The overall goal of the following experiment is to record the electrophysiological activity of cultured cells via a planar patch clamp chip. This is achieved by sterilizing, priming and testing the patch clamp chips to allow the growth of cell culture and the subsequent monitoring of its electrophysiological activity. After testing chips can be stored until they are primed again in a cell biology lab.
As a next step, neurons are extracted from limbs, staus, and cultured onto the chips. Finally, the chips are connected to a patch clamp amplifier in order to monitor the electrophysiological activity of the cells. The main advantage of this technology over the existing planner patch clamp chips is that it is suitable for use with neurons establishing culture, allowing the interrogation of synaptically communicating, communicate in neurals.
This method and the approach will allow better understanding of ion channel functions of excitable cells embedded within a neuronal network under both normal and pathological conditions. The implications of this method extend to pharmacology where it can be used to perform medium throughput, high information content, drug screenings for ion channels and receptor proteins. We have chosen to demonstrate this method using snail neurons because these neurons can be easily positioned and cultured.
And because these neurons will reform network in culture, they make an excellent mod studies to transformation. More importantly, these chips can also be used in other cell systems such as cardiac and cancer cells. The main challenge with using planar patch clamp chips is to obtain chips with clean apertures, plating and culturing cells.
Unplugged chips would be counterproductive, and therefore all chips are tested before being Used. The most critical point of electrophysiological assessment comes once the cells have been cultured. It consists in the formation of the giga seal between the cell membrane and the cheap upper two rim.
All plantar patch clamp chips produced date used isolated cell in suspension and rely on suction to draw cells to the aperture to form a giga seal. GigE formation. Using our chips occurs spontaneously.
While the neuron are culture and does not rely on suction, I will demonstrate the chip sterilization and screening step. Tanya will explain the loading in a cell biology lab, Colin will illustrate cell preparation and plating. Finally, Marcia will show our recordings are taken To begin this procedure, sterilize the chips in a heric basic plasma cleaner with 0.1 to 0.3 millibars of residual air pressure for 15 minutes at the maximum power of 18 watts.
The plasma treatment also renders the chip's hydrophilic, which facilitates priming. Next, fit the glass tubes with a sterile cel elastic laboratory Silicone tubing, three inches on one end and one inch on the other end. Then fill the tubes with sterile standard phosphate buffer solution, making sure that no bubble is trapped.
Clip the long silicone tube while pressurizing the PBS from the short end at one atmosphere. Then release the pressure in the fluidic channel. After that, fill the vial with PBS using a syringe.
Immerse a silver, silver chloride electrode in the vial and a counter electrode in the short tube. Use an impedance meter to confirm that the access resistance is between 300 kilo ohms and three mega ohms, and the shunt capacitance is between 10 pico ferriday and 25 pico ferriday. Next, flush the fluidic channel with sterile deionized water.
Empty the top vial and repeat the rinsing process twice. Subsequently, immerse the package to chip with the tubes in fresh, sterile deionized water for 30 minutes. Then load other chips in a sterile container filled with sterile deionized water.
This ensures that chips remain hydrophilic and protected from contamination. Open a chip container under a HEPA filtered laminar flow hood and remove the chips with the top vial facing down to avoid contamination from packaging debris floating on the water surface. To remove any residual debris from the chip surface, rinse the top vial twice with filtered sterile deionized water Connect one end of the elastic tubing attached to the fluidic channel to a pressurized syringe containing sterile filtered deionized water.
In this system, the water in the syringe is pressurized by a compressed air tank set to one atmosphere to ensure sterility air is filtered prior to entering the syringe. This system is also equipped with two on-off valves that allow the control of pressure when needed. Clamp the output end of the fluidics with a hemostat.
Next, open the compressed air tank valve to pressurize the solution. Then open the hemostat at the output end of the fluidics to flush the chip with fresh sterile deionized water. Clamp the output end of the fluidics with a hemostat to force the water up through the aperture.
To avoid water drainage, clamp the input of the fluidics with the hemostat and close the on-off valves. Then detach the input end of the tubing from the syringe. Plug both ends of the tubing and remove the hemostats.
Fill the top vial with the filtered sterile deionized water. Next, place the lid of a 35 millimeter sterile dish on the base of a 100 millimeter sterile dish. Subsequently, place a chip on top of the 35 millimeter lid and cover with a 100 millimeter dish lid.
To ensure that the membrane and aperture of the chips are free of debris or air bubbles, the chips are imaged. If debris or air bubbles are present. The fluidic channel and the top vial are washed repeatedly with sterile water.
If the debris or air bubbles cannot be removed, the chip is discarded. Once the chips are imaged, bring the chips back to the laminar flow hood. Unplug both ends of the tubing.
Attach one end of the tubing to a pressurized syringe containing physiological media and clamp the output end of the tubing with the hemostat to rinse the subterranean fluidic with physiological media. Open the on off valves and remove the hemostat from the output end of the tubing to force the physiological media up through the aperture clamp the output end of the tubing with the hemostat clamp, the input end of the tubing with the hemostat and close the on off valves. Next, remove the input end of the tubing from the syringe and plug both ends of the tubing with glass plugs.
Then remove the hemostats. Subsequently, remove water from the top vial. Fill the top vial with filtered sterile media.
Place the chip back in the 100 millimeter dish. After that, place the base of the 35 millimeter dish in the 100 millimeter dish and fill it with filtered sterile deionized water to enrich humidity. Then cover the dish until the time of plating.
To begin this procedure, remove the outer shell from two to three month old li sags with a pair of blunt forceps. Then anesthetize the animals in limba saline containing 10%Listerine in a laminar airflow hood. Pin the snails into a cell guard dish filled with limba saline and remove the entire brain.
Next, treat the isolated brains in defined media with trypsin enzyme for 18 minutes. After that, treat them in defined media and trypsin inhibitor. For 15 minutes, pin the brains to a small cell guard dish filled with high osmolarity defined media using a pair of fine forceps and a pair of dissection scissors.
Remove the outer and inner sheath of the ganglia of interest and expose the neurons. Next, attach a fire polished glass pipette filled with high osmolarity defined media to a micros syringe gently applies suction near the cell of interest until the neuron det attaches from the brain and is suspended in the pipette. Having changed the media in the fluidics of the neuro chip to an appropriate recording solution, tip the glass pipette into a culture vial and gently expose the neuron on top of the aperture of the chip.
Allow the cells to sit undisturbed for a minimum of two hours at room temperature to promote their attachment to the chip surface surrounding the aperture. Replace the solution in the culture dish with the appropriate recording solution For stability. Glue the chip onto a glass slide and place it under a microscope to connect the chips to the amplifier.
Cut the long end of the tubing and insert the silver wire connected to the head stage. Next place the reference electrode in the chip culture dish. The chip is now ready for recording.
Here shows an example of the voltage responses of a left pedal dorsal one neuron to graded series of intracellular current pulses. After watching this video, you should have a good understanding of how to record the electrophysiological activity of cultured cells with a planar patch clamp chip. Remember that in the first step we screen chips to ensure that non-used to plate cells have a plugged up Ture biology users start from the loading step and typically still find a few block chips for every batch.
This new method represents an important breakthrough in developing a cheap technology that provides for the interrogation with an unprecedented level of resolution of in vitro neural networks. This technology can lead to advanced assays for therapeutic target identification, drug development, and diagnostic assessment. Further development of this technology will provide new means for health researchers to explore novel therapies for neurological diseases or disorders.
Its potential extends to cardiac cells, cancer, and other cells that have electrogenic or properties.
