Name: Optical Control of Living Cells Electrical Activity by Conjugated Polymers (Video) | JoVE Journal
Uploaded: 2016-01-28T14:00:00.000+00:00
Duration: 10 min 16 s
Description: Learn optical control of cell membrane potential using light-sensitive conjugated polymers for in vitro neuroscience research. This method offers a non-invasive alternative to optogenetics without requiring gene transfer.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 the coverslips with a nitrogen gun. Put the samples in a plasma cleaner at 100 W power for 10 min.</li>
	<li>Deposit a thin film of the active layer on the cleaned substrates via spin-coating.
	<ol>
		<li>With a diamond tip, cut a microscope glass slide, (thickness &#8776; 1 mm) to the same lateral dimensions of the ITO-coated substrates. Remove the P3HT:PCBM solution from the hotplate and let it cool to ambient temperature.</li>
		<li>Set a two-steps spin-coater program: i) 30 sec at 800 rpm; ii) 30 sec at 1,600 rpm. Place the slide on the chuck of the spin-coater and start the vacuum to fix it. Put a drop of acetone on the slide and then the cleaned substrate, with the ITO-coated side facing upwards. Start the spin-coater rotation to get rid of the excess acetone.</li>
		<li>Put 150 &#181;l of P3HT:PCBM solution on the substrates and start again the spin-coater (use the same protocol described in 1.3.2).</li>
		<li>After the spin-coater has finished, remove the sample and carefully detach the coated substrate from the glass slide. Clean the back of the substrate with a cleanroom swab dipped in acetone. 
		Note: The photoactive substrates can be stored for few days in a dark box until used. For longer periods, keep them in a glovebox with inert gas.</li>
	</ol>
	</li>
</ol>
2. Preparation of Cell Culture Medium and Electrophysiology Solutions
<ol>
	<li>Prepare the complete growth medium (CGM) for HEK-293 cells: Dulbecco Modified Eagle&#39;s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) not heat-inactivated, 100 U/ml penicillin and 100 &#181;g/ml streptomycin. Sterilize the medium with a 0.2 &#181;m filtration system and store at 4 &#176;C.</li>
	<li>Prepare the extracellular solution in ultrapure water (concentrations in mM): 135 NaCl, 5.4 KCl, 5 HEPES, 10 Glucose, 1.8 CaCl2, 1 MgCl2. Adjust pH to 7.4 with NaOH. Sterilize the solution with a 0.2 &#181;m filtration system and store at 4 &#176;C.</li>
	<li>Prepare the intracellular solution in ultrapure water (concentrations in mM): 12 KCl, 125 K-Gluconate, 1 MgCl2, 0.1 CaCl2, 10 EGTA, 10 HEPES, 10 ATP-Na2. Note that ATP-Na2 is heat-sensitive and add it last. Adjust pH to 7.4 with NaOH. Sterilize the solution with a 0.2 &#181;m filtration system, aliquot in 1 ml tubes and store at -20 &#176;C.</li>
</ol>
3. Plating HEK-293 Cells on the Active Substrates
<ol>
	<li>Place the photoactive substrate in a glass beaker covered with aluminum or a closed Petri dish and put in an oven at 120 &#176;C for 2 hr for sterilization. From this moment onwards, perform all operations in sterile conditions. Put the samples in a sterile hood and let them cool down.</li>
	<li>Coat the photoactive substrate with an adhesion layer to reduce the surface hydrophobicity and to promote cell adhesion.
	<ol>
		<li>Place the active substrates in a P35 Petri dish or a 6 multiwell plate. Given the hydrophobicity of the conjugated polymer layer, make a polydimethylsiloxane (PDMS) well to fix the sample and confine the solutions.
		<ol>
			<li>Mix PDMS and catalyst in a 10:1 proportion with a glass rod to obtain a viscous slurry.</li>
			<li>Pour the slurry in a 6 multiwell plate, dispensing an amount sufficient to cover half of each well.</li>
			<li>Wait for PDMS polymerization at RT.</li>
			<li>Cut the cured PDMS in the middle for obtaining a square shape of the same dimension of the polymer sample.</li>
			<li>Sterilize the PDMS wells by immersion in a 70% ethanol-water mixture for at least 30 min.</li>
		</ol>
		</li>
		<li>If using the PDMS well, scratch the photoactive layer along the entire border of the well with pointed tweezers, in order to avoid detachment of the entire layer when removing the PDMS well after cell culturing.</li>
		<li>Cover the entire surface of each sample with a fibronectin solution (2 mg/L in PBS). A volume between 500-750 &#181;l per sample is usually sufficient if a PDMS well is used (clear surface of the well: 15x15 mm2). Without the PDMS well, about 2 ml of fibronectin solution are necessary to completely immerse the sample; pay attention that the substrate does not float while adding the solution. Incubate the samples at 37 &#176;C for at least 1 hr.</li>
		<li>Remove the fibronectin solution with a glass pipette and wash once with 2 ml of PBS.</li>
	</ol>
	</li>
	<li>Plate HEK-293 cells on the fibronectin-treated samples at a density of 15,000 cells/cm2 in complete growth medium. Use a volume of 750-1,000 &#181;l of CGM for each sample if a PDMS well is used, otherwise 2-3 ml of CGM are necessary. Incubate the cells at 37 &#176;C and 5% CO2 for 24-48 hr.</li>
</ol>
4. Optical Stimulation Protocol and Electrophysiological Measurements
<ol>
	<li>Put the extracellular solution in a water bath at 37 &#176;C to thermalize; thaw the intracellular solution, put it in a 1 ml syringe with a 34 gauge needle and keep it in contact with ice to avoid degradation of the ATP.</li>
	<li>Take a cell-coated substrate from the incubator, carefully removing the PDMS well if present. Remove the growth medium with a glass pipette and rinse the sample with extracellular solution. Proceed slowly to avoid cell detachment.</li>
	<li>Put the device into the sample-holder of the electrophysiology station with extracellular solution and the reference electrode. Prepare fresh glass micropipettes for patch-clamp with a puller (ideal resistance is in the range 2-4 M&#937;). Fill half of the pipette with intracellular solution and mount it on the micromanipulator.</li>
	<li>Look for a healthy cell to patch on the device. To avoid unwanted excitation of the photoactive substrate, use IR illumination for imaging, placing a long-wavelength pass filter (for example, a cut wavelength &#955; = 750 nm can be used for polymers absorbing in the visible) in front of the microscope illuminator.</li>
	<li>Patch the selected cell and apply the desired protocol for optical stimulation by delivering light to the sample via the fluorescence excitation path of the microscope (Figure 1). A detailed description of the patch-clamp protocol can be found in reference 20.
	<ol>
		<li>Position the patch pipette in close proximity to the cell membrane with a micrometric manipulator. During the positioning, apply overpressure to the pipette in order to avoid dirt sticking on the tip. The pipette should be few microns above the cell.</li>
		<li>Start lowering the pipette towards the cell membrane while controlling the pipette resistance on the patch control software. When the pipette resistance has increased about 1 M&#8486;, remove the overpressure from the pipette while applying a gentle suction, in order to form the gigaseal between the pipette and the cell membrane (i.e., the measured resistance should increase at values greater than 1 G&#8486;).</li>
		<li>Apply a negative potential to the pipette close to the expected cell resting potential (for HEK293 cell a potential of -40 mV can be used) to help stabilize the seal. Compensate the capacitive transients due to the pipette capacitance with the relative commands on the patch amplifier and/or the patch control software.</li>
		<li>Break the membrane to allow electrical access to the cell cytoplasm by applying a brief and intense suction pulse to the pipette. Set the patch amplifier to current clamp mode (I = 0, i.e., with no current injected into the cell) to track the cell membrane potential. Wait few minutes to see if the cell potential is stable.</li>
		<li>Apply the desired illumination protocol to the cell and record the effect on the membrane potential. 
		Note: The illumination can be delivered to the sample either from the bottom or from the top, depending on the microscope architecture.
		<ol>
			<li>Select an excitation wavelength that is well absorbed by the active material; for the P3HT:PCBM blend, a wavelength in the 450-600 nm range can be used. Suitable photoexcitation densities are in the range 10-100 mW/mm2. 
			Note: Short pulses of light (below 10-20 msec) result in a transient depolarization of the cell, while with prolonged illumination (hundreds of milliseconds or longer pulses) a sustained hyperpolarization is observed until the light is switched off.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Alivisatos, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanotools+for+Neuroscience+and+Brain+Activity+Mapping%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">Nanotools for Neuroscience and Brain Activity Mapping.</a> ACS Nano. 7 (3), 1850-1866 (2013).</li>
<li>Spira, M. E., Hai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-electrode+array+technologies+for+neuroscience+and+cardiology%5BTitle%5D)+AND+%22Nat.+Nanotechnol%22%5BJournal%5D)">Multi-electrode array technologies for neuroscience and cardiology.</a> Nat. Nanotechnol. 8 (2), 83-94 (2013).</li>
<li>Scanziani, M., Häusser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophysiology+in+the+age+of+light%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Electrophysiology in the age of light.</a> Nature. 461 (7266), 930-939 (2009).</li>
<li>Bareket-Keren, L., Hanein, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+interfaces+for+light+directed+neuronal+stimulation%3A+advances+and+challenges%5BTitle%5D)+AND+%22Int.+J.+Nanomed%22%5BJournal%5D)">Novel interfaces for light directed neuronal stimulation: advances and challenges.</a> Int. J. Nanomed. 9 (1), 65-83 (2014).</li>
<li>Antognazza, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shedding+Light+on+Living+Cells%5BTitle%5D)+AND+%22Adv.+Mater%22%5BJournal%5D)">Shedding Light on Living Cells.</a> Adv. Mater. , (2014).</li>
<li>Martino, N., Ghezzi, D., Benfenati, F., Lanzani, G., Antognazza, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organic+semiconductors+for+artificial+vision%5BTitle%5D)+AND+%22J.+Mater.+Chem.+B%22%5BJournal%5D)">Organic semiconductors for artificial vision.</a> J. Mater. Chem. B. 1 (31), 3768-3780 (2013).</li>
<li>Antognazza, M. R., Scherf, U., Monti, P., Lanzani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organic-based+tristimuli+colorimeter%5BTitle%5D)+AND+%22Appl.+Phys.+Lett%22%5BJournal%5D)">Organic-based tristimuli colorimeter.</a> Appl. Phys. Lett. 90 (16), 163509(2007).</li>
<li>Liao, C., Zhang, M., Yao, M. Y., Hua, T., Li, L., Yan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flexible+Organic+Electronics+in+Biology%3A+Materials+and+Devices%5BTitle%5D)+AND+%22Adv.+Mater%22%5BJournal%5D)">Flexible Organic Electronics in Biology: Materials and Devices.</a> Adv. Mater. , (2014).</li>
<li>Khodagholy, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NeuroGrid%3A+recording+action+potentials+from+the+surface+of+the+brain%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">NeuroGrid: recording action potentials from the surface of the brain.</a> Nat. Neurosci. 18 (2), 310-315 (2015).</li>
<li>Campana, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrocardiographic+Recording+with+Conformable+Organic+Electrochemical+Transistor+Fabricated+on+Resorbable+Bioscaffold%5BTitle%5D)+AND+%22Adv.+Mater%22%5BJournal%5D)">Electrocardiographic Recording with Conformable Organic Electrochemical Transistor Fabricated on Resorbable Bioscaffold.</a> Adv. Mater. 26 (23), 3874-3878 (2014).</li>
<li>Bonetti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Lysinated+Thiophene-Based+Semiconductor+as+a+Multifunctional+Neural+Bioorganic+Interface%5BTitle%5D)+AND+%22+Adv.+Healthc.+Mater%22%5BJournal%5D)">A Lysinated Thiophene-Based Semiconductor as a Multifunctional Neural Bioorganic Interface.</a> Adv. Healthc. Mater. , (2015).</li>
<li>Benfenati, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+transparent+organic+transistor+structure+for+bidirectional+stimulation+and+recording+of+primary+neurons%5BTitle%5D)+AND+%22Nat.+Mater%22%5BJournal%5D)">A transparent organic transistor structure for bidirectional stimulation and recording of primary neurons.</a> Nat. Mater. 12 (7), 672-680 (2013).</li>
<li>Rivnay, J., Owens, R. M., Malliaras, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Rise+of+Organic+Bioelectronics%5BTitle%5D)+AND+%22Chem.+Mater%22%5BJournal%5D)">The Rise of Organic Bioelectronics.</a> Chem. Mater. 26 (1), 679-685 (2014).</li>
<li>Pires, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+stem+cell+differentiation+by+electrical+stimulation+using+a+cross-linked+PEDOT+substrate%3A+Expanding+the+use+of+biocompatible+conjugated+conductive+polymers+for+neural+tissue+engineering%5BTitle%5D)+AND+%22BBA-Gen.+Subjects%22%5BJournal%5D)">Neural stem cell differentiation by electrical stimulation using a cross-linked PEDOT substrate: Expanding the use of biocompatible conjugated conductive polymers for neural tissue engineering.</a> BBA-Gen. Subjects. 1850 (6), 1158-1168 (2015).</li>
<li>Ghezzi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+hybrid+bioorganic+interface+for+neuronal+photoactivation%5BTitle%5D)+AND+%22Nat.+Commun%22%5BJournal%5D)">A hybrid bioorganic interface for neuronal photoactivation.</a> Nat. Commun. 2 (166), 1-7 (2011).</li>
<li>Ghezzi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+polymer+optoelectronic+interface+restores+light+sensitivity+in+blind+rat+retinas%5BTitle%5D)+AND+%22Nat.+Photonics%22%5BJournal%5D)">A polymer optoelectronic interface restores light sensitivity in blind rat retinas.</a> Nat. Photonics. 7 (5), 400-406 (2013).</li>
<li>Benfenati, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photostimulation+of+Whole-Cell+Conductance+in+Primary+Rat+Neocortical+Astrocytes+Mediated+by+Organic+Semiconducting+Thin+Films%5BTitle%5D)+AND+%22Adv.+Healthc.+Mater%22%5BJournal%5D)">Photostimulation of Whole-Cell Conductance in Primary Rat Neocortical Astrocytes Mediated by Organic Semiconducting Thin Films.</a> Adv. Healthc. Mater. 3 (3), 392-399 (2014).</li>
<li>Martino, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photothermal+cellular+stimulation+in+functional+bio-polymer+interfaces%5BTitle%5D)+AND+%22Sci.+Rep%22%5BJournal%5D)">Photothermal cellular stimulation in functional bio-polymer interfaces.</a> Sci. Rep. 5 (8911), (2015).</li>
<li>Antognazza, M. R., Ghezzi, D., Musitelli, D., Garbugli, M., Lanzani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+hybrid+solid-liquid+polymer+photodiode+for+the+bioenvironment%5BTitle%5D)+AND+%22Appl.+Phys.+Lett%22%5BJournal%5D)">A hybrid solid-liquid polymer photodiode for the bioenvironment.</a> Appl. Phys. Lett. 94 (24), 243501(2009).</li>
<li><a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Essentials+of+Neuroscience.+Patch+Clamp+Electrophysiology%5BTitle%5D)+AND+%22JoVE+Science+Education+Database%22%5BJournal%5D)">Essentials of Neuroscience. Patch Clamp Electrophysiology.</a> JoVE Science Education Database. , JoVE. Cambridge, MA. (2015).</li>
<li>Scarpa, G., Idzko, A. L., Götz, S., Thalhammer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biocompatibility+Studies+of+Functionalized+Regioregular+Poly%283-hexylthiophene%29+Layers+for+Sensing+Applications%5BTitle%5D)+AND+%22Macromol.+Biosci%22%5BJournal%5D)">Biocompatibility Studies of Functionalized Regioregular Poly(3-hexylthiophene) Layers for Sensing Applications.</a> Macromol. Biosci. 10 (4), 378-383 (2010).</li>
<li>Bellani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversible+P3HT%2FOxygen+Charge+Transfer+Complex+Identification+in+Thin+Films+Exposed+to+Direct+Contact+with+Water%5BTitle%5D)+AND+%22J.+Phys.+Chem.+C%22%5BJournal%5D)">Reversible P3HT/Oxygen Charge Transfer Complex Identification in Thin Films Exposed to Direct Contact with Water.</a> J. Phys. Chem. C. 118 (12), 6291-6299 (2014).</li>
<li>Wells, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+stimulation+of+neural+tissue+in+vivo%5BTitle%5D)+AND+%22Opt.+Lett%22%5BJournal%5D)">Optical stimulation of neural tissue in vivo.</a> Opt. Lett. 30 (5), 504-506 (2005).</li>
<li>Shapiro, M. G., Homma, K., Villarreal, S., Richter, C. P., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infrared+light+excites+cells+by+changing+their+electrical+capacitance%5BTitle%5D)+AND+%22Nat.+Commun%22%5BJournal%5D)">Infrared light excites cells by changing their electrical capacitance.</a> Nat. Commun. 3 (736), 1-10 (2012).</li>
<li>Duke, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+and+selective+suppression+of+neural+activity+with+infrared+light%5BTitle%5D)+AND+%22Sci.+Rep%22%5BJournal%5D)">Transient and selective suppression of neural activity with infrared light.</a> Sci. Rep. 3 (2600), 1-8 (2013).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>rr-P3HT</td><td>Sigma Aldrich</td><td>698989-5G</td><td></td></tr><tr><td>ITO-coated substrates</td><td>Nano-CS</td><td>IT10300100</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F1141</td><td></td></tr><tr><td>chlorobenzene</td><td>Sigma Aldrich</td><td>319996</td><td></td></tr><tr><td>PCBM</td><td>Nano-C</td><td>Nano-CPCBM-BF</td><td></td></tr><tr><td>acetone&amp;nbsp;</td><td>Sigma Aldrich</td><td>270725</td><td></td></tr><tr><td>isopropyl alcohol</td><td>Sigma Aldrich</td><td>563935</td><td></td></tr><tr><td>HEK cells</td><td>LGC standards srl</td><td>ATCC-CRL-1573</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H0887</td><td></td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>P5244</td><td></td></tr><tr><td>E-MEM</td><td>LGC standards srl</td><td>ATCC-30-2003</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E8008-</td><td></td></tr><tr><td>FBS</td><td>LGC standards srl</td><td>ATCC-30-2020</td><td></td></tr></tbody></table>

optical control of living cells electrical activity by conjugated polymers

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...

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.

optical-control-living-cells-electrical-activity-conjugated

Research

JoVE Journal

Bioengineering

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

A protocol for the analysis of gels formed from the optoelectronic conjugated polymer poly(3-hexylthiophene-2,5-diyl) (P3HT) using small and ultra-small angle neutron scattering in both the presence and absence of illumination is presented.

Monitoring the Effects of Illumination on the Structure of Conjugated Polymer Gels Using Neutron Scattering

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

This protocol involves transfecting cAMP sensors and bPAC-nLuc, an optogenetic protein, to accurately track its cellular distribution and response to light stimulation. The innovative approach of creating a cAMP response map using a point scanning system holds the potential for advancing research with optogenetic proteins in different fields.

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

Learn optical control of cell membrane potential using light-sensitive conjugated polymers for in vitro neuroscience research. This method offers a non-invasive alternative to optogenetics without requiring gene transfer.

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Explore More Videos