The overall goal of this procedure is to study thermal dose-dependent cell biological responses in vitro by heating cultured cells with precise temperature and time control. This method can help answer key questions in fields investigating cell or tissue responses to hyperthermia such as the study of retinal laser treatment. The main advantage of this technique is a quick heating of cells and a precise control over heating duration allowing a high reproducible thermal exposure during experiments.
Though this method can provide insight into the responses of cultured retinal pigment epithelial cells to thermal laser irradiation, it can also be applied to other models such as cancer cells. We first had the idea for this method when heating melanin containing cells with visible laser light. However, this resulted in varying temperature profiles owing to variations in pigmentation.
To begin to procedure, place a hot plate in an adjustable metal lab stand on a clean bench. Set the hot plate to 39 degrees Celsius. Secure a 0.22 NA fiber with a core diameter of 365 micrometers to the horizontal arm of the stand.
Position the metal arm over the hot plate and adjust the stand so that the fiber tip is 12 centimeters above the hot plate. Connect a 1.94 micrometer 20 watt Thulium laser with a 365 nanometer one milliwatt aiming beam to the fiber tip. Place a culture dish 30 millimeters in diameter on the hot plate.
Carefully trace the circumference of the dish on the hot plate surface and then remove the dish. Turn on the aiming beam and adjust the stand until the center of the beam spot on the hot plate matches the traced circle. Then turn off the aiming beam.
To begin the temperature calibration procedure, heat the tip of a 20 gauge needle with a Bunsen burner. With the heated needle tip, pierce the 30 millimeter diameter culture dish close to the bottom at four points 90 degrees apart to make holes about 300 micrometers in diameter. Place electrical tape over the holes.
Use a fine needle to punch an approximately 200 micrometer diameter hole through the tape covering each hole. On the underside of the culture dish, draw a line between each pair of holes 180 degrees apart. Starting at the intersection of the lines, mark every three millimeters radially along each line to designate the 21 measurement points.
Then place exactly 1.2 milliliters of fresh culture medium pre-warmed to 37 degrees Celsius in the dish. Cover the dish and set the dish in the traced circle on the hot plate. Connect a thermocouple with a diameter of 200 micrometers to the laser control computer and open the temperature calibration protocol.
Insert the thermocouple into one of the holes in the dish so that the tip is on the bottom of the dish at one of the mark's locations. It is very important to check if the tip of the thermocouple is touching the bottom of the dish at the measured precision because temperature may vary according to depth. Wait for the culture medium to stabilize at 37 degrees Celsius.
Then turn on the Thulium laser and set the laser power to the desired starting level. Verify that the preheating durations are set to 140 and eight seconds and then start the preheating irradiation sequence. After 140 seconds as cued by an audible beep, immediately remove the cover of the culture dish.
Wait while the preheating finishes and the 10-second irradiation occurs. Then immediately cover the dish. Measure the temperature at the 21 marks locations in this way.
Perform this procedure three times each at three watt power intervals spanning the laser power range. Once finished, turn off the thulium laser. Export and analyze the temperature data.
Prior to the experiment, culture rental pigment epithelial cells in 30 diameter culture dishes. Maintain the cell cultures at 37 degrees Celsius in a 5%carbon dioxide atmosphere and check them using a live cell imaging system. One hour before irradiation, replace the culture medium in each dish with exactly 1.2 milliliters of fresh culture medium.
Return the cultures to the incubator. Verify that the irradiation station is ready and that the hot plate is set to 39 degrees Celsius. When ready to begin irradiation, start the thulium laser at the desired power.
Open the experiment protocol and verify that the preheating durations are set to 140 and eight seconds. Place an RPE cell culture in a closed dish on the marked position on the hot plate and immediately start the 140 and eight second preheating irradiation sequence. Remove the dish cover after 140 seconds of preheating.
Wait while the preheating finishes and the 10-second irradiation occurs. Immediately cover the dish, wait seven seconds, and then return the dish to the incubator. Irradiate additional samples at increasing laser power levels.
When finished, turn off the thulium laser. To begin the cell viability assessment, wash the irradiated cells with PBS. Obtain a kit capable of differentiating live apoptotic and necrotic cells.
Then in low light, combine 500 microliters of binding buffer with 25 microliters of ethidium homodimer-3, FITC annexin and Hoechst 33342. Incubate the cells in this staining solution at room temperature for 15 minutes. Then wash the cells with 1X binding buffer twice.
Replace the binding buffer with PBS and place the cell on the stage of a fluorescence microscope that has DAPI, FITC and TRITC filters. Set the microscope light path to the ocular lens and select the DAPI filter. Turn on the illumination light and find the focus plane with a 4X objective lens.
Set the light path to the microscope camera. Use the microscope imaging software to center and focus the image. Define the parameters for image acquisition using the DAPI filter.
Repeat the focusing process and image acquisition setup with the FITC and TRITC filters. Then use the imaging software to acquire multiple images with three filter sets across the dish and combine them into a single image of the whole culture dish. Use analytical software to determine the temperature thresholds for apoptosis and cell death.
The proportional relationship between the maximal temperature increase at the center of a cell culture dish and the laser power was determined by the described thermal calibration procedure. The maximal temperature distribution across the cell culture dish was then fit to a Gaussian function. Three staining patterns were observed following irradiation.
No viability change was observed in cultures subjected to temperatures at or below 43 degrees Celsius. Apoptosis was first observed three hours after irradiation at 47 degrees Celsius. Cell death was observed later than three hours after 51 degrees Celsius and higher.
The mean threshold temperatures for apoptosis and cell death were then determined by applying the average of measured radii of the dead and apoptotic areas to the Gaussian function describing the temperature distribution. Following this procedure, irradiations can be performed under many different conditions such as different laser profiles, spot sizes and irradiation times. This can help to understand and investigate questions related to individual timed temperature causes on the cellular level.
After watching this video, you should have good understanding of how to heat the cell culture with good control over temperature and time. This may ride on the possibilities for studying biological responses to different thermal irradiation conditions. Don't forget that working with lasers can be very hazardous for your eyes and skin.
Stay informed about laser irradiation safety training. Always wear appropriate safety glasses, lab coat and gloves when performing this procedure.
