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Biology

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

Published: June 30th, 2017

DOI:

10.3791/54326

1Institute of Biomedical Optics, University of Luebeck, 2Medical Laser Center Luebeck GmbH, University of Leubeck, 3Department of Ophthalmology, University of Luebeck

An original experimental setup for heating cells in a culture dish using 1.94 µm 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 µm 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 µm 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.

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 ....

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1. RPE Cell Culture

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

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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 (°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.......

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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.......

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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)

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

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