The overall goal of the following experiment is to show the cytotoxic efficacy of photodynamic therapy in osteosarcoma cells. This is achieved by first measuring the intracellular uptake of the photosensitizer M-T-H-P-C by osteosarcoma cells. In the second step, laser light is applied to the cells to activate the M-T-H-P-C and to produce cytotoxic oxygen radicals.
Finally, cell metabolism and cell number are measured by a water soluble tetra oleum one or WST one assay. Ultimately, the WST one assay, as well as direct cell counting, can be used to measure the cytotoxicity of the photodynamic therapy on the osteosarcoma cells. The implications of this technique extend towards the therapy of osteosarcoma as it allows the measurement of photodynamic therapy efficacy in vitro and resembles the technique currently used in patients.
To compare the M-T-H-P-C uptake between osteosarcoma cell lines begin by plating 0.2 times 10 to the sixth Hoss and 143 B cells per well. In triplicate in six well plates let the cells adhere overnight to an 80 to 90%co fluency, and then on the following day, incubate the cells with a fixed concentration of M-T-H-P-C for different time periods in the dark to evaluate the time dependent uptake of the photosensitizer by the cells. After the incubations, aspirate the media and wash the cells three times with PBS, then detach the cells with 0.5 milliliters of tripsin EDTA and count them.
Next, spin down the cells for five minutes at 400 times G and room temperature. Aspirate the medium and resuspend the pellet in PBS to a final density of two times 10 to the fifth cells per milliliter. Pipette 100 microliters of the cell suspension into each well of a 96.
Well plate and measure the fluorescence of the cells in a fluorescence spectrophotometer set to 417 nanometers excitation, and 652 nanometers emission. Then prepare a standard curve by plating 100 microliters of zero to four milligrams per milliliter concentrations of M-T-H-P-C in triplicate. In a 96 well plate and measure the fluorescence as just demonstrated to generate a standard curve for calculating the relative fluorescence unit of each cell containing well from the previously measured plate.
To measure the phototoxicity of the photo sensitizer first seed 3000 cells per well in triplicate per each M-T-H-P-C concentration in a 96 well plate, including dark toxicity control cells for each PHOTOSENSITIZER concentration. Let the cells adhere overnight to a 50 to 60%co fluency the following day. Incubate the cells with a range of M-T-H-P-C concentrations in the dark for five hours following the incubation.
Aspirate the medium and wash the cells twice with PBS and add fresh media without M-T-H-P-C then specific for the M-T-H-P-C absorption spectrum, wearing proper eye protection. Set a 652 nanometer diode laser to a 21.88 milliwatts per square centimeter power at a height of 13.5 centimeters. Distance from the cells to get an energy dose of five joules per square centimeter.
Then illuminate the cells except for the dark toxicity control cells for 230 seconds after the illumination. Keep the cells in the dark at 37 degrees Celsius for 24 hours. Subsequently add 10 microliters of wst, one reagent per 100 microliters of medium three hours after addition of the wst one reagent measure the absorbance of the individual wells at 415 nanometers.
Finally, calculate the number of surviving cells in addition to the WST one assay. Direct counting of the cells is a second method for measuring cell toxicity. To calculate the percentage of viable cells, seed two times 10 to the fourth cells per well in 2 24 well plates and let them adhere overnight.
The next day incubate the cells for five hours in the dark with M-T-H-P-C and illuminate them as just demonstrated. Keep dark control cells in the dark after 24 hours, collect the supernatant, wash the cells once with PBS and trypsin eyes them finally spin down the cells and resuspend the pellet in 200 microliters of fresh medium for counting. The results illustrated in this graph show in the top panel the uptake of M-T-H-P-C in a time dependent manner with a fluorescence intensity of the cell suspension representing the intracellular levels of the M-T-H-P-C.
In the bottom panel. The dose dependent M-T-H-P-C uptake can be assessed by measuring the fluorescence of M-T-H-P-C with a fluorescence spectrophotometer. This graph illustrates the uptake of a fixed concentration of M-T-H-P-C in a time dependent manner with the fluorescence intensity of the cell suspension representing the intracellular levels of the M-T-H-P-C.
In this graph, the dose dependent M-T-H-P-C uptake in Haass and 143 B cells after a five hour incubation is demonstrated. The uptake in the high metastatic cell line 143 B was significantly higher than in the low metastatic parental host cell line. The dose dependent dark toxicity of the osteosarcoma cells is demonstrated.
143 B cells were treated with different M-T-H-P-C dosages and compared to untreated cells and a dose dependent decrease of the metabolic activity and cell number measured by wst one assay was observed. Decreased cell viability was recognized at an M-T-H-P-C concentration equal to and higher than 2.5 micrograms per milliliter. In the bottom graph, the dose dependent toxicity of M-T-H-P-C is illustrated.
The cells were treated with different M-T-H-P-C doses and subsequent illumination at five joules per square centimeter. A consequential decrease in cell metabolic activity as well as a decrease in cell number was observed. The development of this technique paved the way for researchers in the field of cancer biology to apply this methods to in vivo models of osteosarcoma as well as for other preclinical investigations.
