The overall goal of this method is to control the cell membrane potential upon stimulation by visible light pulses. The phototransduction process is mediated by a light sensitive conjugated polymer. This method provides a new tool in the technologies field seeing as it represents a valuable alternative to existing methods such as optogenetics and thermal stimulation for optical control of living cells activity.
The main advantages of this technique are the prompt integrability with any electrophysiologist type, the potentially high special and temporal resolution, they reduce the invasivity. More over, it allows to avoid a gene transfer which is required by optogenetics. Our method is described here for photostimulation of in vitro cell culture.
But it might open also interesting opportunities for in vivo applications. Demonstrating the procedure will be Caterina Bossio and Susana Vaquero Morata to post-docs from our labs with biological and physical chemistry backgrounds respectively. To begin, prepare a solution of the light sensitive conjugated polymer by mixing P3HT and PCBM in chlorobenzine at a one to one relative concentration.
Mix the solution with a magnetic stirrer for at least four hours at 60 degrees Celsius. Next, clean indium tin oxide-coated glass slides with consecutive 10-minute baths of deionized water, acetone, and isopropanol in a sonicator. Dry the cover slips with a nitrogen gun, and then treat the substrates by a plasma cleaner.
Once clean, set the slide on the chuck of a spincoater, ITO-coated side facing upwards. Next, add 150 microliters of the P3HT PCBM solution to the slide and spincoat the sample. After the spincoater is finsihed, remove the sample, and use a cleanroom swab dipped in acetone to clean the back of the substrate.
Sterilize the photoactive substrates by placing them into a covered heat resistant container and heating them at 120 degrees Celsius for two hours. Then transfer the samples to a sterile hood and let them cool. From this point on, maintain the sterility of the samples.
Place the photoactive substrates into a six well plate. Given the hydrophobicity of the conjugated polymer layer, make a polydimethylsiloxane well to fix the sample, and confine the solutions as described in the accompanying text protocol. Then, place the photoactive substrates into the wells of the plate, and cover the entire surface of each sample with 500 to 750 microliters of a two milligrams per liter solution of fibronectin in PBS.
Incubate the samples at 37 degrees Celsius for at least one hour. After incubation, use a glass pipette to remove the fibronectine solution, and then wash the substrates once with two milliliters of PBS. First, add a total volume of 750 to 1, 000 microliters of complete growth medium per well.
Next, plate 15, 000 AGK293 cells per square centimeter of photoactive substrate into each well. Then, incubate the cells for 24 to 48 hours. Cells can be easily cultured on the photoactive substrates, provided that a suitable adhesion layer, like fibronectin, is depositited.
To begin, prepare both the extracellular and intracellular solutions as described in the accompanying text protocol. Sterilize the solutions by passing them through a 0.2 micron filter. Then, prewarm the extracellular solution by placing it into a water bath at 37 degrees Celsius.
Also, fill a one milliliter syringe with the intracellular solution and equip it with a 34-gauge needle. Keep the intracellular solution in contact with ice to avoid degradation of the ATP. Next, take a cell-coated substrate from the incubator and carefully remove the PDMS well.
Then remove the growth medium with a glass pipette and rinse the sample slowly with extracellular solution to avoid cell detachment. Add three milliliters of extracellular solution to each well and then transfer the plate to the sample holder of the electrophysiology station. Once in place, set the reference electrode into the well that is to be tested.
Next, prepare a fresh glass micropipette for patch clamp using a micropipette puller. Fill half of the pipette with intracellular solution. Use the micromanipulator to position the patch pipette in close proximity to the selected cell membrane.
During the positioning, apply over pressure to the pipette in order to avoid dirt sticking on the tip. With the pipette a few microns above the cell, start lowering the pipette towards the cell membrane while contolling the pipette resistance on the patch control software. When the pipette resistance increases about one megaohm, remove the over pressure and apply a gentle suction in order to form the gigaseal between the pipette and the cell membrane.
Next, apply a negative potential of negative 40 millivolts to the pipette to help stabilize the seal. Compensate for the capacitive transience due to the pipette capacitance with the relative commands on the patch amplifier and/or the patch control software. Apply a brief and intense suction impulse to the pipette in order to break the membrane and to allow electrical access to the cell cytoplasm.
Then, set the patch amplifier to current clamp mode so that no current is injected into the cell. In current clamp mode, track the cell membrane potential and wait a few minutes for it to stabilize. Once stable, apply the desired illumination protocol to the cell by illuminating the photoactive membrane under the cell with light in the range of 450 to 600 nanometers.
Record the effect of the light on the membrane potential. Short pulses of light around 10 to 20 milliseconds result in a transient depolarization of the cell, while with prolonged illumination in order of hundreds of milliseconds are sustained, hyperbolization is observed until the light is switched off. Optical stimulation of cells mediated by the photoactive substrates can result in different effects on the cell membrane, depending on the duration of the light stimulus.
A fast spike, like the one shown here, is followed by a transient depolarization on the order of tens of milliseconds. For short pulses of light as the light is switched off, an opposite behavior is observed. These signals have been attributed to a variation in membrane capacitance due to local heating following light absorption.
For prolonged illumination, the initial depolarization turns into a cell hyperpolarization. This phenomenon has again a thermal origin, but in this case, it is related to a variation of the membrane equilibrium potential due to a change in the ion channel's conductivities induced by the increased temperature. It is important to note that this technique is quite flexible and can be adapted to the particular needs of a specific experiment, for instance, by changing the photoxic polymer, the fabrication process, or the illumination protocol.
However, before changing the protocol, one should first verify the important properties of the active polymer like its biocompatability, suitability to the sterilization process, electrochemical and temporal stability. After watching this video, you should have a good understanding of how to realize a hybrid interface between connective polymers and living cells and how to use it for optical control of cell activity. We believe that this technique also promises to become a complimentary tool in neuroscience for in vitro investigations and also to open a new perspective in vivo applications.
To refine the protocol, however, a strong and joint effort from material scientists and neuroscientists, physicists, and medical doctors is mandatory.
