The work was supported by EU through project FP7-PEOPLE-212-ITN 316832-OLIMPIA, 
Telethon - Italy (grants GGP12033 and GGP14022), Fondazione Cariplo (grant ID 2013-0738).

1. Preparation of Photoactive Substrates
<ol>
	<li>Prepare a P3HT:PCBM solution (1:1 w/w) in chlorobenzene at a P3HT concentration of 20 g/L. Mix the solution with a magnetic stirrer for at least 4 hr at 60 &#176;C. Consider a volume of 150 &#181;l of solution for each substrate to be prepared.</li>
	<li>Clean ITO-coated glass slides (Rs = 10 &#937;/sq, 18x18 mm2, thickness 170 &#181;m) with consecutive baths of deionized water, acetone and isopropanol in a sonicator (10 min for each step). Dry th...

<ol>
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Critical steps of the reported protocol for in-vitro optical stimulation of cells mainly concern the choice of the light-sensitive polymer, the thermal sterilization parameters, the intensity and the duration of light stimuli. A P3HT:PCBM thin film was selected here, since it guarantees good temporal and electrochemical stability. However, one should notice that not all light-sensitive polymers can offer analogue performances,22 more specifically upon illumination. In addition, in this case selected t...

The possibility to manipulate the cellular activity with a precise spatial and temporal resolution represents a key strategy in neuroscientific research and in the treatment of neurological and psychiatric disorders.1 Traditional methods are based on electrical stimulation of cells using electrodes positioned in proximity or in contact with the targeted system,2 which can be of different complexity (single cell, cellular network, brain slices, in-vivo brain tissues). During the past century, the use of patch-clamp, metal and substrate-integrated electrodes have provided a detailed picture of the physiology and pathophysiology of single neurons and of the functioning mechanisms of neural networks. However, electrical stimulation suffers from important limitations. The first one is related to a generally poor spatial resolution due to the physical dimensions of the electrodes and their fixed geometry, which cannot be readily adapted to complex organized systems like biological tissues. Also, problems related to the electrodes impedance and of cross-talk between stimulation and recording systems may deteriorate the final signal-to-noise ratio of the measurements.3 On the other hand, the use of light for stimulation may help to overcome many limitations of the electrical approach. First of all, it offers unprecedented spatial (&#60; 1 &#181;m) and temporal resolution (&#60; 1 msec), making it possible to target specific cell types or even sub-cell compartments. In addition it is highly non-invasive since it avoids any physical contact with the tissue of interest and disentangles stimulation from recording. Moreover, both light intensity and wavelength can be precisely regulated and thus diverse stimulation protocols can be applied.3,4
However, the vast majority of animal cells do not present any specific sensitivity to light. Several strategies for optical stimulation have thus been proposed, either exploiting light-sensitive molecular mediators nearby or within the cells, or using photoactive device placed externally, close to the cell. The former category refers to endogenous mechanisms like the stimulation via visible or infrared (IR) light, as well as the use of either photoisomerizable/photocleavable compounds or the genetic expression of photosensitive molecular actuators (optogenetics). The latter class includes techniques for exogenous stimulation achieved with the use of inorganic nano/micro-particles or photoconductive silicon substrates.5 Nevertheless, all these systems have bright sides and drawbacks. In particular, endogenous absorption of cells in the visible range is weak and not reliable, and the concomitant generation of reactive oxygen species may be harmful to the cell. In general, IR is used for inducing local thermal heating due to water absorption, but the extinction coefficient of water is small, thus requiring strong infrared light (from tens to hundreds of W/mm2) that is difficult to deliver via standard microscope optics and may pose safety concerns for in-vivo applications. On the other hand, photo-switchable caging compounds have a time limited action and often require UV light that is hard to deliver due to limited tissue penetration. In addition they suffer from diffusion problems of the activated compounds upon photolysis outside the illuminated area. Finally, optogenetic tools have allowed scientists to target specific cellular subpopulation and sub-compartments and are rapidly emerging as one of the key technologies in neuroscientific research. However, inserting an exogenous DNA segment via a viral vector raises important safety issues, especially in view of adoption on human patients.5,6 For these reasons, research on new materials and devices capable of cell optical manipulation is an extremely hot topic.
Recently, a novel approach based on the use of light-sensitive conjugated polymers, able to efficiently transduce an optical stimulus into a modulation of cell electrical activity, has been proposed. The Cell Stimulation by Polymer Photoexcitation (CSPP) technique exploits many key-enabling features typical of organic semiconductors: they are intrinsically sensitive to light in the visible range;7 they are biocompatible, soft and conformable and their mechanical flexibility allows an intimate interface with tissue both in-vitro and in-vivo8-10. Apart from that, they can be easily functionalized to better adapt to the interface with living cells, and to enable specific excitation, probing and sensing capabilities.11,12 Moreover, they support electronic as well as ionic transport, making them ideal for the combination of electronics ad biology.13,14 Interestingly, they can work in photovoltaic mode, avoiding the need to apply an external bias for efficient cell optical stimulation.15 
The reliability of CSPP technique has been previously demonstrated in several systems, including primary neurons,15,16 astrocytes,17 secondary cell lines18 and explanted retinal tissues.16 In this work, all the steps necessary to fabricate a light-sensitive bio-polymer interface19 for optical stimulation of in-vitro systems are described in detail. As a study case, a prototypical organic photovoltaic blend of region-regular poly(3-hexylthiophene) (rr-P3HT), functioning as the electron donor, and phenyl-C61-butyric-acid-methyl ester (PCBM), acting as the electron acceptor is employed. As the biological system, Human Embryonic Kidney (HEK-293) cells are used. An example of a photostimulation protocol with the relative recording of cell activity via electrophysiological measurements is provided.
The described platform is however of general validity, and it can be easily extended to the use of other conjugated polymers (by properly adjusting the solution preparation process and the deposition parameters), different cell types (by properly changing the cell culture protocol, plating procedure and time requested for cell seeding and proliferation) and different stimulation protocols (light wavelength, stimuli frequency and duration, photoexcitation density).

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">rr-P3HT</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">698989-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ITO-coated substrates</td>
			<td style="width:120px;">Nano-CS</td>
			<td style="width:191px;">IT10300100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibronectin</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chlorobenzene</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td align="right" style="width:191px;">319996</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PCBM</td>
			<td style="width:120px;">Nano-C</td>
			<td style="width:191px;">Nano-CPCBM-BF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">acetone&#160;</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td align="right" style="width:191px;">270725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isopropyl alcohol</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td align="right" style="width:191px;">563935</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEK cells</td>
			<td style="width:120px;">LGC standards srl</td>
			<td style="width:191px;">ATCC-CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">P5244</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">E-MEM</td>
			<td style="width:120px;">LGC standards srl</td>
			<td style="width:191px;">ATCC-30-2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">E8008-</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FBS</td>
			<td style="width:120px;">LGC standards srl</td>
			<td style="width:191px;">ATCC-30-2020</td>
			<td></td>
		</tr>
	</tbody>
</table>

optical control of living cells electrical activity by conjugated polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

Cells can be easily cultured on P3HT:PCBM substrates, provided that a suitable adhesion layer is deposited (like the fibronectin used in step 3.2 of the described protocol). P3HT:PCBM optical absorption peaks in the green part of the visible spectrum; however other light-sensitive conjugated polymers can be selected, according to the preferred photostimulation wavelengths range (Figure 2). Biocompatibility of these substrates has been demonstrated not only with cell lines...

Watch this Scientific Journal Video about Optical Control of Living Cells Electrical Activity by Conjugated Polymers at JoVE.com

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

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.

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Research

JoVE Journal

Bioengineering

7.5K vistas.  Istituto Italiano di Tecnologia. 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 ...