture Automated or manual (visual) cell selection Recovery speed: ~30 sec./single cell or 20 singe cells in 10 min. First-cell-out-time: 2 min (manual selection in BF) or min (automated scanning/selection, time depends on the protocol) Isolating individual single cells or pooling cells in groups Isolating both single cells and clusters Bright field, phase contrast or fluorescence detection Picking accuracy of >95%. If a given picking event fails, the target cell can be always re-picked using new picking parameters Isolate pure intact cells ready for molecular characterization or cell culture (cells spend <30 seconds in the capillary) The cells are deposited in a buffer volume down to 0.1 µl Source and destination vessels to fit user s upstream and downstream applications; picking from/to any plates, dishes, slides or chips Fastest rare single cell isolation system on the market! 38

38 Thank you! For more information: Constantin Nelep, PhD cnelep ALS Automated Lab Solutions GmBH Stockholmer Straße 10 Gewerbegebiet Lobeda/Süd Jena - Germany 39