research activities in 2007 - CSEM

Isolation and Reversible Immobilization of Single Cells
S. F. Graf, P. Schmid, H. F. Knapp
An innovative system to meet the need of drug researchers is under development. The novel approach integrates new technologies including
microrobotics and microfluidics to achieve a high throughput screening rate which will replace time consuming and costly manual operations used
today. To this end, a multipurpose robotic system called ‘CellBot’ was developed consisting of the high-precision robot ‘µDelta’, the fully automated
inverse reflected light microscope ‘iMic’, the tool stage, the fixed working platform and the “Carousel” for reversible immobilizing of the cells. By
using this visual feedback controlled setup an automated isolation and reversible immobilization of single Xenopus laevis oocytes could be
demonstrated.
Cell-based assays are set to become the preferred choice of
screening in drug discovery research, potentially overtaking
more traditional approaches that include animal models. New
target screening often requires the use of cell assays to detect
specific cellular pathways of chemical compounds, therapeutic
proteins, siRNA agents and other structures of interest. Insight
from these assays could help more efficient discovery of
effective drugs, thus saving time and costs as well as the
need for future secondary screens. The emphasis now for
cell-based assay manufacturers is to develop easy-to-use and
highly sensitive cell systems as an alternative to current
rodent bioassays. High throughput screening (HTS) using cellbased
assays will particularly become increasingly needed for
both industrial and scientific applications. Introduction of DNA,
siRNA, or other substances into cells is one important
micromanipulation technology applied to develop and optimize
various cellular systems, which enables cell systems either to
more closely approximate in vivo testing or to become more
competent or more specific for various in vitro applications.
However, the pharmaceutical industry needs a highthroughput,
efficient, and automated system for direct delivery
of substances (including compounds, DNA, siRNA and mAbs)
into a large number of cells for HTS use.
To address this need, the development of an automated
microinjection system is in process. Microinjection is a
technique where a glass capillary filled with substances is
controlled by a micromanipulator. Cells have to be individually
immobilized while the capillary with an apex of 0.5 to 10 µm
diameter penetrates the cell and a pressuring device injects
the substances.
Therefore, as a first step, the isolation of single Xenopus
laevis oocytes with a following reversible immobilization is
demonstrated. The “CellBot” [1] setup is used, consisting of a
high precision robot “µDelta” equipped with a glass capillary
connected to a peristaltic pump, a fully automated inverted
light microscope “iMic” and a fixed working platform with a
Petri dish and a “Carousel” for further cell manipulations
(Figure 1). To start the process, the user has to place a
suspension of Xenopus laevis oocytes into a Petri dish. Via
the user interface the routine can be started: The iMic scans
the Petri dish (Figure 2) and cells of interest are automatically
identified via a pattern matching software based on a set of
given parameters If a cell is detected, the glass capillary is
automatically guided by vision feedback to pick up the cell and
places it into the carousel. The carousel contains specially
designed cone structures for immobilizing the cells, with or
without negative pressure. By rotating the “carousel” the cell is
moved to the microinjection position, which will be
implemented at a later stage. Finally the carousel can be
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rotated to two additional positions, where the cells can be
released into a collection or waste container, respectively. The
release of the cells is aided by pressure pulses. The collection
container can be removed by the user for further processing.
collectio
n
carousel
pipette
petri
Figure 1: Workspace of the CellBot where the micropipette is about
to pick a cell to transfer it to the carousel for immobilization.
Figure 2: A special combination of bright– and darkfield illumination
enables simultaneous detection of several parameters of the
Xenopus laevis oocytes, such as shape, size, and coloration, that will
identify viable cells.
This work was partly funded by the EU (project NMP2-CT-
2006-026622) and the cantons of central Switzerland and the
MCCS (Micro Center Central Switzerland). CSEM thanks
them for their support.
[1] T. Stöckli, et al., “High precision robotics for automated cell
handling“, CSEM Scientific and Technical Report 2006, page 83