Automated Single Cell Sorting System

mmi CellEctor Plus

Molecular Machines & Industries

Molecular analysis of pure enriched cell populations or even of single cells is an important prerequisite of medical genomics and proteomics and requires efficient selection strategies for single cell isolation. Therefore, mmi have developed and implemented a new microscopy based, fully automated single cell sorting device specifically for this purpose: the mmi CellEctor Plus.

LEAD THE WAY TO MICROSCOPY BASED SINGLE CELL SORTING

The mmi CellEctor Plus is the starting point for a new integrated work flow for the isolation of single and rare cells from tiny sample sources. It facilitates the manual or automated isolation of single and rare cells in three steps: 1. Cell Recognition 2. Cell Acquisition 3. Cell Deposition

all under full Software control (mmi CellExplorer). Aquisition and depositi on are controlled by the high precision mmi CellPump. The deposition of cells is possible onto a wide range of devices, either commercially available reaction grids, e.g. such as Ampligrid from Beckmann Coulter, an IBIDI-Chamber, a PCR-tube or a microfluidic device to facilitate the downstream applications.

The mmi CellEctor Plus is an adjustable and open platform for any assay format. It closes the gap between fre quent and rare cell sorting in a range of fields: Cancer Research & Oncology, Molecular Pathology, Immunology, Stem Cell Research, and many other applications in: Biomedical Research, Cellular Diagnostics Microbiology & Virology, Environ mental Ecology, Forensics, Material Sciences.

Currently the mmi CellEctor Plus is widely utilised for the enrichment and characterisation of all rare cells i.e. circulating tumour cells (CTC), endothelial, histiocy te, stem, and many other cell types from blood, bone marrow, or any other sour ces. Blood and bone marrow is treated by erythrocyte lysis, to remove present erythrocytes. In a first enrichment step, paramagnetic beads coated with an appropriate surface-antibody were used for the immu-nomagnetic enrichment. In the second step the mmi CellEctor Plus technology is used for individual cell picking. Numerous researchers are using the system moving towards the stream-lining of clinical applications.

All methods for assessing molecular changes require a pure cell population, for example the analysis of DNA, copy number variations or single nucleotide polymorphisms or changes of RNA expression. Protein patterns are analyzed with spectroscopic or biochemical protocols, and epigenetic changes are detected by changes in methylation patterns. Among the state-of-the-art methods to isolate single cells are FACS – sorting and bead-based protocols. Also a wide variety of physical and immunological methods are employed. The mmi CellEctor Plus was designed to over-come the hurdles and drawbacks of the above mentioned methods.

1 Preparation of a cell suspension: Disaggregate lymph node & filter cells

2 Lyse erythrocytes from blood or bone marrow samples

3 Add specific immuno magnetic beads ...

5 Magnetic separation of immunomagnetically

rosetted cells - micro-scopic evaluation &

CellEctor medi-ated cell picking

4 ... and incubate under rotation

Example Work Flow

6 Lymphnode metastatic cells targeted with an anti EP-CAM antibody

Pictures are a courtesy of H.K.Hoifodt,G. Maelandsmo & Prof. O. Fodstad, Riks hospitalet, Oslo, Norway

Reference 1: Tveito, S. | Maelandsmo, M. G. | Hoifodt, K. H. | Rasmussen, H. | Fodstad, O.:Specific isolation of disseminated cancer cells: a new method permitting sensitive detection of target molecules of diagnostic and therapeutic value. Clin Exp Metastatis 5 (24), 317 (2007)

Another two step selection set up was used by Ren et al., to isolate phagocytic histiocytes from patient blood as a prerequisite for EBV-infection diagnosis in individual cells. The phagocytic histiocytes were fed with fluorescent beads prior to image recognition analysis and cell picking.

Reference 2: Ren, X. | Niederfuehr, A. | Alunni, M. | Goerner, K. | Mann, W. | Piechotka, M. | Plaschke-Schluetter, A. | Brill, N. | Schneider, M.: States of EBV Infection in Patients with acute and recurrent hemophagocytosis.Abstract & oral presentation at 24th Annual Meeting of the Histiocyte Society Berlin (2008)

Reference 3: Tveito, S. | Piechotka, M. | Curtis, A. | Seeger, S. | Plaschke-Schluetter, A.: CellEctor™: a microscopy based and fully automated single cell sorting device for molecular and cellular analysis. Rikshospitalet-Radiumhospitalet Medical Center, Depart -ment of Tumor Biology, Oslo, Norway; Molecular Machines & Industries Zuerich, Switzerland; Uni ver si ty Zurich, Dep. of Physical Chemistry

• Based on an inverted microscope from Nikon or Olympus • Cell recognition unit (mmi CellExplorer)• Choice of three independent working modes: manual, semi and fully automated • Fully automated workflow under video image for total process control• Fully motorized and software controlled high precision mmi CellPump with

patented design for easy fill and flush• mmi 3D CellRobot, 3-axis fully motorized and Software controlled that can be

swivelled into parking position, to keep all stage space free for lasermicrodissection applications

• mmi CapFix patented capillary protection and quick fix system with mmi Smart Capillary construction with movable protection cap for optimal work security

• Compatibility with a wide range of accessories i.e. AmpliGrid, IBIDI Slides and Microfluidic devices

• A motorized x/y stage • Modular workstation, upgradeable with Laser Microdissection and Optical Tweezer

for integrated workflows

MMI CELLECTOR PLUS FEATURES

REFERENCES

mmi 3D CellRobot with mmi SmartCapillary

mmi CellPump

Hummingbirds represent the smallest genera of birds on earth. Nevertheless, they de velo ped conspicuous proper ties: Did you know, that humming birds are the on ly kind of bird on earth that can fly back wards? Some species, especially those with unusual bill shapes, coevolved with a small number of flower species. It is their capillary – like shaped bill, that allows them to feed on the nectar of specific plants. Nature itself is a model for coevolution and technical perfection and is our paradigm for in-house technical developments of the MMI micromanipulation devices.

FOLLOW NATURE

mmi CellExplorer Software

Example for Cell Deposition

mmi SmartCapillary operated by

mmi 3D CellRobot

The manufacturer reserves the right to make technical changes without prior notice. 2011

MMI CELLECTOR PLUS SPECIFICATIONS

MMI GmbHBreslauer Strasse 285386 Eching, GermanyPhone : +49 89 319 048 40Fax : +49 89 319 048 59Mail : info@molecular-machines.comwww.molecular-machines.com

MMI AGFlughofstrasse 378152 Glattbrugg, SwitzerlandPhone: +41 44 809 10 10Fax: +41 44 809 10 11Mail: info@molecular-machines.comwww.molecular-machines.com

MMI Inc.P.O. Box 348Haslett, MI 48840, USAPhone: +1 603 629 9536Fax: +1 321 978 0304Mail: sales_us@molecular-machines.comwww.molecular-machines.com

www.molecular-machines.com

mmi CellEctor Plus Specificationmmi CellEctor Plus An integrated instrumentation solution for the fully automated recognition, aspiration and deposition

of single cells in suspension onto cell culture and molecular biology devices | A modular microscopy

based sorting technology under full optical control and integrable with lasermicrodissection on the

same platform | Fully automated and software controlled manipulator

Automated Manipulator Fully automated manipulator for a glass capillary with motorized 3D movement | Customized tube

adaptor | Three high resolution step motors, software controlled | Adjustable holder for glass capillary

Set of 25 custom Tip Type III inner diameter 40μm

mmi CellPump Fully motorized and software controlled high precision pump to aquire and deposit cells or particles in defined

volumes consisting of: Cell tram vario pump with 2 nanoliter resolution | Patented design for easy fill and flush

mmi CellRobot 3 Axis fully motorized and software controlled | Easy swivel device to allow full access to the stage

Patented capillary protection and easy adjustment

Microscope adaptation Microscope mounting and adapters for integration with mmi CellCut Plus based on Ti-E Nikon or IX81 Olympus microscope

Microscopic systems Nikon Ti-E:

Inverted and motorized Microscope body | bright field | 100W illumination arm | Motorized revolver and z-focus

ELWD condenser | Plan Fluor Objectives: 4x | 10x | 20x | 40x

Olympus IX81:

Motorized Microscope body | bright field | 100W illumination arm | Motorized revolver and z-focus

IX-ULWCD condenser Phase Contrast Objectives: 4x UPlanFLN | 10x UPlanFLN | 20x LUCPlanFLN | 40x LUCPlanFLN

Fluorescence attachment Fluorescence attachment with cassette | shutter | x-cite | 3 fluorescence filter cubes with filters

Computer controlled xy-table 120x100 mm travelling path 0,025μm Step width for high precision cutting | 1μm repositioning accuracy

mmi CellEctor Plus Software mmi CellEctor software integrated into the mmi CellTools platform | mmi CellExplorer cell recognition

software integrated into the mmi CellTools platform | Integrated CellTools Software for video interface

and system control

Software features Fully automated or manual acquisition and deposition of cells | Choice of three automation levels

including full automation with scanning of the sample and cell recognition | Complex movement

modes of the capillary (skipping of the sample container wall, user-defined immersion and deceleration

distance, deposition at vertical walls) | User-defined spacing between the capillary tip and the slide

with a dynamic tilt correction | Automated capillary refocusing options: on the microscope slide

or on the capillary tip (user-selectable) | Five user-defined pump parameter sets for different

capillary diameters (10, 15, 20, 30 and 40 μm)

Options:Pen Screen system operation Sensitive 22” HD touch screen monitor for user-friendly system operation

to allow direct target identification with a special pen

mmi CellExplorer A cell identification software based on colour recognition which automatically identifies and cuts out cells

Possible mmi CellEctor Plus system upgrademmi CellManipulator Plus optical tweezer Ultra precise, contact-free manipulation of microscopic particles on a high-quality Nd: YAG infrared laserwith two level

options, allowing multibeam trapping on two Z-levels and 10 time shared traps per level.

mmi CellCut Plus laser microdissection system The ultimate laser microdissection tool to isolate single cells, groups of cells and life cells with the patented

mmi CapLift technology.