Cell Stretcher CS-10 Series - a device for simultaneous live cell imaging with motion compensation for uni-axial mechanical straining or compression
Numerous different types of cells in the body experience mechanical forces and the effect of cell stretching, compression or other mechanical stimuli is becoming a new and surprising scientific focus. Many machines have already been designed to stretch cells. Cells are typically grown on elastic membranes and the whole membrane is deformed to deform the attached cells. This approach has been shown to be useful but suffers one major disadvantage: When cells need to be imaged during or after the membrane is deformed, the cells are shifted relative to the optical axis of the microscope and microscopic examination is virtually impossible. As can be seen in figure below, only the centre part (red X) remains stable relative to the microscope target.
The dcCS10-compensation transfers the entire membrane throughout the stretch to combat this displacement. As a consequence, the area of interest is still precisely located above the microscope objective during (and after) the run. The benefit: the cells can be imaged even during stretching!
Once cells are developed on an elastic membrane and the pressure is applied to the membrane, only the centre portion of the membrane can be used for imaging as only that section remains at its original position relative to the objective of the microscope. This is a huge drawback, as only certain cells are eligible for microscopic examination that have formed in this central portion of the membrane. We have created a computer-controlled stretch device that overcomes this obstacle while using active movement compensation. With our new stretcher cells, either uni-axial strain or strain relaxation (compression) can occur and the cells can be simultaneously imaged at high optical resolution. This is possible at any place on the membrane when strain or compression is applied. Multiple other improvements have been introduced to ensure efficient handling of cells – both in the incubator and on the microscope during prolonged cell culture.
Detailed Description of the Device
This convenient cell strain system enables people to conduct life-cell imaging studies whereby rapid imaging is required, such as detecting changes in intracellular concentration of Ca 2 + when cells are stimulated mechanically. This convenient cell strain system enables people to conduct life-cell imaging studies whereby rapid imaging is required, such as detecting changes in intracellular concentration of Ca 2 + when cells are stimulated mechanically. The commercially produced PDMS substrates are thin enough to use even oil immersion targets. The cell strain device is not controlled by a microscope software, but the programme runs on a separate computer – a small laptop or even a net book with an adequate interface is suitable. It helps the researcher to use the stretcher on any microscope and irrespective of the image processing software installed. Of example, the tool can also be used without the need for a microscope.
The diagram of the unit is shown in Figure 1. The strain / compression system consists of two sections with various functions. One part deforms the membrane, allowing the user to strain the installed membrane chamber or to compress the pre-stretched membrane chamber. The other portion needs to compensate for the unfavorable lateral displacement by moving the entire carrier plate (see Figure 1A) along the strain / compression axis. The synchronous interaction of these two components holds every desired area of the membrane in the same place when applying pressure or compression. The deformation portion consists of two sliding blocks where the membrane chamber is placed during the study. To extend the membrane, the two blocks move in opposing directions on a linear motion map, increasing the length of the membrane chamber and deforming the cells. One sliding block is directly moved by the SC motor through a ball screw, while the other sliding block is pulled in the opposite direction by a metal band and a pulley mechanism (see also figure 1A). Any motion of the motor-driven block leads to the very same amount of displacement of the other block by the pulley mechanism. Both sliding blocks are attached to the spring to provide a permanent pulling force. This provides the necessary counterforce to keep the metal band under tension. Expanding the distance between both the blocks needs the motors, while the passive, attractive force of the spring allows the distance to be reduced (see arrows in Figure 1A).
Figure 1. Schematic of the strain/compression (SC) device.
A: The SC-device with the attached membrane chamber is assembled on the microscope. The carrier plate can be seen in grey with all the components attached in matching colours. The SC motor is fitted on the carrier plate and deforms the mounted membrane chamber by shifting the sliding blocks through a ball screw and a metal band (see also text). To account for the stretch-induced lateral displacement, the carrier plate is shifted sideways with the ball screw operated by the relief engine (both in blue). The red arrows indicate the movements of the holders of the membrane during deformation and the movements of the carrier plate to compensate for the lateral displacement.
B: The membrane chamber forms the elastic membrane into a tray used to grow the cells. Before assembly, one of the holders is shown in the view on the top right. Two dual hooks of the pre-train holder (bottom) carry the membrane chamber and can be pushed freely along the rail plate and affixed to any location with two screws. Assessment of the membrane chamber with only a regular cell culture microscope via the observation window enables the inspection of the cells during the cultivation of the cells without removing the cells from the prestrain holder.
C: The membrane chamber and the pre-stress holder are quickly connected. The next steps are typically the autoclaving and coating of the membrane followed by the seeding of the cells in the tray.
To accommodate for the lateral movement of the membrane, the carrier plate is shifted laterally by the compensation motor along the linear motion guide (not shown in Figure 1). Since all parts of the deforming component are mounted to the carrier plate, this component is transferred as a whole. The two DC servo motors (compensation and SC-motor) are attached to an exterior control unit and operated by a custom-written software computer (see also below). The programme not only measures how much the carrier plate has to be shifted to compensate for the stretch-induced displacement, but also provides a convenient graphical user interface for the stretch protocol design.
1) Any required strain protocol may be programmed including cyclic protocols with different characteristics (linear, sinusoidal, sigmoidal). It involves pauses or loops for adding static steps, or repeating a particular sequence.
2) The location of the membrane chamber along the stretch axis may be adjusted separately from the motion control movements.
3) The width of the two sliding blocks of the part SC can be modified for mounting the membrane chambers. Thus it is possible to mount membrane chambers of any length (within the limits of the device) which also allows the mounting of prestrained membranes and the compression of cells.
Membrane Chamber Including Prestrain Holder
The membrane chamber consists of the membrane holders and the translucent membrane (127 microns thick). The membrane holders secure the membrane in such a way that it assumes a flat bottom with two side walls (ca. 60 ° angle). Thus a tray is shaped which allows the chamber to be used as a daily crop dish. A lateral view of the membrane chamber during assembly is shown in Figure 1B (top right). One of the two membrane holders has been shown to demonstrate the mounting process before the membrane is clamped. Only after the membrane chamber is mounted it is hooked onto the prestrain holder where it usually remains throughout the rest of the procedure (autoclaving, surface coating, cell seeding). The membrane chamber fits perfectly into a standard 15 cm crop bowl, which enables sterile processing outside a sterile work bench and even tracking cell growth with a low magnification objective in a traditional cell culture microscope. Two metal rods in the membrane holders are used for positioning the membrane chamber onto the SC component's two sliding blocks. The rods and sliding blocks are constructed to comfortably mount and unmount the membrane holder from the unit and perform all appropriate steps avoiding deformation of the membrane which could lead to undesirable cell deformation before the actual stretch. The two membrane holders will move along a platform rail (see Figure 1B) and be locked with two locking screws at any desired prestrain. For a later compression of the cells in the SC system, mounting the membranes with prestrain is not only essential, but typically the membrane is installed with a slight prestrain (about 20 per cent) to flatten the bottom of the membrane. When cells need to be compressed, of course, the prestrain needs to be sufficiently high to keep the membrane under stress after relaxation. The upper limit (and therefore compression) for the prestrain is ca. 100%, defined by the total strain that can be introduced mechanically to the membrane and by the membrane stretch sensitivity.
Cultivated cells react in various ways to a mechanical stimulus and the visualisation of these responses has become an important tool for investigating mechanotransduction. The installed chamber holder fits in a standard 150 mm cell culture dish enabling the sterile handling and cell incubation in a traditional CO2 incubator. In addition, the cells in the dish can be examined for brief nspection with simple cell culture microscope at low magnification, without removing the holder from the container. Microscopic techniques are constantly improving, particularly fluorescence detection methods, and countless fluorescence dyes and labelling methods are available to resolve cell-biological issues related to mechanical cell stimulation.
- Software based on "JAVA"
- Windows 2000 or above
- Pentium 4
- 1024 MB RAM
- 100 MB free space on disc
- 1024 x 768 screen resolution
Measurements: 18cm (w) x 11.5cm (t) x 2.5cm (h)
Power supply: 1 battery, SR44 and 4 units of holder/mounting included