A Spheroid Killing Assay by CAR T Cells

This method can help to answer key question in the immunotherapy field about the assessment of immune cell cytotoxicity against 3D structure tumor cells. The main advantages of this method are that it is straightforward and it combines advanced imaging technology and a sensitive immunological assay. Begin by washing the colorectal cancer cell line of interest with five milliliters of PBS and removing the cells from the flask bottom with 0.5 milliliters of trypsin for five minutes at 37 degrees Celsius.

After confirming detachment under a microscope, neutralize the cell dissociation enzyme with 10 milliliters of complete medium and collect the cells by centrifugation. Resuspend the pellet in five milliliters of fresh complete medium for counting and resuspend the cells at a 1.5 times 10 to the third cells per milliliter concentration. Then seed 200 microliters of cells into each well of 96-well round bottom plate and place the plate in an automated imaging apparatus inside a 37 degree Celsius incubator with 5%CO2 and 95%humidity.

Log in to the acquisition software of the apparatus and select Schedule to Acquire, Launch Add Vessel, Scan on Schedule, and Create Vessel New. Next, select scan type Spheroid and select the appropriate bright-field and fluorescence channels of interest. Set the magnification to 10 times and select the plate model and its position within the imaging apparatus drawer.

Select the position of the wells to be imaged and enter the description of the experiment including the name, type of cells, and number of cells. For the analysis setup, select Defer Analysis Until Later, right click on the timeline, and select Set Selected Scan Group Interval and set Add Scans Every to four hours and For a Total of to 24 hours. Then set the starting time to at least one hour after the incubation in the automated imaging apparatus.

To check the spheroid growth progress, every two days, log in to the imaging software and select View Recent Scans. Double click on the experiment of interest and select Brightfield in the Image Channels panel. Then use the Measure Image Features tool to measure the diameter of the spheroids, adding 50 microliters of complete medium per well at day four to limit any medium evaporation effects.

When the spheroids reach the appropriate experimental size, carefully angle the plate and use a multi-channel pipette to gently remove 150 microliters of complete medium from each well without disturbing the spheroids. Next, replace the discarded medium with 50 microliters of a one to 200 solution of Annexin V Red and place the plate in a cell culture incubator for 15 minutes. Centrifuge transduced CAR CD19 T cells in a 15 milliliter conical tube and resuspend the pellet in two milliliters of complete medium.

After counting, dilute the cells to a two times 10 to the fifth cells per milliliter of complete medium concentration. Then add 100 microliters of CAR CD19 T cells to each spheroid-containing well and return the plate to the automated imaging apparatus. In the acquisition software, select Schedule to Acquire and right click on the scan timeline.

Select Edit Timeline and right click on the scan group to delete it. Right click on the timeline again and select Set Selected Scan Group Interval and set Add Scans Every to 1 1/2 hours and For a Total of to 24 hours. Then set the imaging start time to at least one hour after the start of the incubation in the automated imaging apparatus and select Save Schedule Scans.

For automated image analysis, in the scan software, select View Recent Scans and Launch Analysis. Select Create New Analysis Definition and analysis type Spheroid and set the image channels to be analyzed. Select at least 10 representative images and preview the default analyze procedure on the entire image stack.

Modify the parameters for the bright-field mask and preview the image stack, confirming that the selected parameters detect the spheroids accurately. Modify the parameters for green mask and preview the image stack to confirm that the selected parameters detect the spheroids accurately. Modify the parameters for red mask and preview the image stack to confirm that the selected parameters detect the spheroids accurately.

Take care to achieve the best signal over noise and sensitivity over specificity ratios, keeping in mind that you won't be able to find a set of parameters that will permit to capture every event at every time point. Then launch the analyzer. When the analysis is complete, extract the measurement of interest and select the analyzed file and the graph metrics option.

Select the metrics of interest, the scan, and the well. Click Export Data to extract the selected metrics in several file formats. Then, to extract the images, select the analyzed file and select Export Images and Movies.

CD19 CAR expression on transduced T cells and CD19 expression on transduced colorectal cancer cells can be confirmed by flow cytometry. Here, the outcome of a typical spheroid experiment can be observed. Unlike mock T cells, CD19 CAR T cells are able to specifically kill the colorectal cancer cell line-derived spheroids, greatly diminishing the number of live tumor cells.

Tracking of the evolution of the total green and red signals within the spheroid boundaries over time reveals that shortly after CD19 CAR T cell injection, the size of the spheroids shrinks quickly as the apoptosis signal increases quickly. Following this procedure, other methods like toxicity assays, cell migration assays, or spheroid structure measurements can be performed to answer additional questions about drug screening, T cell motility, or spheroid formation, respectively.