Name: continuous-wave thulium laser for heating cultured cells to investigate cellular thermal effects
Uploaded: 2017-06-30T13:00:00
Description: Watch this Scientific Journal Video about Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects at JoVE.com

This work was supported by a research grant from the German Federal Ministry of Education and Research (BMBF) (grant #13GW0043C) and and a European Office of Aerospace Research and Development (EOARD, grant # FA9550-15-1-0443)

1. RPE Cell Culture
<ol>
	<li>Isolation of RPE cells from porcine eyes
	<ol>
		<li>Obtain freshly enucleated porcine eyes from the local slaughterhouse. Keep them cool (4 &#176;C) and in a dark environment.</li>
		<li>Remove extracellular tissues with scissors and soak the eyes in an antiseptic solution for 5 min.</li>
		<li>Place the eyes in sterilized phosphate-buffered saline without calcium and magnesium (PBS (-)) until use.</li>
		<li>Using a scalpel, penetrate the sclera at about 5 mm posterior t...

<ol>
	<li>Inagaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+efficacy+of+pure+yellow+(577-nm)+and+810-nm+subthreshold+micropulse+laser+photocoagulation+combined+with+yellow+(561-577-nm)+direct+photocoagulation+for+diabetic+macular+edema.">Comparative efficacy of pure yellow (577-nm) and 810-nm subthreshold micropulse laser photocoagulation combined with yellow (561-577-nm) direct photocoagulation for diabetic macular edema.</a> Jpn J Ophthalmol. 59 (1), 21-28 (2015).</li><li>Roider, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+retina+therapy+(SRT)+for+clinically+significant+diabetic+macular+edema.">Selective retina therapy (SRT) for clinically significant diabetic macular edema.</a> Graefes Arch Clin Exp Ophthalmol. 248 (9), 1263-1272 (2010).</li><li>Brinkmann, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+temperature+determination+during+retinal+photocoagulation+on+patients.">Real-time temperature determination during retinal photocoagulation on patients.</a> J Biomed Opt. 17 (6), 061219 (2012).</li><li>Yoshimura, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocoagulated+human+retinal+pigment+epithelial+cells+produce+an+inhibitor+of+vascular+endothelial+cell+proliferation.">Photocoagulated human retinal pigment epithelial cells produce an inhibitor of vascular endothelial cell proliferation.</a> Invest Ophthalmol Vis Sci. 36 (8), 1686-1691 (1995).</li><li>Denton, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage+Thresholds+for+Exposure+to+NIR+and+Blue+Lasers+in+an+In+Vitro+RPE+Cell+System.">Damage Thresholds for Exposure to NIR and Blue Lasers in an In Vitro RPE Cell System.</a> Invest Ophthalmol Vis Sci. 47 (7), 3065-3073 (2006).</li><li>Shrestha, R., Choi, T. Y., Chang, W., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-precision+micropipette+sensor+for+cellular-level+real-time+thermal+characterization.">A high-precision micropipette sensor for cellular-level real-time thermal characterization.</a> Sensors (Basel). 11 (9), 8826-8835 (2011).</li><li>Gao, F., Ye, Y., Zhang, Y., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+bath+hyperthermia+reduces+stemness+of+colon+cancer+cells.">Water bath hyperthermia reduces stemness of colon cancer cells.</a> Clin Biochem. 46 (16-17), 1747-1750 (2013).</li><li>Jansen, E. D., van Leeuwen, T. G., Motamedi, M., Borst, C., Welch, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependence+of+the+absorption+coefficient+of+water+for+midinfrared+laser+radiation.">Temperature dependence of the absorption coefficient of water for midinfrared laser radiation.</a> Lasers Surg Med. 14 (3), 258-268 (1994).</li><li>Iwami, H., Pruessner, J., Shiraki, K., Brinkmann, R., Miura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+a+laser-induced+sub-lethal+temperature+rise+on+RPE+cells+from+oxidative+stress.">Protective effect of a laser-induced sub-lethal temperature rise on RPE cells from oxidative stress.</a> Exp Eye Res. 124, 37-47 (2014).</li><li>Denton, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+correlated+microthermography+maps+threshold+temperature+in+laser-induced+damage.">Spatially correlated microthermography maps threshold temperature in laser-induced damage.</a> J Biomed Optics. 16 (3), (2011).</li><li>Morgan, C. M., Schatz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrophic+creep+of+the+retinal+pigment+epithelium+after+focal+macular+photocoagulation.">Atrophic creep of the retinal pigment epithelium after focal macular photocoagulation.</a> Ophthalmology. 96 (1), 96-103 (1989).</li></ol>

In discussing temperature-related biological cellular responses, not only the temperature, but also the time duration of the increased temperature, is of importance, since most biochemical processes are time-dependent. Particularly in the field of laser-induced hyperthermia in ophthalmology, due to the short time range-from milliseconds to seconds-it is difficult to investigate cellular thermal effects with precise temperature control. Therefore, a laser irradiation setup suitable for the cell culture model and with an o...

Understanding temperature-dependent cell biological responses is of great importance to successful hyperthermia treatments. Retinal laser photocoagulation with a thermal laser, used in ophthalmology, is one of the most established laser treatments in medicine. Visible light, mostly from green to yellow wavelengths, is used in retinal laser treatment. The light is highly absorbed by the melanin in retinal pigment epithelial (RPE) cells, which form the outermost cell monolayer of the retina. There has been recent interest among physicians and researchers in very mild thermal irradiation (sub-visible photocoagulation) as a new therapeutic strategy for different kinds of retinal disorders1,2. Following this trend, our interest is in sub-lethally heating RPE cells under precise temperature control, a technique called temperature-controlled photothermal therapy (TC-PTT).
Recent optoacoustic technology from our institute has allowed for the real-time measurement of temperature increases at irradiated sites in the retina. This enables control over the temperature increase during irradiation3. However, since sub-lethal hyperthermia on the retina, caused by heating RPE cells sub-lethally, has not been previously considered due to the impossibility of measuring and controlling the temperature, the temperature-dependent cell responses of RPE cells following thermal laser irradiation has been studied very little to date. Moreover, not only has the temperature difference not been discussed in detail, but also the difference in the cell behavior of the surviving cells after sub-lethal and lethal irradiation. Therefore, to gather scientific evidence on TC-PTT-based treatments, we aim to elucidate the temperature-dependent RPE cell biological responses and their mechanisms using in vitro experimental setups.
For this purpose, it is necessary to establish a cell-heating setup that meets the following conditions: 1) a possibility for fast temperature increases, 2) a precisely controlled time and temperature, and 3) a relatively high number of examined cells for biological experiments. Regarding the heating method, a clinical laser, such as a frequency-doubled Nd.YAG laser (532 nm), is unfortunately unsuitable for cell culture heating. This is because of the strongly reduced number of melanosomes in cultured RPE cells. The laser light absorption might be inhomogeneous, and the temperature increase at the cellular level is variable between experiments, even when irradiated with same radiation power. Several previous studies have reported the use of black paper beneath the dish bottom during irradiation4 or the use of additional melanosomes that are phagocytized by the culture cells before the experiments5,6. Many of the in vitro biological studies to assess hyperthermia-induced cell responses have been performed using a hot plate, a water bath, or a CO2 incubator with a temperature setting7. These methods require a long heating period because it takes some time (i.e., several minutes) to reach the desired temperature. Furthermore, using these methods, it is difficult to obtain a detailed thermal history (i.e., temperature multiplied by time) at the cellular level. Moreover, the temperature among the cells at different positions in one culture dish may differ due to variable temperature diffusion. In most cases, this temporal and spatial temperature information during hyperthermia has not been taken into consideration for biological analyses, even though biological cell response may be critically affected by the temperature and the time duration of the increased temperature.
To overcome these problems, a continuous-wave thulium laser was used here to heat the cells. Thulium laser radiation (&#955; = 1.94 &#181;m) is strongly absorbed by water8, and the cells at the bottom of the culture dish are thermally stimulated solely through thermal diffusion. The laser fiber with a 365-&#181;m diameter is set about 12 cm above the culture dish, without any optics in between. The laser beam diameter diverges such that it is almost equivalent to the inner diameter of the culture dish (30 mm) at the surface of the culture medium.With a consistent amount of culture medium, it is possible to irradiate the cells with the temperature increase of high repeatability. Variable power settings enable irradiation with up to 20 W, and the medium temperature at the cellular level may be increased up to &#916;T &#8776; 26 &#176;C in 10 s.
By modifying the irradiation conditions, it is also possible to change the laser beam profile to vary the temperature distribution in a culture dish. For example, it is possible to investigate with a Gaussian-like temperature distribution, as in the current study, or with a homogeneous temperature distribution. The latter may be advantageous for investigating the effects of temperature-dependent cell responses more specifically for sub-lethal temperature increases, but not for cell death stress or wound healing responses.
Altogether, thulium laser irradiation may enable the investigation of different kinds of biological factors, such as gene/protein expression, cell death kinetics, cell proliferation, and cell functionality development, after different thermal exposures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle's Medium - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5796-500ML</td>
			<td>Add (2)-(4) before use. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>A5955-100ML</td>
			<td>Containing 10000 units penicillin, 10 mg streptomycin and 25 &#956;g Amphotericin B in 1ml. Add 5.5 ml in 500 ml medium bottle (1) before use.</td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM)</td>
			<td>Sigma-Aldrich</td>
			<td>S8636-100ML</td>
			<td>Add 5.5 ml in 500 ml medium bottle (1) before use (final concentration: 1 mM)</td>
		</tr>
		<tr>
			<td>Porcine serum</td>
			<td>Sigma-Aldrich</td>
			<td>12736C-500ML</td>
			<td>Add 50 ml in 500 ml medium bottole (1) before use (final: 10%)</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>T4799-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>ED-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF Quantikine ELISA Kit</td>
			<td>R&#38;D System</td>
			<td>DVE00</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxiselect Total Glutathione Assay Kit</td>
			<td>Cell Biolabs, Inc</td>
			<td>STA-312</td>
			<td></td>
		</tr>
		<tr>
			<td>Apoptotic/Necrotic/Healthy Cells Detection Kit</td>
			<td>PromoKine</td>
			<td>PK-CA707-30018</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thulium laser</td>
			<td>Starmedtec GmbH</td>
			<td>Prototype</td>
			<td>0-20 W</td>
		</tr>
		<tr>
			<td>365 mm core diameter fiber</td>
			<td>LASER COMPONENTS Germany</td>
			<td>CF01493-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocouple</td>
			<td>Omega Engineering Inc</td>
			<td>HYP-0- 33-1-T-G-60-SMPW-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>MEDAX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader (spectrofluorometer)</td>
			<td>Molecular Device</td>
			<td>Spectramax M4</td>
			<td></td>
		</tr>
		<tr>
			<td>cell homogenizer</td>
			<td>QIAGEN</td>
			<td>TissueLyser LT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>mathematical software program</td>
			<td>The Mathworks. Inc</td>
			<td>MATLAB Release 2015b</td>
			<td></td>
		</tr>
		<tr>
			<td>system-design platform</td>
			<td>National Instrument</td>
			<td>Labview</td>
			<td>Laboratory Virtual Instrument Engineering Workbench</td>
		</tr>
	</tbody>
</table>

continuous-wave thulium laser for heating cultured cells to investigate cellular thermal effects

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser radiation is strongly absorbed by water, and the cells at the bottom of the culture dish are heated through thermal diffusion. A laser fiber with a diameter of 365 &#181;m is set about 12 cm above the culture dish, without any optics, such that the laser beam diameter is almost equivalent to the inner diameter of the culture dish (30 mm). By keeping a consistent amount of culture medium in each experiment, it is possible to irradiate the cells with a highly reproducible temperature increase.
To calibrate the temperature increase and its distribution in one cell culture dish for each power setting, the temperature was measured during 10 s of irradiation at different positions and at the cellular level. The temperature distribution was represented using a mathematical graphics software program, and its pattern across the culture dish was in Gaussian form. After laser irradiation, different biological experiments could be performed to assess temperature-dependent cell responses. In this manuscript, viability staining (i.e., distinguishing live, apoptotic, and dead cells) is introduced to help determine the threshold temperatures for cell apoptosis and death after different points in time.
The advantages of this method are the preciseness of the temperature and the time of heating, as well as its high efficiency in heating cells in a whole cell culture dish. Furthermore, it allows for study with a wide variety of temperatures and time durations, which can be well-controlled by a computerized operating system.

Temperature distribution after different power settings
All temperature developments for each single irradiation were monitored in the temperature calibration. From this data, the maximal temperature at the measured point was obtained and defined as Tmax (&#176;C). As shown in Figure 3A, the program was executed at the time point when the culture dish was placed on the heating plate. After t...

Watch this Scientific Journal Video about Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects at JoVE.com

Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

An original experimental setup for heating cells in a culture dish using 1.94 &#181;m continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser ...

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