This protocol provides experimental settings to perform controllable 2D and 3D co-cultures in micro-devices to visualize and monitor tumor-immune cell crosstalks and analyze the effects of anti-cancer treatments. This technology provides real-time and easy visualization of immune cell recruitment and interactions and can be employed for human and patient-derived samples. It is compatible with most state-of-the-art microscopies.
Demonstrating the procedure will be Francesco Noto, PhD student at the Department of Oncology and Molecular Medicine, Istituto Superiore di Sanita, and Nicoleta Manduca and Esther Maccafeo, PhD students at Universita Cattolica del Sacro Cuore, Department of Translational Medicine and Surgery. Chips are previously plasma-activated in a plasma cleaner or reactive iron-etching machine in clean room. Murine-spleen cells and a tumor cell line is used here for mimicking the protocols described in the text.
Before loading cell suspensions, withdraw media from all six reservoirs. Then slowly load 1 x 10 to the 5th tumor cells resuspended in 10 to 50 microliters of growth medium in the upper left-hand reservoir and lower well. On the right side, gently pipette 1 x 10 to the 6th floating immune cells resuspended in 50 microliters of growth medium into two wells.
When all the cells have been added, fill all six reservoirs of each chip with up to 100 to 150 microliters of growth medium and check that the cells have been correctly distributed within each culture compartment. Incubate the chip for one hour to allow the system to stabilize before the time-lapse recording. For live cell imaging, mount the tumor immune on the chip plate on the microscope stage.
Customize the acquisition workflow setting in the microscope software interface such as Incucyte Live-Cell Analysis Software. Select windows of observation, the optimal frame rate and time duration, depending on the appropriate parameters for the experiment and cell types under study. Image the cells for the appropriate experimental time period.
Prepare two aliquots of matrigel diluted with medium containing a drug or a combination of drugs for the two experimental conditions using cold tips. Gently pipette live-compatible, fluorescent dye-stained tumor cells suspended in matrix solution on ice to obtain a homogenous distribution of the cells. With the chip plate on a cooling block or on a basket with ice, slowly inject the drug-treated tumor cell matrix solution into the left and right gel ports using cold 10 microliter micropipette tips.
Apply gentle pressure to push the matrix solution from one side of the channel to the other. When all tumor cells have been loaded, place the device into the incubator in the upright position for 30 minutes. At the end of the incubation, once the matrix gelation is completed, fill the medium channels of all six reservoirs with 50 microliters of culture medium.
Check the polymerized gel integrity and tumor cell distribution under a microscope. Then place the chips in an incubator until immune cell preparation is complete. After incubation, aspirate the medium from each well.
Place the tip near the inlet of the middle medium channel and use moderate pressure to gently inject 10 microliters of 10 to the 6th immune cells labeled with a contrasting fluorescent dye. Inject 50 to 100 microliters of the medium into each of the four wells of lateral channels, 40 to 90 microliters of the medium into the upper central well, and 50 to 100 microliters of the medium into the lower central well. When all the wells have been loaded, use a microscope to confirm that the immune cell distribution has remained confined to the central chamber.
Then carefully place the device on a level surface in the cell culture incubator until imaging. To calculate the extent of the fluorescently-stained live immune cells infiltrating the tumor compartments, image the device at specific time points of interest by fluorescence microscopy and set the appropriate camera parameters in the microscope imaging software, such as Nikon NIS-Elements. Also set the parameters for labeled immune cells.
The movement of leukocytes through suitably built microchannel bridges in the microfluidic platform toward their target cells can be tracked by video microscopy. In this tracking analysis, individual PBMCs were challenged with dying doxorubicin-treated or live PBS-treated cancer cells. Relevant chemotaxis values and migration plots were automatically generated, and a different migratory profile for the immune cells was observed when co-loaded with breast cancer cells exposed to doxorubicin or PBS.
When the PBMCs were confronted with apoptotic cancer cells, they crossed microchannels toward the dying and dead cells but did not cross to live, untreated cells. A fraction of leukocytes with increasing density over 24 to 48 hours exhibited long-term contacts with doxo-treated cancer cells. In this analysis, a novel 3D immunocompetent tumor model was used to quantify the recruitment of immune cells in response to anti-cancer combinations of epigenetic drugs.
Red-dye-labeled PBMCs were then distributed homogeneously into the central fluidic chamber. The ability of the two tumor masses to attract the PBMCs was then compared, with the PBMCs being robustly recruited in this experiment into the right-side microchannel. For the 3D model, mix the solution with hydrogel and cells on ice, avoiding bubbles and pre-polymerization.
Inject it slowly without excessive pressure. Tune polymerization steps according to the matrix used. Live/dead cell assays and cytokine secretion profiling from supernatants can be implemented.
Immunofluorescence for confocal imaging can be performed to classify immune subtypes and for expression markers of activation and exhaustion maturation.
