This protocol demonstrated a standardized method of constructing three-dimensional tumors of spheroids. And this methodology provided a high circuit and high content analysis of 3D tumor constructs. By using a standard method of tumorous spheroid construction and a high throughput imaging and analysis system, the effectiveness and accuracy of drug tests formed on three-dimensional spheroids can be dramatically increased.

In this protocol, we used AMG510 to treat NCI-H23 spheroid as an example. From the experiment, we could observe a significant effect of cancerous targeted drug on the tumor spheroids. To begin, pipette 100 microliters of anti adhesion reagent into each well of a 48 well plate with a U-shaped well bottom and keep for 10 minutes.

After 10 minutes, aspirate the coating reagent and wash twice with sterilized PBS. Place the culture plate in an incubator at 37 degrees Celsius in humidified air with 5%carbon dioxide until use. Observe the cells under the microscope.

Then, wash the cells cultured in a T25 flask twice with PBS to remove the culture medium and treat the expanded cells with one milliliter of 0.25%trypsin EDTA for one to two minutes in an incubator at 37 degrees Celsius, 5%carbon dioxide. Confirm the cell shape under the microscope. And then stop the trypsin treatment by aspirating the used trypsin EDTA suspension in the T25 flask and washing the cells with four milliliters of fresh medium.

Transfer all of the suspension to a 15 milliliter tube and use one milliliter of fresh medium to wash down the residual cells and add it to the tube. Centrifuge the cells at 186.48 G for five minutes at room temperature and discard the supernatant. Add 10 milliliters of fresh medium to the cell pellet and gently pipette until the cells are in a homogeneous suspension.

Aspirate 0.1 milliliters of cell suspension into a new centrifuge tube. Add 0.9 milliliters of fresh medium and then pipette the suspension well. Extract 10 microliters of the cell suspension for cell counting.

Repeat this process once or twice and take an average value. Dilute the suspension to reach a final seeding density of 50, 000 cells per milliliter. Next, add 200 microliters of the cell suspension to each well of a 48 well U-bottom plate.

Wrap the sealing film around the plate and centrifuge it at 119.35 G for five minutes at room temperature. After centrifugation, pull off the protective film and add five to eight milliliters of sterilized water into the water channel surrounding the wells. Incubate the plate at 37 degrees Celsius for five days.

Do not change or supplement any water to the water channel during this period and observe the cell aggregation during the following five days. For the gel embedding, after taking out the frozen gel from the minus 20 degrees Celsius fridge, place it on an ice box for the whole time during the experiment. Observe the cell spheroids under the microscope.

Before the gel embedding begins, the status of the spheroids should again be checked. Carefully remove 150 microliters of the medium and embed each spheroid in the gel by slowly adding the liquid gel from the wall side of the well while moving the pre-cooled pipette tip around and inside the well. Wait for five minutes and if the gel does not spread evenly, gently pipette the gel with a 10 microliter pipette tip.

Each well contains a tumor spheroid, 25 microliters of 3.5 milligrams per milliliter gel, and 50 microliters of complete culture medium. Add 75 microliters of medium to the controls as well. Incubate the plate at 37 degrees Celsius for 30 minutes until the hydrogelation is fully completed.

Confirm the gelation status under the microscope. Overlay 125 microliters of fresh medium on each sample and culture the spheroids for another 7 to 10 days. Prepare groups of spheroids with four to six wells each and choose at least three of them for analysis.

Dissolve the drug according to the manufacturer's instructions and prepare 100 fold working solutions with DMSO. Use 0.1%DMSO as a positive control and add 125 microliters of drug-treated medium to each well. Place the plate back into the incubator at 37 degrees Celsius in humidified air with 5%carbon dioxide.

At this stage, each well contains a tumor spheroid, 25 microliters of 3.5 milligrams per milliliter gel, and 175 microliters of the medium. The controls contain 200 microliters of the medium. Measure the spheroid viability using an alamarBlue assay kit according to the manufacturer's guidelines.

Aspirate 100 microliters of the supernatant medium from each well to a new test plate. Then, measure the viability using a microplate photometer. Measure it on day one, day four, day seven, and day 10 after embedding the spheroids in the gel.

Add 80 microliters of fresh medium to each well of the culture plate. Then replace another 100 microliters of the drug-treated medium. Ensure there are no remains of alamarBlue in the well.

Aspirate 100 microliters of the medium out before imaging and place the plate on the stage. Obtain digital images of the spheroids using an automated microscope with a tenfold objective. The microscope can focus and centralize these spheroids automatically.

Wait for the automatic imaging. Four images are acquired for each spheroid. An integrated image is formed and processed with the software connected to the high content imaging system.

Click on the image patch process button and choose the integrated images in the software. Choose U net model and type in the conversion rate. Click at the bottom of the screen to start the image processing.

Save the diameter, perimeter, and roughness data in the spreadsheet software. Finally, add 100 microliters of fresh medium with the drug and place the plate back into the incubator at 37 degrees Celsius in humidified air with 5%carbon dioxide. The brightfield images of NCI-H23 cell spheroids treated with different concentrations of AMG510 and automatically captured by a high content microscope are shown in this figure.

The columns represent different days and the rows represent different drug concentrations. The results include three spheroids for each condition. Tumor spheroid viability of the AMG510 treated sample groups was measured on day one, day four, day seven, and day 10.

The terminal cell viability of the samples with concentration gradients is shown in this figure. The tumor spheroid diameters were measured on day one, day four, day seven, and day 10. The spheroid growth ratio was defined as the terminal volume relative to the original volume and calculated using the spheroid diameters.

The spheroid growth inhibition ratio was defined relative to the volume and calculated using the spheroid diameters. The tumor roughness was measured by the software on day one, day four, day seven, and day 10, indicating the invasiveness of the tumor spheroids. The spheroid's perimeter was located and drawn by the software using deep learning algorithms.

The spheroid areas at the focal plane were then measured by image J and an excess perimeter index was calculated based on these data. Although this message is easy to follow, the samples still need to be handled carefully.