Our protocol provides a simple method for 3D cell culture and is highly amenable to downstream analysis. This facilitates the evaluation of heterogeneous patient samples and their specific drug responses. The advantage of this technique is that spheroids can be formed from small cell numbers, allowing for both preservation of scarce primary samples, and high throughput screening for patient-specific drug responses.
The hanging drop model creates a physiological environment with 3D drug diffusion and does responses corresponding to tumor stage. This allows for the development of targeted therapies and patient-specific treatments. This method is ideal for studying ovarian cancer, heterogeneity and the development of chemo resistance.
However, it can also be used to study other cancers and drug-resistant cell populations. When plating spheroids, it is important to pipette precise volumes. This ensures consistency between spheroids.
The spheroids and their respective droplets are inherently fragile and easily lost without proper handling, therefore we will show how to appropriately plate, maintain and analyze a hanging drop plate. Demonstrating this procedure will be Michael Bregenzer, a graduate student from our laboratory. Start by filling each well of the six-well plate with four to five milliliters of autoclave deionized water and sandwiching the hanging drop plate between the lid and the bottom of the plate.
Add 800 to 1000 microliters of water around the rim of the hanging drop plate to provide a humid environment and minimize evaporation. Next, collect 2D grown cells by detaching them with trypsin, and then adding six or eight milliliters of medium containing FBS. Aspirate the cells with the 10 milliliters serological pipette, and deposit them in a 15 milliliter conical tube.
Use a hemocytometer to count the cells. Prepare cell suspension according to manuscript directions, and mix gently with the pipette to ensure homogenous distribution. Place the tip of the pipette in the well at an angle of 45 degrees and pipette 20 microliters of suspension into each hanging drop well.
Place the lid of the six-well plate back on and use a stretchy thermoplastic strip to seal the edges. Incubate in a standard carbon dioxide humidified incubator and feed the hanging drops every two to three days by adding two to three microliters of cell culture medium to each spheroid-containing well. To quantify proliferation and viability of the cells, add two microliters of filtered resazurin-based solution to the wells designated for proliferation analysis and incubate according to manuscript direction.
After the incubation period, open the hanging drop sandwich in a biosafety cabinet and bring the 384 well plate with the lid still in place to the plate reader. Place it in the plate reader and read the plate. Save the experiment in the popup window and export the data into a spreadsheet.
Return the plate back to the six-well base and place it in the incubator. Once all time points have been read for the day, reseal the plate before incubating. To prepare spheroids for flow cytometry analysis, use a 1000 microliter pipette to collect them from each well and deposit them into a 15 milliliter conical tube for disaggregation.
Aliquot cell suspension into five micro centrifuge tubes so that each tube contains at least 50, 000 cells. Centrifuge the tubes at 400 times G for five minutes in a microcentrifuge, then aspirate the supernatant and re-suspend pellets in 100 microliters of Aldefluor buffer. Label the tubes according to manuscript directions.
Add 0.5 microliters of APC isotype antibody to the APC ISO tube and one microliter of CD133 antibody to the ALDH CD133 tube. Then add five microliters of DEAB reagent and 0.5 microliters of ALDH to the DEAB tube and one microliter of ALDH to the ALDH CD133 tube. Vortex all tubes for a few seconds and incubate them at 37 degrees celsius for 45 minutes.
After the incubation, vortex all tubes again and centrifuge them at 400 times G for five minutes. Label FACS tubes according to manuscript directions and fill an insulated foam container with ice. Aspirate the supernatant from the microcentrifuge tubes.
Then re-suspend the unstained control in 400 microliters of FACS buffer and the rest of the cells in 400 microliters of FACS DAPI buffer. Place the tubes on the ice until they can be analyzed on a flow cytometer. Use FlowJo to analyze the data from the flow cytometer.
Double click the unstained file and set the Y-axis to side scatter height or SSCH and the X-axis to forward scatter height or FSCH. Click the T button next to each axis to adjust the scale and maximize separation between different cell populations. Next, click on the Polygon Gating button, draw polygon gate around the cell population and label the population Cells.
Double click the cell's population in the workspace, and then change the FSC axis to FSC width and the SSC axis to the FSC height. Choose the rectangle gate to only draw a rectangle around the leftmost dense population of cells, spanning the entire Y-axis. Label this gate Single Cells.
Then right click and copy this Cells in the nested Single Cells gate and paste them under each sample in the workspace. Double click the Single Cells gate nested under the DAPI sample to view the single cell population from that sample tube. Change the FSC axis channel to the DAPI area channel and change the SSC axis channel to histogram.
Click on the T button next to the DAPI axis and click on Customize Axis to adjust the scale and maximize separation between DAPI positive and DAPI negative peaks. Click Apply in the popup window and choose the Range Gate button. Spread it over the DAPI negative peak and label this gate Live Cells.
Copy the Live Cells gate and paste it under the Single Cells gate, under the APC ISO DEAB and ALDH CD133 tubes, to select the same portion of live cells in each tube. Then double click the Live Cells population nested under the APC ISO sample file, and switch the X-axis to ALDH area and the Y-axis to APC area. Apply Quadrant Gate and adjust the intersection of the gate such as that approximately 0.5%of the population lies in the upper left of the plot window.
Then name the quadrants as desired, then copy and paste the quadrant gates onto the live cells population nested under the DEAB file and adjust the vertical line so that approximately 0.15%of the cell population lies within the ALDH positive quadrant. Finally, copy and paste the quadrant gates to the ALDH CD133 files Live Cells population and evaluation cancer stem cell proportions based on ALDH and CD133 proportions. This protocol can be used for high throughput analysis of patient-derived cancer stem cells.
As little as 10 cells per well can form reliable spheroids and as the cells proliferate, the spheroids expand in size. Proliferation capacity can be easily quantified with a resazurin-based florescence assay. The effect of drugs on spheroid morphology can be visualized with phase contrast imaging and quantified by comparing resazurin florescence in untreated and treated cells.
Cell death can be validated via addition of Calcein-AM and ethidium homodimer I to spheroids, followed by imaging on a confocal microscope. Finally, spheroids can be harvested and dispersed into a single cells suspension for analysis with flow cytometry, which makes it possible to discern the effectiveness of different drug treatments on specific cell populations. To avoid droplet loss, medium evaporation and variation in spheroid size, it is important to handle your plates carefully, pipette with precision and completely seal your hanging drop plates.
To evaluate changes in gene expression and soluble signaling due to drug treatment, single cell RNA sequencing and enzyme-linked immunosorbent assays, can be used to facilitate the development of therapeutics. This technique allows researches in oncology, precision medicine and drug development to explore intra and inter patient heterogeneity as well chemo resistance through high throughput drug screening of small biopsies. When performing this protocol, be sure to wear all standard personal protective equipment.
Including gloves, safety glasses, lab coats, pants and close-toed shoes.
