5. Select the desired stretch ratio and frequency on the Control Unit. Programs are
outlined in Section 3.
6. Press the START button to start the stretch cycle. During the action cycle, the green
light will be illuminated. To STOP movement at any time, press the STOP button.
7.
Do not change the Stretch Ratio or Frequency parameters during the operation of
a stretch cycle. Press the STOP button and wait for the program to complete the
final stretch-contract sequence (returning to the original start position) before
initiating the next stretch parameter.
Changing parameters during a stretch cycle may
damage the motor.
8. After a few minutes of running a stretch program, stop the cycle and check the condition
of the cells. If the cells have not detached from the membrane, proceed with your
experiment. If the cells are detached, the chamber coating was probably insufficient.
Recoat the chambers.
Culturing Cells in the Silicone Chambers
1. Seed cells at the appropriate concentration in a freshly coated chamber.
Important:
It is critical to not over expose the cells to dissociation enzymes. Cells should
be treated in the same manner (type and concentration of enzyme, temperature, and
exposure time) for all experiments.
Important:
Cells should not be seeded at a high density in the chambers. For example,
epithelial cells often form a cell-sheet and the cell-cell adhesion seems to be stronger
than a cell-surface adhesion. When this happens cells may detach from the chamber.
Additionally, cultures that are grown over a week in the chambers may detach.
2. After an overnight incubation without stretching, inspect the cells with a microscope to
ensure they have adhered to the chamber.
Preparation of Silicone Chambers
Before using the chambers, they should be sterilized and coated with a cell adhesion
matrix. The coating procedures can be adapted for use with other matrices, such as
elastin, pronectin, and laminin.
Sterilize the chambers in an autoclave for 20 minutes at 121°C. The silicone chambers can
withstand temperatures up to 180°C. Use of an autoclave is preferable. However, if an
autoclave is not available, the chambers may be sterilized by submerging them in 70%
ethanol, rinsing with water, then drying in a sterile environment.
Place the sterile chambers in a Petri dish in preparation for coating.
The PDMS (silicone) chamber
is very hydrophobic with two methyl-bases on the surface; therefore the chamber
must be coated with a cell adhesion matrix such as fibronectin or collagen. In the presence of fibronectin or
collagen, cells adhere to the matrix by integrins. Integrin attachment is a cell specific interaction unlike cell
attachment to plastic or glass dishes, where cells non-specifically attach due to a charged surface.
