Análise alto teor de triagem para avaliar os efeitos toxicológicos de Constituintes nocivas e potencialmente prejudiciais (HPHC)

The overall goal of this approach, which combines an Impedance-Based Real Time Cell Analyzer, and a High Content Screening-Based Platform, is to provide information on effective doses, toxicity, and biological effects, to obtain the Tox Profile of the tested compounds. This approach can help answer key question in the field of Toxicology. Addressing, for example, cytotoxiticity, genotoxicity, and cellular stress responses.

Moreover, not only provide information about multiple biochemical and morphological parameters simultaneously, it can also be applied to a different range of cell types, and cell culture models. Due to jurisdiction and limitation, the entire approach will be performed while assessing only a short piece of compound. For the same reason, only one protocol will be shown for demonstrating the High Content Screening Assessment.

Demonstrating the procedure will be Stefano Acali and Alexander Laurent, technicians from my laboratory. To see the normal human bronchial epithelial cells, or NHBE cells, program the instrument to define the number and duration of impedance measurements. In this study, data is recorded every 15 minutes, for 48 hours.

Measure the plate background by pipetting 50 microliters of pre-warmed medium into each well of a 96-well RTCA plate. This step represents a technical requirement for the instrument to calculate the medium electrical resistance to use as a baseline reference for cell-based calculations. Next, add 50 microliters of cell suspension at a concentration of 144, 000 cells per milliliter to the medium already in each well of the RTCA plate.

Once we cell has heated, it's very important to leave the plate at room temperature for 40 minutes to allow the cells to attach and obtain homogeneous cell distribution. Incubate the RTCA plates in the cradle in the incubator, and start recording the data for the next 24, plus or minus two hours, prior to dosing. For the positive control, dilute the 10 millimolar staurosporine stock solution, one to 10, in DMSO.

Add five microliters of the dilution to 195 microliters of medium to obtain a 5x working solution. For the harmful and potentially harmful constituents, dissolve each of them in the vehicle to generate a one molar stock solution. Then, dilute each test substance stock solution, one to 10, in medium to generate a 100 millimolar solution.

Generate the compound master plate by first adding medium plus 10%vehicle in wells from B to F, and the 100 millimolar solution in well A.Perform a five step, one to 10, serial dilution to obtain the 5x working solutions. For dosing, pause the RTCA instrument, open the cradle to remove the exposure plate, and place the plate in the RTCA temperature tool to avoid cooling the cells, which could impact the impedance measurement. Add 25 microliters of 5x solution from the Compound master plate to the cells in triplicate, maintaining the same dosing order as in the Compound master plate.

Do not remove the existing cell culture medium. Then, add 25 microliters of 5x Positive control solution to the cells in the right half of the bottom row, without removing existing culture medium. Similarly, add 25 microliters of medium to the cells in the left half of the bottom row.

After sealing the plate with the plate-sealer, place the plate back in the RTCA cradle, and lock it. Restart the data recording for the desired exposure time. Following data recording, analyze the RTCA cell viability data to determine which test substance to use in the HCS analysis.

In this experiment, 1-aminonaphtalene, Arsenic, Chromium, and Crotonaldehyde are selected for HCS analysis. To see the NHBE cells, add 100 microliters of a cell suspension at 120, 000 cells per milliliter to each well of a 96-well black plate. After leaving the HCS plate a room temperature for 30 minutes, to allow cells to attach to the wells, incubate at 37 degrees Celsius and 5%CO2 for 24, plus or minus two hours, prior to dosing.

For the positive control, dispense 40 microliters of the stock solution of each positive control in column two. Then, dispense 20 microliters of the vehicle in columns three and five. And 40 microliters of the vehicle in column four.

Withdraw 12 microliters from the wells in column two, dispense it to the wells in column three, and mix. Continue until a final serial dilution with three doses and vehicle for each positive control compound is obtained. To generate the positive control plate, prepare a one to 40 dilution in media of the three doses and vehicle.

To make the compound master plate, re-suspend and dilute each test substance stock solution, one to 10 in medium for a concentration of 100 millimolar. Perform dilutions using medium with 10%vehicle to obtain the selected 5x doses for each test substance. Both positive controls and compound plate are now ready to be dosed over the exposure plate.

Next, add 25 microliters of 5x solutions from the Compound master plate to the cells in triplicate, maintaining the same dosing order as in the Compound master plate. Do not remove the existing cell culture medium. Then, add 25 microliters of the 5x solution from the Positive control plate to the cells in the bottom row, maintaining the same dosing order as in the Positive control plate.

Prepare a sufficient volume of the live-cell staining solution as described in the text protocol. Then, dilute both the mitochondria dye and the membrane permeability dye in the live-cell staining solution, according to the vendor's instructions. Add 50 microliters of the live-cell staining solution to each well of the plate designated Cytotoxicity Assay.

Do not remove cell culture medium, and incubate for 30 minutes at 37 degrees Celsius, 5%CO2. Gently aspirate the medium and staining solution and add 100 microliters of Fixation Solution to each each well. Then, incubate the plate for 20 minutes at room temperature, in the dark.

Following incubation, gently aspirate the Fixation Solution and wash once with 100 microliters per well of wash buffer. Remove the wash buffer, and add 100 microliters per well of 1x permeabilization buffer to each well, before incubating for 10 minutes at room temperature, in the dark. Aspirate the permeabilization buffer, and wash the plate twice with 100 microliters, per well, of wash buffer.

Then, aspirate the wash buffer and add 100 microliters of 1x Blocking buffer to each well. Then, incubate for 15 minutes at room temperature, in the dark. Dilute the Anti-Cytochrome C Antibody, one to 250, in Primary Antibody Solution.

Next, aspirate the Blocking buffer and add 50 microliters, per well, of Primary Antibody Solution to each well, before incubating for 60 minutes at room temperature, in the dark. Dilute the Anti-Mouse Antibody, one to 500, in Secondary Antibody and Nuclear Solution. Also, dilute the nuclear dye, one to 1, 000, in Secondary Antibody and Nuclear Solution.

After aspirating the Primary Antibody Solution, wash the plate three times with 100 microliters, per well, of wash buffer, using the plate washer. Aspirate the wash buffer, and add 50 microliters, per well, of Secondary Antibody and Nuclear Solution to each well of the plate. Now, incubate the plate for 60 minutes at room temperature, in the dark.

Following aspiration of the Secondary Antibody and Nuclear Solution, wash the plate three times with 100 microliters of, per well, of wash buffer using the plate washer. Then, add 100 microliters, per well, of wash buffer. The plate is now ready to be evaluated on the HCS instrument and the acquistion is started.

Representative RTCA cell viability results of test substance treatment are shown. A toxic dose-dependent effect is obtained over 24 hours of exposure only with 1-aminonaphtalene, Arsenic, Chromium, and Crotonaldehyde, as they all displayed a computed LD50 of less than 20 millimolar. No toxic effect was obtained with the other compounds.

Shown here, are representative HCS results only for 1-aminonaphtalene treatment. The toxic effect of 1-aminonaphtalene is inducing a severe damage of the cell membrane, which leads to an increased cell membrane permeability in a dose-dependent manner, after four hours of exposure. The compound is also affecting the mitochondria, inducing a mass increase after four hours of exposure, along with the release of Cytochrome C, which is then detectable in the nuclei after four and 24 hours of exposure.

Once mastered, this technique can be executed within two to three days, as most of them require about an hour per day of hands-on time for setting and dosing. Depending on the protocol selected, the High Content Screening requires a maximum of three and 1/2 hours for the execution of the entire staining procedure. While attempting this procedure, keep in mind that although selection of a broader range of endpoints add complexity to the workflow, it will not substantially increase the time.

With an optimal workflow, in fact, several plates can be assessed in parallel. Continued development of the High Content Screening Platform, including fully automated cell setting, compound dilution, dosing and staining, and the addition of new endpoint, will further expand the capability of the High Content Screening Platform as a powerful tool for toxicological profiling. Moreover, the combination of multiple, single toxicological endpoints in such high throughput will bring us one step further in our effort to reduce chemical testing in animals.