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 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 (λ = 1.94 µm) 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-µm 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 ΔT ≈ 26 °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.
1. RPE Cell Culture
2. Thulium Laser Irradiation
Figure 1: Schematic Image of the Thulium Laser Irradiation Station. A culture dish is placed on the heating plate. The cells are placed 12 cm below the thulium laser fiber tip so that the beam size is almost identical to the inner diameter of the culture dish (about 30 mm). The laser irradiation procedure is controlled by a time-controlled routine of the custom-made system design platform. The power setting must be determined before the irradiation program is started. Please click here to view a larger version of this figure.
Figure 2: The Points for Temperature Calibration in One Cell Culture Dish. The temperature data was measured in the center and at 5 radial points over 4 different angles (blue dots). Please click here to view a larger version of this figure.
3. Biological Assessments for Cell Responses after Different Thermal Irradiations
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 the 140 s "pre-heating time 1," which was needed to archive a stable medium temperature at 37 °C, the cover of the culture dish was removed during the 8 s "pre-heating time 2." At the end of "pre-heating time 2," laser emission started automatically. This curve is a representative temperature progression for a 10 s irradiation. During irradiation, the temperature increased, and immediately after the laser emission was turned off, temperature started to decrease. The maximal temperature at the center of the culture dish was defined in this study as Tmax (°C). The Tmax was proportional to the laser power (Figure 3B). Figure 3C shows the distribution of the maximal temperature for each power across the culture dish. The distributions are bell-shaped, as shown in Figure 3C, and fit to a Gaussian function according to the following formula:
t(r)= tbase + A・
where r, tbase, A, and w stand for the distance from the center (mm), the lowest temperature for the curve, the amplitude, and the width of the curve, respectively. The parameters (tbase, A, and w) of the fitted Gaussian curve for each power setting, namely for each Tmax,are shown in the table next to the graph.
Figure 3: Temperature Calibration Data. A Representative Temperature Development at the Central Position after a Single Irradiation at 4.9 W (Tmax = 43°C) (A), the Proportional Relationship Between the Laser Power and the Maximal ΔT at the Central Position of a Cell Culture (B), and Temperature Distributions Across the Culture Dish after Different Power Settings (C). (A) The program is executed from the time point at which the culture dish is placed on the heating plate. After 140 s of "pre-heating time 1," which is needed to archive a stable medium temperature at 37 °C, the cover of the culture dish is removed for the 8-s "pre-heating time 2." At the end of "pre-heating time 2," the laser emission starts automatically. This curve is a representative temperature progression at the central position during a 10-s irradiation at 4.9 W. During irradiation, the temperature increases, and immediately after the laser emission is turned off, the temperature starts to decrease. The maximal temperature is obtained at the end of the irradiation, which is defined in this study as Tmax (°C). (B) The laser power and the maximal temperature increase (ΔTmax) are proportional. (C) The fitted Gaussian functions of the measured temperature distributions across the culture dish. The parameters for the functions, determined with a mathematical software program, are shown in the table at the side of the graph. Please click here to view a larger version of this figure.
Cell viability after thermal irradiation
As shown in Figure 4A, there are three different staining patterns indicating cell viability after laser irradiation: 1) no annexin V/ethidium homodimer III-positive (i.e., only live), 2) annexin V-positive at the center (i.e., almost only early apoptosis), and 3) ethidium homodimer III-positive at the center (i.e., dead cells) surrounded by apoptotic cells at the border between dead and live cells (Figure 4B). The size of the dead/apoptotic area is generally dependent on Tmax and the post-irradiation time up to 48 h after irradiation. No apparent viability change was detected in the cultures irradiated with Tmax≤43 °C. The only apoptotic change could be observed at an early point in time (3 h), followed by a late cell death after an irradiation with Tmax = 47 °C. Immediate or early cell death (up to 3 h) was found in the cultures irradiated with Tmax≥ 51 °C (Table 1).
Figure 4: Pattern of Viability Staining After Different Power Settings (A) and an Exemplary Image at the Rim of the Dead and Apoptotic Area after Lethal Laser Irradiations (B).
(A) Three patterns of staining can occur, depending on the temperature. (B) The apoptotic zone (FITC-annexin V-positive: green) around the dead area (ethidium homodimer III-positive: red). All cells are positive for Hoechst 33342 (blue), and the cells with blue nuclei are the live cells. The image was taken 24 h after an irradiation at Tmax= 59 °C. Bar = 100 µm. Please click here to view a larger version of this figure.
Table 1: Annexin V and Ethidium Homodimer III Responses at Various Temperatures and Times.
Determination of the threshold temperature for cell death
The average radii of the dead area (red) and the apoptotic area (green) were measured and applied to the Gaussian function of the temperature distribution to determine the threshold peak temperatures for cell death and apoptosis after 10 s of irradiation. According to this analysis, the mean threshold temperatures for complete cell death 3 h, 24 h, and 48 h after irradiation were 54.0 °C, 50.9 °C, and 50.1 °C, respectively. The mean threshold temperature for cell apoptotic change were lower by about 2 – 3 °C , with the threshold temperatures for 3 h, 24 h, and 48 h at 51.7 °C, 48.0 °C, and 47.0 °C, respectively (Figure 5).
Figure 5: Threshold Temperatures for Apoptosis and Cell Death.
Mean threshold temperatures for complete cell death (positive for Hoechst 33342, annexin V, and ethidium homodimer III) and for apoptosis (positive only for Hoechst 33342 and annexin V) at different time points in after irradiation, calculated from the results of fluorescence viability staining. Please click here to view a larger version of this figure.
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 operation system that enables strict temperature and time control is desired. The biological assessment of cell responses after thermal exposure, such as protein expression or secretion, requires repeated quantitative evaluations on a sufficient number of affected cells. This has been an obstacle to studies using laser spots several hundred micrometers in diameter, as in clinical treatments. Quantitative analysis at a single laser spot is quite burdensome. In this study, attempts have been made to fulfill these demands as much as possible. By using a 1.94 µm wavelength continuous-wave thulium laser with an irradiation control program, a temporal temperature rise could be conducted in a whole cell culture within a short timeframe. Since the temperature distribution may be adjusted by changing the light path, different kinds of hyperthermia-related experiments can be conducted using this setup.
The limitation of the presented technique is the impossibility of conducting simultaneous temperature measurements during the laser irradiation of the cells. Since the use of thermocouples is not suitable for sterilized cell cultures, temperature calibration must be conducted separately from cell irradiation. Considering the possible variations in the laser-power output, real-time temperature measurements during each laser irradiation would be ideal to directly assess cellular responses corresponding to the thermal dose. Moreover, the temperature distribution used here was created through data interpolation based on the measurements at 21 points on a culture dish and at several different power settings. Therefore, to overcome these limitations and critical points, it is our goal to develop an alternative method that allows for the measurement of the temperature of the culture dish while laser irradiation is being performed. We also aim obtain the spatial temperature information at once. Infrared imaging (thermography) is one possible method to measure the temperature during laser irradiation10. The great advantage of this method is the real-time temperature measurement at the cellular level for each irradiation; subsequent cell biological responses can always be individually compared to the temperature history during irradiation. Considering cost-effectiveness and usability, however, using thermography for cell heating experiments is not possible for every laboratory.
In the method using a thulium laser at a wavelength of 1.94 µm, the water in the cell culture dish is heated at its surface, and thermal diffusion and convection are used to heat the cells. The height of the culture medium in this irradiation setup, with 1.2 mL of culture medium, is 935 µm at the central position (from a previous measurement using optical coherence tomography). The level of absorption of the thulium laser in water is very high (absorption coefficient: 127 cm-1 at 35 °C), and 72% of the light is absorbed in the first 100 µm of the culture medium. There is almost no absorption (0.0007%) at a depth of 935 µm.
It is important to note that one of the critical points in the protocol is to add the same amount of medium (1,200 µL) for each irradiation. Using different amounts of culture medium may lead to the height differences, which may cause differences in the temperature increase of the cells. The second critical point relates to the timing of the opening of the culture dish. It must be done at the same time-in this study, 8 s before the start of the irradiation, when the system makes a sound. Differences in this timing may vary the base temperature due to the cooling caused by the surrounding air (about 23 °C). This can lead to significant differences in the laser-induced temperature.
For temperature calibration, the same amount of medium (1.2 mL) that was used in the experiments was used to measure the temperature distribution at the bottom of cell-free culture dishes. However, the medium height with a cell monolayer may be different than the one without cells, even with the same volume of medium added. The measurement using optical coherence tomography revealed that there is a 58-µm difference at the central position between dishes with and without a confluent cell monolayer (877 µm without cells, compared to 935 µm with cells). This difference is potentially due to the capillary action of the cells. The 58-µm difference in height at the central position may be caused by approximately 40 µL of medium (measured data). It was also confirmed that this difference in height did not cause significant differences in Tmax at all power settings. Therefore, we have concluded that this difference does not significantly influence the results of the analyses done in this study. Nevertheless, to gather more precise temperature information, as written above, a method to calibrate the temperature using a cell culture dish containing a cell monolayer should be developed. Moreover, the mathematical modeling of thermal diffusion and convection in the whole culture medium is also required.
In this study, the cells were heated with a Gaussian temperature distribution. There are several possible methods to heat the whole medium more uniformly over time. One is to use a laser source with a lower absorption coefficient in water. However, the drawback is that, in this case, the lasers must have a higher power, since only a small percentage of the light is absorbed over about 0.9 mm. Another possibility is to image the distal fiber tip of a multimode optical fiber that transmits the laser light into the plane of the culture dish; the magnification can be chosen arbitrarily by the optics.
The second highlight of this protocol is its ability to determine the threshold temperature for cell death and apoptosis using the fluorescent image of viability staining and the lateral temperature distribution. A long-term aim is not only to determine cell viability, but also to elucidate the temperature range for cell biological responses relating to cell functionality, such as protein expression and cell proliferation. The determination of the cell death threshold temperature is of great interest to researchers10. Using this method, it might be possible to determine the critical factors for cell death, including apoptosis. Critical factors for thermal laser-induced cell death might be determined not only through the temperature history, but also through the endogenous factors (i.e., intra/extra-cellular factors at the molecular level). Answering these questions might pave the way for understanding cell death mechanisms and kinetics after different thermal exposures and in different retinal pathologies. Furthermore, it can also help to clarify clinically observed issues, such as the inter-individual difference in response to laser treatment or the variability in scar size after retinal photocoagulation, even when the initial spot size was almost identical ("atrophic creep")11.
The final aim of this study is to aid in developing temperature-controlled photothermal therapy of the retina. To achieve this, in parallel with the technical advancement of the temperature measurement3, further elucidation of RPE cell behavior after thermal exposure, determined using this method, will be of great benefit.
The authors have nothing to disclose.
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)
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 |