Fig. 2: 4D-Table robot with 10 μl Hamilton syringe (1); gel preparation unit (2); dual injection unit (3), this unit will be replaced by the tissue carrier system and the multimodal incubator; cleaning device (4) and sample store (5).
Fig. 1: 4D-Table robot (1), temperature controlled housing. Temperature range: 11°C - 40°C (2), temperature control unit (3), PC for robot control (4)
Fig. 3: Probe handling system for MRI 11,7 T Imaging System (1), Positioning system for high frequency probe exchange (2), Bruker BioSpec MRI system (3), Temperature and gas supply for the incubator (4).
Fig. 5: 19F image at 470 MHz, Sequence: 3D-FLASH TR: 100 ms, TE: 4,4 ms spatial esolution: 125 μm acquisition time: 20 min
Fig. 4: MRI 1H image at 500MHz, spatial resolution: 170 μm acquisition time: 6 min
3 x 3 injections of Hexafluorobenzene
10 nl - 4 μl
Fig. 6: Fluorescence image (λ=685 nm),
(Photon Imager, Biospace) of varying amounts of mCherry marked tumor cells (9660 cells/μl) in gelatine matrix (1.25% gelatine).
Fig. 7: MR image of Endorem marked tumor cells in gel (1.25% gelatine) FLASH 3D sequence, TR: 150 ms, TE: 7 ms,
Resolution: 43x43x62.5 μm
Methodologies and techniques
For in-vitro cell transplantation ProbRob (Fig. 1) will be used, a computer controlled table robot unit, with implemented temperature control (11°- 40°C). ProbRob works with a 3D spatial accuracy of 25 μm. Up to 18 different samples (cells or fluids) can be injected. Injection volume can be adjusted down to 50 nl /injection directly into one to two samples at the same time. This procedure will be modified to transplant cells into tissue samples.
We would also like to draw on MPInF’s experiences in developing applications with complex user interfaces, e.g. VINCI, http://www.nf.mpg.de/vinci, and pursue similar strategies, one of which is to use Trolltech’s Qt toolkit, http://www.trolltech.com/qt, because of the excellent developer support, graphics capabilities, test framework and inhouse expertise (MPINF). The Qt toolkit will also allow us to develop in parallel on modern flavours of MS Windows and MacOS X (optionally also Linux), an approach that improves software quality by being able to run automated tests on different platforms.
The development of therapeutics administered on a cellular level strongly benefits from highly resolving imaging techniques. First MRI investigations show the potential of F19 as a cellular marker that selectively allows to image only the labelled cells. The observation of cellular mechanisms or cell tracking in vivo still needs further increase of SNR and optimisation of labelling conditions. We therefore demonstrate basic phantom experiments based on a robotic implantation technique.
The figures 6 and 7 show a matrix of 3x3 injections of Gli36-DEGFR-human glioblastoma cells labelled with Endorem, expressing mCherry in an area of 4x4mm. MRI images were acquired on a 11.7 T MRI scanner (Bruker BioSpec 117/16USR, Ettlingen) using a transmit/receive surface coil of optimal size (22 mm) for high SNR. This coil can be tuned to any frequency between 200 and 500 MHz.
Human embryonic stem cells derived cardiomyocytes (hESCMs) have been suggested for cardiac cell replacement therapy. Several in vitro studies assessed the mechanical and functional properties of transplanted hESCMs. This approach is severely limited by a missing standardization and quantification of the transplantation method. Therefore an automated technology is paramount to establish a basis for reproducible and quantifiable experiments.
We have developed a method of robotic probe preparation (ProbRob) with a multimodal imaging incubator, which ensures both high accuracy, reproduce ability and minimum preparation time.
ProbRob (Fig. 1) has been used to prepare multimodal MRI, luminescence and fluorescence cell phantoms. For transplanting cells into tissue slices, a tissue carrier module will be developed and integrated into a multimodal incubator system. This incubator will serve as a module and can be used to acquire high resolution (a few μm isotropic) 3 dimensional anatomical data (MRI), metabolic information (NMR) and functional parameters (fluorescence and luminescence imaging).
Several studies indicate that electromechanical stimuli can influence the formation, alignment and force development of hESCMs. Therefore the tissue carrier system will be able to exert a defined static or dynamic strain on the tissue before and after transplantation. The force will be generated by piezo crystal devices controlled by the robot control software.
The method works fast and precisely, and guarantees comparability between different measurements. It is therefore a highly valuable tool for quality management. As each step is automated, conditions can be adjusted as close to real in-vivo experiments as possible.
In Vitro Cell Transplantation Technology
Cell transplantation is generally performed manually in most laboratories and is therefore very time-consuming.
Reliable statistical data can not be achieved with manual handling for two reasons:
Therefore it is highly desirable to minimize time and errors by establishing standardized equipment and preparing in-vitro transplantations with automated processes. We established a new method of robotic probe preparation (ProbRob) with a multimodal imaging incubator, which ensures high accuracy, reproducibility and minimum preparation time. Tissue samples of standardized diameter will be embedded into a special tissue carrier system. This system allows to exert a defined static or dynamic strain to the tissue at any time point of the experiment. After optical detection multiple transplantation points at individual depth can be defined with the robot control software. A predefined quantity of cells can then be injected automatically. For reasons of quality control, all steps are recorded precisely. The carrier unit can be integrated into a multimodal bioreactor system. This multimodal approach gives the opportunity to perform in-vitro cell transplantation under incubator conditions and then investigate the transplant with optical microscopy, MRI, PET, FLI and luminescence imaging giving access to a wide spectrum of parameters.