Establishment of a Co-culture System of Patient-Derived Colorectal Tumor Organoids and Tumor-Infiltrating Lymphocytes (TILs)

The research aim to explore tumor-infiltrating Lymphocytes interactions with colorectal cancer organoids, and the therapeutic potential of TIL based therapy for personalized colorectal cancer treatments. Our protocol simplified tumor-infiltrating Lymphocytes prevention from Lymphocytes and enables their co-culture with colorectal cancer organoids overcoming inferior efficacy and common morbidity in patients.

[Narrator] To begin prepare the experimental reagents and lab wear for use. Then rinse the primary colorectal cancer tumor tissue three times with five milliliters of PBS followed by tissue wash buffer. With tissue scissors, mince the tissue into small fragments. Transfer the minced tissue into the CRC digestion buffer. Incubate the sample in a water bath set to 37 degrees Celsius for 30 minutes until the tissue is fully dissociated. Then add an equal volume of Ad DMEM to the digested sample to neutralize the reaction. Centrifuge the suspension at 380G for five minutes at 25 degrees Celsius. Now discard the supernatant from the centrifuged sample using a pipette. Pipette one to three milliliters of prewarmed erythrocyte lysis buffer to the pellet and incubate the sample at 25 degrees Celsius for 10 minutes. Add an equal volume of Ad DMEM to terminate lysis. Centrifuge the tube at 380G for five minutes at approximately 25 degrees Celsius. After discarding the supernatant re suspend the cell pellet in an appropriate volume of Ad DMEM. Add trypan blue to the suspension to quantify viable cells and obtain a single cell suspension of the tumor tissue.

Divide the single cell suspension of tumor tissue into two portions. Use one portion for establishing the patient derived organoid model and the other for culturing tumor-infiltrating lymphocytes. After counting the cells mix 80,000 cells per well with Matrigel at a one-to-one ratio for organoid culture. Then re suspend the remaining cell suspension in complete medium in a 24 well plate. Collect the tumor-infiltrating lymphocytes into a centrifuge tube. Rinse the well plate with fresh medium to recover any remaining cells. Transfer them to the same centrifuge tube and centrifuge. After discarding the supernatant, wash the pellet with two milliliters of PBS and centrifuge again. Next, use CD3 positive magnetic beads to sort and isolate CD3 positive T cells. Place the sorting column into the magnetic stand. Then transfer the cell suspension to the column and allow magnetic bead labeled cells to remain in the column. Once sorting is complete, remove the sorting column from the magnet. Then add buffer to elute the retained positive cells. Re suspend the eluted cells in two milliliters of PBS and centrifuge. Discard the supernatant using a pipette. Re suspend the cells in a 12 well plate with fresh EMEM medium at a concentration of 1 million cells per milliliter. Add CD3 antibody to a final concentration of five micrograms per milliliter. Add interferon gamma to the organoid medium to enhance antigen presentation. Incubate the culture at 37 degrees Celsius with five percent carbon dioxide for 24 hours. Dilute the anti CD28 antibody with PBS to a concentration of two micrograms per milliliter. Coat each well of a 96 well plate by adding 50 microliters of the antibody solution. Then seal the plate with sealing film and incubate. After 24 hours, use TrypLE to dissociate the organoids into single cells and count them. Then wash the cells twice with PBS. Collect the paired tumor-infiltrating lymphocytes. Count the cells using a cell counter or hemocytometer and wash them twice with PBS. Inoculate organoids into each well at 10,000 cells per well. Inoculate tumor-infiltrating lymphocytes at 100,000 cells per well to achieve an effector to target ratio of 10 to one. Perform the co-culture in supplemented RPMI 1640 medium. Incubate the co-culture for three days and capture images for observation and documentation. At the end of the co-culture period collect the patient derived organoids and tumor-infiltrating lymphocytes into a centrifuge tube. Centrifuge the collected cells twice in PBS at 300G for five minutes each time. Then, prepare the antibody cocktail containing CD3 FITC, CD4 APC-Cyanine7, CD8 APC, CD107 APE-Cyanine7, CD279 PE and 7-AAD. Re suspend the washed cells in 100 microliters of the antibody cocktail and incubate for 20 minutes. Add one milliliter of PBS to wash the cells. Then centrifuge the tube at 300G for five minutes. Discard the supernatant using a pipette. Finally, re suspend the cell pellet in 500 microliters of PBS before performing flow cytometric analysis. Patient derived organoids showed visible expansion over a 12 day culture period with small clusters observed on day one, larger and more numerous structures by day six and well-defined mature organoids by day 12. Organoids exhibited similar histological architecture to colorectal cancer tissue as shown by hematoxylin and eosin staining. Immuno staining revealed cytokeratin-7 expression in both tissue and organoids confirming epithelial identity with cytokeratin-20 also showing strong expression in both sample types. Similar expression patterns were also observed for Ki-67, a proliferation marker and caudal type homeobox 2, an intestinal differentiation marker. Tumor-infiltrating lymphocytes showed progressive expansion from sparse distribution on day one to noticeable clustering by day 14 and dense aggregation by day 22. Immunofluorescence staining showed strong CD45 and CD3 signals in tumor-infiltrating lymphocytes confirming the immune origin of the cells. Absence of EpCAM staining in the tumor-infiltrating lymphocytes population confirmed that the expanded cells were free of epithelial tumor cells.