Fully automated illumination study series for modern cancer drug development
In vitro cell viability and dose escalation study for developing photosensitive or photoactivated drugs using Modulight ML7710 medical laser and ML8500 automated illumination system.
STUDY PLAN
The goal was to investigate the effect of irradiance and light dose on a cancer cell line while keeping the photosensitive drug dosing constant. The well-by-well dose escalation plan was done in tabular format.
TARGET
Cancer cell line incubated at 37°C in acidic culture
DRUG TYPE
Photosensitive or photoactivated cancer drug (constant)
ILLUMINATION
Variable dose of
0/10/20/25/50/75/100 J/cm2
IRRADIANCE
100 and 600 mW/cm2
Standard 96-well microplate
A standard 96-well microplate was divided so that half of the wells were illuminated with irradiance of 600 mW/cm2 (columns 1-6), and the other half with 100 mW/cm2 (columns 7-12).
The dose was increased by the row, using doses of 0 – 5 – 10 – 25 – 50 – 75 – 100 J/cm2.
This way, there were 6 technical replicates of each combination of dose & irradiance, which allows calculation of standard deviation for each data point.
Dose (J/cm2) / Irradiance (mW/cm2)
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | 0 600 |
0 600 |
0 600 |
0 600 |
0 600 |
0 600 |
0 100 |
0 100 |
0 100 |
0 100 |
0 100 |
0 100 |
| B | 5 600 |
5 600 |
5 600 |
5 600 |
5 600 |
5 600 |
5 100 |
5 100 |
5 100 |
5 100 |
5 100 |
5 100 |
| C | 10 600 |
10 600 |
10 600 |
10 600 |
10 600 |
10 600 |
10 100 |
10 100 |
10 100 |
10 100 |
10 100 |
10 100 |
| D | 15 600 |
15 600 |
15 600 |
15 600 |
15 600 |
15 600 |
15 100 |
15 100 |
15 100 |
15 100 |
15 100 |
15 100 |
| E | 25 600 |
25 600 |
25 600 |
25 600 |
25 600 |
25 600 |
25 100 |
25 100 |
25 100 |
25 100 |
25 100 |
25 100 |
| F | 50 600 |
50 600 |
50 600 |
50 600 |
50 600 |
50 600 |
50 100 |
50 100 |
50 100 |
50 100 |
50 100 |
50 100 |
| G | 75 600 |
75 600 |
75 600 |
75 600 |
75 600 |
75 600 |
75 100 |
75 100 |
75 100 |
75 100 |
75 100 |
75 100 |
| H | 100 600 |
100 600 |
100 600 |
100 600 |
100 600 |
100 600 |
100 100 |
100 100 |
100 100 |
100 100 |
100 100 |
100 100 |
The dose is displayed on the first row and the irradiance on the second row.
Experimental set up
Cell counting
Nexcelom Cellometer Auto2000 cell viability counter
STUDY EXECUTION
The study of photosensitive or photoactivated drugs requires a controlled and systematic sample illumination process. Modulight ML8500 automated illumination system and ML7710 medical laser ensure the repeatable and accurate illumination of each sample. ML7710 supports up to 8 wavelengths allowing research of multiple drugs, or combining fluorescence measurements to the study plan.
Extracting and counting the cells with Cellometer Auto2000 (unit / ml)
Diluting the cells to 1 million / ml
Centrifuging and separating the cancer cells with cell stripper
Adding saline based new medium and serum
Mixing and incubating the sample
Washing the cells from free-floating drug
Confirming the drug bindings
Dispensing the cells to be radiated on 96-well sample plate
Illuminating the 96-well sample plate with Modulight ML8500 and ML7710
Incubating to eliminate close-to-death cells for appr. 1.5 h
ML8500 automated illumination system enables the researchers to investigate the effect of multiple independently variable parameters easily and reliably.
- Increase in dose (J/cm2) and in the overall cell death reached saturation at 50 J/cm2
- The efficacy of the drug was dependent on the irradiance. High irradiance (600 mW/cm2) resulted in decreased killing ratio at lower light dose range, which is supported by the literature and suspected to be caused by oxygen depletion in the sample [1] or less effective exhaustion of cell antioxidant responses [2].
- ML8500 automated illumination system enabled variation of 2 independent parameters (dose and irradiance) over 96-well plate in a single experiment.
| 100 mW/cm2 | 600 mW/cm2 | |
|---|---|---|
| Log LLD50 | 0.9908 | 1.317 |
| HillSlope | -2.431 | -4.106 |
| LLD50 | 9.79 | 20.75 |
Fully automated testing
Better validability and reliability
Optimized drug & light dose for the patient
Cited Publications
[1] Henderson, Barbara W. et al. “Fluence Rate as a Modulator of PDT Mechanisms.”Lasers in Surgery and Medicine 38.5 (2006): 489–493. [2] Dos Santos, Ancély F et al. “Fluence Rate Determines PDT Efficiency in Breast Cancer Cells Displaying Different GSH Levels.”
Photochemistry and photobiology (2019).
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