Experiment

Regular mechanical stimulation, respectively movement, is essential for human health. Too little mechanical load on the human body has a dramatic effect, which can be observed by astronauts and bedridden patients. Biological cells have several mechanisms with which mechanical forces can be detected. One of these mechanisms are responsive ion channels in the cell membrane which react to mechanical load. The topic of this research project is the study of such mechanosensitive ion channels under normal gravity and microgravity. This approach is promising to find clues on how cells perceive external forces. To investigate this, a host cell that expresses a large amount of such ion channels is required. A suitable host cell for this research project is the egg of the South African clawed frog (Xenopus laevis). This egg is particularly well suited for this purpose because it is easy to handle and very robust. In addition to this, it is relatively large with approximately one millimeter in diameter. For the implementation of the biological experiment various processes must be optimized and the composition of the used chemicals have to be determined. Since the measurement is fully automated, the used protocol during the flight must be worked out and tested.

 


 

The measuring chamber

The used measuring chamber is an further development of an existing measuring chamber which was successfully testet on parabolic flights. In the previous measurement chamber, the frog egg was placed in a small funnel manufactured of hard polymethylmethacrylate (also known as PMMA or under the market name Plexiglas). In the new design, this part is replaced by a silicone chip. Due to this modification, a better signal to noise ratio is expected. Because silicone itself is not stable enough, the chip is stabilized by a 0.3 mm thick glass plate. The biggest challenge so far was the integration of the thin glass plate into the silicone chip, which is manufactured by casting. The hole in the glass plate must be precisely aligned to the cavity in the silicone chip. Because the glass plate often cracks during demolding from the mold, the glass also needs to be reinforced before the casting.


 

The measurement

The mechanosensitive ion channels which are embedded in the cell membrane allow ions to pass through the cell membrane. The electrical conductivity of the cell membrane depends on how many ion channels are open or closed. By applying a voltage across the cell membrane, this conductivity can be determined with the measured current. Such measurements are usually performed with electrodes, which are stuck into the egg through the cell membrane. For the experiment on the sounding rocket, the less invasive patch-clamp technique is applied. In this technique, the egg is placed in a cavity in a silicone chip with a small hole at the lower end (see Figure 1).

Messkamme_schema_DE
Figure 1: Scheme of the measuring chamber

The oocyte is placed in a cavity in a silicone chip (green). This is stabilized by a glass plate (dark green). A hole in the glass plate isolates a small piece of the cell membrane. With four electrodes, the conductivity can be measured within this piece of membrane. 

 

By applying an overpressure in the upper part of the chamber the egg is pressed onto the tiny hole. The membrane body lying on this hole is electrically isolated from the remaining cell membrane. The conductivity measurement is now carried out on this small patch of the cell membrane. For the measurement four electrodes are used. The upper two regulate the voltage across the membrane at a desired value. The two lower electrodes measure the corresponding current. For further information about the measurement procedure, see Electronics.

 

The measured conductivity depends not exclusively on the analyzed ion channels. Therefore, the background signal must also be determined. The isolated membrane body is in contact with a microfluidic chip, where three different liquids can be applied to the cell membrane. By applying pharmacological drugs the ion channels can be specifically blocked. Thereof the background signal can be determined. By the subsequent subtraction of the measurement with blockers from the measurement without the blocker the actual conductivity of the ion channels can be determined.